U.S. patent application number 17/727519 was filed with the patent office on 2022-08-04 for method of treating colorectal cancers using a pkc inhibitor.
The applicant listed for this patent is UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC., UNIVERSITY OF SOUTH FLORIDA. Invention is credited to Mildred ACEVEDO-DUNCAN, Anisul ISLAM, David A. OSTROV.
Application Number | 20220241226 17/727519 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220241226 |
Kind Code |
A1 |
ACEVEDO-DUNCAN; Mildred ; et
al. |
August 4, 2022 |
Method of Treating Colorectal Cancers Using a PKC Inhibitor
Abstract
The subject invention pertains to methods of treating colorectal
cancer by administering an atypical PKC inhibitor. The inhibitors
of aPKC useful in the methods of the instant invention include
ACPD, ICA-1, DNDA and .zeta.-Stat. Also provided are methods of
measuring the susceptibility of colon cancer cells of a subject to
inhibitors of aPKCs.
Inventors: |
ACEVEDO-DUNCAN; Mildred;
(Plant City, FL) ; ISLAM; Anisul; (Tampa, FL)
; OSTROV; David A.; (Gainesville, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF SOUTH FLORIDA
UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC. |
Tampa
Gainesville |
FL
FL |
US
US |
|
|
Appl. No.: |
17/727519 |
Filed: |
April 22, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15643117 |
Jul 6, 2017 |
11337944 |
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17727519 |
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62359496 |
Jul 7, 2016 |
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International
Class: |
A61K 31/185 20060101
A61K031/185; C12Q 1/6886 20060101 C12Q001/6886; G01N 33/50 20060101
G01N033/50; C12Q 1/48 20060101 C12Q001/48; A61K 31/675 20060101
A61K031/675; A61K 31/122 20060101 A61K031/122 |
Claims
1-6. (canceled)
7. A method of measuring the susceptibility of colon cancer cells
of a subject suffering from colon cancer to an aPKC inhibitor, the
method comprising: obtaining a colon cancer cell sample from a
subject suffering from colon cancer, wherein the colon cell sample
comprises colon cancer cells; exposing a portion of the colon
cancer cells of the colon cell sample to an inhibitor of an aPKC,
while leaving another portion of the colon cancer cells of the
colon cell sample untreated; detecting cell proliferation,
expression, and activation of one or more aPKCs in treated and
untreated cells; selecting a subject having a cancer susceptible to
an aPKC inhibitor by detecting (i) the reduction of proliferation,
expression and activation of one or more aPKCs in treated cells
compared to untreated cells, wherein reduction indicates the
subject's cancer is susceptible to aPKC inhibitors.
8. The method, according to claim 7, wherein the one or more aPKC
comprises PKC-.tau..
9. The method, according to claim 7, wherein the one or more aPKC
comprises PKC-.zeta..
10. The method, according to claim 7, wherein the activation of one
or more aPKC is detected as increased phosphorylation of the
aPKC.
11. The method, according to claim 10, wherein the increased
phosphorylation is increased phosphorylation of PKC-.tau..
12. The method, according to claim 10, wherein the increased
phosphorylation is increased phosphorylation of PKC-.zeta..
13. The method, according to claim 7, wherein the marker of
apoptosis is Caspase-3.
14. The method, according to claim 7, wherein the marker of
anti-apoptosis is BCL-XL.
15. The method, according to claim 7, wherein the marker of
anti-apoptosis is survivin.
16-19. (canceled)
20. The method of claim 7, wherein when the subject's cancer is
susceptible to aPKC inhibitors, further comprising: administering
to the subject a therapeutically effective amount of an atypical
PKC (aPKC) inhibitor selected from the group consisting of ACPD,
ICA-1, DNDA, and .zeta.-Stat.
21. The method of claim 20, wherein the aPKC inhibitor is
.zeta.-Stat.
22. The method of claim 7, wherein when the subject's cancer is not
susceptible to aPKC inhibitors, further treating the subject with
surgery, radiation therapy, or immunotherapy.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/359,496, filed Jul. 7, 2016, the disclosure
of which is hereby incorporated by reference in its entirety,
including all figures, tables and drawings.
FIELD OF THE INVENTION
[0002] This invention relates to treating cancer with inhibitors of
atypical protein kinase C. Specifically, the invention describes
methods of treating colorectal cancers using a protein kinase C
(PKC) inhibitor such as 2-acetyl-1,3-cyclopentanedione (ACPD),
[4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl]methyl
dihydrogen phosphate (ICA-1), 3,4, diamino-2,7-naphthalene
disulfonic acid (DNDA), or 8-hydroxy-1,3,6-naphthalenetrisulfonic
acid (.zeta.-Stat).
BACKGROUND OF THE INVENTION
[0003] Colorectal cancer is one of the most common and lethal
cancers in the world. Globally, the incidence of colon cancer
varies over 10-fold [1]. It is the second leading cause of
cancer-related mortality in the United States. Over the past
decade, colorectal cancer incidence and mortality rates have
decreased in all racial/ethnic populations except American
Indians/Alaska Natives. According to the US National Cancer
Institute's Surveillance Epidemiology and End Results (SEER)
database, the risk of developing colorectal cancer is lin 21
(4.69%) in man adn 1 in 23 (4.35%) in women based on the incidence
and mortality rate from 2010 to 2012. Also, the risk of dying from
colorectal cancer is 1 in 50 (1.99%) in male and 1.55 (1.81%) in
females [61]. Men and women have similar incidence rates through
age 39; at and above age 40, rates are higher in men [2].
[0004] Colorectal cancer or Colorectal adenocarcinoma or CRC is the
development of cancer in the colon or rectum due to the abnormal
growth of cells that have the ability to invade or spread to other
parts of the body [4]. Colorectal cancer often begins as a growth
called a polyp, which may form on the inner wall of the colon or
rectum (FIG. 1). Over time, polyps may become malignant [2].
Colorectal cancers are mostly adenocarcinomas (cancers that begin
in cells that make and release mucus and other fluids. Other
colorectal cancers can include: Gastrointestinal carcinoid tumor,
slow-growing cancer that forms in the neuroendocrine cell (a nerve
cell that also creates hormones) in the lining of the
gastrointestinal tract; Gastrointestinal stromal tumor, also known
as GISTs, this is a rare type of colorectal cancer that starts in a
special cell found in the lining of the gastrointestinal (GI) tract
called interstitial cells of Cajal (ICCs); Primary colorectal
lymphoma, a type of non-Hodgkin lymphoma (NHL), lymphomas are
cancers that develop in the lymphatic system from cells called
lymphocytes; Leiomyosarcoma, another form of sarcoma,
leiomyosarcomas essentially means cancer of smooth muscle;
Melanoma, though most commonly associated with the skin, melanomas
can occur anywhere, including the colon or rectum and Squamous cell
carcinoma, cancer of the squamous cells of the upper esophagus and
the end of the anus [62]. Based on the depth and severity of the
disease progress, CRC can be divided into five stages: stage-0:
only innermost lining of the colon or rectum has been affected,
Stage-I: cancer grown up to the muscle layer, Stage II: Outermost
layer of the colon has been affected, Stage III: the disease has
spread into one or more lymph nodes, Stage-IV: cancer becomes
malignant and metastasized to other part of the body such as liver,
lungs or bone. 9 (FIG. 2) [63]. The development of colorectal
carcinogenesis is a complex multistep process accompanied by the
disruption of the intestinal epithelial-cell growth, proliferation,
differentiation, apoptosis, and survival mechanism [3]. The
etiological understanding of this life threatening disease is still
unknown. Tumor progression is usually acquired by a number of
characteristic alterations. These alterations can include the
uncontrolled proliferation of cells independently of extracellular
promoting or inhibitory signals, invasiveness to nearby tissues and
metastasize to distant sites, elicitation of an angiogenic
response, and obstacles in the mechanisms that limit cell
proliferation, such as apoptosis and replicative senescence. These
properties reflect alterations in the cellular signaling pathways.
Many proteins that are important component of the cell signaling
pathways are currently under investigation as possible target for
cancer therapy [5]. One of those most important proteins that is
involved in controlling crucial cellular signal transduction
pathways is Protein Kinase C (known as PKC) [6].
[0005] Protein kinase C (PKC) is a family of isozymes, also known
as phospholipid dependent serine/threonine kinases that transduce
signals and control other proteins through phosphorylation. PKC is
a family of fourteen known isozymes which are found in varying
ratios in the cytostolic and membrane fraction of cells depending
on the type of tissue and its physiological state. (Nishizuka, Y.
Intracellular signally by hydrolysis of phospholipids and
activation of protein kinase C. Science. (1992); 258: 607-614.) The
PKCs sit at the cross roads of many transmembrane signal
transduction pathways that can be triggered by various external and
internal stimuli [8]. PKCs are associated with many cell functions
including survival, proliferation, motility and apoptosis [9]. The
activation of some PKCs require calcium, lipid metabolites such as
Diacylglycerol (DAG), co-factors like phosphotidyl serine (PS) and
secondary messengers (phorbol esters) [10,11].
[0006] PKC isozymes can be classified into three groups based on
their structure and requirements (i.e. activators and
co-activators) for activation. Group I includes conventional
isozymes: cPKC-.alpha., cPKC-.beta.l, cPKC-.beta.ll, and
cPKC-.gamma. all of which are activated by calcium and
Diacylglycerol (DAG). Novel isozymes in group II (nPKC-.epsilon.,
nPKC-.delta., nPKC-.eta. and PKC-.theta.) are Ca2+ independent but
require Diacylglycerol (DAG) and phospholipids to get activated.
Group III includes the atypical PKCs (aPKC-.tau., aPKC-.zeta.,
aPKC-.zeta.ll, aPKC-.mu. (protein kinase D) and aPKC-.nu.) which
are insensitive to both diacylglycerol and calcium and neither bind
to nor are activated by phorbol esters. PKC Lamda (.lamda.) in
mouse is equivalent to aPKC-.tau. in human. Another distant member
of the PKC family is PKC related kinases (PRK 1-3), known as PKN
[13].
[0007] PKC is a single polypeptide consisting of a regulatory N
terminal (approximately 20-40 kDa) and a catalytic C terminal
(approximately 45 kDa) (FIG. 1). All PKC isozymes have similar
highly conserved catalytic domain, also known as kinase domain
consisting of motifs for ATP/substrate binding and catalysis but
differ in their regulatory domain that maintain the enzyme in an
inactive form. The regulatory domain also has a pseudosubstrate
region (a sequence that has Alanin in place of Serine/Threonine
phosphogroup acceptor which otherwise mimic a PKC substrate) and
two discrete membrane targeting modules, termed C1 and C2.
Conventional PKCs have a C1 domain termed as C1A and C1B (each
consisting of 6 cysteine and 2 histidine residues coordinate with
two Zn2+) that serve as a binding site for tumor promoting phorbol
ester and diacylgycerol (DAG), C2 domain that binds anionic
phospholipid in a calcium dependent manner. Novel PKCs also have
twin C1 domain but they lack critical calcium coordinating acidic
residues even though they have a so called C2 domain. The
activation mechanism of PKCs involves two steps, first release of
pseudosubstrate and, second, phosphorylation of kinase domain
[15].
