U.S. patent application number 14/470488 was filed with the patent office on 2014-12-18 for membrane resident peptide in anti-cancer peptides causes tumor cell necrosis rather than apoptosis of cancer cells.
This patent application is currently assigned to THE RESEARCH FOUNDATION OF STATE UNIVERSITY OF NEW YORK. The applicant listed for this patent is Josef Michl, Matthew R. Pincus, Ehsan Sarafraz-Yazdi. Invention is credited to Josef Michl, Matthew R. Pincus, Ehsan Sarafraz-Yazdi.
Application Number | 20140371156 14/470488 |
Document ID | / |
Family ID | 41800793 |
Filed Date | 2014-12-18 |
United States Patent
Application |
20140371156 |
Kind Code |
A1 |
Pincus; Matthew R. ; et
al. |
December 18, 2014 |
MEMBRANE RESIDENT PEPTIDE IN ANTI-CANCER PEPTIDES CAUSES TUMOR CELL
NECROSIS RATHER THAN APOPTOSIS OF CANCER CELLS
Abstract
An aspect of the invention provides a method of selectively
necrosing cells, comprising: providing a plurality cells, including
at least one cancer cell and at least one non-cancerous cell; and
administering to the cells a compound, including an HDM-2 targeting
component and a cytotoxic component attached to the HDM-2 targeting
component, wherein said compound comprises a membrane-active
form.
Inventors: |
Pincus; Matthew R.;
(Brooklyn, NY) ; Michl; Josef; (Little Neck,
NY) ; Sarafraz-Yazdi; Ehsan; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pincus; Matthew R.
Michl; Josef
Sarafraz-Yazdi; Ehsan |
Brooklyn
Little Neck
New York |
NY
NY
NY |
US
US
US |
|
|
Assignee: |
THE RESEARCH FOUNDATION OF STATE
UNIVERSITY OF NEW YORK
Albany
NY
|
Family ID: |
41800793 |
Appl. No.: |
14/470488 |
Filed: |
August 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13122256 |
Sep 14, 2011 |
8822419 |
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PCT/US2009/059380 |
Oct 2, 2009 |
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14470488 |
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61102590 |
Oct 3, 2008 |
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Current U.S.
Class: |
514/18.9 ;
435/29; 435/34; 435/375; 435/7.23; 435/7.4; 506/10; 514/19.3 |
Current CPC
Class: |
G01N 2500/10 20130101;
A61K 45/06 20130101; C07K 14/4746 20130101; A61P 35/00 20180101;
G01N 2333/4748 20130101; G01N 33/57484 20130101; G01N 33/5011
20130101; G01N 2333/904 20130101; G01N 33/57407 20130101; G01N
33/57492 20130101 |
Class at
Publication: |
514/18.9 ;
435/375; 435/29; 506/10; 435/34; 435/7.23; 435/7.4; 514/19.3 |
International
Class: |
C07K 14/47 20060101
C07K014/47; G01N 33/50 20060101 G01N033/50; G01N 33/574 20060101
G01N033/574 |
Goverment Interests
FUNDING STATEMENT
[0002] This invention was made with government support by a
Veteran's Administration Grant (WBB) and The American College of
Surgeon's Faculty Research Fellowship Award 2007-2009 (WBB).
Claims
1. A method of selectively necrosing cells, comprising: providing a
plurality cells, including at least one cancer cell and at least
one non-cancerous cell; administering to the cells a compound,
including an HDM-2 targeting component and a cytotoxic component,
said cytotoxic component attached to said HDM-2 targeting
component, wherein said compound comprises a membrane-active
form.
2. The method of claim 1, wherein the cytotoxic component is
selected from the group consisting of: a membrane resident peptide,
a toxin, a drug, a radionuclide, a whole antibody, an antibody
fragment, and combinations thereof.
3. The method of claim 1, wherein the HDM-2 targeting component is
selected from the group consisting of: a small molecule, a peptide,
a protein, a glycoprotein, a whole antibody, an antibody fragment,
and combinations thereof.
4. The method of claim 1, further comprising the step of observing
in a cell medium a level of LDH.
5. The method of claim 1, further comprising the step of observing
necrosis in the cancer cells.
6. The method of claim 1, further comprising the step of observing
a non-response in the non-cancerous cell, wherein the non-response
indicates the non-cancerous cell is unaffected.
7. The method of claim 1, wherein the administering step further
comprises administering a PNC-27, a PNC-28 peptide, or combinations
thereof.
8. A method of causing membranolysis in at least one cancer cell,
comprising: administering to at least one cancer cell a compound
comprising an HDM-2 targeting component and a pore-forming
component attached to said HDM-2 targeting component, wherein said
administering step results in at least one transmembrane pore in a
cancer cell membrane.
9. The method of claim 8, further wherein the administering step
further comprises administering said compound at a dosage.
10. The method of claim 8, further comprising the step of observing
membranolysis in the cancer cell by: detecting an LDH amount,
performing electron microscopy, observing cell morphology, and
combinations thereof.
11. The method of claim 8, further comprising the step of observing
necrosis of the cancer cell.
12. A method of treating cancer in a subject in need thereof,
comprising: administering to the subject in need thereof a
therapeutically effective amount of a compound having an HDM-2
targeting component and a cytotoxic component, said HDM-2 targeting
component and said cytotoxic component having a membrane-active
form.
13. The method of claim 12, wherein the cytotoxic component is
selected from the group consisting of: a membrane resident peptide,
a toxin, a drug, a radionuclide, a whole antibody, an antibody
fragment, and combinations thereof.
14. The method of claim 12, wherein the HDM-2 targeting component
is selected from the group consisting of: a small molecule, a
peptide, a protein, a glycoprotein, a whole antibody, an antibody
fragment, and combinations thereof.
15. The method of claim 12 further comprising the step of
determining whether a plurality of cancerous cells have undergone
membranolysis.
16. The method of claim 12, further comprising the step of, after
the administering step, determining whether a plurality subsequent
cancer cells exists.
17. A method of screening cancer treatments, comprising: providing
a plurality of cancerous cells; each of said cells having HDM-2 in
said cellular membranes; administering a candidate cancer treatment
to the plurality of cancer cells; and measuring the level of LDH
present in a cellular medium.
18. The method of claim 17, further comprising the step of
determining whether the candidate cancer treatment has membrane
active conformation.
19. The method of claim 17, further comprising the step of
determining whether the candidate cancer treatment includes an
HDM-2 targeting component.
20. The method of claim 17, further comprising the step of
observing the cancer cell membranes for an area of pore
formation.
21. A method of selectively necrosing cancer cells, comprising:
providing a plurality of cells, including at least one cancer cell
and at least one non-cancerous cell; and contacting the plurality
of cells with an HDM-2 targeting compound which includes a membrane
resident peptide (MRP), wherein the HDM-2 targeting compound
colocalizes to HDM-2 present in at least one cancer cell membrane,
binding to a cell membrane of the at least one cancer cell and
adopting a membrane-resident conformation within said cancer cell
membrane.
22. The method of claim 21, wherein adopting the membrane-resident
conformation further includes forming a pore in said cancer cell
membrane.
23. The method of claim 21, comprising observing necrosis in the
plurality of cancer cells.
24. The method of claim 21, comprising observing a non-result in
the non-cancerous cells, indicative of non-targeting of said
non-cancerous cells.
25. A method of identifying cancer cells, comprising: providing a
plurality of cells, wherein at least one of said cells is a
candidate cancer cell; and administering to the plurality of cells
an HDM-2 recognition agent.
26. The method of claim 25, further comprising of the step of
observing the plurality of cells for the HDM-2 recognition agent
colocalization with at least one cell membrane.
27. The method of claim 25, wherein said HDM-2 recognition agent
further comprises an HDM-2 targeting component tagged with an
observation aid, said observation aid selected from the group
consisting of: a dye, an enzyme, a fluorescing agent, a radiopaque
material, a radioactive isotope and combinations thereof.
28. The method of claim 25, further comprising identifying at least
one cancer cell.
29. The method of claim 25, further comprising mapping a plurality
of cancer cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of and priority
to U.S. Provisional Patent Application Ser. No. 61/102,590 filed on
Oct. 3, 2008, the contents of which are incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
[0003] The invention relates to methods of effectively treating
various forms of cancer and screening candidate cancer treatments
and compounds. Specifically, the present invention is directed to
the use of novel compounds and methods to treat cancer and
non-cancerous cells and cause necrosis only to cancer cells.
RELATED ART
[0004] Cancer treatments which target the p53 protein within the
cancer cells have been developed recently. However, some types of
cancer cells do not have p53, while others exhibit p53 in a
mutated, and/or inactive form. Thus, these p53 targeting cancer
treatments are limited since they do not cause cell death in these
types of cancer cells. Thus, p53-targeting cancer treatments are
ineffective at treating various types of cancer.
SUMMARY OF THE INVENTION
[0005] The embodiments of the present invention are directed to the
surprising discovery that cancer cells have approximately several
times as much HDM-2 in their cellular, mitochondrial, and nuclear
membranes than non-cancerous cells. Thus, HDM-2 targeting compounds
are successful cancer treatments, causing necrosis to cancer cells
while leaving adjacent non-cancerous cells unaffected. HDM-2
targeting treatments thus represent a body of wide-acting cancer
treatments that are more effective than the current, limited
p53-targeting treatments.
[0006] An aspect of the invention provides a method of selectively
necrosing cancer cells, but not untransformed or normal cells. The
method includes the steps of: administering to the cells a
compound, wherein the compound includes an HDM-2 targeting
component and a cytotoxic component, where the cytotoxic component
may be attached to said HDM-2 targeting component such that the
compound comprises a membrane-active form. An example of an HDM-2
targeting component may include, for example, one or more small
molecules, a peptide, a protein, a glycoprotein, an antibody
(including whole and fragment antibodies), and combinations
thereof. Examples of a cytotoxic component may include: a membrane
resident peptide (MRP), a toxin, a drug, a radionuclide, an
antibody (including whole and/or fragment), and combinations
thereof, as may be desired. One or more of the cytotoxic
components, including the toxin, drug, radionuclide, antibodies,
and combinations thereof, may be known and/or used in the art, for
their cytotoxic affects to cells, optionally, cancer cells.
[0007] Optionally, the HDM-2 targeting may be a peptide. Where the
HDM-2 targeting component and the MRP are both peptides, the MRP is
preferably attached to the carboxyl terminal end of the
peptide.
[0008] Optionally, the method may further include the step of
observing the release (from the cancer cell) of an increased LDH
amount as compared to an initial LDH amount from the cancer cell,
observing necrosis in the cancer cells, and/or observing a
non-response in the normal cell. The non-response of a normal cell
may indicate that the normal cell is unaffected by the cancer
treatment.
[0009] Another aspect of the present invention provides a method of
causing membranolysis in at least one cancer cell. The method
includes the step of administering to at least one cancer cell a
compound including an HDM-2 targeting component and a MRP, the MRP
attached to the HDM-2 targeting component.
[0010] Optionally, the method may further include, for example,
observing membranolysis in the cancer cell by microscopy. Observing
necrosis of the cancer cell may also be included as a step in the
present invention.
[0011] Still another aspect of the present invention provides a
method of treating cancer in a subject (or patient) in need
thereof. The method includes the steps of administering to the
subject in need thereof a therapeutically effective amount of a
compound having an HDM-2 targeting component and a MRP, the MRP
attached to the HDM-2 targeting component. The subject may include,
for example, mammals including dogs, cats, chimpanzees, and rats.
Optionally, the method may further include the step of correlating
a result thereof of the administration step.
[0012] Still yet another aspect of the present invention provides a
method of screening candidate cancer treatments. The method may
include the steps of: providing a plurality of cancerous cells;
administering a candidate cancer treatment to the plurality of
cancerous cells; and measuring the level of LDH released from said
cells. LDH is measurable in the cell medium, once it is released
from the cells.
[0013] Optionally, the method includes administering the candidate
cancer treatment which may include a compound including an HDM-2
targeting component and an MRP. As the method employs screening
compounds for their potential abilities as (1) binding affinity for
HDM-2 and (2) membrane transport character, one or both of these
characteristics may be desired in various drug candidates that are
screened with the present method. The screening process, may aid in
identifying components that act within the desired parameters and
with the preferable characteristics as effective cancer treatments.
Additionally desirable characteristics of cancer treatment,
including causation of membranolysis and ultimately, cancer cell
necrosis may be observed or otherwise measured after each candidate
compound is administered. Thus, the efficacy of each candidate may
be screened.
[0014] Optionally, the method may also contain the steps of
observing the cells for LDH, and/or correlating the level of LDH in
the cellular medium to a standard. Thus, necrosis, and the level
thereof may be identified for each candidate, as it may correlate
to the level and/or amount of LDH released for a given sample.
[0015] Still yet another aspect of the present invention provides a
method of selectively necrosing cancer cells, including the steps
of: providing at least one cancer cell and at least one
non-cancerous cell; and contacting the cells with a compound, where
the compound includes an HDM-2 targeting compound having an MRP
attached thereto, wherein the compound binds to a cancer cell
membrane and configures to a membrane active form, binding to the
cancer cell membrane. This binding site is preferably at a site of
HDM-2, and results in trans-membrane pore formation in the cancer
cell membrane.
[0016] The method optionally includes the steps of measuring a
level of LDH from the cancer cell, observing necrosis of the cancer
cell, and/or observing a non-result in the non-cancerous cell as a
result of the administering step.
[0017] A further aspect of the present invention provides a method
of identifying cancer cells from a plurality of cells, including:
providing a plurality of cells, wherein at least one of the cells
is a candidate cancer cell; administering to the plurality of cells
an HDM-2 recognition compound; and observing the plurality of cells
for the HDM-2 recognition compound to bind to a cell membrane of at
least one of the cells, where binding to the cell membrane is
indicative of a cancerous cell.
