U.S. patent application number 12/778010 was filed with the patent office on 2011-05-26 for methods for the diagnosis of metabolic disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmental influencers.
Invention is credited to John Patrick McCook, Niven Rajin Narain, Rangaprasad Sarangarajan.
Application Number | 20110123986 12/778010 |
Document ID | / |
Family ID | 43085533 |
Filed Date | 2011-05-26 |
United States Patent
Application |
20110123986 |
Kind Code |
A1 |
Narain; Niven Rajin ; et
al. |
May 26, 2011 |
METHODS FOR THE DIAGNOSIS OF METABOLIC DISORDERS USING EPIMETABOLIC
SHIFTERS, MULTIDIMENSIONAL INTRACELLULAR MOLECULES, OR
ENVIRONMENTAL INFLUENCERS
Abstract
Methods and formulations for diagnosing metabolic disorders in
humans using epimetabolic shifters, multidimensional intracellular
molecules or environmental influencers are described.
Inventors: |
Narain; Niven Rajin;
(Cambridge, MA) ; McCook; John Patrick; (Frisco,
TX) ; Sarangarajan; Rangaprasad; (Rutland,
MA) |
Family ID: |
43085533 |
Appl. No.: |
12/778010 |
Filed: |
May 11, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61177241 |
May 11, 2009 |
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61177243 |
May 11, 2009 |
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61177244 |
May 11, 2009 |
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61177245 |
May 11, 2009 |
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61177246 |
May 11, 2009 |
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Current U.S.
Class: |
435/6.11 ;
435/6.19; 436/501 |
Current CPC
Class: |
A61P 1/16 20180101; A61P
9/00 20180101; C12Q 2600/16 20130101; A61K 31/00 20130101; C12Q
1/6883 20130101; A61P 9/04 20180101; A61P 3/04 20180101; A61P 43/00
20180101; C12Q 2600/136 20130101; A61K 31/122 20130101; A61P 9/10
20180101; C12Q 2600/158 20130101; G01N 33/57484 20130101; A61P 3/10
20180101; G01N 33/5735 20130101; G01N 2570/00 20130101; A61P 35/02
20180101; C12Q 2600/112 20130101; G01N 2800/52 20130101; A61P 3/06
20180101; A61P 35/04 20180101; C12Q 1/6886 20130101; A61P 7/02
20180101; A61P 9/12 20180101; A61P 3/08 20180101; A61P 13/12
20180101; A61P 35/00 20180101; C12Q 2600/106 20130101; G01N
2800/042 20130101; A61P 3/00 20180101 |
Class at
Publication: |
435/6 ;
436/501 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53 |
Claims
1. A method of assessing whether a subject is afflicted with a
metabolic disorder, the method comprising: (1) determining the
level of expression of a marker present in a biological sample
obtained from the subject, wherein the marker is selected from the
group consisting of the markers listed in Tables 2-4 & 6-29 and
64-69; and (2) comparing the level of expression of the marker
present in the biological sample obtained from the subject with the
level of expression of the marker present in a control sample,
wherein a modulation in the level of expression of the marker in
the biological sample obtained from the subject relative to the
level of expression of the marker in the control sample is an
indication that the subject is afflicted with a metabolic disorder,
thereby assessing whether the subject is afflicted with a metabolic
disorder.
2. A method of assessing whether a subject is afflicted with a
metabolic disorder, the method comprising: (1) determining the
level of expression of a marker present in a biological sample
obtained from the subject, wherein the expression of the marker is
modulated in a disease cell of the metabolic disorder induced to
undergo a cellular metabolic energy shift towards normal
mitochondrial oxidative phosphorylation; and (2) comparing the
level of expression of the marker present in the biological sample
obtained from the subject with the level of expression of the
marker present in a control sample, wherein a modulation in the
level of expression of the marker in the biological sample obtained
from the subject relative to the level of expression of the marker
in the control sample is an indication that the subject is
afflicted with a metabolic disorder, thereby assessing whether the
subject is afflicted with a metabolic disorder.
3. A method of prognosing whether a subject is predisposed to
developing a metabolic disorder, the method comprising: (1)
determining the level of expression of a marker present in a
biological sample obtained from the subject, wherein the marker is
selected from the group consisting of the markers listed in Tables
2-4 & 6-29 and 64-69; and (2) comparing the level of expression
of the marker present in the biological sample obtained from the
subject with the level of expression of the marker present in a
control sample, wherein a modulation in the level of expression of
the marker in the biological sample obtained from the subject
relative to the level of expression of the marker in the control
sample is an indication that the subject is predisposed to
developing a metabolic disorder, thereby prognosing whether the
subject is predisposed to developing a metabolic disorder.
4. A method of prognosing whether a subject is predisposed to
developing a metabolic disorder, the method comprising: (1)
determining the level of expression of a marker present in a
biological sample obtained from the subject, wherein the expression
of the marker is modulated in a disease cell of the metabolic
disorder induced to undergo a cellular metabolic energy shift
towards normal mitochondrial oxidative phosphorylation; and (2)
comparing the level of expression of the marker present in the
biological sample obtained from the subject with the level of
expression of the marker present in a control sample, wherein a
modulation in the level of expression of the marker in the
biological sample obtained from the subject relative to the level
of expression of the marker in the control sample is an indication
that the subject is predisposed to developing a metabolic disorder,
thereby prognosing whether the subject is predisposed to developing
a metabolic disorder.
5. A method for assessing the efficacy of a therapy for treating a
metabolic disorder in a subject, the method comprising: comparing
(1) the level of expression of a marker present in a first sample
obtained from the subject prior to administering at least a portion
of the treatment regimen to the subject, wherein the marker is
selected from the group consisting of the markers listed in Tables
2-4 & 6-29 and 64-69; with (2) the level of expression of the
marker present in a second sample obtained from the subject
following administration of at least a portion of the treatment
regimen, wherein a modulation in the level of expression of the
marker in the second sample as compared to the first sample is an
indication that the therapy is efficacious for treating the
metabolic disorder in the subject.
6. A method for assessing the efficacy of a therapy for treating a
metabolic disorder in a subject, the method comprising: comparing
(1) the level of expression of a marker present in a first sample
obtained from the subject prior to administering at least a portion
of the treatment regimen to the subject, wherein the expression of
the marker is modulated in a disease cell of the metabolic disorder
induced to undergo a cellular metabolic energy shift towards normal
mitochondrial oxidative phosphorylation; with (2) the level of
expression of the marker present in a second sample obtained from
the subject following administration of at least a portion of the
treatment regimen, wherein a modulation in the level of expression
of the marker in the second sample as compared to the first sample
is an indication that the therapy is efficacious for treating the
metabolic disorder in the subject.
7. A method of assessing the efficacy of an environmental
influencer compound for treating a metabolic disorder to in a
subject in need thereof, the method comprising: (1) determining the
level of expression of one or more markers present in a biological
sample obtained from the subject, wherein the biological sample is
exposed to the environmental influencer compound, and wherein the
marker is selected from the group consisting of the markers listed
in Tables 2-4 & 6-29 and 64-69 with a positive fold change
and/or with a negative fold change; (2) determining the level of
expression of the one or more markers present in a second
biological sample obtained from the subject, wherein the sample is
not exposed to the environmental influencer compound; and (3)
comparing the level of expression of the one of more markers in the
biological sample exposed to the environmental influencer compound
and the level of expression of the one of more markers in the
biological sample not exposed to the environmental influencer
compound, (4) wherein a decrease in the level of expression of the
one or more markers with a negative fold change present in the
biological sample exposed to the environmental influencer compound
relative to the level of expression of the one or more markers
present in the second sample is an indication that the
environmental influencer compound is efficacious for treating the
metabolic disorder in the subject in need thereof, and, wherein an
increase in the level of expression of the one or more markers with
a positive fold change present in the biological sample exposed to
the environmental influencer compound relative to the level of
expression of the one or more markers present in the second sample
is an indication that the environmental influencer compound is
efficacious for treating the metabolic disorder in the subject in
need thereof, thereby assessing the efficacy of the environmental
influencer compound for treating the metabolic disorder.
8. A method of assessing the efficacy of an environmental
influencer compound for treating a metabolic disorder to in a
subject in need thereof, the method comprising: (1) determining the
level of expression of one or more markers present in a biological
sample obtained from the subject, wherein the biological sample is
exposed to the environmental influencer compound, and wherein the
expression of the marker is up- or down-regulated, in a disease
cell of the metabolic disorder induced to undergo a cellular
metabolic energy shift towards normal mitochondrial oxidative
phosphorylation; (2) determining the level of expression of the one
or more markers present in a second biological sample obtained from
the subject, wherein the sample is not exposed to the environmental
influencer compound; and (3) comparing the level of expression of
the one of more markers in the biological sample exposed to the
environmental influencer compound and the level of expression of
the one of more markers in the biological sample not exposed to the
environmental influencer compound, (4) wherein a decrease, in the
biological sample exposed to the environmental influencer compound,
in the level of expression of the one or more down-regulated
markers relative to the level of expression of the one or more
markers present in the second sample is an indication that the
environmental influencer compound is efficacious for treating the
metabolic disorder in the subject in need thereof, and, wherein an
increase, in the biological sample exposed to the environmental
influencer compound, in the level of expression of the one or more
up-regulated markers relative to the level of expression of the one
or more markers present in the second sample is an indication that
the environmental influencer compound is efficacious for treating
the metabolic disorder in the subject in need thereof, thereby
assessing the efficacy of the environmental influencer compound for
treating the metabolic disorder.
9. A method of identifying a compound for treating a metabolic
disorder in a subject, the method comprising: (1) obtaining a
biological sample from the subject; (2) contacting the biological
sample with a test compound; (3) determining the level of
expression of one or more markers present in the biological sample
obtained from the subject, wherein the marker is selected from the
group consisting of the markers listed in Tables 2-4 & 6-29 and
64-69 with a positive fold change and/or with a negative fold
change; (4) comparing the level of expression of the one of more
markers in the biological sample with a control sample not
contacted by the test compound; and (5) selecting a test compound
that decreases the level of expression of the one or more markers
with a negative fold change present in the biological sample and/or
increases the level of expression of the one or more markers with a
positive fold change present in the biological sample, thereby
identifying a compound for treating a metabolic disorder in a
subject.
10. A method of identifying a compound for treating a metabolic
disorder in a subject, the method comprising: (1) obtaining a
biological sample from the subject; (2) contacting the biological
sample with a test compound; (3) determining the level of
expression of one or more markers present in the biological sample
obtained from the subject, wherein the expression of the marker is
up- or down-regulated, in a disease cell of the metabolic disorder
induced to undergo a cellular metabolic energy shift towards normal
mitochondrial oxidative phosphorylation; (4) comparing the level of
expression of the one of more markers in the biological sample with
a control sample not contacted by the test compound; and (5)
selecting a test compound that decreases the level of expression,
in the biological sample, of the one or more down-regulated
markers, and/or increases the level of expression, in the
biological sample, of the one or more up-regulated markers, thereby
identifying a compound for treating a metabolic disorder in a
subject.
11. The method of any one of claims 1-10, wherein the metabolic
disorder is a disorder selected from the group consisting of
diabetes, obesity, pre-diabetes, hypertension, cardiovascular
disease, metabolic syndrome, and any key elements of a metabolic
disorder.
12. The method of any one of claims 1-11, wherein said marker(s)
selectively elicits, in a disease cell of the subject, a cellular
metabolic energy shift towards normalized mitochondrial oxidative
phosphorylation.
13. The method of any one of claims 1-12, wherein the sample
comprises a fluid obtained from the subject.
14. The method of claim 13, wherein the fluid is selected from the
group consisting of blood fluids, vomit, saliva, lymph, and
urine.
15. The method of claim 14, wherein the sample is a blood sample or
a component thereof.
16. The method of any one of claims 1-12, wherein the sample
comprises a tissue or component thereof obtained from the
subject.
17. The method of claim 16, wherein the tissue is selected from the
group consisting of bone, connective tissue, cartilage, lung,
liver, kidney, muscle tissue, heart, pancreas, and skin.
18. The method of any one of claims 1-17, wherein the subject is a
human.
19. The method of any one of claims 1-18, wherein the level of
expression of the marker in the biological sample is determined by
assaying a transcribed polynucleotide or a portion thereof in the
sample.
20. The method of claim 19, wherein assaying the transcribed
polynucleotide comprises amplifying the transcribed
polynucleotide.
21. The method of any one of claims 1-18, wherein the level of
expression of the marker in the subject sample is determined by
assaying a protein or a portion thereof in the sample.
22. The method of any one of claims 1-18, wherein the marker is
assayed using a reagent which specifically binds the marker.
23. The method of claim 22, wherein the reagent is labeled.
24. The method of claim 22, wherein the reagent is selected from
the group consisting of an antibody and an antigen-binding antibody
fragment.
25. The method of any one of claims 1-24, wherein the level of
expression of the marker in the sample is determined using a
technique selected from the group consisting of polymerase chain
reaction (PCR) amplification reaction, reverse-transcriptase PCR
analysis, single-strand conformation polymorphism analysis (SSCP),
mismatch cleavage detection, heteroduplex analysis, Southern blot
analysis, Northern blot analysis, Western blot analysis, in situ
hybridization, array analysis, deoxyribonucleic acid sequencing,
restriction fragment length polymorphism analysis, and combinations
or sub-combinations thereof, of said sample.
26. The method of any one of claims 1-24, wherein the level of
expression of the marker in the sample is determined using a
technique selected from the group consisting of
immunohistochemistry, immunocytochemistry, flow cytometry, ELISA
and mass spectrometry.
27. The method of any one of claims 1-26, wherein the marker is a
marker selected from the group consisting of HNF4-alpha, Bcl-xl,
Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11 (Bim), XIAP, BRAF, Bax,
c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1, COQ3, COQ6,
prenyltransferase, 4-hydrobenzoate, neutrophil cytosolic factor 2,
nitric oxide synthase 2A, superoxide dismutase 2, VDAC, Bax
channel, ANT, Cytochrome c, complex 1, complex II, complex III,
complex IV, Foxo 3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II.
28. The method of any one of claims 1-26, wherein the marker is a
marker associated with apoptosis.
29. The method of any one of claims 1-26, wherein the marker is a
marker associated with oxidative stress.
30. The method of any one of claims 1-26, wherein the marker is a
marker associated with heat shock.
31. The method of any one of claims 1-26, wherein the marker is a
marker associated with angiogenesis.
32. The method of any one of claims 1-26, wherein the marker is a
marker associated with diabetes.
33. The method of any one of claims 1-26, wherein the marker is a
marker associated with hypertension.
34. The method of any one of claims 1-26, wherein the marker is a
marker associated with cardiovascular disease.
35. The method of any one of claims 1-26, wherein the level of
expression of a plurality of markers is determined.
36. The method of any one of claims 5-8, wherein the subject is
being treated with a therapy selected from the group consisting of
an environmental influencer compound, a sulfonylurea compound, a
meglitinide compound, prandin, a nateglinide compound, a biguanide
compound, a thiazolidinedione compound, precose, symlin, Byetta, a
DPP-IV inhibitor, and insulin.
37. The method of claim 5 or claim 6, wherein the therapy comprises
an environmental influencer compound.
38. The method of claim 36 or 37, wherein the therapy further
comprises a treatment regimen selected from the group consisting of
treatment with a sulfonylurea compound, treatment with a
meglitinide compound, treatment with prandin, treatment with a
nateglinide compound, treatment with a biguanide compound,
treatment with a thiazolidinedione compound, treatment with
precose, treatment with symlin, treatment with Byetta, treatment
with a DPP-IV inhibitor, and treatment with insulin.
39. The method of claim 36 or 37, wherein the environmental
influencer compound is a multidimensional intracellular molecule
(MIM) or epimetabolic shifter (epi-shifter).
40. The method of claim 36 or 37, wherein the environmental
influencer compound is CoQ-10.
41. The method of claim 36 or 37, wherein the environmental
influencer compound is vitamin D3.
42. The method of claim 36 or 37, wherein the environmental
influencer compound is a compound selected from the group
consisting of acetyl Co-A, palmityl, L-carnitine, tyrosine,
phenylalanine, cysteine and a small molecule.
43. The method of claim 36 or 37, wherein the environmental
influencer compound is a compound selected from the group
consisting of fibronectin, TNF-alpha, IL-5, IL-12, IL-23, an
angiogenic factor and an apoptotic factor.
44. A kit for assessing whether a subject is afflicted with a
metabolic disorder, the kit comprising reagents for determining the
level of expression of at least one marker selected from the group
consisting of the markers listed in Tables 2-4 & 6-29 and
64-69, and instructions for use of the kit to assess whether the
subject is afflicted with the metabolic disorder.
45. A kit for prognosing whether a subject is predisposed to
developing a metabolic disorder, the kit comprising reagents for
determining the level of expression of at least one marker selected
from the group consisting of the markers listed in Tables 2-4 &
6-29 and 64-69, and instructions for use of the kit to prognose
whether the subject is predisposed to developing the metabolic
disorder.
46. A kit for assessing the efficacy of a therapy for treating a
metabolic disorder, the kit comprising reagents for determining the
level of expression of at least one marker selected from the group
consisting of the markers listed in Tables 2-4 & 6-29 and
64-69, and instructions for use of the kit to assess the efficacy
of the therapy for treating the metabolic disorder.
47. A kit for assessing the efficacy of an environmental influencer
compound for treating a metabolic disorder to in a subject having a
metabolic disorder, the kit comprising reagents for determining the
level of expression of at least one marker selected from the group
consisting of the markers listed in Tables 2-4 & 6-29 and
64-69, and instructions for use of the kit to assess the efficacy
of the environmental influencer compound for treating the metabolic
disorder in the subject having the metabolic disorder.
48. The kit of any one of claims 44-47, further comprising means
for obtaining a biological sample from a subject.
49. The kit of any one of claims 44-48, further comprising a
control sample.
50. The kit of any one of claims 44-49, wherein the means for
determining the level of expression of at least one marker
comprises means for assaying a transcribed polynucleotide or a
portion thereof in the sample.
51. The kit of any one of claims 44-49, wherein the means for
determining the level of expression of at least one marker
comprises means for assaying a protein or a portion thereof in the
sample.
52. The kit of any one of claims 44-51, further comprising an
environmental influencer compound.
53. The kit of any one of claims 44-52, wherein the kit comprises
reagents for determining the level of expression of a plurality of
markers.
54. A method of assessing whether a subject is afflicted with a
CoQ10 responsive state, the method comprising: (1) determining the
level of expression of a marker present in a biological sample
obtained from the subject, wherein the marker is selected from the
group consisting of the markers listed in Tables 2-4 & 6-29 and
64-69; and (2) comparing the level of expression of the marker
present in the biological sample obtained from the subject with the
level of expression of the marker present in a control sample,
wherein a modulation in the level of expression of the marker in
the biological sample obtained from the subject relative to the
level of expression of the marker in the control sample is an
indication that the subject is afflicted with the CoQ10 responsive
state, thereby assessing whether the subject is afflicted with a
CoQ10 responsive state.
55. The method of claim 54, wherein the CoQ10 responsive state is a
metabolic disorder.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/177,241, filed May 11, 2009, entitled
"Methods for Treatment of Oncological Disorders Using an
Epimetabolic Shifter (Coenzyme Q10)" (Attorney Docket No.:
117732-00601), U.S. Provisional Application Ser. No. 61/177,243,
filed May 11, 2009, entitled "Methods for Treatment of Oncological
Disorders Using Epimetabolic Shifters, Multidimensional
Intracellular Molecules or Environmental Influencers" (Attorney
Docket No.: 117732-00701), U.S. Provisional Application Ser. No.
61/177,244, filed May 11, 2009, entitled "Methods for the Diagnosis
of Oncological Disorders Using Epimetabolic Shifters,
Multidimensional Intracellular Molecules or Environmental
Influencers" (Attorney Docket No.: 117732-00801), U.S. Provisional
Application Ser. No. 61/177,245, filed May 11, 2009, entitled
"Methods for Treatment of Metabolic Disorders Using Epimetabolic
Shifters, Multidimensional Intracellular Molecules or Environmental
Influencers" (Attorney Docket No.: 117732-00901), and U.S.
Provisional Application Ser. No. 61/177,246, filed May 11, 2009,
entitled "Methods for the Diagnosis of Metabolic Disorders Using
Epimetabolic Shifters, Multidimensional Intracellular Molecules or
Environmental Influencers" (Attorney Docket No.: 117732-01001). The
entire contents of each of the foregoing applications are hereby
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates to the diagnosis of metabolic
disorders, such as diabetes and obesity.
[0003] As the levels of blood glucose rise postprandially, insulin
is secreted and stimulates cells of the peripheral tissues
(skeletal muscles and fat) to actively take up glucose from the
blood as a source of energy. Loss of glucose homeostasis as a
result of dysregulated insulin secretion or action typically
results in metabolic disorders such as diabetes, which may be
co-triggered or further exacerbated by obesity. Because these
conditions are often fatal, strategies to restore adequate glucose
clearance from the bloodstream are required.
[0004] Although diabetes may arise secondary to any condition that
causes extensive damage to the pancreas (e.g., pancreatitis,
tumors, administration of certain drugs such as corticosteroids or
pentamidine, iron overload (e.g., hemochromatosis), acquired or
genetic endocrinopathies, and surgical excision), the most common
forms of diabetes typically arise from primary disorders of the
insulin signaling system. There are two major types of diabetes,
namely type 1 diabetes (also known as insulin dependent diabetes
(IDDM)) and type 2 diabetes (also known as insulin independent or
non-insulin dependent diabetes (NIDDM)), which share common
long-term complications in spite of their different pathogenic
mechanisms.
[0005] Type 1 diabetes, which accounts for approximately 10% of all
cases of primary diabetes, is an organ-specific autoimmune disease
characterized by the extensive destruction of the insulin-producing
beta cells of the pancreas. The consequent reduction in insulin
production inevitably leads to the deregulation of glucose
metabolism. While the administration of insulin provides
significant benefits to patients suffering from this condition, the
short serum half-life of insulin is a major impediment to the
maintenance of normoglycemia. An alternative treatment is islet
transplantation, but this strategy has been associated with limited
success.
[0006] Type 2 diabetes, which affects a larger proportion of the
population, is characterized by a deregulation in the secretion of
insulin and/or a decreased response of peripheral tissues to
insulin, i.e., insulin resistance. While the pathogenesis of type 2
diabetes remains unclear, epidemiologic studies suggest that this
form of diabetes results from a collection of multiple genetic
defects or polymorphisms, each contributing its own predisposing
risks and modified by environmental factors, including excess
weight, diet, inactivity, drugs, and excess alcohol consumption.
Although various therapeutic treatments are available for the
management of type 2 diabetes, they are associated with various
debilitating side effects. Accordingly, patients diagnosed with or
at risk of having type 2 diabetes are often advised to adopt a
healthier lifestyle, including loss of weight, change in diet,
exercise, and moderate alcohol intake. Such lifestyle changes,
however, are not sufficient to reverse the vascular and organ
damages caused by diabetes.
[0007] Coenzyme Q10, also referred to herein as CoQ10, Q10,
ubiquinone, or ubidecarenone, is a popular nutritional supplement
and can be found in capsule form in nutritional stores, health food
stores, pharmacies, and the like, as a vitamin-like supplement to
help protect the immune system through the antioxidant properties
of ubiquinol, the reduced form of CoQ10. CoQ10 is art-recognized
and further described in International Publication No. WO
2005/069916, the entire disclosure of which is incorporated by
reference herein.
[0008] CoQ10 is found throughout most tissues of the human body and
the tissues of other mammals. The tissue distribution and redox
state of CoQ10 in humans has been reviewed in a review article by
Bhagavan and Chopra (2006 Free Radical Research 40(5):445-453). The
authors report that "as a general rule, tissues with high-energy
requirements or metabolic activity such as the heart, kidney, liver
and muscle contain relatively high concentrations of CoQ10." The
authors further report that "[a] major portion of CoQ10 in tissues
is in the reduced form as the hydroquinone or uniquinol, with the
exception of brain and lungs," which "appears to be a reflection of
increased oxidative stress in these two tissues." In particular,
Bhagavan report that in heart, kidney, liver, muscle, intenstine
and blood (plasma), about 61%, 75%, 95%, 65%, 95% and 96%,
respectively, of CoQ10 is in the reduced form. Similarly,
Ruiz-Jiminez et al. (2007 J. Chroma A, 1175, 242-248) report that
when human plasma was evaluated for Q10 and the reduced form of Q10
(Q10H2), the majority (90%) of the molecule was found in the
reduced form.
[0009] CoQ10 is very lipophilic and, for the most part, insoluble
in water. Due to its insolubility in water, limited solubility in
lipids, and relatively large molecular weight, the efficiency of
absorption of orally administered CoQ10 is poor. Bhagavan and
Chopra report that "in one study with rats it was reported that
only about 2-3% of orally-administered CoQ10 was absorbed."
Bhagavan and Chopra further report that "[d]ata from rat studies
indicate that CoQ10 is reduced to ubiquinol either during or
following absorption in the intestine."
[0010] Given that the strategies currently available for the
management of diabetes are suboptimal, there is a compelling need
for treatments that are more effective and are not associated with
such debilitating side-effects.
SUMMARY OF THE INVENTION
[0011] The present invention is based, at least in part, on the
discovery that application of endogenous Coenzyme Q10 (also
referred to as CoQ10 or Q10 herein) to cells results in the
restoration of apoptotic potential. The apoptotic response is
preferentially induced in cancer cells. A time and dose response of
mitochondrial Q10 levels was observed, wherein after 48 hours, the
level of Q10 in cell mitochondria was increased by six fold. The
invention is further based on the surprising and unexpected
discovery that the Q10 is maintained in the supplied oxidized form
(pro-oxidant) and not converted to the reduced (anti-oxidant) form
of Q10H2 in any significant amounts. The invention is based on the
further discovery that a significant number of proteins and mRNA
levels are modulated in cells treated with Q10. These modulated
proteins were found to be clustered into several cellular pathways,
including apoptosis, cancer biology and cell growth, glycolysis and
metabolism, molecular transport, and cellular signaling.
[0012] Applicants' data described herein has provided insight into
the mechanism of action of Q10. In particular, while not wishing to
be bound by theory, Applicants' discoveries indicate that Q10
induces a metabolic shift to the cell microenvironment. Many
diseases are known to be associated with an altered metabolic
state. For example, differential metabolism is known to occur in
cancer cells (the Warurg effect), whereby most cancer cells
predominantly produce energy by glycolysis followed by lactic acid
fermentation in the cytosol, rathe than by oxidative
phosphorylation (oxidation of pyruvate) in the mitochondria. In
another example, metabolic disorders, such as diabetes and obesity,
are associated with an altered glucose metabolism.
[0013] Accordingly, in certain aspects, the present invention is
directed to methods of assessing whether a subject is afflicted
with a metabolic disorder. Such methods include (1) determining the
level of expression of a marker present in a biological sample
obtained from the subject, wherein the marker is selected from the
group consisting of the markers listed in Tables 2-4 & 6-29 and
64-69; and (2) comparing the level of expression of the marker
present in the biological sample obtained from the subject with the
level of expression of the marker present in a control sample,
wherein a modulation in the level of expression of the marker in
the biological sample obtained from the subject relative to the
level of expression of the marker in the control sample is an
indication that the subject is afflicted with a metabolic disorder,
thereby assessing whether the subject is afflicted with a metabolic
disorder.
[0014] In certain aspects, the present invention is directed to
methods of assessing whether a subject is afflicted with a
metabolic disorder. Such methods include (1) determining the level
of expression of a marker present in a biological sample obtained
from the subject, wherein the expression of the marker is modulated
in a disease cell of the metabolic disorder induced to undergo a
cellular metabolic energy shift towards normal mitochondrial
oxidative phosphorylation; and (2) comparing the level of
expression of the marker present in the biological sample obtained
from the subject with the level of expression of the marker present
in a control sample, wherein a modulation in the level of
expression of the marker in the biological sample obtained from the
subject relative to the level of expression of the marker in the
control sample is an indication that the subject is afflicted with
a metabolic disorder, thereby assessing whether the subject is
afflicted with a metabolic disorder.
[0015] In certain aspects, the present invention is directed to
methods of prognosing whether a subject is predisposed to
developing a metabolic disorder. Such methods include (1)
determining the level of expression of a marker present in a
biological sample obtained from the subject, wherein the marker is
selected from the group consisting of the markers listed in Tables
2-4 & 6-29 and 64-69; and (2) comparing the level of expression
of the marker present in the biological sample obtained from the
subject with the level of expression of the marker present in a
control sample, wherein a modulation in the level of expression of
the marker in the biological sample obtained from the subject
relative to the level of expression of the marker in the control
sample is an indication that the subject is predisposed to
developing a metabolic disorder, thereby prognosing whether the
subject is predisposed to developing a metabolic disorder.
[0016] In certain aspects, the present invention is directed to
methods of prognosing whether a subject is predisposed to
developing a metabolic disorder. Such methods include (1)
determining the level of expression of a marker present in a
biological sample obtained from the subject, wherein the expression
of the marker is modulated in a disease cell of the metabolic
disorder induced to undergo a cellular metabolic energy shift
towards normal mitochondrial oxidative phosphorylation; and (2)
comparing the level of expression of the marker present in the
biological sample obtained from the subject with the level of
expression of the marker present in a control sample, wherein a
modulation in the level of expression of the marker in the
biological sample obtained from the subject relative to the level
of expression of the marker in the control sample is an indication
that the subject is predisposed to developing a metabolic disorder,
thereby prognosing whether the subject is predisposed to developing
a metabolic disorder.
[0017] In certain aspects, the present invention is directed to
methods for assessing the efficacy of a therapy for treating a
metabolic disorder in a subject. Such methods include comparing (1)
the level of expression of a marker present in a first sample
obtained from the subject prior to administering at least a portion
of the treatment regimen to the subject, wherein the marker is
selected from the group consisting of the markers listed in Tables
2-4 & 6-29 and 64-69; with (2) the level of expression of the
marker present in a second sample obtained from the subject
following administration of at least a portion of the treatment
regimen, wherein a modulation in the level of expression of the
marker in the second sample as compared to the first sample is an
indication that the therapy is efficacious for treating the
metabolic disorder in the subject.
[0018] In certain aspects, the present invention is directed to
methods for assessing the efficacy of a therapy for treating a
metabolic disorder in a subject. Such methods include comparing (1)
the level of expression of a marker present in a first sample
obtained from the subject prior to administering at least a portion
of the treatment regimen to the subject, wherein the expression of
the marker is modulated in a disease cell of the metabolic disorder
induced to undergo a cellular metabolic energy shift towards normal
mitochondrial oxidative phosphorylation; with (2) the level of
expression of the marker present in a second sample obtained from
the subject following administration of at least a portion of the
treatment regimen, wherein a modulation in the level of expression
of the marker in the second sample as compared to the first sample
is an indication that the therapy is efficacious for treating the
metabolic disorder in the subject.
[0019] In certain aspects, the present invention is directed to
methods of assessing the efficacy of an environmental influencer
compound for treating a metabolic disorder to in a subject in need
thereof. Such methods include (1) determining the level of
expression of one or more markers present in a biological sample
obtained from the subject, wherein the biological sample is exposed
to the environmental influencer compound, and wherein the marker is
selected from the group consisting of the markers listed in Tables
2-4 & 6-29 and 64-69 with a positive fold change and/or with a
negative fold change; (2) determining the level of expression of
the one or more markers present in a second biological sample
obtained from the subject, wherein the sample is not exposed to the
environmental influencer compound; and (3) comparing the level of
expression of the one of more markers in the biological sample
exposed to the environmental influencer compound and the level of
expression of the one of more markers in the biological sample not
exposed to the environmental influencer compound, wherein a
decrease in the level of expression of the one or more markers with
a negative fold change present in the biological sample exposed to
the environmental influencer compound relative to the level of
expression of the one or more markers present in the second sample
is an indication that the environmental influencer compound is
efficacious for treating the metabolic disorder in the subject in
need thereof, and, wherein an increase in the level of expression
of the one or more markers with a positive fold change present in
the biological sample exposed to the environmental influencer
compound relative to the level of expression of the one or more
markers present in the second sample is an indication that the
environmental influencer compound is efficacious for treating the
metabolic disorder in the subject in need thereof, thereby
assessing the efficacy of the environmental influencer compound for
treating the metabolic disorder.
[0020] In certain aspects, the present invention is directed to
methods of assessing the efficacy of an environmental influencer
compound for treating a metabolic disorder to in a subject in need
thereof. Such methods include (1) determining the level of
expression of one or more markers present in a biological sample
obtained from the subject, wherein the biological sample is exposed
to the environmental influencer compound, and wherein the
expression of the marker is up- or down-regulated, in a disease
cell of the metabolic disorder induced to undergo a cellular
metabolic energy shift towards normal mitochondrial oxidative
phosphorylation; (2) determining the level of expression of the one
or more markers present in a second biological sample obtained from
the subject, wherein the sample is not exposed to the environmental
influencer compound; and (3) comparing the level of expression of
the one of more markers in the biological sample exposed to the
environmental influencer compound and the level of expression of
the one of more markers in the biological sample not exposed to the
environmental influencer compound, wherein a decrease, in the
biological sample exposed to the environmental influencer compound,
in the level of expression of the one or more down-regulated
markers relative to the level of expression of the one or more
markers present in the second sample is an indication that the
environmental influencer compound is efficacious for treating the
metabolic disorder in the subject in need thereof, and, wherein an
increase, in the biological sample exposed to the environmental
influencer compound, in the level of expression of the one or more
up-regulated markers relative to the level of expression of the one
or more markers present in the second sample is an indication that
the environmental influencer compound is efficacious for treating
the metabolic disorder in the subject in need thereof, thereby
assessing the efficacy of the environmental influencer compound for
treating the metabolic disorder.
[0021] In certain aspects, the present invention is directed to
methods of identifying a compound for treating a metabolic disorder
in a subject. Such methods include (1) obtaining a biological
sample from the subject; (2) contacting the biological sample with
a test compound; (3) determining the level of expression of one or
more markers present in the biological sample obtained from the
subject, wherein the marker is selected from the group consisting
of the markers listed in Tables 2-4 & 6-29 and 64-69 with a
positive fold change and/or with a negative fold change; (4)
comparing the level of expression of the one of more markers in the
biological sample with a control sample not contacted by the test
compound; and (5) selecting a test compound that decreases the
level of expression of the one or more markers with a negative fold
change present in the biological sample and/or increases the level
of expression of the one or more markers with a positive fold
change present in the biological sample, thereby identifying a
compound for treating a metabolic disorder in a subject.
[0022] In certain aspects, the present invention is directed to
methods of identifying a compound for treating a metabolic disorder
in a subject. Such methods include (1) obtaining a biological
sample from the subject; (2) contacting the biological sample with
a test compound; (3) determining the level of expression of one or
more markers present in the biological sample obtained from the
subject, wherein the expression of the marker is up- or
down-regulated, in a disease cell of the metabolic disorder induced
to undergo a cellular metabolic energy shift towards normal
mitochondrial oxidative phosphorylation; (4) comparing the level of
expression of the one of more markers in the biological sample with
a control sample not contacted by the test compound; and (5)
selecting a test compound that decreases the level of expression,
in the biological sample, of the one or more down-regulated
markers, and/or increases the level of expression, in the
biological sample, of the one or more up-regulated markers, thereby
identifying a compound for treating a metabolic disorder in a
subject.
[0023] In some embodiments, the metabolic disorder is a disorder
selected from the group consisting of diabetes, obesity,
pre-diabetes, hypertension, cardiovascular disease, metabolic
syndrome, and any key elements of a metabolic disorder.
[0024] In some embodiments, the marker(s) selectively elicits, in a
disease cell of the subject, a cellular metabolic energy shift
towards normalized mitochondrial oxidative phosphorylation.
[0025] In some embodiments, the sample comprises a fluid obtained
from the subject, e.g., a fluid selected from blood fluids, vomit,
saliva, lymph, cystic fluid, urine, fluids collected by bronchial
lavage, fluids collected by peritoneal rinsing, and gynecological
fluids. In some embodiments, the sample is a blood sample or a
component thereof. In some embodiments, the sample comprises a
tissue or component thereof obtained from the subject, e.g., tissue
selected from bone, connective tissue, cartilage, lung, liver,
kidney, muscle tissue, heart, pancreas, and skin.
[0026] In some embodiments, the subject is a human.
[0027] In some embodiments, the level of expression of the marker
in the biological sample is determined by assaying a transcribed
polynucleotide or a portion thereof in the sample. In some
embodiments, assaying the transcribed polynucleotide comprises
amplifying the transcribed polynucleotide. In some embodiments, the
level of expression of the marker in the subject sample is
determined by assaying a protein or a portion thereof in the
sample. In some embodiments, the marker is assayed using a reagent,
e.g., a labeled reagent, which specifically binds with the marker.
Reagents may include, for example, an antibody and an
antigen-binding antibody fragment.
[0028] In some embodiments, the level of expression of the marker
in the sample is determined using a technique selected from the
group consisting of polymerase chain reaction (PCR) amplification
reaction, reverse-transcriptase PCR analysis, single-strand
conformation polymorphism analysis (SSCP), mismatch cleavage
detection, heteroduplex analysis, Southern blot analysis, Northern
blot analysis, Western blot analysis, in situ hybridization, array
analysis, deoxyribonucleic acid sequencing, restriction fragment
length polymorphism analysis, and combinations or sub-combinations
thereof, of said sample. In some embodiments, the level of
expression of the marker in the sample is determined using a
technique selected from the group consisting of
immunohistochemistry, immunocytochemistry, flow cytometry, ELISA
and mass spectrometry.
[0029] In some embodiments, the marker is a marker selected from
the group consisting of HNF4-alpha, Bcl-xl, Bcl-xS, BNIP-2, Bcl-2,
Birc6, Bcl-2-L11 (Bim), XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, cMyc,
transaldolase 1, COQ1, COQ3, COQ6, prenyltransferase,
4-hydrobenzoate, neutrophil cytosolic factor 2, nitric oxide
synthase 2A, superoxide dismutase 2, VDAC, Bax channel, ANT,
Cytochrome c, complex 1, complex II, complex III, complex IV, Foxo
3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II. In some embodiments, the
marker is a marker associated with apoptosis. In some embodiments,
the marker is a marker associated with oxidative stress. In some
embodiments, the marker is a marker associated with heat shock. In
some embodiments, the marker is a marker associated with
angiogenesis. In some embodiments, the marker is a marker
associated with diabetes. In some embodiments, the marker is a
marker associated with hypertension. In some embodiments, the
marker is a marker associated with cardiovascular disease.
[0030] In some embodiments, the level of expression of a plurality
of markers is determined.
[0031] In some embodiments, the subject is being treated with a
therapy selected from an environmental influencer compound, a
sulfonylurea compound, a meglitinide compound, prandin, a
nateglinide compound, a biguanide compound, a thiazolidinedione
compound, precose, symlin, Byetta, a DPP-IV inhibitor, and insulin.
In some embodiments, the therapy comprises an environmental
influencer compound. Environmental influencer compounds can be, for
example, multidimensional intracellular molecules (MIMs) or
epimetabolic shifters (epi-shifters). In some embodiments, the
environmental influencer compound is CoQ-10. In some embodiments,
the environmental influencer compound is vitamin D3. In some
embodiments, the environmental influencer compound is a compound
selected from acetyl Co-A, palmityl, L-carnitine, tyrosine,
phenylalanine, cysteine and a small molecule. In some embodiments,
the environmental influencer compound is a compound selected from
fibronectin, TNF-alpha, IL-5, IL-12, IL-23, an angiogenic factor
and an apoptotic factor. In some embodiments, the therapy further
comprises a treatment regimen selected from treatment with a
sulfonylurea compound, treatment with a meglitinide compound,
treatment with prandin, treatment with a nateglinide compound,
treatment with a biguanide compound, treatment with a
thiazolidinedione compound, treatment with precose, treatment with
symlin, treatment with Byetta, treatment with a DPP-IV inhibitor,
and treatment with insulin.
[0032] In certain aspects, the present invention is directed to
kits for assessing whether a subject is afflicted with a metabolic
disorder. Such kits include reagents for determining the level of
expression of at least one marker selected from the group
consisting of the markers listed in Tables 2-4 & 6-29 and
64-69, and instructions for use of the kit to assess whether the
subject is afflicted with the metabolic disorder.
[0033] In certain aspects, the present invention is directed to
kits for prognosing whether a subject is predisposed to developing
a metabolic disorder. Such kits include reagents for determining
the level of expression of at least one marker selected from the
group consisting of the markers listed in Tables 2-4 & 6-29 and
64-69, and instructions for use of the kit to prognose whether the
subject is predisposed to developing the metabolic disorder.
[0034] In certain aspects, the present invention is directed to
kits for assessing the efficacy of a therapy for treating a
metabolic disorder. Such kits include reagents for determining the
level of expression of at least one marker selected from the group
consisting of the markers listed in Tables 2-4 & 6-29 and
64-69, and instructions for use of the kit to assess the efficacy
of the therapy for treating the metabolic disorder.
[0035] In certain aspects, the present invention is directed to
kits for assessing the efficacy of an environmental influencer
compound for treating a metabolic disorder to in a subject having a
metabolic disorder. Such kits include reagents for determining the
level of expression of at least one marker selected from the group
consisting of the markers listed in Tables 2-4 & 6-29 and
64-69, and instructions for use of the kit to assess the efficacy
of the environmental influencer compound for treating the metabolic
disorder in the subject having the metabolic disorder.
[0036] In some embodiments, the kit further comprises means for
obtaining a biological sample from a subject. In some embodiments,
the kit further comprises a control sample. In some embodiments,
further comprises an environmental influencer compound. In some
embodiments, the kit comprises reagents for determining the level
of expression of a plurality of markers.
[0037] In some embodiments, the means for determining the level of
expression of at least one marker comprises means for assaying a
transcribed polynucleotide or a portion thereof in the sample. In
some embodiments, the means for determining the level of expression
of at least one marker comprises means for assaying a protein or a
portion thereof in the sample.
[0038] In certain aspects, the present invention is directed to
methods of assessing whether a subject is afflicted with a CoQ10
responsive state. Such methods include (1) [0039] determining the
level of expression of a marker present in a biological sample
obtained from the subject, wherein the marker is selected from the
group consisting of the markers listed in Tables 2-4 & 6-29 and
64-69; and (2) comparing the level of expression of the marker
present in the biological sample obtained from the subject with the
level of expression of the marker present in a control sample,
wherein a modulation in the level of expression of the marker in
the biological sample obtained from the subject relative to the
level of expression of the marker in the control sample is an
indication that the subject is afflicted with the CoQ10 responsive
state, thereby assessing whether the subject is afflicted with a
CoQ10 responsive state. In some embodiments, the CoQ10 responsive
state is a metabolic disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1: Sensitivity of SK-MEL-28 to 24 hours of Q10
treatment measured by the amount of early and late apoptotic
cells.
[0041] FIG. 2: Sensitivity of SKBR3 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0042] FIG. 3: Sensitivity of PaCa2 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0043] FIG. 4: Sensitivity of PC-3 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0044] FIG. 5: Sensitivity of HepG2 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0045] FIG. 6: Sensitivity of MCF-7 to 24 hours of Q10 treatment
measured by the amount of early and late apoptotic cells.
[0046] FIG. 7: Measurement of apoptotic cells upon 24 hour
treatment with Q10, as measured by Apostrand ELISA method.
[0047] FIG. 8: Example gel analysis of 2-D gel electrophoresis.
Spots excised for identification are marked.
[0048] FIG. 9: Network of interaction between proteins identified
by 2-D gel electrophoresis as being modulated by Q10 in SK-MEL-28
cells.
[0049] FIG. 10: The pentose phosphate pathway adapted from
Verhoeven et al. (Am. J. Hum. Genet. 2001 68(5):1086-1092).
[0050] FIG. 11: 2-D gel of the mitochondrial enriched material of
SK-MEL-28 cells. Spots excised and identified by mass spectrometry
characterization are marked.
[0051] FIG. 12: Comparative plot of the relative amounts of Q10
present in SK-MEL-28 mitochondria following the exogenous addition
of 100 .mu.M Q10 into the culture medium.
[0052] FIG. 13: Apoptosis pathway mapping known processes.
[0053] FIG. 14: Western blot analysis of Bcl-xl.
[0054] FIG. 15: Western blot analysis of SK-MEL-28 sample set
proved with a Vimentin antibody.
[0055] FIG. 16: Western blot analysis of cell lysis from a number
of cell lines, evaluated with five antibodies targeting oxidative
phosphorylation complexes (MitoSciences #MS601).
[0056] FIG. 17: Western blot comparison of F1-alpha levels.
[0057] FIG. 18: Western blot comparison of Q10 response with
C-III-Core 2.
[0058] FIG. 19: Western blot comparison of Q10 response with
C-II-30.
[0059] FIG. 20: Western blot comparison of Q10 response with
C-IV-COX II.
[0060] FIG. 21: Western blot comparison of Q10 response with C-1-20
(ND6).
[0061] FIG. 22: Western blot analysis of a variety of cell types
against five mitochondrial protein.
[0062] FIG. 23: Western blot comparison of Q10 response with
Complex V protein C-V-.alpha..
[0063] FIG. 24: Western blot comparison of Q10 response with
C-III-Core 1.
[0064] FIG. 25: Western blot comparison of Q10 response with Porin
(VDAC1).
[0065] FIG. 26: Western blot comparison of Q10 response with
Cyclophilin D
[0066] FIG. 27: Western blot comparison of Q10 response with
Cytochrome C.
[0067] FIG. 28: Theoretical model of Q10 (spheres) inserted into
the lipid binding channel of HNF4alpha (1M7W.pdb) in the Helix 10
open conformation.
[0068] FIG. 29: OCR in HDFa cells in various glucose conditions in
normoxic and hypoxic conditions.
[0069] FIG. 30: OCR in HASMC cells in various glucose conditions in
normoxic and hypoxic conditions.
[0070] FIG. 31: OCR values in MCF-7 breast cancer cells in the
absence and presence of CoQ10 and stressors.
[0071] FIG. 32: OCR values in PaCa-2 pancreatic cancer cells in the
absence and presence of CoQ10 and stressors.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0072] As used herein, each of the following terms has the meaning
associated with it in this section.
[0073] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0074] The term "including" is used herein to mean, and is used
interchangeably with, the phrase "including but not limited
to".
[0075] The term "or" is used herein to mean, and is used
interchangeably with, the term "and/or," unless context clearly
indicates otherwise.
[0076] The term "such as" is used herein to mean, and is used
interchangeably, with the phrase "such as but not limited to".
[0077] A "patient" or "subject" to be treated by the method of the
invention can mean either a human or non-human animal, preferably a
mammal. As used herein, a "subject" or a "patient" includes,
without limitation, any animal (e.g., a human), including horses,
dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs,
rats, mice, lizards, snakes, sheep, cattle, fish, and birds.
[0078] As used herein, "survival" refers to the continuation of
life of a subject which has been treated for a metabolic disorder.
In one embodiment, survival refers to the failure of a metabolic
disorder to recur.
[0079] "Preventing" or "prevention" refers to a reduction in risk
of acquiring a disease or disorder (i.e., causing at least one of
the clinical symptoms of the disease not to develop in a patient
that may be exposed to or predisposed to the disease but does not
yet experience or display symptoms of the disease).
[0080] As used herein, the term "amount", refers to either (a) an
absolute amount as measured in molecules, moles or weight per unit
volume or cell or (b) a relative amount as designated, for example,
by a numerical rating from 0 to 5.
[0081] The term "control amount", as used herein, refers to the
amount of marker in a cell or a sample derived from a subject not
afflicted with a metabolic disorder. The "control amount" may, for
example, be determined by calculating the average amount of marker
present in cells or tissues that are known to express the marker,
e.g., express these proteins at high levels, intermediate levels
and low levels.
[0082] "Therapeutically effective amount" means the amount of a
compound that, when administered to a patient for treating a
disease, is sufficient to effect such treatment for the disease.
When administered for preventing a disease, the amount is
sufficient to avoid or delay onset of the disease. The
"therapeutically effective amount" will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the patient to be treated.
[0083] "Preventing" or "prevention" refers to a reduction in risk
of acquiring a disease or disorder (i.e., causing at least one of
the clinical symptoms of the disease not to develop in a patient
that may be exposed to or predisposed to the disease but does not
yet experience or display symptoms of the disease).
[0084] The term "prophylactic" or "therapeutic" treatment refers to
administration to the subject of one or more of the subject
compositions. If it is administered prior to clinical manifestation
of the unwanted condition (e.g., disease or other unwanted state of
the host animal) then the treatment is prophylactic, i.e., it
protects the host against developing the unwanted condition,
whereas if administered after manifestation of the unwanted
condition, the treatment is therapeutic (i.e., it is intended to
diminish, ameliorate or maintain the existing unwanted condition or
side effects therefrom).
[0085] The term "therapeutic effect" refers to a local or systemic
effect in animals, particularly mammals, and more particularly
humans caused by a pharmacologically active substance. The term
thus means any substance intended for use in the diagnosis, cure,
mitigation, treatment or prevention of disease or in the
enhancement of desirable physical or mental development and
conditions in an animal or human. The phrase
"therapeutically-effective amount" means that amount of such a
substance that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. In
certain embodiments, a therapeutically-effective amount of a
compound will depend on its therapeutic index, solubility, and the
like. For example, certain compounds discovered by the methods of
the present invention may be administered in a sufficient amount to
produce a reasonable benefit/risk ratio applicable to such
treatment.
[0086] The term "expression" is used herein to mean the process by
which a polypeptide is produced from DNA. The process involves the
transcription of the gene into mRNA and the translation of this
mRNA into a polypeptide. Depending on the context in which used,
"expression" may refer to the production of RNA, protein or
both.
[0087] The terms "level of expression of a gene in a cell" or "gene
expression level" refer to the level of mRNA, as well as pre-mRNA
nascent transcript(s), transcript processing intermediates, mature
mRNA(s) and degradation products, encoded by the gene in the
cell.
[0088] The term "modulation" refers to upregulation (i.e.,
activation or stimulation), downregulation (i.e., inhibition or
suppression) of a response, or the two in combination or apart. A
"modulator" is a compound or molecule that modulates, and may be,
e.g., an agonist, antagonist, activator, stimulator, suppressor, or
inhibitor.
[0089] A "higher level of expression", "higher level of activity",
"increased level of expression" or "increased level of activity"
refers to an expression level and/or activity in a test sample that
is greater than the standard error of the assay employed to assess
expression and/or activity, and is preferably at least twice, and
more preferably three, four, five or ten or more times the
expression level and/or activity of the marker in a control sample
(e.g., a sample from a healthy subject not afflicted with an
oncological disorder) and preferably, the average expression level
and/or activity of the marker in several control samples.
[0090] A "lower level of expression", "lower level of activity",
"decreased level of expression" or "decreased level of activity"
refers to an expression level and/or activity in a test sample that
is greater than the standard error of the assay employed to assess
expression and/or activity, but is preferably at least twice, and
more preferably three, four, five or ten or more times less than
the expression level of the marker in a control sample (e.g., a
sample that has been calibrated directly or indirectly against a
panel of oncological disorders with follow-up information which
serve as a validation standard for prognostic ability of the
marker) and preferably, the average expression level and/or
activity of the marker in several control samples.
[0091] As used herein, "antibody" includes, by way of example,
naturally-occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE)
and recombinant antibodies such as single-chain antibodies,
chimeric and humanized antibodies and multi-specific antibodies, as
well as fragments and derivatives of all of the foregoing, which
fragments and derivatives have at least an antigenic binding site.
Antibody derivatives may comprise a protein or chemical moiety
conjugated to an antibody.
[0092] As used herein, "known standard" or "control" refers to one
or more of an amount and/or mathematical relationship, as
applicable, with regard to a marker of the invention, and the
presence or absence of a metabolic disorder. Reagents for
generating a known standard include, without limitation, cells from
a patient without a metabolic disorder and optionally labeled
antibodies. Known standards may also include tissue culture cell
lines (including, but not limited to, cell lines that have been
manipulated to express specific marker proteins or to not express
specific marker proteins, or samples that either constitutively
contain constant amounts of marker protein, or can be manipulated
(e.g., by exposure to a changed environment, where such changed
environment may include but not limited to growth factors,
hormones, steroids, cytokines, antibodies, various drugs and
anti-metabolites, and extracellular matrices) to express a marker
protein. Cell lines may be mounted directly on glass slides for
analysis, fixed, embedded in paraffin directly as a pellet, or
suspended in a matrix such as agarose, then fixed, embedded in
paraffin, sectioned and processed as tissue samples. The standards
must be calibrated directly or indirectly against a panel of
patient samples with follow-up information which serve as a
validation standard for prognostic ability of the marker
proteins.
[0093] "Primary treatment" as used herein, refers to the initial
treatment of a subject afflicted with a metabolic disorder. Primary
treatments include, without limitation, treatment with a
sulfonylurea compound, a meglitinide compound, prandin, a
nateglinide compound, a biguanide compound, a thiazolidinedione
compound, precose, symlin, Byetta, a DPP-IV inhibitor, or
insulin.
[0094] A metabolic disorder is "treated" if at least one symptom of
the metabolic disorder is expected to be or is alleviated,
terminated, slowed, or prevented. As used herein, an metabolic
disorder is also "treated" if recurrence or progression of the
metabolic disorder is reduced, slowed, delayed, or prevented.
[0095] A kit is any manufacture (e.g. a package or container)
comprising at least one reagent, e.g. a probe, for specifically
detecting a marker of the invention, the manufacture being
promoted, distributed, or sold as a unit for performing the methods
of the present invention.
[0096] "Metabolic pathway" refers to a sequence of enzyme-mediated
reactions that transform one compound to another and provide
intermediates and energy for cellular functions. The metabolic
pathway can be linear or cyclic.
[0097] "Metabolic state" refers to the molecular content of a
particular cellular, multicellular or tissue environment at a given
point in time as measured by various chemical and biological
indicators as they relate to a state of health or disease.
[0098] The term "microarray" refers to an array of distinct
polynucleotides, oligonucleotides, polypeptides (e.g., antibodies)
or peptides synthesized on a substrate, such as paper, nylon or
other type of membrane, filter, chip, glass slide, or any other
suitable solid support.
[0099] The terms "disorders" and "diseases" are used inclusively
and refer to any deviation from the normal structure or function of
any part, organ or system of the body (or any combination thereof).
A specific disease is manifested by characteristic symptoms and
signs, including biological, chemical and physical changes, and is
often associated with a variety of other factors including, but not
limited to, demographic, environmental, employment, genetic and
medically historical factors. Certain characteristic signs,
symptoms, and related factors can be quantitated through a variety
of methods to yield important diagnostic information.
[0100] In some embodiments, the present invention provides methods
for diagnosing or prognosing a Coenzyme Q10 responsive state in a
subject in need thereof. The language "Coenzyme Q10 responsive
state," or "CoQ10 responsive state," includes diseases, disorders,
states and/or conditions which can be treated, prevented, or
otherwise ameliorated by the administration of Coenzyme Q10.
Without wishing to be bound by any particular theory, and as
described further herein, it is believed that CoQ10 functions, at
least partially, by inducing a metabolic shift to the cell
microenvironment, such as a shift towards the type and/or level of
oxidative phosphorylation in normal state cells. Accordingly, in
some embodiments, CoQ10 responsive states are states that arise
from an altered metabolism of cell microenvironment. Coenzyme Q10
responsive states include, for example, oncological disorders,
which, for example, may be biased towards glycolysis and lactate
biosynthesis. In some embodiments, CoQ10 responsive oncological
disorders include liver cancer, pancreatic cancer, breast cancer,
prostate cancer, liver cancer, or bone cancer, squamous cell
carcinomas, basal cell carcinomas, melanomas, and actinic
keratosis, among others. Coenzyme Q10 responsive states also
include, for example, metabolic disorders such as obesity,
diabetes, pre-diabetes, Metabolic Syndrome, satiety, and endocrine
abnormalities. Coenzyme Q10 responsive states further include other
metabolic disorders as described herein.