[0008] Atypical PKC:
[0009] Atypical PKC (aPKC), PKC-.tau. and PKC.zeta., are different
from other PKC isozymes both structurally and functionally.
PKC-.tau. and PKC-.zeta. are the human homologs of the mouse
PKC-.lamda. (lamda). PKC-.tau. and PKC-.zeta. have significantly
different structures from the other two groups of PKCs. Like other
PKCs, the aPKCs also have a regulatory domain and a catalytic
domain, the N-terminus of regulatory has a protein-protein
interacting PB1 (Phox and Bem 1) domain (required for interaction
with other PB1 containing scaffolding proteins such as p62,
partitioning defective-6, called PAR-6 & MEK-5), a
pseudo-substrate sequence, an atypical C1 domain of single Cysteine
rich zinc finger motif that can bind Phosphatidylinositol 3,4,5
triphosphate (PIP3) and ceramide but the key residue that maintain
the C2 fold is absent in this category of PKC isozyme and the
C-terminus contains a kinase domain (FIG. 2). The structural
differences make them insensitive to Ca.sup.2+, DAG and phorbol
ester. Atypical PKC activity is regulated primarily by
protein-protein interactions and phosphorylation by
phosphoinositide-dependent kinase-1, also called PDK-1 and Protein
Kinase B, also known as PKB/Akt (PDK-1 and Akt, isozymes of AGC
kinase family that contains a pleckstrin homology (PH domain),
which can carry PIP-3 required for enzyme activity) [39,40]. The
primary structure of atypical PKCs indicates that they are 72%
homologues in regulatory and 84% homologues in catalytic domain.
Atypical PKCs are of particular interest because of their
involvement in cell cycle disruption, cell survival, apoptosis and
tumor progression [14].
[0010] Role of PKC in cell malignancies:
[0011] PKC regulates cellular functions, metabolism and
proliferation by phosphorylating proteins in response to
transmembrane signals from hormones, growth factors,
neuro-transmitters, and pharmacological agents. Activation of PKC
by various agonists (including radiation) results in altered
transcription of a considerable number of genes. PKC is the major
receptor for tumor promoting phorbol esters, but the extent of PKC
involvement in cellular malignancy is not clearly defined. Various
studies indicate that increased tumorigenicity results from
dysregulation of PKC activity, or changes in PKC concentration, or
both (Persons D A, Wilkison W O, Bell R M. et al. Altered growth
regulation and enhanced tumorigenicity of NIH 3T3 fibroblasts
transfected with protein kinase C-I DNA. Cell 1988; 52:447-458;
Housey G M, Johnson M D, Hsiao W L M, et al. Overproduction of
protein kinase C causes disordered growth control in rat
fibroblasts. Cell 1988; 52:343-354; Kamata T, Sullivan N F and
Wooten M W. Reduced protein kinase C activity in a ras-resistant
cell line derived from Ki-MSV transformed cells. Oncogene 1987;
1:37-46; Weyman C M, Taparowsky E J, Wolfson M, et al. Partial
down-regulation of protein kinase C in C3H10t1/2 mouse fibroblasts
transfected with the human Ha-ras oncogene. Cancer Res. 1988;
48:6535-6541; Mizuguhi J, Makabayashi H, Yoshida Y. Et al.
Increased degradation of protein kinase C without diminution of
mRNA level after treatment of WEHI-231 B lymphoma cells with
phorbol esters. Biochem. Biophy. Res. Commun. 1988;
155:1311-1317).
[0012] PKC's are involved in normal cell functioning but when they
are disrupted they can cause many adverse effects. PKC's have been
discovered to play a role in tumor growth and formation when the
levels of PKC are vastly altered.
[0013] PKC-.tau. is a member of the PKC family located in
chromosome 3 at 3q26.2 and has been shown to be a human oncogene.
There is an expanding body of knowledge regarding the status of
atypical PKC-.tau. which does not contain a Ca 2+-binding region,
has one zinc finger-like motif and is the human homolog of the
mouse PKC-.lamda.. (Bradford M M. A rapid and sensitive method for
the quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 1976; 72,
248-254).
[0014] PKC's have been shown to be overexpressed in a variety of
cancers including ovarian, lung, head, neck, and prostate. Elder et
al. provided evidence for the role of PKC-.tau. in cell
proliferation by showing that increased PKC-.tau. protein levels
were associated with increased cyclin E protein expression and
proliferation of ovarian cancers (Laemmli U K. Cleavage of
structural proteins during the assembly of the head of
bacteriophage T4. Nature 1970; 337:680-685). In non-serous ovarian
cancers they demonstrated that increased PKC-.tau. protein levels
markedly decreased overall survival. Work from Field's laboratory
indicated that PKC-.tau. is critical for non-small cell lung cancer
proliferation in-vivo by activation of Rac1/Pak/Mek1,2/Erk1,2
signaling pathway which has been implicated in tumor cell
proliferation and concluded that PKC-.tau. is an oncogene in human
non-small cell lung cancer (Towbin H, Staehelin T, and Gordon P E.
Electrophoretic transfer of proteins from polyacrylamide gels to
nitrocellulose sheets: Procedure and some applications. Proc. Natl.
Acad. Sci. USA 1979; 76:4350-4354; Allred D C, Harvey J M, Berardo
M, et al: Prognostic and predictive factors in breast cancer by
immunohistochemical analysis. Mod Pathol 1998; 11:155-168).
[0015] Additional powerful evidence of PKC-C's importance in
cellular malignancy includes its exclusive association with
transformed phenotype of human melanomas in vivo and in vitro, it's
overexpression in human non-small lung cancer cell lines,
cholangiocarcinoma, and its presence in the transformed growth of
human lung adenocarcinoma A549 cell line in vitro and
tumorigenicity in vivo (Towbin H, Staehelin T, and Gordon P E.
Electrophoretic transfer of proteins form polyacrylamide gels to
nitrocellulose sheets: Procedure and some applications. Proc. Natl.
Acad. Sci. USA 1979; 76:4350-4354; Allred D C, Harvey J M, Berardo
M, et al: Prognostic and predictive factors in breast cancer by
immunohistochemical analysis. Mod Pathol 1998; 11:155-168;
Diaz-Meco, M. T., Municio, M. M., Sanchez, P., Lozano, J. and
Moscat, J. Lamda-interacting protein, a novel protein that
specifically interacts with the Zinc finger domain of the atypical
Protein Kinase C isotype lambda/iota and stimulates its kinase
activity in vitro and in vivo. Molec. and Cell Bio., 1996;
16:105-114; Eder, A. M, Sui, X., Rosen, D. G., Nolden. L. K., Chen,
K. W., Lahad, J. P., Kango-Singh, M., Lu, K. H., Warneke, C. L.,
Atkinson, E. N., Bedrosian, I., Keyomarsi, K., Kuo, W-l., Gray, J.
W., Yin, J. C. P., Lui, J., Halder, G., Mills, G. B. Atypical PKC
iota contributes to poor prognosis through loss of apical-basal
polarity and Cyclin E overexpression in ovarian cancer. PNAS 2005;
102:12519-12524).
[0016] PKC-.tau. has been shown to be exclusively associated with
the transformed phenotype of gliomas, benign and malignant
meningiomas (Regala, R. P., Weems, C., Jamison, L., Copland, J. A.
Thompson, E. A., Fields, A. P. Atypical protein kinase C iota plays
a critical role in human lung cancer cell growth and
tumorigenicity. J. Biol. Chem. 2005; 280:31109-31115). Elevated
levels of PKC-.tau. have been correlated with poorer prognosis.
Lung cancer patients who had elevated levels of PKC-.tau. during
the early stages are 10 times more likely to die from the disease
than those who had low levels of PKC-.tau.. PKC-.tau. has also been
found to be involved in many oncogenic signaling pathways.
PKC-.alpha. and PKC-.delta. are involved in the apoptosis of
androgen dependent prostate cancer cells [16]. UV light induced
overexpression of PKC-.epsilon. cause human squamous cell carcinoma
[17]. PKC-.tau. is involved in androgen dependent prostate
carcinoma and glioblastoma [18,19]. PKC-.beta., PKC-.gamma.,
PKC-.delta. and PKC-.zeta. are also found to be involved in chronic
lymphocytic leukemia [20].
[0017] Inhibitors of aPKC include, but are not limited to,
2-acetyl-1,3-cyclopentanedione (ACPD), 3,4, diamino-2,7-naphthalene
disulfonic acid (DNDA),
[4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl]methyl
dihydrogen phosphate (ICA-1), and
8-hydroxy-1,3,6-naphthalenetrisulfonic acid (.zeta.-Stat).
[0018] ACPD is an inhibitor of atypical PKCs PKC-.tau. and PKC zeta
(PKC-.zeta.). PKC-iota is a phosphorylating protein that plays a
role in mediating apoptosis. PKC-zeta has been shown to contribute
to survival of some cancer cells.
[0019] ICA-1 is an inhibitor of PKC-.tau.. DNDA, similar to ACPD,
is a pan atypical PKC inhibitor, inhibiting PKC-.zeta. by about 80%
and PKC-.tau. by about 20%.
[0020] .zeta.-Stat is an inhibitor of PKC-.zeta..
[0021] A linkage of the PKC family of proteins to colon cancer
remains unknown. Given the implications of atypical PKCs in tumor
formation and growth in general and the need for more efficient
treatment of human colon cancer in particular, the instant
invention provides methods for treating colon cancer by
administering inhibitors of aPKCs.
BRIEF SUMMARY OF THE INVENTION
[0022] Provided herein are novel methods of treating colorectal
cancers using atypical PKC (aPKC) inhibitors. In preferred
embodiments, the instant invention relates to methods of treating
colorectal cancer by administering to a subject in need thereof, a
composition comprising a therapeutically effective amount of an
inhibitor of an aPKC, including, but not limited to, PKC-.zeta. and
PKC-.tau.. An inhibitor of an aPKC useful in the methods of the
invention can inhibit PKC-.zeta. and/or PKC-.tau.. Non-limiting
examples of inhibitors of PKC-.zeta. and/or PKC-.tau. useful in the
methods of the instant invention include, but are not limited to,
ACPD, DNDA, ICA-1, and .zeta.-Stat.
[0023] In further embodiments, the instant invention provides
methods of measuring the susceptibility of colon cancer cells of a
subject suffering from colon cancer to an aPKC inhibitor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] For a fuller understanding of the invention, reference
should be made to the following detailed description, taken in
connection with the accompanying drawings, in which:
[0025] FIG. 1 shows an image of the development of colorectal
polyps that can lead to cancer over time.
[0026] FIG. 2 shows an image of the stages of colorectal
cancer.
[0027] FIG. 3 shows a series of images of the domain structure of
protein kinase C (PKC) isoforms.
[0028] FIG. 4 shows a schematic representation of the domain
structures of aPKC.
[0029] FIG. 5 shows the effect of ACPD on the HT-29 colon cancer
cell line. The number of HT-29 cells decreases as the concentration
of ACPD increases over time. Blue diamond, control; orange square,
0.25 .mu.M ACPD; gray triangle, 0.5 .mu.M ACPD; yellow X, 0.75
.mu.M ACPD; blue X, 1.0 .mu.M ACPD; and green circle, 1.5 .mu.M
ACPD.