[0018] Optionally, the method may include the step of fluorescing
the HDM-2 recognition molecule with an observable agent. The
observable agent may include, for example, various known dyes,
enzyme-substrate combinations, radiopaque materials, fluorescing
agents, and combinations thereof. The observable agents may be
visually observable, observable with filtered light through various
scopes, and/or identified through X-ray and or other medical
instrumentation photography. With HDM-2 targeting compounds used in
conjunction with observable agents, cancerous areas may be
identified, topographically mapped, and better understood than with
previous cancer identification and visualization techniques.
[0019] The method may further include the step of classifying an
identified cancer cell as a type of cancer. This may be done in
vivo, as part of a diagnostic for cancer. Alternatively, the
fluorescent-labeled MRP attached to an HDM-2 targeting component or
HDM-2 recognition agent may be administered to a candidate surgical
area. Such a use may provide derivative identifiers to a surgeon of
the highly cancerous tissue, less cancerous tissue, and
non-cancerous tissue for surgical removal and/or intervention
purposes. As such, the embodiments and features of the present
invention may provide a great aid in surgical pathology, helping
pathologists to distinguish cancer from non-cancer.
[0020] The description of the elements and features of the present
invention and equivalents thereof may be better understood through
a study of the following drawings, description, and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is experimental data which illustrates that PNC-28 is
cytotoxic to MiaPaCa-2 cells. Panel A shows untreated MiaPaCa-2
cells incubated for 24 h. Panel B represents MiaPaCa-2 cells
treated for 24 h with 75 .mu.mol/ml of PNC-28. Panel C shows
MiaPaCa-2 cells treated with 75 .mu.mol/ml PNC-29 negative control
peptide for 48 h.
[0022] FIG. 2 is a chart of experimental data which illustrates
that PNC-28 (diamonds) is cytotoxic to MiaPaCa-2 cells in a
dose-dependent manner over a dose range of 0 .mu.mol/ml up to 160
.mu.mol/ml. Specifically, a dosage of 20 .mu.mol/ml caused roughly
35% cell death, while a dosage of 80 .mu.mol/ml caused over 80%
cell death at 48 h. The effect of the negative control PNC-29
(squares) is also shown. The effective dose range for PNC-28 at 48
h from 20 to 80 .mu.mol/ml is strongly statistically significant
(P<0.001).
[0023] FIG. 3 is a chart of experimental data which illustrates
that the carboxyl terminal attached MRP to residues 17-26 (PNC-28)
is required for cytotoxicity to cancer cells. The chart depicts the
number of remaining cells after 48 hours have passed, shown as a
function of treatments administered. MiaPaCa-2 cell death following
treatment with no peptide (condition 1), 75 .mu.mol/ml of PNC-28
(condition 3), PNC-26 (condition 2), and negative control PNC-29
(condition 4) after 48 h of treatment.
[0024] FIG. 4A is a chart of experimental data which illustrates
that PNC-28 induces cellular death by necrosis in MiaPaCa-2 cells.
LDH activity (measured as absorbance/optical density at 492 nm) was
recorded for MiaPaCa-2 cells incubated with 25 .mu.mol/ml of PNC-28
(condition 2), no peptide (condition 3), and PNC-29 (condition 4)
at 24 h. Maximal LDH release is shown after treatment with known
lysis buffer (condition 1).
[0025] FIG. 4B is experimental data which illustrates comparative
electron micrographs of MiaPaCa-2 cells that were untreated (right
panel) versus treated (left panel) with 25 .mu.mol/ml PNC-28 for 15
min. The arrows in the left panel point to gaps or holes in the
cell membrane induced by PNC-28.
[0026] FIG. 4C is a chart of experimental data which illustrates
that PNC-28-induced cell death not caused by apoptosis. Caspase 3
activity, an apoptosis indicator, was recorded for MiaPaCa-2 cells
incubated with 25 .mu.mol/ml of PNC-28 (condition 2), no peptide
(condition 3), and PNC-29 (condition 4) at 24 h. Maximal caspase
release is shown after treatment with TNF-.alpha. (condition 1)
known to induce apoptosis. Caspase 3 activity is measured by
luminescence (UL), as shown on the y-axis.
[0027] FIG. 4D is a chart of experimental data which illustrates
that PNC-28 induces cell death by causing tumor cell necrosis, and
not apoptosis, over its entire effective concentration range. At
each dose, both LDH and caspase activity were measured after
incubation with PNC-28 after 24 h. For the points on the abscissa,
two numbers are separated by a dash. The first number refers to the
concentration of peptide; the second number refers to the
particular peptide, e.g., "28" refers to PNC-28, while "29" refers
to the negative control, PNC-29. Optical Density is shown as a
function of treatment.
[0028] FIG. 5 is experimental data in the form of a chart which
illustrates cell death (number of dead cells divided by total cell
count) as measured by trypan blue dye exclusion in MiaPaCa-2 and
BMRPA1 cells transfected with either p53 17-26-V or control p53
17-26-scrm-V plasmid, 48 h post-transfection. Condition 1:
MiaPaCa-2 cells transfected with p53 17-26-V (black); condition 2:
Mia-PaCa-2 cells transfected with p53 17-26-scrm-V (white);
condition 3: BMRPA1 cells transfected with p53 17-26-V (black);
condition 4, BMRPA1 cells transfected with p53 17-26-scrm-V
(white).
[0029] FIG. 6 is experimental data which shows the effects of
expression of the p53 17-26 peptide, following transfection of its
expression vector into MiaPaCa-2 cells, on expression of p53 and
waf.sup.p21, a cell cycle inhibitor protein induced by activated
wild-type p53, as a function of time, measured by immunoblotting
and on caspase activity. Peptide expression in cells was measured
by blotting for the peptide with the anti-p53 monoclonal antibody
DO-1 that recognizes the p53 17-26 sequence expressed by the
plasmid. For comparison, the effects of incubating PNC-28 with
MIA-PaCa-2 cells on induction of these proteins and on caspase
activity are also shown. Intracellular PNC-28 level was determined
using the same DO-1 antibody. The left ordinate shows the
absorbance results for the caspase activity assay while the right
ordinate shows the band intensity for each Western blot; the actual
immunoblots are shown above each bar graph for the two proteins,
p53, waf.sup.p21 and each peptide (p53 17-26 and PNC-28). The left
side of the figure shows the results for 0 time, after Mia-PaCa-2
cells were transfected with the plasmid (labeled as "transfection"
in the figure) and immediately after PNC-28 was added to the
incubation medium (labeled in the figure as "PNC-28."). The right
side of the figure shows the results after close to 100 percent of
the cells were killed by the plasmid-expressed peptide (labeled in
the figure as "transfection") and by PNC-28 (labeled in the figure
as "PNC-28"). Actin controls were the same for all four conditions
(not shown).
[0030] FIG. 7 is experimental data which illustrates that
transfection of p53 17-26 vector induces apoptosis in MiaPaCa-2 but
not BRMPA1 cells. Confocal microscopy demonstrating green
fluorescence following transfection of control vectors into
MiaPaCa-2 and BMRPA1 cells (Panels A and C) Annexin V binding to
phosphatidyl serine (red staining) detected in p53 17-26-V-treated
MiaPaCa-2 cells (Panel B) but not in 17-26-V-treated BMRPA1 cells
(D).
[0031] FIG. 8A is a chart of experimental data which illustrates
that p53 17-26 induces cellular death by apoptosis. Caspase 3, 7
activity recorded (luminescence as labeled on the ordinate) for
MiaPaCa-2 cells transfected with p53 17-26-V (condition 3), empty
vector (condition 2), PNC-28 (condition 4), and PNC-29 (condition
5). Maximal caspase release is shown after treatment with
TNF-.alpha. condition 1).
[0032] FIG. 8B is a chart of experimental data which illustrates
that p53 17-26 induced cell death does not cause necrosis. LDH
activity recorded for MiaPaCa-2 cells transfected with p53 17-26-V
(condition 3), empty vector (condition 2), PNC-28 (condition 4),
and PNC-29 (condition 5). Maximal LDH release is shown after
treatment with lysis known buffer (condition 1).
[0033] FIG. 9 depicts a table summarizing the efficiency of
transfection of plasmids into MIA-PaCa-2 and BMRPA1 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0034] p53 is the gene that is most commonly disrupted in cancer.
p53 acts as the guardian of the genome, as it guards against
copying of the DNA. It was previously established that p53 gene
within the cells was a target treatment for cancer. However, p53
targeting treatment in cancer cells has various problems associated
with it that limits the use of p53 targeting treatments. For
example, not all cancers exhibit p53 in the cell. Targeting
treatments for these types of cancers would not work, as there is
no p53 for the targeting compounds to bind to. Also, some cancers
exhibit a mutated form of p53, which is inactive. As the p53 is
inactive in these cancers, targeting compounds also do not work for
these cancers. Thus, p53 dependent treatment mechanisms are
ineffective against these types of cancer.
[0035] The materials and methods of the present invention provide
novel methods of treatment that are directed to a common
characteristic in many various forms of cancer. Such materials and
methods may be used as a targeted treatment to many various forms
of cancer, which, up until now, may have very different and less
effective and predictable treatment options and avenues. These new
methods, materials, and screening methods provide effective
treatments, screening methods for additional novel drug candidates,
and other benefits and advantages over the current medical
technology.
[0036] As used herein, cancer includes any disease or disorder
associated with uncontrolled cellular proliferation, survival,
growth, or motility. Cancers that may be treated or prevented by
the present invention include any cancer whose cells have increased
expression of HDM-2 in their plasma membranes. Such cancers may
include, for example, pancreatic cancer, breast cancer, colon
cancer, gastric cancer, prostate cancer, thyroid cancer, ovarian
cancer, endometrial cancer, glioblastoma, astrocytoma, renal
carcinoma, lung cancer, sarcoma, including osteogenic sarcoma,
mesothelioma, sporadic nonfamilial tumors, lymphoma, and others
including hematologic cancers such chronic myelogenous leukemia.
Precancerous conditions, where cells exhibit high amounts of HDM-2
in the plasma membrane, are also included as treatable with the
compositions and methods of the present invention.
[0037] The present invention is directed to the surprising
discovery of the inventors that cancer cell membranes and nuclear
membranes have a large amount of HDM-2 as compared to normal
non-cancerous cells. HDM-2 and MDM-2 (human double minute vs. mouse
double minute) each have a p53 binding domain. HDM-2 and MDM-2 are
found in the cell and nuclear membranes of cancer cells, but not in
normal, healthy cells. The compounds of the present invention
include a HDM-2 targeting component and a Membrane Resident Peptide
(MRP), where the MRP is attached to the carboxyl terminal end of
the HDM-2 targeting component. The MRP may include the residues
that are shared by the p53 binding domain (or region) of HDM-2. As
such, when the compound is administered or otherwise contacted to
at least one cancer cell, the HDM-2 targeting component, which has
a p53 binding domain, may bind to the HDM-2 in the cancer cell
membrane. The presence of the MRP on the end allows the compound to
become membrane-active and to form well-defined pores in the cell
membrane (membranolysis), which allow for extrusion of the
intracellular contents and compromise the integrity of the cell.
Pores in the cancer cell membrane are formed as an immediate result
of administration of the compound. After the pores are formed, cell
necrosis, or cell death, results within a short time frame. Thus,
cancer treatments having an HDM-2 targeting component and an MRP
are effective treatments for cancer.
[0038] Cancer cells have approximately five times as much HDM-2 as
normal cells in their cellular membranes and nuclear membranes.
Though the cancers may not exhibit similar characteristics or
typical treatment avenues, as many cancers each have HDM-2 in their
cell membranes, these cancers are likewise susceptible to the
methods of treatment of the present invention. Cancers that have
been identified as having a large amount of HDM-2 in the cell
membrane include, for example: MIA-PaCa-2 human pancreatic cancer
cells, MCF-7 human breast cancer cells, B 16 mouse melanoma, and a
human melanoma cell line A2025. The compounds which have both an
HDM-2 targeting component and an MRP thus have a high affinity for
cancer cells, and will thus only bind to and cause necrosis of
cancer cells when administered to a combination of cancer cells and
normal, healthy cells.
[0039] The present inventors have discovered methods and uses of
the compounds containing an HDM-2 binding domain having a MRP
attached at its end, where the compound is specifically designed to
target cancer cells and not target normal, healthy cells of a
sample. Where both the HDM-2 targeting component and the MRP are
peptides, the MRP is desirably attached to the carboxyl terminal
end of the HDM-2 targeting component.
[0040] Thus, these methods may be used to treat a sample of cells
containing both healthy, normal cells and cancer cells. Such
samples would include cell lines, tissue samples, tumors, and/or a
subject having cancer in need of treatment. As the methods of
treatment do not cause cell death of normal cells, these methods of
treatment are focused on the cancer cells, irrespective of the mode
of administration to the cell sample. Thus, these methods of
treatment may be used for tumors or cancers that are widespread,
inoperable, or otherwise not easily treated with conventional means
or combination therapies.
[0041] The methods of the present invention kill cancer cells by
necrosis. Necrosis is induced by the combined action of the
compound, which acts both to bind to HDM-2 and to form pores on the
cellular membrane. The pores ultimately cause the cell membrane to
lose its integrity such that intracellular contents leak from the
cell, and the cell undergoes necrosis.
[0042] The present invention provides methods of using HDM-2
targeting cancer compounds which correspond to all or a portion of
amino acid residues 12-26 of human p53 protein. When fused to a
MRP, the peptides are lethal to malignant or transformed cells. The
subject cancer treatment compounds may be useful in treating
neoplastic disease in an animal, preferably a human.
[0043] The compounds of the present invention may include, for
example, PNC-27 and PNC-28. Additionally, one or more compounds may
be used, where a compound may have an HDM-2 targeting component.
The HDM-2 targeting components may be, for example, the residues of
p53 which bind to HDM-2. Further, the compound may include a
membrane resident peptide, or, MRP. Both PNC-27 and PNC-28 are
examples of p53-derived peptides from the human double minute
binding domain (HDM-2) that are attached to MRP. These compounds
induce tumor cell necrosis of cancer cells, but not normal cells.