[0101] In some embodiments, the compounds of the present invention,
e.g., the MIMs or epi-shifters described herein, share a common
activity with Coenzyme Q10. As used herein, the phrase "share a
common activity with Coenzyme Q10" refers to the ability of a
compound to exhibit at least a portion of the same or similar
activity as Coenzyme Q10. In some embodiments, the compounds of the
present invention exhibit 25% or more of the activity of Coenzyme
Q10. In some embodiments, the compounds of the present invention
exhibit up to and including about 130% of the activity of Coenzyme
Q10. In some embodiments, the compounds of the present invention
exhibit about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,
40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%,
104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%,
115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%,
126%, 127%, 128%, 129%, or 130% of the activity of Coenzyme Q10. It
is to be understood that each of the values listed in this
paragraph may be modified by the term "about." Additionally, it is
to be understood that any range which is defined by any two values
listed in this paragraph is meant to be encompassed by the present
invention. For example, in some embodiments, the compounds of the
present invention exhibit between about 50% and about 100% of the
activity of Coenzyme Q10. In some embodiments, the activity shared
by Coenzyme Q10 and the compounds of the present invention is the
ability to induce a shift in cellular metabolism. In certain
embodiments, the activity shared by of CoQ10 and the compounds of
the present invention is measured by OCR (Oxygen Consumption Rate)
and/or ECAR (ExtraCellular Acidification Rate).
[0102] A kit is any manufacture (e.g. a package or container)
comprising at least one reagent, e.g. a probe, for specifically
detecting a marker of the invention, the manufacture being
promoted, distributed, or sold as a unit for performing the methods
of the present invention.
[0103] The term "intermediate of the coenzyme biosynthesis pathway"
as used herein, characterizes those compounds that are formed
between the chemical/biological conversion of tyrosine and
Acetyl-CoA to uqiquinone. Intermediates of the coenzyme
biosynthesis pathway include 3-hexaprenyl-4-hydroxybenzoate,
3-hexaprenyl-4,5-dihydroxybenzoate,
3-hexaprenyl-4-hydroxy-5-methoxybenzoate,
2-hexaprenyl-6-methoxy-1,4-benzoquinone,
2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,
2-hexaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,
3-Octaprenyl-4-hydroxybenzoate, 2-octaprenylphenol,
2-octaprenyl-6-metholxyphenol,
2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,
2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,
2-decaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,
2-decaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,
2-decaprenyl-6-methoxy-1,4-benzoquinone,
2-decaprenyl-6-methoxyphenol,
3-decaprenyl-4-hydroxy-5-methoxybenzoate,
3-decaprenyl-4,5-dihydroxybenzoate, 3-decaprenyl-4-hydroxybenzoate,
4-hydroxy phenylpyruvate, 4-hydroxyphenyllactate,
4-hydroxy-benzoate, 4-hydroxycinnamate and
hexaprenydiphosphate.
[0104] As used herein, the phrase "anaerobic use of glucose" or
"anaerobic glycolysis" refers to cellular production of energy by
glycolysis followed by lactic acid fermentation in the cytosol. For
example, many cancer cells produce energy by anaerobic
glycolysis.
[0105] As used herein, the phrase "aerobic glycolysis" or
"mitochondrial oxidative phosphorylation" refers to cellular
production of energy by glycolysis followed by oxidation of
pyruvate in mitochondria.
[0106] As used herein, the phrase "capable of blocking anaerobic
use of glucose and augmenting mitochondrial oxidative
phosphorylation" refers to the ability of an environmental
influencer (e.g., an epitmetabolic shifter) to induce a shift or
change in the metabolic state of a cell from anaerobic glycolysis
to aerobic glycolysis or mitochondrial oxidative
phosphorylation.
I. Metabolic Disorders
[0107] The present invention provides methods for diagnosing or
prognosing a metabolic disorder. A "metabolic disorder", as used
herein, refers to any pathological condition resulting from an
alteration in a patient's metabolism. Such disorders include those
associated with aberrant whole-body glucose, lipid and/or protein
metabolism and pathological consequences arising therefrom.
Metabolic disorders include those resulting from an alteration in
glucose homeostasis resulting, for example, in hyperglycemia.
According to this invention, an alteration in glucose levels is
typically an increase in glucose levels by at least 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relative to such
levels in a healthy individual. Metabolic disorders can
detrimentally affect cellular functions such as cellular
proliferation, growth, differentiation, or migration, cellular
regulation of homeostasis, inter- or intracellular communication;
tissue function, such as liver function, muscle function, or
adipocyte function; systemic responses in an organism, such as
hormonal responses (e.g., insulin response). Metabolic disorders
include, but are not limited to, obesity, diabetes (also referred
to herein as diabetes mellitus) (e.g., diabetes type I, diabetes
type II, MODY, and gestational diabetes), pre-diabetes, Metabolic
Syndrome, satiety, and endocrine abnormalities, e.g., of aging.
Further examples of metabolic disorders include, but are not
limited to, hyperphagia, hypophagia, triglyceride storage disease,
Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi
syndrome, Kearns-Sayre syndrome, anorexia, medium chain acyl-CoA
dehydrogenase deficiency, and cachexia. In some embodiments, the
metabolic disorder is a Coenzyme Q10 responsive state.
[0108] By "treating, reducing, or preventing a metabolic disorder"
is meant ameliorating such a condition before or after it has
occurred. As compared with an equivalent untreated control, such
reduction or degree of prevention is at least 5%, 10%, 20%, 40%,
50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard
technique. Diabetes mellitus is a heterogeneous group of metabolic
diseases which lead to chronic elevation of glucose in the blood
(hyperglycemia). Diabetes is characterized by pancreatic islet
destruction or dysfunction leading to loss of glucose regulation.
The two major types of diabetes mellitus are Type I, also known as
"insulin-dependent diabetes" ("IDDM") or "juvenile-onset diabetes",
and Type II, also known as "non-insulin dependent" ("NIDDM") or
"maturity-onset diabetes".
[0109] "Type I diabetes" refers to a condition that results from an
autoimmune-mediated destruction of pancreatic .beta. cells with
consequent loss of insulin production, which results in
hyperglycemia. Type I diabetics require insulin replacement therapy
to ensure survival. While medications such as injectable insulin
and oral hypoglycemics allow diabetics to live longer, diabetes
remains the third major killer, after heart disease and cancer.
However, these medications do not control blood sugar levels well
enough to prevent swinging between high and low blood sugar levels,
with resulting damage to the kidneys, eyes, and blood vessels. Data
from the Diabetes Control and Complications Trial (DCCT) show that
intensive control of blood glucose significantly delays
complications of diabetes, such as retinopathy, nephropathy, and
neuropathy, compared with conventional therapy consisting of one or
two insulin injections per day. Intensive therapy in the DCCT
included multiple injection of insulin three or more times per day
or continuous subcutaneous insulin infusion (CSII) by external
pump. Insulin pumps are one of a variety of alternative approaches
to subcutaneous multiple daily injections (MDI) for approximating
physiological replacement of insulin.
[0110] "Type 2 diabetes" refers to the condition in which a patient
has a fasting blood glucose or serum glucose concentration greater
than 125 mg/dl (6.94 mmol/L). Type II diabetes is characterized by
hyperglycemia in the presence of higher-than-normal levels of
plasma insulin (hyperinsulinemia) and represents over 90% of all
cases and occurs most often in overweight adults over 40 years of
age. Progression of Type II diabetes is associated with increasing
concentrations of blood glucose, coupled with a relative decrease
in the rate of glucose-induced insulin secretion. In Type II
diabetes, tissue processes which control carbohydrate metabolism
are believed to have decreased sensitivity to insulin and therefore
occur not from a lack of insulin production, but a decreased
sensitivity to increased glucose levels in the blood and an
inability to respond by producing insulin. Alternatively, diabetes
may result from various defects in the molecular machinery that
mediate the action of insulin on its target cells, such as a lack
of insulin receptors on their cell surfaces. Treatment of Type II
diabetes therefore frequently does not require administration of
insulin but may be based on diet and lifestyle changes, augmented
by therapy with oral hypoglycemic agents such as, for example,
sulfonylurea.
[0111] "Pre-diabetes" refers to a condition where a patient is
pre-disposed to the development of type 2 diabetes. Pre-diabetes
extends the definition of impaired glucose tolerance to include
individuals with a fasting blood glucose within the high normal
range.gtoreq.100 mg/dL (Meigs et al., Diabetes 2003 52:1475-1484)
and fasting hyperinsulinemia (elevated plasma insulin
concentration).
[0112] "Obesity" refers to the condition where a patient has a BMI
equal to or greater than 30 kg/m.sup.2. "Visceral obesity" refers
to a waist to hip ration of 1.0 in male patients and 0.8 in female
patients. In another aspect, visceral obesity defines the risk for
insulin resistance and the development of pre-diabetes.
[0113] "Overweight" refers to a patient with a BMI greater than or
25 kg/m.sup.2 and less than 30 kg/m.sup.2. "Weight gain" refers to
the increase in body weight in relationship to behavioral habits or
addictions, e.g., overeating or gluttony, smoking cessation, or in
relationship to biological (life) changes, e.g., weight gain
associated with aging in men and menopause in women or weight gain
after pregnancy.
[0114] "Metabolic Syndrome" (MS), also referred to as Syndrome X,
refers to a metabolic disorder that affects other pathways and
systems in the body. Originally, Metabolic Syndrome was defined as
a cluster of metabolic disorders (including obesity, insulin
resistance, hypertension, and dyslipidemia primarily
hypertriglyceridemia), that synergize to potentiate cardiovascular
disease. More recently (2001), the U.S. National Cholesterol
Education Program (NCEP) has classified "Metabolic Syndrome" as
meeting any three out of the following five criteria: fasting
glucose level of at least 110 mg/dl, plasma triglyceride level of
at least 150 mg/dl (hypertriglycerdemia), HDL cholesterol below 40
mg/dl in men or below 50 mg/dl in women, blood pressure at least
130/85 mm Hg (hypertension), and central obesity, with central
obesity being defined as abdominal waist circumference greater than
40 inches for men and greater than 35 inches for women. Presently,
there are three other internationally recognized definitions for
Metabolic Syndrome as follows: 1) World Health Organization 2)
American Heart Association/National Heart, Lung and blood Institute
(AHA/NHLBI) and 3) International Diabetes Federation (IDF). The
definitions of Metabolic Syndrome by the WHO, AHA/NHLBI and IDF are
very similar to the definition of the NECP and all use the same
metabolic parameters to define the syndrome, but the WHO also
includes assessment of insulin fasting insulin levels (Moebus S et
al, Cardiovascular Diabetology, 6: 1-10, 2007; Athyros V G et al,
Int. J. Cardiology, 117: 204-210, 2007). Yet subtle differences in
the thresholds for these metabolic parameters required to be
classified as having the syndrome among these different definitions
can result in different classification of a particular subject as
having or not having the syndrome according to these different
definitions. Also, the prevalence of cardiovascular disease (CVD)
with MS varies by the definition used. (Moebus S et al,
Cardiovascular Diabetology, 6: 1-10, 2007; Athyros V G et al, Int.
J. Cardiology, 117: 204-210, 2007). The American Diabetes
Association estimates that 1 in every 5 overweight people suffer
from Metabolic Syndrome.
[0115] In other aspects, the metabolic syndrome is described by
accepted synonyms, which includes, but is not limited to, syndrome
X, insulin resistance syndrome, insulin-resistant hypertension, the
metabolic hypertensive syndrome, dysmetabolic syndrome. Components
of the metabolic syndrome include, but are not limited to, glucose
intolerance, impaired glucose tolerance, impaired fasting serum
glucose, impaired fasting blood glucose, hyperinsulinemia,
pre-diabetes, obesity, visceral obesity, hypertriglyceridemia,
elevated serum concentrations of free fatty acids, elevated serum
concentrations of C-reactive protein, elevated serum concentrations
of lipoprotein(a), elevated serum concentrations of homocysteine,
elevated serum concentrations of small, dense low-density
lipoprotein (LDL)-cholesterol, elevated serum concentrations of
lipoprotein-associated phospholipase (A2), reduced serum
concentrations of high density lipoprotein (HDL)-cholesterol,
reduced serum concentrations of HDL(2b)-cholesterol, reduced serum
concentrations of adiponectin, and albuminuria (see: Pershadsingh H
A. Peroxisome proliferator-activated receptor-gamma: therapeutic
target for diseases beyond diabetes: quo vadis? Expert Opin
Investig Drugs. (2004) 13:215-28, and references cited
therein).
[0116] The "key elements" of the foregoing metabolic disorders
include but are not limited to, impaired fasting glucose or
impaired glucose tolerance, increased waist circumference,
increased visceral fat content, increased fasting plasma glucose,
increased fasting plasma triglycerides, decreased fasting high
density lipoprotein level, increased blood pressure, insulin
resistance, hyperinsulinemia, cardiovascular disease (or components
thereof such as arteriosclerosis, coronary artery disease,
peripheral vascular disease, or cerebrovascular disease),
congestive heart failure, elevated plasma norepinephrine, elevated
cardiovascular-related inflammatory factors, elevated plasma
factors potentiating vascular endothelial dysfunction,
hyperlipoproteinemia, arteriosclerosis or atherosclerosis,
hyperphagia, hyperglycemia, hyperlipidemia, and hypertension or
high blood pressure, increased plasma postprandial triglyceride or
free fatty acid levels, increased cellular oxidative stress or
plasma indicators thereof, increased circulating hypercoagulative
state, hepatic steatosis, hetaptic steatosis, renal disease
including renal failure and renal insufficiency.
[0117] "Insulin resistance" refers to a condition in which
circulating insulin levels in excess of the normal response to a
glucose load are required to maintain the euglycemic state (Ford et
al., JAMA. 2002, 287:356-9). Insulin resistance and the response of
a patient with insulin resistance to therapy, may be quantified by
assessing the homeostasis model assessment to insulin resistance
(HOMA-IR) score, a reliable indicator of insulin resistance
(Katsuki et al., Diabetes Care 2001, 24:362-5). An estimate of
insulin resistance by the homeostasis assessment model (HOMA)-IR
score may be calculated by a formula disclosed in Galvin et al.,
Diabet Med 1992, 9:921-8 where HOMA-IR=[fasting serum insulin
(.mu.U/mL)].times.[fasting plasma glucose (mmol/L)/22.5].
[0118] "Hyperinsulinemia" is defined as the condition in which a
subject with insulin resistance, with or without euglycemia, in
which the fasting or postprandial serum or plasma insulin
concentration is elevated above that of normal, lean individuals
without insulin resistance, having a waist-to-hip ration <1.0
(for men) or <0.8 (for women).
[0119] The term "impaired glucose tolerance" (IGT) is used to
describe a person who, when given a glucose tolerance test, has a
blood glucose level that falls between normal and hyperglycemic.
Such a person is at a higher risk of developing diabetes although
they are not considered to have diabetes. For example, impaired
glucose tolerance refers to a condition in which a patient has a
fasting blood glucose concentration or fasting serum glucose
concentration greater than 110 mg/dl and less than 126 mg/dl (7.00
mmol/L), or a 2 hour postprandial blood glucose or serum glucose
concentration greater than 140 mg/dl (7.78 mmol/L) and less than
200 mg/dl (11.11 mmol/L).
[0120] The condition of "hyperglycemia" (high blood sugar) is a
condition in which the blood glucose level is too high. Typically,
hyperglycemia occurs when the blood glucose level rises above 180
mg/dl. Symptoms of hyperglycemia include frequent urination,
excessive thirst and, over a longer time span, weight loss.
[0121] The condition of "hypoglycemia" (low blood sugar) is a
condition in which the blood glucose level is too low. Typically,
hypoglycemia occurs when the blood glucose level falls below 70
mg/dl. Symptoms of hypoglycemia include moodiness, numbness of the
extremities (especially in the hands and arms), confusion,
shakiness or dizziness. Since this condition arises when there is
an excess of insulin over the amount of available glucose it is
sometimes referred to as an insulin reaction.
[0122] The methods and compositions of the present invention are
useful for diagnosing any patient that is at risk of having a
metabolic disorder, such as diabetes. A patient in whom the
development of a metabolic disorder (e.g., diabetes or obesity) is
being prevented may or may not have received such a diagnosis. One
in the art will understand that patients of the invention may have
been subjected to standard tests or may have been identified,
without examination, as one at high risk due to the presence of one
or more risk factors.
[0123] Diagnosis of metabolic disorders previously has been
performed using any standard method known in the art, such as those
described herein. Methods for diagnosing diabetes are described,
for example, in U.S. Pat. No. 6,537,806, hereby incorporated by
reference. Diabetes may be diagnosed and monitored using, for
example, urine tests (urinalysis) that measure glucose and ketone
levels (products of the breakdown of fat); tests that measure the
levels of glucose in blood; glucose tolerance tests; and assays
that detect molecular markers characteristic of a metabolic
disorder in a biological sample (e.g., blood, serum, or urine)
collected from the mammal (e.g., measurements of Hemoglobin Alc
(HbAlc) levels in the case of diabetes).
[0124] A patient who is being treated for a metabolic disorder is
one who a medical practitioner has diagnosed as having such a
condition. Diagnosis may be performed by any suitable means, such
as those described herein. A patient in whom the development of
diabetes or obesity is being prevented may or may not have received
such a diagnosis. One in the art will understand that patients of
the invention may have been subjected to standard tests or may have
been identified, without examination, as one at high risk due to
the presence of one or more risk factors, such as family history,
obesity, particular ethnicity (e.g., African Americans and Hispanic
Americans), gestational diabetes or delivering a baby that weighs
more than nine pounds, hypertension, having a pathological
condition predisposing to obesity or diabetes, high blood levels of
triglycerides, high blood levels of cholesterol, presence of
molecular markers (e.g., presence of autoantibodies), and age (over
45 years of age). An individual is considered obese when their
weight is 20% (25% in women) or more over the maximum weight
desirable for their height. An adult who is more than 100 pounds
overweight, is considered to be morbidly obese. Obesity is also
defined as a body mass index (BMI) over 30 kg/m.sup.2.
[0125] Patients may be diagnosed as being at risk or as having
diabetes if a random plasma glucose test (taken at any time of the
day) indicates a value of 200 mg/dL or more, if a fasting plasma
glucose test indicates a value of 126 mg/dL or more (after 8
hours), or if an oral glucose tolerance test (OGTT) indicates a
plasma glucose value of 200 mg/dL or more in a blood sample taken
two hours after a person has consumed a drink containing 75 grams
of glucose dissolved in water. The OGTT measures plasma glucose at
timed intervals over a 3-hour period. Desirably, the level of
plasma glucose in a diabetic patient that has been treated
according to the invention ranges between 160 to 60 mg/dL, between
150 to 70 mg/dL, between 140 to 70 mg/dL, between 135 to 80 mg/dL,
and preferably between 120 to 80 mg/dL.
[0126] Optionally, a hemoglobin Alc (HbAlc) test, which assesses
the average blood glucose levels during the previous two and three
months, may be employed. A person without diabetes typically has an
HbAlc value that ranges between 4% and 6%. For every 1% increase in
HbAlc, blood glucose levels increases by approximately 30 mg/dL and
the risk of complications increases. Preferably, the HbAlc value of
a patient being treated according to the present invention is
reduced to less than 9%, less than 7%, less than 6%, and most
preferably to around 5%. Thus, the HbAlc levels of the patient
being treated are preferably lowered by 10%, 20%, 30%, 40%, 50%, or
more relative to such levels prior to treatment.
[0127] Gestational diabetes is typically diagnosed based on plasma
glucose values measured during the OGTT. Since glucose levels are
normally lower during pregnancy, the threshold values for the
diagnosis of diabetes in pregnancy are lower than in the same
person prior to pregnancy. If a woman has two plasma glucose
readings that meet or exceed any of the following numbers, she has
gestational diabetes: a fasting plasma glucose level of 95 mg/dL, a
1-hour level of 180 mg/dL, a 2-hour level of 155 mg/dL, or a 3-hour
level of 140 mg/dL.
[0128] Ketone testing may also be employed to diagnose type I
diabetes. Because ketones build up in the blood when there is not
enough insulin, they eventually accumulate in the urine. High
levels of blood ketones may result in a serious condition called
ketoacidosis.
[0129] According to the guidelines of the American Diabetes
Association, to be diagnosed with Type 2 diabetes, an individual
must have a fasting plasma glucose level greater than or equal to
126 mg/dl or a 2-hour oral glucose tolerance test (OGTT) plasma
glucose value of greater than or equal to 200 mg/dl (Diabetes Care,
26:S5-S20, 2003).
[0130] A related condition called pre-diabetes is defined as having
a fasting glucose level of greater than 100 mg/dl but less than 126
mg/dl or a 2-hour OGTT plasma glucose level of greater than 140
mg/dl but less than 200 mg/dl. Mounting evidence suggests that the
pre-diabetes condition may be a risk factor for developing
cardiovascular disease (Diabetes Care 26:2910-2914, 2003).
Prediabetes, also referred to as impaired glucose tolerance or
impaired fasting glucose is a major risk factor for the development
of type 2 diabetes mellitus, cardiovascular disease and mortality.
Much focus has been given to developing therapeutic interventions
that prevent the development of type 2 diabetes by effectively
treating prediabetes (Pharmacotherapy, 24:362-71, 2004).
[0131] Obesity (commonly defined as a Body Mass Index of
approximately >30 kg/m.sup.2) is often associated with a variety
of pathologic conditions such as hyperinsulinemia, insulin
resistance, diabetes, hypertension, and dyslipidemia. Each of these
conditions contributes to the risk of cardiovascular disease.
[0132] Along with insulin resistance, hypertension, and
dyslipidemia, obesity is considered to be a component of the
Metabolic Syndrome (also known as Syndrome X) which together
synergize to potentiate cardiovascular disease. More recently, the
U.S. National Cholesterol Education Program has classified
Metabolic Syndrome as meeting three out of the following five
criteria: fasting glucose level of at least 110 mg/dl, plasma
triglyceride level of at least 150 mg/dl (hypertriglycerdemia), HDL
cholesterol below 40 mg/dl in men or below 50 mg/dl in women, blood
pressure at least 130/85 mm Hg (hypertension), and central obesity,
with central obesity being defined as abdominal waist circumference
greater than 40 inches for men and greater than 35 inches for
women.
[0133] The skilled artisan will recognize that the use of any of
the above tests or any other tests known in the art may be used to
monitor the efficacy of the therapeutic treatments of the
invention. Since the measurements of hemoglobin Alc (HbAlc) levels
is an indication of average blood glucose during the previous two
to three months, this test may be used to monitor a patient's
response to diabetes treatment.
[0134] The therapeutic methods of the invention are effective in
reducing glucose levels or lipid levels in a patient. By "reducing
glucose levels" is meant reducing the level of glucose by at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative
to an untreated control. Desirably, glucose levels are reduced to
normoglycemic levels, i.e., between 150 to 60 mg/dL, between 140 to
70 mg/dL, between 130 to 70 mg/dL, between 125 to 80 mg/dL, and
preferably between 120 to 80 mg/dL. Such reduction in glucose
levels may be obtained by increasing any one of the biological
activities associated with the clearance of glucose from the blood.
Accordingly, an agent having the ability to reduce glucose levels
may increase insulin production, secretion, or action. Insulin
action may be increased, for example, by increasing glucose uptake
by peripheral tissues and/or by reducing hepatic glucose
production. Alternatively, the agent of the invention may reduce
the absorption of carbohydrates from the intestines, alter glucose
transporter activity (e.g., by increasing GLUT4 expression,
intrinsic activity, or translocation), increase the amount of
insulin-sensitive tissue (e.g., by increasing muscle cell or
adipocyte cell differentiation), or alter gene transcription in
adipocytes or muscle cells (e.g., altered secretion of factors from
adipocytes expression of metabolic pathway genes). Desirably, the
agent of the invention increases more than one of the activities
associated with the clearance of glucose. By "reducing lipid
levels" is meant reducing the level of lipids by at least 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative
to an untreated control.
[0135] By "alter insulin signaling pathway such that glucose levels
are reduced" is meant to alter (by increasing or reducing) any one
of the activities involved in insulin signaling such that the
overall result is an increase in the clearance of glucose from
plasma. For example, the env-influencer of the invention alters the
insulin signaling pathway causing an increase in insulin
production, secretion, or action, an increase in glucose uptake by
peripheral tissues, a reduction in hepatic glucose production, or a
reduction in the absorption of carbohydrates from the
intestines.
[0136] The ability of an environmental influencer, e.g.,
epi-shifter, to reduce glucose levels and thereby treat a metabolic
disorder may be assessed using standard assays known in the art.
For example, cell-based screening assays that identify agents that
increase glucose uptake may be employed. In particular,
differentiated adipocytes in cell culture can be employed to assess
the ability of the epi-shifter to increase glucose uptake upon
insulin stimulation, as detected by radiolabeled glucose. In
another exemplary assay, human myoblasts obtained by the
conditional immortalization of cells derived from a non-diabetic
subject can be used to screen the effect of agents on glycogen
synthesis, using insulin as a positive control. Prior to treatment,
cells are serum-starved, and are then incubated either with the
epi-shifter or control for a period of two hours in serum-free
media containing radiolabeled glucose, after which, glycogen
synthesis is measured. Exemplary assays are further described in
the Examples.
II. Environmental Influencers
[0137] The present invention provides methods of treating metabolic
disorders by administration of an Environmental influencer.
"Environmental influencers" (Env-influencers) are molecules that
influence or modulate the disease environment of a human in a
beneficial manner allowing the human's disease environment to
shift, reestablish back to or maintain a normal or healthy
environment leading to a normal state. Env-influencers include both
Multidimensional Intracellular Molecules (MIMs) and Epimetabolic
shifters (Epi-shifters) as defined below.
1. Multidimensional Intracellular Molecule (MIM)
[0138] The term "Multidimensional Intracellular Molecule (MIM)", is
an isolated version or synthetically produced version of an
endogenous molecule that is naturally produced by the body and/or
is present in at least one cell of a human. A MIM is characterized
by one or more, two or more, three or more, or all of the following
functions. MIMs are capable of entering a cell, and the entry into
the cell includes complete or partial entry into the cell, as long
as the biologically active portion of the molecule wholly enters
the cell. MIMs are capable of inducing a signal transduction and/or
gene expression mechanism within a cell. MIMs are multidimensional
in that the molecules have both a therapeutic and a carrier, e.g.,
drug delivery, effect. MIMs also are multidimensional in that the
molecules act one way in a disease state and a different way in a
normal state. For example, in the case of CoQ-10, administration of
CoQ-10 to a melanoma cell in the presence of VEGF leads to a
decreased level of Bcl2 which, in turn, leads to a decreased
oncogenic potential for the melanoma cell. In contrast, in a normal
fibroblast, co-administration of CoQ-10 and VEFG has no effect on
the levels of Bcl2. Preferably, MIMs selectively act in cells of a
disease state, and have substantially no effect in (matching) cells
of a normal state. Preferably, MIMs selectively renders cells of a
disease state closer in phenotype, metabolic state, genotype,
mRNA/protein expression level, etc. to (matching) cells of a normal
state.
[0139] In one embodiment, a MIM is also an epi-shifter. In another
embodiment, a MIM is not an epi-shifter. The skilled artisan will
appreciate that a MIM of the invention is also intended to
encompass a mixture of two or more endogenous molecules, wherein
the mixture is characterized by one or more of the foregoing
functions. The endogenous molecules in the mixture are present at a
ratio such that the mixture functions as a MIM.
[0140] MIMs can be lipid based or non-lipid based molecules.
Examples of MIMs include, but are not limited to, CoQ10, acetyl
Co-A, palmityl Co-A, L-carnitine, amino acids such as, for example,
tyrosine, phenylalanine, and cysteine. In one embodiment, the MIM
is a small molecule. In one embodiment of the invention, the MIM is
not CoQ10. MIMs can be routinely identified by one of skill in the
art using any of the assays described in detail herein.
[0141] In some embodiments, MIMs include compounds in the Vitamin B
family, or nucleosides, mononucleotides or dinucleotides that
comprise a compound in the Vitamin B family. Compounds in the
vitamin B family include, for example, thiamine (vitamin B1),
niacin (also known as nicotinic acid or Vitamin B3), or pyridoxine
(vitamin B6) as well as provitamins such as panthenol (provitamin
B5). In some embodiments, the MIM is selected from thiamine, niacin
and pyridoxine. Nucleosides, mononucleotides or dinucleotides that
comprise a compound in the vitamin B family include, for example,
nucleosides, mononucleotides or dinucleotides which include an
adenine or a niacin (nicotinic acid) molecule. In some embodiments,
the MIM is selected from adenosine, adenosine diphosphate (ADP),
flavin adenosine dinucleotide (FAD, which comprises parts of
vitamin B2 and ADP) and nicotinic acid dinucleotide.
[0142] In other embodiments, the MIMs include amino acids. Examples
of amino acids include, for example, tyrosine (e.g., L-tyrosine),
cysteine, phenylalanine (e.g., L-phenylalanine) and alanine. In
some embodiments, the amino acid is phenylalanine or alanine. In
some embodiments, the MIMs include amino acid derivatives such as
4-hydroxyphenylpyruvate or acetylglycine.
[0143] In some embodiment, the MIM is a glucose analog, e.g., a
glucose molecule wherein one --OH or --CH.sub.2OH substituent has
been replaced with a --COOH, a --COO.sup.- or an --NH.sub.2
substituent. Examples of glucose analogs include glucosamine,
glucuronic acid, glucuronide and glucuronate.
[0144] In some embodiments, the MIM is selected from compounds of
formula (I):
##STR00001##
[0145] wherein
[0146] n is an integer of 0 or 1;
[0147] R.sup.1, R.sup.2, R.sup.3 and R.sup.4, when present, are
each independently selected from hydrogen and hydroxyl or R.sup.1
and R.sup.2 are taken together with the carbon on which they are
attached to form a carbonyl (C.dbd.O) group;
[0148] W is --COOH or --N(CH.sub.3).sub.3.sup.+; and
[0149] X is hydrogen, a negative charge or a alkali metal cation,
such as Na.sup.+ or.
[0150] It is to be understood that when n is 0, the CHR.sup.3 group
is bonded to the W substituent.
[0151] In some embodiments, W is --N(CH.sub.3).sub.3.sup.+. In some
embodiments, the MIM is a carnitine, such as L-carnitine.
[0152] In some embodiments, the MIM is a dicarboxylic acid. In some
embodiments, W is --COOH. In some embodiments, R.sup.3 is hydrogen.
In some embodiments, n is 0. In some embodiments, R.sup.1 and
R.sup.2 are each independently hydrogen. In some embodiments, W is
--COOH, R.sup.3 is hydrogen, n is 0 and R.sup.1 and R.sup.2 are
each independently hydrogen. In some embodiments, n is 1. In some
embodiments R.sup.1 and R.sup.2 are taken together with the carbon
on which they are attached to form a carbonyl (C.dbd.O) group. In
some embodiments, R.sup.4 is hydrogen. In some embodiments, R.sup.4
is hydroxyl. In some embodiments, W is --COOH, R.sup.3 is hydrogen,
n is 1 and R.sup.1 and R.sup.2 are taken together with the carbon
on which they are attached to form a carbonyl (C.dbd.O) group.
[0153] In some embodiments, the MIM is an intermediate of the Krebs
Cycle, the excess of which drives the Krebs Cycle towards
productive oxidative phosphorylation. Exemplary Krebs Cycle
intermediates that are MIMs include succinic acid or succinate,
malic acid or malate, and .alpha.-ketoglutaric acid or
.alpha.-ketoglutarate.
[0154] In some embodiments, the MIM is a building block of CoQ10,
which has the following structure:
##STR00002##
[0155] Thus, building blocks of CoQ10 include, but are not limited
to, phenylalanine, tyrosine, 4-hydroxyphenylpyruvate,
phenylacetate, 3-methoxy-4-hydroxymandelate, vanillic acid,
4-hydroxybenzoate, mevalonic acid, farnesyl,
2,3-dimethoxy-5-methyl-p-benzoquinone, as well as the corresponding
acids or ions thereof. In some embodiments, the MIM is selected
from phenylalanine, tyrosine, 4-hydroxyphenylpyruvate,
phenylacetate and 4-hydroxybenzoate.
(i) Methods of Identifying MIMS
[0156] The present invention provides methods for identifying a
MIM. Methods for identifying a MIM involve, generally, the
exogenous addition to a cell of an endogenous molecule and
evaluating the effect on the cell, e.g., the cellular
microenvironment profile, that the endogenous molecule provides.
Effects on the cell are evaluated at one or more of the cellular,
mRNA, protein, lipid, and/or metabolite level to identify
alterations in the cellular microenvironment profile. In one
embodiment, the cells are cultured cells, e.g., in vitro. In one
embodiment, the cells are present in an organism. The endogenous
molecule may be added to the cell at a single concentration or may
be added to the cell over a range of concentrations. In one
embodiment, the endogenous molecule is added to the cells such that
the level of the endogenous molecule in the cells is elevated
(e.g., is elevated by 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5
fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold,
4.0 fold, 5.0 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35
fold, 40 fold, 45 fold, 50 fold or greater) as compared to the
level of the endogenous molecule in a control, untreated cell.
[0157] Molecules that induce a change in the cell as detected by
alterations in, for example, any one or more of morphology,
physiology, and/or composition (e.g., mRNA, protein, lipid,
metabolite) may be evaluated further to determine if the induced
changes to the cellular microenvironment profile are different
between a disease cellular state and a normal cellular state. Cells
(e.g., cell culture lines) of diverse tissue origin, cell type, or
disease state may be evaluated for comparative evaluation. For
example, changes induced in the cellular microenvironment profile
of a cancer cell may be compared to changes induced to a
non-cancerous or normal cell. An endogenous molecule that is
observed to induce a change in the microenvironment profile of a
cell (e.g., induces a change in the morphology, physiology and/or
composition, e.g., mRNA, protein, lipid or metabolite, of the cell)
and/or to differentially (e.g., preferentially) induce a change in
the microenvironment profile of a diseased cell as compared to a
normal cell, is identified as a MIM.
[0158] MIMs of the invention may be lipid based MIMs or non-lipid
based MIMs. Methods for identifying lipid based MIMs involve the
above-described cell based methods in which a lipid based
endogenous molecule is exogenously added to the cell. In a
preferred embodiment, the lipid based endogenous molecule is added
to the cell such that the level of the lipid based endogenous
molecule in the cell is elevated. In one embodiment, the level of
the lipid based endogenous molecule is elevated by 1.1 fold, 1.2
fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold,
1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10 fold, 15 fold,
20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold or
greater as compared to the level in an untreated control cell.
Formulation and delivery of the lipid based molecule to the cell is
dependent upon the properties of each molecule tested, but many
methods are known in the art. Examples of formulation and delivery
of lipid based molecules include, but are not limited to,
solubilization by co-solvents, carrier molecules, liposomes,
dispersions, suspensions, nanoparticle dispersions, emulsions,
e.g., oil-in-water or water-in-oil emulsions, multiphase emulsions,
e.g., oil-in-water-in-oil emulsions, polymer entrapment and
encapsulation. The delivery of the lipid based MIM to the cell can
be confirmed by extraction of the cellular lipids and
quantification of the MIM by routine methods known in the art, such
as mass spectrometry.
[0159] Methods for identifying non-lipid based MIMs involve the
above-described cell based methods in which a non-lipid based
endogenous molecule is exogenously added to the cell. In a
preferred embodiment, the non-lipid based endogenous molecule is
added to the cell such that the level of the non-lipid based
endogenous molecule in the cell is elevated. In one embodiment, the
level of the non-lipid based endogenous molecule is elevated by 1.1
fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold,
1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10
fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45
fold, 50 fold or greater as compared to the level in an untreated
control cell. Formulation and delivery of the non-lipid based
molecule to the cell is dependent upon the properties of each
molecule tested, but many methods are known in the art. Examples of
formulations and modes of delivery of non-lipid based molecules
include, but are not limited to, solubilization by co-solvents,
carrier molecules, active transport, polymer entrapment or
adsorption, polymer grafting, liposomal encapsulation, and
formulation with targeted delivery systems. The delivery of the
non-lipid based MIM to the cell may be confirmed by extraction of
the cellular content and quantification of the MIM by routine
methods known in the art, such as mass spectrometry.
2. Epimetabolic Shifters (Epi-Shifters)
[0160] As used herein, an "epimetabolic shifter" (epi-shifter) is a
molecule (endogenous or exogenous) that modulates the metabolic
shift from a healthy (or normal) state to a disease state and vice
versa, thereby maintaining or reestablishing cellular, tissue,
organ, system and/or host health in a human. Epi-shifters are
capable of effectuating normalization in a tissue microenvironment.
For example, an epi-shifter includes any molecule which is capable,
when added to or depleted from a cell, of affecting the
microenvironment (e.g., the metabolic state) of a cell. The skilled
artisan will appreciate that an epi-shifter of the invention is
also intended to encompass a mixture of two or more molecules,
wherein the mixture is characterized by one or more of the
foregoing functions. The molecules in the mixture are present at a
ratio such that the mixture functions as an epi-shifter. Examples
of epi-shifters include, but are not limited to, coQ-10; vitamin
D3; ECM components such as fibronectin; immunomodulators, such as
TNFa or any of the interleukins, e.g., IL-5, IL-12, IL-23;
angiogenic factors; and apoptotic factors.
[0161] In some embodiments, the epi-shifter is an enzyme, such as
an enzyme that either directly participates in catalyzing one or
more reactions in the Krebs Cycle, or produces a Krebs Cycle
intermediate, the excess of which drive the Krebs Cycle. In some
embodiments, the enzyme is an enzyme of the non-oxidative phase of
the pentose phosphate pathway, such as transaldolase, or
transketolase. In other embodiments, the enzyme is a component
enzyme or enzyme complex that facilitates the Krebs Cycle, such as
a synthase or a ligase. Exemplary enzymes include succinyl CoA
synthase (Krebs Cycle enzyme) or pyruvate carboxylase (a ligase
that catalyzes the reversible carboxylation of pyruvate to form
oxaloacetate (OAA), a Krebs Cycle intermediate).
[0162] In some embodiments, the epi-shifter is a building block of
CoQ10. Building blocks of CoQ10 include, but are not limited to,
phenylalanine, tyrosine, 4-hydroxyphenylpyruvate, phenylacetate,
3-methoxy-4-hydroxymandelate, vanillic acid, 4-hydroxybenzoate,
mevalonic acid, farnesyl, 2,3-dimethoxy-5-methyl-p-benzoquinone, as
well as the corresponding acids or ions thereof. In some
embodiments, the epi-shifter is selected from phenylalanine,
tyrosine, 4-hydroxyphenylpyruvate, phenylacetate and
4-hydroxybenzoate.
[0163] In some embodiments, the epi-shifter is a compound in the
Vitamin B family. Compounds in the vitamin B family include, for
example, riboflavin (vitamin B2), or analogs thereof. Epi-shifters
also include any analogs or pro-drugs that may be metabolized in
vivo to any of the endogenous MIMs, such as those described
herein.
[0164] In one embodiment, the epi-shifter also is a MIM. In one
embodiment, the epi-shifter is not CoQ10. Epi-shifters can be
routinely identified by one of skill in the art using any of the
assays described in detail herein.
(i) Methods of Identifying Epi-Shifters
[0165] Epimetabolic shifters (epi-shifter) are molecules capable of
modulating the metabolic state of a cell, e.g., inducing a
metabolic shift from a healthy (or normal) state to a disease state
and vice versa, and are thereby capable of maintaining or
reestablishing cellular, tissue, organ, system and/or host health
in a human. Epi-shifters of the invention thus have utility in the
diagnostic evaluation of a diseased state. Epi-shifters of the
invention have further utility in therapeutic applications, wherein
the application or administration of the epi-shifter (or modulation
of the epi-shifter by other therapeutic molecules) effects a
normalization in a tissue microenvironment and the disease
state.
[0166] The identification of an epimetabolic shifter involves,
generally, establishing a molecular profile, e.g., of metabolites,
lipids, proteins or RNAs (as individual profiles or in
combination), for a panel of cells or tissues that display
differential disease states, progression, or aggressiveness A
molecule from the profile(s) for which a change in level (e.g., an
increased or decreased level) correlates to the disease state,
progression or aggressiveness is identified as a potential
epi-shifter.
[0167] In one embodiment, an epi-shifter is also a MIM. Potential
epi-shifters may be evaluated for their ability to enter cells upon
exogenous addition to a cell by using any number of routine
techniques known in the art, and by using any of the methods
described herein. For example, entry of the potential epi-shifter
into a cell may be confirmed by extraction of the cellular content
and quantification of the potential epi-shifter by routine methods
known in the art, such as mass spectrometry. A potential
epi-shifter that is able to enter a cell is thereby identified as a
MIM.
[0168] To identify an epi-shifter, a potential epi-shifter is next
evaluated for the ability to shift the metabolic state of a cell.
The ability of a potential epi-shifters to shift the metabolic
state of the cell microenvironment is evaluated by introducing
(e.g., exogenously adding) to a cell a potential epi-shifter and
monitoring in the cell one or more of: changes in gene expression
(e.g., changes in mRNA or protein expression), concentration
changes in lipid or metabolite levels, changes in bioenergetic
molecule levels, changes in cellular energetics, and/or changes in
mitochondrial function or number. Potential epi-shifters capable of
shifting the metabolic state of the cell microenvironment can be
routinely identified by one of skill in the art using any of the
assays described in detail herein. Potential epi-shifters are
further evaluated for the ability to shift the metabolic state of a
diseased cell towards a normal healthy state (or conversely, for
the ability to shift the metabolic state of a normal cell towards a
diseased state). A potential epi-shifter capable of shifting the
metabolic state of a diseased cell towards a normal healthy state
(or of shifting the metabolic state of healthy normal cell towards
a diseased state) is thus identified as an Epi-shifter. In a
preferred embodiment, the epi-shifter does not negatively impact
the health and/or growth of normal cells.
[0169] Epimetabolic shifters of the invention include, but are not
limited to, small molecule metabolites, lipid-based molecules, and
proteins and RNAs. To identify an epimetabolic shifter in the class
of small molecule endogenous metabolites, metabolite profiles for a
panel of cells or tissues that display differential disease states,
progression, or aggressiveness are established. The metabolite
profile for each cell or tissue is determined by extracting
metabolites from the cell or tissue and then identifying and
quantifying the metabolites using routine methods known to the
skilled artisan, including, for example, liquid-chromatography
coupled mass spectrometry or gas-chromatography couple mass
spectrometry methods. Metabolites for which a change in level
(e.g., an increased or decreased level) correlates to the disease
state, progression or aggressiveness, are identified as potential
epi-shifters.
[0170] To identify epimetabolic shifters in the class of endogenous
lipid-based molecules, lipid profiles for a panel of cells or
tissues that display differential disease states, progression, or
aggressiveness are established. The lipid profile for each cell or
tissue is determined by using lipid extraction methods, followed by
the identification and quantitation of the lipids using routine
methods known to the skilled artisan, including, for example,
liquid-chromatography coupled mass spectrometry or
gas-chromatography couple mass spectrometry methods. Lipids for
which a change in level (e.g., an increase or decrease in bulk or
trace level) correlates to the disease state, progression or
aggressiveness, are identified as potential epi-shifters.
[0171] To identify epimetabolic shifters in the class of proteins
and RNAs, gene expression profiles for a panel of cells or tissues
that display differential disease states, progression, or
aggressiveness are established. The expression profile for each
cell or tissue is determined at the mRNA and/or protein level(s)
using standard proteomic, mRNA array, or genomic array methods,
e.g., as described in detail herein. Genes for which a change in
expression (e.g., an increase or decrease in expression at the mRNA
or protein level) correlates to the disease state, progression or
aggressiveness, are identified as potential epi-shifters.
[0172] Once the molecular profiles described above are established
(e.g., for soluble metabolites, lipid-based molecules, proteins,
RNAs, or other biological classes of composition), cellular and
biochemical pathway analysis is carried out to elucidate known
linkages between the identified potential epi-shifters in the
cellular environment. This information obtained by such cellular
and/or biochemical pathway analysis may be utilized to categorize
the pathways and potential epi-shifters.
[0173] The utility of an Epi-shifter to modulate a disease state
can be further evaluated and confirmed by one of skill in the art
using any number of assays known in the art or described in detail
herein. The utility of an Epi-shifter to modulate a disease state
can be evaluated by direct exogenous delivery of the Epi-shifter to
a cell or to an organism. The utility of an Epi-shifter to modulate
a disease state can alternatively be evaluated by the development
of molecules that directly modulate the Epi-shifter (e.g., the
level or activity of the Epi-shifter). The utility of an
Epi-shifter to modulate a disease state can also be evaluated by
the development of molecules that indirectly modulate the
Epi-shifter (e.g., the level or activity of the Epi-shifter) by
regulating other molecules, such as genes (e.g., regulated at the
RNA or protein level), placed in the same pathway as the
Epi-shifter.
[0174] The Epimetabolomic approach described herein facilitates the
identification of endogenous molecules that exist in a cellular
microenvironment and the levels of which are sensed and controlled
through genetic, mRNA, or protein-based mechanisms. The regulation
response pathways found in normal cells that are triggered by an
Epi-shifter of the invention may provide a therapeutic value in a
misregulated or diseased cellular environment. In addition, the
epimetabolic approach described herein identifies epi-shifters that
may provide a diagnostic indication for use in clinical patient
selection, a disease diagnostic kit, or as a prognostic
indicator.
III. Assays Useful for Identifying MIMs/Epi-Shifters
[0175] Techniques and methods of the present invention employed to
separate and identify molecules and compounds of interest include
but are not limited to: liquid chromatography (LC), high-pressure
liquid chromatography (HPLC), mass spectroscopy (MS), gas
chromatography (GC), liquid chromatography/mass spectroscopy
(LC-MS), gas chromatography/mass spectroscopy (GC-MS), nuclear
magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier
Transform InfraRed (FT-IR), and inductively coupled plasma mass
spectrometry (ICP-MS). It is further understood that mass
spectrometry techniques include, but are not limited to, the use of
magnetic-sector and double focusing instruments, transmission
quadrapole instruments, quadrupole ion-trap instruments,
time-of-flight instruments (TOF), Fourier transform ion cyclotron
resonance instruments (FT-MS) and matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS).
Quantification of Bioenergetic Molecule Levels:
[0176] Environmental influencers (e.g., MIMs or Epi-shifters) may
be identified by changes in cellular bioenergetic molecule levels
(e.g., ATP, pyruvate, ADP, NADH, NAD, NADPH, NADP, acetylCoA,
FADH2) of cells to which a candidate epi-shifter has been applied.
Exemplary assays of bioenergetic molecule levels use colorometric,
fluorescence, and/or bioluminescent-based methods. Examples of such
assays are provided below.
[0177] Levels of ATP within cells can be measured with a number of
assays and systems known in the art. For example, in one system,
cytoplasmic ATP released from lysed cells reacts with luciferin and
the enzyme luciferase to produce light. This bioluminescence is
measured by a bioluminometer and the intracellular ATP
concentration of the lysed cells can be calculated (EnzyLight.TM.
ATP Assay Kit (EATP-100), BioAssay Systems, Hayward, Calif.). In
another system, for example, both ATP and its dephosphorylated
form, ADP, are calculated via bioluminescence; after ATP levels are
calculated, ADP is transformed into ATP and then detected and
calculated using the same luciferase system (ApoSENSOR.TM. ADP/ATP
Ratio Assay Kit, BioVision Inc., Mountain View, Calif.).
[0178] Pyruvate is an important intermediate in cellular metabolic
pathways. Pyruvate may be converted into carbohydrate via
gluconeogenesis, converted into fatty acid or metabolized via
acetyl CoA, or converted into alanine or ethanol, depending upon
the metabolic state of a cell. Thus detection of pyruvate levels
provides a measure of the metabolic activity and state of a cell
sample. One assay to detect pyruvate, for example, uses both a
colorimetric and fluorimetric to detect pyruvate concentrations
within different ranges (EnzyChrom.TM. Pyruvate Assay Kit (Cat#
EPYR-100), BioAssay Systems, Hayward, Calif.).
[0179] Environmental influencers (e.g., MIMs or Epi-shifters) may
influence the process of oxidative phosphorylation carried out by
mitochondria in cells, which are involved in the generation and
maintenance of bioenergetic molecules in cells. In addition to
assays that detect changes in cellular energetics in cell cultures
and samples directly (described below), assays exist that detect
and quantify the effects of compounds on discrete enzymes and
complexes of mitochondria in cells. For example, the MT-OXC
MitoTox.TM. Complete OXPHOS Activity Assay (MitoSciences Inc.,
Eugene, Oreg.) can detect and quantify the effects of compounds
applied directly to complexes Ito V extracted from mitochondria.
Assays for the detection and quantification of effects on
individual mitochondrial complexes such as NADH dehydrogenase
(Complex I), cytochrome c oxidase (Complex IV) and ATP synthase
(Complex V) are also available (MitoSciences Inc., Eugene,
Oreg.).
Measurement of Cellular Energetics:
[0180] Environmental influencers (e.g., MIMs or Epi-shifters) may
also be identified by changes in cellular energetics. One example
of the measurement of cellular energetics are the real-time
measures of the consumption of molecular oxygen and/or the change
in pH of the media of a cell culture. For example, the ability of a
potential epi-shifter to modulate the metabolic state of a cell may
be analyzed using, for example, the XF24 Analyzer (Seahorse, Inc.).
This technology allows for real time detection of oxygen and pH
changes in a monolayer of cells in order to evaluate the
bioenergetics of a cell microenvironment. The XF24 Analyzer
measures and compares the rates of oxygen consumption (OCR), which
is a measure of aerobic metabolism, and extracellular acidification
(ECAR), which is a measure of glycolysis, both key indicators of
cellular energetics.
Measurement of Oxidative Phosphorylation and Mitochondrial
Function
[0181] Oxidative Phosphorylation is a process by which ATP is
generated via the oxidation of nutrient compounds, carried out in
eukaryotes via protein complexes embedded in the membranes of
mitochondria. As the primary source of ATP in the cells of most
organisms, changes in oxidative phosphorylation activity can
strongly alter metabolism and energy balance within a cell. In some
embodiments of the invention, environmental influencers (e.g., MIMs
or Epi-shifters) may be detected and/or identified by their effects
on oxidative phosphorylation. In some embodiments, environmental
influencers (e.g., MIMs or Epi-shifters) may be detected and/or
identified by their effects on specific aspects of oxidative
phosphorylation, including, but not limited to, the electron
transport chain and ATP synthesis.
[0182] The membrane-embedded protein complexes of the mitochondria
that carry out processes involved in oxidative phosphorylation
perform specific tasks and are numbered I, II, III and IV. These
complexes, along with the trans-inner membrane ATP synthase (also
known as Complex V), are the key entities involved in the oxidative
phosphorylation process. In addition to assays that can examine the
effects of environmental influencers (e.g., MIMs or Epi-shifters)
on mitochondrial function in general and the oxidative
phosphorylation process in particular, assays are available that
can be used to examine the effects of an epi-shifter on an
individual complex separately from other complexes.
[0183] Complex I, also known as NADH-coenzyme Q oxidoreductase or
NADH dehydrogenase, is the first protein in the electron transport
chain. In some embodiments, the detection and quantification of the
effect of an epi-shifter on the production of NAD.sup.+ by Complex
I may be performed. For example, the complex can be immunocaptured
from a sample in a 96-well plate; the oxidation of NADH to
NAD.sup.+ takes place concurrently with the reduction of a dye
molecule which has an increased absorbance at 450 nM (Complex I
Enzyme Activity Microplate Assay Kit, MitoSciences Inc., Eugene,
Oreg.).
[0184] Complex IV, also known as cytochrome c oxidase (COX), is the
last protein in the electron transport chain. In some embodiments,
the detection and quantification of the effect of an epi-shifter on
the oxidation of cytochrome c and the reduction of oxygen to water
by Complex IV may be performed. For example, COX can be
immunocaptured in a microwell plate and the oxidation of COX
measured with a colorimetric assay (Complex IV Enzyme Activity
Microplate Assay Kit, MitoSciences Inc., Eugene, Oreg.).
[0185] The final enzyme in the oxidative phosphorylation process is
ATP synthase (Complex V), which uses the proton gradient created by
the other complexes to power the synthesis of ATP from ADP. In some
embodiments, the detection and quantification of the effect of an
epi-shifter on the activity of ATP synthase may be performed. For
example, both the activity of ATP synthase and the amount of ATP
synthase in a sample may be measured for ATP synthase that has been
immunocaptured in a microwell plate well. The enzyme can also
function as an ATPase under certain conditions, thus in this assay
for ATP synthase activity, the rate at which ATP is reduced to ADP
is measured by detecting the simultaneous oxidation of NADH to
NAD.sup.+. The amount of ATP is calculated using a labeled antibody
to ATPase (ATP synthase Duplexing (Activity+Quantity) Microplate
Assay Kit, MitoSciences Inc., Eugene, Oreg.). Additional assays for
oxidative phosphorylation include assays that test for effects on
the activity of Complexes II and III. For example, the MT-OXC
MitoTox.TM. Complete OXPHOS System (MitoSciences Inc., Eugene,
Oreg.) can be used to evaluate effects of a compound on Complex II
and III as well as Complex I, IV and V, to provide data on the
effects of a compound on the entire oxidative phosphorylation
system.
[0186] As noted above, real-time observation of intact cell samples
can be made using probes for changes in oxygen consumption and pH
in cell culture media. These assays of cell energetics provide a
broad overview of mitochondrial function and the effects of
potential environmental influencers (e.g., MIMs or Epi-shifters) on
the activity of mitochondria within the cells of the sample.
[0187] Environmental influencers (e.g., MIMs or Epi-shifters) may
also affect mitochondrial permeability transition (MPT), a
phenomena in which the mitochondrial membranes experience an
increase in permeability due to the formation of mitochondrial
permeability transition pores (MPTP). An increase in mitochondrial
permeability can lead to mitochondrial swelling, an inability to
conduct oxidative phosphorylation and ATP generation and cell
death. MPT may be involved with induction of apoptosis. (See, for
example, Halestrap, A. P., Biochem. Soc. Trans. 34:232-237 (2006)
and Lena, A. et al. Journal of Translational Med. 7:13-26 (2009),
hereby incorporated by reference in their entirety.)
[0188] In some embodiments, the detection and quantification of the
effect of an environmental influencer (e.g., MIM or epi-shifter) on
the formation, discontinuation and/or effects of MPT and MPTPs are
measured. For example, assays can detect MPT through the use of
specialized dye molecules (calcein) that are localized within the
inner membranes of mitochondria and other cytosolic compartments.
The application of another molecule, CoCl.sub.2, serves to squelch
the fluorescence of the calcein dye in the cytosol. CoCl.sub.2
cannot access, however, the interior of the mitochondria, thus the
calcein fluorescence in the mitochondria is not squelched unless
MPT has occurred and CoCl.sub.2 can access the interior of the
mitochondria via MPTPs. Loss of mitochondrial-specific fluorescence
signals that MPT has occurred. Flow cytometry can be used to
evaluate cellular and organelle fluorescence (MitoProbe.TM.
Transition Pore Assay Kit, Molecular Probes, Eugene, Oreg.).
Additional assays utilize a fluorescence microscope for evaluating
experimental results (Image-iT.TM. LIVE Mitochondrial Transition
Pore Assay Kit, Molecular Probes, Eugene, Oreg.).
Measurement of Cellular Proliferation and Inflammation
[0189] In some embodiments of the invention, environmental
influencers (e.g., MIMs or Epi-shifters) may be identified and
evaluated by their effects on the production or activity of
molecules associated with cellular proliferation and/or
inflammation. These molecules include, but are not limited to,
cytokines, growth factors, hormones, components of the
extra-cellular matrix, chemokines, neuropeptides,
neurotransmitters, neurotrophins and other molecules involved in
cellular signaling, as well as intracellular molecules, such as
those involved in signal transduction.
[0190] Vascular endothelial growth factor (VEGF) is a growth factor
with potent angiogenic, vasculogenic and mitogenic properties. VEGF
stimulates endothelial permeability and swelling and VEGF activity
is implicated in numerous diseases and disorders, including
rheumatoid arthritis, metastatic cancer, age-related macular
degeneration and diabetic retinopathy.