[0030] FIG. 6 shows the accumulated effect of ACPD treatment on the
HT-29 colon cancer cells line after three days of treatment. At
this time, HT-29 cells had a slight decrease in proliferation at
0.25 .mu.M and a dramatic decrease in proliferation at 1.5 .mu.M
concentration of ACPD.
[0031] FIG. 7 shows the percentage of inhibition of HT-29 cell
growth by ACPD. The percentage of inhibition of the HT-29 colon
cancer cells by ACPD increases as a function of ACPD. The growth of
the cells decreases by 50 percent (half, IC.sub.50) at
approximately 1.0 .mu.M.
[0032] FIG. 8 shows the effect of DNDA on the HT-29 colon cancer
cell line. The number of HT-29 cells decreases as the concentration
of DNDA increases over time. Blue diamond, control; orange square,
0.25 .mu.M DNDA; gray triangle, 0.5 .mu.M DNDA; yellow X, 0.75
.mu.M DNDA; blue X, 1.0 .mu.M DNDA; and green circle, 1.5 .mu.M
DNDA.
[0033] FIG. 9 shows the accumulated effect of DNDA treatment on the
HT-29 colon cancer cell line after three days of treatment. At this
time, HT-29 cells showed a slight decrease in proliferation at 0.25
.mu.M of DNDA and a dramatic decrease in proliferation at 1.5 .mu.M
concentration of DNDA.
[0034] FIG. 10 shows the percentage of inhibition of HT-29 cell
growth by DNDA. The percentage inhibition of the HT-29 colon cancer
cells by DNDA increases as a function of DNDA. The growth of the
cells decreases by 50 percent (half, IC.sub.50) at approximately
0.78 .mu.M.
[0035] FIG. 11 shows the effect of ICA-1 on the HT-29 colon cancer
cell line. The number of HT-29 cells decreases as the concentration
of ICA-I increases over time. Blue diamond, control; orange square,
0.25 .mu.M ICA-1; gray triangle, 0.5 .mu.M ICA-1; yellow X, 0.75
.mu.M ICA-1; blue X, 1.0 .mu.M ICA-1; and green circle, 1.5 .mu.M
ICA-1.
[0036] FIG. 12 shows the accumulated effect of ICA-1 treatment on
the HT-29 colon cancer cell line after three days of treatment. At
this time, ICA-1 had a slight increase in cell proliferation at
0.25 .mu.M and a dramatic decrease in cell proliferation at 1.5
.mu.M of ICA-1.
[0037] FIG. 13 shows the percentage of inhibition of HT-29 cell
growth by ICA-1. The percentage of inhibition of the HT-29 colon
cancer cells increases as a function of ICA-1. The growth of the
cells reduces by 50 percent (half, IC.sub.50) at approximately 0.95
.mu.M.
[0038] FIG. 14 shows the effect of .zeta.-Stat on HT-29 colorectal
cancer cell line. The number of HT-29 cells decreases as the
concentration of .zeta.-Stat increases over time. Blue diamond,
control; orange square, 1.0 .mu.M .zeta.-Stat; gray triangle, 1.5
.mu.M .zeta.-Stat; yellow X, 2.0 .mu.M .zeta.-Stat; and green
circle, 3.0 .mu.M .zeta.-Stat.
[0039] FIG. 15 shows percentage inhibition of HT-29 cell growth by
.zeta.-Stat.
[0040] FIG. 16 shows the effect of .zeta.-Stat on the expression
and phosphorylation of PKC-.tau. and PKC-.zeta. in the HT-29
colorectal cancer cell line.
[0041] FIG. 17 shows the effect of specific aPKC inhibition on
HT-29 colorectal cancer cell survival.
[0042] FIG. 18 shows the effect of .zeta.-Stat on the normal colon
epithelial cell line CCD18CO. The number of HT-29 cells did not
change with increasing concentrations of .zeta.-Stat. Blue diamond,
control; orange square, 1.0 .mu.M .zeta.-Stat; gray triangle, 1.5
.mu.M .zeta.-Stat; yellow X, 2.0 .mu.M .zeta.-Stat; and green
circle, 3.0 .mu.M .zeta.-Stat.
[0043] FIG. 19 shows the percentage of inhibition of CCD18CO cell
growth by .zeta.-Stat.
[0044] FIG. 20 shows the effects of specific aPKC inhibition by
ICA-1 and .zeta.-Stat on CCD18CO normal colorectal cell
survival.
DETAILED DISCLOSURE OF THE INVENTION
[0045] In the following detailed description of the preferred
embodiments, reference is made to the accompanying drawings, which
form a part hereof, and within which are shown by way of
illustration specific embodiments by which the invention may be
practiced. It is to be understood that other embodiments by which
the invention may be practiced. It is to be understood that other
embodiments may be utilized and structural changes may be made
without departing from the scope of the invention.
[0046] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
described herein. All publications mentioned herein are
incorporated herein by reference in their entirety to disclose and
describe the methods and/or materials in connection with which the
publications are cited. It is understood that the present
disclosure supercedes any disclosure of an incorporated publication
to the extent there is a contradiction.
[0047] Provided herein are methods of treating colorectal cancers
using atypical PKC (aPKC) inhibitors. Further provided are methods
of assessing the effectiveness of aPKC inhibitors in the
effectiveness of inhibiting aPKCs in colon cancer cells of a
subject suffering from colon cancer and the treatment of colon
cancer in the subject in colon cancer cells obtained from a subject
suffering from colon cancer.
[0048] In preferred embodiments, the instant invention provides
methods of treating colorectal cancer by administering to a subject
in need thereof, a composition comprising a therapeutically
effective amount of an inhibitor of an aPKC.
[0049] The term "therapeutically effective amount," as used herein,
refers to an amount that is capable of treating or ameliorating a
disease or condition or otherwise capable of producing an intended
therapeutic effect. In preferred embodiments, a therapeutically
effective amount is an amount that is useful for treating a colon
cancer disease.
[0050] The quantity to be administered, both according to number of
treatments and unit dose, depends on the colon cancer disease and
stage of colon cancer disease to be treated, the subject to be
treated, the state of the subject and the susceptibility of the
colon cancer disease to an inhibitor of an atypical PKC.
[0051] Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Generally, the dosage of the composition of the subject
invention will vary depending upon such factors as the patient's
age, weight, height, sex, general medical condition and previous
medical history.
[0052] In some embodiments of the subject invention, the method
comprises administration of multiple doses of the composition. The
method may comprise administration of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, or more therapeutically effective doses
of a composition comprising at least one of an atypical PKC as
described herein. In some embodiments, doses are administered over
the course of a short period of about 1 week to a long period of
more than about 12 months. In some embodiment, the compositions
according to the instant invention are administered for a period of
about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 10
weeks, 14 weeks, 20 weeks, 30 weeks, 40 weeks, 52 weeks or more
than about 52 weeks.
[0053] For example, the compositions of the instant invention are
administered from a low of about 1 week to a high of about 12
months; from about 2 weeks to about 50 weeks; from about 4 weeks to
about 40 weeks; from about 6 weeks to about 30 weeks; from about 8
weeks to about 25 weeks; from about 10 weeks to about 20 weeks;
from about 11 weeks to about 18 weeks; from about 12 weeks to about
17 weeks; or from about 14 to about 16 weeks.
[0054] The dosage of a composition according to the instant
invention administered can be an amount from a low of about 0.1
.mu.g/kg, about 0.5 .mu.g/kg or about 1 .mu.g/kg to a high of about
4000 mg/kg, about 4500 mg/kg or about 5000 mg/kg.
[0055] For example the amount of an inhibitor of an aPKC
administered can be from about 0.1 .mu.g/kg to about 5000 mg/kg;
about 0.1 .mu.g/kg to about 4500 mg/kg; about 0.1 .mu.g/kg to about
4000 mg/kg; about 0.1 .mu.g/kg to about 3000 mg/kg; about 0.1
.mu.g/kg to about 2000 mg/kg; about 0.1 .mu.g/kg to about 1500
mg/kg; about 0.1.mu./kg to about 1000 mg/kg; about 0.1 .mu.g/kg to
about 750 mg/kg; about 0.1 .mu.g/kg to about 500 mg/kg; about 0.1
.mu.g/kg to about 250 mg/kg; about 0.1 .mu.g/kg to about 100 mg/kg;
about 0.1 .mu.g/kg to about 75 mg/kg; about 0.1 .mu.g/kg to about
50 mg/kg; about 0.1 .mu.g/kg to about 25 mg/kg; about 0.1 .mu.g/kg
to about 10 mg/kg; about 0.1 .mu.g/kg to about 7.5 mg/kg; about 0.1
.mu.g/kg to about 5 mg/kg; about 0.1 .mu.g/kg to about 2.5 mg/kg;
about 0.1 .mu.g/kg to about 1 mg/kg; about 0.1 .mu.g/kg to about
750 .mu.g/kg; about 0.1 .mu.g/kg to about 500 .mu.g/kg; about 0.1
.mu.g/kg to about 250 .mu.g/kg; about 0.1 .mu.g/kg to about 100
.mu.g/kg; about 0.1 .mu.g/kg to about 75 .mu.g/kg; about 0.1
.mu.g/kg to about 50 .mu.g/kg; about 0.1 .mu.g/kg to about 25
.mu.g/kg; about 0.1 .mu.g/kg to about 10 .mu.g/kg; about 0.1
.mu.g/kg to about 7.5 .mu.g/kg; about 0.1 .mu.g/kg to about 5
.mu.g/kg; about 0.1 .mu.g/kg to about 2.5 .mu.g/kg; about 0.1
.mu.g/kg to about 1 .mu.g/kg; about 0.1 .mu.g/kg to about 0.75
.mu.g/kg; about 0.1 .mu.g/kg to about 0.5 .mu.g/kg; about 0.1
.mu.g/kg to about 0.25 .mu.g/kg.
[0056] The amount of an inhibitor of an aPKC administered can also
be from about 0.2 .mu.g/kg to about 5000 mg/kg; about 0.2 .mu.g/kg
to about 4500 mg/kg; about 0.2 .mu.g/kg to about 4000 mg/kg; about
0.2 .mu.g/kg to about 3000 mg/kg; about 0.2 .mu.g/kg to about 2000
mg/kg; about 0.2 .mu.g/kg to about 1500 mg/kg; about 0.2 .mu.g/kg
to about 1000 mg/kg; about 0.2 .mu.g/kg to about 750 mg/kg; about
0.2 .mu.g/kg to about 500 mg/kg; about 0.2 .mu.g/kg to about 250
mg/kg; about 0.2 .mu.g/kg to about 100 mg/kg; about 0.2 .mu.g/kg to
about 75 mg/kg; about 0.2 .mu.g/kg to about 50 mg/kg; about 0.2
.mu.g/kg to about 25 mg/kg; about 0.2 .mu.g/kg to about 10 mg/kg;
about 0.2 .mu.g/kg to about 7.5 mg/kg; about 0.2 .mu.g/kg to about
5 mg/kg; about 0.2 .mu.g/kg to about 2.5 mg/kg; about 0.2 .mu.g/kg
to about 1 mg/kg; about 0.2 .mu.g/kg to about 750 .mu.g/kg; about
0.2 .mu.g/kg to about 500 .mu.g/kg; about 0.2 .mu.g/kg to about 250
.mu.g/kg; about 0.2 .mu.g/kg to about 100 .mu.g/kg; about 0.2
.mu.g/kg to about 75 .mu.g/kg; about 0.2 .mu.g/kg to about 50
.mu.g/kg; about 0.2 .mu.g/kg to about 25 .mu.g/kg; about 0.2
.mu.g/kg to about 10 .mu.g/kg; about 0.2 .mu.g/kg to about 7.5
.mu.g/kg; about 0.2 .mu.g/kg to about 5 .mu.g/kg; about 0.2
.mu.g/kg to about 2.5 .mu.g/kg; about 0.2 .mu.g/kg to about 1
.mu.g/kg; about 0.2 .mu.g/kg to about 0.75 .mu.g/kg; about 0.2
.mu.g/kg to about 0.5 .mu.g/kg; about 0.2 .mu.g/kg to about 0.25
.mu.g/kg.
