U.S. patent application number 13/821725 was filed with the patent office on 2016-04-21 for use of pkc-iota inhibitors for the treatment of breast cancer.
This patent application is currently assigned to UNIVERSITY OF SOUTH FLORIDA. The applicant listed for this patent is Mildred Enid Acevedo-Duncan, Diondra Denise Hill, David A. Ostrov. Invention is credited to Mildred Enid Acevedo-Duncan, Diondra Denise Hill, David A. Ostrov.
Application Number | 20160106767 13/821725 |
Document ID | / |
Family ID | 45832290 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160106767 |
Kind Code |
A9 |
Acevedo-Duncan; Mildred Enid ;
et al. |
April 21, 2016 |
USE OF PKC-IOTA INHIBITORS FOR THE TREATMENT OF BREAST CANCER
Abstract
The subject invention pertains to uses of PKC-iota inhibitors
for treatment of breast cancer. In one embodiment, the subject
invention provides novel uses of 1H-imidazole-4-carboxamide,
5-amino-1-[2,3 -dihydroxy-4-[(phosphonooxy)
methyl]cyclopentyl]-,[1R-(1.alpha., 2.beta., 3.beta., 4.alpha.)]
(ICA-1) and related compounds for treatment of breast cancer. The
compounds of the subject invention have potent anti-proliferative
effects against human breast cancer cells. The compounds of the
subject invention also inhibit the phosphorylation of
IKK-.alpha./IKK-.beta., induce chromatin condensation, and/or
induce DNA fragmentation in cancer cells.
Inventors: |
Acevedo-Duncan; Mildred Enid;
(Plant City, FL) ; Hill; Diondra Denise; (Tampa,
FL) ; Ostrov; David A.; (Gainesville, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Acevedo-Duncan; Mildred Enid
Hill; Diondra Denise
Ostrov; David A. |
Plant City
Tampa
Gainesville |
FL
FL
FL |
US
US
US |
|
|
Assignee: |
UNIVERSITY OF SOUTH FLORIDA
Tampa
FL
THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF
VETERANS AFFAIRS
Washington
DC
UNIVERSITY OF FLORIDA RESEARCH FOUNDATION INC.
Gainesville
FL
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130189257 A1 |
July 25, 2013 |
|
|
Family ID: |
45832290 |
Appl. No.: |
13/821725 |
Filed: |
September 19, 2011 |
PCT Filed: |
September 19, 2011 |
PCT NO: |
PCT/US2011/052147 PCKC 00 |
371 Date: |
April 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12210576 |
Sep 15, 2008 |
8461192 |
|
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13821725 |
|
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61384023 |
Sep 17, 2010 |
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60993814 |
Sep 13, 2007 |
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Current U.S.
Class: |
424/134.1 ;
424/139.1; 424/158.1; 514/44A; 514/94 |
Current CPC
Class: |
A61K 31/66 20130101;
A61K 39/39558 20130101; A61K 31/675 20130101; C12N 2310/14
20130101; A61K 31/7088 20130101; A61P 35/00 20180101; C12N 15/1137
20130101; A61K 31/4164 20130101; C12Y 207/11013 20130101 |
International
Class: |
A61K 31/675 20060101
A61K031/675; A61K 31/7088 20060101 A61K031/7088; A61K 39/395
20060101 A61K039/395 |
Claims
1. A method of treating breast cancer or tumor comprising
administering, to a subject in need of such treatment, an effective
amount of a PKC-iota inhibitor, and, optionally, a pharmaceutical
acceptable carrier.
2. The method according to claim 1, wherein the subject is a
human.
3. The method according to claim 1, wherein the PKC-iota inhibitor
is selected from: a) a compound of Formula (I) or a salt thereof,
wherein the compound of formula (I) is: ##STR00004## wherein
R.sub.1 and R.sub.2 are, independently, --NH.sub.2 or alkylamino,
and wherein R.sub.3 and R.sub.4 are, independently, --H, --OH,
alkoxy, or --OC(O)R', wherein R' is a linear saturated monovalent
radical of one to eight carbon atoms or a branched saturated
monovalent of three to eight carbon atoms; b) an antibody, aptamer,
or binding partner that binds specifically to PKC-, or a fusion
construct comprising said antibody, aptamer, or binding partner; or
c) an antisense polynucleotide that specifically targets PKC-
mRNA.
4. The method of claim 3, wherein the PKC-iota inhibitor is a
compound of Formula (I), or a salt thereof.
5. The method of claim 4, wherein the PKC-iota inhibitor is
(1H-imidazole-4-carboxamide,5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)met-
hyl]cyclopentyl]-,[1R-(1.alpha., 2.beta., 3.beta., 4.alpha.)]
(ICA-1), or a salt thereof.
6. The method of claim 3, wherein the PKC-iota inhibitor is an
antibody that binds specifically to a PKC- polypeptide comprising
an amino acid sequence having at least 90% identity to SEQ ID NO:1,
wherein the PKC- polypeptide comprises a catalytic domain that is
amino acid residues 469-475 of SEQ ID NO:1 (glutamine-469,
isoleucine-470, arginine-471, isoleucine-472, proline-473,
arginine-474, serine- 475).