The anti-cancer activity and mechanism of PNC-28 (p53 aa17-26-MRP)
was specifically studied by the inventors of the present invention
as against human pancreatic cancer, though uses and applications
are included with the various methods of the present invention.
[0044] The inventors show with the present invention and supporting
experimental examples that the MRP is necessary for this action
since expression of the naked p53 sequence without MRP in cancer
cells causes wild-type p53-dependent apoptosis, or programmed cell
death, not tumor cell necrosis.
[0045] Preferably, the MRP includes predominantly positively
charged amino acid residues since a positively charged leader
sequence, which may stabilize the alpha helix of a subject peptide.
Examples of MRPs which may be useful to the HDM-2 targeting
compounds of the present invention are described in Futaki, S. et
al (2001) Arginine-Rich Peptides, J. Biol. Chem. 276,:5836-5840,
and include but are not limited to the following MRPs in the TABLE
1, below. The MRP may be, for example, peptides included in SEQ ID
NO:1, or 9-29. The numbering of the amino acid residues making up
the MRP is indicated parenthetically immediately after the name of
the component in most of the examples in most of the sequence
listings.
TABLE-US-00001 TABLE I SEQ ID NO: Sequence NAME SEQ ID NO: 1
KKWKMRRNQFWVKVQRG Membrane resident peptide (MRP), reverseomer of
Antennapedia SEQ ID NO: 2 PPLSQETFSDLWKLL PNC-27 KKWKMRRNQFWVKVQRG
SEQ ID NO: 3 ETFSDLWKLLKKWKMRRNQFWVKVQRG PNC-28 SEQ ID NO: 4
MPFSTGKRIMLGEKKWKMRRNQFWVKV PNC-29 QRG SEQ ID NO: 5 MPFSTGKRIMLGE
peptide from cytochrome P450 (aka ''X13'') SEQ ID NO: 6
TIEDSYRKQVVIDKKWKMRRNQFWVKV PNC-7 QRG SEQ ID NO: 7 TIEDSYRKQVVID
ras-p21 residues 35-47 SEQ ID NO: 8 PPLSQETFSDLWKLL PNC-26,
residues 12-26 of the HDM-2 binding domain of p53 SEQ ID NO: 9
YGRKKRRQRRRPPQ HIV-1 TAT(47-60), membrane resident peptide SEQ ID
NO: 10 GRKKRRQRRRPPQ D-TAT, membrane resident peptide SEQ ID NO: 11
GAAAAAAAAAPPQ R-TAT G(R).sub.9PPQ, membrane resident peptide SEQ ID
NO: 12 PKKKRKV SV40-NLS, membrane resident peptide SEQ ID NO: 13
KRPAAIKKAGQAKKKK nucleoplasmin-NLS, membrane resident peptide SEQ
ID NO: 14 TRQARRNRRRRWRERQR HIV REV (34-50), membrane resident
peptide SEQ ID NO: 15 RRRRNRTRRNRRRVR FHV (35-49) coat, membrane
resident peptide SEQ ID NO: 16 KMTRAQRRAAARRNRWTAR BMV GAG (7-25),
membrane resident peptide SEQ ID NO: 17 TRRQRTRRARRNR HTLV-II REX
4-16, membrane resident peptide SEQ ID NO: 18 KLTRAQRRAAARKNKRNTR
CCMV GAG (7-25), membrane resident peptide SEQ ID NO: 19
NAKTRRHERRRKLAIER P22 N (14-30), membrane resident peptide SEQ ID
NO: 20 MDAQTRRRERRAEKQAQWKAAN LAMBDA N(1-22), membrane resident
peptide SEQ ID NO: 21 TAKTRYKARRAELIAERR Phi N (12-29), membrane
resident peptide SEQ ID NO: 22 TRRNKRNRIQEQLNRK YEAST PRP6
(129-124), membrane resident peptide SEQ ID NO: 23 SQMTRQARRLYV
HUMAN U2AF, membrane resident peptide SEQ ID NO: 24
KRRIRRERNKMAAAKSRNRRRELTDT HUMAN C-FOS (139-164), membrane resident
peptide SEQ ID NO: 25 RIKAERKRMRNRIAASKSRKRKLERIA R HUMAN C-JUN
(252-279), membrane resident peptide SEQ ID NO: 26
KRARNTEAARRSRARKLQRMKQ YEAST GCN4, membrane resident peptide SEQ ID
NO: 27 KLALKLALKALKAALKLA Example membrane resident peptide (MRP)
SEQ ID NO: 28 LLIILRRRIRKQAKAHSK p-vec, membrane resident peptide
SEQ ID NO: 29 RRRRRRRR (Arg).sub.8 or any poly-R from
(R).sub.4-(R).sub.16, membrane resident peptide SEQ ID NO: 30
GCCACCATGG Kozak sequence SEQ ID NO: 31
AGTCGAATTCGCCACCATGGAAACATTT sense strand sequence
TCAGACCTATGGAAACTACTTTGAGCGG of cDNA encoding the CCGCAGTC p53
17-26 sequence SEQ ID NO: 32 ETFSDLWKLL residues 17-26 of HDM-2
binding domain of p53
[0046] Other MRP materials may also be used. Such sequences are
described e.g., in Scheller et al. (2000) Eur. J. Biochem.
267:6043-6049, and Elmquist et al., (2001) Exp. Cell Res.
269:237-244, the contents of which are incorporated herein by
reference in its entirety.
[0047] Desirably, the positively charged MRP may include the amino
acid sequence: KKWKMRRNQFWVKVQRG (SEQ ID NO: 1), which is related
to the reverseomer sequence of the antennapedia sequence.
Preferably, the MRP is attached to the carboxyl terminal end of a
subject compound (e.g. peptide).
[0048] Cell death can occur by either necrosis or apoptosis.
p53-targeting treatments typically cause cell death through
apoptosis, while the compounds and methods of the present invention
cause cell death by necrosis. Necrosis is not genetically
controlled, while apoptosis is genetically controlled. Apoptosis is
the deliberate cellular response to specific environmental and
developmental stimuli or programmed cell death. Cells undergoing
apoptosis exhibit cell shrinkage, membrane blebbing, chromatin
condensation and fragmentation. Necrosis involves the destruction
of cytoplasmic organelles and a loss of plasma membrane integrity.
Though apoptosis does not have the inflammation which results when
cancer cells die through necrosis, p53 targeting treatments fail to
treat those cancers that do not exhibit p53, or, through mutations,
exhibit an inactive p53 form that is unresponsive to p53 targeted
treatments. After the DNA damage in the caspase enzyme pathway,
there are a series of events which occur that involve calcium
activation and calpain enzymes which further leads to other
cellular changes and regulation of cytoplasmic enzymes. During
p53-dependent apoptosis, there is a sequential expression of
annexin V-binding membrane phospho-Serine, Bax waf.sup.p21, and
caspases; these proteins are used as markers for p53-dependent
apoptosis.
[0049] A major difference between necrosis and apoptosis in vivo is
the complete elimination of the apoptotic cell before an
inflammatory response is seen. Necrosis usually causes
inflammation. Though apoptosis can be thought of as a clean and
neat process, the p53 targeting treatments do not result in
apoptosis in all types of cancer cases. Though necrosis may
typically cause an inflammatory response to a treatment site
directed at targeting HDM-2, HDM-2-targeting treatments are more
effective against various forms of cancer, including those where
p53 is not present in the cancer cells, or where p53 is in a
mutated or an inactive form.
[0050] Human pancreatic cancer cells, MiaPaCa-2 cells, were treated
with PNC-28. Necrosis was determined by measuring lactate
dehydrogenase (LDH) as well as elevation of pro-apoptotic proteins.
Mutant PNC-compound (PNC-29) and HDM-2-binding domain p53 aa12-26
without MRP (PNC-26) were controls. PNC-29 and PNC-26 are both used
as controls, as PNC-29 includes a non-p53 peptide bound to the MRP,
and PNC-26 includes aa 12-26 of the p53 binding domain but no
MRP.
[0051] Since the inventors have discovered evidence that MRP is
required for anti-cancer activity, the inventors of the present
invention tested "naked" p53 peptide without MRP by transfecting a
plasmid that encodes p53 aa17-26 segment of PNC-28 into MiaPaCa-2
and an untransformed rat pancreatic acinar cell line, BMRPA1.
Time-lapse electron microscopy was employed to further elucidate
anti-cancer mechanism.
[0052] The inventors acquired the following results from the above
experiment. Treatment with PNC-28 does not result in the elevation
of pro-apoptotic proteins found in p53-induced apoptosis, but
elicits rapid release of LDH, which is indicative of tumor cell
necrosis. Accordingly, using transmission electron microscopy, the
inventors of the present invention observed membrane pore formation
and dose-dependent killing. In direct contrast, MiaPaCa-2 cells,
that were transfected with a vector expressing p53 aa 17-53, as in
PNC-28, underwent apoptosis, and not necrosis, as evidenced by
expression of high levels of caspases-3, 7 and annexin V with
background levels of LDH.
[0053] These results suggest that PNC-28 may be effective in
treating human pancreatic cancer. More particularly, these results
suggest that compounds having an HDM-2 binding domain which is
attached to an MRP at the carboxyl terminal end may be effective in
treating human pancreatic cancer. The MRP appears responsible for
the fundamental change in the mechanism of action inducing rapid
necrosis initiated by membrane pore formation. Cancer cell death by
apoptosis was observed in the absence of MRP. Thus, PNC-28 and
compounds of similar form and function will cause cancer cell
necrosis by a cell membrane pore formation mechanism, rather than a
p53 targeted treatment within the cell, which causes necrosis.
[0054] The inventors of the present invention have developed two
peptides, PNC-27 and PNC-28, that contain p53 protein residues
12-26 and 17-26, respectively, attached to a MRP. Although
originally conceived to block the binding of p53 to HDM-2 in cancer
cells, thereby increasing the half-life of p53 preventing its
ubiquitination and proteosomic degradation, it was determined that
these compounds caused cancer cell death even in cells that lacked
p53 expression (1,3). The principals further observed that in
cancer cells treated with these compounds, there was no increase in
expression of p53-induced pro-apoptotic proteins such as caspase
and Bax (1-3). Rather, these compounds induced tumor cell necrosis
as evidenced by the rapid release of lactate dehydrogenase (LDH)
from treated cancer cells (2, 3).
[0055] Interestingly, fluorescent probe-labeled PNC-27 was detected
at early stages after treatment in the cell and nuclear membranes
(2). Time-lapse electron microscopy studies later revealed that
both compounds induced pore formation in the cell and nuclear
membranes, consistent with the compounds being membrane active (2).
Furthermore, consistent with this activity, the three-dimensional
structure of PNC-27 by two-dimensional NMR was determined to be an
amphipathic alpha-helix-loop-alpha-helix, a structural motif
similar to that of a number of membrane-active peptides (4, 5).
Importantly, the principals devised a double-fluorescent-labeled
PNC-27 that contained a green fluorescent probe (fluorescein
isothiocyanate, i.e., FITC) on the amino terminal end and a red
fluorescent probe (rhodamine) on the carboxyl terminal end
(MRP-end). When this double-labeled PNC-27 was incubated with
MIA-PaCa-2 and MCF-7 cancer cells, a high density of yellow
fluorescence confined to the cell membrane after 1 hour of
incubation was identified. The only manner in which yellow
fluorescence could be obtained is if the amino and carboxyl
terminal ends of the compound stay together, i.e., there is no
splitting of PNC-27 into HDM-2 targeting and MRP
portions/components. During this time, there is a maximal release
of LDH into the incubation medium, indicating maximal cell membrane
damage. Thus the full PNC-27 compound is required for cell membrane
lysis and cancer cell death.
[0056] Though these compounds induce tumor cell necrosis among a
wide range of different human tumors, including TUC-3 metastatic
pancreatic cancer cells (6), they remarkably have no effect on the
growth and viability of a number of normal cell lines. These
include rat pancreatic acinar cells, called BMRPA1 (1,3), the
normal counterpart of TUC-3 cells, human breast epithelial
(MCF-10-2A) cells (2), and cord blood-derived human stem cells (1).
These compounds also have no effect on the growth or viability of
human keratinocytes and human fibroblasts. As previously shown,
both compounds appear to induce the killing of cancer but not
normal cells by a novel membranolytic mechanism (2).
[0057] In contrast, several studies (7-12) reported p53-dependent
apoptosis of treated cancer cells when synthesized peptide
sequences targeted to bind to intranuclear HDM-2 were attached to
leader sequences on their amino terminal ends. In one such study
(12), twelve residues from the h (or m) dm-2 binding domain of p53
were synthesized and attached at their amino termini to a TAT
leader peptide. This peptide was found to induce apoptosis of uveal
melanoma and retinoblastoma cell lines, both containing wild-type
p53. Although active against cell lines homozygous for mutant p53,
this peptide was not tested against p53-null cells. Interestingly,
placement of the MRP on the amino terminal end of the p53 17-26
peptide resulted in a marked diminution in the cytotoxicity of the
compound to cancer cells (M Kanovsky, M R Pincus & J Michl,
unpublished observations).
[0058] Since PNC-27 and PNC-28 both contain p53 sequences involved
in the binding of p53 to HDM-2 but induce cancer cell death via
membranolysis in a p53-independent manner and display a structural
motif of a membrane active component that depends on the presence
of the MRP on the carboxyl terminal end of the p53 sequence, the
present inventors inquired as to whether MRP plays an essential
role in the membranolytic activity of these compounds.
[0059] To investigate this question, the "naked" p53 17-26 peptide,
i.e., with no MRP, was introduced into a human pancreatic cancer
cell line, MiaPaCa-2, by transfecting a plasmid encoding this
peptide into these cells in which peptide expression occurs. The
inventors also treated these cells with PNC-28. In both conditions,
the inventors measured expression of markers for apoptosis and
necrosis to explore whether the naked peptide induces apoptosis in
contrast to the same peptide linked to MRP on its carboxyl terminal
end (which induces necrosis).
[0060] PNC-28, but not Negative Control Peptide PNC-29, is
Cytotoxic to MiaPaCa-2 Cells.