[0057] The amount of an inhibitor of an aPKC administered can
further be from about 0.5 .mu.g/kg to about 5000 mg/kg; about 0.5
.mu.g/kg to about 4500 mg/kg; about 0.5 .mu.g/kg to about 4000
mg/kg; about 0.5 .mu.g/kg to about 3000 mg/kg; about 0.5 .mu.g/kg
to about 2000 mg/kg; about 0.5 .mu.g/kg to about 1500 mg/kg; about
0.5 .mu.g/kg to about 1000 mg/kg; about 0.5 .mu.g/kg to about 750
mg/kg; about 0.5 .mu.g/kg to about 500 mg/kg; about 0.5 .mu.g/kg to
about 250 mg/kg; about 0.5 .mu.g/kg to about 100 mg/kg; about 0.5
.mu.g/kg to about 75 mg/kg; about 0.5 .mu.g/kg to about 50 mg/kg;
about 0.5 .mu.g/kg to about 25 mg/kg; about 0.5 .mu.g/kg to about
10 mg/kg; about 0.5 .mu.g/kg to about 7.5 mg/kg; about 0.5 .mu.g/kg
to about 5 mg/kg; about 0.5 .mu.g/kg to about 2.5 mg/kg; about 0.5
.mu.g/kg to about 1 mg/kg; about 0.5 .mu.g/kg to about 750
.mu.g/kg; about 0.5 .mu.g/kg to about 500 .mu.g/kg; about 0.5
.mu.g/kg to about 250 .mu.g/kg; about 0.5 .mu.g/kg to about 100
.mu.g/kg; about 0.5 .mu.g/kg to about 75 .mu.g/kg; about 0.5
.mu.g/kg to about 50 .mu.g/kg; about 0.5 .mu.g/kg to about 25
.mu.g/kg; about 0.5 .mu.g/kg to about 10 .mu.g/kg; about 0.5
.mu.g/kg to about 7.5 .mu.g/kg; about 0.5 .mu.g/kg to about 5
.mu.g/kg; about 0.5 .mu.g/kg to about 2.5 .mu.g/kg; about 0.5
.mu.g/kg to about 1 .mu.g/kg; about 0.5 .mu.g/kg to about 0.75
.mu.g/kg.
[0058] The amount of an inhibitor of an aPKC administered can
further be from about 1 .mu.g/kg to about 5000 mg/kg; about 1
.mu.g/kg to about 4500 mg/kg; about 1 .mu.g/kg to about 4000 mg/kg;
about 1 .mu.g/kg to about 3000 mg/kg; about 1 .mu.g/kg to about
2000 mg/kg; about 1 .mu.g/kg to about 1500 mg/kg; about 1 .mu.g/kg
to about 1000 mg/kg; about 1 .mu.g/kg to about 750 mg/kg; about 1
.mu.g/kg to about 500 mg/kg; about 1 .mu.g/kg to about 250 mg/kg;
about 1 .mu.g/kg to about 100 mg/kg; about 1 .mu.g/kg to about 75
mg/kg; about 1 .mu.g/kg to about 50 mg/kg; about 1 .mu.g/kg to
about 25 mg/kg; about 1 .mu.g/kg to about 10 mg/kg; about 1
.mu.g/kg to about 7.5 mg/kg; about 1 .mu.g/kg to about 5 mg/kg;
about 1 .mu.g/kg to about 2.5 mg/kg; about 1 .mu.g/kg to about 1
mg/kg; about 1 .mu.g/kg to about 750 .mu.g/kg; about 1 .mu.g/kg to
about 500 .mu.g/kg; about 1 .mu.g/kg to about 250 .mu.g/kg; about 1
.mu.g/kg to about 100 .mu.g/kg; about 1 .mu.g/kg to about 75
.mu.g/kg; about 1 .mu.g/kg to about 50 .mu.g/kg; about 1 .mu.g/kg
to about 25 .mu.g/kg; about 1 .mu.g/kg to about 10 .mu.g/kg; about
1 .mu.g/kg to about 7.5 .mu.g/kg; about 1 .mu.g/kg to about 5
.mu.g/kg; about 1 .mu.g/kg to about 2.5 .mu.g/kg.
[0059] The amount of an inhibitor of an aPKC administered can
further be from about 2/kg to about 5000 mg/kg; about 2 .mu.g/kg to
about 4500 mg/kg; about 2 .mu.g/kg to about 4000 mg/kg; about 2
.mu.g/kg to about 3000 mg/kg; about 2 .mu.g/kg to about 2000 mg/kg;
about 2 .mu.g/kg to about 1500 mg/kg; about 2 .mu.g/kg to about
1000 mg/kg; about 2 .mu.g/kg to about 750 mg/kg; about 2 .mu.g/kg
to about 500 mg/kg; about 2 .mu.g/kg to about 250 mg/kg; about 2
.mu.g/kg to about 100 mg/kg; about 2 .mu.g/kg to about 75 mg/kg;
about 21 .mu.g/kg to about 50 mg/kg; about 2 .mu.g/kg to about 25
mg/kg; about 2 .mu.g/kg to about 10 mg/kg; about 2 .mu.g/kg to
about 7.5 mg/kg; about 2 .mu.g/kg to about 5 mg/kg; about 2
.mu.g/kg to about 2.5 mg/kg; about 2 .mu.g/kg to about 1 mg/kg;
about 21 .mu.g/kg to about 750 .mu.g/kg; about 2 .mu.g/kg to about
500 .mu.g/kg; about 2 .mu.g/kg to about 250 .mu.g/kg; about 2
.mu.g/kg to about 100 .mu.g/kg; about 2 .mu.g/kg to about 75
.mu.g/kg; about 2 .mu.g/kg to about 50 .mu.g/kg; about 2 .mu.g/kg
to about 25 .mu.g/kg; about 2 .mu.g/kg to about 10 .mu.g/kg; about
2 .mu.g/kg to about 7.5 .mu.g/kg; about 2 .mu.g/kg to about 5
.mu.g/kg; about 2 .mu.g/kg to about 2.5 .mu.g/kg The amount of an
inhibitor of an aPKC administered can further be from about 5
.mu.g/kg to about 5000 mg/kg; about 5 .mu.g/kg to about 4500 mg/kg;
about 5 .mu.g/kg to about 4000 mg/kg; about 5 .mu.g/kg to about
3000 mg/kg; about 5 .mu.g/kg to about 2000 mg/kg; about 5 .mu.g/kg
to about 1500 mg/kg; about 5 .mu.g/kg to about 1000 mg/kg; about 5
.mu.g/kg to about 750 mg/kg; about 5 .mu.g/kg to about 500 mg/kg;
about 5 .mu.g/kg to about 250 mg/kg; about 5 .mu.g/kg to about 100
mg/kg; about 5 .mu.g/kg to about 75 mg/kg; about 5 .mu.g/kg to
about 50 mg/kg; about 5 .mu.g/kg to about 25 mg/kg; about 5
.mu.g/kg to about 10 mg/kg; about 5 .mu.g/kg to about 7.5 mg/kg;
about 5 .mu.g/kg to about 5 mg/kg; about 5 .mu.g/kg to about 2.5
mg/kg; about 5 .mu.g/kg to about 1 mg/kg; about 5 .mu.g/kg to about
750 .mu.g/kg; about 5 .mu.g/kg to about 500 .mu.g/kg; about 5
.mu.g/kg to about 250 .mu.g/kg; about 5 .mu.g/kg to about 100
.mu.g/kg; about 5 .mu.g/kg to about 75 .mu.g/kg; about 5 .mu.g/kg
to about 50 .mu.g/kg; about 5 .mu.g/kg to about 25 .mu.g/kg; about
5 .mu.g/kg to about 10 .mu.g/kg; about 5 .mu.g/kg to about 7.5
.mu.g/kg.
[0060] The amount of an inhibitor of an aPKC administered can
further be from about 10 .mu.g/kg to about 5000 mg/kg; about 10
.mu.g/kg to about 4500 mg/kg; about 10 .mu.g/kg to about 4000
mg/kg; about 10 .mu.g/kg to about 3000 mg/kg; about 10 .mu.g/kg to
about 2000 mg/kg; about 10 .mu.g/kg to about 1500 mg/kg; about 10
.mu.g/kg to about 1000 mg/kg; about 10 .mu.g/kg to about 750 mg/kg;
about 10 .mu.g/kg to about 500 mg/kg; about 10 .mu.g/kg to about
250 mg/kg; about 10 .mu.g/kg to about 100 mg/kg; about 10 .mu.g/kg
to about 75 mg/kg; about 10 .mu.g/kg to about 50 mg/kg; about 10
.mu.g/kg to about 25 mg/kg; about 10 .mu.g/kg to about 10 mg/kg;
about 10 .mu.g/kg to about 7.5 mg/kg; about 10 .mu.g/kg to about 5
mg/kg; about 10 .mu.g/kg to about 2.5 mg/kg; about 10 .mu.g/kg to
about 1 mg/kg; about 10 .mu.g/kg to about 750 .mu.g/kg; about 10
.mu.g/kg to about 500 .mu.g/kg; about 10 .mu.g/kg to about 250
.mu.g/kg; about 10 .mu.g/kg to about 100 .mu.g/kg; about 10
.mu.g/kg to about 75 .mu.g/kg; about 10 .mu.g/kg to about 50
.mu.g/kg; about 10 .mu.g/kg to about 25 .mu.g/kg.
[0061] The amount of an inhibitor of an aPKC administered can
further be from about 20 .mu.g/kg to about 5000 mg/kg; about 20
.mu.g/kg to about 4500 mg/kg; about 20 .mu.g/kg to about 4000
mg/kg; about 20 .mu.g/kg to about 3000 mg/kg; about 20 .mu.g/kg to
about 2000 mg/kg; about 20 .mu.g/kg to about 1500 mg/kg; about 20
.mu.g/kg to about 1000 mg/kg; about 20 .mu.g/kg to about 750 mg/kg;
about 20 .mu.g/kg to about 500 mg/kg; about 20 .mu.g/kg to about
250 mg/kg; about 20 .mu.g/kg to about 100 mg/kg; about 20 .mu.g/kg
to about 75 mg/kg; about 20 .mu.g/kg to about 50 mg/kg; about 20
.mu.g/kg to about 25 mg/kg; about 20 .mu.g/kg to about 10 mg/kg;
about 20 .mu.g/kg to about 7.5 mg/kg; about 20 .mu.g/kg to about 5
mg/kg; about 20 .mu.g/kg to about 2.5 mg/kg; about 20 .mu.g/kg to
about 1 mg/kg; about 20 .mu.g/kg to about 750 .mu.g/kg; about 20
.mu.g/kg to about 500 .mu.g/kg; about 20 .mu.g/kg to about 250
.mu.g/kg; about 20 .mu.g/kg to about 100 .mu.g/kg; about 20
.mu.g/kg to about 75 .mu.g/kg; about 20 .mu.g/kg to about 50
.mu.g/kg; about 20 .mu.g/kg to about 25 .mu.g/kg.