[0191] In some embodiments of the invention, an environmental
influencer (e.g., MIM or Epi-shifter) may be identified and
characterized by its effects on the production of VEGF. For
example, cells maintained in hypoxic conditions or in conditions
mimicking acidosis will exhibit increased VEGF production. VEGF
secreted into media can be assayed using an ELISA or other
antibody-based assays, using available anti-VEGF antibodies
(R&D Systems, Minneapolis, Minn.). In some embodiments of the
invention, an Epi-shifter may be identified and/or characterized
based on its effect(s) on the responsiveness of cells to VEGF
and/or based on its effect(s) on the expression or activity of the
VEGF receptor.
[0192] Implicated in both healthy immune system function as well as
in autoimmune diseases, tumor necrosis factor (TNF) is a key
mediator of inflammation and immune system activation. In some
embodiments of the invention, an Epi-shifter may be identified and
characterized by its effects on the production or the activity of
TNF. For example, TNF produced by cultured cells and secreted into
media can be quantified via ELISA and other antibody-based assays
known in the art. Furthermore, in some embodiments an environmental
influencer may be identified and characterized by its effect(s) on
the expression of receptors for TNF (Human TNF RI Duoset, R&D
Systems, Minneapolis, Minn.).
[0193] The components of the extracellular matrix (ECM) play roles
in both the structure of cells and tissues and in signaling
processes. For example, latent transforming growth factor beta
binding proteins are ECM components that create a reservoir of
transforming growth factor beta (TGF.beta.) within the ECM.
Matrix-bound TGF.beta. can be released later during the process of
matrix remodeling and can exert growth factor effects on nearby
cells (Dallas, S. Methods in Mol. Biol. 139:231-243 (2000)).
[0194] In some embodiments, an environmental influencer (e.g., MIM
or Epi-shifter) may be identified or characterized by its effect(s)
on the creation of ECM by cultured cells. Researchers have
developed techniques with which the creation of ECM by cells, as
well as the composition of the ECM, can be studied and quantified.
For example, the synthesis of ECM by cells can be evaluated by
embedding the cells in a hydrogel before incubation. Biochemical
and other analyses are performed on the ECM generated by the cells
after cell harvest and digestion of the hydrogel (Strehin, I. and
Elisseeff, J. Methods in Mol. Bio. 522:349-362 (2009)).
[0195] In some embodiments, the effect of environmental influencer
(e.g., MIM or epi-shifter) on the production, status of or lack of
ECM or one of its components in an organism may be identified or
characterized. Techniques for creating conditional knock-out (KO)
mice have been developed that allow for the knockout of particular
ECM genes only in discrete types of cells or at certain stages of
development (Brancaccio, M. et al. Methods in Mol. Bio. 522:15-50
(2009)). The effect of the application or administration of an
epi-shifter or potential epi-shifter on the activity or absence of
a particular ECM component in a particular tissue or at a
particular stage of development may thus be evaluated.
Measurement of Plasma Membrane Integrity and Cell Death
[0196] Environmental influencers (e.g., MIMs or Epi-shifters) may
be identified by changes in the plasma membrane integrity of a cell
sample and/or by changes in the number or percentage of cells that
undergo apoptosis, necrosis or cellular changes that demonstrate an
increased or reduced likelihood of cell death.
[0197] An assay for lactate dehydrogenase (LDH) can provide a
measurement of cellular status and damage levels. LDH is a stable
and relatively abundant cytoplasmic enzyme. When plasma membranes
lose physical integrity, LDH escapes to the extracellular
compartment. Higher concentrations of LDH correlate with higher
levels of plasma membrane damage and cell death. Examples of LDH
assays include assays that use a colorimetric system to detect and
quantify levels of LDH in a sample, wherein the reduced form of a
tetrazolium salt is produced via the activity of the LDH enzyme
(QuantiChrom.TM. Lactate Dehydrogenase Kit (DLDH-100), BioAssay
Systems, Hayward, Calif.; LDH Cytotoxicity Detection Kit, Clontech,
Mountain View, Calif.).
[0198] Apoptosis is a process of programmed cell death that may
have a variety of different initiating events. A number of assays
can detect changes in the rate and/or number of cells that undergo
apoptosis. One type of assay that is used to detect and quantify
apoptosis is a capase assay. Capases are aspartic acid-specific
cysteine proteases that are activated via proteolytic cleavage
during apoptosis. Examples of assays that detect activated capases
include PhiPhiLux.RTM. (OncoImmunin, Inc., Gaithersburg, Md.) and
Caspase-Glo.RTM. 3/7 Assay Systems (Promega Corp., Madison, Wis.).
Additional assays that can detect apoptosis and changes in the
percentage or number of cells undergoing apoptosis in comparative
samples include TUNEL/DNA fragmentation assays. These assays detect
the 180 to 200 base pair DNA fragments generated by nucleases
during the execution phase of apoptosis. Exemplary TUNEL/DNA
fragmentation assays include the In Situ Cell Death Detection Kit
(Roche Applied Science, Indianapolis, Ind.) and the DeadEnd.TM.
Colorimetric and Fluorometric TUNEL Systems (Promega Corp.,
Madison, Wis.).
[0199] Some apoptosis assays detect and quantify proteins
associated with an apoptotic and/or a non-apoptotic state. For
example, the MultiTox-Fluor Multiplex Cytotoxicity Assay (Promega
Corp., Madison, Wis.) uses a single substrate, fluorimetric system
to detect and quantify proteases specific to live and dead cells,
thus providing a ratio of living cells to cells that have undergone
apoptosis in a cell or tissue sample.
[0200] Additional assays available for detecting and quantifying
apoptosis include assays that detect cell permeability (e.g.,
APOPercentage.TM. APOPTOSIS Assay, Biocolor, UK) and assays for
Annexin V (e.g., Annexin V-Biotin Apoptosis Detection Kit,
BioVision Inc., Mountain View, Calif.).
IV. Uses of the Invention
[0201] The invention provides methods for assaying whether a
subject is afflicted with a metabolic disorder. The methods of the
present invention can be practiced in conjunction with any other
method used by the skilled practitioner to prognose a metabolic
disorder and/or the survival of a subject being treated for a
metabolic disorder. For example, the methods of the invention may
be performed in conjunction with a morphological or cytological
analysis of the sample obtained from the subject. Cytological
methods would include immunohistochemical or immunofluorescence
detection (and quantitation if appropriate) of any other molecular
marker either by itself, in conjunction with other markers, and/or
in conjunction with the Shc markers. Other methods would include
detection of other markers by in situ PCR, or by extracting tissue
and quantitating other markers by real time PCR. PCR is defined as
polymerase chain reaction.
[0202] Methods for assessing the efficacy of a treatment regimen
for treating a metabolic disorder in a subject are also provided.
In these methods the amount of marker in a pair of samples (a first
sample not subjected to the treatment regimen and a second sample
subjected to at least a portion of the treatment regimen) is
assessed.
[0203] Using the methods described herein, a variety of molecules,
particularly including molecules sufficiently small to be able to
cross the cell membrane, may be screened in order to identify
molecules which modulate, e.g., increase the expression and/or
activity of a marker of the invention. Compounds so identified can
be provided to a subject in order to inhibit a metabolic disorder
in the subject or to treat a metabolic disorder in the subject.
V. Markers of the Invention
[0204] The invention relates to markers (hereinafter "biomarkers",
"markers" or "markers of the invention"), which are listed in
Tables 2-4 & 6-29. The invention provides nucleic acids and
proteins that are encoded by or correspond to the markers
(hereinafter "marker nucleic acids" and "marker proteins,"
respectively). These markers are particularly useful in screening
for the presence of a metabolic disorder, prognosing whether a
subject is predisposed to developing a metabolic disorder,
identifying a compound for treating a metabolic disorder and
assessing the efficacy of a therapy or of an environmental
influencer compound for treating a metabolic disorder.
[0205] In some embodiments of the present invention, one or more
biomarkers is used in connection with the methods of the present
invention. As used herein, the term "one or more biomarkers" is
intended to mean that at least one biomarker in a disclosed list of
biomarkers is assayed and, in various embodiments, more than one
biomarker set forth in the list may be assayed, such as two, three,
four, five, ten, twenty, thirty, forty, fifty, more than fifty, or
all the biomarkers in the list may be assayed.
[0206] A "marker" is a gene whose altered level of expression in a
tissue or cell from its expression level in normal or healthy
tissue or cell is associated with a disease state, such as a
metabolic disorder. A "marker nucleic acid" is a nucleic acid
(e.g., mRNA, cDNA) encoded by or corresponding to a marker of the
invention. Such marker nucleic acids include DNA (e.g., cDNA)
comprising the entire or a partial sequence of any of SEQ ID NO
(nts) or the complement of such a sequence. The marker nucleic
acids also include RNA comprising the entire or a partial sequence
of any SEQ ID NO (nts) or the complement of such a sequence,
wherein all thymidine residues are replaced with uridine residues.
A "marker protein" is a protein encoded by or corresponding to a
marker of the invention. A marker protein comprises the entire or a
partial sequence of any of the SEQ ID NO (AAs). The terms "protein"
and "polypeptide` are used interchangeably.
[0207] A "marker associated with apoptosis" is a marker involved in
an apoptotic pathway. For example, markers associated with
apoptosis include, but are not limited to, the markers listed in
Tables 6A, 6B, 7-9, 25 and 28. Specifically, markers associated
with apoptosis include Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6,
Bcl-2-L11 (Bim), XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, and cMyc.
[0208] A "marker associated with oxidative stress" is a marker
involved in an oxidative stress pathway. For example, markers
associated with oxidative stress include, but are not limited to,
the markers listed in Tables 10-12. Specifically, markers
associated with oxidative stress include Neutrophil cytosolic
factor 2, nitric oxide synthase 2A, and superoxide dismutase 2
(mitochondrial).
[0209] A "marker associated with heat shock" is a marker involved
in heat shock. For example, markers associated with heat shock
include, but are not limited to, the markers listed in Table
13.
[0210] A "marker associated with angiogenesis" is a marker involved
in an angiogenesis pathway. For example, markers associated with
angiogenesis include, but are not limited to, the markers listed in
Tables 24 and 27.
[0211] A "marker associated with diabetes" is a marker involved in
diabetes. For example, markers associated with diabetes include,
but are not limited to, the markers listed in Tables 14-17, 23 and
26.
[0212] A "metabolic disorder-associated" body fluid is a fluid
which, when in the body of a patient, contacts or passes through
metabolic cells or into which cells or proteins shed from metabolic
cells are capable of passing. Exemplary metabolic
disorder-associated body fluids include blood fluids (e.g. whole
blood, blood serum, blood having platelets removed therefrom), and
are described in more detail below. Many metabolic
disorder-associated body fluids can have metabolic cells therein,
particularly when the cells are metastasizing. Cell-containing
fluids which can contain metabolic cells include, but are not
limited to, whole blood, blood having platelets removed therefrom,
lymph, and urine.
[0213] The "normal" level of expression of a marker is the level of
expression of the marker in cells of a human subject or patient not
afflicted with a metabolic disorder.
[0214] An "over-expression" or "higher level of expression" of a
marker refers to an expression level in a test sample that is
greater than the standard error of the assay employed to assess
expression, and is preferably at least twice, and more preferably
three, four, five, six, seven, eight, nine or ten times the
expression level of the marker in a control sample (e.g., sample
from a healthy subject not having the marker associated disease,
i.e., metabolic disorder) and preferably, the average expression
level of the marker in several control samples.
[0215] A "lower level of expression" of a marker refers to an
expression level in a test sample that is at least twice, and more
preferably three, four, five, six, seven, eight, nine or ten times
lower than the expression level of the marker in a control sample
(e.g., sample from a healthy subjects not having the marker
associated disease, i.e., a metabolic disorder) and preferably, the
average expression level of the marker in several control
samples.
[0216] A "transcribed polynucleotide" or "nucleotide transcript" is
a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such
RNA or cDNA) which is complementary to or homologous with all or a
portion of a mature mRNA made by transcription of a marker of the
invention and normal post-transcriptional processing (e.g.
splicing), if any, of the RNA transcript, and reverse transcription
of the RNA transcript.
[0217] "Complementary" refers to the broad concept of sequence
complementarity between regions of two nucleic acid strands or
between two regions of the same nucleic acid strand. It is known
that an adenine residue of a first nucleic acid region is capable
of forming specific hydrogen bonds ("base pairing") with a residue
of a second nucleic acid region which is antiparallel to the first
region if the residue is thymine or uracil. Similarly, it is known
that a cytosine residue of a first nucleic acid strand is capable
of base pairing with a residue of a second nucleic acid strand
which is antiparallel to the first strand if the residue is
guanine. A first region of a nucleic acid is complementary to a
second region of the same or a different nucleic acid if, when the
two regions are arranged in an antiparallel fashion, at least one
nucleotide residue of the first region is capable of base pairing
with a residue of the second region. Preferably, the first region
comprises a first portion and the second region comprises a second
portion, whereby, when the first and second portions are arranged
in an antiparallel fashion, at least about 50%, and preferably at
least about 75%, at least about 90%, or at least about 95% of the
nucleotide residues of the first portion are capable of base
pairing with nucleotide residues in the second portion. More
preferably, all nucleotide residues of the first portion are
capable of base pairing with nucleotide residues in the second
portion.
[0218] "Homologous" as used herein, refers to nucleotide sequence
similarity between two regions of the same nucleic acid strand or
between regions of two different nucleic acid strands. When a
nucleotide residue position in both regions is occupied by the same
nucleotide residue, then the regions are homologous at that
position. A first region is homologous to a second region if at
least one nucleotide residue position of each region is occupied by
the same residue. Homology between two regions is expressed in
terms of the proportion of nucleotide residue positions of the two
regions that are occupied by the same nucleotide residue. By way of
example, a region having the nucleotide sequence 5'-ATTGCC-3' and a
region having the nucleotide sequence 5'-TATGGC-3' share 50%
homology. Preferably, the first region comprises a first portion
and the second region comprises a second portion, whereby, at least
about 50%, and preferably at least about 75%, at least about 90%,
or at least about 95% of the nucleotide residue positions of each
of the portions are occupied by the same nucleotide residue. More
preferably, all nucleotide residue positions of each of the
portions are occupied by the same nucleotide residue.
[0219] "Proteins of the invention" encompass marker proteins and
their fragments; variant marker proteins and their fragments;
peptides and polypeptides comprising an at least 15 amino acid
segment of a marker or variant marker protein; and fusion proteins
comprising a marker or variant marker protein, or an at least 15
amino acid segment of a marker or variant marker protein.
[0220] The invention further provides antibodies, antibody
derivatives and antibody fragments which specifically bind with the
marker proteins and fragments of the marker proteins of the present
invention. Unless otherwise specified herewithin, the terms
"antibody" and "antibodies" broadly encompass naturally-occurring
forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant
antibodies such as single-chain antibodies, chimeric and humanized
antibodies and multi-specific antibodies, as well as fragments and
derivatives of all of the foregoing, which fragments and
derivatives have at least an antigenic binding site. Antibody
derivatives may comprise a protein or chemical moiety conjugated to
an antibody.
[0221] In some embodiments, the biomarker is a regulator of the
insulin receptor pathway. In some embodiments, the biomarker binds
the insulin receptor. In some embodiments, the biomarker is a
diabetes related gene. Diabetes related genes include, for example,
the genes listed in Table 23. In some embodiments, the biomarker is
involved in oxidative stress. In some embodiments, the biomarker is
a caspase modulator, e.g., a caspase activator or a caspase
inhibitor. In some embodiments, the biomarker is involved in cell
growth. In other embodiments, the biomarker is involved in cell
cycle regulation and DNA synthesis. In still other embodiments, the
biomarker is involved in glycolysis and metabolism, e.g., pentose
phosphate pathway and mitochondrial oxidative metabolism. In
further embodiments, the biomarker is involved in molecular
transport. In some embodiments, the biomarker is involved in cell
signaling. In other embodiments, the biomarker is involved in
diabetes and oxidative stress, e.g., glycolytic pathways and
insulin processing. In still other embodiments, the biomarker is
involved in 14-3-3 mediated signaling. In further embodiments, the
biomarker is involved in ceramide signaling. In some embodiments,
the biomarker is involved in mitochondrial protein transport. In
other embodiments, the biomarker is involved in adipocyte
differentiation. In still other embodiments, the biomarker is
involved in lipid and cholesterol metabolism. In some embodiments,
the biomarker is involved in membrane fluidity. In other
embodiments, the biomarker is involved in immunomodulation. In
still other embodiments, the biomarker is involved in genomic
stability. In further embodiments, the biomarker is involved in
extracellular matrix protein integrity. In some embodiments, the
biomarker is involved in membrane transport. In other embodiments,
the biomarker is involved in oxidative control. In some
embodiments, the biomarker is involved in the pentose phosphate
pathway. In some embodiments, the biomarker is a member of the
tumor necrosis factor receptor superfamily. In some embodiments,
the biomarker is involved in arachidonic acid metabolism. In some
embodiments, the biomarker is involved in two or more of the
pathways indicated hereinabove. In some embodiments, the biomarker
is involved in three or more, four or more, five or more, etc. of
the pathways indicated hereinabove. In some embodiments, more than
one biomarker is utilized in connection with the present invention.
In these embodiments, the biomarkers may each individually be
involved in one or more, two or more, three or more, four or more,
five or more, etc. of the pathways indicated hereinabove.
[0222] In certain embodiments, where a particular listed gene is
associated with more than one treatment conditions, such as at
different time periods after a treatment, or treatment by different
concentrations of a potential environmental influencer (e.g.,
CoQ10), the fold change for that particular gene refers to the
longest recorded treatment time. In other embodiments, the fold
change for that particular gene refers to the shortest recorded
treatment time. In other embodiments, the fold change for that
particular gene refers to treatment by the highest concentration of
env-influencer (e.g., CoQ10). In other embodiments, the fold change
for that particular gene refers to treatment by the lowest
concentration of env-influencer (e.g., CoQ10). In yet other
embodiments, the fold change for that particular gene refers to the
modulation (e.g., up- or down-regulation) in a manner that is
consistent with the therapeutic effect of the env-influencer.
[0223] In certain embodiments, the positive or negative fold change
refers to that of any gene listed in any of the Tables 2-4 &
6-29 and 64-69. In certain embodiments, the positive or negative
fold change refers to that of any gene listed in any of the Tables
2-4 & 6-29 and 64-69, except for one of the tables (e.g.,
except for Table 1, except for Table 5, etc.). In certain
embodiments, the positive or negative fold change refers to that of
any gene listed in any of the Tables 2-4 & 6-29 and 64-69,
except for any two of the tables (e.g., except for Tables 1 and 5,
except for Table 2 & 16, etc.). In certain embodiments, the
positive or negative fold change refers to that of any gene listed
in any of the Tables 2-4 & 6-29 and 64-69, except for any three
of the tables; or except for any four of the tables; or except for
any 5, 6, 7, 8, 9, 10, or more of the tables. In certain
embodiments, the positive or negative fold change refers to that of
any gene listed in any of the Tables 2-4 & 6-29 and 64-69,
except for tables 1, 5, 9, 12, and 59.
[0224] As used herein, "positive fold change" refers to
"up-regulation" or "increase (of expression)" of a gene that is
listed in the relevant tables.
[0225] As used herein, "negative fold change" refers to
"down-regulation" or "decrease (of expression)" of a gene that is
listed in the relevant tables.
[0226] Various aspects of the invention are described in further
detail in the following subsections.
1. Isolated Nucleic Acid Molecules
[0227] One aspect of the invention pertains to isolated nucleic
acid molecules, including nucleic acids which encode a marker
protein or a portion thereof. Isolated nucleic acids of the
invention also include nucleic acid molecules sufficient for use as
hybridization probes to identify marker nucleic acid molecules, and
fragments of marker nucleic acid molecules, e.g., those suitable
for use as PCR primers for the amplification or mutation of marker
nucleic acid molecules. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid
molecule can be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0228] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. In one embodiment,
an "isolated" nucleic acid molecule is free of sequences
(preferably protein-encoding sequences) which naturally flank the
nucleic acid (i.e., sequences located at the 5' and 3' ends of the
nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For example, in various embodiments, the
isolated nucleic acid molecule can contain less than about 5 kB, 4
kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences
which naturally flank the nucleic acid molecule in genomic DNA of
the cell from which the nucleic acid is derived. In another
embodiment, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. A nucleic acid molecule that is
substantially free of cellular material includes preparations
having less than about 30%, 20%, 10%, or 5% of heterologous nucleic
acid (also referred to herein as a "contaminating nucleic
acid").
[0229] A nucleic acid molecule of the present invention can be
isolated using standard molecular biology techniques and the
sequence information in the database records described herein.
Using all or a portion of such nucleic acid sequences, nucleic acid
molecules of the invention can be isolated using standard
hybridization and cloning techniques (e.g., as described in
Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0230] A nucleic acid molecule of the invention can be amplified
using cDNA, mRNA, or genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, nucleotides corresponding to all or a portion of a
nucleic acid molecule of the invention can be prepared by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
[0231] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
has a nucleotide sequence complementary to the nucleotide sequence
of a marker nucleic acid or to the nucleotide sequence of a nucleic
acid encoding a marker protein. A nucleic acid molecule which is
complementary to a given nucleotide sequence is one which is
sufficiently complementary to the given nucleotide sequence that it
can hybridize to the given nucleotide sequence thereby forming a
stable duplex.
[0232] Moreover, a nucleic acid molecule of the invention can
comprise only a portion of a nucleic acid sequence, wherein the
full length nucleic acid sequence comprises a marker nucleic acid
or which encodes a marker protein. Such nucleic acids can be used,
for example, as a probe or primer. The probe/primer typically is
used as one or more substantially purified oligonucleotides. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7,
preferably about 15, more preferably about 25, 50, 75, 100, 125,
150, 175, 200, 250, 300, 350, or 400 or more consecutive
nucleotides of a nucleic acid of the invention.
[0233] Probes based on the sequence of a nucleic acid molecule of
the invention can be used to detect transcripts or genomic
sequences corresponding to one or more markers of the invention.
The probe comprises a label group attached thereto, e.g., a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as part of a diagnostic test kit
for identifying cells or tissues which mis-express the protein,
such as by measuring levels of a nucleic acid molecule encoding the
protein in a sample of cells from a subject, e.g., detecting mRNA
levels or determining whether a gene encoding the protein has been
mutated or deleted.
[0234] The invention further encompasses nucleic acid molecules
that differ, due to degeneracy of the genetic code, from the
nucleotide sequence of nucleic acids encoding a marker protein
(e.g., protein having the sequence of the SEQ ID NO (AAs)), and
thus encode the same protein.
[0235] It will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid
sequence can exist within a population (e.g., the human
population). Such genetic polymorphisms can exist among individuals
within a population due to natural allelic variation. An allele is
one of a group of genes which occur alternatively at a given
genetic locus. In addition, it will be appreciated that DNA
polymorphisms that affect RNA expression levels can also exist that
may affect the overall expression level of that gene (e.g., by
affecting regulation or degradation).
[0236] As used herein, the phrase "allelic variant" refers to a
nucleotide sequence which occurs at a given locus or to a
polypeptide encoded by the nucleotide sequence.
[0237] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
encoding a polypeptide corresponding to a marker of the invention.
Such natural allelic variations can typically result in 1-5%
variance in the nucleotide sequence of a given gene. Alternative
alleles can be identified by sequencing the gene of interest in a
number of different individuals. This can be readily carried out by
using hybridization probes to identify the same genetic locus in a
variety of individuals. Any and all such nucleotide variations and
resulting amino acid polymorphisms or variations that are the
result of natural allelic variation and that do not alter the
functional activity are intended to be within the scope of the
invention.
[0238] In another embodiment, an isolated nucleic acid molecule of
the invention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150,
200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200,
1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000,
4500, or more nucleotides in length and hybridizes under stringent
conditions to a marker nucleic acid or to a nucleic acid encoding a
marker protein. As used herein, the term "hybridizes under
stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least
60% (65%, 70%, preferably 75%) identical to each other typically
remain hybridized to each other. Such stringent conditions are
known to those skilled in the art and can be found in sections
6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989). A preferred, non-limiting example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C.
[0239] In addition to naturally-occurring allelic variants of a
nucleic acid molecule of the invention that can exist in the
population, the skilled artisan will further appreciate that
sequence changes can be introduced by mutation thereby leading to
changes in the amino acid sequence of the encoded protein, without
altering the biological activity of the protein encoded thereby.
For example, one can make nucleotide substitutions leading to amino
acid substitutions at "non-essential" amino acid residues. A
"non-essential" amino acid residue is a residue that can be altered
from the wild-type sequence without altering the biological
activity, whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are not
conserved or only semi-conserved among homologs of various species
may be non-essential for activity and thus would be likely targets
for alteration. Alternatively, amino acid residues that are
conserved among the homologs of various species (e.g., murine and
human) may be essential for activity and thus would not be likely
targets for alteration.
[0240] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding a variant marker protein that
contain changes in amino acid residues that are not essential for
activity. Such variant marker proteins differ in amino acid
sequence from the naturally-occurring marker proteins, yet retain
biological activity. In one embodiment, such a variant marker
protein has an amino acid sequence that is at least about 40%
identical, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identical to the amino acid sequence of a marker
protein.
[0241] An isolated nucleic acid molecule encoding a variant marker
protein can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence
of marker nucleic acids, such that one or more amino acid residue
substitutions, additions, or deletions are introduced into the
encoded protein. Mutations can be introduced by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), non-polar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded protein can be expressed recombinantly and the activity of
the protein can be determined.
[0242] The present invention encompasses antisense nucleic acid
molecules, i.e., molecules which are complementary to a sense
nucleic acid of the invention, e.g., complementary to the coding
strand of a double-stranded marker cDNA molecule or complementary
to a marker mRNA sequence. Accordingly, an antisense nucleic acid
of the invention can hydrogen bond to (i.e. anneal with) a sense
nucleic acid of the invention. The antisense nucleic acid can be
complementary to an entire coding strand, or to only a portion
thereof, e.g., all or part of the protein coding region (or open
reading frame). An antisense nucleic acid molecule can also be
antisense to all or part of a non-coding region of the coding
strand of a nucleotide sequence encoding a marker protein. The
non-coding regions ("5' and 3' untranslated regions") are the 5'
and 3' sequences which flank the coding region and are not
translated into amino acids.
[0243] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in
length. An antisense nucleic acid of the invention can be
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. Examples of modified nucleotides which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been sub-cloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0244] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a marker protein to thereby inhibit expression of the
marker, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. Examples of a
route of administration of antisense nucleic acid molecules of the
invention includes direct injection at a tissue site or infusion of
the antisense nucleic acid into a metabolic disorder-associated
body fluid. Alternatively, antisense nucleic acid molecules can be
modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[0245] An antisense nucleic acid molecule of the invention can be
an .alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric
nucleic acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .alpha.-units,
the strands run parallel to each other (Gaultier et al., 1987,
Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid
molecule can also comprise a 2'-O-methylribonucleotide (Inoue et
al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA
analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
[0246] The invention also encompasses ribozymes. Ribozymes are
catalytic RNA molecules with ribonuclease activity which are
capable of cleaving a single-stranded nucleic acid, such as an
mRNA, to which they have a complementary region. Thus, ribozymes
(e.g., hammerhead ribozymes as described in Haselhoff and Gerlach,
1988, Nature 334:585-591) can be used to catalytically cleave mRNA
transcripts to thereby inhibit translation of the protein encoded
by the mRNA. A ribozyme having specificity for a nucleic acid
molecule encoding a marker protein can be designed based upon the
nucleotide sequence of a cDNA corresponding to the marker. For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved (see Cech et
al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.
5,116,742). Alternatively, an mRNA encoding a polypeptide of the
invention can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules (see, e.g.,
Bartel and Szostak, 1993, Science 261:1411-1418).
[0247] The invention also encompasses nucleic acid molecules which
form triple helical structures. For example, expression of a marker
of the invention can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the gene encoding the
marker nucleic acid or protein (e.g., the promoter and/or enhancer)
to form triple helical structures that prevent transcription of the
gene in target cells. See generally Helene (1991) Anticancer Drug
Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and
Maher (1992) Bioassays 14(12):807-15.
[0248] In various embodiments, the nucleic acid molecules of the
invention can be modified at the base moiety, sugar moiety or
phosphate backbone to improve, e.g., the stability, hybridization,
or solubility of the molecule. For example, the deoxyribose
phosphate backbone of the nucleic acids can be modified to generate
peptide nucleic acids (see Hyrup et al., 1996, Bioorganic &
Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide
nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA
mimics, in which the deoxyribose phosphate backbone is replaced by
a pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to allow for
specific hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670-675.
[0249] PNAs can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antigene agents for
sequence-specific modulation of gene expression by, e.g., inducing
transcription or translation arrest or inhibiting replication. PNAs
can also be used, e.g., in the analysis of single base pair
mutations in a gene by, e.g., PNA directed PCR clamping; as
artificial restriction enzymes when used in combination with other
enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or as probes or
primers for DNA sequence and hybridization (Hyrup, 1996, supra;
Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA
93:14670-675).
[0250] In another embodiment, PNAs can be modified, e.g., to
enhance their stability or cellular uptake, by attaching lipophilic
or other helper groups to PNA, by the formation of PNA-DNA
chimeras, or by the use of liposomes or other techniques of drug
delivery known in the art. For example, PNA-DNA chimeras can be
generated which can combine the advantageous properties of PNA and
DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and
DNA polymerases, to interact with the DNA portion while the PNA
portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.
For example, a DNA chain can be synthesized on a solid support
using standard phosphoramidite coupling chemistry and modified
nucleoside analogs. Compounds such as
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite can be
used as a link between the PNA and the 5' end of DNA (Mag et al.,
1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled
in a step-wise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al., 1996, Nucleic Acids Res.
24(17):3357-63). Alternatively, chimeric molecules can be
synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et
al., 1975, Bioorganic Med. Chem. Lett. 5:1119-11124).
[0251] In other embodiments, the oligonucleotide can include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.,
1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g.,
Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide
can be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent,
hybridization-triggered cleavage agent, etc.
[0252] The invention also includes molecular beacon nucleic acids
having at least one region which is complementary to a nucleic acid
of the invention, such that the molecular beacon is useful for
quantitating the presence of the nucleic acid of the invention in a
sample. A "molecular beacon" nucleic acid is a nucleic acid
comprising a pair of complementary regions and having a fluorophore
and a fluorescent quencher associated therewith. The fluorophore
and quencher are associated with different portions of the nucleic
acid in such an orientation that when the complementary regions are
annealed with one another, fluorescence of the fluorophore is
quenched by the quencher. When the complementary regions of the
nucleic acid are not annealed with one another, fluorescence of the
fluorophore is quenched to a lesser degree. Molecular beacon
nucleic acids are described, for example, in U.S. Pat. No.
5,876,930.
2. Isolated Proteins and Antibodies
[0253] One aspect of the invention pertains to isolated marker
proteins and biologically active portions thereof, as well as
polypeptide fragments suitable for use as immunogens to raise
antibodies directed against a marker protein or a fragment thereof.
In one embodiment, the native marker protein can be isolated from
cells or tissue sources by an appropriate purification scheme using
standard protein purification techniques. In another embodiment, a
protein or peptide comprising the whole or a segment of the marker
protein is produced by recombinant DNA techniques. Alternative to
recombinant expression, such protein or peptide can be synthesized
chemically using standard peptide synthesis techniques.
[0254] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the protein is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated
from chemical precursors or other chemicals which are involved in
the synthesis of the protein. Accordingly such preparations of the
protein have less than about 30%, 20%, 10%, 5% (by dry weight) of
chemical precursors or compounds other than the polypeptide of
interest.
[0255] Biologically active portions of a marker protein include
polypeptides comprising amino acid sequences sufficiently identical
to or derived from the amino acid sequence of the marker protein,
which include fewer amino acids than the full length protein, and
exhibit at least one activity of the corresponding full-length
protein. Typically, biologically active portions comprise a domain
or motif with at least one activity of the corresponding
full-length protein. A biologically active portion of a marker
protein of the invention can be a polypeptide which is, for
example, 10, 25, 50, 100 or more amino acids in length. Moreover,
other biologically active portions, in which other regions of the
marker protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of the native form of the marker protein.
[0256] Preferred marker proteins are encoded by nucleotide
sequences comprising the sequence of any of the SEQ ID NO (nts).
Other useful proteins are substantially identical (e.g., at least
about 40%, preferably 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99%) to one of these sequences and retain the
functional activity of the corresponding naturally-occurring marker
protein yet differ in amino acid sequence due to natural allelic
variation or mutagenesis.
[0257] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. Preferably, the
percent identity between the two sequences is calculated using a
global alignment. Alternatively, the percent identity between the
two sequences is calculated using a local alignment. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In one embodiment the two sequences are the
same length. In another embodiment, the two sequences are not the
same length.
[0258] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the BLASTN and BLASTX
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the BLASTN program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the invention. BLAST protein
searches can be performed with the BLASTP program, score=50,
wordlength=3 to obtain amino acid sequences homologous to a protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, a newer version of the BLAST algorithm called
Gapped BLAST can be utilized as described in Altschul et al. (1997)
Nucleic Acids Res. 25:3389-3402, which is able to perform gapped
local alignments for the programs BLASTN, BLASTP and BLASTX.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules. When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., BLASTX and BLASTN) can
be used. See http://www.ncbi.nlm nih gov. Another preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller,
(1988) CABIOS 4:11-17. Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used. Yet
another useful algorithm for identifying regions of local sequence
similarity and alignment is the FASTA algorithm as described in
Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448.
When using the FASTA algorithm for comparing nucleotide or amino
acid sequences, a PAM120 weight residue table can, for example, be
used with a k-tuple value of 2.
[0259] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0260] The invention also provides chimeric or fusion proteins
comprising a marker protein or a segment thereof. As used herein, a
"chimeric protein" or "fusion protein" comprises all or part
(preferably a biologically active part) of a marker protein
operably linked to a heterologous polypeptide (i.e., a polypeptide
other than the marker protein). Within the fusion protein, the term
"operably linked" is intended to indicate that the marker protein
or segment thereof and the heterologous polypeptide are fused
in-frame to each other. The heterologous polypeptide can be fused
to the amino-terminus or the carboxyl-terminus of the marker
protein or segment.
[0261] One useful fusion protein is a GST fusion protein in which a
marker protein or segment is fused to the carboxyl terminus of GST
sequences. Such fusion proteins can facilitate the purification of
a recombinant polypeptide of the invention.
[0262] In another embodiment, the fusion protein contains a
heterologous signal sequence at its amino terminus. For example,
the native signal sequence of a marker protein can be removed and
replaced with a signal sequence from another protein. For example,
the gp67 secretory sequence of the baculovirus envelope protein can
be used as a heterologous signal sequence (Ausubel et al., ed.,
Current Protocols in Molecular Biology, John Wiley & Sons, NY,
1992). Other examples of eukaryotic heterologous signal sequences
include the secretory sequences of melittin and human placental
alkaline phosphatase (Stratagene; La Jolla, Calif.). In yet another
example, useful prokaryotic heterologous signal sequences include
the phoA secretory signal (Sambrook et al., supra) and the protein
A secretory signal (Pharmacia Biotech; Piscataway, N.J.).
[0263] In yet another embodiment, the fusion protein is an
immunoglobulin fusion protein in which all or part of a marker
protein is fused to sequences derived from a member of the
immunoglobulin protein family. The immunoglobulin fusion proteins
of the invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between a ligand (soluble or membrane-bound) and a
protein on the surface of a cell (receptor), to thereby suppress
signal transduction in vivo. The immunoglobulin fusion protein can
be used to affect the bioavailability of a cognate ligand of a
marker protein Inhibition of ligand/receptor interaction can be
useful therapeutically, both for treating proliferative and
differentiative disorders and for modulating (e.g. promoting or
inhibiting) cell survival. Moreover, the immunoglobulin fusion
proteins of the invention can be used as immunogens to produce
antibodies directed against a marker protein in a subject, to
purify ligands and in screening assays to identify molecules which
inhibit the interaction of the marker protein with ligands.
[0264] Chimeric and fusion proteins of the invention can be
produced by standard recombinant DNA techniques. In another
embodiment, the fusion gene can be synthesized by conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor
primers which give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be annealed and
re-amplified to generate a chimeric gene sequence (see, e.g.,
Ausubel et al., supra). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A nucleic acid encoding a polypeptide of the
invention can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the polypeptide of the
invention.
[0265] A signal sequence can be used to facilitate secretion and
isolation of marker proteins. Signal sequences are typically
characterized by a core of hydrophobic amino acids which are
generally cleaved from the mature protein during secretion in one
or more cleavage events. Such signal peptides contain processing
sites that allow cleavage of the signal sequence from the mature
proteins as they pass through the secretory pathway. Thus, the
invention pertains to marker proteins, fusion proteins or segments
thereof having a signal sequence, as well as to such proteins from
which the signal sequence has been proteolytically cleaved (i.e.,
the cleavage products). In one embodiment, a nucleic acid sequence
encoding a signal sequence can be operably linked in an expression
vector to a protein of interest, such as a marker protein or a
segment thereof. The signal sequence directs secretion of the
protein, such as from a eukaryotic host into which the expression
vector is transformed, and the signal sequence is subsequently or
concurrently cleaved. The protein can then be readily purified from
the extracellular medium by art recognized methods. Alternatively,
the signal sequence can be linked to the protein of interest using
a sequence which facilitates purification, such as with a GST
domain.
[0266] The present invention also pertains to variants of the
marker proteins. Such variants have an altered amino acid sequence
which can function as either agonists (mimetics) or as antagonists.
Variants can be generated by mutagenesis, e.g., discrete point
mutation or truncation. An agonist can retain substantially the
same, or a subset, of the biological activities of the naturally
occurring form of the protein. An antagonist of a protein can
inhibit one or more of the activities of the naturally occurring
form of the protein by, for example, competitively binding to a
downstream or upstream member of a cellular signaling cascade which
includes the protein of interest. Thus, specific biological effects
can be elicited by treatment with a variant of limited function.
Treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein can have fewer side effects in a subject relative to
treatment with the naturally occurring form of the protein.
[0267] Variants of a marker protein which function as either
agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the protein of the invention for agonist or antagonist
activity. In one embodiment, a variegated library of variants is
generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of variants can be produced by, for example, enzymatically ligating
a mixture of synthetic oligonucleotides into gene sequences such
that a degenerate set of potential protein sequences is expressible
as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display). There are a variety of
methods which can be used to produce libraries of potential
variants of the marker proteins from a degenerate oligonucleotide
sequence. Methods for synthesizing degenerate oligonucleotides are
known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3;
Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al.,
1984, Science 198:1056; Ike et al., 1983 Nucleic Acid Res.
11:477).
[0268] In addition, libraries of segments of a marker protein can
be used to generate a variegated population of polypeptides for
screening and subsequent selection of variant marker proteins or
segments thereof. For example, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of the coding sequence of interest with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes amino
terminal and internal fragments of various sizes of the protein of
interest.
[0269] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. The most widely used techniques, which
are amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify variants of a protein of the invention (Arkin and Yourvan,
1992, Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.,
1993, Protein Engineering 6(3):327-331).
[0270] Another aspect of the invention pertains to antibodies
directed against a protein of the invention. In preferred
embodiments, the antibodies specifically bind a marker protein or a
fragment thereof. The terms "antibody" and "antibodies" as used
interchangeably herein refer to immunoglobulin molecules as well as
fragments and derivatives thereof that comprise an immunologically
active portion of an immunoglobulin molecule, (i.e., such a portion
contains an antigen binding site which specifically binds an
antigen, such as a marker protein, e.g., an epitope of a marker
protein). An antibody which specifically binds to a protein of the
invention is an antibody which binds the protein, but does not
substantially bind other molecules in a sample, e.g., a biological
sample, which naturally contains the protein. Examples of an
immunologically active portion of an immunoglobulin molecule
include, but are not limited to, single-chain antibodies (scAb),
F(ab) and F(ab').sub.2 fragments.
[0271] An isolated protein of the invention or a fragment thereof
can be used as an immunogen to generate antibodies. The full-length
protein can be used or, alternatively, the invention provides
antigenic peptide fragments for use as immunogens. The antigenic
peptide of a protein of the invention comprises at least 8
(preferably 10, 15, 20, or 30 or more) amino acid residues of the
amino acid sequence of one of the proteins of the invention, and
encompasses at least one epitope of the protein such that an
antibody raised against the peptide forms a specific immune complex
with the protein. Preferred epitopes encompassed by the antigenic
peptide are regions that are located on the surface of the protein,
e.g., hydrophilic regions. Hydrophobicity sequence analysis,
hydrophilicity sequence analysis, or similar analyses can be used
to identify hydrophilic regions. In preferred embodiments, an
isolated marker protein or fragment thereof is used as an
immunogen.
[0272] An immunogen typically is used to prepare antibodies by
immunizing a suitable (i.e. immunocompetent) subject such as a
rabbit, goat, mouse, or other mammal or vertebrate. An appropriate
immunogenic preparation can contain, for example,
recombinantly-expressed or chemically-synthesized protein or
peptide. The preparation can further include an adjuvant, such as
Freund's complete or incomplete adjuvant, or a similar
immunostimulatory agent. Preferred immunogen compositions are those
that contain no other human proteins such as, for example,
immunogen compositions made using a non-human host cell for
recombinant expression of a protein of the invention. In such a
manner, the resulting antibody compositions have reduced or no
binding of human proteins other than a protein of the
invention.
[0273] The invention provides polyclonal and monoclonal antibodies.
The term "monoclonal antibody" or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope. Preferred
polyclonal and monoclonal antibody compositions are ones that have
been selected for antibodies directed against a protein of the
invention. Particularly preferred polyclonal and monoclonal
antibody preparations are ones that contain only antibodies
directed against a marker protein or fragment thereof.
[0274] Polyclonal antibodies can be prepared by immunizing a
suitable subject with a protein of the invention as an immunogen
The antibody titer in the immunized subject can be monitored over
time by standard techniques, such as with an enzyme linked
immunosorbent assay (ELISA) using immobilized polypeptide. At an
appropriate time after immunization, e.g., when the specific
antibody titers are highest, antibody-producing cells can be
obtained from the subject and used to prepare monoclonal antibodies
(mAb) by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature
256:495-497, the human B cell hybridoma technique (see Kozbor et
al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see
Cole et al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., 1985) or trioma techniques. The technology for
producing hybridomas is well known (see generally Current Protocols
in Immunology, Coligan et al. ed., John Wiley & Sons, New York,
1994). Hybridoma cells producing a monoclonal antibody of the
invention are detected by screening the hybridoma culture
supernatants for antibodies that bind the polypeptide of interest,
e.g., using a standard ELISA assay.
[0275] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody directed against a protein of the
invention can be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e.g., an antibody phage
display library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display
Kit, Catalog No. 240612). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening
antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.
WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No.
WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No.
WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No.
WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; Griffiths et al. (1993) EMBO J.
12:725-734.
[0276] The invention also provides recombinant antibodies that
specifically bind a protein of the invention. In preferred
embodiments, the recombinant antibodies specifically binds a marker
protein or fragment thereof. Recombinant antibodies include, but
are not limited to, chimeric and humanized monoclonal antibodies,
comprising both human and non-human portions, single-chain
antibodies and multi-specific antibodies. A chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region. (See,
e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al.,
U.S. Pat. No. 4,816,397, which are incorporated herein by reference
in their entirety.) Single-chain antibodies have an antigen binding
site and consist of a single polypeptide. They can be produced by
techniques known in the art, for example using methods described in
Ladner et. al U.S. Pat. No. 4,946,778 (which is incorporated herein
by reference in its entirety); Bird et al., (1988) Science
242:423-426; Whitlow et al., (1991) Methods in Enzymology 2:1-9;
Whitlow et al., (1991) Methods in Enzymology 2:97-105; and Huston
et al., (1991) Methods in Enzymology Molecular Design and Modeling:
Concepts and Applications 203:46-88. Multi-specific antibodies are
antibody molecules having at least two antigen-binding sites that
specifically bind different antigens. Such molecules can be
produced by techniques known in the art, for example using methods
described in Segal, U.S. Pat. No. 4,676,980 (the disclosure of
which is incorporated herein by reference in its entirety);
Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;
Whitlow et al., (1994) Protein Eng. 7:1017-1026 and U.S. Pat. No.
6,121,424.
[0277] Humanized antibodies are antibody molecules from non-human
species having one or more complementarity determining regions
(CDRs) from the non-human species and a framework region from a
human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No.
5,585,089, which is incorporated herein by reference in its
entirety.) Humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using
methods described in PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science
229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0278] More particularly, humanized antibodies can be produced, for
example, using transgenic mice which are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunized in the normal fashion with a selected antigen, e.g.,
all or a portion of a polypeptide corresponding to a marker of the
invention. Monoclonal antibodies directed against the antigen can
be obtained using conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the transgenic mice rearrange
during B cell differentiation, and subsequently undergo class
switching and somatic mutation. Thus, using such a technique, it is
possible to produce therapeutically useful IgG, IgA and IgE
antibodies. For an overview of this technology for producing human
antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol.
13:65-93). For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., U.S. Pat. No.
5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S.
Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In addition,
companies such as Abgenix, Inc. (Freemont, Calif.), can be engaged
to provide human antibodies directed against a selected antigen
using technology similar to that described above.
[0279] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a murine antibody, is used to guide the selection
of a completely human antibody recognizing the same epitope
(Jespers et al., 1994, Bio/technology 12:899-903).
[0280] The antibodies of the invention can be isolated after
production (e.g., from the blood or serum of the subject) or
synthesis and further purified by well-known techniques. For
example, IgG antibodies can be purified using protein A
chromatography. Antibodies specific for a protein of the invention
can be selected or (e.g., partially purified) or purified by, e.g.,
affinity chromatography. For example, a recombinantly expressed and
purified (or partially purified) protein of the invention is
produced as described herein, and covalently or non-covalently
coupled to a solid support such as, for example, a chromatography
column. The column can then be used to affinity purify antibodies
specific for the proteins of the invention from a sample containing
antibodies directed against a large number of different epitopes,
thereby generating a substantially purified antibody composition,
i.e., one that is substantially free of contaminating antibodies.
By a substantially purified antibody composition is meant, in this
context, that the antibody sample contains at most only 30% (by dry
weight) of contaminating antibodies directed against epitopes other
than those of the desired protein of the invention, and preferably
at most 20%, yet more preferably at most 10%, and most preferably
at most 5% (by dry weight) of the sample is contaminating
antibodies. A purified antibody composition means that at least 99%
of the antibodies in the composition are directed against the
desired protein of the invention.
[0281] In a preferred embodiment, the substantially purified
antibodies of the invention may specifically bind to a signal
peptide, a secreted sequence, an extracellular domain, a
transmembrane or a cytoplasmic domain or cytoplasmic membrane of a
protein of the invention. In a particularly preferred embodiment,
the substantially purified antibodies of the invention specifically
bind to a secreted sequence or an extracellular domain of the amino
acid sequences of a protein of the invention. In a more preferred
embodiment, the substantially purified antibodies of the invention
specifically bind to a secreted sequence or an extracellular domain
of the amino acid sequences of a marker protein.
[0282] An antibody directed against a protein of the invention can
be used to isolate the protein by standard techniques, such as
affinity chromatography or immunoprecipitation. Moreover, such an
antibody can be used to detect the marker protein or fragment
thereof (e.g., in a cellular lysate or cell supernatant) in order
to evaluate the level and pattern of expression of the marker. The
antibodies can also be used diagnostically to monitor protein
levels in tissues or body fluids (e.g. in a metabolic
disorder-associated body fluid) as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by the use of an
antibody derivative, which comprises an antibody of the invention
coupled to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0283] Antibodies of the invention may also be used as therapeutic
agents in treating metabolic disorders. In a preferred embodiment,
completely human antibodies of the invention are used for
therapeutic treatment of human patients with metabolic disorders,
particularly those having diabetes or obesity. In another preferred
embodiment, antibodies that bind specifically to a marker protein
or fragment thereof are used for therapeutic treatment. Further,
such therapeutic antibody may be an antibody derivative or
immunotoxin comprising an antibody conjugated to a therapeutic
moiety such as a cytotoxin, a therapeutic agent or a radioactive
metal ion. A cytotoxin or cytotoxic agent includes any agent that
is detrimental to cells. Examples include taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof. Therapeutic agents include, but are not limited
to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0284] The conjugated antibodies of the invention can be used for
modifying a given biological response, for the drug moiety is not
to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as ribosome-inhibiting
protein (see Better et al., U.S. Pat. No. 6,146,631, the disclosure
of which is incorporated herein in its entirety), abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, .alpha.-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator; or, biological response modifiers such as, for example,
lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"),
or other growth factors.
[0285] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Anton et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
[0286] Accordingly, in one aspect, the invention provides
substantially purified antibodies, antibody fragments and
derivatives, all of which specifically bind to a protein of the
invention and preferably, a marker protein. In various embodiments,
the substantially purified antibodies of the invention, or
fragments or derivatives thereof, can be human, non-human, chimeric
and/or humanized antibodies. In another aspect, the invention
provides non-human antibodies, antibody fragments and derivatives,
all of which specifically bind to a protein of the invention and
preferably, a marker protein. Such non-human antibodies can be
goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.
Alternatively, the non-human antibodies of the invention can be
chimeric and/or humanized antibodies. In addition, the non-human
antibodies of the invention can be polyclonal antibodies or
monoclonal antibodies. In still a further aspect, the invention
provides monoclonal antibodies, antibody fragments and derivatives,
all of which specifically bind to a protein of the invention and
preferably, a marker protein. The monoclonal antibodies can be
human, humanized, chimeric and/or non-human antibodies.
[0287] The invention also provides a kit containing an antibody of
the invention conjugated to a detectable substance, and
instructions for use. Still another aspect of the invention is a
pharmaceutical composition comprising an antibody of the invention.
In one embodiment, the pharmaceutical composition comprises an
antibody of the invention and a pharmaceutically acceptable
carrier.
3. Predictive Medicine
[0288] The present invention pertains to the field of predictive
medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for determining the level of
expression of one or more marker proteins or nucleic acids, in
order to determine whether an individual is at risk of developing a
metabolic disorder. Such assays can be used for prognostic or
predictive purposes to thereby prophylactically treat an individual
prior to the onset of the disorder.
[0289] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs or other compounds
administered either to inhibit a metabolic disorder or to treat or
prevent any other disorder {i.e. in order to understand any
carcinogenic effects that such treatment may have}) on the
expression or activity of a marker of the invention in clinical
trials. These and other agents are described in further detail in
the following sections.
[0290] A. Diagnostic Assays
[0291] An exemplary method for detecting the presence or absence of
a marker protein or nucleic acid in a biological sample involves
obtaining a biological sample (e.g. a metabolic disorder-associated
body fluid) from a test subject and contacting the biological
sample with a compound or an agent capable of detecting the
polypeptide or nucleic acid (e.g., mRNA, genomic DNA, or cDNA). The
detection methods of the invention can thus be used to detect mRNA,
protein, cDNA, or genomic DNA, for example, in a biological sample
in vitro as well as in vivo. For example, in vitro techniques for
detection of mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of a marker
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations and immunofluorescence. In
vitro techniques for detection of genomic DNA include Southern
hybridizations. In vivo techniques for detection of mRNA include
polymerase chain reaction (PCR), Northern hybridizations and in
situ hybridizations. Furthermore, in vivo techniques for detection
of a marker protein include introducing into a subject a labeled
antibody directed against the protein or fragment thereof. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0292] A general principle of such diagnostic and prognostic assays
involves preparing a sample or reaction mixture that may contain a
marker, and a probe, under appropriate conditions and for a time
sufficient to allow the marker and probe to interact and bind, thus
forming a complex that can be removed and/or detected in the
reaction mixture. These assays can be conducted in a variety of
ways.
[0293] For example, one method to conduct such an assay would
involve anchoring the marker or probe onto a solid phase support,
also referred to as a substrate, and detecting target marker/probe
complexes anchored on the solid phase at the end of the reaction.
In one embodiment of such a method, a sample from a subject, which
is to be assayed for presence and/or concentration of marker, can
be anchored onto a carrier or solid phase support. In another
embodiment, the reverse situation is possible, in which the probe
can be anchored to a solid phase and a sample from a subject can be
allowed to react as an unanchored component of the assay.
[0294] There are many established methods for anchoring assay
components to a solid phase. These include, without limitation,
marker or probe molecules which are immobilized through conjugation
of biotin and streptavidin. Such biotinylated assay components can
be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques known in the art (e.g., biotinylation kit, Pierce
Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical). In certain
embodiments, the surfaces with immobilized assay components can be
prepared in advance and stored.
[0295] Other suitable carriers or solid phase supports for such
assays include any material capable of binding the class of
molecule to which the marker or probe belongs. Well-known supports
or carriers include, but are not limited to, glass, polystyrene,
nylon, polypropylene, nylon, polyethylene, dextran, amylases,
natural and modified celluloses, polyacrylamides, gabbros, and
magnetite.
[0296] In order to conduct assays with the above mentioned
approaches, the non-immobilized component is added to the solid
phase upon which the second component is anchored. After the
reaction is complete, uncomplexed components may be removed (e.g.,
by washing) under conditions such that any complexes formed will
remain immobilized upon the solid phase. The detection of
marker/probe complexes anchored to the solid phase can be
accomplished in a number of methods outlined herein.
[0297] In a preferred embodiment, the probe, when it is the
unanchored assay component, can be labeled for the purpose of
detection and readout of the assay, either directly or indirectly,
with detectable labels discussed herein and which are well-known to
one skilled in the art.
[0298] It is also possible to directly detect marker/probe complex
formation without further manipulation or labeling of either
component (marker or probe), for example by utilizing the technique
of fluorescence energy transfer (see, for example, Lakowicz et al.,
U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.
4,868,103). A fluorophore label on the first, `donor` molecule is
selected such that, upon excitation with incident light of
appropriate wavelength, its emitted fluorescent energy will be
absorbed by a fluorescent label on a second `acceptor` molecule,
which in turn is able to fluoresce due to the absorbed energy.
Alternately, the `donor` protein molecule may simply utilize the
natural fluorescent energy of tryptophan residues. Labels are
chosen that emit different wavelengths of light, such that the
`acceptor` molecule label may be differentiated from that of the
`donor`. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, spatial
relationships between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in the assay should be
maximal. An FET binding event can be conveniently measured through
standard fluorometric detection means well known in the art (e.g.,
using a fluorimeter).
[0299] In another embodiment, determination of the ability of a
probe to recognize a marker can be accomplished without labeling
either assay component (probe or marker) by utilizing a technology
such as real-time Biomolecular Interaction Analysis (BIA) (see,
e.g., Sjolander, S, and Urbaniczky, C., 1991, Anal. Chem.
63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol.
5:699-705). As used herein, "BIA" or "surface plasmon resonance" is
a technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore). Changes
in the mass at the binding surface (indicative of a binding event)
result in alterations of the refractive index of light near the
surface (the optical phenomenon of surface plasmon resonance
(SPR)), resulting in a detectable signal which can be used as an
indication of real-time reactions between biological molecules.
[0300] Alternatively, in another embodiment, analogous diagnostic
and prognostic assays can be conducted with marker and probe as
solutes in a liquid phase. In such an assay, the complexed marker
and probe are separated from uncomplexed components by any of a
number of standard techniques, including but not limited to:
differential centrifugation, chromatography, electrophoresis and
immunoprecipitation. In differential centrifugation, marker/probe
complexes may be separated from uncomplexed assay components
through a series of centrifugal steps, due to the different
sedimentation equilibria of complexes based on their different
sizes and densities (see, for example, Rivas, G., and Minton, A.
P., 1993, Trends Biochem Sci. 18(8):284-7). Standard
chromatographic techniques may also be utilized to separate
complexed molecules from uncomplexed ones. For example, gel
filtration chromatography separates molecules based on size, and
through the utilization of an appropriate gel filtration resin in a
column format, for example, the relatively larger complex may be
separated from the relatively smaller uncomplexed components.