[0061] PNC-28 was incubated with 6.times.10.sup.6 Mia-PaCa-2 cells
for 5 days at concentrations ranging from 12.5-75 .mu.mol/ml. The
anti-cancer effect on MiaPaCa-2 cells incubated with PNC-28 is
shown in FIG. 1(A-C). FIG. 1A demonstrates untreated MiaPaCa-2
cells that are not contact-inhibited and spindle-shaped, many
becoming multinucleated. After 24 h of treatment with 75 .mu.mol/ml
PNC-28, these cells appear necrotic demonstrated by membrane
blebbing and disruption, forming cell clumps coalescing into
aggregates of cellular debris (FIG. 1B). At 48 h, there was near
100 percent cell death as measured by trypan blue dye uptake. In
contrast, negative control peptide PNC-29 at 75 .mu.mol/ml had no
effect on cellular growth, morphology, and viability (FIG. 1C).
Thus, FIGS. 1A through 1C illustrate that peptide sequence that
correlates to p53 aa residues 17-26 (PNC-28 peptide sequence) with
the MRP, as opposed to non-p53 peptide sequence with MRP, causes
necrosis, resulting in mere cell clumps and cellular debris when
administered to the human pancreatic cancer cell line MiaPaCa-2.
Thus, the MRP causes pore formation in the membrane of cancer cells
when in combination with a p53 aa residue, which binds to HDM-2 in
the cell membrane.
[0062] In FIG. 2, inhibition of proliferation was obtained after
only 48 h of peptide treatment. The effective compound dose ranged
between 20 and 75 .mu.mol/ml. It should be noted that doses of
PNC-28 between 20 and 75 .mu.mol/ml induced virtually 100 percent
cell death; the times required for cell killing decreased as dose
increased. For example, 80 mmol/ml induced near total cell death in
48 h while 40 .mu.mol/ml induced similar cell death in 4 days, and
20 .mu.mol/ml induced cell death in 1 week. FIG. 2 illustrates
that, as the dosage of compound PNC-28 increases, so too does the
percent of cell death measured in a 48 hour period. While it is
possible to administer a dosage above 160 .mu.mol/ml, the cancer
cells will not be necrosed at any greater of a speed. Thus, 160
.mu.mol/ml is the desired upper limit of the dosage for the cell
sample sized used with these experiments.
[0063] FIG. 3 summarizes the cytotoxic effects on Mia-PaCa-2 cells
of PNC-28 but not control compounds including PNC-29 and the
"naked" p53 17-26 peptide (without the MRP), PNC-26, which cannot
traverse the cell membrane since it lacks the MRP. Since PNC-29
contains the MRP, but not the p53, sequence and has no effect on
cell growth, the MRP itself does not induce the observed
cytotoxicity. FIG. 3 illustrates that the number of cells in the
sample untreated as compared to treated with the two controls is
relatively the same; in contrast, PNC-28 cell death of cytotoxicity
is far greater than the negative controls and control. Thus, to
cause necrosis by the proposed HDM-2 mechanism, the compound
preferably includes both an HDM-2 binding site (HDM-2 targeting
component) and a MRP.
[0064] As a further control, the inventors' original experiment (1)
by incubating 75 mmol/ml PNC-28 with untransformed BMRPA1 acinar
cells (1) and with the untransformed breast epithelial cell line,
MCF-10-2A (2). There was no growth inhibition or cytotoxicity found
(data not shown; see refs. 1 and 2). These results suggest that
PNC-28 is lethal specifically to cancer cells and does not
interfere with normal cell growth, as concluded in the inventors'
previous studies (1,2).
[0065] Markers for Necrosis and Apoptosis in MiaPaCa-2 Cells
Treated with PNC-28.
[0066] In previous studies by the present inventors, it was found
that PNC-28 induced cancer cell death in a variety of human cancer
cells (1,2) by inducing tumor cell necrosis rather than apoptosis
(2). This was manifested in baseline expressions of caspase but
high levels of LDH within 24 h in the medium indicative of membrane
lysis. Therefore, the expression of LDH and caspase in MiaPaCa-2
cells treated with PNC-28 was investigated. FIG. 4A shows that LDH
activity is elevated in cells treated with PNC-28 (condition 2)
almost to the same extent as cells that were lysed with lysis
buffer (condition 1). On the other hand, only baseline levels of
LDH were found for cells treated with negative control PNC-29. As
is shown in FIG. 4A, when PNC-28 is administered to cancer cells,
it exhibits similar lysis causing conditions as the administration
of a lysis buffer; whereas the PNC-29 which has no HDM-2 binding
domain and does not cause lysis (and exhibits an optical density
closer to the control where no compound--or peptide--is
administered). Along the Y axis, OD refers to optical density, or
absorption, of LDH, the necrosis indicating factor.
[0067] The premise that PNC-28 induced tumor cell necrosis is
supported by electron micrographs of MiaPaCa-2 cells treated with
this compound in a study that is identical to the one performed on
breast cancer cells by the present inventors (2). As shown in FIG.
4B, MiaPaCa-2 cells treated with PNC-28 (left panel) exhibit lysis
of their plasma membranes as previously determined for breast
cancer cell lines (2), in contrast to untreated cells (right panel)
that have their plasma membranes intact. This pattern is
characteristic of tumor cell necrosis (2). Thus, PNC-28 is able to
both bind to the cell membrane and also transport at least part of
the compound molecule through the cell membrane, which results in
the pore formation, or lysis, shown in FIG. 4B.
[0068] In contrast, as can be seen in FIG. 4C, only baseline levels
of caspase were expressed in MiaPaCa-2 cells treated with PNC-28
and were identical to the level expressed in cells treated with
control, PNC-29. This finding confirms the conclusion that PNC-28
does not induce apoptosis. Thus PNC-28 induces tumor cell necrosis
in MiaPaCa-2 cells as found for other cancer cell lines (1,2).
TNF-.alpha. is a necrosis inducing agent. On the Figure, UL refers
to units luminescence, or luminescence intensity.
[0069] The results shown in FIGS. 4A-C were obtained using 25
.mu.mol/ml PNC-28. As shown in FIG. 4D, the same results were
obtained with all doses of compound that were used over the 20-75
.mu.mol/ml range, i.e., early release of LDH (necrosis indicating)
but only background levels of caspase (apoptosis indicating),
suggesting that tumor cell necrosis is induced at all
concentrations of PNC-28 and that this mechanism of induction of
cell death is not dependent on PNC-28 concentration. As is shown on
FIG. 4D, at 25 umol/ml PNC-28, caspase was approximately 0.03,
while LDH was approximately 0.235 OD; at 50 umol/ml PNC-28, caspase
was approximately 0.02, while LDH was approximately 0.44 OD; at 75
umol/ml PNC-28, caspase was approximately 0.015, while LDH was
approximately 0.35 OD; and at 75 umol/ml PNC-29 (negative control),
caspase was approximately 0.05, while LDH was approximately
0.01.
[0070] Transfection of MiaPaCa-2 Cells with a Plasmid that Encodes
the p53 17-26 Sequence.
[0071] Results of Transfection of MiaPaCa-2 and BMRPA1 Cells.
[0072] After 2 hours post-transfection, cell counts were performed
on slides using light microscopy and then counted the number of
cells exhibiting green fluorescence from GFP (Green Fluorescent
Protein). On this basis, it was found that between 30 and 45
percent of the cells expressed GFP as summarized in FIG. 9. The
highest transfection rates were found for MiaPaCa-2 cells whether
transfected with empty vector (EV) or p53 17-26-encoding vector
(p53 17-26-V).
[0073] Morphological Examination of Transfected Cells, as
Visualized by Inverted Light Microscopy:
[0074] In the initial set of transfection experiments, cells were
observed by light microscopy beginning 18 h post-transfection.
MiaPaCa-2 cells transfected with p53 17-26-V were visibly
hypertrophic, many showing membrane blebbing and some were
necrotic. In contrast, BMRPA1 cells transfected in the same way as
MiaPaCa-2 by either EV or p53 17-26-V showed little alteration in
morphology. This observation was confirmed 90 h post-transfection,
as the cells by this time point resumed their normal polygonal
epithelial cell morphology, identical to that of untreated
cells.
[0075] Cell Viability Post-Transfection.
[0076] FIG. 5 shows the effect of transfection of the plasmid p53
17-26-V encoding the p53 17-26 peptide on cell viability for
MiaPaCa-2 and untransformed BMRPA1 cells. As can be seen in this
FIG. 5, within 48 h, transfection of this plasmid induces 60
percent cell death (condition 1) while transfection of p53
17-26-scrm-V control plasmid results in a much lower level of cell
death, i.e., 20 percent as shown in condition 2. This is a baseline
level since this is the level of cell death observed for
untransformed BMRPA1 cells transfected with the same control
vector, condition 4. In condition 3 of this FIG. 5, it can be seen
that expressed p53 17-26 peptide has a much less pronounced effect
on BMRPA1 cells, resulting in the same baseline level of cell death
seen in control plasmid-transfected BMRPA1 cells (condition 4).
Thus, expression of the compound induces cell death in cancer, but
not in untransformed, cells.
[0077] Effects of the p53 17-26 Peptide on MiaPaCa-2 Cells.
[0078] MiaPaCa-2 cells expressing GFP that had been transfected
with EV or p53 17-26-V were lysed and blotted for p53, waf.sup.p21,
a protein that is induced by a p53-dependent pathway, and the p53
17-26 peptide itself. In these experiments, the DO-1 anti-p53
antibody that recognizes a determinant that contains residues 17-26
of p53 was used (2). In addition, caspase activity in these cells
was measured. For comparison, the same set of experiments was
performed on Mia-PaCa-2 cells treated with 75 .mu.mol/ml of PNC-28.
As can be seen in FIG. 6, at 0 time (left side of FIG. 6, labeled
"0 time") after transfection or incubation with PNC-28, peptide,
p53, waf.sup.p21 and caspase activity are all expressed at baseline
levels. At times when cell death was near 100 percent at 96 hr for
transfected cells, 48 hr for PNC-28-treated cells, peptide levels
were found to be high in both transfected and PNC-28-treated cells
(FIG. 6, right side, labeled "100% cell death"). However, in the
transfected cells, it can be seen that there are elevated levels of
p53, waf.sup.p21 and caspase activity (labeled "transfection" on
the right side of the figure) that are not observed in the
PNC-28-treated cells (labeled "PNC-28" on the right side of the
figure). For controls, actin was blotted for and it was found that
the levels were the same for all four conditions in FIG. 6 (not
shown). These results suggest that the p53 17-26 peptide induces
increased intracellular expression of p53 protein with consequent
apoptosis of MiaPaCa-2 cells, as evidenced by the concomitant
increased expression of waf.sup.p21 that does not occur in cells
treated with PNC-28. Along the right axis, luminescence intensity
is measured at 405 NM. Along the left axis, 450 NM measures the
band intensity of the Western Blot. Interestingly, when lysates
from untransformed BMRPA1 cells transfected either with EV or with
p53 17-26-V were blotted, only low levels of expression of p53 were
found. In addition, only a low level of expression of p53 17-26
peptide in p53 17-26-V-transfected cells was found (results not
shown). Since GFP was expressed at high levels in these cells, it
appears that expressed peptide is unstable in these untransformed
cells.
[0079] Expressed p53 17-26 Peptide Induces Apoptosis, not Necrosis,
of MiaPaCa-2 Cells.
[0080] Since expression of p53 and waf.sup.p21 was elevated in
cells transfected with p53 17-26-V to much higher levels than in
cells transfected with control vector, it was concluded that the
peptide was inducing apoptosis in contrast to its counterpart
PNC-28 peptide as discussed above. Further confirmation of
peptide-induced apoptosis was sought. In the early stages of
apoptosis, phosphatidyl serine (PS), normally present in the inner
leaflet of the bilayer membrane of intact cells, is found on the
external plasma membrane of cells undergoing apoptosis Annexin V
binds PS and can be located by a probe that carries the red
fluorescent TRITC probe. Consequently, cells that had been
transfected approx 48 h earlier were processed for staining with
Annexin V-biotin followed by streptavidin-TRITC. FIG. 7 shows the
confocal microscopic results for MiaPaCa-2 cells transfected with
control vector (upper left), showing green fluorescent cells with
no red staining, and cells transfected with p53 17-26-V that show
green fluorescent cells with strong red staining for PS. On the
right side of the figure are the results for BMRPA1 control cells
that have been transfected with control vector (upper right) or p53
17-26-V (lower right). As can be seen in FIG. 7, neither panel is
positive for PS in the normal control cells. As discussed above,
expression of p53 17-26 peptide is low in this cell line, possibly
causing the absence of signs of apoptosis. Thus the p53 17-26
peptide induces apoptosis in the cancer cell line only.
[0081] Caspase and LDH in Transfected MiaPaCa-2 Cells.
[0082] To compare the results with those from MiaPaCa-2 cells
treated with PNC-28 peptide (FIG. 4) p53 17-26-V-transfected cells
were assayed for caspase and LDH release. As shown in FIG. 8A,
condition 3, caspase expression is over four-fold higher in these
treated cells than in untreated cells (condition 2) and has the
same fold-increase over that in cells treated with PNC-28 and
control PNC-29. In contrast, as shown in FIG. 8B, LDH release from
these transfected cells (condition 3) is at the baseline level
found for untreated cells (condition 2) and is about five-fold
lower than release from cells treated with PNC-28 (condition 4).
Thus, presence of the MRPin PNC-28 results in a change in the
mechanism of action of the p53 17-26 peptide; without MRP, the
peptide induces apoptosis, while, with MRP on its carboxyl terminal
end, the peptide induces tumor cell necrosis.
[0083] Prior Evidence that the MRP in PNC-27 and PNC-28 is Required
for Induction of Tumor Cell Necrosis.