[0062] The amount of an inhibitor of an aPKC administered can
further be from about 50 .mu.g/kg to about 5000 mg/kg; about 50
.mu.g/kg to about 4500 mg/kg; about 50 .mu.g/kg to about 4000
mg/kg; about 50 .mu.g/kg to about 3000 mg/kg; about 50 .mu.g/kg to
about 2000 mg/kg; about 50 .mu.g/kg to about 1500 mg/kg; about 50
.mu.g/kg to about 1000 mg/kg; about 50 .mu.g/kg to about 750 mg/kg;
about 50 .mu.g/kg to about 500 mg/kg; about 50 .mu.g/kg to about
250 mg/kg; about 50 .mu.g/kg to about 100 mg/kg; about 50 .mu.g/kg
to about 75 mg/kg; about 50 .mu.g/kg to about 50 mg/kg; about 50
.mu.g/kg to about 25 mg/kg; about 50 .mu.g/kg to about 10 mg/kg;
about 50 .mu.g/kg to about 7.5 mg/kg; about 50 .mu.g/kg to about 5
mg/kg; about 50 .mu.g/kg to about 2.5 mg/kg; about 50 .mu.g/kg to
about 1 mg/kg; about 50 .mu.g/kg to about 750 .mu.g/kg; about 50
.mu.g/kg to about 500 .mu.g/kg; about 50 .mu.g/kg to about 250
.mu.g/kg; about 50 .mu.g/kg to about 100 .mu.g/kg; about 50
.mu.g/kg to about 75 .mu.g/kg.
[0063] The amount of an inhibitor of an aPKC administered can
further be from about 100 .mu.g/kg to about 5000 mg/kg; about 100
.mu.g/kg to about 4500 mg/kg; about 100 .mu.g/kg to about 4000
mg/kg; about 100 .mu.g/kg to about 3000 mg/kg; about 100 .mu.g/kg
to about 2000 mg/kg; about 100 .mu.g/kg to about 1500 mg/kg; about
100 .mu.g/kg to about 1000 mg/kg; about 100 .mu.g/kg to about 750
mg/kg; about 100 .mu.g/kg to about 500 mg/kg; about 100 .mu.g/kg to
about 250 mg/kg; about 100 .mu.g/kg to about 100 mg/kg; about 100
.mu.g/kg to about 75 mg/kg; about 100 .mu.g/kg to about 50 mg/kg;
about 100 .mu.g/kg to about 25 mg/kg; about 100 .mu.g/kg to about
10 mg/kg; about 100 .mu.g/kg to about 7.5 mg/kg; about 100 .mu.g/kg
to about 5 mg/kg; about 100 .mu.g/kg to about 2.5 mg/kg; about 100
.mu.g/kg to about 1 mg/kg; about 100 .mu.g/kg to about 750
.mu.g/kg; about 100 .mu.g/kg to about 500 .mu.g/kg; about 100
.mu.g/kg to about 250 .mu.g/kg.
[0064] The amount of an inhibitor of an aPKC administered can
further be from about 20 .mu.g/kg to about 5000 mg/kg; about 200
.mu.g/kg to about 4500 mg/kg; about 200 .mu.g/kg to about 4000
mg/kg; about 200 .mu.g/kg to about 3000 mg/kg; about 200 .mu.g/kg
to about 2000 mg/kg; about 200 .mu.g/kg to about 1500 mg/kg; about
200 .mu.g/kg to about 1000 mg/kg; about 200 .mu.g/kg to about 750
mg/kg; about 200 .mu.g/kg to about 500 mg/kg; about 200 .mu.g/kg to
about 250 mg/kg; about 200 .mu.g/kg to about 100 mg/kg; about 200
.mu.g/kg to about 75 mg/kg; about 200 .mu.g/kg to about 50 mg/kg;
about 200 .mu.g/kg to about 25 mg/kg; about 200 .mu.g/kg to about
10 mg/kg; about 200 .mu.g/kg to about 7.5 mg/kg; about 200 .mu.g/kg
to about 5 mg/kg; about 200 .mu.g/kg to about 2.5 mg/kg; about 200
.mu.g/kg to about 1 mg/kg; about 200 .mu.g/kg to about 750
.mu.g/kg; about 200 .mu.g/kg to about 500 .mu.g/kg; about 200
.mu.g/kg to about 250 .mu.g/kg.
[0065] The amount of an inhibitor of an aPKC administered can
further be from about 500 .mu.g/kg to about 5000 mg/kg; about 500
.mu.g/kg to about 4500 mg/kg; about 500 .mu.g/kg to about 4000
mg/kg; about 500 .mu.g/kg to about 3000 mg/kg; about 500 .mu.g/kg
to about 2000 mg/kg; about 500 .mu.g/kg to about 1500 mg/kg; about
500 .mu.g/kg to about 1000 mg/kg; about 500 .mu.g/kg to about 750
mg/kg; about 500 .mu.g/kg to about 500 mg/kg; about 500 .mu.g/kg to
about 250 mg/kg; about 500 .mu.g/kg to about 100 mg/kg; about 500
.mu.g/kg to about 75 mg/kg; about 500 .mu.g/kg to about 50 mg/kg;
about 500 .mu.g/kg to about 25 mg/kg; about 500 .mu.g/kg to about
10 mg/kg; about 500 .mu.g/kg to about 7.5 mg/kg; about 500 .mu.g/kg
to about 5 mg/kg; about 500 .mu.g/kg to about 2.5 mg/kg; about 500
.mu.g/kg to about 1 mg/kg; about 500 .mu.g/kg to about 750
.mu.g/kg.
[0066] The amount of an inhibitor of an aPKC administered can
further be from about 1 mg/kg to about 5000 mg/kg; about 1 mg/kg to
about 4500 mg/kg; about 1 mg/kg to about 4000 mg/kg; about 1 mg/kg
to about 3000 mg/kg; about 1 mg/kg to about 2000 mg/kg; about 1
mg/kg to about 1500 mg/kg; about 1 mg/kg to about 1000 mg/kg; about
1 mg/kg to about 750 mg/kg; about 1 mg/kg to about 500 mg/kg; about
1 mg/kg to about 250 mg/kg; about 1 mg/kg to about 100 mg/kg; about
1 mg/kg to about 75 mg/kg; about 1 mg/kg to about 50 mg/kg; about 1
mg/kg to about 25 mg/kg; about 1 mg/kg to about 10 mg/kg; about 1
mg/kg to about 7.5 mg/kg; about 1 mg/kg to about 5 mg/kg; about 1
mg/kg to about 2.5 mg/kg.
[0067] Advantageously, the administration of up to 5000 mg/kg of a
composition comprising a aPKC inhibitor according to the instant
invention in vivo in mice does not cause any toxicity.
[0068] The composition of the instant invention can be administered
at once or several times per day. It is within the knowledge of the
skilled clinician to determine the frequency and duration of
administration of compositions of the instant invention.
[0069] In some embodiments, the PKC inhibitor is specific to a
single aPKC. In certain embodiments, the aPKC inhibitor specific
for a single aPKC is specific for PKC-.tau.. In preferred
embodiments, the specific inhibitor of PKC-.tau. is ICA-1.
[0070] In other embodiments, the PKC inhibitor is a pan-aPKC
inhibitor. In some embodiments, the pan-aPKC inhibitor is ACPD. In
other embodiments, the pan-aPKC inhibitor is DNDA.
[0071] In certain preferred embodiments, the inhibitor is specific
to PKC-.zeta.. In further preferred embodiments, the aPKC inhibitor
specific for PKC-.zeta. is .zeta.-Stat.
[0072] In some embodiments, the methods of the instant invention
provide treatment of a subject suffering from a colon cancer by
administering a composition according to the invention, wherein the
subject is a subject that suffers from colon cancer and the subject
has cancer cells that are responsive to an inhibitor of an atypical
PKC.
[0073] The colon cancers treated by methods of the instant
invention include, but are not limited to, adenocarcinomas and
squamous cell carcinomas.
[0074] The adenocarcinomas treated by the instant methods include,
but are not limited to, carcinoid tumors, gastrointestinal stromal
tumors including soft tissue sarcomas and sarcomas of blood vessels
and connective tissue, and lymphomas of the colon.
[0075] The squamous cell carcinomas treated by the instant methods
include, but are not limited to, carcinomas developing from blood
vessels or smooth muscle cells of the colon, including
leiomyosarcomas and angiosarcomas.
[0076] Further included in the instant methods of treating colon
cancer are methods of treating colon cancer metastases, e.g. cancer
cells that originate in the colon and spread to other organs in the
subject's body. Also included are metastases to the colon, e.g.
from cancers in other part of the intestine, e.g. the rectum, or
other organs, which cells metastasize into the colon.
[0077] Further included in the instant methods of treating colon
cancer are methods treating right colon cancer and left colon
cancer.
[0078] The instant invention also provides methods of determining
the susceptibility of the colon cancer cells of a subject to
inhibitors of an aPKC.
[0079] In certain embodiments, the method of measuring the
susceptibility of colon cancer cells of a subject suffering from
colon cancer to an aPKC inhibitor, the method comprising: [0080]
obtaining a colon cell sample from a subject suffering from colon
cancer, wherein the colon cell sample comprises cells suspected of
being colon cancer cells and adjacent normal colon epithelial
cells; [0081] measuring the expression of an atypical PKC in the
cells of the colon cell sample that are suspected of being colon
cancer cells; [0082] measuring the expression of an atypical PKC in
the cells of the colon cell sample that are normal colon epithelial
cells; [0083] in the case of a higher expression of at least one
atypical PKC in the cells of the colon cancer sample compared to
the normal colon epithelial cells, exposing a portion of the colon
cancer cells of the colon cell sample to an inhibitor of an aPKC
according to the methods of the instant invention, while leaving
another prion of the colon cancer cells of the colon cell sample
untreated; [0084] measuring cell proliferation, expression and
activation of aPKCs and markers of apoptosis and anti-apoptosis in
treated and untreated cells; [0085] assigning the colon cancer
cells of the subject as susceptible to methods of treatment by
administering aPKC inhibitors according to the methods of the
instant invention; and [0086] treating the subject according to the
methods of the instant invention.
[0087] In certain embodiments, the methods of measuring the
susceptibility of colon cancer cells of a subject suffering from
colon cancer to an aPKC inhibitor include assigning the colon
cancer cells of the subject as susceptible to an inhibitor of a
aPKC when a decrease in the expression of an aPKC upon treatment of
the cells with an inhibitor of a PKC is determined.