Similarly, the relatively different charge properties of the
marker/probe complex as compared to the uncomplexed components may
be exploited to differentiate the complex from uncomplexed
components, for example through the utilization of ion-exchange
chromatography resins. Such resins and chromatographic techniques
are well known to one skilled in the art (see, e.g., Heegaard, N.
H., 1998, J. Mol. Recognit. Winter 11(1-6):141-8; Hage, D. S., and
Tweed, S. A. J Chromatogr B Biomed Sci Appl 1997 Oct. 10;
699(1-2):499-525). Gel electrophoresis may also be employed to
separate complexed assay components from unbound components (see,
e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,
John Wiley & Sons, New York, 1987-1999). In this technique,
protein or nucleic acid complexes are separated based on size or
charge, for example. In order to maintain the binding interaction
during the electrophoretic process, non-denaturing gel matrix
materials and conditions in the absence of reducing agent are
typically preferred. Appropriate conditions to the particular assay
and components thereof will be well known to one skilled in the
art.
[0301] In a particular embodiment, the level of marker mRNA can be
determined both by in situ and by in vitro formats in a biological
sample using methods known in the art. The term "biological sample"
is intended to include tissues, cells, biological fluids and
isolates thereof, isolated from a subject, as well as tissues,
cells and fluids present within a subject. Many expression
detection methods use isolated RNA. For in vitro methods, any RNA
isolation technique that does not select against the isolation of
mRNA can be utilized for the purification of RNA from metabolic
disorder cells (see, e.g., Ausubel et al., ed., Current Protocols
in Molecular Biology, John Wiley & Sons, New York 1987-1999).
Additionally, large numbers of tissue samples can readily be
processed using techniques well known to those of skill in the art,
such as, for example, the single-step RNA isolation process of
Chomczynski (1989, U.S. Pat. No. 4,843,155).
[0302] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length cDNA, or a portion thereof, such as an oligonucleotide
of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length
and sufficient to specifically hybridize under stringent conditions
to a mRNA or genomic DNA encoding a marker of the present
invention. Other suitable probes for use in the diagnostic assays
of the invention are described herein. Hybridization of an mRNA
with the probe indicates that the marker in question is being
expressed.
[0303] In one format, the mRNA is immobilized on a solid surface
and contacted with a probe, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probe(s) are immobilized on a solid surface and the mRNA is
contacted with the probe(s), for example, in an Affymetrix gene
chip array. A skilled artisan can readily adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the markers of the present invention.
[0304] An alternative method for determining the level of mRNA
marker in a sample involves the process of nucleic acid
amplification, e.g., by RT-PCR (the experimental embodiment set
forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193),
self sustained sequence replication (Guatelli et al., 1990, Proc.
Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification
system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988,
Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,
U.S. Pat. No. 5,854,033) or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers. As
used herein, amplification primers are defined as being a pair of
nucleic acid molecules that can anneal to 5' or 3' regions of a
gene (plus and minus strands, respectively, or vice-versa) and
contain a short region in between. In general, amplification
primers are from about 10 to 30 nucleotides in length and flank a
region from about 50 to 200 nucleotides in length. Under
appropriate conditions and with appropriate reagents, such primers
permit the amplification of a nucleic acid molecule comprising the
nucleotide sequence flanked by the primers.
[0305] For in situ methods, mRNA does not need to be isolated from
the cells prior to detection. In such methods, a cell or tissue
sample is prepared/processed using known histological methods. The
sample is then immobilized on a support, typically a glass slide,
and then contacted with a probe that can hybridize to mRNA that
encodes the marker.
[0306] As an alternative to making determinations based on the
absolute expression level of the marker, determinations may be
based on the normalized expression level of the marker. Expression
levels are normalized by correcting the absolute expression level
of a marker by comparing its expression to the expression of a gene
that is not a marker, e.g., a housekeeping gene that is
constitutively expressed. Suitable genes for normalization include
housekeeping genes such as the actin gene, or epithelial
cell-specific genes. This normalization allows the comparison of
the expression level in one sample, e.g., a patient sample, to
another sample, e.g., a non-metabolic disorder sample, or between
samples from different sources.
[0307] Alternatively, the expression level can be provided as a
relative expression level. To determine a relative expression level
of a marker, the level of expression of the marker is determined
for 10 or more samples of normal versus metabolic disorder
isolates, preferably 50 or more samples, prior to the determination
of the expression level for the sample in question. The mean
expression level of each of the genes assayed in the larger number
of samples is determined and this is used as a baseline expression
level for the marker. The expression level of the marker determined
for the test sample (absolute level of expression) is then divided
by the mean expression value obtained for that marker. This
provides a relative expression level.
[0308] Preferably, the samples used in the baseline determination
will be from tissues associated with a metabolic disorder. The
choice of the cell source is dependent on the use of the relative
expression level. Using expression found in normal tissues as a
mean expression score aids in validating whether the marker assayed
is metabolic disorder specific (versus normal cells). In addition,
as more data is accumulated, the mean expression value can be
revised, providing improved relative expression values based on
accumulated data.
[0309] In another embodiment of the present invention, a marker
protein is detected. A preferred agent for detecting marker protein
of the invention is an antibody capable of binding to such a
protein or a fragment thereof, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment or derivative thereof
(e.g., Fab or F(ab').sub.2) can be used. The term "labeled", with
regard to the probe or antibody, is intended to encompass direct
labeling of the probe or antibody by coupling (i.e., physically
linking) a detectable substance to the probe or antibody, as well
as indirect labeling of the probe or antibody by reactivity with
another reagent that is directly labeled. Examples of indirect
labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with fluorescently
labeled streptavidin.
[0310] Proteins from cells can be isolated using techniques that
are well known to those of skill in the art. The protein isolation
methods employed can, for example, be such as those described in
Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.).
[0311] A variety of formats can be employed to determine whether a
sample contains a protein that binds to a given antibody. Examples
of such formats include, but are not limited to, enzyme immunoassay
(EIA), radioimmunoassay (RIA), Western blot analysis and enzyme
linked immunoabsorbent assay (ELISA). A skilled artisan can readily
adapt known protein/antibody detection methods for use in
determining whether cells express a marker of the present
invention.
[0312] In one format, antibodies, or antibody fragments or
derivatives, can be used in methods such as Western blots or
immunofluorescence techniques to detect the expressed proteins. In
such uses, it is generally preferable to immobilize either the
antibody or proteins on a solid support. Suitable solid phase
supports or carriers include any support capable of binding an
antigen or an antibody. Well-known supports or carriers include
glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
amylases, natural and modified celluloses, polyacrylamides,
gabbros, and magnetite.
[0313] One skilled in the art will know many other suitable
carriers for binding antibody or antigen, and will be able to adapt
such support for use with the present invention. For example,
protein isolated from metabolic disorder cells can be run on a
polyacrylamide gel electrophoresis and immobilized onto a solid
phase support such as nitrocellulose. The support can then be
washed with suitable buffers followed by treatment with the
detectably labeled antibody. The solid phase support can then be
washed with the buffer a second time to remove unbound antibody.
The amount of bound label on the solid support can then be detected
by conventional means.
[0314] The invention also encompasses kits for detecting the
presence of a marker protein or nucleic acid in a biological
sample. Such kits can be used to determine if a subject is
suffering from or is at increased risk of developing a metabolic
disorder such as diabetes or obesity. For example, the kit can
comprise a labeled compound or agent capable of detecting a marker
protein or nucleic acid in a biological sample and means for
determining the amount of the protein or mRNA in the sample (e.g.,
an antibody which binds the protein or a fragment thereof, or an
oligonucleotide probe which binds to DNA or mRNA encoding the
protein). Kits can also include instructions for interpreting the
results obtained using the kit.
[0315] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a marker protein; and, optionally, (2) a second, different
antibody which binds to either the protein or the first antibody
and is conjugated to a detectable label.
[0316] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding a marker protein or (2) a pair of primers useful for
amplifying a marker nucleic acid molecule. The kit can also
comprise, e.g., a buffering agent, a preservative, or a protein
stabilizing agent. The kit can further comprise components
necessary for detecting the detectable label (e.g., an enzyme or a
substrate). The kit can also contain a control sample or a series
of control samples which can be assayed and compared to the test
sample. Each component of the kit can be enclosed within an
individual container and all of the various containers can be
within a single package, along with instructions for interpreting
the results of the assays performed using the kit.
[0317] B. Pharmacogenomics
[0318] The markers of the invention are also useful as
pharmacogenomic markers. As used herein, a "pharmacogenomic marker"
is an objective biochemical marker whose expression level
correlates with a specific clinical drug response or susceptibility
in a patient (see, e.g., McLeod et al. (1999) Eur. J. Cancer
35(12): 1650-1652). The presence or quantity of the pharmacogenomic
marker expression is related to the predicted response of the
patient and more particularly the patient's metabolic disorder to
therapy with a specific drug or class of drugs. By assessing the
presence or quantity of the expression of one or more
pharmacogenomic markers in a patient, a drug therapy which is most
appropriate for the patient, or which is predicted to have a
greater degree of success, may be selected. For example, based on
the presence or quantity of RNA or protein encoded by specific
markers in a patient, a drug or course of treatment may be selected
that is optimized for the treatment of the specific metabolic
disorder likely to be present in the patient. The use of
pharmacogenomic markers therefore permits selecting or designing
the most appropriate treatment for each metabolic disorder patient
without trying different drugs or regimes.
[0319] Another aspect of pharmacogenomics deals with genetic
conditions that alters the way the body acts on drugs. These
pharmacogenetic conditions can occur either as rare defects or as
polymorphisms. For example, glucose-6-phosphate dehydrogenase
(G6PD) deficiency is a common inherited enzymopathy in which the
main clinical complication is hemolysis after ingestion of oxidant
drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0320] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, a PM will show no therapeutic
response, as demonstrated for the analgesic effect of codeine
mediated by its CYP2D6-formed metabolite morphine. The other
extreme are the so called ultra-rapid metabolizers who do not
respond to standard doses. Recently, the molecular basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
[0321] Thus, the level of expression of a marker of the invention
in an individual can be determined to thereby select appropriate
agent(s) for therapeutic or prophylactic treatment of the
individual. In addition, pharmacogenetic studies can be used to
apply genotyping of polymorphic alleles encoding drug-metabolizing
enzymes to the identification of an individual's drug
responsiveness phenotype. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and thus enhance therapeutic or prophylactic efficiency when
treating a subject with a modulator of expression of a marker of
the invention.
[0322] C. Monitoring Clinical Trials
[0323] Monitoring the influence of agents (e.g., drug compounds) on
the level of expression of a marker of the invention can be applied
not only in basic drug screening, but also in clinical trials. For
example, the effectiveness of an agent to affect marker expression
can be monitored in clinical trials of subjects receiving treatment
for a metabolic disorder. In a preferred embodiment, the present
invention provides a method for monitoring the effectiveness of
treatment of a subject with an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or
other drug candidate) comprising the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of one or more
selected markers of the invention in the pre-administration sample;
(iii) obtaining one or more post-administration samples from the
subject; (iv) detecting the level of expression of the marker(s) in
the post-administration samples; (v) comparing the level of
expression of the marker(s) in the pre-administration sample with
the level of expression of the marker(s) in the post-administration
sample or samples; and (vi) altering the administration of the
agent to the subject accordingly. For example, increased expression
of the marker gene(s) during the course of treatment may indicate
ineffective dosage and the desirability of increasing the dosage.
Conversely, decreased expression of the marker gene(s) may indicate
efficacious treatment and no need to change dosage.
[0324] D. Arrays
[0325] The invention also includes an array comprising a marker of
the present invention. The array can be used to assay expression of
one or more genes in the array. In one embodiment, the array can be
used to assay gene expression in a tissue to ascertain tissue
specificity of genes in the array. In this manner, up to about 7600
genes can be simultaneously assayed for expression. This allows a
profile to be developed showing a battery of genes specifically
expressed in one or more tissues.
[0326] In addition to such qualitative determination, the invention
allows the quantitation of gene expression. Thus, not only tissue
specificity, but also the level of expression of a battery of genes
in the tissue is ascertainable. Thus, genes can be grouped on the
basis of their tissue expression per se and level of expression in
that tissue. This is useful, for example, in ascertaining the
relationship of gene expression between or among tissues. Thus, one
tissue can be perturbed and the effect on gene expression in a
second tissue can be determined. In this context, the effect of one
cell type on another cell type in response to a biological stimulus
can be determined. Such a determination is useful, for example, to
know the effect of cell-cell interaction at the level of gene
expression. If an agent is administered therapeutically to treat
one cell type but has an undesirable effect on another cell type,
the invention provides an assay to determine the molecular basis of
the undesirable effect and thus provides the opportunity to
co-administer a counteracting agent or otherwise treat the
undesired effect. Similarly, even within a single cell type,
undesirable biological effects can be determined at the molecular
level. Thus, the effects of an agent on expression of other than
the target gene can be ascertained and counteracted.
[0327] In another embodiment, the array can be used to monitor the
time course of expression of one or more genes in the array. This
can occur in various biological contexts, as disclosed herein, for
example development of a metabolic disorder, progression of a
metabolic disorder, and processes, such a cellular transformation
associated with a metabolic disorder.
[0328] The array is also useful for ascertaining the effect of the
expression of a gene on the expression of other genes in the same
cell or in different cells. This provides, for example, for a
selection of alternate molecular targets for therapeutic
intervention if the ultimate or downstream target cannot be
regulated.
[0329] The array is also useful for ascertaining differential
expression patterns of one or more genes in normal and abnormal
cells. This provides a battery of genes that could serve as a
molecular target for diagnosis or therapeutic intervention.
VI. Methods for Obtaining Samples
[0330] Samples useful in the methods of the invention include any
tissue, cell, biopsy, or bodily fluid sample that expresses a
marker of the invention. In one embodiment, a sample may be a
tissue, a cell, whole blood, serum, plasma, buccal scrape, saliva,
cerebrospinal fluid, urine, stool, or bronchoalveolar lavage. In
preferred embodiments, the tissue sample is a metabolic disorder
sample, including a blood or urine sample.
[0331] Body samples may be obtained from a subject by a variety of
techniques known in the art including, for example, by the use of a
biopsy or by scraping or swabbing an area or by using a needle to
aspirate bodily fluids. Methods for collecting various body samples
are well known in the art.
[0332] Tissue samples suitable for detecting and quantitating a
marker of the invention may be fresh, frozen, or fixed according to
methods known to one of skill in the art. Suitable tissue samples
are preferably sectioned and placed on a microscope slide for
further analyses. Alternatively, solid samples, i.e., tissue
samples, may be solubilized and/or homogenized and subsequently
analyzed as soluble extracts.
[0333] In one embodiment, a freshly obtained tissue sample is
frozen using, for example, liquid nitrogen or
difluorodichloromethane. The frozen sample is mounted for
sectioning using, for example, OCT, and serially sectioned in a
cryostat. The serial sections are collected on a glass microscope
slide. For immunohistochemical staining the slides may be coated
with, for example, chrome-alum, gelatine or poly-L-lysine to ensure
that the sections stick to the slides. In another embodiment,
samples are fixed and embedded prior to sectioning. For example, a
tissue sample may be fixed in, for example, formalin, serially
dehydrated and embedded in, for example, paraffin.
[0334] Once the sample is obtained any method known in the art to
be suitable for detecting and quantitating a marker of the
invention may be used (either at the nucleic acid or at the protein
level). Such methods are well known in the art and include but are
not limited to western blots, northern blots, southern blots,
immunohistochemistry, ELISA, e.g., amplified ELISA,
immunoprecipitation, immunofluorescence, flow cytometry,
immunocytochemistry, mass spectrometrometric analyses, e.g.,
MALDI-TOF and SELDI-TOF, nucleic acid hybridization techniques,
nucleic acid reverse transcription methods, and nucleic acid
amplification methods. In particular embodiments, the expression of
a marker of the invention is detected on a protein level using, for
example, antibodies that specifically bind these proteins.
[0335] Samples may need to be modified in order to make a marker of
the invention accessible to antibody binding. In a particular
aspect of the immunocytochemistry or immunohistochemistry methods,
slides may be transferred to a pretreatment buffer and optionally
heated to increase antigen accessibility. Heating of the sample in
the pretreatment buffer rapidly disrupts the lipid bi-layer of the
cells and makes the antigens (may be the case in fresh specimens,
but not typically what occurs in fixed specimens) more accessible
for antibody binding. The terms "pretreatment buffer" and
"preparation buffer" are used interchangeably herein to refer to a
buffer that is used to prepare cytology or histology samples for
immunostaining, particularly by increasing the accessibility of a
marker of the invention for antibody binding. The pretreatment
buffer may comprise a pH-specific salt solution, a polymer, a
detergent, or a nonionic or anionic surfactant such as, for
example, an ethyloxylated anionic or nonionic surfactant, an
alkanoate or an alkoxylate or even blends of these surfactants or
even the use of a bile salt. The pretreatment buffer may, for
example, be a solution of 0.1% to 1% of deoxycholic acid, sodium
salt, or a solution of sodium laureth-13-carboxylate (e.g.,
Sandopan LS) or and ethoxylated anionic complex. In some
embodiments, the pretreatment buffer may also be used as a slide
storage buffer.
[0336] Any method for making marker proteins of the invention more
accessible for antibody binding may be used in the practice of the
invention, including the antigen retrieval methods known in the
art. See, for example, Bibbo, et al. (2002) Acta. Cytol. 46:25-29;
Saqi, et al. (2003) Diagn. Cytopathol. 27:365-370; Bibbo, et al.
(2003) Anal. Quant. Cytol. Histol. 25:8-11, the entire contents of
each of which are incorporated herein by reference.
[0337] Following pretreatment to increase marker protein
accessibility, samples may be blocked using an appropriate blocking
agent, e.g., a peroxidase blocking reagent such as hydrogen
peroxide. In some embodiments, the samples may be blocked using a
protein blocking reagent to prevent non-specific binding of the
antibody. The protein blocking reagent may comprise, for example,
purified casein. An antibody, particularly a monoclonal or
polyclonal antibody that specifically binds to a marker of the
invention is then incubated with the sample. One of skill in the
art will appreciate that a more accurate prognosis or diagnosis may
be obtained in some cases by detecting multiple epitopes on a
marker protein of the invention in a patient sample. Therefore, in
particular embodiments, at least two antibodies directed to
different epitopes of a marker of the invention are used. Where
more than one antibody is used, these antibodies may be added to a
single sample sequentially as individual antibody reagents or
simultaneously as an antibody cocktail. Alternatively, each
individual antibody may be added to a separate sample from the same
patient, and the resulting data pooled.
[0338] Techniques for detecting antibody binding are well known in
the art. Antibody binding to a marker of the invention may be
detected through the use of chemical reagents that generate a
detectable signal that corresponds to the level of antibody binding
and, accordingly, to the level of marker protein expression. In one
of the immunohistochemistry or immunocytochemistry methods of the
invention, antibody binding is detected through the use of a
secondary antibody that is conjugated to a labeled polymer.
Examples of labeled polymers include but are not limited to
polymer-enzyme conjugates. The enzymes in these complexes are
typically used to catalyze the deposition of a chromogen at the
antigen-antibody binding site, thereby resulting in cell staining
that corresponds to expression level of the biomarker of interest.
Enzymes of particular interest include, but are not limited to,
horseradish peroxidase (HRP) and alkaline phosphatase (AP).
[0339] In one particular immunohistochemistry or
immunocytochemistry method of the invention, antibody binding to a
marker of the invention is detected through the use of an
HRP-labeled polymer that is conjugated to a secondary antibody.
Antibody binding can also be detected through the use of a
species-specific probe reagent, which binds to monoclonal or
polyclonal antibodies, and a polymer conjugated to HRP, which binds
to the species specific probe reagent. Slides are stained for
antibody binding using any chromagen, e.g., the chromagen
3,3-diaminobenzidine (DAB), and then counterstained with
hematoxylin and, optionally, a bluing agent such as ammonium
hydroxide or TBS/Tween-20. Other suitable chromagens include, for
example, 3-amino-9-ethylcarbazole (AEC). In some aspects of the
invention, slides are reviewed microscopically by a
cytotechnologist and/or a pathologist to assess cell staining,
e.g., fluorescent staining (i.e., marker expression).
Alternatively, samples may be reviewed via automated microscopy or
by personnel with the assistance of computer software that
facilitates the identification of positive staining cells.
[0340] Detection of antibody binding can be facilitated by coupling
the anti-marker antibodies to a detectable substance. Examples of
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S, .sup.14C, or
.sup.3H.
[0341] In one embodiment of the invention frozen samples are
prepared as described above and subsequently stained with
antibodies against a marker of the invention diluted to an
appropriate concentration using, for example, Tris-buffered saline
(TBS). Primary antibodies can be detected by incubating the slides
in biotinylated anti-immunoglobulin. This signal can optionally be
amplified and visualized using diaminobenzidine precipitation of
the antigen. Furthermore, slides can be optionally counterstained
with, for example, hematoxylin, to visualize the cells.
[0342] In another embodiment, fixed and embedded samples are
stained with antibodies against a marker of the invention and
counterstained as described above for frozen sections. In addition,
samples may be optionally treated with agents to amplify the signal
in order to visualize antibody staining. For example, a
peroxidase-catalyzed deposition of biotinyl-tyramide, which in turn
is reacted with peroxidase-conjugated streptavidin (Catalyzed
Signal Amplification (CSA) System, DAKO, Carpinteria, Calif.) may
be used.
[0343] Tissue-based assays (i.e., immunohistochemistry) are the
preferred methods of detecting and quantitating a marker of the
invention. In one embodiment, the presence or absence of a marker
of the invention may be determined by immunohistochemistry. In one
embodiment, the immunohistochemical analysis uses low
concentrations of an anti-marker antibody such that cells lacking
the marker do not stain. In another embodiment, the presence or
absence of a marker of the invention is determined using an
immunohistochemical method that uses high concentrations of an
anti-marker antibody such that cells lacking the marker protein
stain heavily. Cells that do not stain contain either mutated
marker and fail to produce antigenically recognizable marker
protein, or are cells in which the pathways that regulate marker
levels are dysregulated, resulting in steady state expression of
negligible marker protein.
[0344] One of skill in the art will recognize that the
concentration of a particular antibody used to practice the methods
of the invention will vary depending on such factors as time for
binding, level of specificity of the antibody for a marker of the
invention, and method of sample preparation. Moreover, when
multiple antibodies are used, the required concentration may be
affected by the order in which the antibodies are applied to the
sample, e.g., simultaneously as a cocktail or sequentially as
individual antibody reagents. Furthermore, the detection chemistry
used to visualize antibody binding to a marker of the invention
must also be optimized to produce the desired signal to noise
ratio.
[0345] In one embodiment of the invention, proteomic methods, e.g.,
mass spectrometry, are used for detecting and quantitating the
marker proteins of the invention. For example, matrix-associated
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) or surface-enhanced laser desorption/ionization
time-of-flight mass spectrometry (SELDI-TOF MS) which involves the
application of a biological sample, such as serum, to a
protein-binding chip (Wright, G. L., Jr., et al. (2002) Expert Rev
Mol Diagn 2:549; Li, J., et al. (2002) Clin Chem 48:1296; Laronga,
C., et al. (2003) Dis Markers 19:229; Petricoin, E. F., et al.
(2002) 359:572; Adam, B. L., et al. (2002) Cancer Res 62:3609;
Tolson, J., et al. (2004) Lab Invest 84:845; Xiao, Z., et al.
(2001) Cancer Res 61:6029) can be used to detect and quantitate the
PY-Shc and/or p66-Shc proteins. Mass spectrometric methods are
described in, for example, U.S. Pat. Nos. 5,622,824, 5,605,798 and
5,547,835, the entire contents of each of which are incorporated
herein by reference.
[0346] In other embodiments, the expression of a marker of the
invention is detected at the nucleic acid level. Nucleic acid-based
techniques for assessing expression are well known in the art and
include, for example, determining the level of marker mRNA in a
sample from a subject. Many expression detection methods use
isolated RNA. Any RNA isolation technique that does not select
against the isolation of mRNA can be utilized for the purification
of RNA from cells that express a marker of the invention (see,
e.g., Ausubel et al., ed., (1987-1999) Current Protocols in
Molecular Biology (John Wiley & Sons, New York). Additionally,
large numbers of tissue samples can readily be processed using
techniques well known to those of skill in the art, such as, for
example, the single-step RNA isolation process of Chomczynski
(1989, U.S. Pat. No. 4,843,155).
[0347] The term "probe" refers to any molecule that is capable of
selectively binding to a marker of the invention, for example, a
nucleotide transcript and/or protein. Probes can be synthesized by
one of skill in the art, or derived from appropriate biological
preparations. Probes may be specifically designed to be labeled.
Examples of molecules that can be utilized as probes include, but
are not limited to, RNA, DNA, proteins, antibodies, and organic
molecules.
[0348] Isolated mRNA can be used in hybridization or amplification
assays that include, but are not limited to, Southern or Northern
analyses, polymerase chain reaction analyses and probe arrays. One
method for the detection of mRNA levels involves contacting the
isolated mRNA with a nucleic acid molecule (probe) that can
hybridize to the marker mRNA. The nucleic acid probe can be, for
example, a full-length cDNA, or a portion thereof, such as an
oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to marker genomic DNA.
[0349] In one embodiment, the mRNA is immobilized on a solid
surface and contacted with a probe, for example by running the
isolated mRNA on an agarose gel and transferring the mRNA from the
gel to a membrane, such as nitrocellulose. In an alternative
embodiment, the probe(s) are immobilized on a solid surface and the
mRNA is contacted with the probe(s), for example, in an Affymetrix
gene chip array. A skilled artisan can readily adapt known mRNA
detection methods for use in detecting the level of marker
mRNA.
[0350] An alternative method for determining the level of marker
mRNA in a sample involves the process of nucleic acid
amplification, e.g., by RT-PCR (the experimental embodiment set
forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193),
self sustained sequence replication (Guatelli et al. (1990) Proc.
Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification
system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988)
Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,
U.S. Pat. No. 5,854,033) or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers. In
particular aspects of the invention, marker expression is assessed
by quantitative fluorogenic RT-PCR (i.e., the TaqMan.TM. System).
Such methods typically utilize pairs of oligonucleotide primers
that are specific for a marker of the invention. Methods for
designing oligonucleotide primers specific for a known sequence are
well known in the art.
[0351] The expression levels of a marker of the invention may be
monitored using a membrane blot (such as used in hybridization
analysis such as Northern, Southern, dot, and the like), or
microwells, sample tubes, gels, beads or fibers (or any solid
support comprising bound nucleic acids). See U.S. Pat. Nos.
5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are
incorporated herein by reference. The detection of marker
expression may also comprise using nucleic acid probes in
solution.
[0352] In one embodiment of the invention, microarrays are used to
detect the expression of a marker of the invention. Microarrays are
particularly well suited for this purpose because of the
reproducibility between different experiments. DNA microarrays
provide one method for the simultaneous measurement of the
expression levels of large numbers of genes. Each array consists of
a reproducible pattern of capture probes attached to a solid
support. Labeled RNA or DNA is hybridized to complementary probes
on the array and then detected by laser scanning Hybridization
intensities for each probe on the array are determined and
converted to a quantitative value representing relative gene
expression levels. See, U.S. Pat. Nos. 6,040,138, 5,800,992 and
6,020,135, 6,033,860, and 6,344,316, which are incorporated herein
by reference. High-density oligonucleotide arrays are particularly
useful for determining the gene expression profile for a large
number of RNA's in a sample.
[0353] The amounts of phosphorylated marker, and/or a mathematical
relationship of the amounts of a marker of the invention may be
used to calculate the survival of a subject being treated for a
metabolic disorder, the efficacy of a treatment regimen for
treating a metabolic disorder, and the like, using the methods of
the invention, which may include methods of regression analysis
known to one of skill in the art. For example, suitable regression
models include, but are not limited to CART (e.g., Hill, T, and
Lewicki, P. (2006) "STATISTICS Methods and Applications" StatSoft,
Tulsa, Okla.), Cox (e.g., www.evidence-based-medicine.co.uk),
exponential, normal and log normal (e.g.,
www.obgyn.cam.ac.uk/mrg/statsbook/stsurvan.html), logistic (e.g.,
www.en.wikipedia.org/wiki/Logistic_regression or
http://faculty.chass.ncsu.edu/garson/PA765/logistic.htm),
parametric, non-parametric, semi-parametric (e.g.,
www.socserv.mcmaster.ca/jfox/Books/Companion), linear (e.g.,
www.en.wikipedia.org/wiki/Linear_regression or
http://www.curvefit.com/linear_regression.htm), or additive (e.g.,
www.en.wikipedia.org/wiki/Generalized_additive_model or
http://support.sas.com/rnd/app/da/new/dagam.html).
[0354] In one embodiment, a regression analysis includes the
amounts of phosphorylated marker. In another embodiment, a
regression analysis includes a marker mathematical relationship. In
yet another embodiment, a regression analysis of the amounts of
phosphorylated marker, and/or a marker mathematical relationship
may include additional clinical and/or molecular co-variates. Such
clinical co-variates include, but are not limited to, treatment
regime, clinical outcome (e.g., disease-specific survival, therapy
failure), and/or clinical outcome as a function of time after
diagnosis, time after initiation of therapy, and/or time after
completion of treatment.
[0355] In another embodiment, the amounts of phosphorylated marker,
and/or a mathematical relationship of the amounts of a marker may
be used to calculate the survival of a subject being treated for a
metabolic disorder, the efficacy of a treatment regimen for
treating a metabolic disorder, and the like, using the methods of
the invention, which may include methods of regression analysis
known to one of skill in the art. For example, suitable regression
models include, but are not limited to CART (e.g., Hill, T, and
Lewicki, P. (2006) "STATISTICS Methods and Applications" StatSoft,
Tulsa, Okla.), Cox (e.g., www.evidence-based-medicine.co.uk),
exponential, normal and log normal (e.g.,
www.obgyn.cam.ac.uk/mrg/statsbook/stsurvan.html), logistic (e.g.,
www.en.wikipedia.org/wiki/Logistic_regression or
http://faculty.chass.ncsu.edu/garson/PA765/logistic.htm),
parametric, non-parametric, semi-parametric (e.g.,
www.socserv.mcmaster.ca/jfox/Books/Companion), linear (e.g.,
www.en.wikipedia.org/wiki/Linear_regression or
http://www.curvefit.com/linear_regression.htm), or additive (e.g.,
www.en.wikipedia.org/wiki/Generalized_additive_model or
http://support.sas.com/rnd/app/da/new/dagam.html).
[0356] In one embodiment, a regression analysis includes the
amounts of phosphorylated marker. In another embodiment, a
regression analysis includes a marker mathematical relationship. In
yet another embodiment, a regression analysis of the amounts of
phosphorylated marker, and/or a marker mathematical relationship
may include additional clinical and/or molecular co-variates. Such
clinical co-variates include, but are not limited to, treatment
regime, clinical outcome (e.g., disease-specific survival, therapy
failure), and/or clinical outcome as a function of time after
diagnosis, time after initiation of therapy, and/or time after
completion of treatment.
VII. Kits
[0357] The invention also provides compositions and kits for
prognosing a metabolic disorder or survival of a subject being
treated for a metabolic disorder. These kits include one or more of
the following: a detectable antibody that specifically binds to a
marker of the invention, a detectable antibody that specifically
binds to a marker of the invention, reagents for obtaining and/or
preparing subject tissue samples for staining, and instructions for
use.
[0358] The kits of the invention may optionally comprise additional
components useful for performing the methods of the invention. By
way of example, the kits may comprise fluids (e.g., SSC buffer)
suitable for annealing complementary nucleic acids or for binding
an antibody with a protein with which it specifically binds, one or
more sample compartments, an instructional material which describes
performance of a method of the invention and tissue specific
controls/standards.
VIII. Screening Assays
[0359] The invention also provides methods (also referred to herein
as "screening assays") for identifying modulators, i.e., candidate
or test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs), which
modulate a metabolic disorder by modulating the expression and/or
activity of a marker of the invention. Such assays typically
comprise a reaction between a marker of the invention and one or
more assay components. The other components may be either the test
compound itself, or a combination of test compounds and a natural
binding partner of a marker of the invention. Compounds identified
via assays such as those described herein may be useful, for
example, for modulating, e.g., inhibiting, ameliorating, treating,
or preventing a metabolic disorder.
[0360] The test compounds used in the screening assays of the
present invention may be obtained from any available source,
including systematic libraries of natural and/or synthetic
compounds. Test compounds may also be obtained by any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; peptoid libraries (libraries
of molecules having the functionalities of peptides, but with a
novel, non-peptide backbone which are resistant to enzymatic
degradation but which nevertheless remain bioactive; see, e.g.,
Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); spatially
addressable parallel solid phase or solution phase libraries;
synthetic library methods requiring deconvolution; the `one-bead
one-compound` library method; and synthetic library methods using
affinity chromatography selection. The biological library and
peptoid library approaches are limited to peptide libraries, while
the other four approaches are applicable to peptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, 1997,
Anticancer Drug Des. 12:145).
[0361] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0362] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria and/or spores, (Ladner, U.S. Pat. No. 5,223,409), plasmids
(Cull et al, 1992, Proc Natl Acad Sci USA 89:1865-1869) or on phage
(Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science
249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci.
87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner,
supra.).
[0363] The screening methods of the invention comprise contacting a
biological sample from a subject with a test compound and
determining the ability of the test compound to modulate the
expression and/or activity of a marker of the invention in the
sample. The expression and/or activity of a marker of the invention
can be determined as described herein.
[0364] In another embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
marker of the invention or biologically active portions thereof. In
yet another embodiment, the invention provides assays for screening
candidate or test compounds which bind to a marker of the invention
or biologically active portions thereof. Determining the ability of
the test compound to directly bind to a marker can be accomplished,
for example, by coupling the compound with a radioisotope or
enzymatic label such that binding of the compound to the marker can
be determined by detecting the labeled marker compound in a
complex. For example, compounds (e.g., marker substrates) can be
labeled with .sup.131I, .sup.125I, .sup.35S, .sup.14C, or .sup.3H,
either directly or indirectly, and the radioisotope detected by
direct counting of radioemission or by scintillation counting.
Alternatively, assay components can be enzymatically labeled with,
for example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0365] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent capable of modulating the expression and/or activity of a
marker of the invention identified as described herein can be used
in an animal model to determine the efficacy, toxicity, or side
effects of treatment with such an agent. Alternatively, an agent
identified as described herein can be used in an animal model to
determine the mechanism of action of such an agent. Furthermore,
this invention pertains to uses of novel agents identified by the
above-described screening assays for treatment as described
above.
[0366] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references and published patents and patent applications cited
throughout the application are hereby incorporated by
reference.
Exemplification of the Invention:
[0367] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention, as one skilled in the art would recognize from
the teachings hereinabove and the following examples, that other
assays, cell types, agents, constructs, or data analysis methods,
all without limitation, can be employed, without departing from the
scope of the invention as claimed.
[0368] The contents of any patents, patent applications, patent
publications, or scientific articles referenced anywhere in this
application are herein incorporated in their entirety.
[0369] The practice of the present invention will employ, where
appropriate and unless otherwise indicated, conventional techniques
of cell biology, cell culture, molecular biology, transgenic
biology, microbiology, virology, recombinant DNA, and immunology,
which are within the skill of the art. Such techniques are
described in the literature. See, for example, Molecular Cloning: A
Laboratory Manual, 3rd Ed., ed. by Sambrook and Russell (Cold
Spring Harbor Laboratory Press: 2001); the treatise, Methods In
Enzymology (Academic Press, Inc., N.Y.); Using Antibodies, Second
Edition by Harlow and Lane, Cold Spring Harbor Press, New York,
1999; Current Protocols in Cell Biology, ed. by Bonifacino, Dasso,
Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons,
Inc., New York, 1999; and PCR Protocols, ed. by Bartlett et al.,
Humana Press, 2003.
Example 1
Identification of CoQ10 as a MIM
[0370] In order to evaluate CoQ10 as a potential MIM, CoQ10 in
oxidized form was exogenously added to a panel of cell lines,
including both cancer cell lines and normal control cell lines, and
the changes induced to the cellular microenvironment profile for
each cell line in the panel were assessed. Changes to cell
morphology/physiology, and to cell composition, including both mRNA
and protein levels, were evaluated and compared for the diseased
cells as compared to normal cells. The results of these experiments
identified CoQ10 and, in particular, the oxidized form of CoQ10, as
a MIM.
[0371] In a first set of experiments, changes to cell
morphology/physiology were evaluated by examining the sensitivy and
apoptotic response of cells to CoQ10. A panel of skin cell lines
including a control cell lines (primary culture of keratinocytes
and melanocytes) and several skin cancers cell lines (SK-MEL-28, a
non-metastatic skin melanoma; SK-MEL-2, a metastatic skin melanoma;
or SCC, a squamous cell carcinoma; PaCa2, a pancreatic cancer cell
line; or HEP-G2, a liver cancer cell line) were treated with
various levels of Coenzyme Q10. The results of these experiments
demonstrated that the cancer cell lines exhibited an altered dose
dependent response as compared to the control cell lines, with an
induction of apoptosis and cell death in the cancer cells only.
Exemplary experiments are described in detail in Example 3
below.
[0372] Assays were next employed to assess changes in the
composition of the cell following treatment with CoQ10. Changes in
gene expression at the mRNA level were analyzed using Real-Time PCR
array methodology. Exemplary experiments are described in detail in
Examples 6 and 9-13 below. In complementary experiments, changes in
gene expression at the protein level were analyzed by using
antibody microarray methodology, 2-dimensional gel electrophoresis
followed by protein identification using mass spectrometry
characterization, and by western blot analysis. Exemplary
experiments are described in detail below in Examples 4, 7 and 8,
respectively. The results from these assays demonstrated that
significant changes in gene expression, both at the mRNA and
protein levels, were induced in the cell lines examined due to the
addition of the oxidized form of CoQ10. Genes modulated by CoQ10
treatment were found to be clustered into several cellular
pathways, including apoptosis, cancer biology and cell growth,
glycolysis and metabolism, molecular transport, and cellular
signaling.
[0373] Experiments were carried out to confirm the entry of CoQ10
into cells and to determine the level and form of CoQ10 present in
the cells. In particular, the level of Coenzyme Q10, as well as the
form of CoQ10 (i.e., oxidized or reduced), present in the
mitochondria was determined by analyzing mitochondrial enriched
preparations from cells treated with CoQ10. The level of Coenzyme
Q10 present in the mitochondria was confirmed to increase in a time
and dose dependent manner with the addition of exogenous Q10. In a
surprising and unexpected result, CoQ10 was determined to be
present in the mitochondria primarily in oxidized form. In
addition, changes in levels of proteins from mitochondria enriched
samples were analyzed by using 2-D gel electrophoresis and protein
identification by mass spectrometry characterization. The results
from these experiments demonstrated that the levels of the oxidized
form of CoQ10 in the mitochondria over the time course examined
correlated with a wide variety of cellular changes, as evidenced by
the modulation of mRNA and protein levels for specific proteins
related to metabolic and apoptotic pathways. Exemplary experiments
are described in detail in Example 5 below.
[0374] The results described by Applicants herein identified the
endogenous molecule CoQ10 and, in particular, the oxidized form of
CoQ10, as a MIM. For example, the results identified CoQ10 as a
MIM, since CoQ10 was observed to induce changes in gene expression
at both the mRNA and protein level. The results identified CoQ10 as
having multidimensional character, since CoQ10 induced differential
changes in cell morphology/physiology and cell composition (e.g.,
differential changes in gene expression at both the mRNA and
protein level), in a disease state (e.g., cancer) as compared to a
normal (e.g., non-cancerous) state. Moreover, the results
identified CoQ10 as having multidimensional character in that CoQ10
was capable of entering a cell, and thus exhibited both therapeutic
and carrier effects.
Example 2
Methods for Identifying Disease Relevant Processes and Biomarkers
for Metabolic Disorders
[0375] From the cell based assays in which cell lines were treated
with a molecule of interest, the differences in treated vs
non-treated cells is evaluated by mRNA arrays, protein antibody
arrays, and 2D gel electrophoresis. The proteins identified from
comparative sample analysis to be modulated by the MIM or
Epi-shifter, are evaluated from a Systems Biology perspective with
pathway analysis (Ingenuity IPA software) and a review of the known
literature. Proteins identified as potential therapeutic or
biomarker targets are submitted to confirmatory assays such as
Western blot analysis, siRNA knock-down, or recombinant protein
production and characterization methods.
Materials and Methods for Examples 3-8
[0376] Coenzyme Q10 stock
[0377] A 500 .mu.M Coenzyme Q10 (5% isopropanol in cell growth
media) was prepared as follows. A 10 mL 500 .mu.M Coenzyme Q10
stock was made fresh every time. Molecular Weight: 863.34
(0.0005 mol/L)(0.010 L)(863.34 g/mol)=0.004317 g
[0378] To make 10 mL of 500 .mu.M stock, 4.32 mg Coenzyme Q10 was
weighted out in a 15 mL falcon tube, and 500 .mu.L isopropanol was
added. The solution was warmed in a 50-60.degree. C. water bath
while swirling to dissolve completely. To this solution, 9.5 mL of
media (the same media in which the cells are grown) was added.
Cell Culture
[0379] Cells were obtained from the American Type Culture
Collection or Gibco. Cells were grown in DMEM/F-12 media
supplemented with 5% fetal bovine serum, 0.25 ug/mL Amphotericin,
100 ug/mL Streptomycin, and 100 U mL-1 penicillin. Cells were
maintained in an atmosphere of 95% air and 5% CO2 at 37 degrees
C.
Coenzyme Q10 Treatment and Total Protein Isolation
[0380] Cells were grown to 85% confluency prior to exposure with
Q10. Supplemented media was conditioned with Q10 to 50 and 100
micro molar concentrations. Flasks were treated with control, 50
.mu.M Q10, and 100 .mu.M Q10 in triplicate. Protein was isolated
from the treated and control flask after 4, 8, 12, and 24 hours.
For isolation of proteins, cells were washed three times with 5 mL
of ice cold PBS at a pH of 7.4. The cells were then scraped in 3 mL
of PBS, pelleted by centrifuge, and re-suspended in a lysis buffer
at pH 7.4 (80 mM TRIS-HCl, 1% SDS, with protease and phosphotase
inhibitors). Protein concentrations were quantified using the BCA
method.
Cell Lines
[0381] The cell lines listed below were propagated and a cell bank
established for each. Large scale production of cells for various
assays were performed and the material harvested for analysis. In
general, when a cell specific media was not required for
maintenance of cell lines, the media used for cell growth was
DMEMF-12 with 5% serum. Cells were typically grown to 75-80%
confluence (clear spacing) prior to splitting and use in cell
assays and standard practice methods followed. The following cell
lines were established for experiments: [0382] SK-MEL-28
(non-metastatic skin melanoma) [0383] SK-MEL-2 (metastatic skin
melanoma) [0384] HEKa (keratinocytes, skin control) [0385] HEMa
(melanocyte, skin control) [0386] nFIB (neonatal fibroblasts)
[0387] HEP-G2 (liver cancer) [SBH cell line] [0388] SkBr-3 (breast
cancer, Her2 overexpressed) [0389] MCF-7 (breast cancer, p53
mutation) [0390] PC-3 (prostate cancer) [SBH cell line] [0391]
SkBr-3 (human breast adenocarcinoma) [0392] NCI-ES-0808 [0393] SCC
(squamous cell carcinoma) [0394] PaCa-2 [0395] NIH-3T3
Cell Culture:
[0396] Cells were obtained for the American Type Culture Collection
or Gibco. Cells were grown in DMEM/F-12 media supplemented with 5%
fetal bovine serum, 0.25 ug/mL Amphotericin, 100 ug/mL
Streptomycin, and 100 U mL-1 penicillin. Cells were maintained in
an atmosphere of 95% air and 5% CO2 at 37 degrees C.
[0397] Skin malignant melanoma SK-MEL28 cells were grown and
maintained in DMEM/F12 with Glutamax (Invitrogen, Carlsbad Calif.)
supplemented with 5% FBS, amphotericin and penicillin/streptomycin.
Cells were grown at 37.degree. C. with 5% CO2. Details of
additional cell line and growth conditions are outlined in the
table below.
TABLE-US-00001 TABLE 1 Cell lines analyzed for sensitivity to Q10.
Cell Line Description Growth Conditions PaCa2 Pancreatic DMEM/F12
with Glutamax + 10% FBS, Carcinoma 2.5% Horse Serum, amphotericin,
penicillin/streptomycin. HepG2 Hepatocellular MEM with Earles Salts
Carcinoma supplemented with 10% FBS, amphotericin, penicillin/
streptomycin, sodium pyruvate and non-essential amino acids. PC3
Prostate DMEM/F12 with Glutamax, Adenocarcinoma supplemented with
5% FBS, amphotericin and penicillin/streptomycin. SKBr3 Breast
Cancer DMEM/F12 with Glutamax supplemented with 5% FBS and
amphotericin, penicillin/streptomycin. MCF-7 Breast Cancer DMEM/F12
with Glutamax supplemented with 5% FBS and amphotericin,
penicillin/streptomycin.
Q10 Treatment of SKMEL28 Cells:
[0398] SK-MEL28 cells were treated with 100 .mu.M Q10 or the
control vehicle. The formulation of the Q10 was as follows. In a 15
mL capped tube, 4.32 mg of Q10 (supplied by Cytotech) was
transferred and then dissolved by the addition of 500 .mu.L of
isopropanol. The resulting solution was warmed in a 65.degree. C.
water bath and vortexed at high speed. The Q10/isopropanol solution
was made to a volume of 10 mL with the addition of equilibrated
cell culture media. The stock solution was then vortexed to ensure
maximum solubility of Q10. The stock solution was diluted (2 mL of
stock with 8 mL of media) to obtain a final concentration of 100
.mu.M Q10. For the control vehicle, 9.5 mL of media was added to
500 .mu.L of isopropanol. The control stock was further diluted (2
mL of stock) with 8 mL of media. Cells were harvested 6, 16, 24, 48
or 72 hours after the start of the treatment.
Q10 Treatment of SCC Cells:
[0399] SCC cells were treated with 100 .mu.M Q10 (prepared as
described above) either for 6 hours or 24 hours. The control cells
were untreated cells. Cells were harvested and pelleted at the
different times after treatment and the pellets were flash frozen
and stored at -80.degree. C. until the RNA was isolated at XTAL as
described below.
RNA Isolation:
[0400] Cells were lysed for RNA isolation at different treatment
times using the RNeasy Mini kit (Qiagen, Inc., Valencia Calif.) kit
following the manufacturer's instructions. RNA was quantified by
measuring Optical Density at 260 nm.
First Strand Synthesis:
[0401] First strand cDNA was synthesized from 1 .mu.g of total RNA
using the RT2 First Strand Synthesis kit (SABiosciences, Frederick
Md.) as per manufacturer's recommendations.
Real-Time PCR:
[0402] Products from the first strand synthesis were diluted with
water, mixed with the SYBR green master mix (SABiosciences,
Frederick Md.) and loaded onto PCR arrays. Real time PCR was run on
the PCR Arrays (Apoptosis Arrays, Diabetes Arrays, Oxidative stress
and Antioxidant defense Arrays and Heat Shock Protein Arrays.)
(SABiosciences, Frederick Md.) on a Biorad CFX96.
Determining Cell Line Sensitivity to Coenzyme Q10 by Nexin Assay
for Apoptosis:
[0403] The percentage of cells in early and late apoptosis was
quantified following 24 hours of Coenzyme Q10 treatment. Early and
late apoptosis was used as a marker to understand the differences
in sensitivity of various cancer cell lines to Coenzyme Q10. The
different cell lines tested were PaCa2, HepG2, PC-3, SKBr3, MCF-7
and SK-MEL28. Cells were allowed to adhere overnight in 96-well
plates. These cells were treated with either control vehicle, 50
.mu.M Q10 or 100 .mu.M Coenzyme Q10. After 24 hours, the presence
of apoptotic cells was estimated on a PCA96 flow cytometer (Guava
Technologies, Hayward, Calif.). In addition, some cells were
treated with 4 .mu.M Staurosporine for 2 hours as a positive
control for apoptosis. Cells were first washed with PBS and
detached with 50 .mu.L of Accumax (Innovative Cell Technologies,
San Diego, Calif.) at room temperature. The dissociation was
stopped by addition of culture medium containing 1% Pluronic F-68
(Sigma-Aldrich, St. Louis, Mo.). Then 100 .mu.L of Nexin reagent
(Guava Technologies, Hayward, Calif.) was added to each of the
wells. After 20 minutes of incubation in the dark, the assay was
performed in low binding plates to minimize reattachment of cells
to the substrate. The Nexin Reagent contains two dyes. Annexin-V-PE
which detects phosphatidyl serine on the outside of a cell; a
characteristic of early apoptotic cells. The second dye, 7-AAD
permeates only late apoptotic cells while being excluded from live
(healthy) and early apoptotic cells. The percentage of four
populations of cells; live, early apoptotic, late apoptotic and
debris was determined using the Cytosoft 2.5.7 software (Guava
Technologies, Hayward, Calif.).
Immunoblotting
[0404] Approximately 50 .mu.g of protein were assayed per sample by
immunoblotting. All treatments were run in triplicate with
controls. Proteins were separated on 12% TRIS-HCl gels, transferred
via electrophoresis to nitro-cellulose membranes and blocked using
a 5% milk and TBST solution prior to incubation with primary
antibodies. The primary antibodies were incubated overnight at 4
degrees C. in a 5% BSA and TBST solution. Secondary antibodies were
incubated for one hour at 4 degrees. All antibodies were purchased
from Cell Signaling Technology. Antibodies were used at a ratio of
1:1000, with the exception of .beta.Actin at a ratio of 1:5000.
Blots were developed and results were quantified using the NIH Java
based densitometer analysis software Image J. All blots were also
probed for and normalized to their respective .beta.Actin
expression.
Two-Dimensional Electrophoresis
[0405] Before isoelectric focusing (IEF), samples were solubilized
in 40 mM Tris, 7 M urea, 2 M thiourea, and 1% C7 zwitterionic
detergent, reduced with tributylphosphine, and alkylated with 10 mM
acrylamide for 90 min at room temperature. After the sample was run
through a 10-kDa cutoff Amicon Ultra device with at least 3 volumes
of the resuspension buffer, consisting of 7 M urea, 2 M thiourea,
and 2% CHAPS to reduce the conductivity of the sample. One hundred
micrograms of protein were subjected to IEF on 11-cm pH 3 to 10, pH
4 to 7 or pH 6 to 11 immobilized pH gradient strips (GE, Amersham,
USA) to 100,000 volts hour. After IEF, immobilized pH gradient
strips were equilibrated in 6 M urea, 2% SDS, 50 mM Tris-acetate
buffer, pH 7.0, and 0.01% bromphenol blue and subjected to
SDS-polyacrylamide gel electrophoresis on 8 to 16% Tris-HCl Precast
Gel, 1 mm (Bio-Rad, USA). The gels were run in duplicate. They were
either fixed, stained in SYPRO Ruby, 80 mL/gel (Invitrogen, USA)
and imaged on Fuji FLA-5100 laser scanner or transferred onto PVDF
membrane.
[0406] Additional information was obtained for a control sample to
test the utility of protein identification through the use of
methods that utilize dPC (Protein Forest Inc.) selective pI
fractionation, followed by trypsin digestion of the dPC plug with
mass spec identification and semi-quantization (Nanomate or
LC/LTQ/MS). The dPC analysis performed with a control sample
demonstrated its utility in identifying a large subset of proteins.
The materials produced during the studies were archived so that
they may be utilized as a resource should the future need arise
2D Gel Image Analysis:
[0407] Analysis of all gel images was performed using Progenesis
Discovery and Pro (Nonlinear Dynamics Inc., Newcastle upon Tyne,
UK). After spot detection, matching, background subtraction,
normalization, and filtering, data for SYPRO Ruby gel images was
exported. Pairwise comparisons between groups were performed using
the Student's t test in Progenesis Discovery to identify spots
whose expression was significantly altered (p>0.05).
Antibody Array:
[0408] An antibody microarray (Panorama XP725 Antibody Array,
Sigma) was utilized to screen over 700 protein antibodies to assess
changes at the protein concentration level in Q10 treated cells
(SK-MEL-28, SCC). The expression of a protein in a cell extract is
detected when it is bound by a corresponding antibody spotted on
the slide. Prior to binding, the proteins are directly labeled with
a fluorescent dye which is used for fluorescent visualization and
quantitative analysis. The array is used for comparing protein
expression profiles of two samples (test versus reference samples),
each labeled with a different CyDye (Cy3 or Cy5) and the two
samples are applied simultaneously at equal protein concentrations
on the array. Fluorescent signal intensity for each sample is then
recorded individually at the wavelength corresponding to the dye
label of the sample and compared.
[0409] High doses of Coenzyme Q10 regulates expression of genes
involved in the apoptotic, diabetic and oxidative stress pathways
in cultured SKMEL-28 cells. Experimental details: SKMEL-28 cells
(ATCC Catalog #HTB-72) are non metastatic, skin melanoma cells that
were cultured in DMEM-F12 containing Glutamax (Invitrogen Cat#
10565-042) supplemented with 5% FBS, Penicillin, Streptomycin and
Amphotericin, were treated with the vehicle or 100 uM Coenzyme Q10
for varying amounts of time. Any changes in gene expression
consequent to Coenzyme Q10 treatment were quantified using Real
time PCR Arrays (Apoptosis Cat #PAHS-12, Diabetes Cat #PAHS-023 and
Oxidative Stress Cat #PAHS-065). (SABiosciences, Frederick,
Md.).
[0410] A stock concentration of 500 uM Coenzyme Q10 was prepared by
dissolving 4.32 mg in 500 ul of isopropanol which was further
diluted to 10 ml by addition of media. Alternate vortexing and
heating to 65 oC dissolved the Coenzyme Q10. 2 ml of the stock
solution was diluted to 10 ml with media to get a 100 uM Q10
containing media that was used to treat cells. A vehicle was
prepared in parallel with a similar protocol except that the
Coenzyme Q10 was not added.
[0411] SKMEL-28 cells were plated at a density of 1.times.10.sup.5
cells/well in a 6-well plate. After 24 hours, when cells had
attached and were at 50% confluence, either the vehicle or 100 uM
Q10 was added. Cells were harvested by at 6, 16, 24, 48 or 72 hours
after Q10 treatment while the vehicle treated cells were harvested
after 24 hours. Cells were lysed for RNA isolation at different
treatment times using the RNeasy Mini kit (Qiagen, Inc., Valencia
Calif. Cat #74104) kit following the manufacturer's instructions
using a spin column and on-column DNase treatment. RNA was
quantified by measuring absorbance at 260 nm.
[0412] Real time PCR was preceded by first strand cDNA synthesis
using 0.4-lug of total RNA as the template using the RT2 First
Strand Synthesis kit (SABiosciences, Frederick Md. Cat# C-03) with
a genomic DNA elimination step as per manufacturer's
recommendations. Products from the first strand synthesis were
diluted with water, mixed with the SYBR green master mix
(SABiosciences, Frederick Md. Cat#PA-010-12) and loaded onto PCR
arrays that contain primer assays for 84 different genes linked
within a common pathway, 5 housekeeping genes used for
normalization, reverse transcription and PCR controls. Real time
PCR was run on a Biorad Cfx96. The amplification was initiated with
a hot start to activate the enzyme, followed by 40 cycles each of
(95.degree. C.-15 second denaturation step and 60.degree. C.-1
minute annealing and extension step) followed by a melting curve
program. Ct values, the output from the PCR thermocycler for all
treatment groups were organized on an excel spreadsheet and loaded
onto the comparative analysis software available at
http://www.sabiosciences.com/pcdarrayanalysis.php.
Purification of Mitochondria Enriched Samples:
[0413] Experimental details: SKMEL-28, NCI-ES0808 and NIH-3T3 cells
that were treated with 100 .mu.M Q10 for 24 or 48 hours along with
cells that were harvested at t=0 were harvested by washing and
scraping from T160 flasks. Cells were centrifuged, pelleted, flash
frozen and stored at -80 oC until the mitochondria were isolated.