7. The method of claim 6, wherein the PKC- polypeptide comprises
SEQ ID NO:1.
8. The method of claim 3, wherein the PKC-iota inhibitor is an
antisense polynucleotide that specifically targets PKC- mRNA,
wherein the PKC- mRNA encodes a PKC- polypeptide comprising an
amino acid sequence having at least 90% identity to SEQ ID NO:1,
wherein the PKC- polypeptide comprises a catalytic domain that is
amino acid residues 469-475 of SEQ ID NO:1 (glutamine-469,
isoleucine-470, arginine-471, isoleucine-472, proline-473,
arginine-474, serine- 475).
9. The method of claim 8, wherein the PKC- polypeptide comprises
SEQ ID NO:1.
10. The method of claim 1, wherein the subject has breast
cancer.
11. The method of claim 10, wherein the subject has ductal
carcinoma in-situ (DCIS), invasive ductal carcinoma (IDC), lobular
carcinoma in-situ (LCIS), invasive lobular carcinoma (LCIS),
medullary carcinoma, malignant phyllode tumor, tubular carcinoma,
mucinous carcinoma, metastatic adenocarcinoma, or inflammatory
breast cancer.
12. The method of claim 11, wherein the subject has invasive ductal
carcinoma, invasive lobular carcinoma, or metastatic
adenocarcinoma.
13. A method of inhibiting proliferation of breast cancer or tumor
cells, comprising administering to the breast cancer or tumor cells
an effective amount of a PKC-iota inhibitor, and optionally, a
pharmaceutically acceptable carrier.
14. The method according to claim 13, wherein the PKC-iota
inhibitor is selected from: a) a compound of formula (I) or a salt
thereof, wherein the compound of formula (I) is: ##STR00005##
wherein R.sub.1 and R.sub.2 are, independently, --NH.sub.2 or
alkylamino, and wherein R.sub.3 and R.sub.4 are, independently,
--H, --OH, alkoxy, or --OC(O)R', wherein R' is a linear saturated
monovalent radical of one to eight carbon atoms or a branched
saturated monovalent of three to eight carbon atoms; b) an
antibody, aptamer, or binding partner that binds specifically to
PKC-, or a fusion construct comprising said antibody, aptamer, or
binding partner; or c) an antisense polynucleotide that
specifically targets PKC- mRNA.
15. The method of claim 14, wherein the PKC-iota inhibitor is a
compound of Formula (I), or a salt thereof.
16. The method of claim 15, wherein the PKC-iota inhibitor is
(1H-imidazole-4-carboxamide, 5-amino-1-[2,3
-dihydroxy-4-[(phosphonooxy) methyl]cyclopentyl][1R-(1.alpha.,
2.beta., 3.beta., 4.alpha.)] (ICA-1), or a salt thereof.
17. The method of claim 14, wherein the PKC-iota inhibitor is an
antibody that binds specifically to a PKC- polypeptide comprising
an amino acid sequence having at least 90% identity to SEQ ID NO:1,
wherein the PKC- polypeptide comprises a catalytic domain that is
amino acid residues 469-475 of SEQ ID NO:1 (glutamine-469,
isoleucine-470, arginine-471, isoleucine-472, proline-473,
arginine-474, serine- 475).
18. The method of claim 14, wherein the PKC-iota inhibitor is an
antisense polynucleotide that specifically targets PKC- mRNA,
wherein the PKC- mRNA encodes a PKC- polypeptide comprising an
amino acid sequence having at least 90% identity to SEQ ID NO:1,
wherein the PKC- polypeptide comprises a catalytic domain that is
amino acid residues 469-475 of SEQ ID NO:1 (glutamine-469,
isoleucine-470, arginine-471, isoleucine-472, proline-473,
arginine-474, serine- 475).
19. The method of claim 13, wherein the breast cancer or tumor
cells are of human origin.
20. The method of claim 19, wherein the breast cancer or tumor
cells are selected from non-invasive ductal carcinoma cells,
invasive ductal carcinoma cells, non-invasive lobular carcinoma
cells, invasive lobular carcinoma cells, medullary carcinoma cells,
malignant phyllode tumor cells, tubular carcinoma cells, mucinous
carcinoma cells, metastatic adenocarcinoma cells, or inflammatory
breast cancer cells.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
application Ser. No. 61/384,023, filed Sep. 17, 2010, which is
herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Breast cancer is the most common female malignancy and the
leading cause of cancer-related death among women. The 2009 cancer
statistics estimated that 181,000 new cases of invasive breast
cancer were diagnosed that year and would result in 46,300 new
deaths. In North America, breast cancer accounts for about 27% of
all female cancers and 15% -20% of all female cancer mortalities.
While advancement in the diagnosis and treatment of breast cancer
has prolonged patient survival, alternative therapeutic agents for
treatment of breast cancer are needed.