[0088] In certain embodiments, the methods of measuring the
susceptibility of colon cancer cells of a subject suffering from
colon cancer to an aPKC inhibitor include assigning the colon
cancer cells of the subject as susceptible to an inhibitor of a
aPKC when a decrease in the expression of an aPKC upon treatment of
the cells with an inhibitor of a PKC is determined.
[0089] The phrase "susceptible to an aPKC inhibitor" indicates that
upon administration of the colon cancer therapy comprising an aPKC
inhibitor according to the instant invention, the subject exhibits
beneficial or desired results including but not limited to
therapeutic benefit; eradication, amelioration, delay or prevention
of one or more of the symptoms of colon cancer; prolonging the
life-span of the subject; or improvement in the quality of life of
the subject. An improvement may be observed in the patient,
notwithstanding that the patient may still be afflicted with colon
cancer.
[0090] The phrase "non-susceptible to an aPKC inhibitor" according
to the instant invention indicates that upon administration of the
colon cancer therapy, the subject does not exhibit beneficial or
desired results such as, therapeutic benefit; eradication,
amelioration, delay or prevention of one or more of the symptoms of
colon cancer; prolonging the life-span of the subject; or
improvement in the quality of life of the subject.
[0091] It was advantageously determined that a subject having a
colon cancer wherein the colon cancer cells from the subject
exhibit a higher level and/or activity of mRNA or protein for
PKC-.zeta. and/or PKC-.tau. is responsive to a colon cancer therapy
with an inhibitor of PKC-.zeta. and/or PKC-.tau..
[0092] The methods of measuring susceptibility of colon cancer
cells to treatment with an inhibitor of an aPKC as provided herein
are based on the measurement of a decreased expression of an aPKC
in the colon cancer cells upon exposure to an aPKC inhibitor.
[0093] The methods of measuring susceptibility of colon cancer
cells to treatment with an inhibitor of an aPKC as provided herein
are further based on the measurement of a decreased phoshorylation
of an aPKC in the colon cancer cells upon exposure to an aPKC
inhibitor.
[0094] The methods of measuring susceptibility of colon cancer
cells to treatment with an inhibitor of an aPKC as provided herein
are also based on the measurement of a decreased expression of a
caspase protein, including, but not limited to, caspase 3 in the
colon cancer cells upon exposure to an aPKC inhibitor.
[0095] The methods of measuring susceptibility of colon cancer
cells to treatment with an inhibitor of an aPKC as provided herein
are further based on the measurement of a decreased expression of a
survivin protein in the colon cancer cells upon exposure to an aPKC
inhibitor. The methods of measuring susceptibility of colon cancer
cells to treatment with an inhibitor of an aPKC as provided herein
are yet further based on the measurement of a decreased expression
of a BCL protein, including, but not limited to, BCL-XL in the
colon cancer cells upon exposure to an aPKC inhibitor.
[0096] Accordingly, in preferred embodiments of the subject
invention, methods are provided for identifying a subject as
responsive or non-responsive to a colon cancer therapy with an
inhibitor of PKC-.zeta. and/or PKC-.tau. based on higher level
and/or activity of mRNA or protein for PKC-.zeta. and/or PKC-.tau.
in the colon cancer cells from the subject compared to control
normal colon epithelial cell o the subject or normal colon
epithelial cells of a control subject that is free of colon cancer
cells.
[0097] In a certain embodiment, the subject invention provides a
method of identifying a subject suffering from a colon cancer as
responsive or non-responsive to a therapy with an inhibitor of
PKC-.zeta. and/or PKC-.tau.. The method comprises the steps of:
[0098] (a) determining the level and/or activity of PKC-.zeta.
and/or PKC-.tau. mRNA or protein in a test sample obtained from the
subject, [0099] (b) optionally, determining the level and/or
activity of PKC-.zeta. and/or PKC-.tau. mRNA or protein in a
control sample, or obtaining a reference value corresponding to the
level of PKC-.zeta. and/or PKC-.tau. mRNA or protein; and [0100]
(i) identifying the subject as being responsive to the colon cancer
therapy with the inhibitor of PKC-.zeta. and/or PKC-.tau. based on
the level and/or activity of PKC-.zeta. and/or PKC-.tau. mRNA or
protein in the test sample as compared to the level and/or activity
of PKC-.zeta. and/or PKC-.tau. mRNA or protein in the control
sample, and optionally, administering the colon cancer therapy with
the inhibitor of PKC-.zeta. and/or PKC-.tau. to the subject
identified as being responsive; or [0101] (ii) identifying the
subject as being non-responsive to the colon cancer therapy with
the inhibitor of PKC-.zeta. and/or PKC-.tau. based the level and/or
activity of PKC-.zeta. and/or PKC-.tau. mRNA or protein in the test
sample as compared to the level and/or activity of PKC-.zeta.
and/or PKC-.tau. mRNA or protein in the control sample, and
optionally, administering a colon cancer therapy other than the
colon cancer therapy with the inhibitor of PKC-.zeta. and/or
PKC-.tau. to the subject identified as being non-responsive.
[0102] In specific embodiments, the test samples comprise the colon
cancer cells from the subject.
[0103] It was advantageously determined that a subject's colon
cancer cells are responsive to a colon cancer therapy with an
inhibitor of PKC-.zeta. and/or PKC-.tau. if the colon cancer cells
in the subject contain a higher level and/or activity of mRNA or
protein for PKC-.zeta. and/or PKC-.tau. in the colon cancer cells
compared to non-cancer normal colon epithelial cells.
[0104] It was also determined that a subject's colon cancer is
non-responsive to a colon cancer therapy with an inhibitor of
PKC-.zeta. and/or PKC-.tau. if the colon cancer cells in the
subject contain a similar or lower level and/or activity of mRNA or
protein for PKC-.zeta. and/or PKC-.tau. in the colon cancer cells
from the subject compared to the control normal colon epithelial
cells of the subject.
[0105] Various techniques are known to a person of ordinary skill
in the art to determine the level of mRNA in a sample. Non-limiting
examples of such techniques include microarray analysis, real-time
polymerase chain reaction (PCR), Northern blot, in situ
hybridization, solution hybridization, or quantitative reverse
transcription PCR (qRT-PCR). Methods of carrying out these
techniques are routine in the art. Additional methods of
determining the level of mRNA in a sample are also well known to a
person of ordinary skill in the art and such embodiments are within
the purview of the invention.
[0106] Various techniques are known to a person of ordinary skill
in the art to determine the level of PKC-.zeta. and/or PKC-.tau.
protein in a sample. Non-limiting examples of such techniques
include protein array analysis, Western blot analysis,
enzyme-linked immunosorbent assay (ELISA), radio-immune assay
(RIA), etc. Methods of carrying out these techniques are routine in
the art. Additional methods of determining the level of PKC-.zeta.
and/or PKC-.tau. protein in a sample are also known to a person of
ordinary skill in the art and such embodiments are within the
purview of the invention.
[0107] Similarly, various techniques are known to a person of
ordinary skill in the art to determine the activity of PKC-.zeta.
and/or PKC-.tau. protein in a sample. Methods of carrying out
activity assays of PKC-.zeta. and/or PKC-.tau. protein are known in
the art and are within the purview of the invention.
[0108] A reference value corresponding to the level of mRNA or
protein for PKC-.zeta. and/or PKC-.tau. indicates the level of mRNA
or protein for PKC-.zeta. and/or PKC-.tau. in the colon cancer
cells of a subject that is responsive or non-responsive to a colon
cancer therapy with an inhibitor of PKC-.zeta. and/or PKC-.tau.. As
such, a reference value corresponding to the level of mRNA or
protein for PKC-.zeta. and/or PKC-.tau. may be indicative of the
subject being responsive or non-responsive to a colon cancer
therapy with an inhibitor of PKC-.zeta. and/or PKC-.tau..
[0109] To practice the methods of the subject invention described
herein for identifying a subject as being responsive or
non-responsive to a melanoma therapy with an inhibitor of
PKC-.zeta. and/or PKC-.tau., a control sample can be one or more of
the following: [0110] a) normal colon epithelial cells from an
individual belonging to the same species as the subject and not
having colon cancer, [0111] b) normal colon epithelial cells from
the subject prior to getting melanoma, [0112] c) normal colon
epithelial cells from the subject not affected by colon cancer,
[0113] d) colon cancer cells from an individual known to be
responsive to a colon cancer therapy with an inhibitor of
PKC-.zeta. and/or PKC-.tau., or [0114] e) colon cancer cells from
an individual known to be non-responsive to a colon cancer therapy
with an inhibitor of PKC-.zeta. and/or PKC-.tau..
[0115] Additional examples of control samples that can be used in
the invention can be designed by a person of ordinary skill in the
art and such embodiments are within the purview of the
invention.
[0116] In certain embodiments, the subject used in the invention is
a mammal. Non-limiting examples of mammals include human, ape,
canine, pig, bovine, rodent, or feline.
[0117] Once a subject is identified as being responsive to a colon
cancer treatment with an inhibitor of PKC-.zeta. and/or PKC-.tau.
based on the methods of the subject invention, the steps of
treating and/or managing colon cancer include administering to the
subject one or more inhibitors of PKC-.zeta. and/or PKC-.tau.
described herein.
[0118] In preferred embodiments, a colon cancer treatment with an
inhibitor of PKC-.zeta. and/or PKC-.tau. is administered to the
subject in combination with one or more additional colon cancer
therapies. Such additional melanoma therapies can be therapies that
do not inhibit PKC-.zeta. and/or PKC-.tau., a surgery, a radiation
therapy, an immunotherapy or a combination thereof.
[0119] In other preferred embodiments, two inhibitors of inhibitor
of PKC-.zeta. and/or PKC-.tau. are administered to the subject in
combination.
[0120] In further preferred embodiments, the two inhibitors of
PKC-.zeta. and/or PKC-.tau. that are administered to the subject in
combination are administered in a way that each inhibitor is
administered at a sub-therapeutic level.
[0121] Once a subject is identified as being non-responsive to a
colon cancer treatment with an inhibitor of PKC-.zeta. and/or
PKC-.tau. based on the methods described herein, the step of
treating and/or managing colon cancer includes administering to the
subject one or more colon cancer therapies other than an inhibitor
of PKC-.zeta. and/or PKC-.tau.. Examples of colon cancer therapies
other than an inhibitor of PKC-.zeta. and/or PKC-.tau. include a
surgery, a radiation therapy, an immunotherapy or a combination
thereof.
[0122] In specific preferred embodiments, the subject invention
provides a novel atypical PKC inhibitor that is specific to PKC-Q.
In further preferred embodiments, the novel atypical PKC inhibitor
specific to PKC-.zeta. is 8-hydroxy-1,3,6-naphthalenetrisulfonic
acid (.zeta.-Stat). Advantageously, the novel PKC-.zeta. specific
inhibitor can decrease the levels of total and phosphorylated
PKC-.zeta. without affecting PKC-.tau.. In preferred embodiments of
the subject invention, the PKC-.zeta. specific inhibitor
.zeta.-Stat inhibits cellular signaling pathways that are involved
in endothelial-to-mesenchymal transition (EMT) of colon cancer
cells. Further advantageously, the PKC-.zeta. specific inhibitor
.zeta.-Stat effectively suppresses PKC-.zeta. mediated EMT and
induces apoptosis in colon cancer cells.