[0084] The purpose of this study was to define the role of the MRP
in PNC-28 in inducing tumor cell necrosis. In previous studies with
this MRP and its compound, PNC-27, it was found that both PNC-27
and PNC-28 induced tumor cell necrosis, not apoptosis, and caused
necrosis even in cancer cells in which p53 protein was absent
(1-3). These findings suggested that both PNC-27 and PNC-28 exerted
their effects independently of p53 activation. They contrasted with
the results of studies in which similar p53 sequences, were
attached to MRP on their amino terminal ends, and induced
p53-dependent tumor cell apoptosis, not necrosis (7-11). This
results from the binding of these peptides to HDM-2 in place of the
p53 protein; these peptides are not themselves ubiquitinated since
the sites for p53 ubiquitination lie outside this domain (16).
[0085] Further evidence that the presence of the MRP on the
carboxyl terminal end of PNC-27 and 28 is essential for its
induction of tumor cell necrosis is the three-dimensional structure
of PNC-27. PNC-27 was found to have a highly amphipathic
alpha-helix-loop-alpha-helix structure that is found in
membrane-active peptides (4). Disruption of this structure, as may
occur by placement of the MRP on the amino terminal end of the
compound, would be expected to change the activity of the compound.
It was found that the p53 17-26 peptide containing the MRP on its
amino terminus, called reverse or r-PNC-28, has much lower activity
in cell killing than does PNC-28 itself (M. Kanovsky, J. Michl and
M. R. Pincus, unpublished observations). These findings support the
conclusion that the MRP is critical to the activity of the compound
but leaves open the question as to whether the "naked" p53 peptide
itself can induce necrosis or apoptosis of cancer cells.
[0086] Cells Transfected with pTracer-SV40 Plasmid Encoding p53
17-26 "Naked" Peptide Express this Peptide.
[0087] To define the role of the MRP definitively, it was sought to
determine the effects of the p53 17-26 peptide itself on tumor cell
growth, i.e., whether even without the MRP, it could induce tumor
cell necrosis. To accomplish this goal, the p53 peptide was
introduced into MiaPaCa-2 cells via transfection using the
pTracer-SV40 plasmid that constitutively expressed this peptide.
Then, the expression of markers for apoptosis and necrosis in the
transfected cells and compared the levels of these markers with
those found in MiaPaCa-2 cells treated with PNC-28 was
determined.
[0088] As can be seen in FIG. 9, transfection efficiencies were
relatively high. MiaPaCa-2 cells transfected with the p53
peptide-expressing plasmid expressed high levels of the p53 peptide
and expressed high levels of GFP as revealed by Western blots over
this time period. By 90 hours, when at least two-thirds of the
cells were killed (FIG. 7), peptide expression decreased to barely
detectable levels while GFP levels also decreased significantly
(FIG. 6). This phenomenon may have been caused by cell death and
release of proteases causing peptide degradation. On the other
hand, BMRPA1 cells transfected with the same peptide-encoding
plasmid expressed much lower levels of this peptide. Since these
cells expressed high levels of GFP, which is expressed under the
same promoter, it is not likely that p53 peptide was not also being
synthesized in these cells. One possible explanation for this
observation is the status of HDM-2. Recently, it was found by the
present inventors that this protein is expressed at barely
detectable levels in untransformed cells, including BMRPA1 cells,
but is expressed at high levels in transformed cells. If binding of
the small p53 decapeptide to HDM-2 blocks its degradation in cells,
absence of HDM-2 may make the peptide susceptible to intracellular
proteases resulting in its degradation.
[0089] The p53 17-26 Peptide Induces Apoptosis of Cancer Cells.
[0090] As summarized in FIG. 6, expression of the p53 17-26 peptide
in MiaPaCa-2 cells induces increasing expression of p53 with
concomitant increasing expression of waf.sup.p21 over a time period
in which cell death increases. These results are compatible with
binding of the peptide to hdm-2, blocking the binding of p53 to
this protein, resulting in prolongation of the half-life of p53.
This would result in its increased expression in MiaPaCa-2 cells,
allowing it to cause apoptosis, explaining the increasingly
elevated levels of waf.sup.p21. As shown in FIG. 8A, caspase, a
marker for p53-dependent apoptosis, is elevated to almost five
times the background level (condition 2) in p53 17-26
peptide-expressing MiaPaCa-2 cells (condition 3). Likewise, it is
elevated above background in cells incubated with PNC-28 (condition
4). Also, in virtually all MiaPaCa-2 cells treated with p53 17-26
peptide-expressing cells, there was strong expression of
annexin-V-binding phosphatidyl serine in the membranes of
transfected MiaPaCa-2 cells, a known early phenomenon in apoptosis
(15), but not in MiaPaCa-2 cells transfected with empty vector
(FIG. 7). These results suggest that the naked p53 17-26 peptide
causes cancer cell death by inducing apoptosis. Furthermore, the
p53 peptide-expressing MiaPaCa-2 cells do not release LDH in 24 h
(condition 3, FIG. 8B) as would be expected if the peptide induced
tumor cell necrosis. In contrast, treatment of MiaPaCa-2 cells with
PNC-28 resulted in high levels of LDH (condition 2, FIG. 4A and
condition 4 in FIG. 8B) over this time course that began as early
as several minutes after treatment, confirming that the peptide
induces tumor cell necrosis and not apoptosis. This effect is
independent of the concentration of PNC-28, suggesting that its
mechanism of action does not change in a concentration-related
manner. This finding, that expression of the p53 17-26 peptide in a
cancer cell line induces apoptosis, is in agreement with the
results of prior studies that showed that similar peptides from the
HDM-2 binding domain of p53 likewise induce apoptosis (7-12).
[0091] Overall, experimental results set forth herein strongly
suggest that the p53 17-26 peptide induces tumor cell apoptosis and
not necrosis. On the other hand, presence of the MRP to the
carboxyl terminal end of the p53 17-26 peptide plays a critical
role in changing the mechanism of cell killing of this peptide from
apoptosis to tumor cell necrosis.
[0092] The p53 17-26 Peptide Induces Apoptosis in Tumor but not
Normal Cells.
[0093] As shown in FIG. 5, transfection of empty vector in
MiaPaCa-2 cells causes a low level of cell death. However, as shown
in FIG. 7, transfection of empty vector into MiaPaCa-2 cells causes
no exposure of phosphatidyl serine as revealed by absence of
annexin V binding suggesting that the transfection does not induce
apoptosis. On the other hand, transfection of the vector inducing
expression of the p53 17-26 sequence into these cells induces
higher rates of cell death (FIG. 5). In all of these cells, as
shown in FIG. 7, there is strong annexin binding suggesting that
these cells are all undergoing apoptosis.
[0094] In contrast, transfection of the p53 peptide-expressing
vector into untransformed BMRPA1 cells does not result in an
increase in cell death over the background (FIG. 7). Furthermore,
neither transfection of empty vector or of p53 peptide-expressing
vector results in any exposure of annexin-binding phosphatidyl
serine (FIG. 8). These findings may be due at least partly to the
low level of expression of the p53 17-26 peptide. Nonetheless, the
p53 peptide-encoding plasmid does not induce apoptosis in
untransformed BMRPA1 cells. Thus, like PNC-27 and PNC-28, the p53
17-26 peptide appears to be selective for killing cancer but not
untransformed cells.
[0095] In summary, the MRPon the carboxyl terminal end of PNC-28
causes it to induce tumor cell necrosis. Removal of the MRP from
the p53 17-26 peptide in cancer cells results likewise in
cytotoxicity to these cells except by apoptosis of the tumor cells.
Thus presence of the MRP on the carboxyl terminal end of p53
peptide results in a fundamental change in the mechanism of action
of the compound in causing tumor cell death. Like the full peptide,
PNC-28, the naked p53 17-26 peptide appears to be selective to
inducing apoptosis of cancer cells but not of untransformed cells.
This may be due in part to low levels of expression of the peptide
in untransformed cells that express low levels of HDM-2 that may
shield expressed peptide from protease degradation. This finding
implies that introduction of the naked peptide into cells can
induce tumor cell apoptosis while leaving normal cells
unaffected.
[0096] These results underscore the importance of elucidating the
p53-hdm-2 interaction in the cancer cell, whereby it is possible to
gain a better understanding of the manner of which this complex
potentiates a possible cross-talk between necrotic and apoptotic
pathways that may lead to novel approaches for directed therapy. As
a result, such with HDM-2 targeting component(s) and MRP(s)
therapeutics may deliver selective cytotoxic small molecules to
cancer cells targeting pathways inducing necrosis, apoptosis or
both.
[0097] A method of selectively necrosing cells is provided. The
method includes the steps of providing a plurality cells, including
at least one cancer cell and at least one non-cancerous cell; and
administering to the cells a compound, including an HDM-2 targeting
component and a cytotoxic component, said cytotoxic component
attached to said HDM-2 targeting component, wherein said compound
comprises a membrane-active form. The cytotoxic component may
desirably be, for example, a membrane resident peptide (MRP), a
toxin, a drug, a radionuclide, a whole antibody, an antibody
fragment, and combinations thereof. The HDM-2 targeting component
may be, for example, a small molecule, a peptide, a protein, a
glycoprotein, a whole antibody, an antibody fragment, and
combinations thereof.
[0098] One or more of the methods may optionally include the step
of observing, in a cell medium, a level of LDH. A level of LDH may
be an amount of LDH measured as omitted from a number of cells. LDH
is known necrosis indicator, thus, the presence of LDH in the cell
medium of cells treated with the compound of the present method
indicates that at least some of the cells have undergone necrosis,
cell death. Necrosis may similarly be observed in a sample of cells
by microscopically inspecting the cells to determine whether the
cell membranes are intact or have transmembrane pore formation
therein/thereon.
[0099] Though the compound of the present method selectively
necroses cancer cells, the compound has no observable effect on
non-cancerous cells. Thus, the method may further comprise the step
of observing for a non-response in a non-cancerous cell. The cells
may be observed for pore formation, cell breakdown, and the like.
However, observation and/or analysis of the non-cancerous cells
will yield no effect on the non-cancerous cells. Similarly, this no
effect may be referred to as a non-response by the non-cancerous
cells to the cancer treatment (compound) administered. As such, the
present method results in necrosis of cancer cells, while
non-cancerous cells are unaffected.
[0100] A method of causing membranolysis in at least one cancer
cell is provided. The method of causing membranolysis in at least
one cancer cell further includes administering to at least one
cancer cell a compound comprising an HDM-2 targeting component and
a pore forming component attached to said HDM-2 targeting
component, wherein said administering step results in at least one
transmembrane pore in a cancer cell membrane. The pore forming
component may include, for example, any chemical moiety with a pore
forming character when put into association with a cell membrane,
desirably, a cancer cell membrane. The pore forming component may
include, for example, a membrane resident peptide (MRP).
[0101] Causing membranolysis in cancer cells is a desirable basis
for cancer treatments. The present method may be employed to more
readily understand the dosing effectiveness of the cancer treatment
and/or compound. Thus, administration of multiple dosages may be
completed in order to more readily understand the upper and lower
limits of effectiveness, if any. Further, the effectiveness of
various treatment plans, repeating administration, may be studied
utilizing this method. Also, various types of cancer cells and/or
pre-cancerous cells (atypical cells) may be studied to analyze and
determine varying levels of aggressiveness, progression,
treatability, and/or responsiveness to dosages of the compound of
the present method.
[0102] The method of causing membranolysis in at least one cancer
cell may further include observing membranolysis in the cancer cell
by: detecting an LDH amount, performing electron microscopy,
observing cell morphology, and combinations thereof. By observing
membranolysis, the effectiveness of various dosages and,
potentially, the effectiveness combination therapies, may be better
understood. The observation of membranolysis is linked to necrosis
in the cancer cells, as the structural integrity of the cell is no
longer maintained once transmembrane pores trigger membranolysis.
However, varying degrees of necrosis may be observed. This may be
due, in part, to the natural breakdown of the compound within a
sample or a subject. Further, this may be linked to dosage,
strength of the cancer cells, or duration of treatment.
[0103] A method of treating cancer in a subject in need thereof is
provided. The method includes administering to the subject in need
thereof a therapeutically effective amount of a compound having an
HDM-2 targeting component and a membrane resident peptide (MRP),
said HDM-2 targeting component and said MRP having a
membrane-active form. After the administration step, optionally,
the method may include determining whether a plurality of cancerous
cells have undergone membranolysis.
[0104] The cytotoxic component may include, for example, a membrane
resident peptide, a toxin, a drug, a radionuclide, a whole
antibody, an antibody fragment, and combinations thereof. The HDM-2
targeting component is selected from the group consisting of: a
small molecule, a peptide, a protein, a glycoprotein, a whole
antibody, an antibody fragment, and combinations thereof.
[0105] A method of screening cancer treatments is provided. The
method includes providing a plurality of cancerous cells; each of
said cells having HDM-2 in said cellular membranes; administering a
candidate cancer treatment to the plurality of cancer cells; and
measuring the level of LDH present in a cellular medium. LDH is a
known necrosis factor, thus, the measurement of a level or amount
of LDH present after administering a candidate cancer treatment to
the cancer cells with detect whether the cancer treatment was
successful in causing cell death to cancer cells.
[0106] Optionally, the candidate cancer treatment may be screened
to determine whether the candidate cancer treatment has membrane
active conformation. The membrane active conformation may refer to
the desired shape of the candidate cancer treatment in solution.
The conformation may be directly linked to the candidate cancer
treatment's ability to colocalize with HDM-2 in the cancer cell
membrane and be retained within the cancer cell membrane, causing
the formation of pores therein. Desirably, candidate cancer
treatments may have a three dimensional shape or conformation in an
alpha-helix-loop-alpha-helix. This is the three-dimensional shape
that has been determined by the present inventors for the PNC-27
and PNC-28 peptide-based compositions. The alpha-helix-loop-helix
conformation allows the composition to advantageously interact with
the cancer cell membrane.
[0107] Optionally, the candidate cancer treatment may be screened
to determine the candidate cancer treatment includes an HDM-2
targeting component. To determine this, it is possible to analyze
the HDM-2 targeting component to determine whether it has any
affinity and or binding capability to HDM-2 in cancer cell
membranes.