[0003] Protein kinase C-iota (PKC-), an atypical protein kinase C
isozyme, plays an essential role in the growth, proliferation and
survival of many types of cancer cells. PKC- has been shown to
promote cell survival in ovarian cancer, non-small cell lung cancer
and prostate cancer. However, PKC- has not previously been reported
to play any role in breast cancer. It also remains unknown whether
agents that inhibit PKC- activity would have any effect on the
treatment of breast cancer.
BRIEF SUMMARY OF THE INVENTION
[0004] The subject invention provides use of PKC-iota (PKC-)
inhibitors for treatment of breast cancer. PKC- inhibitors useful
according to the subject invention include, but are not limited to,
agents that inhibit PKC- activity; and agents that reduce or
inhibit the expression of PKC-iota.
[0005] In certain embodiments, the subject invention can be used to
treat or ameliorate breast cancer including, but not limited to,
ductal carcinoma in-situ (DCIS), invasive ductal carcinoma (IDC),
lobular carcinoma in-situ (LCIS), invasive lobular carcinoma
(LCIS), medullary carcinoma, malignant phyllode tumor, tubular
carcinoma, mucinous carcinoma, metastatic adenocarcinoma, and
inflammatory breast cancer.
[0006] In one embodiment, the subject invention pertains to novel
uses of 1H-imidazole-4-carboxamide,
5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl]-[1R-(1.alph-
a., 2.beta., 3.beta., 4.alpha.)] (ICA-1) and related compounds for
treatment of breast cancer. Surprisingly, the compounds of the
subject invention have potent anti-proliferative effects against
human breast cancer cells. The compounds of the subject invention
also inhibit the phosphorylation of IKK-.alpha./IKK-.beta., induce
chromatin condensation, and/or induce DNA fragmentation in cancer
cells.
[0007] In an embodiment, the subject invention provides a method
for treating breast cancer, comprising administering to a subject
in need of such treatment an effective amount of a compound of
Formula I:
##STR00001##
[0008] wherein R.sub.1 and R.sub.2 are, independently, --NH.sub.2
or alkylamino, and
[0009] wherein R.sub.3 and R.sub.4 are, independently, --H, --OH,
alkoxy, or --OC(O)R',
[0010] wherein R' is a linear saturated monovalent radical of one
to eight carbon atoms or a branched saturated monovalent of three
to eight carbon atoms.
[0011] In an embodiment, the subject invention further comprises
administering to the subject a second therapeutic agent including,
but not limited to, taxanes, e.g., paclitaxel (TAXOL, BRISTOL-MYERS
SQUIBB Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE,
Rhone-Poulenc Rorer, Antony, France), chlorambucil, vincristine,
vinblastine, gemcitabine, ixabepilone, doxorubicin, anti-estrogens
such as tamoxifen and raloxifene, aromatase inhibitors such as
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and toremifene (Fareston), and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin, and immuno-modulating agents.
BRIEF DESCRIPTION OF THE SEQUENCES
[0012] SEQ ID NO: 1 is an amino acid sequence of human Protein
kinase C-iota (PKC-).
[0013] SEQ ID NO: 2 5'-CAAGCCAAGCGUUUCAACA-3' is a single strand of
PKC- siRNA.
[0014] SEQ ID NO: 3 5'-UGUUGAAACGCUUGGCUUG-3' is a single strand of
PKC- siRNA.
[0015] SEQ ID NO: 4 5'-GGAACGAUUGGGUUGUCAU-3' is a single strand of
PKC- siRNA.
[0016] SEQ ID NO: 5 5'-AUGACAACCCAAUCGUUUCC-3' is a single strand
of PKC- siRNA.
[0017] SEQ ID NO: 6 5'-CCCAAUAUCUUCUCUUGUA-3' is a single strand of
PKC- siRNA.
[0018] SEQ ID NO: 7 5'-UACAAGAGAAGAUAUUGGG3' is a single strand of
PKC- siRNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A-B show that PKC- is over-expressed in benign and
malignant breast biopsies. (A) Breast tissue biopsies (50 .mu.g)
were subjected to gel electrophoresis and Western blot analysis.
Western blot was performed using monoclonal antibodies against PKC-
(cat. # 610176, BD Transduction, San Diego, Calif..) at a 1:2000
dilution (5 .mu.g). Secondary antibodies obtained from Accurate
JOM035146, Westbury, N.Y.) were used at a 1.5:10000 dilution (48
.mu.g). To control equal loading of the proteins, .beta.-actin was
probed with goat polyclonal antibodies (SC-1616) at a 2.5:2000
dilution (10 .mu.g) and secondary antibodies (SC-2350) at a 1:2000
dilution (8 .mu.g, Santa Cruz Biotechnology). Du-145 cell lysates
(50 .mu.g) containing PKC- were used as positive control (not
shown). (B) PKC- is over-expressed in malignant breast cancer
tissue as compared to normal and benign breast tissue. Western blot
results from 5 normal breast tissue specimens, 10 benign
fibroadenoma tissue specimens and 11 malignant breast tissue
specimens were quantified by densitometry. The mean plus and minus
the standard error value is presented for each tissue type. Bars
represent means with standard deviations.