Cell pellets were thawed, resuspended and ruptured in Dounce
homogenizer. The homogenate was centrifuged and mitochondria were
isolated using reagents and the protocol recommended by the
Mitochondria Isolation kit for Cultured cells (MitoSciences, Eugene
Oreg., Cat # MS852). The mitochondrial fraction was aliquoted and
stored at -80 oC.
Coenzyme Q10 and Ubiquinol-10 Quantification Method:
[0414] A method for the simultaneous determination of Coenzyme Q10
(Q10) and the reduced form ubiquinol-10 (Q10H2) was implemented
based upon a recently published method (Ruiz-Jimenez, 2007, J.
Chromatogr. A, 1175, 242-248) through the use of LC-MS/MS with
electrospray ionization (ESI) in the positive ion mode. The highly
selective identification and sensitive quantitation of both Q10 and
Q10H2 is possible, along with the identification of other selected
lipids. An aliquot of the mitochondrial enriched samples from
SK-MEL-28 treated with 100 .mu.M Q10 was subjected to a
conventional pre-treatment based on protein precipitation (100
.mu.l of packed cells sonicated in 300 .mu.l of 1-propanol),
liquid-liquid extraction (add 100 .mu.l of water to supernatant and
extract X3 with 200 .mu.l of n-hexane), evaporation of combined
hexane extracts to dryness and reconstitution in 50 .mu.l of 95:5
methanol/hexane (v/v). Analysis was by LC-MS/MS on a Waters Quattro
II triple quadrupole mass spectrometer with a Prism RP 1.times.100
mm, 5 .mu.m particle size column (Keystone Scientific). Isocratic
elution with 4 mM ammonium formate in 20% isopropyl alcohol 80%
methanol at a flow rate of 50 .mu.l/min Ten .mu.l of each sample
was injected. MRM analysis was performed using m/z 882.7>197.00
(Q10H2) and m/z 880.80>197.00 (Q10) transitions with cone
voltage of 40 and collision energy of 30.
Example 3
Sensitivity of Cell Lines to CoQ10
[0415] A number of cell lines were tested for their sensitivity to
Q10 after 24 hours of application by using a reagent (Nexin
reagent) that contains a combination of two dyes, 7AAD and
Annexin-V-PE. The 7AAD dye will enter into cells with permeabilized
cell membranes; primarily those cells that are in late apoptosis.
Annexin-V-PE is a dye that binds to Phosphotidyl serine, which is
exposed on the outer surface of the plasma membrane in early
apoptotic cells. The Nexin reagent thus can be used to
differentiate between different populations of apoptotic cells in a
flow cytometer.
[0416] PaCa2 cells showed an increase in both early and late
apoptotic cells (between 5-10% of gated cells) with 50 .mu.M Q10
and 100 .mu.M Q10 after 24 hours of Q10 application. PC-3 cells
also showed an increase in both early and late apoptotic population
with 50 .mu.M and 100 .mu.M Q10, although the increase was less
when compared to PaCa2 cells. MCF-7 and SK-MEL28 cells showed an
increase only in early apoptotic population with 50 .mu.M and 100
.mu.M Q10. HepG2 cells were also sensitive to 50 .mu.M Q10
treatment, where there was an increase of about 20% of the gated
populated in the late apoptotic and early apoptotic stages. SKBr3
was the only cell line tested that did not show any significant
increases of early and late apoptosis with either 50 .mu.M or 100
.mu.M Q10 treatment. The results are depicted in FIGS. 1-6.
[0417] To provide additional confirmation that Q10 treatment causes
an apoptotic response in HepG2 liver cancer cells, a second
apoptosis assay was evaluated using the ApoStrand.TM. ELISA based
method that measures single-stranded DNA. The ApoStrand.TM. ELISA
is based on the sensitivity of DNA in apoptotic cells to formamide
denaturation and the detection of the denatured DNA with a
monoclonal antibody to single-stranded DNA (ssDNA). Treatment of
the liver cancer cell line HepG2 with 50 and 100 .mu.M Q10 resulted
in detectable apoptosis, with a dose-response of 17% and 32%,
respectively (FIG. 7). These results are consistent with the
observation of Q10 inducing apoptosis in other cancer cell lines
from other tissues (e.g., SCC, SKMEL-28, MCF-7, and PC-3).
Example 4
Proteomic Analysis of Cells Treated with Q10
[0418] Cell pellets of samples treated with Q10 were analyzed using
proteomic methods. The cell pellets were lysed and treated for use
in 2-D gel and Western blot analysis. Three cell types (SKMEL-28,
SCC, and nFib) were treated with Q10 and submitted to proteomic
characterization by 2-D gel electrophoresis.
Proteomic Analysis of SKMEL-28 Cells Treated with Q10
[0419] The first experimental set processed and evaluated by
Western blot and 2-D gel electrophoresis was the skin cancer cell
line SKMEL-28. This experimental set involved SK-MEL-28 cells
treated at 3, 6, 12, and 24 hours with 0, 50 or 100 .mu.M Q10.
[0420] The set of Q10 treated SK-MEL-28 samples were subjected to
2-D gel electrophoreses (FIG. 8) and were analyzed to identify
protein-level changes relative to the control samples. A
comparative analysis of 943 spots across all twenty-four gels was
performed, comparing the control sample against all of the treated
samples. The analysis included the identification of spot changes
over the time course due to increase, decrease, or
post-translational modification.
[0421] The analysis found thirty-two statistically significant
differential spot changes. From this, twenty non-redundant spots
were excised and submitted for protein identification by trypsin
digestion and mass spectrometry characterization. The characterized
peptides were searched against protein databases with Mascot and
MSRAT software analysis to identify the protein (Table 2).
TABLE-US-00002 TABLE 2 Proteins identified to have a differential
response to Q10 treatment in SKMEL-28 cell. Time Q10 Conc. 2D Spot
(hr) (.mu.M) # Expression Difference Protein Name Type 3 50 528
down 1.234 cathepsin D CTSD peptidase 3 50 702 down 1.575
chaperonin containing TCP1, CCT3 other subunit 3 3 50 74 down 1.383
eukaryotic translation EIF3G translation initiation factor 3
regulator 3 50 829 down 1.074 Ribosomal protein P2 RPLP2 other 3 50
368 down 1.121 transaldolase 1 TALDO1 enzyme 5 50 452 up -1.464
Eukaryotic translation EIF6 translation initiation factor 6
regulator 5 50 175 up -1.32 Stomatin; HSPC322 STOM other 5 50 827
up -1.457 lyrosine 3/Tryptophan 5- YWHAZ enzyme monooxygenase
activation protein 6 50 139 up -1.628 Vimentin VIM other 6 50 218
up -1.416 Vimentin VIM other 6 50 218 up -1.212 Vimentin VIM other
5 50 139 up -1.036 Vimentin VIM other 5 50 507 down 1.379 Lamin B1
LMNB1 other 6 50 571 down 1.832 mitochandrial import receptor
TOMM22 transporter Tom22 12 50 166 up -1.171 ALG-2 interacting
protein 1 PDCD6IP other 12 50 550 up -1.747 peptidylprolyl
isomerase A PPIA enzyme 12 50 613 down 1.802 galectin-1 LGALS1
other 12 50 242 down 1.373 Phosphoglycerate mutase; PGAM2
phosphatase Posphomannomutase 2 24 50 326 down 1.385 glycyl-tRNA
synthase GARS enzyme 24 50 419 down 1.451 Mago-nashi homolog MAGOH
other 3 100 528 down -1.036 cathepsin D CTSD peptidase 3 100 702
down 1.151 chaperonin containing TCP1, CCT3 other subunit 3 3 100
74 down 1.122 Eukaryotic translation EIF3G translation initiation
factor 3 regulator 3 100 829 down 1.145 Ribosomal protein P2 RPLP2
other 3 100 368 down 1.209 transaldolase 1 TALDO1 enzyme 6 100 139
up -1.829 Vimentin VIM other 6 100 218 up -1.761 Vimentin VIM other
5 100 452 down 1.134 Eukaryotic translation EIF6 translation
initiation factor 6 regulator 6 100 252 down 1.4 Sec 13 protein,
Keratin II ? 6 100 827 down 1.12 Fyrosine 3/Tryptophan 5- YWHAZ
enzyme monooxygenase activation protein 12 100 76 up -1.679
galectin-1; keratin II LGALS1 other
[0422] A key finding in this experiment was the decrease of
Transaldolase 1, which supports the premise that Q10 acts by
altering the metabolic state within the cancer cell. Transaldolase
1 is an enzyme in the pentose phosphate pathway (also known as the
hexose monophosphate shunt). Transaldolase (EC:2.2.1.2) catalyses
the reversible transfer of a three-carbon ketol unit from
sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate to form
erythrose 4-phosphate and fructose 6-phosphate. This enzyme,
together with transketolase, provides a link between the glycolytic
and pentose-phosphate pathways. This is relevant to nucleotide and
NADPH synthesis, to facilitate production of reducing equivalents
for biosynthetic reactions and maintenance of a reducing
environment.
[0423] A recent publication (Basta, P., et. al. August 2008, Cancer
Detect Prevention, 32, 200-208) provided evidence of genetic
polymorphism in Transaldolase and was linked to squamous cell
carcinoma of the head and neck. Another recent publication (Qian,
Y., et. al. May 2008, Biochem J, 415, 123-134) identified
transaldolase deficiency as a modulator of mitochondrial
homeostasis, Ca2+ fluxing and apoptosis.
[0424] From these initial results, the other proteins identified by
2-D gel electrophoresis as being modulated by Q10 in SK-MEL-28 were
analyzed for known relationships (FIG. 9). A functional evaluation
of these proteins revealed that there was a group involved in
14-3-3-mediated signaling (PDCP6IP, YWHAZ, and VIM), along with
individual proteins linked to a variety of processes [cell cycle;
pentose phosphate pathway (TALDO1); ceramide signaling (CTSD);
aminoacyl-tRNA biosynthesis (GARS), and mitochondrial protein
import (TOM22)].
Proteomic Analysis of SCC Cells Treated with Q10
[0425] Another skin cancer cell line, Squamous Cell Carcinoma
(SCC), was also prepared and analyzed by 2-D gel electrophoreses as
a follow-up experiment the previous SK-MEL-28 analysis The SCC
cells were treated with 100 .mu.M Q10 for 6 hour or 24 hours before
harvesting. A control of untreated cells was also harvested. The
cell pellets were lysed and the samples were subjected to 2-D
electrophoresis (in duplicate). Analysis of over six hundred
protein spots in the comparative study was performed, comparing the
control sample against the six hour and twenty-four hour
treatments.
[0426] The top twenty-five statistically significant differential
spot changes were evaluated from the comparative analysis of the
2-D electrophoresis gels. From this, twelve spots were excised and
submitted for identification by trypsin digestion and mass
spectrometry characterization (results summarized in Table 3
below).
TABLE-US-00003 TABLE 3 Proteins identified to have a differential
response to 100 .mu.M Q10 treatment in SCC cells at 6 and 24 hours.
Spot Cellular Response # Protein Name localization Function (fold
change) 331 Transaldolase 1 TALDO1 Cytoplasm Enzyme Decrease (1.5)
at 6 and 14 hr 23 Human BSCv C20ORF3 Plasma strictosidine Decrease
(chromosome 20 reading membrane synthase (2.1) at 6 frame 3) and 24
hr 54 NM23 protein NME1 Nucleus, Kinase Increase (mitochondria?)
(-1.2) at 6 hr, decrease at 24 hr 116 two Human ESTs from HSP70
Decrease MCF7 breast cancer cell (2.6) at 6 hr, line (HSP 70)
further decrease at 24 hr 176 Heat shock 27 kDa HSPB1 Cytoplasm
Response to Increase protein 1 environmental (-1.9) at 6 stresses
and 24 hr 135 Keratin I KRT1 Cytoplasm intermediate Decrease
filaments (2.3) at 6 and 24 hr 50 Keratin 14 KRT14 Cytoplasm
intermediate Increase filaments (-1.6) at 6 and 24 hr 68 Keratin 13
KRT13 Cytoplasm intermediate Increase filaments (-1.5) at 6 and 24
hr 49 Proteasome Beta 7 PSMB7 Cytoplasm Proteasome Decrease subunit
(1.6) at 24 hr only 93 Proteasome activator PSME3 Cytoplasm
peptidase Decrease subunit 3 (1.3) at 24 hr only 66 Rho GDP
dissociation ARHGDIA Cytoplasm Inhibitor Decrease inhibitor (GDI)
alpha (1.5) at 6 hr only 1 Unknown? Decrease (9.5)
[0427] Transaldolase 1: As previously observed in the SKMEL-28
cells treated with Q10, the enzyme Transaldolase 1 was modulated
with a decrease in levels. This provides an independent
confirmation of the previously observation of a linkage between Q10
and alterations in transaldolase (and thus the metabolic state of
the cell).
[0428] Transaldolase is an enzyme in the non-oxidative phase of the
pentose phosphate pathway (FIG. 10). The pentose phosphate pathway
is critical in the metabolic state of cells for the generation of
nicotinamide adenine dinucleotide phosphate (reduced NADH), for
reductive biosynthesis, and in the formation of ribose which is an
essential component of ATP, DNA, and RNA. Transaldolase also links
the pentose phosphate pathway to glycolysis. Glycolysis is the
metabolic pathway by which cancer cells obtain the energy needed
for cell survival, as the mitochondrial process of oxidative
phosphorylation is not utilized. Q10 is an essential coenzyme
factor required for oxidative phosphorylation and mitochondrial ATP
production.
[0429] BSCv: Spot 23 was a novel human protein from Chromosome 20
named BSCv. BSCv protein is also known as Adipocyte plasma
membrane-associated protein (Gene names: APMAP or C20orf3) and is
predicted to be a single-pass type II membrane protein with
sequence similarity to the strictosidine synthase family of
proteins. Q10 treatment caused a reduction in the levels of this
protein. This protein is not well characterized, nor has its
homology with strictosidine synthases been confirmed.
Interestingly, this protein has been associated with a role in
adipocyte differentiation (Albrektsen et al., 2001). Recent
proteomic studies of human omental adipose tissue identified BSCv
as one of nine proteins with differential expression for polcystic
ovary syndrome (PCOS) from morbidly obese women (Corton, 2008 Hum.
Reprod. 23: 651-661). As a cell surface protein that responds to
Q10, an antibody against BSCv would be useful as a biomarker. Based
on the current results and the literature available, BSCv may a
have a potential role in cancer and diabetes.
[0430] NM23A: Non-metastatic cells 1, protein (NM23A, also known as
NME1) is thought to be a metastasis suppressor. This gene (NME1)
was identified because of its reduced mRNA transcript levels in
highly metastatic cells. The protein has activity as a nucleoside
diphosphate kinase (NDK) and exists as a hexamer composed of `A`
(encoded by this gene) and `B` (encoded by NME2) isoforms.
Mutations in this gene have been identified in aggressive
neuroblastomas. NDK activities maintain an equilibrium between the
concentrations of different nucleoside triphosphates such as, for
example, when GTP produced in the citric acid (Krebs) cycle is
converted to ATP. The NDK complex is associated with p53 through
interaction with STRAP. It is noteworthy that STRAP is linked to
HNF4A. Thus, NM23A is a potential protein involved in pathways
important for cell control and disease treatment.
[0431] Rho GDP dissociation inhibitor (GDI) alpha: GDI Regulates
the GDP/GTP exchange reaction of the Rho proteins by inhibiting the
dissociation of GDP from them, and the subsequent binding of GTP to
them. The protein is upregulated in cancer cells.
Example 5
Mitochondrial Enrichment Analysis
[0432] Several lines of evidence suggested that a closer evaluation
of the role of mitochondrial proteins and cancer biology and Q10
response was warranted. First, there is the essential role of Q10
in the mitochondrial oxidative phosphorylation process for energy
production in normal cells. However, the metabolic shift that
occurs in cancer cells is to energy production through the
alternative pathway of glycolysis, which does not require Q10.
Second, the apoptotic response of cells requires mitochondrial
proteins to occur. Q10 has been established as stimulating
apoptosis in cancer cells (Bcl-2 family proteins, cytochrome c).
Finally, new mitochondrial proteins were identified as being
modulated by Q10 treatment, as exemplified by the modulation in
protein levels of the mitochondrial import receptor protein TOM22
(see experiments described herein).
Production of Mitochondrial Enriched Samples
[0433] The skin cancer SKMEL-28 cells were treated with 100 .mu.M
Q10 or a mock vehicle for 6, 19, or 48 hours. The cells were
harvested by washing and scraping the cells from T-160 flasks (4
for each time point). The cells were collected by centrifugation
and the pellets flash frozen and stored at -80.degree. C. The cell
pellets were resuspended and ruptured using a 2 mL Dounce
homogenizer. The reagents and method were obtained from a
Mitochondria Isolation Kit for Cultured Cells (MitoSciences, Cat#
MS852). The resultant mitochondria samples were divided into 75
.mu.L aliquots (4-5 aliquots per sample) and stored at -80.degree.
C.
Proteomic Analysis of Mitochondria Enriched Samples Isolated from
SK-MEL-28 Cells Treated with Q10
[0434] 2-D gel electrophoresis was performed on proteins
solubilized from two aliquots of the SK-MEL-28 mitochondria
enriched samples treated with 100 .mu.M Q10 for 6, 19, and 48 hours
(along with the corresponding mock vehicle controls). The samples
were subjected to 2-D electrophoresis (in duplicate). Analysis of
525 protein spots in the comparative study was performed, comparing
the control samples against the other time point samples (FIG.
11).
[0435] The nine statistically significant differential spot changes
were selected from the comparative analysis of the 2-D
electrophoresis gels. From these, 9 spots were excised and
submitted for identification by trypsin digestion and mass
spectrometry characterization
TABLE-US-00004 TABLE 4 Proteins identified to have a differential
response to Q10 treatment in SKMEL-28 mitochondria. Spot Response #
Protein Name Function (fold change) 11 Unknown protein 7 7 Up (1.3)
at 6 hr, drop to low levels after this 131 Unknown, same ? ? Down
(1.3) at 6 hr, as spot drops more for 19 #11, modified and 48 hr
279 acyl-CoA thioesterase ACOT7 Cleaves fatty acyl-CoA's Down (1.3)
at 6 hr, 7 isoform hBACHb into free fatty acids and back to normal
at CoA 48 hr 372 Pyruvate kinase PKM2 catalyzes the production Up
(1.5) at 6 hr, of phosphoenolpyruvate back to normal at from
pyruvate and ATP 48 hr 110 ER60 protein PDIA3 Protein disulfide Up
at 19 and 48 hr isomerase 185 Keratin 10 KRT10 intermediate
filament Up only at 19 hr 202 Beta-Actin Structural protein Up only
at 19 hr 246 Malectin MLEC carbohydrate-binding Up only at 19 hr
protein of the endoplasmic reticulum and a candidate player in the
early steps of protein N-glycosylation 75 Coiled-coil domain CCDC58
Conserved hypothetical Up at 48 hr containing 58 protein - nuclear
pore forming
[0436] Acyl-CoA thioesterase 7: Acyl-CoA thioesterase 7 (ACOT7) is
a member of the enzyme family that catalyzes the hydrolysis of
fatty acyl-CoA to free fatty acid and CoA. This enzyme thus has a
role in the regulation of lipid metabolism and cellular signaling.
ACOT7 has a preference for long-chain acyl-CoA substrates with
fatty acid chains of 8-16 carbon atoms (C8-C16). The exact cellular
function is ACOT7 is not fully understood. The transcription of
this gene is activated by sterol regulatory element-binding protein
2, thus suggesting a function in cholesterol metabolism.
[0437] The results in this Example indicate that ACOT7 is
potentially involved in the metabolism of Q10, either directly or
indirectly. Thus, targeting ACOT7 could facilitate modulation of
intercellular levels of Q10 and thus impact cellular Q10
effects.
[0438] Pyruvate kinase: Pyruvate kinase is an enzyme involved in
the last step of glycolysis. It catalyzes the transfer of a
phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding one
molecule of pyruvate and one molecule of ATP.
##STR00003##
[0439] The protein is presumably that of PKM2, the type 2 isoform,
as this was identified from the mitochondria enriched SK-MEL-28
sample. This isoform is well known to be involved in tumor cell
formation and regulation.
Quantification of Q10 Levels in Mitochondria
[0440] A method for the simultaneous determination of Coenzyme Q10,
(Q10) and the reduced form ubiquinol-10 (Q10H2) was implemented
based upon a recently published method (Ruiz-Jimenez, 2007, J.
Chroma A, 1175, 242-248) through the use of LC-MS-MS with
electrospray ionization (ESI) in the positive mode. The highly
selective identification and sensitive quantitation of both Q10 and
Q10H2 is possible, along with the identification of other selected
lipids. An aliquot of the mitochondrial enriched samples from
SK-MEL-28 treated with 100 .mu.M Q10 were subject to a conventional
pre-treatment based on protein precipitation, liquid-liquid
extraction, evaporation to dryness and reconstitution with 95:5
methanol/hexane (v/v).
[0441] In this analysis, Q10, Q10H2, and Q9 were quantitated (Table
5). The levels of the related molecule Q9 were low, and near the
level of detection. The level of the untreated samples were
relatively consistent, with the 6 hour Q10 treated sample having
this same level. To control for sample variance in total material,
the levels of cholesterol was also measured to confirm that the
differences were not due to sample size errors. When the Q10 levels
were corrected against total protein values obtained by protein
extraction other aliquots of the same mitochondrial preps, the
relative ratios were comparative. Thus, a significant increase in
Q10 levels was obtained at 19 hours (.about.3-fold) with an even
larger increase by the 48 hour time point (.about.6-fold) (FIG.
12).
TABLE-US-00005 TABLE 5 HPLC-MS Quantification results for the
levels of Q10 present in mitochondrial enriched samples from
SK-MEL-28 cells treated with 100 .mu.M Q10 in the media. Peak Area
ng/Sample .mu.g/sample File Sample Injection Q9 Q10 Q9 Q10
Q10H.sub.2 Cholesterol 081204-05 100 ng Std 245,342 352792
081204-06 6 hr mock#1 10% 2560 32649 1.04 9.25 081204-07 Solvent
Blank#1 5 ul 3781 3174 1.54 0.9 081204-08 Solvent Blank#2 5 ul 2396
4399 0.98 1.25 081204-09 6 hr mock#2 20% 1572 36328 0.64 10.3
081204-10 Solvent Blank#3 10 ul 1722 2504 0.7 0.71 081204-11 48 hr
Q10 treated 20% 4879 164496 1.99 46.63 0.28 13.85 081204-12 48 hr
mock 20% 2412 25552 0.98 7.24 0.09 13.04 081204-13 6 hr Q10 treated
20% 692 25427 0.28 7.21 081204-14 19 hr Q10 treated 20% 1161 59164
0.47 16.27 081204-15 19 hr mock 20% 901 19999 0.37 5.67
[0442] A surprising result from this study was the finding that the
Q10 was supplied to the cells as the oxidized form. For the 48 hour
samples, the reduced form Q10H2 was also measured and found to be
present in significantly lower amounts (0.28 ng/sample of CoQ10H2
as compared to 46.63 ng/sample of CoQ10). There was a general
increase (3-fold) in the levels of Q10H2 in the Q10 treated 48 hour
sample, although the levels were near the presumed detection limit
of the assay. Interestingly, the oxidized form (Q10) can act as a
pro-oxidant in biological systems. According to the literature,
when human plasma was evaluated for Q10 and Q10H2, the majority
(90%) of the molecule was found in the reduced form of Q10H2
(Ruiz-Jimenez, 2007, J. Chroma A, 1175, 242-248) which can act as
an anti-oxidant.
[0443] Thus, these results confirm and quantitate that the levels
of Q10 increase in the mitochondria upon the exogenous addition of
Q10 to the media. A surprising and unexpected discovery was that
Q10 was maintained in the supplied oxidized form (pro-oxidant) and
not converted to the reduced (anti-oxidant) form of Q10H2 in any
significant amounts.
Example 6
Real-Time PCR Arrays
Experiment 1: Apoptosis Array
[0444] As discussed above in Example 3, exposure of cancer cells to
Q10 induces a portion of these cells to die due to apoptotic
processes. To identify proteins that were involved in the Q10
response, real-time polymerase chain reaction (RT-PCR) methods were
employed to identify changes in the level of mRNA for
genes/proteins involved in targeted pathway arrays for
apoptosis.
[0445] Using PCR arrays as a screening tool, a spectrum of
molecular targets that would potentially offer an insight to the
mode of biological action of Q10 within the cells were thus
evaluated. Changes in mRNA levels were evaluated using real-time
PCR quantification to assess mRNA levels in pre-selected subsets
containing 80 pathway specific targets.
[0446] For the interpretation of mRNA results, the genes that were
altered in their mRNA transcription by a two-fold level were
identified and evaluated. The level of gene transcription to
produce mRNA only provides a rough estimate of potential changes in
the level of the expressed protein. The skilled artisan will
appreciate that each mRNA may have different rates at which it is
degraded or its translation inefficiently, thus resulting in
differing amounts of protein.
SkBr-3 Cells Treated with 50 um Q10 for 24 Hours
[0447] The assay method of RT-PCR was utilized to provide a measure
of mRNA level changes to a total of 84 apoptotic pathway related
proteins. The experiments with the real-time PCR apoptosis analysis
on SkBr3 with Q10 (24 hr) identified the following mRNA's being
affected: Bcl2, Bcl2L1, Bcl2L11, Birc6, Bax, Xiap, Hprt1, Apaf1,
Abl1, Braf. These results again provided supporting evidence for
the apoptotic response of cancer cells to Q10 treatment.
TABLE-US-00006 TABLE 6A Up-Down Symbol Regulation Unigene Refseq
Description Gname BCL2L1 13.1957 Hs.516966 NM_138578 BCL2-like 1
BCL-XL/S BNIP2 6.3291 Hs.646490 NM_004330 BCL2/adenovirus E1B
BNIP-2/NIP2 19 kDa interacting protein 2 BCL2 5.4717 Hs.150749
NM_000633 B-cell CLL/lymphoma 2 Bcl-2 BIRC6 4.7966 Hs.150107
NM_016252 Baculoviral IAP APOLLON/ repeat-containing 6 BRUCE
(apollon) BCL2L11 4.6012 Hs.469658 NM_006538 BCL2-like 11 BAM/BIM
(apoptosis facilitator) XIAP 4.3832 Hs.356076 NM_001167 X-linked
inhibitor of API3/BIRC4 apoptosis BRAF 4.3832 Hs.550061 NM_004333
V-raf murine sarcoma B-raf viral oncogene homolog B1 1/BRAF1 BAX
3.896 Hs.631546 NM_004324 BCL2-associated X protein Bax zeta APAF1
2.6244 Hs.708112 NM_001160 Apoptotic peptidase CED4/DKFZp
activating factor 1 781B1145 HPRT1 -160.6748 Hs.412707 NM_000194
Hypoxanthine HGPRT/HPRT phosphoribosyltransferase 1 (Lesch-Nyhan
syndrome)
[0448] Results that are consistent from three independent
experiments from SK-MEL-28 cells are summarized below in Table 6B.
Many genes are regulated in SCC cells as well with 100 .mu.M Q10
treatment. The genes in the Apoptosis array that appear to be
regulated in SCC cells are described in Table 7. We find that many
genes are regulated at 6 hours, both in SK-MEL-28 cells and in SCC
cells. By 24 hours, the regulation is decreased. Genes that appear
to be regulated in both SK-MEL-28 cells and in SCC cells are
described in Table 8.
TABLE-US-00007 TABLE 6B Genes in SK-MEL-28 cells regulated by 100
.mu.M Q10 treatment when analyzed by the Apoptosis Array. Symbol
Description Regulation Location Possible Functions ABL1 C-abl
oncogene 1, Down Regulated at Nucleus Tyrosine Kinase receptor
tyrosine 72 hours kinase BAG1 BCL2-associated Up Regulated at
Cytoplasm Anti-apoptotic, athanogene 48 hours glucocorticoid
receptor pathway BCL2 B-cell CLL/lymphoma 2 Down Regulated at
Cytoplasm Cell death 48 hours BCL2A1 BCL2-related protein A1 Down
Regulated at Cytoplasm Regulates Caspases, 48 hours phosphorylates
TP73 BCL2L1 BCL2-like 1 Down Regulated at Cytoplasm Caspase
Inhibitor 72 hours BCL2L10 BCL2-like 10 Down Regulated at Cytoplasm
Caspase Activator (apoptosis facilitator) 48 hours BCL2L11
BCL2-like 11 Down Regulated at Cytoplasm Pro-Apoptotic, (apoptosis
facilitator) 48 hours Caspase3 Activator BIRC3 Baculoviral IAP Down
Regulated at Cytoplasm Anti-apoptotic repeat-containing 3 6 hours
BIRC8 Baculoviral IAP Down Regulated at Cytoplasm Activates Caspase
repeat-containing 8 48 hours CARD8 Caspase recruitment Down
Regulated at Nucleus Caspase Activator domain family, member 8 48
hours CASP14 Caspase 14, apoptosis- Down Regulated at Cytoplasm
Apoptosis related related cysteine 48 hours cysteine peptidase
peptidase CASP5 Caspase 5, apoptosis- Down Regulated at Cytoplasm
Apoptosis related related cysteine 48 hours cysteine peptidase
peptidase CD40LG CD40 ligand (TNF Down Regulated at Extracellular
CD40 receptor superfamily, member 5, 48 hours Space binding
hyper-IgM syndrome) CIDEA Cell death-inducing Up Regulated at
Cytoplasm Pro-Apoptotic DFFA-like effector a 48 hours FADD Fas
(TNFRSF6)- Down Regulated at Cytoplasm Pro-Apoptotic associated via
death 6 hours domain FAS Fas (TNF receptor Up Regulated at Plasma
Pro-Apoptotic superfamily, member 6) 48 hours Membrane FASLG Fas
ligand (TNF Down Regulated at Extracellular Pro-Apoptotic
superfamily, member 6) 48 hours Space GADD45A Growth arrest and
DNA- Up Regulated at Nucleus Growth Arrest damage-inducible, alpha
48 hours HRK Harakiri, BCL2 Down Regulated at Cytoplasm
Pro-Apoptotic interacting protein 48 hours (contains only BH3
domain) PYCARD PYD and CARD Down Regulated at Cytoplasm Apoptotic
Protease domain containing 6 hours Activator TNF Tumor necrosis
factor Up Regulated at Extracellular TNF receptor binding (TNF
superfamily, 48 hours then Space member 2) down regulated TNFRSF10A
Tumor necrosis factor Up Regulated at Plasma Caspase Activator
receptor superfamily, 48 hours then Membrane member 10a down
regulated TNFRSF10B Tumor necrosis factor Down Regulated at Plasma
p53 signaling, caspase receptor superfamily, 72 hours Membrane
activation. member 10b TNFRSF1A Tumor necrosis factor Down
Regulated at Plasma Pro-apoptotic receptor superfamily, 72 hours
Membrane member 1A TNFRSF21 Tumor necrosis factor Down Regulated at
Plasma Activates Caspase receptor superfamily, 48 hours Membrane
member 21 CD27 CD27 molecule Down Regulated at Plasma Caspase
Inhibitor 48 hours Membrane TNFRSF9 Tumor necrosis factor Down
Regulated at Plasma Pro-apoptotic receptor superfamily, 48 hours
Membrane member 9 TNFSF10 Tumor necrosis factor Upregulated at
Extracellular Pro-apoptotic (ligand) superfamily, 48 hours Space
member 10 TP73 Tumor protein p73 Down Regulated at Nucleus
Transcription factor 48 hours TRAF3 TNF receptor- Down Regulated at
Cytoplasm Zinc-finger domain associated factor 3 48 hours TRAF4 TNF
receptor- Down Regulated at Cytoplasm Zinc-finger domain associated
factor 4 48 hours
TABLE-US-00008 TABLE 7 Genes in SCC cells that are regulated by 100
.mu.M Q10 treatment when analyzed by the Apoptosis Array. Symbol
Description Regulation. AKT1 V-akt murine thymoma viral oncogene
Down regulated at 6 hours and then homolog 1 up regulated at 24
hours. BAG4 BCL2-associated athanogene 4 Up regulated at 24 hours.
BAX BCL2-associated X protein Up regulated at 24 hours. BCL2 B-cell
CLL/lymphoma 2 Up regulated at 24 hours. BCL2L1 BCL2-like 1 Down
regulated at 6 hours and then up regulated at 24 hours. BIRC3
Baculoviral IAP repeat-containing 3 Down regulated at 6 hours.
BNIP3 BCL2/adenovirus E1B 19 kDa Down regulated at 24 hours.
interacting protein 3 CARD6 Caspase recruitment domain family, Down
regulated at 6 hours. member 6 CASP6 Caspase 6, apoptosis-related
cysteine Up regulated at 24 hours. peptidase CASP7 Caspase 7,
apoptosis-related cysteine Up regulated at 24 hours. peptidase CD40
CD40 molecule, TNF receptor Down regulated at 6 hours. superfamily
member 5 FADD Fas (TNFRSF6)-associated via death Up regulated at 24
hours. domain GADD45A Growth arrest and DNA-damage- Up regulated at
24 hours. inducible, alpha HRK Harakiri, BCL2 interacting protein
Up regulated at 24 hours. (contains only BH3 domain) TNFRSF21 Tumor
necrosis factor receptor Down regulated at 6 hours. superfamily,
member 21 TNFRSF25 Tumor necrosis factor receptor Down regulated at
6 hours and then superfamily, member 25 up regulated at 24 hours.
CD27 CD27 molecule Down regulated at 6 hours. TNFRSF9 Tumor
necrosis factor receptor Down regulated at 6 hours. superfamily,
member 9 TNFSF10 Tumor necrosis factor (ligand) Up regulated at 24
hours. superfamily, member 10 CD70 CD70 molecule Down regulated at
6 hours. TP53 Tumor protein p53 Up regulated at 24 hours. TP73
Tumor protein p73 Down regulated at 6 hours and then up regulated
at 24 hours. TRAF2 TNF receptor-associated factor 2 Up regulated at
24 hours.
TABLE-US-00009 TABLE 8 Genes from the apoptosis array regulated
with 100 .mu.M Q10 treatment in both SK-MEL-28 and SCC cells.
Symbol Description BCL2 B-cell CLL/lymphoma 2 BCL2L1 BCL2-like 1
(Bcl-xl) BIRC3 Baculoviral IAP repeat-containing 3 FADD Fas
(TNFRSF6)-associated via death domain GADD45A Growth arrest and
DNA-damage-inducible, alpha TNFRSF21 Tumor necrosis factor receptor
superfamily, member 21 CD27 CD27 molecule TNFRSF9 Tumor necrosis
factor receptor superfamily, member 9 TNFSF10 Tumor necrosis factor
(ligand) superfamily, member 10 TP73 Tumor protein p73 TRAF2 TNF
receptor-associated factor 2
[0449] Interestingly, the altered mRNA levels showed a significant
up-regulation in a series of apoptitic proteins, with Bcl-xl one of
the highest. This was also observed in the protein array
experiments on SK-MEL-28 cells.
[0450] Bcl-xl is a transmembrane molecule in the mitochondria
(Bcl-xl stands for "Basal cell lymphoma-extra large"). It is
involved in the signal transduction pathway of the FAS-L and is one
of several anti-apoptotic proteins which are members of the Bcl-2
family of proteins. It has been implicated in the survival of
cancer cells. However, it is known that alternative splicing of
human Bcl-x mRNA may result in at least two distinct Bcl-x mRNA
species, Bcl-xL and Bcl-xS. The predominant protein product (233
amino acids) is the larger Bcl-x mRNA, Bcl-xL, which inhibits cell
death upon growth factor withdrawal (Boise et al., 1993. Cell 74,
597-608). Bcl-xS, on the other hand, inhibits the ability of Bcl-2
to inhibit cell death and renders cells more susceptible to
apoptotic cell death. The employed assays utilized do not
distinguish which isoform of Bcl-x is being upregulated. The Bcl-x
isoform being upregulated by CoQ10 in these studies may be
determined by routine methods known in the art, e.g., by using
RT-PCR methods to evaluate the ratio of the two mRNA splicing
isoforms (Bcl-xL vs Bcl-sL).
[0451] From the survey of apoptotic related proteins it was
observed multiple pro- and anti-apoptotic factors were in the BCL-2
family or that interact with these factors have modulated
expression levels (BCL2L11, BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1).
These proteins govern mitochondrial outer membrane
permeabilization.
[0452] An early marker for apoptotic response is observed with the
upregulation of Caspase-9 (16 hour) which is consistent with
previous observations of apoptosis with caspase 3/7 proteins.
Induction of stress signaling pathways causes release of cytochrome
c from mitochondria and activation of apaf-1 (apoptosome), which in
turn cleaves the pro-enzyme of caspase-9 into the active form. Once
intiated caspase-9 goes on to cleave procaspase-3 &
procaspase-7 to trigger additional apoptotic pathways.
[0453] There is also a consistent linkage to the tumor necrosis
factor receptor family of proteins being modulated.
[0454] A strong down regulation of tumor protein p73 is also noted.
Analyses of many tumors typically found in humans including breast
and ovarian cancer show a high expression of p73 when compared to
normal tissues in corresponding areas. Recent finding are
suggesting that deregulated over expression of transcription
factors within the body involved in cell cycle regulation and
synthesis of DNA in mammalian cells (i.e.: E2F-1), induces the
expression of p73. The suggestion is that p73 may be an
oncoprotein, but may involve different mechanism that the related
p53 protein. A schematic showing mapping of the apoptosis pathway
is provided in FIG. 13.
SKMEL-28 Cells
[0455] From the survey of apoptotic related proteins it was
observed multiple pro- and anti-apoptotic factors were in the BCL-2
family or that interact with these factors have modulated
expression levels (BCL2L11, BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1).
These proteins govern mitochondrial outer membrane
permeabilization.
[0456] An early marker for apoptotic response is observed with the
upregulation of Caspase-9 (16 hour) which is consistent with
previous observations of apoptosis with caspase 3/7 proteins.
Induction of stress signaling pathways causes release of cytochrome
c from mitochondria and activation of apaf-1 (apoptosome), which in
turn cleaves the pro-enzyme of caspase-9 into the active form. Once
intiated caspase-9 goes on to cleave procaspase-3 &
procaspase-7 to trigger additional apoptotic pathways.
TABLE-US-00010 TABLE 9 Changes in mRNA levels for SKMEL-28 cells
treated with 100 .mu.M A10, evaluated by RT-PCR arrays focused
around apoptotic pathways. 6 hr 16 hr 24 hr 72 hr Refseq
Description Symbol Q10 Q10 Q10 Q10 NM_006538 BCL2-like 11 BCL2L11
2.13 2.41 1.92 2.51 (apoptosis facilitator) NM_000875 Insulin-like
growth IGF1R 1.77 1.09 1.33 1.25 factor 1 receptor NM_004048
Beta-2-microglobulin B2M 1.74 1.76 1.58 3.11 NM_003921 B-cell
CLL/lymphoma 10 BCL10 1.55 1.87 1.48 -3.11 NM_004330
BCL2/adenovirus E1B BNIP2 1.46 1.51 1.57 -1.61 19 kDa interacting
protein 2 NM_005157 C-abl oncogene 1, ABL1 1.42 2.77 -1.22 -2.03
receptor tyrosine kinase NM_004323 BCL2-associated BAG1 1.41 1.44
-1.61 -2.45 athanogene NM_001229 Caspase 9, apoptosis- CASP9 1.32
3.96 1.83 1.14 related cysteine peptidase NM_003806 Harakiri, BCL2
HRK 1.18 4.52 2.73 -1.14 interacting protein (contains only BH3
domain) NM_001924 Growth arrest and GADD45A 1.07 3.34 1.13 -2.36
DNA-damage- inducible, alpha NM_001188 BCL2- BAK1 1.06 2.73 -1.00
-4.54 antagonist/killer 1 NM_004295 TNF receptor- TRAF4 -1.91 2.63
-1.58 -740.66 associated factor 4 NM_003842 Tumor necrosis factor
TNFRSF10B -2.07 1.53 -1.81 -710.49 receptor superfamily, member 10b
NM_000633 B-cell CLL/lymphoma 2 BCL2 -2.98 -1.63 -2.82 -11.36
NM_001242 CD27 molecule CD27 -3.40 -2.38 -1.35 -12.72 NM_014430
Cell death-inducing CIDEB -3.48 1.56 -3.69 -2.59 DFFA-like effector
b NM_001065 Tumor necrosis factor TNFRSF1A -4.53 2.28 -3.30 1.22
receptor superfamily, member 1A NM_005427 Tumor protein p73 TP73
-4.66 -9.80 -8.71 -26.96 NM_003844 Tumor necrosis factor TNFRSF10A
-4.84 -5.26 -4.33 -11.84 receptor superfamily, member 10a NM_138578
BCL2-like 1 BCL2L1 -4.94 -1.80 -6.17 -7.04 NM_001165 Baculoviral
IAP BIRC3 -13.68 -1.98 -2.42 -3.42 repeat-containing 3
[0457] There is a consistent linkage to the tumor necrosis factor
receptor family of proteins being modulated.
[0458] A strong down regulation of tumor protein p73 is also noted.
Analyses of many tumors typically found in humans including breast
and ovarian cancer show a high expression of p73 when compared to
normal tissues in corresponding areas. Recent finding are
suggesting that deregulated over expression of transcription
factors within the body involved in cell cycle regulation and
synthesis of DNA in mammalian cells (i.e.: E2F-1), induces the
expression of p73. The suggestion is that p73 may be an
oncoprotein, but may involve different mechanism that the related
p53 protein
Experiment 2: Real-Time PCR Arrays Using Oxidative Stress and
Antioxidant Defense Array
[0459] To identify proteins that were involved in the Q10 response,
real-time polymerase chain reaction (RT-PCR) methods were employed
to identify changes in the level of mRNA's for genes/proteins
involved in targeted pathway arrays for oxidative stress and
antioxidant defense.
[0460] Table 10 below lists the genes that are regulated in
SK-MEL28 cells with 100 .mu.M Q10 treatment. Results are given only
for those genes that are regulated in two independent experiments.
Although there is a significant amount of gene regulation seen at 6
hours, most significant changes in RNA levels are seen at 48
hours.
TABLE-US-00011 TABLE 10 Genes in SK-MEL-28 cells that are regulated
by 100 .mu.M Q10 treatement as seen in the Oxidative Stress and
Antioxidant Defense Arrays. Symbol Description Regulation Location
Possible Functions. ALB Albumin Down Regulation Extracellular
Carrier protein, anti-apoptotic at 48 hours space AOX1 Aldehyde
oxidase 1 Up regulation Cytoplasm Produces free radicals, drug from
16 hours metabolic process. APOE Apolipoprotein E Down Regulation
Extracellular Lipid metabolism at 48 hours space ATOX1 ATX1
antioxidant protein Down Regulation Cytoplasm Copper metabolism 1
homolog (yeast) at 48 hours BNIP3 BCL2/adenovirus E1B Down
Regulation Cytoplasm Anti-apoptotic 19 kDa interacting protein 3 at
48 hours CSDE1 Cold shock domain containing Down Regulation
Cytoplasm Transcriptional regulation. E1, RNA-binding at 48 hours
CYBA Cytochrome b-245, alpha Down Regulation Cytoplasm Apoptotic,
polypeptide at 48 hours CYGB Cytoglobin Down Regulation Cytoplasm
Peroxidase, Transporter. at 48 hours DHCR24 24-dehydrocholesterol
Down Regulation Cytoplasm Electron carrier, binds to TP53,
reductase at 6 hours involved in apoptosis. DUOX1 Dual oxidase 1 Up
Regulation at Plasma Calcium ion binding, electron 48 hours
Membrane carrier. DUOX2 Dual oxidase 2 Down Regulation Unknown
Calcium ion binding. at 48 hours EPHX2 Epoxide hydrolase 2, Down
Regulation Cytoplasm Arachidonic acide metabolism. cytoplasmic at
48 hours EPX Eosinophil peroxidase Down Regulation Cytoplasm Phenyl
alanine metabolism, at 48 hours apoptosis. GPX2 Glutathione
peroxidase 2 Down Regulation Cytoplasm Electron carrier, binds to
TP53, (gastrointestinal) at 48 hours involved in apoptosis. GPX3
Glutathione peroxidase 3 Up Regulation at Extracellular Arachidonic
acid metabolims, (plasma) 48 hours space up regulated in
carcinomas. GPX5 Glutathione peroxidase 5 Up Regulation at
Extracellular Arachidonic acid metabolism. (epididymal androgen- 48
hours space related protein) GPX6 Glutathione peroxidase 6 Down
Regulation Extracellular Arachidonic acid metabolism. (olfactory)
at 48 hours space GSR Glutathione reductase Down Regulation
Cytoplasm Glutamate and glutathione at 48 hours metabolism,
apoptosis. GTF2I General transcription Down Regulation Nucleus
Transcriptional activator, factor II, i at 6 hours transcription of
fos. KRT1 Keratin 1 (epidermolytic Up Regulation at Cytoplasm Sugar
Binding. hyperkeratosis) 48 hours LPO Lactoperoxidase Down
Regulation Extracellular Phenyl alanine metabolism. at 48 hours
space MBL2 Mannose-binding lectin Down Regulation Extracellular
Complement signaling, pattern (protein C) 2, soluble at 48 hours
space recognition in receptors. (opsonic defect) MGST3 Microsomal
glutathione Upregulation at Cytoplasm Xenobiotic metabolism.
S-transferase 3 16 hours MPO Myeloperoxidase Down Regulation
Cytoplasm Anti-apoptotic, phenyl alanine at 48 hours metabolism.
MPV17 MpV17 mitochondrial Down Regulation Cytoplasm Maintenance of
mitochondrial DNA. inner membrane protein at 6 hours MT3
Metallothionein 3 Down Regulation Cytoplasm Copper ion binding. at
48 hours NCF1 Neutrophil cytosolic factor Down Regulation Cyoplasm
Produces free radicals. 1, (chronic granulomatous from 6 hours
disease, autosomal 1) NCF2 Neutrophil cytosolic factor Up
Regulation at Cytoplasm Electron carrier. 2 (65 kDa, chronic 48
hours granulomatous disease, autosomal 2) NME5 Non-metastatic cells
5, Down Regulation Unknown Kinase, Purine and pyrimidine protein
expressed in at 48 hours metabolism. (nucleoside-diphosphate
kinase) NOS2A Nitric oxide synthase 2A Down Regulation Cytoplasm
Glucocorticoid receptor (inducible, hepatocytes) at 48 hours
signaling, apoptosis. OXR1 Oxidation resistance 1 Down Regulation
Cytoplasm Responds to oxidative stress. at 48 hours PDLIM1 PDZ and
LIM domain 1 Up Regulation at Cytoplasm Transcriptional activator.
(elfin) 48 hours PIP3-E Phosphoinositide-binding Down Regulation
Cytoplasm Peroxidase. protein PIP3-E at 48 hours PRDX2
Peroxiredoxin 2 Down Regulation Cytoplasm Role in phenyl alanine at
6 hours metabolism. Role in cell death. PRDX4 Peroxiredoxin 4 Down
Regulation Cytoplasm Thioredoxin peroxidase. from 24 hours PREX1
Phosphatidylinositol 3,4,5- Down Regulation Cytoplasm Forms oxygen
free radicals. trisphosphate-dependent at 48 hours RAC exchanger 1
PRG3 Proteoglycan 3 Down Regulation Extracellular Role in cell
death. at 48 hours space PTGS1 Prostaglandin- Down Regulation
Cytoplasm arachidonic acid metabolism, endoperoxide synthase 1 at
48 hours prostaglandin synthesis. (prostaglandin G/H synthase and
cyclooxygenase) PTGS2 Prostaglandin- Up Regulation at Cytoplasm
arachidonic acid metabolism, endoperoxide synthase 2 48 hours
prostaglandin synthesis. (prostaglandin G/H synthase and
cyclooxygenase) PXDN Peroxidasin homolog Up Regulation at Unknown
binds to TRAF4, calcium ion (Drosophila) 48 hours binding, iron ion
binding. PXDNL Peroxidasin homolog Down Regulation Unknown
peroxidase, calcium ion (Drosophila)-like at 48 hours binding, iron
ion binding. RNF7 Ring finger protein 7 Up Regulation at Nucleus
apoptotic, copper ion binding, 16 hours ubiquitin pathway. SGK2
Serum/glucocorticoid Down Regulation Cytoplasm Kinase, potasium
channel regulated kinase 2 at 48 hours regulator. SIRT2 Sirtuin
(silent mating type Up regulation at Nucleus Transcription factor.
information regulation 2 16 hours homolog) 2 (S. cerevisiae) SOD1
Superoxide dismutase 1, Up Regulation at Cytoplasm Apoptotic,
Caspase Activator. soluble (amyotrophic 16 hours lateral sclerosis
1 (adult)) SOD2 Superoxide dismutase 2, Up regulation at Cytoplasm
Apoptotic, Regulated by TNF. mitochondrial 16 hours SOD3 Superoxide
dismutase 3, Down Regulation Extracellular Pro-apoptotic
extracellular at 48 hours space SRXN1 Sulfiredoxin 1 homolog Down
Regulation Cytoplasm DNA binding, oxidoreductase (S. cerevisiae) at
48 hours TPO Thyroid peroxidase Down Regulation Plasma iodination
of thyroglobulin, at 48 hours Membrane tyrosine metabolism,
phenylalanine metabolism. TTN Titin Down Regulation Cytoplasm Actin
cytoskeleton signaling, at 48 hours integrin signaling TXNDC2
Thioredoxin domain- Down Regulation Cytoplasm Pyrimidine metabolism
containing 2 at 48 hours (spermatozoa)
[0461] The Neutrophil cytosolic factor 2 (NCF2, 65 kDa, chronic
granulomatous disease, autosomal 2) was one of the initial top
induced mRNA's (observed at 6 hours). Subsequently at the 16 hour
time point and onward, Neutrophil cytosolic factor 1 (NCF1)
(chronic granulomatous disease, autosomal 1) was induced at very
high levels after an initial lag phase.
[0462] Neutrophil cytosolic factor 2 is the cytosolic subunit of
the multi-protein complex known as NADPH oxidase commonly found in
neutrophils. This oxidase produces a burst of superoxide which is
delivered to the lumen of the neutrophil phagosome.
[0463] The NADPH oxidase (nicotinamide adenine dinucleotide
phosphate-oxidase) is a membrane-bound enzyme complex. It can be
found in the plasma membrane as well as in the membrane of
phagosome. It is made up of six subunits. These subunits are: a Rho
guanosine triphosphatase (GTPase), usually Rac1 or Rac2 (Rac stands
for Rho-related C3 botulinum toxin substrate) [0464] Five "phox"
units. (Phox stands for phagocytic oxidase.) [0465] P91-PHOX
(contains heme) [0466] p22phox [0467] p40phox [0468] p47phox (NCF1)
[0469] p67phox (NCF2)
[0470] It is noted that another NADPH oxidase levels do not change.
The enzyme is NOX5, which is a novel NADPH oxidase that generates
superoxide and functions as a H+ channel in a Ca(2+)-dependent
manner
[0471] In addition Phosphatidylinositol
3,4,5-trisphosphate-dependent RAC exchanger 1 (PREX1) was also
upregulated. This protein acts as a guanine nucleotide exchange
factor for the RHO family of small GTP-binding proteins (RACs). It
has been shown to bind to and activate RAC1 by exchanging bound GDP
for free GTP. The encoded protein, which is found mainly in the
cytoplasm, is activated by phosphatidylinositol-3,4,5-trisphosphate
and the beta-gamma subunits of heterotrimeric G proteins.
[0472] The second major early induced protein was Nitric oxide
synthase 2A (inducible, hepatocytes) (NOS2A). Nitric oxide is a
reactive free radical which acts as a biologic mediator in several
processes, including neurotransmission and antimicrobial and
antitumoral activities. This gene encodes a nitric oxide synthase
which is expressed in liver and is inducible by a combination of
lipopolysaccharide and certain cytokines.
[0473] Superoxide dismutase 2, mitochondrial (SOD2) is a member of
the iron/manganese superoxide dismutase family. It encodes a
mitochondrial protein that forms a homotetramer and binds one
manganese ion per subunit. This protein binds to the superoxide
byproducts of oxidative phosphorylation and converts them to
hydrogen peroxide and diatomic oxygen. Mutations in this gene have
been associated with idiopathic cardiomyopathy (IDC), premature
aging, sporadic motor neuron disease, and cancer.
[0474] An example of a down regulated protein is Forkhead box M1
(FOXM1), which is known to play a key role in cell cycle
progression where endogenous FOXM1 expression peaks at S and G2/M
phases. Recent studies have shown that FOXM1, regulates expression
of a large array of G2/M-specific genes, such as Plk1, cyclin B2,
Nek2 and CENPF, and plays an important role in maintenance of
chromosomal segregation and genomic stability. The FOXM1 gene is
now known as a human proto-oncogene. Abnormal upregulation of FOXM1
is involved in the oncogenesis of basal cell carcinoma (BCC). FOXM1
upregulation was subsequently found in the majority of solid human
cancers including liver, breast, lung, prostate, cervix of uterus,
colon, pancreas, and brain. Further studies with BCC and Q10 should
evaluate FOXM1 levels.
[0475] SKMEL-28 Cells
[0476] Further experiments were carried out using SKMEL-28 cells.
The level of mRNA present in SKMEL-28 cells treated with 100 .mu.M
Q10 were compared to the levels in untreated cells at various time
points using real-time PCR methods (RT-PCR). The PCR array
(SABiosciences) is a set of optimized real-time PCR primer assays
on 96-well plates for pathway or disease focused genes as well as
appropriate RNA quality controls. The PCR array performs gene
expression analysis with real-time PCR sensitivity and the
multi-gene profiling capability of a microarray.
TABLE-US-00012 TABLE 11 Listing and classification of mRNA levels
evaluated in the Oxidative Stress and Antioxidant Defense PCR
Array. After six hours of treatment with 100 .mu.M Q10 on SKMEL-28
cells, the largest changes to the mRNA levels are indicated by
highlighting the protein code (increased--bold;
decreased--underlined; or no change--grey). ##STR00004##
TABLE-US-00013 TABLE 12 Time course evaluation of 100 .mu.M
treatment of SKMEL-28. 6 hr 16 hr 24 hr 48 hr 72 hr Refseq Symbol
Description Q10 Q10 Q10 Q10 Q10 NM_000265 NCF1 Neutrophil cytosolic
factor 0 high 3.3829 15.7838 31.5369 1, (chronic granulomatous
disease, autosomal 1) NM_012423 RPL13A Ribosomal protein L13a
-0.9025 3.1857 2.5492 4.9253 7.82 NM_020820 PREX1
Phosphatidylinositol -3.2971 2.867 0.3222 6.3719 7.476
3,4,5-trisphosphate- dependent RAC exchanger 1 NM_012237 SIRT2
Sirtuin (silent mating type -0.9025 4.0829 4.4766 5.7166 6.6257
information regulation 2 homolog) 2 (S. cerevisiae) NM_005125 CCS
Copper chaperone for -0.6206 3.0077 3.452 2.9801 6.1539 superoxide
dismutase NM_181652 PRDX5 Peroxiredoxin 5 -2.995 3.0454 3.5381
4.7955 6.0169 NM_016276 SGK2 Serum/glucocorticoid 0 0 0 0.5995
5.937 regulated kinase 2 NM_003551 NME5 Non-metastatic cells 5,
-0.6652 3.1138 3.3694 3.1549 5.782 protein expressed in
(nucleoside-diphosphate kinase) NM_004417 DUSP1 Dual specificity
-0.6998 0.5902 2.7713 3.321 5.5375 phosphatase 1 NM_001752 CAT
Catalase -0.8589 2.8424 0.1046 3.8557 5.3988 NM_000041 APOE
Apolipoprotein E -0.8212 3.2069 -0.9543 3.7694 5.3315 NM_000101
CYBA Cytochrome b-245, alpha -0.3945 4.3475 3.9208 6.2452 5.0762
polypeptide NM_000433 NCF2 Neutrophil cytosolic factor 1.2266
3.0077 0.0954 5.476 0 2 (65 kDa, chronic granulomatous disease,
autosomal 2) NM_000963 PTGS2 Prostaglandin-endoperoxide -0.6912
2.7046 2.6552 4.0553 -3.3022 synthase 2 (prostaglandin G/H synthase
and cyclooxygenase) NM_183079 PRNP Prion protein (p27-30) -0.2144
3.5236 2.9086 5.0837 -3.9396 (Creutzfeldt-Jakob disease,
Gerstmann-Strausler-Scheinker syndrome, fatal familial insomnia)
NM_004052 BNIP3 BCL2/adenovirus E1B -2.9376 3.3288 4.312 -18.2069
-4.8424 19 kDa interacting protein 3 NM_000242 MBL2 Mannose-binding
lectin -0.3622 -1.9072 -3.0142 -1.1854 -6.4544 (protein C) 2,
soluble (opsonic defect) NM_021953 FOXM1 Forkhead box M1 -0.8135
0.068 -0.9216 3.3655 -10.0953 The mRNA level changes were monitored
by RT-PCR methods and oxidative stress and antioxidant defense
proteins array was evaluated.