[0123] In further embodiments, methods of inhibiting colon cancer
proliferation are provided. In preferred embodiments, the methods
comprise contacting the colon cancer cells with a therapeutically
effective amount of an inhibitor of an aPKC, for example, PKC-.tau.
and PKC-.zeta..
[0124] In some embodiments, the PKC inhibitor administered to
inhibit colon cancer proliferation is specific to a single aPKC,
for example, PKC-.tau.. In a preferred embodiment, the specific
inhibitor of PKC-.tau. is ICA-1.
[0125] In other embodiments, the PKC inhibitor administered to
inhibit colon cancer proliferation is a pan-aPKC inhibitor. In
preferred embodiments, the pan-aPKC inhibitor is ACPD. In some
embodiments, the pan-aPKC inhibitor is DNDA.
[0126] In preferred embodiments, the PKC inhibitor administered to
inhibit colon cancer proliferation is specific to a single aPKC,
for example, PKC-.tau.. In some preferred embodiments, the specific
aPKC inhibitor is ICA-1.
[0127] In further preferred embodiments, the PKC inhibitor
administered to inhibit colon cancer proliferation is specific to
PKC-.zeta.. In certain preferred embodiments, the specific
inhibitor of PKC-.zeta. is .zeta.-Stat.
[0128] An inhibitor of an aPKC useful in the methods of the
invention can inhibit PKC-.zeta. and/or PKC-.tau.. Non-limiting
examples of inhibitors of PKC-.zeta. and/or PKC-.tau. useful in the
methods of the instant invention include, but are not limited to,
ACPD, DNDA, ICA-1, and .zeta.-Stat.
[0129] All numerical designations, such as pH, temperature, time,
concentration, and molecular weight, including ranges, are
approximations which are varied up or down by increments of 1.0 or
0.1, as appropriate. It is to be understood, even if it is not
always explicitly stated that all numerical designations are
preceded by the term "about". It is also to be understood, even if
it is not always explicitly stated, that the reagents described
herein are merely exemplary and that equivalents of such are known
in the art and can be substituted for the reagents explicitly
stated herein.
[0130] The term "about" or "approximately" as used herein refers to
being within an acceptable error range for the particular value as
determined by one of ordinary skill in the art, which will depend
in part on how the value is measured or determined, i.e. the
limitations of the measurement system, i.e. the degree of precision
required for a particular purpose, such as a pharmaceutical
formulation. In some embodiments, the term "about" represents up to
.+-.15%.
[0131] The composition of the subject invention can comprise a
pharmaceutically acceptable carrier and/or excipient comprising
substances, such as an inert vehicle, or pharmaceutical acceptable
adjuvants, and can contain preservatives and additives usual for
injection solutions, such as stabilizing agents, salts or saline,
and/or buffers etc. Examples pharmaceutically acceptable substances
are well known to a person of ordinary skill in the art and such
embodiments are within the purview of the current invention.
[0132] The pharmaceutical composition may be a liquid formulation
or a solid formulation. When the pharmaceutical composition is a
solid formulation it may be formulated as a tablet, a sucking
tablet, a chewing tablet, a chewing gum, a capsule, a sachet, a
powder, a granule, a coated particle, a coated tablet, an
enterocoated tablet, an enterocoated capsule, a melting strip or a
film. When the pharmaceutical composition is a liquid formulation
it may be formulated as an oral solution, a suspension, an emulsion
or syrup. Said composition may further comprise a carrier material
independently selected from, but not limited to, the group
consisting of lactic acid fermented foods, fermented dairy
products, resistant starch, dietary fibers, carbohydrates,
proteins, and glycosylated proteins.
[0133] Pharmaceutical compositions, as disclosed herein, can be
formulated in accordance with standard pharmaceutical practice
(see, e.g., Remington: The Science and Practice of Pharmacy (20th
ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000
and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick
and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a
person skilled in the art. Pharmaceutical composition according to
the invention may also be formulated to release active agents
substantially immediately upon administration or at any
predetermined time or time period after administration.
[0134] Exemplary modes of administration include oral, rectal,
transmucosal, topical, transcervical, transuterine, transurethral,
transdermal, inhalation, parenteral (e.g., intravenous,
subcutaneous, intradermal, intramuscular, and intraarticular)
administration. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions. Alternatively, one may administer the composition of the
subject invention in a local rather than systemic manner, for
example, in a depot or sustained-release formulation.
MATERIALS AND METHODS
Cell Lines and Subculture:
[0135] The colon cancerous cell line, HT-29 and normal colon
epithelial cells, CCD18CO were obtained from American Type Tissue
Culture Collection. The HT-29 cells and CCD18CO cells were
subculture and maintained in T75 flasks containing McCoy's media
and Eagle's Minimum Essential Media respectively, both of the
flasks were supplemented with 10% FBS (Fetal Bovine Serum) and 1%
antibiotics (Penicillin 10 U/ml and streptomycin 10 mg/ml). Cells
were incubated at 37.degree. C. and 5% C02. After few days of
incubation following subculture, cells were used at 70-80%
confluency for the experiment.
Antibodies and Reagents:
[0136] .zeta.-Stat or Compound 50 or
8-hydroxy-1,3,6-naphthalenetrisulfonic acid was purchased from
National Cancer Institute (NCI). It was dissolved in sterile water
before use. PKC-.tau. antibody was purchased from BD Biosciences
(San Jose, Calif.). The antibodies to Phospho PKC-.zeta. (T410),
phosphor AKT (S473), Caspase-3, Cleaved Caspase-3 and BCL-XL were
purchased from Cell signaling Technology (Danvers, Mass.) The
antibodies purchased from Thermo Fisher Scientific were PKC-.zeta.,
.beta.-actin and survivin, phospho PKC-.tau. (T555) was purchased
from Invitrogen Inc. Enhanced Chemiluminescence (Super Signal West
Pico Chemiluminescent Substrate) was Purchased from Pierce
(Rockford, Ill.). McCOY'S 5A medium was purchased from GE
Healthcare Life Sciences (South Logan, Utah), Eagle's minimum
essential medium was procured from Corning (Manassas, Va.) and
Trypsin-EDTA (ethylene diamine tetra-acetic acid) solution was
obtained from Fisher Scientific (Norcross, Ga.).
In-Vitro Treatment of HT-29 Colon Cancer Cell with DNDA, ACPD,
ICA-1, and .zeta.-Stat:
[0137] Cells (3.0.times.10.sup.4/well) were grown as monolayer on 6
wells plate. After incubation for 24 hrs, the cells were treated
with 0.25 .mu.M, 0.50 .mu.M, 0.75 .mu.M, 1.0 .mu.M, 1.5 .mu.M, 2
.mu.M, and 3 .mu.M of ACPD, ICA-1, DNDA or .zeta.-Stat for three
consecutive days. Cells are then washed (2 ml 2.times. DPBS
buffer), trypsinized, resuspended in McCoy's 5A media and counted
with hemocytometer, Scepter & MTT assay.
Scratch Wound Healing Assay:
[0138] Cells were plated into 6-well plates, at approximately 90%
confluent and a line scratched across the cell monolayer using a
100-.mu.L pipette tip. Cells were treated using 0.5 .mu.M, 1.5
.mu.M and 3.0 .mu.M of DNDA, ACPD and ICA-1. The cells that moved
into the interspace of the wound line were counted at 24 hrs, 48
hrs and 72 hrs intervals using a phase contrast microscope (Motic,
China). This assay was repeated 3 times.
Immunoblot Analysis:
[0139] Cells were cultured (9.0.times.10.sup.4) in 100 mm cell
culture flasks. After incubation for 24 hours (to get least 50%
confluency), fresh media was supplied and each experimental flasks
were treated with 0.5 .mu.M, 0.75 .mu.M, 1.0 .mu.M, 1.5 .mu.M, 2.0
.mu.M, 3.0 .mu.M, 5.0 .mu.M and 7.0 .mu.M with .zeta.-Stat, ICA-I,
ACPD, and DNDA. Equal volume of sterile water (vehicle control) as
the volume of drug was added in the control also. Treatment was
continued for three consecutive days. Total cell-lysates were
prepared and subjected to western blot analysis for aPKC
analysis.
[0140] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0141] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Example 1: Effect of Atypical PKC Inhibitors on Colon Cancer
[0142] FIG. 1 shows an image of the development of colorectal
polyps which can lead to cancer over time. FIG. 2 shows an image of
the different stages of colorectal cancer. Colorectal cancer (CRC)
is the development of cancer in the colon or rectum due to the
abnormal growth of cells that have the ability to invade or spread
to other parts of the body [4]. Colorectal cancer often begins as a
growth called a polyp, which may form on the inner wall of the
colon or rectum (FIG. 1.1). Over time, polyps may become malignant
[2]. Colorectal cancers are mostly adenocarcinomas.
[0143] A comparison of the domain structures of protein kinase C
(PKC) isoforms including conventional PKCs (cPKCs), novel PKCs
(nPKCs) and atypical PKCs (aPKCs) indicates the similarities
between the kinase domains and the hydrophobic motif, the turn
motif and the activation loop of the kinase core (FIG. 3). PKCs
have a conserved kinase domain (depicted in teal) and more variable
regulatory domains. All PKC regulatory domains have an
auto-inhibitory pseudosubstrate motif (shown in green) and an
NH.sub.2 terminal to the C1 domain (shown in pink). Tandem C1
domains are the molecular sensors of phorbol 12-myristate
13-acetate (PMA)/diacylglycerol (DAG) in cPKC and nPKC isoforms,
whereas the single aPKC C1 domain does not bind DAG/PMA. The C2
domains (in yellow) function as calcium-dependent phospholipid
binding modules in cPKCs. nPKC C2 domains do not bind calcium; the
PKC.delta.-C2-like domain is a phosphotyrosine interaction module.
PKC isoform variable regions are shown in gray. Bottom: ribbon
diagrams of PKC C1B domain, C2 domain, and kinase domain structures
[14]. After being freed from PS-dependent auto-inhibition, some
lipids play important roles. In case of conventional and novel PKCs
some membrane lipids such as phosphatidylserine, and lipid
metabolites like diacylglycerol (DAG) cause release of
pseudosubstrate from the active sites and resulting in
phosphorylation of PKC substrate proteins [38].
[0144] FIG. 4 shows a schematic representation of the domain
structures of aPKC. The N-terminus of the aPKC has a PB1 domain
required for protein-protein interaction with MEK5, PAR-6 and p62,
a pseudosubstrate, a C1 domain and a Ser/Thr kinase (also called
catalytic) domain in the C-terminus. The kinase domain has ATP
binding site, activation loop, turn motif and hydrophobic motif.
Thr-403 in PKC-.tau. and Thr-410 in PKC-.zeta. in the activation
loop need to be phosphorylated (possibly by PDK-1 or Akt) in order
to activate the enzymes [37].