[0108] Optionally, the method may further include the step of
observing the cancer cell membranes for an area of pore formation.
Pore formation is the mechanism by which membranolysis is caused,
which results in cancer cell death. Pore formation may be
microscopically observed. Also, various assays may be completed,
measuring known necrotic factors, including, for example, LDH.
[0109] A candidate cancer treatment which may be found to have a
membrane active form, cause membranolysis to cancer cells, have a
similar conformation to PNC-27 and/or PNC-28, and have an HDM-2
binding affinity may be termed a material with anti-cancer
"activity". Use of the term "activity" with respect to a cancer
treatment with reference to the embodiments of the present
invention refers to an ability to induce a desirable effect upon in
vitro, ex vivo, or in vivo administration of the compound.
Desirable effects include preventing or reducing the likelihood
(increasing the likelihood or causing) one or more of the following
events: binding to HDM-2 in cancer cells, insertion into the cancer
cells' plasma membrane, assembly and pore foundation, transport
across the cancer cell membrane, causing membranolysis. Materials
with anti-cancer "activity" may be flagged for further testing,
review, and consideration as viable cancer treatments.
[0110] Identifying drug candidates from cancer candidate treatments
flagged as having "activity", typically involves multiple phases.
During the early stages, compounds, preferably large libraries of
compounds are screened or tested in vitro for binding to and/or
biological activity at the cancer cell membrane (with HDM-2 and/or
a membrane resident component characteristic). The compounds that
exhibit activity ("active compounds" or "hits") from this initial
screening process are then tested through a series of other in
vitro and in vivo tests to further characterize the anti-cancer
normal, non-cancerous tissue and organ protective activity of the
compounds.
[0111] The in vivo tests at this phase may include tests in
non-human mammals such as those mentioned above. If a compound
meets the standards for continued development as a drug following
in vitro and in vivo tests, the compound is typically selected for
testing in humans.
[0112] A progressively smaller number of test compounds at each
stage are selected for testing in the next stage. The series of
tests eventually leads to one or a few drug candidates being
selected to proceed to testing in human clinical trials. The human
clinical trials may include studies in a human suffering from a
medical condition that can be treated or prevented by reducing
cancer cells (inducing cancer cell necrosis).
[0113] A method of selectively necrosing cancer cells is provided.
The method includes providing a plurality of cells, including at
least one cancer cell and at least one non-cancerous cell; and
contacting the plurality of cells with an HDM-2 targeting compound
which includes a membrane resident peptide (MRP), wherein the HDM-2
targeting compound colocalizes to HDM-2 present in at least one
cancer cell membrane, binding to a cell membrane of the at least
one cancer cell and adopting a membrane-resident conformation
within said cancer cell membrane.
[0114] The adoption of a membrane-resident conformation may further
include forming a pore in said cancer cell membrane. Optionally,
the method may include observing necrosis in the plurality of
cancer cells, while observing necrosis was previously discussed.
Similarly, the method may include the step of optionally observing
a non-result in the non-cancerous cells, which is indicative of
non-targeting of said non-cancerous cells by the method and its
related compound.
[0115] A method of identifying cancer cells is provided. The method
of identifying cancer cells includes providing a plurality of
cells, wherein at least one of said cells is a candidate cancer
cell; and administering to the plurality of cells an HDM-2
recognition agent.
[0116] Candidate cancer cells, as used herein, may generally refer
to cells, cellular samples, or tissues which may include cancer
calls, pre-cancerous cells, and non-cancerous cells. The method of
the present invention may be used to determine which of the cells,
if any, within the candidate cancer cells are cancerous. The
plurality of candidate cancer cells may be optionally observed to
determine whether the HDM-2 recognition agent colocalizes with at
least one cell membrane. Colocalization may result in the HDM-2
recognition molecule being bound to or transported through the cell
membrane, depending on the form of the HDM-2 recognition agent. If
the HDM-2 recognition agent takes on the form of PNC-27, or a
non-peptide component with an MRP attached thereto, the HDM-2
recognition agent will be taken into the cell membrane of cancer
cells, in a membrane active form. This is indicative of the
recognition agent's tendency to be bound to HDM-2 in a cancer
cell.
[0117] In order to better observe and determine which candidate
cancer cells, if any, are cancerous, it is possible to optionally
tag the HDM-2 recognition agent with an observation aid. The
observation aid may be attached to the HDM-2 recognition agent such
that the observation aid follows the HDM-2 recognition agent and
does not interfere with any colocalization to HDM-2 in the cancer
cell membranes. The observation aid may include one or more
materials, as may be desired. The observation aid may be, for
example, a dye, a fluorescing agent, a radiopaque material, a
radioactive isotope, and combinations thereof. One useful
observation aid may include, for example, horseradish radish
peroxidase.
[0118] Identifying at least one cancer cell may refer to
identifying that the HDM-2 recognition agent has colocalized with,
bound to, or otherwise affiliated with the surface of a cellular
membrane containing HDM-2. This may be based on the premise that
HDM-2 is contained in cancer cells at roughly five times greater
presence than in non-cancerous cells.
[0119] Once the HDM-2 recognition agent has tagged or affiliated to
cancer cell membranes present out of the total candidate cancer
cells (or surrounding non-cancerous tissue), it is possible to
quantify and qualify the size, shape, progression, and general
nature of a plurality of cancer cells. Thus, a plurality of cancer
cells may be mapped or plotted with respect to the surrounding
frame of reference (in a subject, the surrounding anatomy and/or
tissues), in order to better understand the placement and size of
the cancer cells (cancerous tissue). The observation aids,
including, for example, dyes, fluorescing agents, radiopaque
materials, and the like, may aid in visualizing the HDM-2
recognition agent within a sample that may have a large amount of
cells present. Thus, once the HDM-2 recognition agent is
administered to cells, various known visualization techniques,
including filtered scopes to detect light at certain wavelengths of
the electromagnetic spectrum, x-ray, catscan, and the like may be
employed in order to better see and understand the plurality of
cancerous cells. Thus, the map of the cancer cells may be useful in
diagnosing types of cancer, treatments for cancer, surgical removal
thereof, observing the progression, monitoring for relapse of
cancer, and/or the responsiveness to treatments.
[0120] The compounds, agents, and or materials used in conjunction
with one or more of the methods of the present invention may refer
to PNC-27, PNC-28, or combinations thereof, as discussed herein.
Further, it should be readily understood that non-peptide materials
which may desirably have an HDM-2 affinity or binding site may be
used in conjunction with the MRP. Hybrid materials containing
peptide and non-peptide components, along with wholly non-peptide
materials may be used with one or more of the methods of the
present invention. The synthesis of one or more of the compounds
may be subsequently followed by purification, as is commonly done
in the art. The compounds synthesized are preferably in purified
form to be used as the compound and with the methods of the present
invention. Thus, the present invention contemplates the use of
peptide as well as non-peptide materials, and combinations thereof,
to cause selective necrosis to cancer cells, in accordance with the
present invention.
[0121] One or more of the methods referenced herein may optionally
include a reiteration or repeated administration step. That is,
after the administration step, it is possible to determine whether
a plurality of subsequent cancer cells exist and remain intact. If
so, it is possible to complete one or more of the method steps for
each of the methods previously discussed, including the
administration of the compound, HDM-2 recognition agent, and the
like.
[0122] The compounds, agents, and materials used in conjunction
with one or more of the aforementioned methods are desirably in a
purified form. Purified form, as used herein, generally refers to
material which has been isolated under certain desirable conditions
that reduce or eliminate unrelated materials, i.e. contaminants.
Substantially free from contaminants generally refers to free from
contaminants within analytical testing and administration of the
material. Preferably, purified material is substantially free of
contaminants is at least 50% pure, more preferably, at least 90%
pure, and more preferably still at least 99% pure. Purity can be
evaluated by conventional means, e.g. chromatography, gel
electrophoresis, immunoassay, composition analysis, biological
assay, NMR, and other methods known in the art.
[0123] At least one cancer cell, as used herein, may similarly
refer to a plurality of cancer cells. A plurality of cells may
include, a sample of cells, a tissue sample, a tumor, and/or even a
subject having cancer. At least one cell may refer to one cell, a
plurality of cells, a sample of cells, a tissue sample, and/or even
a subject. A plurality of cells including at least one cancer cell
and at least one non-cancerous cell may refer to a mixture of cells
in a sample, an area of tissue including both cancerous and
non-cancerous tissues, and or a subject diagnosed with cancer.
[0124] The term "subject", as used herein may refer to a patient or
patient population diagnosed with, or at risk of developing one or
more forms of cancer. Also, as used herein, a subject may refer to
a living animal, including mammals, which may be given cancer
through transplantation or xenotransplanting which may be
subsequently treated with the methods and compounds of the present
invention or which have developed cancer and need veterinary
treatment. Such subjects may include mammals, for example,
laboratory animals, such as mice, rats, and other rodents; monkeys,
baboons, and other primates, etc. They may also include household
pets or other animals in need of treatments for cancer.
[0125] The terms "therapeutically effective dosage" and "effective
amount" refer to an amount sufficient to kill one or more cancer
cells. A therapeutic response may be any response that a user (e.g.
a clinician will recognize) exhibits as an effective response to
the therapy, including the foregoing symptoms and surrogate
clinical markers. Thus, a therapeutic response will generally be an
amelioration or inhibition of one or more symptoms of a disease or
disorder, e.g. cancer.
[0126] Administering, as referred to by one or more of the methods
of the present invention, may include contacting. The term
"contacting" refers to directly or indirectly bringing the cell and
the compound together in physical proximity. The contacting may be
performed in vitro or in vivo. For example, the cell may be
contacted with the compound by delivering the compound into the
cell through known techniques, such as microinjection into the
tumor directly, injecting the compound into the bloodstream of a
mammal, and incubating the cell in a medium that includes the
compound.
[0127] Any method known to those in the art for contacting a cell,
organ or tissue with a compound may be employed. Suitable methods
include in vitro, ex vivo, or in vivo methods. In vitro methods
typically include cultured samples. For example, a cell can be
placed in a reservoir (e.g., tissue culture dish), and incubated
with a compound under appropriate conditions suitable for inducing
necrosis in cancer cells. Suitable incubation conditions can be
readily determined by those skilled in the art.
[0128] Ex vivo methods typically include cells, organs or tissues
removed from a mammal, such as a human. The cells, organs or
tissues can, for example, be incubated with the compound under
appropriate conditions. The contacted cells, organs or tissues are
normally returned to the donor, placed in a recipient, or stored
for future use. Thus, the compound is generally in a
pharmaceutically acceptable carrier.
[0129] In vivo methods are typically limited to the administration
of a compound, such as those described above, to a mammal,
preferably a human. The compounds useful in the methods of the
present invention are administered to a mammal in an amount
effective in necrosing cancer cells for treating cancer in a
mammal. The effective amount is determined during pre-clinical
trials and clinical trials by methods familiar to physicians and
clinicians.
[0130] The compounds useful in the methods of the invention may
also be administered to mammals by sustained release, as is known
in the art. Sustained release administration is a method of drug
delivery to achieve a certain level of the drug over a particular
period of time. The level typically is measured by serum or plasma
concentration.
[0131] The compounds of one or more of the aforementioned methods
of the present invention may be administered to a human in an
amount effective in achieving its purpose. The effective amount of
the compound to be administered can be readily determined by those
skilled in the art, for example, during pre-clinical trials and
clinical trials, by methods familiar to physicians and clinicians.
Typical daily doses include approximately 1 mg to 1000 mg.
[0132] An effective amount of a compound useful in the methods of
the present invention, preferably in a pharmaceutical composition,
may be administered to a mammal in need thereof by any of a number
of well-known methods for administering pharmaceutical compounds.
The compound may be administered systemically or locally.
[0133] Any formulation known in the art of pharmacy is suitable for
administration of the compounds useful in the methods of the
present invention. For oral administration, liquid or solid
formulations may be used. Some examples of formulations include
tablets, capsules, such as gelatin capsules, pills, troches,
elixirs, suspensions, syrups, wafers, chewing gum and the like. The
compounds can be mixed with a suitable pharmaceutical carrier
(vehicle) or excipient as understood by practitioners in the art.
Examples of carriers and excipients include starch, milk, sugar,
certain types of clay, gelatin, lactic acid, stearic acid or salts
thereof, including magnesium or calcium stearate, talc, vegetable
fats or oils, gums and glycols.
[0134] Formulations of the compounds useful in the methods of the
present inventions may utilize conventional diluents, carriers, or
excipients etc., such as those known in the art to deliver the
compounds. For example, the formulations may comprise one or more
of the following: a stabilizer, a surfactant, preferably a nonionic
surfactant, and optionally a salt and/or a buffering agent. The
compound may be delivered in the form of an aqueous solution, or in
a lyophilized form. Similarly, salts or buffering agents may be
used with the compound.
[0135] The compounds of the present invention may be administered
in therapeutically effective concentrations, to be provided to a
subject in standard formulations, and may include any
pharmaceutically acceptable additives, such as excipients,
lubricants, diluents, flavorants, colorants, buffers, and
disintegrants. Standard formulations are well known in the art.
See, e.g. Remington's pharmaceutical Sciences, 20th edition, Mach
Publishing Company, 2000. The formulation may be produced in useful
dosage units for administration by any route that will permit the
compound to contact the cancer cell membranes. Exemplary routes of
administration include oral, parenteral, transmucosal, intranasal,
insulfation, or transdermal routes. Parenteral routes include
intravenous, intra-arterial, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraductal, intraventricular,
intrathecal, and intracranial administrations.
[0136] The pharmaceutical forms suitable for injection include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. The ultimate solution form in all cases must be
sterile and fluid. Typical carriers include a solvent or dispersion
medium containing, e.g., water buffered aqueous solutions, i.e.,
biocompatible buffers, ethanol, polyols such as glycerol, propylene
glycol, polyethylene glycol, suitable mixtures thereof, surfactants
or vegetable oils. Sterilization may be accomplished utilizing any
art-recognized technique, including but not limited to filtration
or addition of antibacterial or antifungal agents.