[0020] FIGS. 2A-B show the association of cell density with PKC-
concentration in MDA-MB-468 breast cancer cells. (A) shows the
expression of PKC isozymes in 100% and 50% confluent cells. 100%
and 50% confluent cells were harvested at the indicated times and
cell lysates were subjected to Western blot analysis. Equal amounts
of cellular protein (15 .mu.g) were loaded per well. (B) shows PKC-
levels in 50% confluent cells cultured with or without serum.
Independent triplicate total cell lysates from cells at 50%
confluence were grown in the presence or absence of serum for 24
and 48 hours.
[0021] FIGS. 3A-C show the effects of PKC- siRNA on the
proliferation of MDA-MB-468 breast cancer cells and PKC- protein
content. (A) shows that PKC- siRNA significantly reduced the
proliferation of breast cancer cells. Open symbols (O) represent
control (siRNA-A) treated cells. Solid symbols ( ) represent cells
treated with PKC- siRNA. (B) shows Western blot results of PKC-
protein content in cells treated with PKC- siRNA or control siRNA-A
(upper panel). Control .beta.-actin Western blots showed
.beta.-actin immuno-reactive bands at a molecular weight of 42 kD
(lower panel). Band intensity was quantified by densitometry
scanning. Molecular mass standard (kD) are shown on the left. Data
is representative of two independent experiments. (C) shows bar
graphs of PKC- protein content in cells treated with PKC- siRNA or
control siRNA-A at 24 hr, 48 hr and 72 hr post-treatment.
[0022] FIG. 4 shows the effects of the PKC- inhibitor ICA-1 on the
proliferation of MDA-MB-468 breast cancer cells. Five thousand
cells were plated in T-25 cm.sup.2 flasks. Twenty-four hours post
vehicle control or ICA-1 treatment, the number of viable cells was
counted by Trypan blue exclusion. Data shown are the mean .+-.SD of
two independent experiments.
[0023] FIG. 5 displays the molecular docking of ICA-1 on amino acid
residues 469-475 of the catalytic domain of PKC-.
DETAILED DISCLOSURE OF THE INVENTION
[0024] The subject invention provides use of PKC-iota (PKC-)
inhibitors for treatment of breast cancer. In one embodiment, the
method comprises administering, to a subject in need of such
treatment, an effective amount of a PKC-iota (PKC-) inhibitor. PKC-
inhibitors useful according to the subject invention include, but
are not limited to, agents that inhibit PKC- activity; and agents
that reduce or inhibit the expression of PKC-iota.
[0025] In one embodiment, the subject invention pertains to
therapeutic uses of
(1H-imidazole-4-carboxamide,5-amino-1-[2,3-dihydroxy-4-[(phosphon-
ooxy) ethyl]cyclopentyl], [1R-(1.alpha., 2.beta., 3.beta.,
4.alpha.)] (ICA-1) and related compounds for, in an embodiment,
treatment of breast cancer. In an embodiment, the method comprises
administering to a subject in need of such treatment an effective
amount of ICA-1 and/or related compounds, or any salts thereof. The
subject invention further provides therapeutic compositions that
contain a therapeutically effective amount of the compound of the
subject invention and a pharmaceutically acceptable carrier or
adjuvant.
[0026] Surprisingly, it has now been discovered that
(1H-imidazole-4-carboxamide,5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)
methyl]cyclopentyl]-,[1R-(1.alpha., 2.beta., 3.beta., 4.alpha.)]
(ICA-1), an inhibitor of protein kinase C-iota (PKC-), has potent
anti-proliferative effects against human breast cancer cells.
MDA-MB-468 breast cancer cells were treated with 0.1 .mu.M or 0.5
.mu.M ICA-1, and cell viability and cell count were determined 24
hours post-treatment. ICA-1 potently decreased the proliferation of
MDA-MB-468 cells by 77% (0.1 .mu.M) and 50% (0.5 .mu.M; P=0.05). In
addition, ICA-1 inhibits the phosphorylation of
IKK-.alpha./IKK-.beta..
[0027] The present inventors have also discovered that ICA-1 binds
to the catalytic domain of human PKC- (SEQ ID NO:1, GenBank
Accession No. AAB17011) at amino acid residues 469-475
(glutamine-469, isoleucine-470, arginine-471, isoleucine-472,
proline-473, arginine-474, serine- 475). The binding of ICA-1 to
PKC- potently inhibits the activity of PKC-, an oncogenic protein
kinase C (PKC) isozyme that plays a critical role in the
proliferation and survival of cancer cells.
[0028] In addition, the present inventors have discovered that
ICA-1 promotes apoptosis of cancer cells. Specifically, ICA-1
induced chromatin condensation in cancer cells, as shown by
4',6-diamidino-2-phenylindole (DAPI) staining of the nucleus. ICA-1
also induced DNA fragmentation in cancer cells, as indicated by 3'
terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick
end-labeling (TUNEL) assay.
[0029] In an embodiment, the subject invention pertains to
therapeutic use of
1H-imidazole-4-carboxamide,5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)
methyl] cyclopentyl]-,[1R-(1.alpha., 2.beta., 3.beta., 4.alpha.)]