[0145] Between atypical PKCs, PKC-.tau. is proven to be a potential
oncogene in many cancers whereas the role of the highly homologous
aPKC PKC-.zeta. has not explored in terms of carcinogenesis. In
this study, a novel specific PKC-.zeta. inhibitor called
.zeta.-Stat or Compound 50 or
8-hydroxy-1,3,6-naphthalenetrisulfonic acid is used to assess the
role of this particular PKC enzyme in colon carcinogenesis. We
found that the inhibition of PKC-.zeta. by .zeta.-Stat specifically
reduces the risk of colon carcinogenesis. Therefore, it might be
used as a potential chemotherapeutic agent for cancer
treatment.
Example 2: The Effects of Atypical PKC Inhibitor ACPD on a Colon
Cancer Cell Line HT-29
[0146] To determine the understanding behind the complex mechanisms
involved the inventors investigated multiple inhibitors of aPKC:
ACPD and DNDA are pan-aPKC inhibitors and ICA-1 as an inhibitor of
PKC-.tau. in colon cancer cell lines. ACPD is an inhibitor of
atypical PKC-.tau. and PKC-.zeta.. PKC-.tau. is a phosphorylating
protein that plays a role in mediating apoptosis. PKC-.zeta. has
been shown to contribute to survival of some cancer cells. Both
ICA-1 and ACPD are inhibitors of PKC-.tau. and ACPD inhibits both
PKC-.tau. and PKC-.zeta.. DNDA, similar to ACPD, is a pan aPKC
inhibitor, inhibiting PKC-.zeta. by about 80% and PKC-.tau. by
about 20%.
[0147] HT-29 cells were treated with five different concentrations
of ACPD for 24-72 hours. After starting treatment, viable cells
were counted at each 24 hours interval using Tryphan blue exclusion
assay and scepter. The data represents n=4 independent
experiments.
[0148] ACPD decreased cell proliferation in a dose-dependent manner
over an exposure time of 4 days (FIG. 5). Within the first three
days of treatment, the number of HT-29 cells decreased as the
concentration of ACPD increased. As shown in FIG. 6, after three
days post treatment, HT-29 cells had a slight decrease in
proliferation at 0.25 .mu.M and a dramatic decrease in
proliferation at 1.5 .mu.M concentration of ACPD. The percentage of
inhibition of the HT-29 colon cancer cells by ACPD increased as a
function of ACPD; a decrease of growth of HT-29 cells by 50 percent
(half, IC.sub.50) occurred at approximately 1.0 .mu.M. (FIG.
7).
Example 3: The Effects of Atypical PKC Inhibitor DNDA on a Colon
Cancer Cell Line HT-29
[0149] HT-29 cells were treated with five different concentrations
of DNDA for 24-72 hours. After starting treatment, viable cells
were counted at each 24 hours interval using Tryphan blue exclusion
assay and scepter. The data represents n=4 independent
experiments.
[0150] DNDA acts in a similar manner to decrease colon cancer cell
proliferation in a dose-dependent manner as illustrated in FIGS.
8-10. The number of HT-29 cells decreased as the concentration of
DNDA increased over time (FIG. 8). After three days post treatment,
HT-29 cells had a slight decrease in proliferation at 0.25 .mu.M of
DNDA and a dramatic decrease in proliferation at 1.5 .mu.M
concentration of DNDA (FIG. 9). The percentage of inhibition of the
HT-29 colon cancer cells by DNDA increased as a function of DNDA; a
decrease of growth of HT-29 cells by 50 percent (half, IC.sub.50)
occurred at approximately 0.78 .mu.M (FIG. 10).
Example 4: The Effects of Atypical PKC Inhibitor ICA-1 on a Colon
Cancer Cell Line HT-29
[0151] HT-29 cells were treated with five different concentrations
of ICA-1 for 24-72 hours. After starting treatment, viable cells
were counted at each 24 hours interval using Tryphan blue exclusion
assay and scepter. The data represents n=4 independent
experiments.
[0152] ICA-1 also acts in a similar manner to decrease colon cancer
cell proliferation in a dose-dependent manner as illustrated in
FIGS. 11-13. The number of HT-29 cells decreased as the
concentration of ICA-I increased over time (FIG. 11). After three
days of post treatment, ICA-1 caused a slight increase in cell
proliferation at 0.25 .mu.M and a dramatic decrease in cell
proliferation at 1.5 .mu.M of ICA-1 (FIG. 12). The percentage of
inhibition of the HT-29 colon cancer cells increased as a function
of ICA-1; a decrease of growth of HT-29 cells by 50 percent (half,
IC.sub.50) occurred at approximately 0.95 .mu.M (FIG. 13).
[0153] Both ACPD and ICA-1 showed a slight increase in cell
proliferation at concentrations of 0.25 .mu.M while at this same
concentration DNDA showed a slight decrease. At low concentrations
ACPD and ICA-1 may be inhibiting phosphorylation of a tumor
suppressor and at higher concentrations ACPD and ICA-1 inhibit
phosphorylation of other substrates. Given the results from the
studies, each drug should be administered at a minimum
concentration of at least the IC.sub.50 of the respective drug.
These results indicate the therapeutic potential of aPKC inhibitors
for the treatment of colon cancer.
Example 5: The Effects of Atypical PKC Inhibitor .zeta.-Stat on a
Colon Cancer Cell Line HT-29
[0154] HT-29 cells were treated with four different concentrations
of .zeta.-Stat for 24-72 hours. After starting treatment, viable
cells were counted at each 24 hours interval using Tryphan blue
exclusion assay and scepter. The data represents n=4 independent
experiments.
[0155] .zeta.-Stat inhibits colon cancer cell proliferation in a
dose-dependent manner. The number of HT-29 cells decreased as the
concentration of .zeta.-Stat increased over time (FIG. 14). After
three days of post treatment, .zeta.-Stat caused a dramatic
decrease in cell proliferation at 1.5 .mu.M of .zeta.-Stat (FIG.
15). The percentage of inhibition of the HT-29 colon cancer cells
increased as a function of .zeta.-Stat; a decrease of growth of
HT-29 cells by 50 percent (half, IC.sub.50) occurred at
approximately 1.4 .mu.M (FIG. 15). The results demonstrate that the
.zeta.-Stat is capable of inhibiting the HT-29 cells by about 60%
upon treatment over three days (FIG. 15).
Example 6: The Effects of Atypical PKC Inhibitor .zeta.-Stat on
aPKC Expression and Phosphorylation in a Colon Cancer Cell Line
HT-29
[0156] Because PKC-.tau. and PKC-.zeta. are homologous (84%) in
terms of amino acids sequence of their catalytic domain [7], the
specificity of the effect of .zeta.-Stat on PKC-.zeta. was tested
by performing western blot analysis. HT-29 colorectal cancerous
cells were treated with seven different concentrations of
.zeta.-Stat for 72 hours, harvested and whole cell extracts were
prepared. Equal amounts (40 .mu.g) of protein were loaded on
SDS-PAGE followed by immunoblotting for pPKC-.tau.(T555),
PKC-.tau., pPKC-.zeta.(T410), PKC.zeta., pAKT(S473) and
.beta.-actin to examine the effect of .zeta.-Stat on those protein
levels. Data represents n=3 independent observations. A selective
decrease in the phosphorylation of PKC-.zeta. was observed upon
increasing concentrations of .zeta.-Stat. The results suggest that
the .zeta.-Stat specifically inhibits PKC-.zeta. not PKC-.tau.
(FIG. 16).
Example 7: The Effects of Atypical PKC Inhibitors ICA-1 and
.zeta.-Stat on HT-29 Colorectal Cancer Cell Survival
[0157] HT-29 colorectal cancer cells were treated with 7 .mu.M
ICA-I or 7 .mu.M .zeta.-Stat for 72 hours, harvested and whole cell
lysates were prepared. Equal amounts (40 .mu.g) of protein were
loaded on SDS-PAGE followed by immunoblotting for Caspase-3,
BCL-XL, Survivin and 3-actin to determine the effects of specific
aPKC inhibition on survival and apoptotic proteins. Data represents
n=3 independent experiments. The results suggest that .zeta.-Stat
also decreases the level of pro-survival proteins (FIG. 17).
Example 8: The Effects of Atypical PKC Inhibitor .zeta.-Stat on
Normal Colon Epithelial Cell Proliferation
[0158] To establish the efficacy of .zeta.-Stat as a specific
inhibitor of PKC.zeta., the effect of .zeta.-Stat on CCD18CO normal
colon epithelial cell line was tested. CCD18CO normal epithelial
cell line cells were treated with four different concentrations of
.zeta.-Stat for three consecutive days followed by incubation with
MTT reagent and taking absorbance reading at each half an hour
interval for three hours at 577 nm using microplate reader. The
data represents n=3 independent experiments. The results indicate
that .zeta.-Stat is not toxic to the normal epithelial (FIGS. 17
and 18).
Example 9: The Effects of Atypical PKC Inhibitor .zeta.-Stat on
Normal Colon Epithelial Cell Growth
[0159] The percentage of inhibition of CCD18CO cell growth was
measured after three hours of MTT incubation. The percent
inhibition of cells growth was less than 10% which is statistically
insignificant. The data represents n=3 independent replicates (FIG.
19).
Example 10: The Effects of Atypical PKC Inhibitor .zeta.-Stat on
Normal Colon Epithelial Cell Survival
[0160] CCD18CO cells were treated with 7 .mu.M ICA-I or 7 .mu.M
.zeta.-Stat, respectively, for 72 hours, harvested and whole cell
lysates were prepared. Equal amounts (40 .mu.g) of protein were
loaded on SDS-PAGE followed by immunoblotting for PKC-.tau.,
PKC.zeta., Caspase-3, BCL-XL, and .beta.-actin to see the effect of
specific aPKC inhibition on survival and apoptotic proteins. Data
represents n=3 independent experiments. The results indicate that
ICA-1 and .zeta.-Stat are not toxic to normal epithelial cells even
at concentrations of 7 .mu.M (FIG. 20).
[0161] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
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[0228] In the preceding specification, all documents, acts, or
information disclosed does not constitute an admission that the
document, act, or information of any combination thereof was
publicly available, known to the public, part of the general
knowledge in the art, or was known to be relevant to solve any
problem at the time of priority.
[0229] The disclosures of all publications cited above are
expressly incorporated herein by reference, each in its entirety,
to the same extent as if each were incorporated by reference
individually. Furthermore, where a definition or use of a term in a
reference, which is incorporated by reference herein, is
inconsistent or contrary to the definition of that term provided
herein, the definition of that term provided herein applies and the
definition of that term in the reference does not apply.
[0230] The advantages set forth above, and those made apparent from
the foregoing description, are efficiently attained. Since certain
changes may be made in the above construction without departing
from the scope of the invention, it is intended that all matters
contained in the foregoing description or shown in the accompanying
drawings shall be interpreted as illustrative and not in a limiting
sense.
[0231] While there has been described and illustrated specific
embodiments of the invention, it will be apparent to those skilled
in the art that variations and modifications are possible without
deviating from the broad spirit and principle of the present
invention. It is also to be understood that the following claims
are intended to cover all of the generic and specific features of
the invention herein described, and all statements of the scope of
the invention which, as a matter of language, might be said to fall
therebetween.
[0232] It is also to be understood that the following claims are
intended to cover all of the generic and specific features of the
invention herein described, and all statements of the scope of the
invention which, as a matter of language, might be said to fall
there between. Now that the invention has been described,
* * * * *
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