[0137] The compounds of the present invention may be administered
as a solid or liquid oral dosage form, e.g. tablet, capsule, or
liquid preparation. The compounds may also be administered by
injection, as a bolus injection or as a continuous infusion. The
compounds may also be administered as a depot preparation, as by
implantation or by intramuscular injection.
[0138] The compounds, agents, and materials referenced in the
present invention may be in a "pharmaceutically acceptable
carrier". A pharmaceutically acceptable carrier includes any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic agents and the like. The use of such
media and agents are well-known in the art. The phase
`pharmaceutically acceptable` refers to molecular entities and
compositions that are physiologically tolerable and do not
typically produce unwanted reactions when administered to a
subject, particularly humans. Preferably, as used herein, the term
"pharmaceutically acceptable" means approved by a regulatory agency
of the federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly, in humans. The term carrier refers
to a diluent, adjuvant, excipient or vehicle with which the
compounds may be administered to facilitate delivery. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, or organic compounds. Water or aqueous solution saline
solutions, and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly as injectable
solutions.
[0139] The synthetic peptides which may include the compounds,
agents, and materials used with the present methods of the present
invention may be synthesized by a number of known techniques. For
example, the peptides may be prepared using the solid-phase
technique initially described by Merrifield (1963) in J. Am. Chem.
Soc. 85:2149-2154. Other peptide synthesis techniques may be found
in M. Bodanszky et al. Peptide Synthesis, John Wiley and Sons, 2d
Ed., (1976) and other references readily available to those skilled
in the art. A summary of polypeptide synthesis techniques may be
found in J. Stuart and J. S. Young, Solid Phase Peptide Synthesis,
Pierce Chemical Company, Rockford, Ill., (1984). Peptides may also
be synthesized by solid phase or solution methods as described in
The Proteins, Vol. II, 3d Ed., Neurath, H. et al., Eds., pp.
105-237, Academic Press, New York, N.Y. (1976). Appropriate
protective groups for use in different peptide syntheses are
described in the texts listed above as well as in J. F. W. McOmie,
Protective Groups in Organic Chemistry, Plenum Press, New York,
N.Y. (1973). The peptides of the present invention may also be
prepared by chemical or enzymatic cleavage from larger portions of
the p53 protein or from the full length p53 protein. Likewise,
membrane-resident sequences for use in the synthetic peptides of
the present invention may be prepared by chemical or enzymatic
cleavage from larger portions or the full length proteins from
which such leader sequences are derived.
[0140] Additionally, the peptides of the present invention may also
be prepared by recombinant DNA techniques. For most amino acids
used to build proteins, more than one coding nucleotide triplet
(codon) can code for a particular amino acid residue. This property
of the genetic code is known as redundancy. Therefore, a number of
different nucleotide sequences may code for a particular subject
peptide selectively lethal to malignant and transformed mammalian
cells. The present invention also contemplates a deoxyribonucleic
acid (DNA) molecule that defines a gene coding for, i.e., capable
of expressing a subject peptide or a chimeric peptide from which a
peptide of the present invention may be enzymatically or chemically
cleaved.
[0141] Thus, the embodiments of the present invention use the
proposed mechanism of interaction between HDM-2 and the compounds
of the present invention. By incorporating a peptide sequence that
shares certain p53 aa residues into the compound, the inventors are
promoting the compound to bind to HDM-2 in the cancer cell
membrane. Further, by combining the HDM-2 targeting component with
an MRP, as the compound is transported over the cancer cell
membrane, the binding of the compound to the cell causes
membranolysis of the cancer cell membrane. This, in turn results in
cell death through necrosis. The inventors of the present invention
have thus invented cancer treatments that kill cancer cells, even
when mixed with healthy cells. The methods of the present invention
may be used to selectively kill cancer cells, thus creating a
pinpointed treatment in a cell sample, tissue sample, or even
within a patient's body. The methods of the present invention thus
target HDM-2 in the cancer cell membrane; rather than p53. Thus,
the HDM-2 targeting treatments of the present invention are
applicable to all cancer cells, including those that have no p53
present, or may have p53 in an inactive (mutated) form.
[0142] It should be readily understood and appreciated that each of
the elements and features of the present invention discussed with
one embodiment may be similarly employed with other embodiments
disclosed herein, and this discussion is by no means deemed
limiting to the various additional permutations that may be
employed, for example, with the methods presented herein.
[0143] As the methods of treatment do not cause cell death of
normal cells, these methods of treatment are focused on the cancer
cells, irrespective of the mode of administration to the cell
sample. Thus, these methods of treatment may be used for tumors or
cancers that are widespread, inoperable, or otherwise not
effectively treated with conventional means or combination
therapies.
EXAMPLES
Materials and Methods
[0144] Peptides.
[0145] The following peptides were synthesized by solid phase
methods and were purified to >95% purity (Biopeptides Corp, La
Jolla, Calif.): PNC-27 containing residues 12-26 (PPLSQETFSDLWKLL)
(SEQ ID NO: 8) from the hdm-2 binding domain of p53 and PNC-28
(ETFSDLWKLL) (SEQ ID NO: 32) containing residues 17-26 from the
hdm-2 binding domain of p53, both attached on their carboxyl
terminal ends to the transmembrane-penetrating sequence which is
related to the reverseomer sequence of the antennapedia sequence,
KKWKMRRNQFWVKVQRG (SEQ ID NO: 1), also called MRP; the control
peptide PNC-26, containing only residues 12-26 of p53 and no MRP;
the control peptide PNC-29, an unrelated peptide from cytochrome
P450 (also called X13) (bold) attached to MRP (italics), whose
sequence is MPFSTGKRIMLGEKKWKMRRNQFWVKVQRG (SEQ ID NO: 4); and
PNC-7, a peptide from the ras-p21 protein containing ras-p21
residues 35-47 (TIEDSYRKQVVID) (SEQ ID NO: 7) attached to the MRP
having SEQ ID NO: 1 on its carboxyl terminal end. In addition, a
fluorescent-labeled form of PNC-27 was synthesized, i.e., PNC-27
peptide conjugated to the fluorescent dye, fluorescein
isothiocyanate (FITC) on its amino terminal end (Biopeptides
Corp.).
[0146] Cells.
[0147] MiaPaCa-2 cells (human pancreatic cancer cells) were
obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.) and were cultured in DMEM supplemented with 10
percent bovine fetal serum and penicillin/streptomycin [100 U/100
ug/ml] as recommended by the ATCC. BMRPA1 cells (untransformed rat
pancreatic acinar cells) were cultured as described previously
(1).
Methods
[0148] Preparation of Plasmids.
[0149] DNA encoding the human p53 amino acid residues 17-26
sequence, corresponding to the p53 sequence in PNC-28, was cloned
into the mammalian pTracer-SV40 (green-fluorescent protein
[GFP]-expressing) expression vector downstream to the SV40
promoter. This vector constitutively expresses a cloned gene
(Invitrogen, Carlsbad, Calif.). Also included in the vector is
another expression cassette which is linked in tandem to the
SV40-p53 17-26-expressing unit. The second expression cassette
contains a CMV promoter driving the expression of the GFP-Zeocin
resistance gene fusion protein. The vector was used to transform
TOP10F' chemically competent E. coli following the Hanahan Method
of transformation (13), and plated on Zeocin-containing agar plates
for overnight growth. Eight colonies were then used to inoculate
cultures in Low Salt Luria Broth (1% bacterial tryptone, 0.5% yeast
extract, 0.5% NaCl, and 25 m/ml Zeocin). Cultures were grown under
constant shaking at 200 rpm for 16 h in a 37.degree. C. incubator,
and plasmids were then extracted using a Qiagen Spin Miniprep
Kit.
[0150] The construct sense and anti-sense strands of the cDNA
encoding the p53 17-26 sequence (Invitrogen, Carlsbad, Calif.) were
synthesized. The sense strand sequence was 5'-AGTCGAATTCGCCACCA
TGGAAACATTTTCAGACCTATGGAAACTACTTTGAGC GGCCGCAGTC-3') (SEQ ID NO:
31). Underlined EcoRI and NotI sites are located in 5' and 3' ends
of the cDNA, respectively. Start and stop codons are in italics.
The p53 17-26 coding sequence is in bold letters. For maximum
protein translation in transfected cell lines, the start codon was
placed within a Kozak sequence, i.e., GCCACCATGG (SEQ ID NO: 30)
(with ATG being the start codon), which is the optimal context for
initiation of translation in vertebrate mRNA (13). The strands (250
nmol/ml) were annealed in annealing buffer by heating to 95.degree.
C., and then cooling to room temperature. The annealed double
stranded p53 17-26-encoding cDNA was then digested with NotI and
EcoRI simultaneously. A total of 20 ug of pTracer-SV40 was digested
with 60 units of NotI and 60 units of EcoRI. Double-digested
pTracer-SV40 and p53 17-26-encoding cDNA were then electrophoresed
through 0.8% and 2.5% agarose gel, respectively. Gel bands
containing DNA of appropriate size were excised, and DNA content
was extracted using the NucleoTrap Gel Extraction kit (ClonTech,
Mountain View, Calif.). Purified vector and cDNA were ligated with
T4 ligase (12 hr, 4.degree. C.) (New England Biolab, Ipswich,
Mass.). Two ul of ligation reaction was then dispensed into a vial
containing 50 ul One Shot TOP10F' competent E. coli (Invitrogen),
and the reaction mixture was incubated on ice for 10 min,
heat-shocked to 42.degree. C. (30 sec) and incubated on ice for
another 2 min. A total of 250 ul SOC medium (Invitrogen) was then
added to the cells which were then shaken at 37.degree. C. (1 hr).
This transformation reaction was then diluted 1:100 or 1:10 using
SOC medium. A total of 50 ul of each was spread on LB plates
containing 12.5 .mu.mol/ml ampicillin that were incubated overnight
at 37.degree. C. Eight colonies were randomly chosen to inoculate
eight 5 ml overnight LB cultures in the presence of 12.5 .mu.mol/ml
ampicillin. Plasmids extracted from each liquid culture were
analyzed by automated DNA sequencing using the fluorescence-based
dideoxy chain termination reaction (Genewiz, North Brunswick,
N.J.). It was found that they all contained the correct p53 17-26
cDNA reading frame associated with a stop codon and a start codon
embedded in the Kozak sequence. This construct plasmid is termed
p53 17-26-V ([expression] vector).
[0151] Precisely the same procedure was followed for preparation of
a plasmid encoding a scrambled p53 peptide sequence (residues
12-26) as follows:
5'-AGTCGAATTCGCCACCATGTGGGACCTGACACTACCCAAACAGCTTCTACCTT
CAAGTTTGAATGAGCGGCCGCAGTC-3'(SEQ ID NO: 31), with start, stop
codons, and restriction enzyme sites denoted as above. This plasmid
construct is called p53-12-26-scrm-V
[0152] Transfection into Cancer Cells.
[0153] Transfection of p53 17-26-V plasmid and the two control
plasmids, one, the p53 12-26-scrm-V vector and the second vector
encoding GFP only, called EV (empty vector) into MiaPaCa-2 and
untransformed BMRPA1 cells was completed. These cells were
evaluated for: viability and expression of: p53 protein, p53 17-26
peptide, caspase, annexin binding to phosphatidyl serine and LDH.
Twenty four hours prior to transfection 5.times.10.sup.5 cells were
seeded in antibiotic-free medium into each well in a six-well
tissue culture dish (TCD) and allowed to adhere overnight. To three
wells, 0.8 ug of p53 17-26-V plasmid were added. To the other three
wells, 0.8 ug of empty vector or p53-12-26-scrm-V vector encoding
control peptide were added. To each of these wells, Lipofectamine
2000 transfection agent (Qiagen) was added such that the ratio of
plasmid DNA (in ug) to Lipofectamine 2000 (in ul) was 1:2 (14).
This ratio was determined in preliminary experiments described in
the next paragraph. Transfections were performed in serum- and
antibiotic-free culture medium at 37.degree. C. for 4 h at which
time the incubation was continued in complete medium, containing
10% fetal bovine serum (FBS) and penicillin and streptomycin (100
U/100 .mu.g/ml). After another 4-5 h, the cells were washed
followed by re-incubation in fresh complete medium. Transfection
efficiency was measured by examining the frequency of GFP
expressing cells in the total cell population 12 h
post-transfection using a Zeiss LSM 410 Confocal Laser Scanning
Microscope.
[0154] For each cell line the effective ratio of DNA:Lipofectamine
2000 reagent was studied and was verified in preliminary
experiments using a checkerboard assay. In these experiments,
transfection efficiency was established by titration of different
concentrations of DNA in the presence of increasing concentrations
of Lipofectamine 2000 on cells that had been seeded onto glass
coverslips. After transfection the GFP-positive (GFP+) cells on the
coverslips were quantitated by counting under a UV Light Zeiss
Epifluorescence Microscope in 3-5 consecutive fields counting
200-400 cells. These preliminary experiments helped to establish
the cell density, the amount of DNA and the DNA:Lipofectamine 2000
ratio to be used, and the time for transfection to proceed.
[0155] Expression of the p53 17-26 Peptide.