[0477] The Neutrophil cytosolic factor 2 (NCF2, 65 kDa, chronic
granulomatous disease, autosomal 2) was one of the initial top
induced mRNA's (observed at 6 hours). Subsequently at the 16 hour
time point and onward, Neutrophil cytosolic factor 1 (NCF1)
(chronic granulomatous disease, autosomal 1) was induced at very
high levels after an initial lag phase.
[0478] Neutrophil cytosolic factor 2 is the cytosolic subunit of
the multi-protein complex known as NADPH oxidase commonly found in
neutrophils. This oxidase produces a burst of superoxide which is
delivered to the lumen of the neutrophil phagosome. The NADPH
oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) is a
membrane-bound enzyme complex. It can be found in the plasma
membrane as well as in the membrane of phagosome. It is made up of
six subunits. These subunits are:
[0479] a Rho guanosine triphosphatase (GTPase), usually Rac1 or
Rac2 (Rac stands for Rho-related C3 botulinum toxin substrate)
[0480] Five "phox" (phagocytic oxidase) units. [0481] P91-PHOX
(contains heme) [0482] p22phox [0483] p40phox [0484] p47phox (NCF1)
[0485] p67phox (NCF2)
[0486] It is noted that another NADPH oxidase levels do not change.
The enzyme is NOX5, which is a novel NADPH oxidase that generates
superoxide and functions as a H+ channel in a Ca(2+)-dependent
manner
[0487] In addition Phosphatidylinositol
3,4,5-trisphosphate-dependent RAC exchanger 1 (PREX1) was also
upregulated. This protein acts as a guanine nucleotide exchange
factor for the RHO family of small GTP-binding proteins (RACs). It
has been shown to bind to and activate RAC1 by exchanging bound GDP
for free GTP. The encoded protein, which is found mainly in the
cytoplasm, is activated by phosphatidylinositol-3,4,5-trisphosphate
and the beta-gamma subunits of heterotrimeric G proteins.
[0488] The second major early induced protein was Nitric oxide
synthase 2A (inducible, hepatocytes) (NOS2A). Nitric oxide is a
reactive free radical which acts as a biologic mediator in several
processes, including neurotransmission and antimicrobial and
antitumoral activities. This gene encodes a nitric oxide synthase
which is expressed in liver and is inducible by a combination of
lipopolysaccharide and certain cytokines.
[0489] An example of a down regulated protein is FOXM1, which is
known to play a key role in cell cycle progression where endogenous
FOXM1 expression peaks at S and G2/M phases. Recent studies have
shown that FOXM1, regulates expression of a large array of
G2/M-specific genes, such as Plk1, cyclin B2, Nek2 and CENPF, and
plays an important role in maintenance of chromosomal segregation
and genomic stability. The FOXM1 gene is now known as a human
proto-oncogene. Abnormal upregulation of FOXM1 is involved in the
oncogenesis of basal cell carcinoma (BCC). FOXM1 upregulation was
subsequently found in the majority of solid human cancers including
liver, breast, lung, prostate, cervix, uterus, colon, pancreas, and
brain.
Experiment 3: Real-Time PCR Arrays Using Heat Shock Array
[0490] Heat Shock Arrays were run for SCC cells and the data of
regulated genes is summarized below in Table 13.
TABLE-US-00014 TABLE 13 Genes from the Heat Shock Protein array
regulated with 100 .mu.M Q10 treatment in SCC cells. Symbol
Description Regulation. Location. Possible functions. CCT6B
Chaperonin Down regulated Cytoplasm Protein folding and protein
containing TCP1, at 24 hours complex assembly. subunit 6B (zeta 2)
DNAJA1 DnaJ (Hsp40) Up regulated at Nucleus Responds to DNA damage
homolog, subfamily 6 hours. and changes in protein A, member 1
folding. DNAJB13 DnaJ (Hsp40) Down regulated Unknown Protein
folding and apoptosis. related, subfamily at 6 hours. B, member 13
DNAJB5 DnaJ (Hsp40) Down regulated Unknown Binds to HSP, involved
in homolog, subfamily at 6 hours. protein folding and in protein B,
member 5 complex assembly. DNAJC12 DnaJ (Hsp40) Down regulated
Unknown Binds to HSP, involved in homolog, subfamily at 6 hours.
protein folding and in protein C, member 12 complex assembly.
DNAJC4 DnaJ (Hsp40) Down regulated Cytoplasm Binds to HSP, involved
in homolog, subfamily at 6 hours. protein folding and in protein C,
member 4 complex assembly. DNAJC5B DnaJ (Hsp40) Down regulated
Unknown Involved in protein folding homolog, subfamily at 6 hours.
responds to changes in protein C, member 5 beta folding. HSPA8 Heat
shock 70 kDa Up regulated at Cytoplasm Regulates TNF, binds BAG1,
protein 8 6 hours. STUB1, TP53, involved in apoptosis. HSPH1 Heat
shock Up regulated at Cytoplasm Binds to HSPA8, important 105
kDa/110 kDa 6 hours. for protein folding, responds protein 1 to
protein unfolding and stress.
Experiment 4: Real-Time PCR Arrays Using Diabetes Array
[0491] The experiments described in this example were performed to
test the overall hypothesis that Q10 would have an impact on
multiple genes and alter the metabolic state of a cell. The mRNA
from SKMEL-28 cells treated with 100 .mu.M Q10 was evaluated by
RT-PCR against a panel of target proteins involved in diabetes and
related pathways. Results from this experiment demonstrate that
several proteins involved in glycolytic pathways and insulin
processing are altered in their mRNA expression levels (summarized
in Table 14).
TABLE-US-00015 TABLE 14 Major mRNA level changes to SKMEL-28 cells
treated with 100 .mu.M Q10 for 16 hours. Fold Change after 16 hours
Refseq Description Symbol (100 .mu.M Q10) NM_000162 Glucokinase
(hexokinase 4) GCK 8.5386 NM_178849 Hepatocyte nuclear factor 4,
alpha HNF4A 8.421 NM_005249 Forkhead box G1 FOXG1 4.6396 NM_000599
Insulin-like growth factor binding IGFBP5 2.2721 protein 5
NM_001101 Actin, beta ACTB -2.0936 NM_002863 Phosphorylase,
glycogen; liver PYGL -2.65 (Hers disease, glycogen storage disease
type VI) NM_001065 Tumor necrosis factor receptor TNFRSF1A -2.8011
superfamily, member 1A NM_021158 Tribbles homolog 3 (Drosophila)
TRIB3 -2.8011 NM_003749 Insulin receptor substrate 2 IRS2 -2.9404
NM_004578 RAB4A, member RAS oncogene family RAB4A -3.1296 NM_004176
Sterol regulatory element binding SREBF1 -3.5455 transcription
factor 1 NM_004969 Insulin-degrading enzyme IDE -4.4878 NM_005026
Phosphoinositide-3-kinase, PIK3CD -6.8971 catalytic, delta
polypeptide NM_000208 Insulin receptor INSR -8.6099 NM_003376
Vascular endothelial growth VEGFA -15.5194 factor A NM_001315
Mitogen-activated protein kinase 14 MAPK14 -74.3366
[0492] The results of this initial experiment show that the mRNA
levels for a variety of insulin related proteins were modulated in
both directions. The results indicate that Q10 would have an impact
on diabetic disease treatment and/or evaluation.
[0493] Further experiments were next conducted to confirm the
results above obtained from SK-MEL-28 cells treated with Q10. Many
of the genes in SK-MEL-28 cells are regulated as early as 6 hours
after Q10 treatment. However, the initial regulation becomes less
evident by 16 and 24 hours. Around 48 hours, we find that many of
the genes in the Diabetes array are again strongly regulated.
Results that are consistent from two or more or independent
experiments are summarized below in Table 15. SCC cells also
appeared to exhibit regulation in some genes, both at 6 and 24
hours after Q10 treatment. These results from SCC cells are
summarized in Table 16 while genes that are regulated both in
SK-MEL-28 cells and in SCC cells are summarized in Table 17.
TABLE-US-00016 TABLE 15 Genes in SK-MEL-28 cells regulated by 100
.mu.M Q10 treatment when analyzed by the Diabetes Array. Symbol
Description Regulation. Location Possible Function ADRB3
Adrenergic, beta-3-, receptor Down Regulated at Plasma cAMP
signaling, 48 hours membrane G-protein signaling CEACAM1
Carcinoembryonic antigen- Down Regulated at Extracellular
Anti-apoptotic, related cell adhesion 48 hours space positive
regulation of molecule 1 (biliary angiogenesis. glycoprotein) CEBPA
CCAAT/enhancer binding Up regulated at Nucleus Glucocorticoid
protein (C/EBP), alpha 48 hours receptor signaling, VDR/RXR
activation. CTLA4 Cytotoxic T-lymphocyte- Down Regulated at Plasma
T cell receptor associated protein 4 48 hours Membrane signaling,
activates CASP8. DUSP4 Dual specificity phosphatase 4 Down
Regulated at Nucleus Phosphatase 48 hours ENPP1 Ectonucleotide Down
Regulated at Plasma Negative regulator of pyrophosphatase/ 48 hours
membrane the insulin receptor phosphodie sterase 1 pathway FOXC2
Forkhead box C2 (MFH-1, Down Regulated at Nucleus Anti-apoptotic,
mesenchyme forkhead 1) 48 hours transcription factor G6PD
Glucose-6-phosphate Up regulated at Cytoplasm Pentose Phosphate
dehydrogenase 48 hours, then Pathway, Glutathione down regulated
metabolism. HMOX1 Heme oxygenase (decycling) 1 Down Regulated at
Cytoplasm Heme oxygenase 48 hours decycling ICAM1 Intercellular
adhesion Down Regulated at Plasma Regulated by molecule 1 (CD54),
human 48 hours membrane atorvastatin, rhinovirus receptor processes
some caspases. IL4R Interleukin 4 receptor Down Regulated at Plasma
Up regulation by 48 hours membrane TP73, binds to IRS1 and IRS2
IRS1 Insulin receptor substrate 1 Up regulated at Plasma Binds
Insulin 48 hours then membrane receptor down regulated IRS2 Insulin
receptor substrate 2 Down Regulated at Plasma IGF-1 signaling 48
hours membrane NSF N-ethylmaleimide-sensitive Down Regulated at
Cytoplasm GABA signaling factor 48 hours PIK3CD
Phosphoinositide-3-kinase, Down Regulated at Cytoplasm Kinase
catalytic, delta polypeptide 48 hours PPARG Peroxisome
proliferator- Down Regulated at Nucleus Transcriptional factor
activated receptor gamma 48 hours PRKCB1 Protein kinase C, beta 1
Down Regulated at Cytoplasm PKC family 48 hours SELL Selectin L
(lymphocyte Down Regulated at Plasma Activates RAS, adhesion
molecule 1) 48 hours membrane MAPK SREBF1 Sterol regulatory element
Up regulated at Nucleus Transcriptional factor binding
transcription factor 1 48 hours then down regulated STXBP1 Syntaxin
binding protein 1 Down Regulated at Cytoplasm Present in myelin 48
hours enriched fraction. TGFB1 Transforming growth factor, Up
regulated at Extracellular Pro-apoptotic beta 1 48 hours then down
space regulated NKX2-1 NK2 homeobox 1 Down Regulated at Nucleus
Transcriptional 48 hours activator TNF Tumor necrosis factor (TNF
Up regulated at Extracellular Pro-apoptotic superfamily, member 2)
48 hours space TNFRSF1A Tumor necrosis factor Down Regulated at
Plasma Pro-apoptotic receptor superfamily, 72 hours membrane member
1A VEGFA Vascular endothelial growth Up regulated at Cytoplasm
Kinase factor A 58 hours then down regulated
TABLE-US-00017 TABLE 16 Genes in SCC cells regulated by 100 .mu.M
Q10 treatment when analyzed by the Diabetes Array. Symbol
Description Regulation. G6PD Glucose-6-phosphate dehydrogenase Down
regulated at 6 hours. ICAM1 Intercellular adhesion molecule 1 Down
regulated at (CD54), human rhinovirus receptor 6 hours. INPPL1
Inositol polyphosphate Down regulated at phosphatase-like 1 6
hours. NOS3 Nitric oxide synthase 3 Down regulated at (endothelial
cell) 6 hours. PIK3CD Phosphoinositide-3-kinase, Down regulated at
catalytic, delta polypeptide 6 hours. PPARA Peroxisome
proliferative activated Down regulated at receptor, alpha 6 hours.
PYGL Phosphorylase, glycogen; liver Down regulated at (Hers
disease, glycogen storage 6 hours. disease type VI) SREBF1 Sterol
regulatory element binding Down regulated at transcription factor 1
6 hours. STXBP2 Syntaxin binding protein 2 Down regulated at 6
hours. TNF Tumor necrosis factor (TNF Down regulated at
superfamily, member 2) 6 hours. TNFRSF1A Tumor necrosis factor
receptor Down regulated at superfamily, member 1A 6 and 24 hours.
VEGFA Vascular endothelial growth Down regulated at factor A 6
hours.
TABLE-US-00018 TABLE 17 Genes from the diabetes array regulated
with 100 .mu.M Q10 treatment for both SK-MEL-28 and SCC cells.
Symbol Description. G6PD Glucose-6-phosphate dehydrogenase ICAM1
Intercellular adhesion molecule 1 (CD54), human rhinovirus receptor
PIK3CD Phosphoinositide-3-kinase, catalytic, delta polypeptide
SREBF1 Sterol regulatory element binding transcription factor 1 TNF
Tumor necrosis factor (TNF superfamily, member 2) TNFRSF1A Tumor
necrosis factor receptor superfamily, member 1A VEGFA Vascular
endothelial growth factor A
[0494] The mRNA levels for a variety of insulin related proteins
were modulated in both directions. Q10 has an impact on regulation
of cellular metabolism, and thus influences metabolic disregulation
diseases such as diabetes. Two proteins that were significantly
modulated are further discussed below.
[0495] Mitogen-activated protein kinase 14 (MAPK14):
Mitogen-activated protein kinase 14 (MAPK14) is a member of the MAP
kinase family. MAP kinases act as an integration point for multiple
biochemical signals, and are involved in a wide variety of cellular
processes such as proliferation, differentiation, transcription
regulation and development. Results from this experiment show that
the MAPK14 was significantly down-regulated.
[0496] Hepatocyte nuclear factor 4, alpha (HNF4A): HNF4 (Hepatocyte
Nuclear Factor 4) is a nuclear receptor protein mostly expressed in
the liver, gut, kidney, and pancreatic beta cells that is critical
for liver development. In humans, there are two isoforms of NHF4,
alpha and gamma encoded by two separate genes HNF4A and HNF4G
respectively. (See, e.g., Chartier F L, Bossu J P, Laudet V,
Fruchart J C, Laine B (1994). "Cloning and sequencing of cDNAs
encoding the human hepatocyte nuclear factor 4 indicate the
presence of two isoforms in human liver". Gene 147 (2):
269-72.)
[0497] HNF4 was originally classified as an orphan receptor.
However HNF4 was found later to be constitutively active by virtue
of being continuously bound to a variety of fatty acids. (See,
e.g., Sladek F (2002). "Desperately seeking . . . something". Mol
Cell 10 (2): 219-221 and Jump D B, Botolin D, Wang Y, Xu J,
Christian B, Demeure O (2005). "Fatty acid regulation of hepatic
gene transcription". J Nutr 135 (11)). The ligand binding domain of
HNF4, as with other nuclear receptors, adopts a canonical alpha
helical sandwich fold (see, e.g., Wisely G B, Miller A B, Davis R
G, Thornquest A D Jr, Johnson R, Spitzer T, Sefler A, Shearer B,
Moore J T, Miller A B, Willson T M, Williams S P (2002).
"Hepatocyte nuclear factor 4 is a transcription factor that
constitutively binds fatty acids". Structure 10 (9): 1225-34 and
Dhe-Paganon S, Duda K, Iwamoto M, Chi Y I, Shoelson S E (2002).
"Crystal structure of the HNF4 alpha ligand binding domain in
complex with endogenous fatty acid ligand". J Biol Chem 277 (41):
37973-6) and interacts with co-activator proteins. (See, e.g., Duda
K, Chi Y I, Shoelson S E (2004). "Structural basis for HNF-4alpha
activation by ligand and coactivator binding". J Biol Chem 279
(22): 23311-6).
[0498] Mutations in the HNF4-a gene have been linked to maturity
onset diabetes of the young (MODY). (See, e.g., Fajans S S, Bell G
I, Polonsky K S (2001). "Molecular mechanisms and clinical
pathophysiology of maturity-onset diabetes of the young". N Engl J
Med 345 (13): 971-80.)
[0499] Hepatocyte nuclear factor 4 (HNF4) is a tissue-specific
transcription factor known to regulate a large number of genes in
hepatocytes and pancreatic cells. Although HNF4 is highly expressed
in some sections of the kidney, little is known about its role in
this organ and about HNF4-regulated genes in the kidney cells. The
abundance and activity of HNF4 are frequently reduced in renal cell
carcinoma (RCC) indicating some tumor suppressing function of HNF4
in renal cells. Interestingly, many of the genes regulated by HNF4
have been shown to be deregulated in RCC microarray studies. These
genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2,
ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for
genes whose activity is changed upon the decrease of HNF4 in
RCC.
[0500] In the structure of the ligand binding domain of HNF4alpha
(1M7W.pdb; Dhe-Paganon (2002) JBC, 277, 37973); a small lipid was
observed and which co-purified from E. coli production. The crystal
contains two conformations of the protein, where the elongated
helix 10 and short helix 12 have alternate conformations. Upon
examination of the lipid binding region, it was interesting to
observe that there are two exits regions. One exit region holds the
small lipids head group, and it is noted that several pocket
regions are co-localized with this exit port. A hypothesis would be
that Q10 binds specifically to this transcription factor. When Q10
in modeled into this lipid binding tunnel, the Q10 ring would fit
into the surface pocket (FIG. 28). A known loss-of-function
mutation (E276Q) would have the potential to order the residues
lining this surface pocket, and thus have a negative impact on the
putative Q10 binding.
[0501] In addition, with this Q10 binding model, the hydrophobic
tail would extend out of the internal cavity and would then
interact with the elongated helix 10. Thus, this interaction could
potential alter the conformation of the helix 10/12 group. This may
then alter the activation/inactivation equilibrium of the
transcription factor activity.
Example 7
Antibody MicroArray Analysis
[0502] The evaluation of protein concentration due to the presence
of Q10 was evaluated through the utilization of antibody microarray
methods. The microarray contained antibodies for over 700 proteins,
sampling a broad range of protein types and potential pathway
markers.
[0503] An initial experiment to assess changes at the protein
concentration level in cells treated with Q10 was conducted with an
antibody microarray (Panorama XP725 Antibody Array, Sigma) and
SK-MEL-28 cells treated for 6 or 24 hour. The cells were harvested
and extracted to obtain a soluble protein supernatant. Two portions
of protein (.about.1 mg total) from each sample (at 1 mg/mL) were
each label with fluorescent dye (Cy3 and Cy5, respectively). The
excess dye was removed from the protein and the material utilized
for the microarray incubations. To compare two time point samples,
equal amounts of protein were mixed, with each sample being of the
different label type (e.g., 3 hour extract labeled with Cy3 was
mixed with the 24 hour extract labeled with Cy5). After incubation
with the microarray chip (according to manufactures recommended
protocols), the chips were washed and dried. The microarrays were
scanned with a fluorescent laser scanner to measure the relative
fluorescence intensity of the Cy3 and Cy5 dyes.
TABLE-US-00019 TABLE 18 Proteins with increased levels in SK-MEL-28
cells after 24 hour treatment with 50 .mu.M Q10 Name Ratio Cdk1 0.1
DcR1 0.1 Protein Kinase Cb2 0.1 Tumor Necrosis Factor Soluble 0.1
Receptor II BAD 0.1 Caspase13 0.2 FBI1 PAKEMON 0.2 Zyxin 0.2 Cdc25A
0.3 PIASx 0.3 Nerve Growth Factor b 0.3 Protein Tyrosine
Phosphatase 0.3 PEST hBRM hSNF2a 0.4 GRP94 0.4 Calmodulin 0.4
Serine Threonine Protein 0.4 Phosphatase 2C a b ARC 0.4 NeurabinII
0.4 Nitric Oxide Synthase bNOS 0.4 Serine Threonine Protein 0.4
Phosphatase 1b Heat Shock Protein 110 0.4 Serine Threonine Protein
0.4 Phosphatase 1g1 COX II 0.5 HSP70 0.5 BLK 0.5 Cytokeratin 8 12
0.5 BUBR1 0.5 FOXC2 0.5 Serine Threonine Protein 0.5 Phosphatase 2
A Bg MSH6 0.5 DR6 0.5 Rad17 0.5 BAF57 0.5 Transforming Growth 0.5
Factorb pan BTK 0.5 SerineThreonine Protein 0.5 Phosphatase 2 A/B
pan2 CNPase 0.5 SynCAM 0.5 Proliferating Cell Nuclear 0.5
Antigen
TABLE-US-00020 TABLE 19 Proteins with increased levels in SK-MEL-28
cells after 24 hour treatment with 50 .mu.M Q10 Name Ratio BclxL
4.2 BID 3.7 Bmf 3.7 PUMA bbc3 3.0 Zip Kinase 2.8 Bmf 2.8 DcR2 2.7
E2F1 2.7 FAK pTyr577 2.5 FKHRL1 FOXO3a 2.5 MTBP 2.5 Connexin 32 2.5
Annexin VII 2.4 p63 2.4 SUMO1 2.4 IAfadin 2.3 MDMX 2.3 Pyk2 2.3 RIP
Receptor Interacting Protein 2.3 RICK 2.3 IKKa 2.3 Bclx 2.3 Afadin
2.2 Proliferating Cell Protein Ki67 2.2 Histone H3 pSer28 2.2 CASK
LIN2 2.2 Centrin 2.2 TOM22 2.1 Nitric Oxide Synthase Endothelial
2.1 eNOS Protein Kinase Ba 2.1 Laminin 2.1 Myosin Ib Nuclear 2.1
Caspase 7 2.1 MAP Kinase 2 ERK2 2.1 KIF17 2.1 Claspin 2.1 GRP75 2.1
Caspase 6 2.1 ILP2 2.1 aActinin 2.1 Vitronectin 2.1 DRAK1 2.1 PTEN
2.1 Grb2 2.1 HDAC4 2.0 HDAC7 2.0 Nitric Oxide Synthase bNOS 2.0
HDAC2 2.0 p38 MAPK 2.0 Reelin 2.0 Protein Kinase Cd 2.0 cerbB3 2.0
hSNF5 INI1 2.0 Protein Kinase Ca 2.0 Glutamate receptor NMDAR 2a
2.0 Leptin 2.0 Dimethyl Histone H3 diMeLys4 2.0 BID 2.0 MeCP2 2.0
Nerve growth factor receptor p75 2.0 Myosin Light Chain Kinase 2.0
cRaf pSer621 2.0 GRP78 BiP 2.0 cMyc 2.0 Raf1 2.0 MTA2 MTA1L 2.0
Sir2 2.0 ATF2 pThr69 71 2.0 Protein Kinase C 2.0 Protein Kinase Cb2
2.0
[0504] In order to confirm the previously observed apoptosis
proteins, and to expand the evaluation into a larger number of
pro-apoptosis and anti-apoptosis proteins, two assay methods were
chosen which were capable of screening the broad family of proteins
potentially involved.
[0505] First, an antibody micro array (Panorama XP725 Antibody
Array, Sigma) was utilized to screen over 700 protein antibodies to
assess changes at the protein concentration level in SK-MEL-28
cells treated for 24 hours with 50 .mu.M Q10.
[0506] From the Antibody array experiments, on SKMEL-28 with Q10
(24 hr), the following are some of the identified proteins with
altered levels: Bcl-xl, Bmf, BTK, BLK, cJun (pSer63), Connexin 32,
PUMA bbc3, BID, Par4, cCb1. The key conclusion from this initial
study was that the expected pro-apoptosis proteins are altered.
Antibody Microarray for SK-MEL-28
[0507] An antibody micro array (Panorama XP725 Antibody Array,
Sigma) was utilized to screen over 700 protein antibodies to assess
changes at the protein concentration level in SK-MEL-28 cells
treated for 24 hours with 50 .mu.M Q10.
TABLE-US-00021 TABLE 20 Changes in protein levels in SKMEL-28
treated with 50 .mu.M Q10 Antibody SKMEL28 Q1Q/ SKMEL28/ HEKa Q10/
Number SKMEL28 HEKa HEKa Name (Sigma) control control control BclxL
B9429 2.46 1.04 1.83 PUMA bbc3 P4743 2.31 1.14 2.14 Bmf B1559 2.23
1.12 2.11 Bmf B1684 2.09 1.13 1.74 cJun pSer63 J2128 1.99 1.14 1.85
BLK B8928 1.94 1.05 1.51
[0508] From the Antibody array experiments, on SKMEL-28 with Q10
(24 hr), the following are some of the identified proteins with
altered levels: Bcl-xl, Bmf, BTK, BLK, cJun (pSer63), Connexin 32,
PUMA bbc3, BID, Par4, cCb1. These data confirm that the levels of
pro-apoptosis proteins are altered upon incubation with elevated
levels of exogenously added Q10.
[0509] Bcl-xl ("Basal cell lymphoma-extra large") is a
transmembrane molecule in the mitochondria. It is involved in the
signal transduction pathway of the FAS-L and is one of several
anti-apoptotic proteins which are members of the Bcl-2 family of
proteins. It has been implicated in the survival of cancer cells.
However, it is known that alternative splicing of human Bcl-x mRNA
may result in at least two distinct Bcl-x mRNA species, Bcl-xL and
Bcl-xS. The predominant protein product (233 amino acids) is the
larger Bcl-x mRNA, Bcl-xL, which inhibits cell death upon growth
factor withdrawal (Boise et al., 1993. Cell 74, 597-608). Bcl-xS,
on the other hand, inhibits the ability of Bcl-2 to inhibit cell
death and renders cells more susceptible to apoptotic cell
death.
TABLE-US-00022 TABLE 21 Proteins with increased levels in SCC cells
after 24 hour treatment with 100 .mu.M Q10. Name Ratio PUMA bbc3
3.81 HDAC7 3.21 BID 3.12 MTBP 3.00 p38 MAP Kinase 2.93 NonActivated
PKR 2.87 TRAIL 2.86 DR5 2.86 Cdk3 2.82 NCadherin 2.71 Reelin 2.68
p35 Cdk5 Regulator 2.63 HDAC10 2.60 RAP1 2.59 PSF 2.56 cMyc 2.55
methyl Histone H3 2.54 MeLys9 HDAC1 2.51 F1A 2.48 ROCK1 2.45 Bim
2.45 FXR2 2.44 DEDAF 2.44 DcR1 2.40 APRIL 2.40 PRMT1 2.36 Pyk2
pTyr580 2.34 Vitronectin 2.33 Synaptopodin 2.32 Caspase13 2.30
Syntaxin 8 2.29 DR6 2.29 BLK 2.28 ROCK2 2.28 Sir2 2.25 DcR3 2.24
RbAp48 RbAp46 2.21 OGlcNAc Transferase 2.21 GRP78 BiP 2.20 Sin3A
2.20 p63 2.20 Presenilin1 2.19 PML 2.18 PAK1pThr212 2.17 HDAC8 2.16
HDAC6 2.15 Nitric Oxide Synthase 2.15 Inducible iNOS Neurofibromin
2.15 Syntaxin 6 2.13 Parkin 2.12 Rad17 2.11 Nitric Oxide Synthase
bNOS 2.10 TIS7 2.09 OP18 Stathmin (stathmin 2.08 1/oncoprotein 18)
phospho-b-Catenin pSer45 2.07 NeurabinII 2.07 e Tubulin 2.07 PKB
pThr308 2.07 Ornithine Decarboxylase 2.07 P53 BP1 2.06 Pyk2 2.05
HDAC5 2.05 Connexin 43 2.05 a1Syntrophin 2.04 MRP1 2.04 cerbB4 2.03
S Nitrosocysteine 2.03 SGK 2.02 Rab5 2.01 Ubiquitin Cterminal 2.01
Hydrolase L1 Myosin Ib Nuclear 2.00 Par4 Prostate Apoptosis 2.00
Response 4
TABLE-US-00023 TABLE 22 Proteins with reduced levels in SCC cells
after 24 hour treatment with 100 .mu.M Q10. Name Ratio AP1 0.68
Centrin 0.55 CUGBP1 0.67 Cystatin A 0.69 Cytokeratin CK5 0.60
Fibronectin 0.63 gParvin 0.70 Growth Factor Independence1 0.63
Nerve Growth Factor b 0.60 ProCaspase 8 0.72 Rab7 0.62 Rab9 0.73
Serine Threonine Protein 0.71 Phosphatase 1g1 Serine Threonine
Protein 0.73 Phosphatase 2 A Bg SKM1 0.70 SLIPR MAGI3 0.67 Spectrin
a and b 0.70 Spred2 0.66 TRF1 0.74
Example 8
Western Blot Analysis
[0510] The first experiment processed and evaluated by Western blot
and 2-D gel electrophoresis was carried out on the skin cancer cell
line SKMEL-28. This experimental set involved SK-MEL-28 cells
treated at 3, 6, 12, and 24 hours with 50 or 100 .mu.M Q10.
[0511] A variety of cell types were evaluated by Western blot
analysis against an antibody for Bcl-xL (FIG. 14), an antibody for
Vimentin (FIG. 15), a series of antibodies for mitochondrial
oxidative phosphorylation function (FIGS. 16-21) and against a
series of antibodies related to mitochondrial membrane integrity
(FIGS. 22-27). The results from these experiments demonstrated that
several of the examined proteins were upregulated or downregulated
as a result of cell treatment with Q10.
Example 9
Diabetes Related Genes Identified as being Modulated at the mRNA
Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100 um
Q10
[0512] Diabetes arrays were run for samples treated with 100 uM Q10
at various times after treatment. Experiments were carried out
essentially as described above. The various genes found to be
modulated upon Q10 treatment are summarized in Table 23 below. The
results showed that the following genes are modulated by Q10
treatment: ABCC8, ACLY, ADRB3, CCL5, CEACAM1, CEBRA, FOXG1, FOXP3,
G6PD, GLP1R, GPD1, HNF4A, ICAM1, IGFBP5, INPPL1, IRS2, MAPK14, MEL
NFKB1, PARP1, PIK3C2B, PIK3CD, PPARGC1B, PRKAG2, PTPN1, PYGL,
SLC2A4, SNAP25, HNF1B, TNRFSF1A, TRIB3, VAPA, VEGFA, IL4R and
IL6.
Table 23
Genes from the Diabetes Array Whose Expression is Regulated with
100 .mu.M Q10 and their Possible Functions in a Cell
Up-Regulated (Grey) and Down-Regulated (White)
TABLE-US-00024 [0513] TABLE 23 Genes from the diabetes array whose
expression is regulated with 100 .mu.M Q10 and their possible
functions in a cell. Up-regulated (grey) and down-regulated
(white). ##STR00005##
Example 10
Angiogenesis Related Genes Identified as being Modulated at the
mRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100
.mu.M Q10
[0514] Angiogenesis arrays were run for samples treated with 100 uM
Q10 at various times after treatment. Experiments were carried out
essentially as described above. The various genes found to be
modulated upon Q10 treatment are summarized in Table 24 below. The
results showed that the following genes are modulated by Q10
treatment: AKT1, ANGPTL4, ANGPEP, CCL2, CDH4, CXCL1, EDG1, EFNA3,
EFNB2, EGF, FGF1, ID3, IL1B, IL8, KDR, NRP1, PECAM1, PROK2,
SERPINF1, SPHK1, STAB1, TGFB1, VEGFA and VEGFB.
TABLE-US-00025 TABLE 24 A list of genes from the angiogenesis array
whose expression is regulated with 100 .mu.M Q10 and their possible
functions in a cell. Up-regulated (grey) and down-regulated
(white). ##STR00006##
Example 11
Apoptosis Related Genes Identified as being Modulated at the mRNA
Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100
.mu.M Q10
[0515] Apoptosis arrays were run for samples treated with 100 uM
Q10 at various times after treatment. Experiments were carried out
essentially as described above. The various genes found to be
modulated upon Q10 treatment are summarized in Table 25 below. The
results showed that the following genes are modulated by Q10
treatment: ABL1, AKT1, Bcl2L1, BclAF1, CASP1, CASP2, CASP6, CIDEA,
FADD, LTA, TNF, TNFSF10A and TNFSF10.
TABLE-US-00026 TABLE 25 A list of genes from the apoptosis array
whose expression is regulated with 100 .mu.M Q10 and their possible
functions in a cell. Up-regulated (Grey) and down-regulated
(white). ##STR00007##
Example 12
PCR Diabetes Arrays on Liver Cancer (HepG2) Cells
[0516] HepG2 (liver cancer) cells were treated with either the
vehicle for 24 hours or 100 .mu.M Q10 for different times. The
treatment was initiated on 1.times.105 cells per well, following
the procedure utilized in the PaCa2 cells (above, Examples 9-11).
However, the total amount of RNA that was extracted from these
samples was lower than expected. Reverse transcription is normally
done using 1 .mu.g of total RNA (determined by measurement at 260
nm). The maximum volume that can be used per reverse transcription
is 8 .mu.l. Since the RNA concentration was low, the RT-PCR array
analysis using the vehicle, and Q10 treated samples from 16 hours
and 48 hours was performed using 0.44 .mu.g of RNA. The arrays
provided an initial analysis of trends and patterns in HepG2 gene
regulation with 100 .mu.M Q10 treatment, as summarized in Table 26
below. The results showed that each of the genes PPARGC1A, PRKAA1
and SNAP25 were downregulated at 16 hours following treatment (by
approximately 20 fold, 6 fold and 5 fold, respectively). At 48
hours following treatment, PPARGC1A and PRKAA1 had normalized or
were slightly upregulated, while SNAP25 was downregulated by
approximately 2 fold.
TABLE-US-00027 TABLE 26 List of genes regulated in the Diabetes
Arrays when HepG2 cells were treated with 100 .mu.M Q10. Gene Gene
name Gene Function. PPARGC1A peroxisome proliferator- Involved in
cell death, activated receptor proliferation, cellular respiration
gamma, coactivator 1 and transmembrane potential. alpha PRKAA1
protein kinase, AMP- Regulates TP53 and is involved activated,
alpha 1 in apoptosis, regulates catalytic subunit glycolysis,
regulates metabolic enzyme activities. SNAP25
synaptosomal-associated Plays in transport, fusion, protein, 25 kDa
exocytosis and release of molecules.
Example 13
PCR Angiogenesis Array on Liver Cancer (HEPG2) Cells
[0517] HepG2 (liver cancer) cells were treated with either the
vehicle for 24 hours or 100 .mu.M Q10 for different times. The
treatment was initiated on 1.times.105 cells per well, following
the procedure utilized in the PaCa2 cells (above Examples 9-11).
However, the total amount of RNA that was extracted from these
samples was lower than expected. Reverse transcription is normally
done using 1 .mu.g of total RNA (determined by measurement at 260
nm). The maximum volume that can be used per reverse transcription
is 8 .mu.l. Since the RNA concentration was low, the RT-PCR array
analysis using the vehicle, and Q10 treated samples from 16 hours
and 48 hours was performed using 0.44 .mu.g of RNA. The arrays
provided an initial analysis of trends and patterns in HepG2 gene
regulation with 100 .mu.M Q10 treatment, as summarized in Table 27
below. The various genes found to be modulated upon Q10 treatment
are summarized in Table 27 below. The results showed that each of
the genes ANGPTL3, ANGPTL4, CXCL1, CXCL3, CXCL5, ENG, MMP2 and
TIMP3 were upregulated at 16 hours following treatment (by
approximately 5.5, 3, 3, 3.2, 3, 3, 1 and 6.5 fold, 6 fold and 5
fold, respectively, over that of control). ID3 was downregulated at
16 hours following Q10 treatment, by approximately 5 fold over
control. At 48 hours following treatment, ANGPTL3, CXCL1, CXCL3,
ENG and TIMP3 were still upregulated (by approximately 3.5, 1.5,
3.175, 2 and 3 fold, respectively, over control), while ANGPTL4,
CXCL5, ID3 and MMP2 were downregulated by approximately 1, 1, 2 and
18 fold, respectively, over control.
TABLE-US-00028 TABLE 27 List of genes regulated in the Angiogenesis
Arrays when HepG2 cells were treated with 100 .mu.M Q10. Gene Gene
Name. Gene Function. ANGPTL3 angiopoietin-like 3 Predominantly
expressed in live, role in cell migration and adhesion, regulates
fatty acid and glycerol metabolism. ANGPTL4 angiopoietin-like 4
Regulated by PPARG, apoptosis inhibitor for vascular endothelial
cells, role lipid and glucose metabolism and insulin sensitivity.
CXCL1 chemokine (C--X--C motif) Role in cell proliferation and
migration ligand 1 (melanoma growth stimulating activity, alpha)
CXCL3 chemokine (C--X--C motif) Chemokine activation, hepatic
stellar cell ligand 3 activation, migration, proliferation. CXCL5
chemokine (C--X--C motif) Produced along with IL8 when stimulated
with ligand 5 IL1 or TNFA. Role in chemotaxis, migration,
proliferation. ENG endoglin Binds to TGFBR and is involved in
migration, proliferation, attachment and invasion. ID3 inhibitor of
DNA binding 3, Regulates MMP2, Regulated by TGFB1, dominant
negative helix- Vitamin D3, Retinoic acid, VEGFA, involved in
loop-helix protein apoptosis, proliferation, differentiation,
migration. MMP2 matrix metallopeptidase 2 Hepatic stellate cell
activation, HIF signaling, (gelatinase A, 72 kDa binds to TIMP3,
involved in tumorigenesis, gelatinase, 72 kDa type IV apoptosis,
proliferation, invasiveness, migration collagenase) and chemotaxis.
TIMP3 TIMP metallopeptidase Regulates MMP2, ICAM1. Regulated by
inhibitor 3 TGFB, EGF, TNF, FGF and TP53. Involved in apoptosis,
cell-cell adhesion and malignancy.
[0518] Proteins known to be involved in the process of angiogenesis
were components in the RT-PCR array. Angiogenesis is a critical
process by which cancer cells become malignant. Some of these
proteins are also implicated in diabetes. ANGPTL3 and ANGPTL4: The
literature related to ANGPTL3 connects this protein to the
regulation of lipid metabolism. In particular, the literature (Li,
C. Curr Opin Lipidol. 2006 April; 17(2):152-6) teaches that both
angiopoietins and angiopoietin-like proteins share similar domain
structures. ANGPTL3 and 4 are the only two members of this
superfamily that inhibit lipoprotein lipase activity. However,
ANGPTL3 and 4 are differentially regulated at multiple levels,
suggesting non-redundant functions in vivo. ANGPTL3 and 4 are
proteolytically processed into two halves and are differentially
regulated by nuclear receptors. Transgenic overexpression of
ANGPTL4 as well as knockout of ANGPTL3 or 4 demonstrate that these
two proteins play essential roles in lipoprotein metabolism:
liver-derived ANGPTL3 inhibits lipoprotein lipase activity
primarily in the fed state, while ANGPTL4 plays important roles in
both fed and fasted states. In addition, ANGPTL4 regulates the
tissue-specific delivery of lipoprotein-derived fatty acids.
ANGPTL4 is thus an endocrine or autocrine/paracarine inhibitor of
lipoprotein lipase depending on its sites of expression.
Lipoprotein lipase is an enzyme that hydrolyzes lipids in
lipoproteins, such as those found in chylomicrons and very
low-density lipoproteins (VLDL), into three free fatty acids and
one glycerol molecule. Lipoprotein lipase activity in a given
tissue is the rate limiting step for the uptake of
triglyceride-derived fatty acids. Imbalances in the partitioning of
fatty acids have major metabolic consequences. High-fat diets have
been shown to cause tissue-specific overexpression of LPL, which
has been implicated in tissue-specific insulin resistance and
consequent development of type 2 diabetes mellitus. The results in
this Example indicate that Q10 is modulating proteins involved in
lipid metabolism and thus warrants further investigation of
ANGPTL3/ANGPTL4 and their related pathways. For example,
ANGPTL3/ANGPTL4 have been implicated to play a role in the
following pathways: Akt, cholesterol, fatty acid, HDL-cholesterol,
HNF1A, ITGA5, ITGA5, ITGAV, ITG83, L-trilodothynonine, LIPG, LPL,
Mapk, Nrth, NR1H3, PPARD, PTK2, RXRA, triacylglerol and
9-cis-retinoic acid.
Example 14
PCR Apoptosis Array on Liver Cancer (HEPG2) Cells
[0519] Apoptosis arrays were run for samples treated with 100 uM
Q10 for 16 and 48 hours as described above. However, the array for
48 hours was run choosing FAM as the fluorophore instead of SYBR.
Both FAM and SYBR fluoresce at the same wavelength.
[0520] The various genes found to be modulated upon Q10 treatment
are summarized in Table 28 below. The results showed that CASP9 was
upregulated at 16 hours following Q10 treatment, by approximately
61 fold over control, while BAG1 and TNFRSF1A were downregulated at
16 hours following treatment by approximately 6 and 4 fold,
respectively, over that of control. At 48 hours following
treatment, CASP9, BAG1 and TNFRSF1A were upregulated by
approximately 55, 1 and 1 fold, respectively, over control.
TABLE-US-00029 TABLE 28 List of genes regulated in the Apoptosis
Arrays when HepG2 cells were treated with 100 .mu.M Q10. Gene Gene
Name Gene Function. BAG1 BCL2-associated athanogene Involved with
Apoptosis CASP9 caspase 9, apoptosis-related Apoptosis through
release cysteine peptidase of cytochrome c. TNFRSF1A tumor necrosis
factor receptor anti-apoptosis, binds many superfamily, member 1A
cell death factors, regulates ICAM1
Example 15
Assessing Ability of Epi-Shifter to Treat Metabolic Disorder
[0521] In order to determine if a selected Epi-shifter, e.g.,
CoQ10, is capable of treating a metabolic disorder, e.g., diabetes,
cell based assays that monitor an increase in insulin-stimulated
glucose uptake in vitro are employed. In particular, differentiated
mouse adipocytes are used to identify agents that have the ability
to increase glucose uptake upon insulin stimulation, as detected by
scintillation counting of radiolabelled glucose (using, for
example, the Perkin Elmer 1450 Microbeta JET reader). These assays
are conducted as follows.
Materials and Methods:
Prees Media
[0522] Complete media, also referred to as "Prees" media, is
prepared as follows. Dulbecco's Modified Eagle's Medium (DMEM) is
supplemented with L-glutamine, penicillin-G and streptomycin
(pen/strep), and heat-inactivated fetal bovine serum (FBS) (heat
inactivated at 65.degree. C. for 30 minutes). Because serum can
affect the growth, adherence, and differentiation of cells, any new
lot of serum was first tested prior to use. Media was equilibrated
in the incubator (5% CO.sub.2) until the pH was within the proper
range (.about.7), as indicated by the red/orange color of the
indicator dye. If the media became pink (indicating a high pH), we
discarded the media as basic conditions can affect cells and
denature the insulin used in the differentiation medium-1 (DM1) and
the differentiation medium-2 (DM2).
Differentiation Medias
[0523] Differentiation media-1 (DM1) was prepared by supplementing
DMEM with 10% FBS, L-glutamine, pen/strep, IBMX (375 .mu.M),
insulin (120 nM), and dexamethasone (188 nM). Differentiation
media-2 (DM2) was prepared by supplementing DMEM with 10% FBS,
L-glutamine, pen/strep, and insulin (120 nM).
Preparation of Gelatinized Plates
[0524] Cell culture plates are gelatinized as follows. Gelatin (1%
w/v in distilled water) was autoclaved and stored at room
temperature. The bottom of each cell culture well was covered
uniformly in the gelatin solution, ensuring that no bubbles are
formed. This solution was removed leaving behind a thin film of
gelatin. These plates are left to dry under the tissue culture
hood. Plates are next washed with PBS, after which a 0.5% glutaric
dialdehyde solution (glutaraldehyde in distilled water) was added
to the cell culture wells. After ten minutes, wells are washed
twice with DMEM containing pen-strep. Each washing step should last
for approximately five minutes.
Cell Culture
[0525] 3T3-L1 pre-adipocyte cells are split approximately every 2-3
days or upon reaching a confluence of approximately 60%.
Overconfluency may affect the ability of these cells to
differentiate into adipocytes.
Other Reagents
[0526] D-(+)-glucose ("cold" glucose, not radiolabeled) was added
to DPBS mix to a final concentration of 10 mM.
[0527] Lysis buffer, a mixture of a base (e.g., sodium hydroxide at
a final concentration of 0.5N) and a detergent (e.g., sodium
dodecyl sulphate (SDS) diluted to a final concentration of 0.1%
w/v) was freshly prepared each time (within one to two hours of
use). Prior to use, lysis buffer was warmed up to a temperature
exceeding that of room temperature for a period of approximately 30
minutes to avoid precipitation of the buffer.
Determination of Glucose Uptake
[0528] Pre-adipocyte 3T3-L1 cells are plated at a density of
approximately 5000 cells/well (in black NUNC 96 well plate). These
cells are differentiated into adipocytes in two separate steps.
Initially, cells are cultured in differentiation medium-1 (DM1)
(day 1 of adipocyte differentiation) for a period of two to three
days. DM1 prevents proliferation and induces the expression of
adipocyte-specific genes. Cells are next cultured in
differentiation medium-2 (DM2) for 3 to 4 days, after which the
culture media is replaced by fresh DM2. The glucose uptake assay is
performed at day 9-15 of differentiation.
[0529] Two days prior to the experiment (at day 7-13 of
differentiation), DM2 is removed and replaced with fresh Prees
media. Candidate compounds are added at this time, allowing an
incubation period of approximately 48 hours. On the day of the
experiment, cells (now at day 9 to 15 of differentiation) are serum
starved for three hours in DPBS, magnesium sulfate (0.8 mM), and
Hepes (10 mM) at pH .about.7. After this incubation period, fresh
DPBS containing insulin (10 nM) is added to the adipocytes. Fresh
DPBS without any insulin are placed on cells that served as a
negative control. Following an incubation period of 25 minutes at
37.degree. C., radioactive glucose (labeled with .sup.14C, at a
final concentration of 0.04 mM, .about.0.26 .mu.Ci .sup.14C-glucose
in each well) is added to the media for a period of 15 minutes at
room temperature. Media is next removed and cells are washed
thoroughly and lysed. Upon lysis, cells form a small, cloudy mass,
detached from the well bottom. 10% glacial acetic acid is added to
each well to neutralize the lysis reaction. Scintillation fluid is
next added to the wells and the incorporation of glucose is
determined by measuring the amount of radioactivity in each well
using the MicroBeta plate reader.
[0530] Using the foregoing experimental protocol, an Epi-shifter is
identified as capable of treating a metabolic disorder, e.g.,
diabetes, when the Epi-shifter enhances, increases or augments
insulin-stimulated glucose uptake in the cells in vitro.
Example 16
Identification of a MIM Associated with an Metabolic Disorder
[0531] In order to evaluate a candidate molecule (e.g.,
environmental influencer) as a potential MIM, the selected
candidate MIM is exogenously added to a panel of cell lines,
including both diseased (cancer) cell lines and normal control cell
lines, and the changes induced to the cellular microenvironment
profile for each cell line in the panel are assessed. Changes to
cell morphology, physiology, and/or to cell composition, including
for example, mRNA and protein levels, are evaluated and compared
for the diseased cells as compared to normal cells.
[0532] Changes to cell morphology/physiology are evaluated by
examining the sensitivy and apoptotic response of cells to the
candidate MIM. These experiments are carried out as described in
detail in Example 3. Briefly, a panel of cell lines consisting of
at least one control cell line and at least one cancer cell line
are treated with various concentrations of the candidate MIM. The
sensitivity of the cell lines to the potential MIM are evaluated by
monitoring cell survival at various times, and over the range of
applied concentrations. The apoptoic response of the cell lines to
the potential MIM are evaluated by using, for example, Nexin
reagent in combination with flow cytometry methodologies. Nexin
reagent contains a combination of two dyes, 7AAD and Annexin-V-PE,
and allows quantification of the population of cells in early and
late apoptosis. An additional apoptosis assay that measures
single-stranded DNA may be used, using for example Apostrand.TM.
ELISA methodologies. The sensitivity and apoptotic response of the
disease and control cell lines are evaluated and compared. A
molecule that displays differential cytotoxicity and/or that
differentially induces the apoptotic response in the diseased cells
as compared to the normal cells is identified as a MIM.
[0533] Changes in the composition of cells following treatment with
the candidate MIM are evaluated. Changes in gene expression at the
mRNA level are analyzed using Real-Time PCR array methodology.
These experiments are carried out as described in detail in
Examples 6 and 9-13. Briefly, the candidate MIM is exogenously
added to one or more cell lines including, for example a diseased
cell and a normal control cell line, and mRNA is extracted from the
cells at various times following treatment. The level of mRNAs for
genes involved in specific pathways are evaluated by using targeted
pathway arrays, including, for example, arrays specific for
apoptosis, oxidative stress and antioxidate defense, angiogenesis,
heat shock or diabetes. The genes that are altered in their mRNA
transcription by a two-fold level or greater are identified and
evaluated. A molecule that induces changes in mRNA levels in cells
and/or that induces differential changes in the level of one or
more mRNAs in the diseased cells as compared to the normal cells is
identified as a MIM.
[0534] In complementary experiments, changes in gene expression at
the protein level are analyzed by using antibody microarray
methodology, 2-dimensional gel electrophoresis followed by protein
identification using mass spectrometry characterization, and by
western blot analysis. These experiments are carried out as
described in detail in Examples 7, 4 and 8, respectively. Briefly,
the candidate MIM is exogenously added to one or more cell lines,
including, for example a diseased cell and a normal control cell
line, and soluble protein is extracted from the cells at various
times, e.g., 6 hours or 24 hours, following treatment. Changes
induced to protein levels by the candidate MIM are evaluated by
using an antibody microarray containing antibodies for over 700
proteins, sampling a broad range of protein types and potential
pathway markers. Further complementary proteomic analysis can be
carried by employing 2-dimensional (2-D) gel electrophoresis
coupled with mass spectrometry methodologies. The candidate MIM is
exogenously added to one or more cell lines, including, for example
a diseased cell and a normal control cell line, and cell pellets
are lysed and subjected to 2-D gel electrophoresis. The gels are
analyzed to identify changes in protein levels in treated samples
relative to control, untreated samples. The gels are analyzed for
the identification of spot changes over the time course of
treatment due to increased levels, decreased levels or
post-translational modification. Spots exhibiting statistically
significant changes are excised and submitted for protein
identification by trypsin digestiona do mass spectrometry
characterization. The characterized peptides are searched against
protein databases with, for example, Mascot and MSRAT software
analysis to identify the proteins. In addition to the foregoing 2-D
gel analysis and antibody microarray experiments, potential changes
to levels of specific proteins induced by the candidate MIM may be
evaluated by Western blot analysis. In all of the proteomic
experiments, proteins with increased or decreased levels in the
various cell lines are identified and evaluated. A molecule that
induces changes in protein levels in cells and/or that induces
differential changes in the level of one or more proteins in the
diseased cells as compared to the normal cells is identified as a
MIM.
[0535] Genes found to be modulated by treatment with a candidate
MIM from the foregoing experiments are subjected to cellular and
biochemical pathway analysis and can thereby be categorized into
various cellular pathways, including, for example apoptosis, cancer
biology and cell growth, glycolysis and metabolism, molecular
transport, and cellular signaling.
[0536] Experiments are carried out to confirm the entry of a
candidate MIM into cells, to determine if the candidate MIM becomes
localized within the cell, and to determine the level and form of
the candidate MIM present in the cells. These experiments are
carried out, for example, as described in detail in Example 5. For
example, to determine the level and the form of the candidate MIM
present in the mitochondria, mitochondrial enriched preparations
from cells treated with the candidate MIM are prepared and
analyzed. The level of the candidate MIM present in the
mitochondria can thereby be confirmed to increase in a time and
dose dependent manner with the addition of exogenous candidate MIM.
In addition, changes in levels of proteins from mitochondria
enriched samples are analyzed by using 2-D gel electrophoresis and
protein identification by mass spectrometry characterization, as
described above for total cell protein samples. Candidate MIMs that
are found to enter the cell and to be present at increased levels,
e.g., in the mitochondria, are identified as a MIM. The levels of
the candidate MIM in the cell, or, for example, specifically in the
mitochondria, over the time course examined can be correlated with
other observed cellular changes, as evidenced by, for example, the
modulation of mRNA and protein levels for specific proteins.
[0537] Candidate MIMs observed to induce changes in cell
composition, e.g., to induce changes in gene expression at the mRNA
or protein level, are identified as a MIM. Candidate MIMs observed
to induce differential changes in cell morphology, physiology or
cell composition (e.g., differential changes in gene expression at
the mRNA or protein level), in a disease state (e.g., diabetes or
obesity) as compared to a normal state are identified as a MIM and,
in particular, as having multidimensional character. Candidate MIMs
found to be capable of entering a cell are identified as a MIM and,
in particular, as having multidimensional character since the
candidate MIM thereby exhibits a carrier effect in addition to a
therapeutic effect.
Example 17
Identification of CoQ10 as an Epi-Shifter Associated with a
Metabolic Disorder
[0538] A panel of skin cell lines consisting of a control cell
lines (primary culture of keratinocytes and melanocytes) and
several skin cancers cell lines (SK-MEL-28, a non-metastatic skin
melanoma; SK-MEL-2, a metastatic skin melanoma; or SCC, a squamous
cell carcinoma; PaCa2, a pancreatic cancer cell line; or HEP-G2, a
liver cancer cell line) were treated with various levels of
Coenzyme Q10. The cancer cell lines exhibited an altered dose
dependent response when compared to the control cell lines, with an
induction of apoptosis and cell death in the cancer cells only.
Detailed exemplary experiments are presented in, e.g., Example 3
herein.
[0539] Assays were employed to assess changes in the mRNA and
protein levels composition of the above-identified cells following
treatment with CoQ10. Changes in mRNA expression were analyzed
using real-time PCR microarrays specific for each of apoptosis,
oxidative stress and antioxidants, angiogenesis and diabetes.
Changes in protein expression were analyzed using antibody
microarray analysis and western blot analysis. The results from
these assays demonstrated that significant changes in gene
expression, both at the mRNA and protein levels, were occurring in
the cell lines due to the addition of the Coenzyme Q10. Numerous
genes known to be associated with or involved in cellular metabolic
processes were observed to be modulated as a result of treatment
with CoQ10. For example, expression of the nuclear receptor protein
HNF4A was found to be upmodulated in cells following Q10 treatment.