(ICA-1), having the following structure:
##STR00002##
[0030] Based on the molecular docking of ICA-1 on amino acid
residues 469-475 of the catalytic domain of PKC- shown in FIG. 5,
the subject invention further contemplates compounds of Formula I,
which reduce or inhibit PKC- activity. The compounds of Formula I
include ICA-1 as well as ester, ether, and alkyl substituted
derivatives of ICA-1, having the following structure:
##STR00003##
[0031] wherein R.sub.1 and R.sub.2 are, independently, --NH.sub.2
or alkylamino, and
[0032] wherein R.sub.3 and R.sub.4 are, independently, --H, --OH,
alkoxy, or --OC(O)R',
[0033] wherein R' is a linear saturated monovalent radical of one
to eight carbon atoms or a branched saturated monovalent of three
to eight carbon atoms. [0034] "Alkyl" means linear saturated
monovalent radicals of one to eight carbon atoms or a branched
saturated monovalent of three to eight carbon atoms. It may include
hydrocarbon radicals of one to four or one to three carbon atoms,
which may be linear. Examples include methyl, ethyl, propyl,
2-propyl, n-butyl, iso-butyl, tert-butyl, pentyl, and the like.
[0035] "Alkylamino" means a radical --NHR or --NR.sub.2 where each
R is independently an alkyl group. Examples include methylamino,
(1-methylethyl)amino, methylamino, dimethylamino, methylethylamino,
di(1-methyethyl)amino, and the like. [0036] "Alkoxy" means the
radical --OR.sub.a , where R.sub.a is an alkyl group. Exemplary
alkoxy groups include methoxy, ethoxy, propoxy, and the like.
[0037] "Carboalkoxy," as used herein, refers to a radical --C(O)R
where R is, for example, hydrogen, alkyl or cycloalkyl,
heterocycloalkyl, or alkyl halo. [0038] "Halo," as used herein,
refers to fluoro, chloro, bromo, or iodo. [0039] "Haloalkyl," as
used herein, refers to alkyl substituted with one or more same or
different halo atoms, e.g., --CH.sub.2Cl, --CH.sub.2Br, --CF.sub.3,
--CH.sub.2CH.sub.2Cl, --CH.sub.2CCl.sub.3, and the like.
[0040] The compounds of Formula I bind to PKC-, preferably, amino
acid residues 469-475 of human PKC- (SEQ ID NO:1). Preferably, the
compounds of Formula I also inhibit the phosphorylation of
IKK-.alpha./IKK-.beta.. Preferably, the compounds of Formula I
induce chromatin condensation in cancer cells. Preferably, the
compounds of Formula I also induce DNA fragmentation in cancer
cells.
[0041] The subject invention also pertains to salt forms of ICA-1
and related compounds including, but not limited to, ammonium
salts, sodium salts, potassium salts, calcium salts, and magnesium
salts.
[0042] PKC- inhibitors useful according to the subject invention
also include, for example, antibodies, peptide aptamers, or binding
partners that bind specifically to PKC-; and antisense
polynucleotides (e.g., siRNAs) that target PKC-iota transcripts.
The skilled artisan would readily appreciate that anti-PKC-
antibodies or peptide aptamers can be designed to bind any PKC-
sequences publically known, and antisense polynucleotides that
target PKC- transcripts can be designed to target any PKC- mRNAs
publically known.
[0043] In one embodiment, the PKC- inhibitor is an antibody or
peptide aptamer that binds specifically to PKC-. In a specific
embodiment, the PKC- inhibitor is an antibody or peptide aptamer
that binds specifically to PKC-. In a further specific embodiment,
the PKC- inhibitor is an antibody or peptide aptamer that binds
specifically to human PKC-. In a further specific embodiment, the
PKC- inhibitor is an antibody or peptide aptamer that binds
specifically to a human PKC- of SEQ ID NO:1.
[0044] In some embodiments, the PKC- inhibitor is an antibody or
peptide aptamer that binds specifically to a naturally-occurring or
recombinant form of PKC- polypeptide, comprising an amino acid
sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% identity
to SEQ ID NO:1, wherein the PKC- polypeptide comprises a catalytic
domain that is amino acid residues 469-475 of SEQ ID NO:1
(glutamine-469, isoleucine-470, arginine-471, isoleucine-472,
proline-473, arginine-474, serine- 475).
[0045] Unless otherwise specified, as used herein, percent sequence
identity and/or similarity of two sequences can be determined using
the algorithm of Karlin and Altschul (1990), modified as in Karlin
and Altschul (1993). Such an algorithm is incorporated into the
NBLAST and XBLAST programs of Altschul et al. (1990). BLAST
searches can be performed with the NBLAST program, score=100,
wordlength=12, to obtain sequences with the desired percent
sequence identity. To obtain gapped alignments for comparison
purposes, Gapped BLAST can be used as described in Altschul et al.
(1997). When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (NBLAST and XBLAST) can be
used. See NCBI/NIH website.
[0046] "Specific binding" or "specificity" refers to the ability of
a protein to detectably bind an epitope presented on a protein or
polypeptide molecule of interest, while having relatively little
detectable reactivity with other proteins or structures.