[0156] For protein analyses, and detection of apoptosis,
2.times.10.sup.6 cells were seeded into 10 cm diameter TCDs and
transfected with DNA:Lipofectamine 2000 proportionally adjusted to
the increased area. When the cell density reached 90-100%, the
cells of the experimental and sham-transfected group were detached
using trypsin and plated into four new TCD's in which they were
allowed to grow in complete medium. At defined time points cells
were released from adherence with 10 mM EDTA in PBS and were lysed
in lysing buffer [1% Triton X-100 in 0.05 M Tris-HCl (pH 8.0), 0.15
mM NaCl, 0.02% Na azide, 0.01 mg/ml phenylmethylsulfonylfluoride
(PMSF), and 0.001 mg/ml Aprotinin]. Protein equivalents of 10.sup.6
cells, i.e., .about.30 g/lane, were then subjected to SDS-PAGE
using 10% Tris-HCl gels and, in some experiments, 16% Tricine
Peptide Gels (Biorad, Hercules, Calif.) to detect PNC-28
(.about.3104 Da) and p53 17-26 (.about.1500 Da). The separated
proteins were then electrophoretically transferred to
nitrocellulose membranes followed by immunoblotting with the mAb
DO-1 to p53 AA residues 11-25, and with mAb B-2 to GFP (each at 1
.mu.g-2.5 .mu.g/ml blotting buffer), respectively (2). After
washing non-reacted mAbs from the membranes, the membranes were
incubated (1 h) with a second enzyme-labeled antibody from the ECL
chemiluminescence kit (Amersham, Piscataway, N.J.) to detect the
presence of p53 and p53 17-26 peptide. In preliminary experiments,
it was noted that identification of p53 protein was easily possible
within 30-90 sec of exposure while clear identifiable binding of
mAbDO-1 to p53 17-26 peptide took much longer time. The membrane
was therefore cut across the 17 kDa marker (kaleidoscope's
polypeptide standard), to allow for the differential exposures. In
addition, a time course of GFP expression was performed in both
MiaPaCa-2 and BMRPA1 cells that established that, during the time
period 48-96 h post-transfection, the cells showed the highest
levels of GFP expression. Semi-quantitation of immunoblotting
results was performed by measuring luminosity of bands in a single
scanned developed x-ray film, using the histogram option of Adobe
Photoshop 5.5. Background was ascertained by measuring average
luminosity of 5 areas of the film outside the blotting region.
Opacity of each band was calculated by the equation,
Opacity=255-Luminosity-background (15).
[0157] Incubation of MiaPaCa-2 Cells with PNC-28.
[0158] Duplicate sets of 6.times.10.sup.6 MiaPaCa-2 cells were
incubated with different concentrations of PNC-28, i.e., 5, 10, 20,
40, 80 and 160 .mu.mol/ml. Duplicate control experiments were also
performed in which 6.times.10.sup.6 MiaPaCa-2 cells were incubated
with the control, PNC-29, present at a concentration of 75 mmol/ml.
All incubations were carried out using a protocol identical to that
described in ref. 1 (1). After the cells had been allowed to adhere
to the tissue culture dish (TCD) for 24 hours the medium was
removed from each TCD, and new medium containing the same or no
peptide concentration was added. Medium from each TCD was removed
every 24 h, and fresh medium with its respective peptide at the
appropriate concentration was added. Cells were inspected daily for
changes in cell growth, morphology, and viability. At the end of
each day over a five-day period, duplicate cell counts were
performed for each incubation using the trypan blue exclusion
method. In addition, cell viability was also determined by
3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium bromide (MTT)
assay according to the manufacturers' instructions (Promega
Corporation, Madison, Wis., USA).
[0159] Incubation of Peptides with BMRPA1 Cells.
[0160] These cells are untransformed rat pancreatic acinar cells
(1). Duplicate 5-day incubations were performed on 6.times.10.sup.6
cells in three circumstances: with no peptide, with PNC-28 at 75
.mu.mol/ml, and with PNC-29 at 75 mmol/ml. Cells were followed for
viability and morphology over this time period. At the end of 5
days, cell counts were performed using the trypan blue exclusion
method.
[0161] Immunocytochemistry for Annexin V-Binding to Phosphatidyl
Serine.
[0162] To determine whether any of the transfected plasmids induced
apoptosis, the cells were evaluated to determine whether the cells
contained phosphatidyl serine in the inner cell membrane,
identified as binding to annexin-V, as a marker for apoptosis (15).
Cells (5.times.10.sup.5) were seeded in 6-well TCDs 24 h prior to
transfection in antibiotic-free medium. Cells were then either
transfected with p53 17-26-V, p53 12-26-scrm-V, EV or were left
untreated. At predetermined time-points post-transfection, the
cells were released using 0.5.times. Trypsin-EDTA, collected and
processed as described in the manufacturer's instructions of the
Annexin V-Biotin Apoptosis Detection Kit (CalBioChem, La Jolla,
Calif.). The stained cells were resuspended in antifade (Molecular
Probes, OR), mounted on glass slides under a glass coverslip and
evaluated for red (TRITC) and green (GFP) fluorescence using
confocal microscopy as described above.
[0163] Evaluation of Cells Treated with PNC-28 for Caspase as a
Marker for Apoptosis and LDH Release as a Marker for Necrosis.
[0164] Cells from culture plates at 18, 44, 66 and 90 h time points
were lysed in situ in cell lysis buffer [1% Triton X-100 in 0.05 M
Tris-HCl (pH 8.0), 0.15 mM NaCl, 0.02% Na azide, 0.1 mg/ml
phenylmethylsulfonylfluoride (PMSF), and 0.001 mg/ml Aprotinin].
Lysates were subjected to 10% SDS-PAGE followed by electrotransfer
to nitrocellulose and immunoblotting with antibodies to GFP and p53
(Santa Cruz Biotechnology, Santa Cruz, Calif.). Antibody-labeled
proteins were identified by chemiluminescence using ECL methodology
(Amersham)(1). Assays for elevated caspase expression were
performed using the Clontech (Palo Alto, Calif.) for caspase
(CPP32) activity (2). As a positive control for the caspase
activity assay, Mia-PaCa-2 cells were incubated with tumor necrosis
factor (TNF) (Sigma, St. Louis, Mo.) at a concentration of 10 ng/ml
for 24 h. In addition, to detect if significant cell necrosis
occurred, the CytoTox96 assay was used (Promega, Madison, Wis.) for
LDH released into the cell culture medium as performed on several
breast cancer cell lines (2).
[0165] Electron Micropscopy of MiaPaCa-2 Cells Treated with
PNC-28.
[0166] Time-lapse electron microscopy (EM) was used to examine the
ultrastructural features of cell death. MiaPaCa-2 cells were grown
for 24 h on Thermanox cover slips (Lux Scientific), and then
treated with 25 .mu.mol of PNC-28 for 1 and 15 min, along with a
corresponding control group without peptide. The cells were washed
with PBS solution and then fixed with 2.5% gluteraldehyde-PBS. The
fixed cultures were rinsed in a 0.1 M phosphate buffer (pH 7.3),
post fixed in 2% (0.08 m) osmium tetroxide-PBS (pH 7.3), dehydrated
in a graded series of ethanol and propylene oxide and embedded in
Epon 812. Sections were cut at 700 .ANG., stained with uranyl
acetate and lead citrate and examined with a Jeol JEM 1010 Electron
Microscope.
[0167] Blotting of Mia-PaCa-2 Cell Lysates for p53 and waf.sup.p21,
a Target for Activated p53.
[0168] Cell lysates were prepared as described in the preceding
paragraph and were subjected to immunoblotting with either DO-1
antibody described in the section above for expression of the p53
17-26 peptide, a (Ab-6) monoclonal anti-p53 antibody (Calbiochem,)
or with polyclonal anti-waf.sup.p21 antibody (Santa Cruz
Biotechnology, Santa Cruz, Calif.) (1:2000 dilution) using a
procedure identical to that described in the same section above.
For controls, for actin was blotted for, using anti-actin
polyclonal antibody (Santa Cruz Biotechnology).
[0169] Statistical Analysis.
[0170] Analysis of growth inhibition and markers for necrosis and
apoptosis were analyzed by a two-tailed Mann-Whitney nonparametric
test or a two-tailed Student T-test where appropriate. A P-value of
less than 0.05 was considered significant.
[0171] Various changes and modifications can be made in the present
invention. It is intended that all such changes and modifications
come within the scope of the invention as set forth in the
following claims.
Sequence CWU 1
1
32117PRTArtificial sequenceMembrane resident peptide (MRP),
reverseomer of Antennapedia 1Lys Lys Trp Lys Met Arg Arg Asn Gln
Phe Trp Val Lys Val Gln Arg 1 5 10 15 Gly 232PRTArtificial
sequencePNC-27 2Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys
Leu Leu Lys 1 5 10 15 Lys Trp Lys Met Arg Arg Asn Gln Phe Trp Val
Lys Val Gln Arg Gly 20 25 30 327PRTArtificial sequencePNC-28 3Glu
Thr Phe Ser Asp Leu Trp Lys Leu Leu Lys Lys Trp Lys Met Arg 1 5 10
15 Arg Asn Gln Phe Trp Val Lys Val Gln Arg Gly 20 25
430PRTArtificial sequencePNC-29 4Met Pro Phe Ser Thr Gly Lys Arg
Ile Met Leu Gly Glu Lys Lys Trp 1 5 10 15 Lys Met Arg Arg Asn Gln
Phe Trp Val Lys Val Gln Arg Gly 20 25 30 513PRTArtificial
sequencepeptide from cytochrome P450 (aka "X13") 5Met Pro Phe Ser
Thr Gly Lys Arg Ile Met Leu Gly Glu 1 5 10 630PRTArtificial
sequencePNC-7 6Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp
Lys Lys Trp 1 5 10 15 Lys Met Arg Arg Asn Gln Phe Trp Val Lys Val
Gln Arg Gly 20 25 30 713PRTArtificial sequenceras-p21 residues
35-47 7Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp 1 5 10
815PRTArtificial sequencePNC-26, residues 12-26 of the HDM-2
binding domain of p53 8Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu
Trp Lys Leu Leu 1 5 10 15 914PRTArtificial sequenceHIV-1
TAT(47-60), membrane resident peptide 9Tyr Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg Pro Pro Gln 1 5 10 1013PRTArtificial sequenceD-TAT,
membrane resident peptide 10Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Pro Pro Gln 1 5 10 1113PRTArtificial sequenceR-TAT G(R)9PPQ,
membrane resident peptide 11Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala
Pro Pro Gln 1 5 10 127PRTArtificial sequenceSV40-NLS, membrane
resident peptide 12Pro Lys Lys Lys Arg Lys Val 1 5
1316PRTArtificial sequencenucleoplasmin-NLS, membrane resident
peptide 13Lys Arg Pro Ala Ala Ile Lys Lys Ala Gly Gln Ala Lys Lys
Lys Lys 1 5 10 15 1417PRTArtificial SequenceHIV REV (34-50),
membrane resident peptide 14Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg
Arg Trp Arg Glu Arg Gln 1 5 10 15 Arg 1515PRTArtificial sequenceFHV
(35-49) coat, membrane resident peptide 15Arg Arg Arg Arg Asn Arg
Thr Arg Arg Asn Arg Arg Arg Val Arg 1 5 10 15 1619PRTArtificial
sequenceBMV GAG (7-25), membrane resident peptide 16Lys Met Thr Arg
Ala Gln Arg Arg Ala Ala Ala Arg Arg Asn Arg Trp 1 5 10 15 Thr Ala
Arg 1713PRTArtificial sequenceHTLV-II REX 4-16, membrane resident
peptide 17Thr Arg Arg Gln Arg Thr Arg Arg Ala Arg Arg Asn Arg 1 5
10 1819PRTArtificial sequenceCCMV GAG (7-25), membrane resident
peptide 18Lys Leu Thr Arg Ala Gln Arg Arg Ala Ala Ala Arg Lys Asn
Lys Arg 1 5 10 15 Asn Thr Arg 1917PRTArtificial sequenceP22 N
(14-30), membrane resident peptide 19Asn Ala Lys Thr Arg Arg His
Glu Arg Arg Arg Lys Leu Ala Ile Glu 1 5 10 15 Arg 2022PRTArtificial
sequenceLAMBDA N(1-22), membrane resident peptide 20Met Asp Ala Gln
Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala 1 5 10 15 Gln Trp
Lys Ala Ala Asn 20 2118PRTArtificial sequencePhi N (12-29),
membrane resident peptide 21Thr Ala Lys Thr Arg Tyr Lys Ala Arg Arg
Ala Glu Leu Ile Ala Glu 1 5 10 15 Arg Arg 2216PRTArtificial
sequenceYEAST PRP6 (129-124), membrane resident peptide 22Thr Arg
Arg Asn Lys Arg Asn Arg Ile Gln Glu Gln Leu Asn Arg Lys 1 5 10 15
2312PRTArtificial sequenceHUMAN U2AF, membrane resident peptide
23Ser Gln Met Thr Arg Gln Ala Arg Arg Leu Tyr Val 1 5 10
2426PRTArtificial sequenceHUMAN C-FOS (139-164), membrane resident
peptide 24Lys Arg Arg Ile Arg Arg Glu Arg Asn Lys Met Ala Ala Ala
Lys Ser 1 5 10 15 Arg Asn Arg Arg Arg Glu Leu Thr Asp Thr 20 25
2528PRTArtificial sequenceHUMAN C-JUN (252-279), membrane resident
peptide 25Arg Ile Lys Ala Glu Arg Lys Arg Met Arg Asn Arg Ile Ala
Ala Ser 1 5 10 15 Lys Ser Arg Lys Arg Lys Leu Glu Arg Ile Ala Arg
20 25 2622PRTArtificial sequenceYEAST GCN4, membrane resident
peptide 26Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg Arg Ser Arg Ala
Arg Lys 1 5 10 15 Leu Gln Arg Met Lys Gln 20 2718PRTArtificial
sequenceExample membrane resident peptide (MRP) 27Lys Leu Ala Leu
Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala
2818PRTArtificial sequencep-vec, membrane resident peptide 28Leu
Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala Lys Ala His 1 5 10
15 Ser Lys 298PRTArtificial sequence(Arg)8 or any poly-R from
(R)4-(R)16, membrane resident peptide 29Arg Arg Arg Arg Arg Arg Arg
Arg 1 5 3010DNAArtificial sequenceKozak sequence 30gccaccatgg
103164DNAArtificial sequencesense strand sequence of cDNA encoding
the p53 17-26 sequence 31agtcgaattc gccaccatgg aaacattttc
agacctatgg aaactacttt gagcggccgc 60agtc 643210PRTArtificial
sequenceresidues 17-26 of HDM-2 binding domain of p53 32Glu Thr Phe
Ser Asp Leu Trp Lys Leu Leu 1 5 10
* * * * *