Expression of transaldolase 1 (TAL) was also modulated in cells
treated with Q10. TAL balances the levels of NADPH and reactive
oxygen intermediate, thereby regulating the mitochondrial
trans-membrande potentional, which is a critical checkpoint of ATP
synthesis and cell survival. Of particular relevance to metabolic
disorders, numerous genes known to be associated with, e.g.,
diabetes, were identified as being regulated by Q10. Detailed
exemplary experiments are presented in, e.g., Examples 4, 6, 7, 8
and 9 herein.
[0540] Q10 is an essential cofactor for exidative phosphorylation
processes in the mitochondria for energy production. The level of
Coenzyme Q10, as well as the form of CoQ10, present in the
mitochondria was determined by analyzing mitochondrial enriched
preparations from cells treated with CoQ10. The level of Coenzyme
Q10 present in the mitochondria was confirmed to increase in a time
and dose dependent manner with the addition of exogenous Q10. The
time course correlated with a wide variety of cellular changes as
observed in modulation of mRNA and protein levels for specific
proteins related to metabolic and apoptotic pathways. Detailed
exemplary experiments are presented in, e.g., Example 5 herein.
[0541] The results described herein identified the endogenous
molecule CoQ10 as an epi-shifter. In particular, the results
identified CoQ10 as inducing a shift in the metabolic state, and
partially restoration of mitochondrial function, in cells. These
conclusions are based on the following interpretation of the data
described herein and the current knowledge in the relevant art.
[0542] Q10 is known to be synthesized, actively transported to,
enriched in, and utilized in the mitochondrial inner membrane. Q10
is also known to be an essential cofactor for oxidative
phosphorylation processes in the mitochondrial for energy
production. However, most cancer cells predominantly produce energy
by glycolysis followed by lactic acid fermentation in the cytosol,
rather than by oxidation of pyruvate in mitochondria like most
normal cells. The oxidative phosphorylation involves the electron
transport complexes and cytochrome c. Apoptosis involves the
disruption of the mitochondria, with permiabilization of the inter
mitochondrial membrane by pro-apoptitic factors. By utilizing a
different metabolic energy synthesis pathway, cancer cells are able
to mitigate the normal apoptosis response to abnormalities in the
cell. While not wishing to be bound by theory, Applicants propose
that Q10 is functioning by upregulating the oxidative
phosphorylation pathway proteins, thus switching the mitochondrial
function back to a state that would recognize the oncogenic defects
and trigger apoptosis. Thus, Q10 is acting as an Epi-shifter by
shifting the metabolic state of a cell.
Example 18
Identification of an Epi-Shifter Associated with a Metabolic
Disorder
[0543] A panel of skin cell lines consisting of control cell lines
(e.g., primary culture of keratinocytes and melanocytes) and cancer
cell lines (e.g., SK-MEL-28, a non-metastatic skin melanoma;
SK-MEL-2, a metastatic skin melanoma; or SCC, a squamous cell
carcinoma; PaCa2, a pancreatic cancer cell line; or HEP-G2, a liver
cancer cell line) are treated with various levels of a candidate
Epi-shifter. Changes to cell morphology/physiology are evaluated by
examining the sensitivy and apoptotic response of cells to the
candidate Epi-shifter. These experiments are carried out as
described in detail in Example 3. Briefly, the sensitivity of the
cell lines to the candidate Epi-shifter are evaluated by monitoring
cell survival at various times, and over a range of applied
concentrations. The apoptoic response of the cell lines to the
candidate Epi-shifter are evaluated by using, for example, Nexin
reagent in combination with flow cytometry methodologies. Nexin
reagent contains a combination of two dyes, 7AAD and Annexin-V-PE,
and allows quantification of the population of cells in early and
late apoptosis. An additional apoptosis assay that measures
single-stranded DNA may be used, using for example Apostrand.TM.
ELISA methodologies. The sensitivity and apoptotic response of the
disease and control cell lines are evaluated and compared.
Candidate Epi-shifters are evaluated based on their ability to
inhibit cell growth preferentially or selectively in cancer cells
as compared to normal or control cells. Candidate Epi-shifters are
further evaluated based on their ability to preferentially or
selectively induce apoptosis in cancer cells as compared to normal
or control cells.
[0544] Assays are employed to assess changes in the mRNA and
protein level composition of the above-identified cells following
treatment with the candidate Epi-shifter. Changes in mRNA levels
are analyzed using real-time PCR microarrays. These experiments are
carried out as described in detail in Examples 6 and 9-13. Briefly,
mRNA is extracted from the cells at various times following
treatment. The level of mRNAs for genes involved in specific
pathways are evaluated by using targeted pathway arrays, including,
arrays specific for apoptosis, oxidative stress and antioxidate
defense, angiogenesis, heat shock or diabetes. The genes that are
altered in their mRNA transcription by a two-fold level or greater
are identified and evaluated.
[0545] Changes in protein expression are analyzed using antibody
microarray analysis, 2-D gel electrophoresis analysis coupled with
mass spectrometry characterization, and western blot analysis.
These experiments are carried out as described in detail in
Examples 7, 4 and 8, respectively. Briefly, soluble protein is
extracted from the cells at various times, e.g., 6 hours or 24
hours, following treatment with the candidate Epi-shifter. Changes
induced to protein levels by the candidate Epi-shifter are
evaluated by using an antibody microarray containing antibodies for
over 700 proteins, sampling a broad range of protein types and
potential pathway markers. Further complementary proteomic analysis
can be carried out by employing 2-dimensional (2-D) gel
electrophoresis coupled with mass spectrometry methodologies. The
candidate Epi-shifter is exogenously added to the cell lines and
cell pellets are lysed and subjected to 2-D gel electrophoresis.
The gels are analyzed to identify changes in protein levels in
treated samples relative to control, untreated samples. The gels
are analyzed for the identification of spot changes over the time
course of treatment due to increased levels, decreased levels or
post-translational modification. Spots exhibiting statistically
significant changes are excised and submitted for protein
identification by trypsin digestion and mass spectrometry
characterization. The characterized peptides are searched against
protein databases with, for example, Mascot and MSRAT software
analysis to identify the proteins. In addition to the foregoing 2-D
gel analysis and antibody microarray experiments, potential changes
to levels of specific proteins induced by the candidate MIM may be
evaluated by Western blot analysis. In all of the proteomic
experiments, proteins with increased or decreased levels in the
various cell lines are identified and evaluated.
[0546] Candidate Epi-shifters are evaluated based on changes
induced to gene expression, at the mRNA and/or protein levels, in
the cell lines due to the addition of the candidate Epi-shifter. In
particular, candidate Epi-shifters are evaluated based on their
ability to modualate genes known to be associated with or involved
in cellular metabolic processes. Of particular relevance to
metabolic disorders, candidate Epi-shifters are evaluated based on
their ability to modulate genes known to be associated with, for
example, diabetes or obesity.
[0547] The level of the candidate Epi-shifter, as well as the form
of the candidate Epi-shifter, present in the cell or a particular
cell location is determined using routine methods known to the
skilled artisan. For example, the level of the candidate
Epi-shifter in mitochondria over time and over a range of doses is
determined by analyzing mitochondrial enriched preparations from
cells treated with the candidate Epi-shifter. The levels of the
candidate Epi-shifter in the mitochondria over the time course can
be compared and correlated with other cellular changes observed,
such as modulation of mRNA and protein levels for specific proteins
related to metabolic and apoptotic pathways.
[0548] Candidate Epi-shifters observed to induce a shift in the
metabolic state of a cell based on the results obtained from the
foregoing experiments are identified as Epi-shifters. For example,
a candidate Epi-shifter that enhances, increases or augments
insulin-stimulated glucose uptake in cells is identified as an
Epi-shifter.
Example 19
Identification of Vitamin D3 as an Epi-Shifter
[0549] Vitamin D3, or 1.alpha., 25-dihydroxyvitamin D3 (also known
as calcitriol), is a vitamin D metabolite that is synthesized from
vitamin D by a two-step enzymatic process. Vitamin D3 interacts
with its ubiquitous nuclear vitamin D receptor (VDR) to regulate
the transcription of a wide spectrum of genes involved in calcium
and phosphate homeostasis as well as in cell division and
differentiation. Vitamin D3 has been reported to have anticancer
effects in numerous model systems, including squamous cell
carcinoma, prostate adenocarcinoma, cancers of the ovary, breast
and lung (reviewed in Deeb et al. 2007 Nature Reviews Cancer
7:684-700).
[0550] The anticancer effects of vitamin D3 are reported to involve
multiple mechanisms, including growth arrest at the G1 phase of the
cell cycle, apoptosis, tumor cell differentiation, disruption of
growth factor-mediated cell survival signals, and inhibition of
angiogenesis and cell adhesion (reviewed in Deeb et al. 2007 Nature
Reviews Cancer 7:684-700). For example, with particular respect to
apoptosis, Vitamin D3 has been reported to induce apoptosis by
regulating key mediators of apoptosis, such as repressing the
expression of the anti-apoptotic, pro-survival proteins BCL2 and
BCL-XL, or inducing the expression of pro-apoptotic proteins (e.g.,
BAX, BAK and BAD) (Deeb et al. 2007). In a further example, with
particular respect to angiogenesis, Vitamin D3 has been reported to
inhibit the proliferation of some tumor-derived endothelial cells
and to inhibit the expression of vascular endothelial growth factor
(VEGF) that induces angiogenesis in tumors (reviewed in Masuda and
Jones, 2006 Mol. Cancer. Ther. 5(4): 797-8070). In another example,
with particular respect to cell cycle arrest, Vitamin D3 has been
reported to induce gene transcription of the cyclin-dependent
kinase inhibitor p21WAFI/CIPI and to induce the synthesis and/or
stabilization of the cyclin-dependent kinase inhibiotor p27KIPI
protein, both of which are critical for induction of G1 arrest.
(Deeb et al. 2007).
[0551] Based on the foregoing observations, Vitamin D3 is
identified as an Epi-shifter, i.e., owing to its ability to shift
the metabolic state of a cell. Vitamin D3 is an Epi-shifter owing
to its ability to induce apoptosis in a cell and, in particular,
based on its ability to differentially inhibit cell growth and
induce the apoptotic response in diseased (cancer) cells as
compared to normal cells (e.g., differentially modulate expression
of proteins, such as BCL-2, BCL-XL, and BAX, involved in apoptosis
in cancer cells as compared to normal cells).
Example 20
Summary of Key Proteins
[0552] In summary, based on the results of experiments described in
the foregoing Examples, the key proteins modulated by Q10 are
summarized in the Table below.
TABLE-US-00030 TABLE 29 Key proteins modulated by Q10. Pathway
Examples Transcription factors HNF4alpha Apoptotic response Bcl-xl,
Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11 (Bim), XIAP, BRAF, Bax,
c-Jun, Bmf, PUMA, cMyc Pentose Phosphate transaldolase 1 Pathway
Biosynthetic COQ1, COQ3, COQ6, prenyltransferase, 4- pathway
hydroxybenzoate Oxidative stress Neutrophil cytosolic factor 2,
nitric oxide synthase (pro-oxidant) 2A, superoxide dismutase 2
(mitochondrial) Membrane VDAC, Bax channel, ANT, Alterations
Oxidative Cytochrome c, complex I, complex II, complex III,
phosphorylation complex IV, metabolism
Example 21
Western Analysis of Cells Treated with Coenzyme Q10
[0553] Over the past five decades enormous volume of information
has been generated implicating endogenous/exogenous factors
influencing specific processes as the underlying cause of malignant
transformations. Clinical and basic literature provides evidence
that changes in the DNA structure and function play a significant
role in the initiation and progression of cancer, defining cancer
as a genetic disease (Wooster, 2010; Haiman, 2010). In the early
1920s, Otto Warburg and other investigators involved in
characterizing fundamental changes in etiology of oncogenesis
described two major observations (a) the ability of cells to
transport and utilize glucose in the generation of ATP for energy
production in the presence of oxygen--also known as Warburg Effect
and (b) alterations in the mitochondrial structure and
function--including changes in the electron transport leading to a
decrease in the production of mitochondrial ATP. The past few years
has seen a resurgence in the investigating the central role of
cellular bioenergetics in the etiology of cancer i.e. viewing
cancer as a metabolic disease.
[0554] Historically, although mutations in genes has been thought
to be responsible for changes in gene expression, there is
accumulating literature in support of epigenetic processes playing
a critical role in influencing gene expression in supporting
carcinogenesis. This is evidenced by the observation that mutation
rate for most genes is low and cannot account for the numerous
(spectrum of) mutations found in the cancer cells. Epigenetic
alteration is regulated by methylation and modification of histone
tails, both changes inherently linked to the energy (nutrient)
status of the cells since they require the availability of
co-factors e.g. acetyl CoA requirement for histone acetylation
(ref). The biosynthesis of acetyl CoA depends on glycolysis and
Kreb's Cycle, directly linking the intracellular energy status to
regulation of gene expression and activity.
[0555] In normal cells, mitochondrial oxidative phosphorylation
generates sufficient ATP to meet the energy demands for maintaining
normal physiological activities and cell survival. A consequence of
mitochondrial energy production is the generation of reactive
oxygen species (ROS), aberrant production of which leads to damage
of mitochondria (refs). It is well established that chronic ROS
generation by the mitochondria leads to cumulative accumulation of
genetic mutations, a phenomenon that has been implicated in the
etiology of carcinogenesis. It has been suggested that cancer cells
decrease mitochondrial respiration to minimize ROS generation, and
switch to glycolysis to sustain energy production. Thus, a
progressive shift of energy generation from oxidative
phosphorylation to glycolysis would be essential for a cell to
maintain energy production to maintain physiological functions and
could be associated with the progression of a normal cell phenotype
to that of a cancer cell. The progressive shift in cellular energy
(bioenergetic) profile in tandem with accumulated alteration
(mutations) in mitochondrial genetic make-up alters the cellular
metabolome. Changes in the whole cell metabolomic profile as a
consequence of mitochondrial phosphorylation to glycolysis
transition corresponds to an abnormal bioenergetic induced
metabolomic profile and is the underlying cause supporting
carcinogenesis. Targeted intervention using an endogenous molecule
to elicit a cellular metabolomic shift towards conditions of a
non-cancerous normal mitochondrial oxidative phosphorylation
associated cellular bioenergetic state represents a therapeutic
endpoint in the treatment of cancer.
Coenzyme Q10 as a MIM Causing an Epi-Metabolomic Shift
[0556] The data presented herein demonstrates that treatment of
normal and cancer cells with Coenzyme Q10 is associated with
changes in the expression of proteins that regulate key biochemical
terminals within the glycolysis--mitochondrial oxidative stress
continuum. The combination of data describing assessment of protein
expression by western blotting and oxygen consumption rates
demonstrates that in normal cells, there is no significant
alteration in normal glycolytic and mitochondrial respiration rates
following exposure to Coenzyme Q10. Thus, the values for expression
of the proteins and mitochondrial respiration rates in normal cell
lines e.g. HDFa (normal human adult fibroblast), HASMC (normal
human aortic smooth muscle cell), nFib (normal fibroblast) and HeKa
(normal human keratinocytes) can be considered as representatives
of baseline physiological state. Any deviation in expression of
proteins and mitochondrial respiration rates in cancer cell lines,
e.g. HepG2 (liver cancer), PaCa-2 (pancreatic cancer), MCF7 (breast
cancer), SK-MEL (melanoma) and SCC-25 (squamous cell carcinoma), is
representative of alteration due to initiation/progression of the
disease, in this case cancer. The experimental evidence provides
support to the hypothesis that exposure of Coenzyme Q10 to cancer
cells is associated with cellular pathophysiological reorganization
that is reminiscent of normal cells. Specifically, the data
provided herein demonstrates that Coenzyme Q10 exposure in cancer
cells is associated with a shift in the glycolytic pathways and
mitochondrial oxidative phosphorylation responsible for induction
of global reorganization of cellular architecture to that observed
in normal cells.
[0557] In normal cells, the end-points of glycolytic output are
linked to mitochondrial oxidative phosphorylation (OXPHOS), i.e.
generation of pyruvate from glucose via the glycolytic pathway for
the entry into the Kreb's Cycle (also known as Tricarboxylic acid
cycle, TCA, or Citric Acid Cycle) to generate reducing equivalents
to support the mitochondrial OXPHOS for ATP production. Thus, in
normal cells the expression and functional orientation of gene
products involved in glycolysis is primed towards adequate
generation of pyruvate and its entry into the Kreb's Cycle.
Dysregulated expression and function of key proteins participating
in glycolysis and Kreb's Cycle pathways in cancer cells results in
enhanced glycolysis with a significant decrease in mitochondrial
function. Exposure of cancer cells to Coenzyme Q10, an endogenous
molecule that selectively influences the mitochondrial respiratory
chain, alters (normalizes) expression of proteins of the glycolyis
and Kreb's Cycle pathways to facilitate a bioenergetic shift such
that energy production (i.e. ATP generation) is restored to the
mitochondria.
Experimental Procedure
Western Blot Experiment 1
[0558] The cells that were used for the experiment were HDFa, and
MCF-7 cells that were treated or not with Coenzyme Q10 at two
different concentrations, 50 .mu.M and 100 .mu.M, and harvested
after 24 hours of treatment. The whole cell pellets were
resuspended one at a time in 1 mL of C7 buffer and transferred to
labeled 15 mL tubes. The samples were then sonicated in the cold
room on ice using 6 sonic pulses with the setting at #14. The
samples were spun for a short time to 2500 g after sonication and
the samples transferred to 2 ml tubes. The pH was verified of each
sample (pH should be 9.0) using the foam remaining in the 50 mL
sample tubes.
[0559] Alkylation and reduction of samples was performed for each
sample by adding 10 ul of 1M acrylamide, 25 ul of tributylphoshene
and incubation for 90 mins with intermittent mixing. After
incubation, 10 ul of 1M DTT was added and the tubes were spun at
20,000 g at 20 deg C. for 10 minutes and transferred the
supernatant to labeled Amicon Ultra centrifugal filter units with a
10 k cut off (Millipore catalog #UFC 801024). The samples were spun
for 15 minutes at 2500 g in 2 intervals. The conductivity was
measured for Chaps alone as well as the samples using a
conductivity meter. If the conductivity of samples is high, then 1
ml of chaps was added for buffer exchange and spun again at 2500 g
until the volume was down to 250 ul. When the conductivity was 200
or less the samples were spun in 5 min intervals at 2500 g until
the volume of the supernatant was between 150-100 ul. The sample
supernatants were transferred to eppendorf tubes and Bradford assay
was performed using BSA as standard.
[0560] The samples were processed as per standard protocols as
described above and the amount of protein in each of the samples
was determined by Bradford assay. Sample volumes equivalent to 10
ug of protein were prepared as shown below with Lamelli Loading dye
(LDS) and MilliQ water were run on a 4-12% Bis-Tris Novex NuPAGE
gel (Invitrogen, cat #NP0323Box)
[0561] The gels were run for 50 minutes using 1.times.MOPS buffer
using a NOVEX Xcell Surelock system at 200 V. The gels were then
transferred for 1 hour using a NOVEX Xcell Surelock wet transfer
protocol at 30 V. The blots were stained with Simply Blue Safestain
from Invitrogen (LC6065).
IDH1 and ATP Citrate Lyase Levels in HDFa and MCF-7 Samples.
[0562] After transfer each of the blots was placed in between 2
Whatman Filter papers and dried for 15-20 minutes. After drying the
blots were labeled with the date, the type of samples and either
blot 1 or blot 2 using a HB pencil. The molecular weight markers
were outlined with the pencil and with single lines for the blue
and a doublet for the colored markers. The blots were activated
with methanol for 5 seconds, washed with water for 5 minutes, and
TBST for 15 minutes. The blots were blocked for 1 hour with 5%
blocking reagent in TBS-T at room temperature and then washed 3
times with TBS-T (1.times.-15'; 2.times.5' each). Blot 1 was probed
with the primary antibody for IDH1 (Cell Signaling # 3997) in TBST
with 5% BSA (at 1:1000 dilutions) and blot 2 with the rabbit
polyclonal antibody for ATP Citrate Lyase in 5% BSA (Cell Signaling
#4332) at 1:1000 dilution by incubation overnight at 4 deg C. with
shaking. After the overnight incubation with primary antibodies,
the blots were washed 3 times with TBS-T (1.times.-15'; 2.times.5'
each) and probed with the secondary antibody (antirabbit; 1:10,000
dilution) for 1 h on the orbital tilting shaker at room
temperature. After 1 h of incubation with secondary antibodies, the
blots were washed 3 times with TBS-T (1.times.-15'; 2.times.5'
each) and then incubated with ECF reagent for 5 mins and then each
blot scanned with 5100 Fuji Laser scanner at 25 uM resolution, 16
bit, green laser, at 400V and at 500 V.
Actin Levels in HDFa and MCF-7 Samples.
[0563] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The 2 blots were scanned in laser scanner to check for complete
stripping. The blots were then activated with methanol for 5
seconds, washed with water for 5 minutes, and TBST for 15 minutes.
The blots were blocked for 1 hour with 5% blocking reagent in TBS-T
at room temperature and then washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the antibody for
Actin in 5% BSA (Sigma catalog #A5316, clone AC-74) at 1:5000
dilutions for 1 hour at room temperature with shaking. After 1 hour
of incubation with primary antibody for Actin, the membranes were
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the secondary antibody (antimouse; 1:10,000 dilution)
for 1 h on the orbital tilting shaker at room temperature. After 1
h of incubation with secondary antibodies, the blots were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and then incubated
with ECF reagent for 5 minutes and then each blot scanned with 5100
Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at
400V and at 500 V.
Western Blot Experiment 2
[0564] The cells used in this experiment were SKMEL28, SCC-25, nFib
and Heka that were treated or not with coenzyme Q10 at two
different concentrations, 50 .mu.M or 100 .mu.M, and harvested
after 3, 6 and/or 24 hours of treatment. The samples were processed
and run on a 4-12% Bis-Tris Novex NuPAGE gel as described above.
The gels were run, transferred and stained essentially as described
above.
Levels of IDH1 for the 4 Cell Lines
[0565] After transfer the blot was dried for 15-20 minutes,
activated with methanol for 5 seconds, washed with water for 5
minutes, and TBST for 15 minutes. The blot was blocked for 1 hour
with 5% blocking reagent in TBS-T at room temperature and then
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each). This was
then probed with the primary antibody for IDH1 (Cell Signaling #
3997) in TBST with 5% BSA (at 1:1000 dilutions) by incubation
overnight at 4 deg C. with shaking. After the overnight incubation
with primary antibody for IDH1, the blot was washed 3 times with
TBS-T (1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antirabbit; 1:10,000 dilution) for 1 h at room
temperature. After 1 h of incubation with secondary antibodies, the
blot was washed 3 times with TBS-T (1.times.-15'; 2.times.5' each)
and then incubated with ECF reagent for 5 mins and then each blot
scanned with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit,
green laser, at 400V and at 500 V.
ATP Citrate Lyase Levels in 4 Different Cell Lines.
[0566] The Isocitrate dehydrogenase blot was stripped by incubating
for 30 minutes with methanol, followed by two 10 minute washes with
TBS-T, then 30 minutes of incubation with stripping buffer at 50
deg C., and followed by two washes with 100 ml or more of TBS-T for
30' each. The blot was scanned in laser scanner to check for
complete stripping. The blot was activated with methanol for 5
seconds, washed with water for 5 minutes, and TBST for 15 minutes.
The blot was blocked for 1 hour with 5% blocking reagent in TBS-T
at room temperature and then washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each). This was then probed with the
rabbit polyclonal antibody for ATP Citrate Lyase in 5% BSA (Cell
Signaling #4332) at 1:1000 dilution overnight at 4 deg C. with
shaking. After the overnight incubation with primary antibody for
ATP Citrate Lyase, the membrane was washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital
tilting shaker at room temperature. After 1 h of incubation with
secondary antibodies, the blot was washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Actin Levels in 4 Different Cell Lines.
[0567] The ATP Citrate Lyase blot was stripped by incubating for 30
minutes with methanol, followed by two 10 minute washes with TBS-T,
then 30 minutes of incubation with Stripping buffer at 50 deg C.,
and followed by two washes with 100 ml or more of TBS-T for 30'
each. The blot was scanned in laser scanner to check for complete
stripping. The blot was activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blot
was blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the antibody for Actin in 5% BSA
(Sigma catalog #A5316, clone AC-74) at 1:5000 dilutions for 1 hour
at room temperature with shaking. After 1 hour of incubation with
primary antibody for Actin, the membranes were washed 3 times with
TBS-T (1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antimouse; 1:10,000 dilution) for 1 h on the orbital
tilting shaker at room temperature. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Western Blot Experiment 3
[0568] The cells used in this experiment were HepG2, HASMC, and
PACA2 cells that were treated or not with Coenzyme Q10 at two
different concentrations (50 .mu.M and 100 .mu.M) and harvested 48
hours of treatment. In this experiment (western blot experiment 3),
and in all of the experiments described below in this Example
(i.e., western blot experiments 4 through 9), the cells were
additionally treated with either 5 mM glucose ("5G") or 22 mM
glucose ("22G"). The samples derived from the cells were processed
and run on a 4-12% Bis-Tris Novex NuPAGE gel as described above.
The gels were run, transferred and stained essentially as described
above.
IDH1, ATP Citrate Lyase and Actin Levels in HASMC vs. PACA2 and
HepG2.
[0569] The levels of IDH1, ATP citrate lyase and actin levels were
determined by probing the blots with primary antibodies for IDH1,
ATP citrate lyase and actin, essentially as described above.
Western Blot Experiment 4
[0570] The cells used in this experiment were HepG2 cells that were
treated or not with Coenzyme Q10 at two different concentrations,
50 or 100 .mu.M, and harvested after 24 or 48 hours of treatment.
The samples were processed and run on a 4-12% Bis-Tris Novex NuPAGE
gel as described above. The gels were run, transferred and stained
essentially as described above.
Lactate Dehydrogenase Levels in HepG2 Cells.
[0571] After transfer each blot was dried for 15-20 minutes,
activated with methanol for 5 seconds, washed with water for 5
minutes, and TBST for 15 minutes. The blots were blocked for 1 hour
with 5% blocking reagent in TBS-T at room temperature and then
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the primary antibody for Lactate Dehydrogenase (abcam
ab2101; polyclonal) in 5% BSA (at 1:1000 dilutions) by incubation
overnight at 4 deg C. with shaking. After the overnight incubation
with primary antibody for Lactate Dehydrogenase, the blots were
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the secondary antibody (rabbit antigoat; 1:10,000
dilution) for 1 h at room temperature. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 mins and then each blot scanned with 5100 Fuji Laser scanner
at 25 uM resolution, 16 bit, green laser, at 400V and at 500 V.
Pyruvate Kinase Muscle Form (PKM2) Levels in HepG2 Cells.
[0572] The lactate dehydrogenase blots were stripped by incubating
for 30 minutes with methanol, followed by two 10 minute washes with
TBS-T, then 30 minutes of incubation with Stripping buffer at 50
deg C., and followed by two washes with 100 ml or more of TBS-T for
30' each. The 2 blots were scanned in laser scanner to check for
complete stripping. The blots were activated with methanol for 5
seconds, washed with water for 5 minutes, and TBST for 15 minutes.
The blots were blocked for 1 hour with 5% blocking reagent in TBS-T
at room temperature and then washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the rabbit
polyclonal antibody for Pyruvate Kinase M2 in 5% BSA (NOVUS
BIOLOGICALS catalog #H00005315-D01P) at 1:500 dilution overnight at
4 deg C. with shaking. After the overnight incubation with primary
antibody for Pyruvate Kinase M2, the membranes were washed 3 times
with TBS-T (1.times.-15'; 2.times.5' each) and probed with the
secondary antibody (antirabbit; 1:10,000 dilution) for 1 h on the
orbital tilting shaker at room temperature. After 1 h of incubation
with secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Pyruvate Dehydrogenase Beta Levels in HepG2 Cells.
[0573] The pyruvate kinase blots were stripped by incubating for 30
minutes with methanol, followed by two 10 minute washes with TBS-T,
then 30 minutes of incubation with Stripping buffer at 50 deg C.,
and followed by two washes with 100 ml or more of TBS-T for 30'
each. The 2 blots were scanned in laser scanner to check for
complete stripping. After making sure stripping of the antibody and
the ECF reagent has worked, the blots were activated with methanol
for 5 seconds, washed with water for 5 minutes, and TBST for 15
minutes. The blots are blocked for 1 hour with 5% blocking reagent
in TBS-T at room temperature and then washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the antibody for
Pyruvate Dehydrogenase in 5% BSA (ABNOVA catalog #H00005162-M03) at
1:500 dilutions) overnight at 4 deg C. with shaking. After the
overnight incubation with primary antibody for Pyruvate
Dehydrogenase, the membranes were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antimouse; 1:10,000 dilution) for 1 h on the orbital
tilting shaker at room temperature. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Actin Levels in HepG2 Cells.
[0574] The Pyruvate Dehydrogenase blots were stripped and then
reprobed for actin, essentially as described above.
Western Blot Experiment 5
[0575] The cells used in this experiment were MIAPACA2 (PACA2)
cells that were treated or not with Coenzyme Q10 at two different
concentrations, 50 or 100 .mu.M, and harvested after 24 or 48 hours
of treatment. The PACA2 samples were processed and the gels were
run, transferred, stained and scanned essentially as described
above.
Lactate Dehydrogenase (LDH) and Pyruvate Dehydrogenase (PDH) Levels
in PaCa2 Cells
[0576] The levels of LDH and PDH were determined by probing the
blots successively with primary antibodies for LDH and PDH,
essentially as described above.
Caspase 3 Levels in PaCa2 Cells.
[0577] The blots were stripped by incubating for 30 minutes with
methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The 2 blots were scanned in laser scanner to check for complete
stripping. The blots were activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blots
were blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the antibody for Caspase 3 in 5%
BSA (Santacruz Biotechnology # sc7272) at 1:200 dilutions)
overnight at 4 deg C. with shaking. After the overnight incubation
with primary antibody for Caspase 3, the membranes were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and probed with
the secondary antibody (antimouse; 1:10,000 dilution) for 1 h on
the orbital tilting shaker at room temperature. After 1 h of
incubation with secondary antibodies, the blots were washed 3 times
with TBS-T (1.times.-15'; 2.times.5' each) and then incubated with
ECF reagent for 5 minutes and then each blot scanned with 5100 Fuji
Laser scanner at 25 uM resolution, 16 bit, green laser, at 400V and
at 500 V.
Western Blot Experiment 6
[0578] The cells that were used for this Western blot experiment
were PC-3, HepG2, MCF-7, HDFa and PACA2 that were treated or not
with a Coenzyme Q10 IV formulation and harvested after 24 hours of
treatment. The samples were processed and the gels were run,
transferred, stained and scanned essentially as described
above.
Capase 3 and Actin Levels in Different Cell Types.
[0579] The levels of Caspase 3 and actin were determined by probing
the blots successively with primary antibodies for Caspase 3 and
actin, essentially as described above.
Western Blot Experiment 7
[0580] The cells used in this experiment were Human Aortic Smooth
Muscle (HASMC) cells that were treated or not with Coenzyme Q10 at
two different concentrations, 50 .mu.M or 100 .mu.M, and harvested
after 24 or 48 hours of treatment. The HASMC samples were processed
and the gels were run, transferred, stained and scanned essentially
as described above.
Experimental Protocol for Actin:
[0581] The levels of actin were determined by probing the blots
with a primary antibody for actin, essentially as described
above.
Experimental Protocol for Hif 1alpha, Caspase3 and PDHB:
[0582] The Actin blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were scanned in laser scanner to check for complete
stripping. The blots were activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blots
were blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for Hif 1
alpha, Caspase 3 or PDHB in 5% BSA (at 1:200 by incubation
overnight at 4 deg C. with gentle shaking. The primary antibody for
Hif 1 alpha (Abcam ab2185; antirabbit) was at 1:500 dilution in 5%
BSA. The primary antibody for Caspase 3 (Santacruz sc7272;
antirabbit) was at 1:200 dilution in 5% BSA. The primary antibody
for Pyruvate Dehydrogenase beta (PDHB) (Novus Biologicals
H00005162-M03; antimouse) was at 1:500 dilution in 5% BSA. After
incubation with primary antibodies, the membranes were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and probed with
the secondary antibody (PDHB antimouse; Hif 1a and Caspase 3
antirabbit; 1:10,000 dilution) for 1 h at room temperature. After 1
h of incubation with secondary antibodies, the blots were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and then incubated
with ECF reagent for 5 minutes and then each blot scanned with 5100
Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at
400V and at 500 V.
Experimental Protocol for PKM2, SDHB and SDHC:
[0583] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were scanned in laser scanner to check for complete
stripping. The blots were activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blots
were blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for PKM2,
SDHB or SDHC in 5% BSA in TBS-T by incubation overnight at 4 deg C.
with gentle shaking. The primary antibody for SDHC (ABNOVA
H00006391-M02; antimouse) was at 1:500 dilution. The primary
antibody for SDHB was from Abcam ab4714-200; antimouse; at 1:1000
dilution. The primary antibody for Pyruvate Kinase M2 (PKM2) was
from Novus Biologicals H00005315-D0IP; antirabbit; at 1:500
dilution. After incubation with primary antibodies, the membranes
were washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the secondary antibody (SDHB & C antimouse; and
PKM2 antirabbit; 1:10,000 dilution) for 1 h on the orbital tilting
shaker at room temperature. After 1 h of incubation, the blots were
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
incubated with ECF reagent for 5 minutes and then each blot scanned
with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green
laser, at 400V and at 500 V.
Experimental Protocol for LDH and Bik:
[0584] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were scanned in laser scanner to check for complete
stripping. The blots were activated with methanol for 5 seconds,
washed with water for 5 minutes, and TBST for 15 minutes. The blots
were blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for LDH or
Bik in 5% BSA in TBS-T by incubation overnight at 4 deg C. with
gentle shaking. The primary antibody for LDH was from Abcam ab2101;
antigoat; at 1:1000 dilution. The primary antibody for Bik was from
Cell Signaling #9942; antirabbit; at 1:1000 dilution. After
incubation with primary antibodies, the membranes were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and probed with
the secondary antibody (LDH antigoat; Jackson Laboratories) and Bik
antirabbit; 1:10,000 dilution) for 1 h on the orbital tilting
shaker at room temperature. After 1 h of incubation, the blots were
washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
incubated with ECF reagent for 5 minutes and then each blot scanned
with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green
laser, at 400V and at 500 V.
Western Blot Experiment 9
[0585] The cells used were HepG2 cells that were treated or not
with Coenzyme Q10 at two different concentrations, 50 .mu.M or 100
.mu.M, and harvested after 24 or 48 hours of treatment. The HepG2
samples processed and the gels were run, transferred, stained and
scanned essentially as described above.
Experimental Protocol for Actin:
[0586] The levels of actin were determined by probing the blots
with a primary antibody for actin, essentially as described
above.
Experimental Protocol for Caspase3 and MMP-6:
[0587] The Actin blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots were
blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for Caspase 3
or MMP-6 in 5% BSA by incubation overnight at 4 deg C. with gentle
shaking. The primary antibody for Caspase 3 (Abcam ab44976-100;
antirabbit) was at 1:500 dilution in 5% BSA. The primary antibody
for MMP-6 (Santacruz scMM0029-ZB5; antimouse) was at 1:100 dilution
in 5% BSA. After incubation with primary antibodies, the membranes
were washed 3 times with TBS-T (1.times.-15'; 2.times.5' each) and
probed with the secondary antibody (MMP-6 antimouse; Caspase 3
antirabbit; 1:10,000 dilution) for 1 h at room temperature. After 1
h of incubation with secondary antibodies, the blots were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and then incubated
with ECF reagent for 5 minutes and then each blot scanned with 5100
Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at
400V and at 500 V.
Experimental Protocol for LDH:
[0588] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots ere blocked
for 1 hour with 5% blocking reagent in TBS-T at room temperature
and then washed 3 times with TBS-T (1.times.-15'; 2.times.5' each)
and probed with the primary antibody for LDH in 5% BSA or 5% milk
by incubation overnight at 4 deg C. with gentle shaking. The
primary antibody for LDH 080309b1 (Abcam ab2101; antigoat) was at
1:1000 dilution in 5% BSA. The primary antibody for LDH 080309b2
(Abcam ab2101; antigoat) was at 1:1000 dilution in 5% milk. After
incubation with primary antibodies, the membranes were washed 3
times with TBS-T (1.times.-15'; 2.times.5' each) and probed with
the secondary antibody (Jackson Immuno Research antigoat; 1:10,000
dilution; 305-055-045) for 1 h. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400V and at
500 V.
Experimental Protocol for Transaldolase and Hif1a:
[0589] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots are blocked
for 1 hour with 5% blocking reagent in TBS-T at room temperature
and then washed 3 times with TBS-T (1.times.-15'; 2.times.5' each)
and probed with the primary antibody for Transaldolase or Hif1a in
5% BSA by incubation overnight at 4 deg C. with gentle shaking. The
primary antibody for Transaldolase (Abcam ab67467; antimouse) was
at 1:500 dilution. The primary antibody for Hif1a (Abcam ab2185;
antirabbit) was at 1:500 dilution. After incubation with primary
antibodies, the membranes were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antimouse or antirabbit; 1:10,000 dilution) for 1 h on
the orbital tilting shaker at room temperature. After 1 h of
incubation with secondary antibodies, the blots were washed 3 times
with TBS-T (1.times.-15'; 2.times.5' each) and then incubated with
ECF reagent for 5 minutes and then each blot scanned with 5100 Fuji
Laser scanner at 25 uM resolution, 16 bit, green laser, at 400
& 500V.
Experimental Protocol for IGFBP3 and TP53:
[0590] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots are blocked
for 1 hour with 5% blocking reagent in TBS-T at room temperature
and then washed 3 times with TBS-T (1.times.-15'; 2.times.5' each)
and probed with the primary antibody for IGFBP3 or TP53 in 5% BSA
by incubation overnight at 4 deg C. with gentle shaking. The
primary antibody for IGFBP3 (Abcam ab76001; antirabbit) was at
1:100 dilution. The primary antibody for TP53 (Sigma Aldrich
AV02055; antirabbit) was at 1:100 dilution. After incubation with
primary antibodies, the membranes were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and probed with the secondary
antibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital
tilting shaker at room temperature. After 1 h of incubation with
secondary antibodies, the blots were washed 3 times with TBS-T
(1.times.-15'; 2.times.5' each) and then incubated with ECF reagent
for 5 minutes and then each blot scanned with 5100 Fuji Laser
scanner at 25 uM resolution, 16 bit, green laser, at 400 &
500V.
Experimental Protocol for Transaldolase and PDHB:
[0591] The above blots were stripped by incubating for 30 minutes
with methanol, followed by two 10 minute washes with TBS-T, then 30
minutes of incubation with Stripping buffer at 50 deg C., and
followed by two washes with 100 ml or more of TBS-T for 30' each.
The blots were activated with methanol for 5 seconds, washed with
water for 5 minutes, and TBST for 15 minutes. The blots were
blocked for 1 hour with 5% blocking reagent in TBS-T at room
temperature and then washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and probed with the primary antibody for
Transaldolase or PDHB in 5% BSA by incubation overnight at 4 deg C.
with gentle shaking. The primary antibody for Transaldolase
(Santacruz sc51440; antigoat) was at 1:200 dilution. The primary
antibody for PDHB (Novus Biologicals H00005162-M03; antimouse) was
at 1:500 dilution. After incubation with primary antibodies, the
membranes were washed 3 times with TBS-T (1.times.-15'; 2.times.5'
each) and probed with the secondary antibody (antigoat or
antimouse; 1:10,000 dilution) for 1 h on the orbital tilting shaker
at room temperature. After 1 h of incubation with secondary
antibodies, the blots were washed 3 times with TBS-T (1.times.-15';
2.times.5' each) and then incubated with ECF reagent for 5 minutes
and then each blot scanned with 5100 Fuji Laser scanner at 25 uM
resolution, 16 bit, green laser, at 400 & 500V.
Results
Isocitrate Dehydrogenase-1 (IDH-1)
[0592] Isocitrate dehydrogenase is one of the enzymes that is part
of the TCA cycle that usually occurs within the mitochondrial
matrix. However, IDH1 is the cytosolic form of the enzyme that
catalyzes the oxidative decarboxylation of isocitrate to
.alpha.-ketoglutarate and generates carbon dioxide in a two step
process. IDH1 is the NADP.sup.+ dependent form that is present in
the cytosol and peroxisome. IDH1 is inactivated by Ser113
phosphorylation and is expressed in many species including those
without a citric acid cycle. IDH1 appears to function normally as a
tumor suppressor which upon inactivation contributes to
tumorigenesis partly through activation of the HIF-1 pathway
(Bayley 2010; Reitman, 2010). Recent studies have implicated an
inactivating mutation in IDH1 in the etiology of glioblasotoma
(Bleeker, 2009; Bleeker, 2010).
[0593] Treatment with Coenzyme Q10 increased expression of IDH1 in
cancer cell lines including MCF-7, SKMEL28, HepG2 and PaCa-2 cells.
There was a moderate increase in expression in the SCC25 cell
lines. In contrast cultures of primary human derived fibroblasts
HDFa, nFIB and the human aortic smooth muscle cells HASMC did not
demonstrate significant changes in the expression pattern of the
IDH1 in response to Coenzyme Q10. .alpha.-ketoglutarate
(.alpha.-KG) is a key intermediate in the TCA cycle, biochemically
synthesized from isocitrate and is eventually converted to succinyl
coA and is a druggable MIM and EpiShifter. The generation of
.alpha.-KG serves as a critical juncture in the TCA cycle as it can
be used by the cell to replenish intermediates of the cycle,
resulting in generation of reducing equivalents to increase
oxidative phosphorylation. Thus, Coenzyme Q10 mediated increase in
IDH1 expression would result in formation of intermediates that can
be used by the mitochondrial TCA cycle to augment oxidative
phosphorylation in cancer cells. The results are summarized in
Tables 30-32, below.
TABLE-US-00031 TABLE 30 IDH1 in HDFa and MCF-7 Composition Average
Normalized Intensity HDF, Media 346 HDF24-50-Coenzyme Q10 519
HDF24-100-Coenzyme Q10 600 MCF, Media 221 MCF24-50-Coenzyme Q10 336
MCF24-100-Coenzyme Q10 649
TABLE-US-00032 TABLE 31 IDH1 in HASMC vs. HepG2 after Treatment
Amount - Composition Normalized Intensity HAS5g48-media 20
HAS5g48-50-Coenzyme Q10 948 HAS5g48-100-Coenzyme Q10 1864
HAS22G48-Media 1917 HAS22G48-50-Coenzyme Q10 1370
HAS22G48-100-Coenzyme Q10 1023 Hep5g48-Media 14892
Hep5g48-50-Coenzyme Q10 14106 Hep5g48-100-Coenzyme Q10 15774
Hep22G48-Media 16558 Hep22G48-50-Coenzyme Q10 15537
Hep22G48-100-Coenzyme Q10 27878
TABLE-US-00033 TABLE 32 IDH1 in HASMC vs. PACA2 after Treatment
Amount - Composition Normalized Intensity HAS5g48-media 562
HAS5g48-50-Coenzyme Q10 509 HAS5g48-100-Coenzyme Q10 627
HAS22G48-Media 822 HAS22G48-50-Coenzyme Q10 1028
HAS22G48-100-Coenzyme Q10 1015 PACA5g48-Media 1095
PACA5g48-50-Coenzyme Q10 1095 PACA5g48-100-Coenzyme Q10 860
PACA22G48-Media 1103 PACA22G48-50-Coenzyme Q10 1503
PACA22G48-100-Coenzyme Q10 1630
ATP Citrate Lyase (ACL)
[0594] ATP citrate Lyase (ACL) is a homotetramer (.about.126 kd)
enzyme that catalyzes the formation of acteyl-CoA and oxaloacetate
in the cytosol. This reaction is a very important first step for
the biosynthesis of fatty acids, cholesterol, and acetylcholine, as
well as for glucogenesis (Towle et al., 1997). Nutrients and
hormones regulate the expression level and phosphorylation status
of this key enzyme. Ser454 phosphorylation of ACL by Akt and PKA
has been reported (Berwick., D C M W et al., 2002; Pierce M W et
al., 1982).
[0595] The data describes the effect of Coenzyme Q10 on ATP citrate
Lyase is that in normal and cancer cells. It is consistently
observed that in cancer cells there is a dose-dependent decrease in
the expression of ACL enzymes. In contrast there appears to be a
trend towards increased expression of ACL in normal cells.
Cytosolic ACL has been demonstrated to be essential for histone
acetylation in cells during growth factor stimulation and during
differentiation. The fact that ACL utilizes cytosolic glucose
derived citrate to generate Acetyl CoA essential for histone
acetylation, a process important in the neoplastic process
demonstrates a role of Coenzyme Q10 induced ACL expression in
influencing cancer cell function. Acetyl CoA generated from citrate
by cytosolic ACL serves as a source for biosynthesis of new lipids
and cholesterol during cell division. Thus, Coenzyme Q10 induced
changes in ACL expression alters Acetyl CoA availability for
synthesis of lipids and cholesterol in normal versus cancer cells.
The results are summarized in tables 33-36 below.
TABLE-US-00034 TABLE 33 ATPCL in HDFa and MCF-7 Composition Average
Normalized Intensity HDF-Media ~15000 HDF-50-Coenzyme Q10 ~17500
HDF-100-Coenzyme Q10 ~25000 MCF-Media ~7500 MCF-50-Coenzyme Q10
~7500 MCF-100-Coenzyme Q10 ~12500
TABLE-US-00035 TABLE 34 ATP Citrate Lysase ~kd band in HASMC vs.
HepG2 Amount - Composition Normalized Intensity HAS5g48-media 24557
HAS5g48-50-Coenzyme Q10 23341 HAS5g48-100-Coenzyme Q10 25544
HAS22G48-Media 27014 HAS22G48-50-Coenzyme Q10 21439
HAS22G48-100-Coenzyme Q10 19491 Hep5g48-Media 28377
Hep5g48-50-Coenzyme Q10 24106 Hep5g48-100-Coenzyme Q10 22463
Hep22G48-Media 24262 Hep22G48-50-Coenzyme Q10 31235
Hep22G48-100-Coenzyme Q10 50588
TABLE-US-00036 TABLE 35 ATP Citrate Lysase ~kd band in HASMC vs.
PACA2 Amount - Composition Normalized Intensity HAS5g48-media 11036
HAS5g48-50-Coenzyme Q10 12056 HAS5g48-100-Coenzyme Q10 15265
HAS22G48-Media 18270 HAS22G48-50-Coenzyme Q10 15857
HAS22G48-100-Coenzyme Q10 13892 PACA5g48-Media 11727
PACA5g48-50-Coenzyme Q10 8027 PACA5g48-100-Coenzyme Q10 4942
PACA22G48-Media 8541 PACA22G48-50-Coenzyme Q10 9537
PACA22G48-100-Coenzyme Q10 14901
TABLE-US-00037 TABLE 36 ATP Citrate Lysase in HepG2 and PACA2 as %
of CTRL Amount - Composition Normalized Intensity PACA5g48-Media
1.00 PACA5g48-50-Coenzyme Q10 0.68 PACA5g48-100-Coenzyme Q10 0.42
PACA22G48-Media 1.00 PACA22G48-50-Coenzyme Q10 1.12
PACA22G48-100-Coenzyme Q10 1.74 Hep5g48-Media 1.00
Hep5g48-50-Coenzyme Q10 0.85 Hep5g48-100-Coenzyme Q10 0.79
Hep22G48-Media 1.00 Hep22G48-50-Coenzyme Q10 1.29
Hep22G48-100-Coenzyme Q10 2.09
Pyruvate Kinase M2 (PKM2)
[0596] Pyruvate Kinase is an enzyme involved in the glycolytic
pathway. It is responsible for the transfer of phosphate from
phosphoenolpyruvate (PEP) to adenosine diphosphophate (ADP) to
generate ATP and pyruvate. PKM2 is an isoenzyme of the glycolytic
pyruvate kinase, expression of which is characterized by the
metabolic function of the tissue i.e. M2 isoenzyme is expressed in
normal rapidly proliferating cells with high energy needs such as
embryonic cells and also expressed in few normal differentiated
tissues such as lung and pancreatic islet cells that require high
rate of nucleic acid synthesis. PKM2 is highly expressed in tumor
cells due to their dependence on glycolytic pathway for meeting
cellular energetic requirements. The PKM2 isoform normally thought
to be embryonically restricted is re-expressed in cancerous cells.
Cells expressing PKM2 favor a stronger aerobic glycolytic phenotype
(show a shift in metabolic phenotype) with increased lactate
production and decreased oxidative phosphorylation. Thus, decrease
in expression of PKM2 in cancer cells would shift or down-regulate
energy generation via the glycolytic pathway, a strategy that is
useful in the treatment of cancer. Data demonstrates variable
expression pattern of PKM2 in normal and cancer cells, with cancer
cells demonstrating higher levels of expression compared to normal.
Treatment of cells with Coenzyme Q10 altered expression pattern of
the PKM2 upper and lower band levels in normal and cancer cells. In
cancer cells tested, there was a dose-dependent decrease in the
PKM2 expression, and no major changes in normal cells were
observed. The results are summarized in tables 37-39, below.
TABLE-US-00038 TABLE 37 Pyruvate Kinase Muscle form 2 Upper Band in
HepG2 Normalized Volume Normalized Amount - Composition (24 h)
Intensity (48 h) 5g-Media 28386 413 5g-50-Coenzyme Q10 29269 303
5g-100-Coenzyme Q10 18307 354 22G-Media 25903 659 22G-50-Coenzyme
Q10 22294 562 22G-100-Coenzyme Q10 19560 601
TABLE-US-00039 TABLE 38 Pyruvate Kinase Muscle form 2 Lower Band
(58 KD) in HepG2 Normalized Normalized Amount - Composition Volume
(24 h) Volume (48 h) 5g-Media 10483 310 5g-50-Coenzyme Q10 11197
185 5g-100-Coenzyme Q10 7642 122 22G-Media 9150 306 22G-50-Coenzyme
Q10 6302 344 22G-100-Coenzyme Q10 6904 465
TABLE-US-00040 TABLE 39 Pyruvate Kinase Muscle form 2 Upper Band in
HASMC Cells after Treatment Amount - Composition Normalized
Intensity 5g48-Media 608 5g48-50-Coenzyme Q10 811 5g48-100-Coenzyme
Q10 611 22G48-Media 516 22G48-50-Coenzyme Q10 595
22G48-100-Coenzyme Q10 496 22G24-Media 301 22G24-50-Coenzyme Q10
477 22G24-100-Coenzyme Q10 701
Lactate Dehydrogenase (LDH)
[0597] LDH is an enzyme that catalyzes the interconversion of
pyruvate and lactate with the simultaneous interconversion of NADH
and NAD.sup.+. It has the ability to convert pyruvate to lactate
(lactic acid) under low cell oxygen tension for generation of
reducing equivalents and ATP generation at the expense of
mitochondrial oxidative phosphorylation. Cancer cells typically
demonstrate increased expression of LDH to maintain the glycolytic
flux to generate ATP and reducing equivalents and reducing
mitochondrial OXPHOS. Thus, reducing the expression of the LDH in
cancer cells would shift metabolism from generation of lactate to
facilitate entry of pyruvate into the TCA cycle. Treatment with
Coenzyme Q10 reduced Lactate Dehydrogenase (LDH) expression in
cancer with minimal effect on normal cells, supporting a role for
Coenzyme Q10 in eliciting a shift in cancer cell bioenergtics for
the generation of ATP from glycolytic to mitochondrial OXPHOS
sources by minimizing the conversion of cytoplasmic pyruvate to
lactic acid. The results are summarized in tables 40-42, below.
TABLE-US-00041 TABLE 40 Lactate Dehydrogenase in HepG2 Normalized
Normalized Amount - Composition Volume (24 h) Volume (48 h)
5g-Media 7981 5997 5g-50-Coenzyme Q10 7900 5188 5g-100-Coenzyme Q10
6616 7319 22G-Media 9171 7527 22G-50-Coenzyme Q10 7550 6173
22G-100-Coenzyme Q10 7124 9141
TABLE-US-00042 TABLE 41 Lactate Dehydrogenase in HepG2 as % Control
from 2 Experiments Average Volume as a Amount - Composition % of
Control 5g24-Media 1.00 5g24-50-Coenzyme Q10 0.64 5g24-100-Coenzyme
Q10 1.06 5g48-Media 1.00 5g48-50-Coenzyme Q10 1.12
5g48-100-Coenzyme Q10 1.21 22G24-Media 1.00 22G24-50-Coenzyme Q10
1.21 22G24-100-Coenzyme Q10 1.44 22G48-Media 1.00 22G48-50-Coenzyme
Q10 0.95 22G48-100-Coenzyme Q10 0.67
TABLE-US-00043 TABLE 42 Lactate Dehydrogenase in PACA2 Normalized
Volume Normalized Amount - Composition (24 h) Volume (48 h)
5g-Media 2122 2360 5g-50-Coenzyme Q10 5068 2978 5g-100-Coenzyme Q10
3675 2396 22G-Media 4499 2332 22G-50-Coenzyme Q10 10218 2575
22G-100-Coenzyme Q10 7158 3557
Pyruvate Dehydrogenase--B (PDH-E1)
[0598] Pyruvate Dehydrogenase beta (PDH-E1) is the first enzyme
component that is part of the pyruvate dehydrogenase complex (PDC)
that converts pyruvate to acetyl CoA. PDH-E1 requires thiamine as
cofactor for its activity, performs the first two biochemical
reactions in the PDC complex essential for the conversion of
pyruvate to acetyl CoA to enter the TCA cycle in the mitochondria.
Thus, concomitant decreases in PKM2 and LDH expression along with
increase in expression of PDH-E1 in cancer cells would enhance the
rate of entry of pyruvate towards augmenting the mitochondrial
OXPHOS for generation of ATP. The data shows that for expression of
PDH-E1 in normal and cancer cell lines, the baseline expressions of
this enzyme is decreased in cancer compared to normal cells.
Treatment with Coenzyme Q10 is associated with progressive increase
in the expression of the PDH-E1 proteins in cancer cells with
minimal changes in the normal cells. The results are summarized in
tables 43-45, below.
TABLE-US-00044 TABLE 43 Pyruvate Dehydrogenase Beta in HepG2
Normalized Normalized Amount - Composition Volume (24 h) Volume (48
h) 5g-Media 517 100 5g-50-Coenzyme Q10 921 123 5g-100-Coenzyme Q10
433 205 22G-Media 484 181 22G-50-Coenzyme Q10 426 232
22G-100-Coenzyme Q10 340 456
TABLE-US-00045 TABLE 44 Pyruvate Dehydrogenase Beta in PACA2
Normalized Normalized Amount - Composition Volume (24 h) Volume (48
h) 5g-Media 323 375 5g-50-Coenzyme Q10 492 339 5g-100-Coenzyme Q10
467 252 22G-Media 572 276 22G-50-Coenzyme Q10 924 279
22G-100-Coenzyme Q10 1201 385
TABLE-US-00046 TABLE 45 Pyruvate Dehydrogenase Beta in HASMC after
Treatment Amount - Composition Normalized Volume 5g48-Media 140
5g48-50-Coenzyme Q10 147 5g48-100-Coenzyme Q10 147 22G48-Media 174
22G48-50-Coenzyme Q10 149 22G48-100-Coenzyme Q10 123 22G24-Media
140 22G24-50-Coenzyme Q10 145 22G24-100-Coenzyme Q10 150
Caspase 3
[0599] Control of the onset of apoptosis is often exerted at the
level of the initiator caspases, caspase-2, -9 and -8/10. In the
extrinsic pathway of apoptosis, caspase-8, once active, directly
cleaves and activates executioner caspases (such as caspase-3). The
active caspase-3 cleaves and activates other caspases (6, 7, and 9)
as well as relevant targets in the cells (e.g. PARP and DFF). In
these studies, the levels of effectors caspase-3 protein were
measured in the cancer cell lines and in normal cell lines in
response to Coenzyme Q10. It should be noted although control of
apoptosis is through initiator caspases, a number of signaling
pathways interrupt instead the transmission of the apoptotic signal
through direct inhibition of effectors caspases. For e.g. P38 MAPK
phosphorylates caspase-3 and suppresses its activity
(Alvarado-Kristensson et al., 2004). Interestingly, activation of
protein phosphates (PP2A) in the same study or protein kinase C
delta (PKC delta) (Voss et al., 2005) can counteract the effect of
p38 MAPK to amplify the caspase-3 activation and bolster the
transmission of the apoptotic signal. Therefore, events at the
level of caspase-3 activation or after Caspase 3 activation may
determine the ultimate fate of the cell in some cases.