Specificity can be relatively determined by binding or competitive
binding assays, using, e.g., Biacore instruments. Specificity can
be exhibited by, e.g., an about 10:1, about 20:1, about 50:1, about
100:1, 10.000:1 or greater ratio of affinity/avidity in binding to
the specific target molecule versus nonspecific binding to other
irrelevant molecules.
[0047] Anti-PKC- antibodies of the subject invention can be in any
of a variety of forms, including intact immunoglobulin molecules,
fragments of immunoglobulin molecules such as Fv, Fab and similar
fragments; multimers of immunoglobulin molecules (e.g., diabodies,
triabodies, and bi-specific and tri-specific antibodies, as are
known in the art; see, e.g., Hudson and Kortt, J. Immunol. Methods
231:177 189, 1999); fusion constructs containing an antibody or
antibody fragment; and human or humanized immunoglobulin molecules
or fragments thereof.
[0048] Antibodies within the scope of the invention can be of any
isotype, including IgG, IgA, IgE, IgD, and IgM. IgG isotype
antibodies can be further subdivided into IgG1, IgG2, IgG3, and
IgG4 subtypes. IgA antibodies can be further subdivided into IgA1
and IgA2 subtypes.
[0049] Antibodies of the subject invention include polyclonal and
monoclonal antibodies. The term "monoclonal antibody," as used
herein, refers to an antibody or antibody fragment obtained from a
substantially homogeneous population of antibodies or antibody
fragments (i.e. the individual antibodies within the population are
identical except for possible naturally occurring mutations that
may be present in a small subset of the antibody molecules).
[0050] A monoclonal antibody composition is typically composed of
antibodies produced by clones of a single cell called a hybridoma
that secretes (produces) only one type of antibody molecule. The
hybridoma cell is formed by fusing an antibody-producing cell and a
myeloma or other self-perpetuating cell line. Such antibodies were
first described by Kohler and Milstein, Nature, 1975, 256:495-497,
the disclosure of which is herein incorporated by reference. An
exemplary hybridoma technology is described by Niman et al., Proc.
Natl. Acad. Sci. U.S.A., 1983, 80:4949-4953. Other methods of
producing monoclonal antibodies, a hybridoma cell, or a hybridoma
cell culture are also well known. See e.g., Antibodies: A
Laboratory Manual, Harlow et al., Cold Spring Harbor Laboratory,
1988; or the method of isolating monoclonal antibodies from an
immunological repertoise as described by Sasatry, et al., Proc.
Natl. Acad. Sci. USA, 1989, 86:5728-5732; and Huse et al., Science,
1981, 246:1275-1281. The references cited are hereby incorporated
herein by reference.
[0051] In one embodiment of the invention, monoclonal antibodies
specific for PKC- can be used as a delivery vehicle for drug or
toxin. Drug or toxin can be conjugated to the antibodies using a
biochemical approach. Monoclonal antibodies specific for the
amino-terminus of PKC- can be used as a delivery vehicle for drug
or toxin. This enables the transport of drug or toxin to tumor
cells with high expression of PKC-.
[0052] In some embodiments, PKC- inhibitors useful according to the
subject invention are agents that reduce or inhibit the expression
of PKC-iota, such as agents that inhibit the transcription,
translation, and/or processing of PKC-iota.
[0053] In an embodiment, the PKC- inhibitor is a PKC- antisense
polynucleotide. In an embodiment, the PKC- inhibitor is an
antisense polynucleotide that targets human PKC- mRNA. In some
embodiments, the PKC- antisense polynucleotides target PKC- mRNAs
of non-human animals including, but not limited to, apes,
chimpanzees, orangutans, monkeys, dogs, cats, horses, cattle, pigs,
sheep, goats, chickens, mice, rats, and guinea pigs. The skilled
artisan would readily appreciate that the antisense polynucleotides
can be designed to target any PKC- mRNAs publically known.
[0054] In some embodiments, the PKC- inhibitor is a siRNA having a
sequence sufficiently complementary to a target PKC- mRNA sequence
to direct target-specific RNA interference (RNAi). In some
embodiments, the PKC- inhibitor is siRNA having a sequence
sufficiently complementary to a target human PKC- mRNA sequence
(such as mRNA encoding SEQ ID NO:1) to direct target-specific RNA
interference.
[0055] In some embodiments, the PKC- inhibitor is a siRNA having a
sequence sufficiently complementary to a target PKC- mRNA sequence,
wherein the target PKC- mRNA sequence encodes a naturally-occurring
or recombinant form of a PKC- polypeptide comprising an amino acid
sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% identity
to SEQ ID NO:1, wherein the PKC- polypeptide comprises a catalytic
domain that is amino acid residues 469-475 of SEQ ID NO:1
(glutamine-469, isoleucine-470, arginine-471, isoleucine-472,
proline-473, arginine-474, serine- 475).
[0056] Examples of siRNA that target human PKC- mRNA include SEQ ID
NOs: 2-7.
[0057] Examples of antisense polynucleotides include, but are not
limited to, single-stranded DNAs and RNAs that bind to
complementary target PKC-iota mRNA and inhibit translation and/or
induce RNaseH-mediated degradation of the target transcript; siRNA
oligonucleotides that target or mediate PKC- mRNA degradation;
ribozymes that cleave PKC- mRNA transcripts; and nucleic acid
aptamers and decoys, which are non-naturally occurring
oligonucleotides that bind to and block PKC- protein targets in a
manner analogous to small molecule drugs.
[0058] The term "nucleotide" refers to a nucleoside having one or
more phosphate groups joined in ester linkages to the sugar moiety.
Exemplary nucleotides include nucleoside monophosphates,
diphosphates and triphosphates. The terms "polynucleotide" and
"nucleic acid molecule" are used interchangeably herein and refer
to a polymer of nucleotides joined together by a phosphodiester
linkage between 5' and 3' carbon atoms. The terms "nucleic acid" or
"nucleic acid sequence" encompass an oligonucleotide, nucleotide,
polynucleotide, or a fragment of any of these, DNA or RNA of
genomic or synthetic origin, which may be single-stranded or
double-stranded and may represent a sense or antisense strand,
peptide nucleic acid (PNA), or any DNA-like or RNA-like material,
natural or synthetic in origin. As will be understood by those of
skill in the art, when the nucleic acid is RNA, the
deoxynucleotides A, G, C, and T are replaced by ribonucleotides A,
G, C, and U, respectively.
[0059] As used herein, the term "RNA" or "RNA molecule" or
"ribonucleic acid molecule" refers generally to a polymer of
ribonucleotides. The term "DNA" or "DNA molecule" or
deoxyribonucleic acid molecule" refers generally to a polymer of
deoxyribonucleotides. DNA and RNA molecules can be synthesized
naturally (e.g., by DNA replication or transcription of DNA,
respectively). RNA molecules can be post-transcriptionally
modified. DNA and RNA molecules can also be chemically synthesized.
DNA and RNA molecules can be single-stranded (i.e., ssRNA and
ssDNA, respectively) or multi-stranded (e.g., double stranded,
i.e., dsRNA and dsDNA, respectively). Based on the nature of the
invention, however, the term "RNA" or "RNA molecule" or
"ribonucleic acid molecule" can also refer to a polymer comprising
primarily (i.e., greater than 80% or, preferably greater than 90%)
ribonucleotides but optionally including at least one
non-ribonucleotide molecule, for example, at least one
deoxyribonucleotide and/or at least one nucleotide analog.
[0060] As used herein, the term "nucleotide analog", also referred
to herein as an "altered nucleotide" or "modified nucleotide"
refers to a non-standard nucleotide, including non-naturally
occurring ribonucleotides or deoxyribonucleotides. Preferred
nucleotide analogs are modified at any position so as to alter
certain chemical properties of the nucleotide yet retain the
ability of the nucleotide analog to perform its intended
function.
[0061] As used herein, the term "RNA interference" ("RNAi") refers
to a selective intracellular degradation of RNA. RNAi occurs in
cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural
RNAi proceeds via fragments cleaved from free dsRNA which direct
the degradative mechanism to other similar RNA sequences.
Alternatively, RNAi can be initiated by the hand of man, for
example, to silence the expression of endogenous target genes, such
as PKC-.
[0062] As used herein, the term "small interfering RNA" ("siRNA")
(also referred to in the art as "short interfering RNAs") refers to
an RNA (or RNA analog) comprising between about 10-50 nucleotides
(or nucleotide analogs) which is capable of directing or mediating
RNA interference.
[0063] As used herein, a siRNA having a "sequence sufficiently
complementary to a target mRNA sequence to direct target-specific
RNA interference (RNAi)" means that the siRNA has a sequence
sufficient to trigger the destruction of the target mRNA (e.g.,
PKC- mRNA) by the RNAi machinery or process. "mRNA" or "messenger
RNA" or "transcript" is single-stranded RNA that specifies the
amino acid sequence of one or more polypeptides. This information
is translated during protein synthesis when ribosomes bind to the
mRNA.
[0064] The subject invention also contemplates vectors (e.g., viral
vectors) and expression constructs comprising the nucleic acid
molecules useful for inhibiting PKC- expression and/or activity. In
an embodiment, the vector comprises a siRNA that targets PKC- mRNA.
In another embodiment, the vector comprises a nucleic acid molecule
encoding an anti-PKC- antibody.
[0065] As used herein, the term "expression construct" refers to a
combination of nucleic acid sequences that provides for
transcription of an operably linked nucleic acid sequence. As used
herein, the term "operably linked" refers to a juxtaposition of the
components described, wherein the components are in a relationship
that permits them to function in their intended manner. In general,
operably linked components are in contiguous relation.
[0066] Expression constructs of the invention will also generally
include regulatory elements that are functional in the intended
host cell in which the expression construct is to be expressed.
Thus, a person of ordinary skill in the art can select regulatory
elements for use in, for example, bacterial host cells, yeast host
cells, mammalian host cells, and human host cells. Regulatory
elements include promoters, transcription termination sequences,
translation termination sequences, enhancers, and polyadenylation
elements.