[0600] Caspase-3 is a cysteine-aspartic acid protease that plays a
central role in the execution phase of cell apoptosis. The levels
of caspase 3 in the cancer cells were increased with Coenzyme Q10
treatment. In contrast the expression of Caspase-3 in normal cells
was moderately decreased in normal cells. The results are
summarized in tables 46-48, below.
TABLE-US-00047 TABLE 46 Caspase 3 in PACA2 Normalized Normalized
Amount - Composition Volume (24 h) Volume (48 h) 5g-Media 324 300
5g-50-Coenzyme Q10 325 701 5g-100-Coenzyme Q10 374 291 22G-Media
344 135 22G-50-Coenzyme Q10 675 497 22G-100-Coenzyme Q10 842
559
TABLE-US-00048 TABLE 47 Caspase 3 in HepG2 cells as % Control from
2 Experiments Normalized Volume as Amount - Composition a % of
Control 5g24-Media 1..00 5g24-50-Coenzyme Q10 1.08
5g24-100-Coenzyme Q10 1.76 5g48-Media 1.00 5g48-50-Coenzyme Q10
1.44 5g48-100-Coenzyme Q10 0.95 22G24-Media 1.00 22G24-50-Coenzyme
Q10 1.39 22G24-100-Coenzyme Q10 1.78 22G48-Media 1.00
22G48-50-Coenzyme Q10 1.50 22G48-100-Coenzyme Q10 1.45
TABLE-US-00049 TABLE 48 Caspase 3 in HASMC after Treatment Amount -
Composition Normalized Volume 5g48-Media 658 5g48-50-Coenzyme Q10
766 5g48-100-Coenzyme Q10 669 22G48-Media 846 22G48-50-Coenzyme Q10
639 22G48-100-Coenzyme Q10 624 22G24-Media 982 22G24-50-Coenzyme
Q10 835 22G24-100-Coenzyme Q10 865
Succinate Dehydrogenase (SDH)
[0601] Succinate dehydrogenase, also known as succinate-coenzyme Q
reductase is a complex of the inner mitochondrial membrane that is
involved in both TCA and electron transport chain. In the TCA, this
complex catalyzes the oxidation of succinate to fumarate with the
concomitant reduction of ubiquinone to ubiquinol. (Baysal et al.,
Science 2000; and Tomlinson et al., Nature Genetics 2002). Germline
mutations in SDH B, C and D subunits were found to be initiating
events of familial paraganglioma or leiomyoma (Baysal et al.,
Science 2000).
[0602] Western blotting analysis was used to characterize
expression of SDH Subunit B in mitochondrial preparations of cancer
cells treated with Coenzyme Q10. The results suggest that Coenzyme
Q10 treatment is associated with increase SDH protein levels in the
mitochondrion of the cells. These results suggest one of the
mechanisms of action of Coenzyme Q10 is to shift the metabolism of
the cell towards the TCA cycle and the mitochondrion by increasing
the levels of mitochondrial enzymes such as SDHB. The results are
summarized in table 49, below.
TABLE-US-00050 TABLE 49 Succinate Dehydrogenase B in NCIE0808
Mitopreps Composition - Time Average Normalized Volume Media 531 50
uM Coezyme Q10, 3 h 634 100 uM Coenzyme Q10, 3 h 964 50 uM Coenzyme
Q10, 6 h 1077 100 uM Coenzyme Q10, 6 h 934
Hypoxia Induced Factor--1
[0603] Hypoxia inducible factor (Hif) is a transcription factor
composed of alpha and beta subunits. Under normoxia, the protein
levels of Hif1 alpha are very low owing to its continuous
degradation via a sequence of post translational events. The shift
between glycolytic and oxidative phosphorylation is generally
considered to be controlled by the relative activities of two
enzymes PDH and LDH that determine the catabolic fate of pyruvate.
Hif controls this crucial bifurgation point by inducing LDH levels
and inhibiting PDH activity by stimulating PDK. Due to this ability
to divert pyruvate metabolism from mitochondrion to cytosol, Hif is
considered a crucial mediator of the bioenergetic switch in cancer
cells.
[0604] Treatment with Coenzyme Q10 decreased Hif1 alpha protein
levels after in mitochondrial preparations of cancer cells. In
whole cell lysates of normal cells, the lower band of Hif1a was
observed and showed a decrease as well. The results are summarized
in tables 50-51, below.
TABLE-US-00051 TABLE 50 Hif1 alpha Lower Band in HASMC Cells after
Treatment Amount - Composition Normalized Volume 5g48-Media 22244
5g48-50-Coenzyme Q10 21664 5g48-100-Coenzyme Q10 19540 22G48-Media
14752 22G48-50-Coenzyme Q10 17496 22G48-100-Coenzyme Q10 23111
22G24-Media 21073 22G24-50-Coenzyme Q10 18486 22G24-100-Coenzyme
Q10 17919
TABLE-US-00052 TABLE 51 Hif1 alpha Upper Band in HepG2 after
Treatment Amount - Composition Normalized Volume 5g24-Media 12186
5g24-50-Coenzyme Q10 8998 5g24-100-Coenzyme Q10 9315 5g48-Media
8868 5g48-50-Coenzyme Q10 8601 5g48-100-Coenzyme Q10 10192
22G24-Media 11748 22G24-50-Coenzyme Q10 14089 22G24-100-Coenzyme
Q10 8530 22G48-Media 8695 22G48-50-Coenzyme Q10 9416
22G48-100-Coenzyme Q10 5608
Example 22
Analysis of Oxygen Consumption Rates (OCR) and Extracellular
Acidification (ECAR) in Normal and Cancer cCells Treated with
CoQ10
[0605] This example demonstrates that exposure of cells to
treatment by a representative MIM/epi-shifter of the
invention--CoQ10--in the absence and/or presence of stressors
(e.g., hyperglycemia, hypoxia, lactic acid), is associated with a
shift towards glycolysis/lactate biosynthesis and mitochondrial
oxidative phosphorylation (as measured by ECAR and OCR values)
representative of values observed in a normal cells under normal
physiological conditions.
[0606] Applicants have demonstrated in the previous section that
treatment with CoQ10 in cancer cells is associated with changes in
expression of specific proteins that enhance mitochondrial
oxidative phosphorylation, with a concomitant decrease in
glycolysis and lactate biosynthesis. This example shows that a
direct measure of mitochondrial oxidative phosphorylation can be
obtained by measuring the oxygen consumption rates (OCR) in cell
lines using the SeaHorse XF analyzer, an instrument that measures
dissolved oxygen and extracellular pH levels in an in vitro
experimental model. (SeaHorse Biosciences Inc, North Billerica,
Mass.).
[0607] The pH of the extracellular microenvironment is relatively
acidic in tumors compared to the intracellular (cytoplasmic) pH and
surrounding normal tissues. This characteristic of tumors serves
multiple purposes, including the ability to invade the
extracellular matrix (ECM), a hallmark attribute of tumor
metastasis that subsequently initiates signaling cascades that
further modulate: [0608] tumor angiogenesis [0609] increased
activation of arrest mechanisms that control cell cycle turn-over
[0610] immuno-modulatory mechanisms that facilitate a cellular
evasion system against immunosurveillance [0611] metabolic control
elements that increase dependency on glycolytic flux and lactate
utilization [0612] dysregulation of key apopototic gene families
such as Bcl-2, IAP, EndoG, AIF that serve to increase
oncogenicity
[0613] While not wishing to be bound by any particular theory, the
acidic pH of the external microenvironment in the tumor is a
consequence of increase in hydrogen ion concentrations extruded
from the tumor cells due to the increased lactate production from
an altered glycolytic phenotype.
[0614] In this experiment, the OCR and extracellular acidification
rate (ECAR) in normal cells lines were obtained in the presence and
absence of CoQ10 to determine baseline values. It was observed that
in its native nutrient environment, the basal OCR rates in normal
cells lines are different, and are usually a function of the
physiological roles of the cells in the body.
[0615] For example, one set of experiments were conducted using the
non-cancerous cell line HDFa, which is a human adult dermal
fibroblast cell line. Fibroblasts are cells that primarily
synthesize and secrete extracellular matrix (ECM) components and
collagen that form the structural framework (stroma) for tissues.
In addition, fibroblasts are known to serve as tissue ambassadors
of numerous functions such as wound healing and localized
immunomodulation. Under normal physiological conditions, energy
requirements in normal fibroblasts are met using a combination of
glycolysis and oxidative phosphorylation--the glycolysis providing
the necessary nutrients for synthesis of ECM.
[0616] In contrast to HDFa, the HASMC (human aortic smooth muscle
cell) is found in arteries, veins, lymphatic vessels,
gastrointestinal tracts, respiratory tract, urinary bladder and
other tissues with the ability to undergo regulated
excitation-contraction coupling. The ability of smooth muscles such
as HASMC cells to undergo contraction requires energy provided by
ATP. These tissues transition from low energy modes wherein ATP may
be supplied from mitochondria to high energy modes (during
exercise/stress) where energy is provided by switching to
glycolysis for rapid generation of ATP. Thus, normal smooth muscle
cells can use a combination of mitochondrial OXPHOS and glycolysis
to meet their energy requirements under normal physiological
environment.
[0617] The differences in their respective physiological roles
(i.e., HDFa and HASMC) were observed in the resting OCR values
measured in these cells lines using the SeaHorse XF analyzer. FIGS.
29 and 30 describe the OCR in HDFa and HASMC cells grown in
physiologically normal glucose (about 4.6 mM) and high glucose
(hyperglycemic) conditions.
[0618] The baseline OCR values for HDFa in the absence of any
treatments under normal oxygen availability is approximately 40
pmoles/min (FIG. 29) in the presence of 5.5 mM glucose. This value
was slightly elevated when the cells were maintained at 22 mM
glucose. In contrast, in HASMC cells, the OCR values at 5.5 mM
glucose is approximately 90 pmoles/min, and the OCR value declined
to approximately 40 pmoles/min while at 22 mM glucose. Thus, under
hyperglycemic conditions, there is a differential response between
HDFa and HASMC, further demonstrating inherent differences in their
respective physiological make-up and function.
[0619] Treatment with CoQ10 in cells is associated with changes in
OCR that is representative of conditions observed at normal (5 mM)
glucose conditions. The complexity of physiological response is
compounded in the presence of low oxygen tension. Thus, CoQ10
exposure is associated with changes in OCR rates in normal cells
towards a physiological state that is native to a particular
cell.
[0620] Table 52, below, describes the ECAR values (mpH/min) in HDFa
cells in the presence or absence of CoQ10 under normoxic and
hypoxic conditions at 5.5 mM and 22 mM glucose. It can be observed
that in normal cells, treatment with CoQ10 had minimal influence on
ECAR values, even though it influenced OCR in these cells. In high
glucose hypoxic conditions, treatment with CoQ10 was associated
with lowering of elevated ECAR to a value that was observed in
untreated normoxic conditions.
TABLE-US-00053 TABLE 52 ECAR values in HDFa cells in the absence
and presence of CoQ10 under normoxic and hypoxic conditions at 5.5
mM and 22 mM glucose Normoxia Hypoxia Normoxia Hypoxia (5.5 mM)
(5.5 mM) (22 mM) (22 mM) Treatment ECAR SEM ECAR SEM ECAR SEM ECAR
SEM Untreated 5 1.32 5 0.62 5 0.62 9 0.81 50 .mu.M 6 1.11 5 0.78 5
0.78 6 0.70 31510 100 .mu.M 6 0.76 5 1.19 5 1.19 8 1.07 31510
[0621] In Table 53 the measured baseline ECAR values (mpH/min) in
HASMC were higher compared to that of HDFa. Induction of hypoxic
conditions caused an increase in ECAR most likely associated with
intracellular hypoxia induced acidosis secondary to increased
glycolysis.
TABLE-US-00054 TABLE 53 ECAR values in HASMC cells in the absence
and presence of CoQ10 under normoxic and hypoxic conditions at 5.5
mM and 22 mM glucose Normoxic Hypoxic Normoxic Hypoxic (5.5 mM)
(5.5 mM) (22 mM) (22 mM) Treatment ECAR SEM ECAR SEM ECAR SEM ECAR
SEM Untreated 9 2.22 11 2.18 22 2.08 19 1.45 50 .mu.M 9 2.13 11
2.54 21 1.72 17 1.60 31510 100 .mu.M 9 1.72 13 2.30 22 1.64 17 1.47
31510
[0622] Treatment with CoQ10 was observed to be associated with a
downward trend of ECAR rates in hyperglycemic HASMC cells in
hypoxic conditions towards a value that would be observed in
normoxic normal glucose conditions. These data demonstrate the
presence of physiological variables that is inherent to the
physiological role of a specific type of cell, alterations observed
in abnormal conditions (e.g. hyperglycemia) is shifted towards
normal when treated with CoQ10.
[0623] In contrast, cancer cells (e.g., MCF-7, PaCa-2) are
inherently primed to culture at higher levels of glucose compared
to normal cells due to their glycolytic phenotype for maintenance
in culture. Treatment with CoQ10 caused a consistent reduction in
OCR values (FIG. 31 and FIG. 32).
[0624] The effects of CoQ10 on OCR values in MCF-7 and PaCa-2 cells
was similar to that of the normal HDFa and HASMC cells, wherein the
variable response was suggestive of a therapeutic response based on
individual metabolic profile of the cancer cell line.
TABLE-US-00055 TABLE 54 ECAR values in PaCa-2 cells in the absence
and presence of CoQ10 under normoxic and hypoxic conditions at 5.5
mM and 22 mM glucose Normoxia Hypoxia Normoxia Hypoxia (17 mM) (17
mM) (22 mM) (22 mM) Treatment ECAR SEM ECAR SEM ECAR SEM ECAR SEM
Untreated 21 5.97 16 3.41 24 4.35 36 5.65 50 .mu.M 13 3.08 12 1.66
20 5.15 25 4.58 31510 100 .mu.M 14 2.14 17 2.59 19 3.38 30 5.62
31510
[0625] Table 54 describes the ECAR values in PaCa-2 cells. In
contrast to normal cells, cancer cells are phenotypically primed to
use high glucose for ATP generation (enhanced glycolysis) resulting
in higher ECAR (Table 54, ECAR for untreated normoxia 17 mM) at 21
mpH/min. Treatment with CoQ10 produces a significant decrease in
ECAR rates under these conditions, most likely associated with a
decrease in the glycolysis generated lactic acid. The associated
decrease in OCR in these cells was likely associated with increased
efficiency of the mitochondrial OXPHOS.
[0626] A similar comparison of OCR and ECAR values (data not shown)
were determined in numerous other normal and cancer cells lines,
including: HAEC (normal human aortic endothelial cells), MCF-7
(breast cancer), HepG2 (liver cancer) and highly metastatic PC-3
(prostate cancer) cell lines. In all of the cell lines tested,
exposure to CoQ10 in the absence and/or presence of stressors
(e.g., hyperglycemia, hypoxia, lactic acid) was associated with a
shift in OCR and ECAR values representative of values observed in a
normal cells under normal physiological conditions. Thus, the
overall effect of CoQ10 in the treatment of cancer, including cell
death, is an downstream effect of its collective influence on
proteomic, genomic, metabolomic outcomes in concert with shifting
of the cellular bioenergetics from glycolysis to mitochondrial
OXPHOS.
Example 23
Building Block Molecules for the Biosynthesis of CoQ10
[0627] This example demonstrates that certain precursors of CoQ10
biosynthesis, such as those for the biosynthesis of the
benzoquinone ring, and those for the biosynthesis of the isoprenoid
repeats and their attachment to the benzoquinone ring ("building
block components"), can be individually administered or
administered in combination to target cells, and effect
down-regulation of the apoptosis inhibitor Bcl-2, and/or
up-regulation of the apoptosis promoter Caspase-3. Certain
precursors or combinations thereof may also inhibit cell
proliferation. The data suggests that such CoQ10 precursors may be
used in place of CoQ10 to achieve substantially the same results as
CoQ10 administration.
[0628] Certain exemplary experimental conditions used in the
experiments are listed below.
[0629] Skmel-28 melanoma cells were cultured in DMEM/F12
supplemented with 5% Fetal Bovine Serum (FBS) and 1.times. final
concentration of Antibiotics. The cells were grown to 85%
confluency and treated with building block components for 3, 6, 12
and 24 hours. The cells were then pelleted and a Western blot
analysis was performed.
[0630] The test building block components included
L-Phenylylalanine, DL-Phenylyalanine, D-Phenylylalanine,
L-Tyrosine, DL-Tyrosine, D-Tyrosine, 4-Hydroxy-phenylpyruvate,
phenylacetate, 3-methoxy-4-hydroxymandelate (vanillylmandelate or
VMA), vanillic acid, 4-hydroxy-benzoate, pyridoxine, panthenol,
mevalonic acid, Acetylglycine, Acetyl-CoA, Farnesyl, and
2,3-Dimethoxy-5-methyl-p-benzoquinone.
[0631] In the Western Blot Analysis, the cells were pelleted in
cold PBS, lysed, and the protein levels were quantified using a BCA
protein assay. The whole cell lysate was loaded in a 4% loading 12%
running Tris-HCl gel. The proteins were then transferred to a
nitrocellulose paper then blocked with a 5% milk Tris-buffered
solution for 1 hour. The proteins were then exposed to primary
antibodies (Bcl-2 and Caspase-3) overnight. The nitrocellulose
paper was then exposed to Pico Chemilluminescent for 5 min and the
protein expression was recorded. After exposure, actin was
quantified using the same method. Using ImageJ the levels of
protein expression were quantified. A t-Test was used to analyze
for statistical significance.
[0632] Illustrative results of the experiments are summarized
below.
[0633] Western Blot Analysis of Building Block component
L-Phenylalanine: Before proceeding to the synthesis pathway for the
quinone ring structure, L-Phenylalanine is converted to tyrosine. A
western blot analysis was performed to quantify any changes in the
expression of the apoptotic proteins in the melanoma cells. The
concentrations tested were 5 .mu.M, 25 .mu.M, and 100 .mu.M.
Initial studies added L-Phenylalanine to DMEM/F12 medium which
contained a concentration of 0.4 M phenylalanine. For the 5 .mu.M,
25 .mu.M, and 100 .mu.M the final concentration of the
L-Phenylalanine in the medium was 0.405 M, 0.425 M, and 0.500 M,
respectively. These final concentrations were tested on the
Skmel-28 cells for incubation periods of 3,6, 12 and 24 hours. The
cells were grown to 80% confluency before adding the treatment
medium and harvested using the western blot analysis procedure as
described above. A statistically significant decrease in Bcl-2 was
observed for the 100 .mu.M L-Phenylalanine after 3 hours and 12
hours incubation. Fr the 5 .mu.M L-phenylalanine, a statistically
significant decrease in Bcl-2 was observed after 6 hours of
incubation. For the 25 .mu.M L-phenylalanine, a statistically
significant decrease in Bcl-2 and a statistically significant
increase in Caspase-3 were observed after 12 hours of incubation. A
statistically significant decrease in Bcl-2 indicates a change in
the apoptotic potential and a statistically significant increase in
Caspase-3 confirms the cells are undergoing apoptosis. There was a
constant trend for the decrease in Bcl-2 compared to the control
even though, due to sample size and standard deviation, these time
points were not statistically significant in this experiment.
[0634] Western Blot Analysis of Building Block component
D-Phenylalanine: D-Phenylalanine, a chemically synthetic form of
the bioactive L-Phenylalanine, was tested for comparison to
L-phenylalanine. For all three concentrations (5 .mu.M, 25 .mu.M,
and 100 .mu.M of D-Phenylalanine, there was a significant reduction
in Bcl-2 expression after 6 hours of incubation. In addition, for
the 5 .mu.M and 25 .mu.M, there was a significant reduction after 3
hours of incubation. For the 5 .mu.M and 100 .mu.M concentrations,
a significant increase in Caspase-3 expression was observed after 6
hours of incubation.
[0635] Western Blot Analysis of Building Block component
DL-Phenylalanine: DL-Phenylalanine was also tested for comparison
to L-Phenylalanine. Again, concentrations of 5 .mu.M, 25 .mu.M, and
100 .mu.M were tested on Skmel-28 cells. The incubation periods
were 3, 6, 12 and 24 hours. A statistically significant increase in
Caspase-3 was observed after 3 hours of incubation. A statistically
significant decrease in Bcl-2 was observed after 24 hours of
incubation. Although a decreasing Bcl-2 and increasing Caspase-3
trend at all other concentrations and incubation time points, they
were not statistically significant in this experiment.
[0636] Western Blot Analysis of Building Block component
L-Tyrosine: L-Tyrosine is a building block component for the
synthesis of quinone ring structure of CoQ10. Initial testing of
L-Tyrosine did not result in a high enough protein concentration
for western blot analysis. From this study concentrations under 25
.mu.M were tested for Western Blot Analysis. The DMEM/F12 medium
used contained L-Tyrosine disodium salt concentration of 0.398467
M. The initial concentration was increased by 500 nM, 5 .mu.M, and
15 .mu.M. A statistically significant increase in Caspase-3 was
observed for the 500 nM concentration after 12 hours of incubation.
A statistically significant increase in Caspase-3 was also observed
for the 5A statistically significant decrease in Bcl-2 was observed
for the 5 .mu.M concentration after 24 hours of incubation. A
statistically significant decrease in Bcl-2 was observed for the
500 .mu.M and 5 .mu.M concentrations after 24 hours of
incubation.
[0637] Western Blot Analysis of Building Block component
D-Tyrosine: D-Tyrosine, a synthetic form of L-Tyrosine, was tested
for comparison against the L-Tyrosine apoptotic effect on the
melanonal cells. Based on initial studies with L-Tyrosine,
concentrations below 25 .mu.M were chosen for the western blot
analysis. The concentrations tested were 1 .mu.m, 5 .mu.M, and 15
.mu.M. D-Tyrosine showed a reduction in Bcl-2 expression for the 5
.mu.M and 15 .mu.M concentrations for 12 and 24 hour time periods.
Caspase-3 was significantly increased for the concentration of 5
.mu.M for 3, 12 and 24 time periods. Also there was an increase in
Caspase-3 expression for the 1 .mu.M for 12 and 24 hour time
period. In addition there is an increase in Caspase-3 expression
for 5 .mu.M for the 12 hour time period.
[0638] Western Blot Analysis of Building Block component
DL-Tyrosine: DL-Tyrosine, a synthetic form of L-Tyrosine, was also
tested for comparison against L-Tyrosine's apoptotic effect on the
cells. There is a statistical decrease in Bcl-2 expression seen in
the 1 .mu.M and 15 .mu.M concentrations after 12 hours incubation
and for the 5 .mu.M after 24 hour of incubation. An increase in
Caspase-3 expression was also observed for the 5 .mu.M and 15 .mu.M
after 12 hours of incubation.
[0639] Western Blot Analysis of Building Block component
4-Hydroxy-phenylpyruvate: 4-Hydroxy-phenylpyruvate is derived from
Tyrosine and Phenylalanine amino acids and may play a role in the
synthesis of the ring structure. The concentration of 1 .mu.M, 5
.mu.M, and 15 .mu.M were tested for Bcl-2 and Caspase-3 expression.
For the 5 .mu.M and 15 .mu.M concentrations there is a significant
reduction in Bcl-2 expression after 24 hours of incubation and a
significant increase in Caspase-3 expression after 12 hours of
incubation.
[0640] Western Blot Analysis of Building Block component
Phenylacetate: Phenylacetate has the potential to be converted to
4-Hydroxy-benzoate, which plays a role in the attachment of the
side chain to the ring structure. The concentration tested were 1
.mu.M, 5 .mu.M, and 15 .mu.M. For phenylacetate there was a
decrease in Bcl-2 expression for the concentration of 5 .mu.M and
15 .mu.M after 12 hours and 24 hours of incubation. An increase in
Caspase-3 expression was observed for the concentration of 5 .mu.M
and 15 .mu.M after 12 hours and 24 hours of incubation.
[0641] Western Blot Analysis of Building Block component
3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA): VMA is an
additional component for the synthesis of the CoQ10 quinone ring
structure. The concentrations tested were 100 nM, 250 nM, 500 nM, 1
.mu.M, 25 .mu.M, 50 .mu.M, and 100 .mu.M. Though no statistically
significant apoptotic effect was observed in this experiment, the
data indicated a downward trend of Bcl-2 expression.
[0642] Western Blot Analysis of Building Block component Vanillic
acid: Vanillic is a precursor for the synthesis of the quinone ring
and was tested at a concentration of 500 nm, 5 .mu.M, and 15 .mu.M.
A western blot analysis measured Bcl-2 and Caspase-3 expression.
Vanillic Acid was shown to significantly reduce Bcl-2 expression
for the concentrations of 500 nM and 5 .mu.M at the 24 hour
incubation time point. For the 15 .mu.M concentration there is a
reduction in Bcl-2 expression after 3 hours of incubation. For the
cells incubated with 15 .mu.M for 24 hours there was a significant
increase in Caspase-3 expression.
[0643] Western Blot Analysis of Building Block component
4-Hydroxybenzoate: 4-Hydroxybenzoate acid plays a role in the
attachment of the isoprenoid side chain to the ring structure. The
concentrations tested were 500 nM, 1 .mu.M, and 50 .mu.M. There was
a significant reduction in Bcl-2 expression for the 15 .mu.M
concentration after 24 hours of incubation.
[0644] Western Blot Analysis of Building Block component
4-Pyridoxine: Pyridoxine is another precursor building block for
the synthesis of the quinone ring structure of CoQ10. The
concentrations tested for this compound are 5 .mu.M, 25 .mu.M, and
100 .mu.M. The cells were assayed for their levels of Bcl-2 and
Caspase-3. Pyridoxine showed a significant reduction in Bcl-2 after
24 hours of incubation in melanoma cells.
[0645] Western Blot Analysis of Building Block component Panthenol:
Panthenol plays a role in the synthesis of the quinone ring
structure of CoQ10. The concentrations tested on melanoma cells
were 5 .mu.M, 25 .mu.M, and 100 .mu.M. This compound showed a
significant reduction in Bcl-2 expression for the 25 .mu.M
concentration.
[0646] Western Blot Analysis of Building Block component Mevalonic:
Mevalonic Acid is one of the main components for the synthesis of
CoQ10. This compound was tested at the concentrations of 500 nM, 1
.mu.M, 25 .mu.m, and 50 .mu.M. There was no significant reduction
in Bcl-2 expression or an increase in Caspase-3 expression in this
experiment.
[0647] Western Blot Analysis of Building Block component
Acetylglycine: Another route for the synthesis of CoQ10 is the
isoprenoid (side chain) synthesis. The addition of Acetylglycine
converts Coenzyme A to Acetyl-CoA which enters the mevalonic
pathway for the synthesis of the isoprenoid synthesis. The
concentrations tested were 5 .mu.M, 25 .mu.M, and 100 .mu.M. The
testing of Acetylglycine showed significant decrease in Bcl-2
expression after 12 hours of incubation for the concentration of 5
.mu.M and 25 .mu.M. A significant decrease in Bcl-2 was recorded
for the 100 .mu.M concentration at the 24 hour incubation time
point.
[0648] Western Blot Analysis of Building Block component
Acetyl-CoA: Acetyl-CoA is a precursor for the mevalonic pathway for
the synthesis of CoQ10. The concentrations tested were 500 nm, 1
.mu.M, 25 .mu.M, and 50 .mu.M. There was no significant observed
reduction in Bcl-2 or increase in Caspase-3 expression for the time
points and concentrations tested.
[0649] Western Blot Analysis of Building Block component L-Tyrosine
in combination with farnesyl: L-Tyrosine is one of the precursors
for the synthesis of the quinone ring structure for CoQ10. Previous
experiment tested the reaction of L-Tyrosine in medium with
L-Phenylalanine and L-Tyrosine. In this study L-Tyrosine was
examined in medium without the addition of L-Phenylalanine and
L-Tyrosine. In this study the final concentrations of L-Tyrosine
tested were 500 nM, 5 .mu.M, and 15 .mu.M. Farnesyl was tested at a
concentration of 50 .mu.M. There was no observed significant
response for the 3 and 6 hour time points.
[0650] Western Blot Analysis of Building Block component
L-Phenylalanine in combination with Farnesyl: L-Phenylalanine, a
precursor for the synthesis of the quinone ring structure, was
examine in combination with farnesyl in medium free of L-Tyrosine
and L-Phenylalanine. A western blot analysis was performed to assay
the expression of Bcl-2 and Caspase-3. The final concentrations of
L-Phenylalanine were: 5 .mu.M, 25 .mu.M, and 100 .mu.M. Farnesyl
was added at a concentration of 50 .mu.M. This study showed a
decrease in Bcl-2 expression for most of the concentrations and
combinations tested as depicted in table 55, below.
TABLE-US-00056 TABLE 55 L- 3 hr 6 hr 12 hr 24 hr Phenylalanine
Bcl-2 Cas-3 Bcl-2 Cas-3 Bcl-2 Cas-3 Bcl-2 Cas-3 5 .mu.M X 5 .mu.M
w/ X X Farnesyl 25 .mu.M X X 25 .mu.M w/ X X Farnesyl 100 .mu.M X X
X 100 .mu.M w/ X Farnesyl
[0651] Cell Proliferation Assay of the Combination of
4-Hydroxy-Benzoate with Benzoquinone: This set of experiments used
a cell proliferation assay to assess the effect of combining
different building block molecules on cell proliferation.
[0652] The first study examined the effect of combining
4-Hydroxy-Benzoate with Benzoquinone. Cells were incubated for 48
hours, after which a cell count was performed for the live cells.
Each test group was compared to the control, and each combination
groups were compared to Benzoquinone control. The compounds were
statistically analyzed for the addition of Benzoquinone. The
following table summarizes the cell count results wherein the X
mark indicates a statistical decrease in cell number.
TABLE-US-00057 TABLE 56 Compared to 4- Hydroxy to Compared to
compound w/o Benzoquinone 4-Hydroxy Compared to Ctrl Benzoquinone
Control 500 nm X 500 nm w/ Benzo X X (35 .mu.M) 500 nm w/ Benzo X X
(70 .mu.M) 1 .mu.m X 1 .mu.m w/ Benzo X X (35 .mu.M) 1 .mu.m w/
Benzo X X (70 .mu.M) 50 .mu.m X 50 .mu.m w/ Benzo X (35 .mu.M) 50
.mu.m w/ Benzo X X X (70 .mu.M)
[0653] There is a significant decrease in cell number for the cells
incubated with 4-Hydroxybenzoic and benzoquinone and in
combination. For the combination of 50 .mu.M 4-Hydroxybenzoate in
combination with 70 .mu.M Benzoquinone there is significant
reduction in cell number compared to the Benzoquinone control. This
suggests a synergistic effect for this molar ratio.
[0654] Additional studies were performed testing additional molar
ratios. For the first test 4-Hydroxybenzoic were tested at
concentrations of 500 nM, 1 .mu.M, and 50 .mu.M. These
concentrations were tested in combination with
2,3-Dimethoxy-5-methyl-p-benzoquinone (Benzo). The concentration of
Benzo tested were 25 .mu.M, 50 .mu.M, and 100 .mu.M. Melanoma cells
were grown to 80% confluency and seeded in 6 well plates at a
concentration of 40K cells per well. The cells were treated with
CoQ10, 4-Hydroxybenzoate, Benzo, and a combination of
4-Hydroxybenzoate/Benzo.
[0655] A T-test was performed with p<0.05 as statistically
significant. An X signifies a statistical decrease in cell
number.
TABLE-US-00058 TABLE 57 Ctrl vs Benzo 25 .mu.M X Ctrl vs Benzo (B)
50 .mu.M Ctrl vs Benzo (B) 100 uM X Ctrl vs 4-Hydroxybenzoate (HB)
500 nm X Ctrl vs HB 1 .mu.M X Ctrl vs HB 50 .mu.M X 500 nM HB vs
500 nM HB w/ 25 B X 500 nM HB vs 500 nM HB w/ 50 B X 500 nM HB vs
500 nM HB w/ 100 B X 1 uM HB vs 1 .mu.M HB w/ 25 B X 1 uM HB vs 1
.mu.M HB w/ 50 B X 1 uM HB vs 1 .mu.M HB w/ 100 B 50 uM HB vs 50
.mu.M HB w/ 25 B X 50 uM HB vs 50 .mu.M HB w/ 50 B X 50 uM HB vs 50
.mu.M HB w/ 100 B 500 nM HB w/ 25 B vs 25 B X 500 nM HB w/ 50 B vs
50 B X 500 nM HB w/ 100 B vs 100 B X 1 .mu.M HB w/ 25 B vs 25 B X 1
.mu.M HB w/ 50 B vs 50 B X 1 .mu.M HB w/ 100 B vs 100 B 50 .mu.M HB
w/ 25 B vs 25 B X 50 .mu.M HB w/ 50 B vs 50 B X 50 .mu.M HB w/ 100
B vs 100 B
[0656] There is a significant decrease in cell proliferation for
the treatment medium containing HB. Moreover the combination of the
HB with benzoquinone showed a significant reduction in cell number
compare to the cells incubated with the corresponding benzoquinone
concentrations.
[0657] A cell proliferation assay was also performed on neonatal
fibroblast cells. The concentrations of HB tested were 500 nM, 5
.mu.M, and 25 .mu.M. HB was also tested in combination with
benzoquinone at a concentrations of 25 .mu.M, 50 .mu.M, and 100
.mu.M. Melanoma cells were seeded at 40 k cells per well and were
treated for 24 hours. The cells were trypsinized and quantified
using a coulter counter.
[0658] Statistical analysis did not show a significant reduction in
fibroblast cells. This indicates minimal to no toxicity in normal
cells.
[0659] Cell Proliferation Assay of the Combination of phenylacetate
and benzoquinone: Phenyl acetate is a precursor for the synthesis
of 4-Hydroxybenzoic acid (facilitates the attachment of the ring
structure. A cell proliferation assay was performed to assay the
effect of incubating phenylacetate in combination with CoQ10 and
Benzoquinone.
TABLE-US-00059 TABLE 58 Ctrl and 25/25 .mu.M Ben X Ctrl and 25/50
.mu.M Ben X Ctrl and 25/100 .mu.M Ben X Ctrl and 25/25 .mu.M Q-10 X
Ctrl and 25/25 .mu.M Q-10 X Ctrl and 25/50 .mu.M Q-10 X Ctrl and
25/100 .mu.M Q-10 X Ctrl and Ben 25 X Ctrl and Ben 50 X Ctrl and
Ben 100 X Ctrl and Q-10 25 Ctrl and Q-10 50 Ctrl and Q-10 100 X Ben
25 .mu.M and 500 nM/25 .mu.M Ben X Ben 25 .mu.M and 5 nM/25 .mu.M
Ben X Ben 25 .mu.M and 25 nM/25 .mu.M Ben X Ben 50 .mu.M and 500
nM/50 .mu.M Ben X Ben 50 .mu.M and 5 nM/50 .mu.M Ben X Ben 50 .mu.M
and 25 nM/50 .mu.M Ben X Ben 100 .mu.M and 500 nM/100 .mu.M Ben Ben
100 .mu.M and 5 nM/100 .mu.M Ben Ben 100 .mu.M and 25 nM/100 .mu.M
Ben Q-10 25 .mu.M and 500 nM/25 .mu.M Q-10 X Q-10 25 .mu.M and 5
nM/25 .mu.M Q-10 X Q-10 25 .mu.M and 25 nM/25 .mu.M Q-10 X Q-10 50
.mu.M and 500 nM/50 .mu.M Q-10 X Q-10 50 .mu.M and 5 nM/50 .mu.M
Q-10 X Q-10 50 .mu.M and 25 nM/50 .mu.M Q-10 X Q-10 100 .mu.M and
500 nM/100 .mu.M Q-10 X Q-10 100 .mu.M and 5 nM/100 .mu.M Q-10 X
Q-10 100 .mu.M and 25 nM/100 .mu.M Q-10 X
[0660] The data indicates the addition of phenylacetate in
combination with benzoquinone significantly decreases the cellular
proliferation. The combination with CoQ10 and phenylacetate
significantly decrease the cell number compared to incubation with
CoQ10 and benzoquinone alone.
[0661] Cell Proliferation Assay of the Combination of
4-Hydroxy-Benzoate with Farnesyl: 4-Hydroxy-Benzoate was incubated
in combination with Farnesyl. The summary of the results are listed
below. 4-Hydroxybenzoate groups were compared to the control and
Farnesyl control groups. The X signifies a statistical decrease in
cell number.
TABLE-US-00060 TABLE 59 Compared to 4- Hydroxy to Compared to
4-Hydroxy- compound w/o Farnesyl Benzoate Compared to Ctrl Farnesyl
Control 500 nm X 500 nm w/ Farnesyl X (35 .mu.M) 500 nm w/ Farnesyl
X (70 .mu.M) 1 .mu.m Error 1 .mu.m w/ Farnesyl Error (35 .mu.M) 1
.mu.m w/ Farnesyl Error (70 .mu.M) 50 .mu.m X 50 .mu.m w/ Farnesyl
X (35 .mu.M) 50 .mu.m w/ Farnesyl X (70 .mu.M)
[0662] Cell Proliferation Assay of the Combination of
L-Phenylalanine with Benzoquinone: A cell proliferation assay was
performed to test the combination of L-Phenylalanine combined with
Benzoquinone. Below is a summary of the results of L-Phenylalanine
compared to the control and Benzoquinone control. The X signifies a
statistical decrease.
Table 60
TABLE-US-00061 [0663] TABLE 60 Compared to L- Phenylalanine to
Compared to compound w/o Benzoquinone L-Phenylalanine Compared to
Ctrl Benzoquinone Control 5 .mu.M 5 .mu.m w/ Benzo X (50 .mu.M) 5
.mu.m w/ Benzo X (100 .mu.M) 25 .mu.m 25 .mu.m w/ Benzo X (50
.mu.M) 25 .mu.m w/ Benzo X (100 .mu.M) 100 .mu.m 100 .mu.m w/ Benzo
X X X (50 .mu.M) 100 .mu.m w/ Benzo X X X (100 .mu.M)
[0664] A similar synergistic role is seen for the L-Phenylalanine
combined with Benzoquinone.
[0665] Cell Proliferation Assay of the Combination of
L-Phenylalanine with Farnesyl: Preliminary results for combination
cell proliferation study of L-Phenylalanine incubated in
combination with Farnesyl. The L-Phenylalanine were compared to the
control and Farnesyl control group. An X signifies a statistical
decrease in cell number.
TABLE-US-00062 TABLE 61 Compared to L- Phenylalanine to Compared to
compound w/o Farnesyl L-Phenylalanine Compared to Ctrl Farnesyl
Control 5 .mu.M 5 .mu.m w/ Farnesyl (50 .mu.M) 5 .mu.m w/ Farnesyl
(100 .mu.M) 25 .mu.m X 25 .mu.m w/ Farnesyl X X X (50 .mu.M) 25
.mu.m w/ Farnesyl X X X (100 .mu.M) 100 .mu.m X 100 .mu.m w/
Farnesyl X X (50 .mu.M) 100 .mu.m w/ Farnesyl X (100 .mu.M)
[0666] Cell Proliferation Assay of the Combination of L-Tyrosine
with Benzoquinone: L-Tyrosine was incubated in combination with
Benzoquinone after which a cell count was performed. The groups
were compared the control groups and Benzoquinone control
group.
TABLE-US-00063 TABLE 62 Compared to L- Tyrosine to Compared to
Compared compound w/o Benzoquinone L-Tyrosine to Ctrl Benzoquinone
Control 500 nm 500 nm w/ Benzo (50 .mu.M) 500 nm w/ Benzo (100
.mu.M) 5 .mu.m X 5 .mu.m w/ Benzo (50 .mu.M) X 5 .mu.m w/ Benzo X
(100 .mu.M) 15 .mu.m X 15 .mu.m w/ Benzo X (50 .mu.M) 15 .mu.m w/
Benzo x (100 .mu.M)
[0667] The addition of Benzoquinone did not amplify the effect of
L-Tyrosine on the cell number.
[0668] Cell Proliferation Assay of the Combination of L-Tyrosine
with Benzoquinone: This study examined the combination of
L-Tyrosine with Farnesyl. The groups were compared to control and
Farnesyl control groups.
TABLE-US-00064 TABLE 63 Compared to L- Tyrosine to Compared to
compound w/o Farnesyl L-Tyrosine Compared to Ctrl Farnesyl Control
500 nm 500 nm w/ Farnesyl (50 .mu.M) 500 nm w/ Farnesyl (50 .mu.M)
5 .mu.m X 5 .mu.m w/ Farnesyl X (50 .mu.M) 5 .mu.m w/ Farnesyl X
(100 .mu.M) 15 .mu.m X 15 .mu.m w/ Farnesyl X (50 .mu.M) 15 .mu.m
w/ Farnesyl X (100 .mu.M)
[0669] Combining L-Tyrosine and Farnesyl does not appear to have a
synergistic effect on reducing the cell number in this
experiment.
[0670] The synthesis of the CoQ10 is divided into two main parts,
which consist of the synthesis of the ring structure and synthesis
of the side chain structure. Here, oncogenic cells were
supplemented with compounds which are precursors for the synthesis
of the side chain and the ring structure components. Our results
have focused the study to 3 main components involved in the
synthesis of the ring structure and two compounds that play a role
in the attachment of the ring structure to the side chain
structure. The three compounds that have shown a significant
reduction in Bcl-2 and increase in Caspase-3 expression are: 1)
L-Phenylalanine, 2) L-Tyrosine and 3) 4-Hydroxyphenylpyruvate. The
two compounds involved with the attachment of the side chain to the
ring structure are: 1) 4-hydroxy benzoate and 2) Phenylacetate.
[0671] Our results also showed that exogenous delivery of these
compounds in combination with 2,3 Dimethoxy-5-methyl-p-benzoquinone
(benzoquinone) significantly inhibits cell proliferation. This
indicates a supplementation of the ring structure with compounds
for the attachment of the side chain to the benzoquinone ring may
supplement an impaired CoQ10 synthesis mechanism. This may also
assist in the stabilization of the molecule to maintain the
functional properties required by cellular processes. Phenylacetate
is a precursor for the synthesis of 4-Hydroxybenzoate, which
exogenous delivery in combination with benzoquinone has a similar
effect in oncogenic cells.
Example 24
Modulation of Gene Expression by Coenzyme Q10 in Cell Model for
Diabetes
[0672] Coenzyme Q10 is an endogenous molecule with an established
role in the maintenance of normal mitochondrial function by
directly influencing oxidative phosphorylation. Experimental
evidence is presented that demonstrates the ability of Coenzyme Q10
in modulating intracellular targets that serve as key indices of
metabolic disorders, such as diabetes, in a manner representative
of therapeutic endpoints.
[0673] In order to understand how Coenzyme Q10 regulates expression
of genes associated with the cause or treatment of diabetes,
immortalized primary kidney proximal tubular cell line derived from
human kidney (HK-2) and primary cultures of the human aortic smooth
muscle cells (HASMC) were used as experimental models. The HK-2 and
HASMC cells are normally maintained in culture at 5.5 mM glucose,
which is a concentration that corresponds to a range considered
normal in human blood. However, in order to simulate a diabetic
environment, both cell lines were subsequently maintained at 22 mM
glucose, which corresponds to the range observed in human blood
associated with chronic hyperglycemia. The cells were subsequently
allowed to propagate over 3 passages so that the intracellular
regulation processes were functionally adapted to mimic a diabetic
state. The choice of cell line was based on the physiologic
influence of diabetes on renal dysfunction and progression to
end-stage renal disease (ESRD) in addition to the progressive
pathophysiology of a compromised cardiovascular function.
Effect of Coenzyme Q10 on Gene Expression in HK-2 Cells Using the
Diabetes PCR Array
[0674] The Diabetes PCR array (SABiosciences) offers a screen for
84 genes simultaneously. The 4 treatments tested in this study
were: [0675] HK-2; [0676] HK-2 H maintained 22 mM glucose; [0677]
HK2(H)+50 .mu.M Coenzyme Q10; and [0678] HK2(H)+100 .mu.M Coenzyme
Q10.
[0679] A stringent analysis of the Real time PCR data of the HK-2
samples on the Diabetes Arrays (Cat # PAHS-023E, SABiosciences
Frederick Md.) was made to exclude all results where gene
regulation was not at least a two-fold regulation over HK-2 normal
untreated cells with a p value of less than 0.05. Genes that were
observed to be regulated either by chronic hyperglycemia or by
Coenzyme Q10 are listed in Table 64 and their functions and
subcellular locations (derived from Ingenuity Pathway Analysis) are
listed in Table 65.
TABLE-US-00065 TABLE 64 HK-2(H)- HK-2(H)- 50 .mu.M 100 .mu.M
HK-2(H) Coenzyme Coenzyme Fold p Q10 Fold p Q10 Fold p Genes
regulation value regulation value regulation value CEACAM1 1.26
0.409 3.47 0.067 5.36 0.032 PIK3C2B 1.48 0.131 2.32 0.115 3.31
0.003 INSR -1.09 0.568 2.51 0.103 2.88 0.024 TNF 2.00 0.005 2.57
0.042 2.81 0.020 ENPP1 -1.50 0.002 1.42 0.238 2.67 0.038 PRKCB
-1.75 0.005 1.82 0.280 2.49 0.042 DUSP4 1.27 0.318 1.24 0.455 2.26
0.060 SELL -1.58 0.219 1.77 0.042 2.06 0.021 SNAP25 -1.00 0.934
1.46 0.377 1.97 0.059
TABLE-US-00066 TABLE 65 Symbol Entrez Gene Name Location Type(s)
CEACAM1 carcinoembryonic antigen- Plasma transmembrane related cell
adhesion Membrane receptor molecule 1 (biliary glycoprotein)
PIK3C2B phosphoinositide-3-kinase, Cytoplasm kinase class 2, beta
polypeptide INSR insulin receptor Plasma kinase Membrane TNF tumor
necrosis factor (TNF Extracellular cytokine superfamily, member 2)
Space ENPP1 ectonucleotide Plasma enzyme pyrophosphatase/ Membrane
phosphodiesterase 1 PRKCB protein kinase C, beta Cytoplasm kinase
DUSP4 dual specificity phosphatase Nucleus phosphatase 4 SELL
selectin L Plasma other Membrane SNAP25 synaptosomal-associated
Plasma transporter protein, 25 kDa Membrane
[0680] Among the detected RNA transcripts with modulated levels,
the Carcino Embryonic Antigen Cell Adhesion Molecule 1 (CEACAM1)
was identified as being highly upregulated in HK2(H) cells,
particularly with 100 .mu.M Coenzyme Q10 treatment. CEACAM-1, also
known as CD66a and BGP-I, is a 115-200 KD type I transmembrane
glycoprotein that belongs to the membrane-bound CEA subfamily of
the CEA superfamily. On the surface of cells, it forms noncovalent
homo- and heterodimers. The extracellular region contains three
C2-type Ig-like domains and one N-terminal V-type Ig-like domain.
Multiple splice variants involving regions C-terminal to the second
C2-type domain (aa 320 and beyond) exist. The lack of intact
CEACAM1 expression in mice has been proposed to promote the
metabolic syndrome associated with diabetes, while an increase in
expression of CEACAM1 is associated with increased insulin
internalization, which suggests an increase in insulin sensitivity
and glucose utilization (e.g., movement of glucose from blood into
the cells), thus mitigating insulin resistance, a hallmark
characteristic of type 2 diabetes mellitus.
[0681] As shown in Table 55, insulin receptor (INSR) expression was
also altered in diabetic HK-2 cells treated with Coenzyme Q10.
Without being bound by theory, the increase in expression of INSR
with Coenzyme Q10 treatment should enhance insulin sensitivity
(either alone or in addition to expression of CEACAM1) with the
potential to reverse a major physiologic/metabolic complication
associated with diabetes.
Effect of Coenzyme Q10 on Gene Expression in HK-2 Cells Using
Mitochondrial Arrays
[0682] Differential expression of mitochondrial genes in diabetes
was assayed using the mitochondria arrays (Cat# PAHS 087E,
SABisociences Frederick Md.). Genes that were regulated by chronic
hyperglycemia and/or Coenzyme Q10 treatment are listed in Table 66
while their functions and location are included in Table 67.
TABLE-US-00067 TABLE 66 HK2 (H) HK-2(H) 50 .mu.M HK-2(H) 100 .mu.M
Genes untreated p value Coenzyme Q10 p value Coenzyme Q10 p value
GRPEL1 -1.5837 0.151255 -2.6512 0.04704 -1.933 0.139161 SLC25A3
-8.6338 0.071951 -8.2059 0.0425 -1.6984 0.995194 TOMM40 -2.3134
0.140033 -1.1567 0.115407 -1.9509 0.038762 TSPO -3.6385 0.111056
-6.7583 0.073769 -2.1104 0.167084
TABLE-US-00068 TABLE 67 Symbol Entrez Gene Name Location Type(s)
GRPEL1 GrpE-like 1, mitochondrial (E. coli) Mitochondria other
SLC25A3 solute carrier family 25 Mitochondrial transporter
(mitochondrial carrier; phosphate membrane. carrier), member 3
TOMM40 translocase of outer mitochondrial Outer membrane of ion
channel membrane 40 homolog (yeast) mitochondria. TSPO translocator
protein (18 kDa) Outer membrane of transmembrane mitochondria.
receptor
[0683] To date, the role of the four mitochondrial genes identified
(Table 3) in diabetic HK-2 cells treated with Coenzyme Q10 in
diabetes is uncharacterized.
Study 2: Effect of Coenzyme Q10 on Gene Expression in HASMC Cells
Using the Diabetes PCR Array
[0684] The Diabetes PCR array (SABiosciences) offers a screen for
84 genes simultaneously. The 4 treatments tested in this study
were: [0685] HASMC; [0686] HASMC H maintained at 22 mM glucose;
[0687] HASMC(H)+50 .mu.M Coenzyme Q10; and [0688] HASMC(H)+100
.mu.M Coenzyme Q10.
[0689] A stringent analysis of the Real time PCR data of the HASMC
cell samples on the Diabetes Arrays (Cat # PAHS-023E, SABiosciences
Frederick Md.) was made to exclude all results where gene
regulation was not at least a two-fold regulation over HASMC normal
untreated cells with a p value of less than 0.05. Genes that were
observed to be regulated either by chronic hyperglycemia or by
Coenzyme Q10 are listed in Table 68.
TABLE-US-00069 TABLE 68 HASMC- HASMC- (H)-50 .mu.M (H)-100 .mu.M
HASMC- Coenzyme Coenzyme Genes (H) p value Q10 p value Q10 p value
AGT 1.3051 0.547507 -1.0169 0.781622 2.3027 0.030195 CCL5 -17.4179
0.013798 -5.3796 0.022489 -4.6913 0.022696 CEACAM1 -5.5629 0.012985
-5.3424 0.014436 -5.8025 0.012948 IL6 2.7085 0.049263 3.8172
0.012685 6.0349 0.000775 INSR 1.4649 0.207788 1.9622 0.081204
2.0801 0.016316 NFKB1 1.482 0.072924 1.3779 0.191191 2.0898
0.027694 PIK3C2B 2.0479 0.218276 1.4331 0.254894 2.6329 0.069422
SELL -1.9308 0.087513 1.2476 0.393904 4.0371 0.000177 TNF -1.814
0.108322 -3.2434 0.043526 -1.8489 0.133757
[0690] In HASMC cells, treatment of hyperglycemic cells with
Coenzyme Q10 resulted in the altered expression of genes involved
in regulating vascular function (AGT), insulin sensitivity
(CEACAM1, INSR, SELL) and inflammation/immune function (IL-6, TNF,
CCL5). Without being bound by theory, an increase in expression of
INSR may be associated with increased insulin sensitivity in HASMC
cells, which is a physiological property that would be beneficial
in the treatment of diabetes, while IL-6, in addition to its
immunoregulatory properties, has been proposed to affect glucose
homeostasis and metabolism, both directly and indirectly, by action
on skeletal muscle cells, adipocytes, hepatocytes, pancreatic
.beta.-cells and neuroendocrine cells. Upon activation, normal
T-cell express and secrete RANTES and chemokine (C-Cmotif) ligand
(CCL5). CCL5 is expressed by adipocytes, and serum levels of RANTES
are increased in obesity and type 2 diabetes. However, as shown in
Table 68, treatment of HASMC cells with Coenzyme Q10 causes a
significant decrease in the expression of CCL5. Based on the
foregoing data, it is expected that administration of Coenzyme Q10
will have a therapeutic benefit in the management of diabetes.
Effect of Coenzyme Q10 on Gene Expression in HASMC Cells Using
Mitochondrial Arrays
[0691] Differential expression of mitochondrial genes in diabetes
was assayed using the mitochondria arrays (Cat# PAHS 087E,
SABisociences Frederick Md.). Genes that were regulated by chronic
hyperglycemia and/or Coenzyme Q10 treatment are shown in Table
69.
TABLE-US-00070 TABLE 69 HASMC- HASMC- (H) 50 uM (H)-100 .mu.M
HASMC- Coenzyme Coenzyme Genes (H) p value Q10 p value Q10 p value
BCL2L1 -1.6558 0.244494 -2.7863 0.008744 -2.3001 0.014537 MFN1
-1.4992 0.317009 -1.2585 0.021185 -2.2632 0.005961 PMAIP1 -4.7816
0.206848 -6.8132 0.000158 -4.352 0.000286 SLC25A1 -2.2051 0.020868
-1.834 0.00581 -3.0001 0.03285 SLC25A13 -2.0527 0.035987 -1.5
0.029019 -1.5245 0.043712 SLC25A19 -1.0699 0.417217 -1.4257
0.104814 -2.1214 0.007737 SLC25A22 -2.1747 0.007344 -1.9839 0.0013
-10.3747 0.003437 TIMM44 -1.3605 0.414909 -2.3214 0.004118 -1.9931
0.010206 TOMM40 -1.1982 0.428061 -2.0922 0.002195 -2.2684 0.003272
TSPO -1.402 0.304875 -2.0586 0.061365 -2.3647 0.044656
[0692] Treatment of hyperglycemic HASMC cells with Coenzyme Q10
resulted in altered expression of genes that regulate programmed
cell death or apoptosis (BCL2L1, PMIAP1 also known as NOXA),
transporter proteins (SLC25A1 [citrate transporter], SLC25A13
[aspartate-glutamate exchanger], SLC25A19 [thiamine pyrophosphate
transporter] and SLC25A22 [glutamate-hydrogen cotransporter]) and
mitochondrial matrix transport proteins (MFN1, TIMM44 and TOMM40).
The activities of these transporters play important role in the
regulation of precursors essential for the Kreb's cycle and
maintenance of mitochondrial oxidative phosphorylation. These
results indicate that exposure of diabetic HASMC cells to Coenzyme
Q10 is associated with changes in expression of cytoplasmic and
mitochondrial genes, which in turn is consistent with Coenzyme Q10
providing a therapeutic benefit in the treatment of diabetes.
[0693] A comparison of the data obtained by treating HASMC cells
and HK-2 cells with Coenzyme Q10 or in a hyperglycemic environment
reveals that 4 genes were commonly regulated by Coenzyme Q10 in
both cell lines (e.g., PIK3C2B and SELL in the gene expression
assay and TOMM40 and TSPO in the mitochondrial array assay). These
results demonstrate that treatment of cells with Coenzyme Q10 in a
diabetic environment is associated with altered expression of genes
that are known to be involved in the cause or treatment of
diabetes.
EQUIVALENTS
[0694] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
* * * * *
References