[0067] An expression construct of the invention can comprise a
promoter sequence operably linked to a polynucleotide sequence
encoding a peptide of the invention. Promoters can be incorporated
into a polynucleotide using standard techniques known in the art.
Multiple copies of promoters or multiple promoters can be used in
an expression construct of the invention. In a preferred
embodiment, a promoter can be positioned about the same distance
from the transcription start site as it is from the transcription
start site in its natural genetic environment. Some variation in
this distance is permitted without substantial decrease in promoter
activity. A transcription start site is typically included in the
expression construct.
[0068] In a further embodiment, the subject invention provides a
method of inhibiting proliferation of breast cancer or tumor cells,
comprising administering to the breast cancer or tumor cells an
effective amount of a PKC-iota inhibitor, and optionally, a
pharmaceutically acceptable carrier.
[0069] Treatment of Breast Cancer
[0070] The subject invention provides use of PKC-iota (PKC-)
inhibitors for treatment of breast cancer. In a specific
embodiment, the subject invention provides a method for treatment
of breast cancer via the administration of ICA-1 and related
compounds to a subject. The method comprises administering to a
subject in need of such treatment an effective amount of a compound
of Formula I (e.g., ICA-1) or salt thereof.
[0071] The term "treatment" or any grammatical variation thereof
(e.g., treat, treating, and treatment etc.), as used herein,
includes but is not limited to, ameliorating or alleviating a
symptom of a disease or condition, reducing, suppressing,
inhibiting, lessening, or affecting the progression and/or severity
of an undesired physiological change or a diseased condition. For
instance, treatment includes, for example, slowing the growth
and/or proliferation of breast cancer cells; reducing breast tumor
size; reducing the number of breast cancer cells; inhibiting or
slowing the invasion of breast cancer cells into surrounding or
neighboring tissues; inhibiting or slowing the metastatic spread of
breast cancer cells into distant parts of the body; alleviating
symptoms associated with breast cancer; and prolonging breast
cancer patient survival.
[0072] The term "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 certain embodiments, the effective amount enables a 5%,
10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, 99% or 100% reduction in
the rate of breast cancer growth and/or proliferation. In certain
embodiments, the effective amount enables a 5%, 10%, 15%, 20%, 25%,
30%, 35% or 40% reduction in breast tumor size.
[0073] The term "subject," as used herein, describes an organism,
including mammals such as primates, to which treatment with the
compositions according to the subject invention can be
administered. Mammalian species that can benefit from the disclosed
methods include, but are not limited to, apes, chimpanzees,
orangutans, humans, monkeys; and other animals such as dogs, cats,
horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea
pigs, and hamsters. Typically, the subject is a human.
[0074] In an embodiment, subjects in need of such treatment are
diagnosed with breast cancer or tumor. By way of example, breast
tumor can be identified by routine diagnostic or screening
techniques such as X rays (e.g., mammography), ultrasound, magnetic
resonance imaging (MRI), needle biopsies, stereotactic breast
biopsies, MRI-guided breast biopsies, and surgical biopsies.
Molecular and phenotypic analysis of cancer cells within a tissue
will usually confirm if the cancer is endogenous to the breast
tissue or if the lesion is due to metastasis from another site.
[0075] In an embodiment, the subject invention provides a method
for treating or ameliorating breast cancer. In an embodiment, the
compounds of the subject invention can be used to treat or
ameliorate primary breast cancer, in which cancer cells originated
from breast tissue have not spread past the breast to distant parts
of the body. In a specific embodiment, the compounds of the subject
invention can be used to treat or ameliorate non-invasive and/or
invasive breast cancer.
[0076] In certain embodiments, the subject invention can be used to
treat or ameliorate breast cancer, including ductal carcinoma
in-situ (DCIS), invasive ductal carcinoma (IDC), lobular carcinoma
in-situ (LCIS), invasive lobular carcinoma (LCIS), medullary
carcinoma, malignant phyllode tumor, tubular carcinoma, mucinous
carcinoma, metastatic adenocarcinoma, and inflammatory breast
cancer. In preferred embodiments, the subject invention can be used
to treat or ameliorate invasive ductal carcinoma and/or invasive
lobular carcinoma.
[0077] In another embodiment, the subject invention can be used to
treat or ameliorate metastatic breast cancer, in which cancer cells
originated from breast tissue have spread past the breast to
distant parts of the body such as the bones, lungs, and liver. In
anther embodiment, the subject invention can be used to treat or
ameliorate recurrent breast cancer.
[0078] While benign breast tumors normally do not increase the risk
of breast cancer and thus are often left untreated, the subject
invention can be used to treat or ameliorate benign breast tumors
such as fibroadenoma.
[0079] In a further embodiment, the compounds of the subject
invention can be used in combination with another anti-cancer
therapy including, but not limited to, surgery, radiation therapy,
chemotherapy, DNA therapy, adjuvant therapy, and gene therapy. In a
specific embodiment, the subject invention comprises administering
to the subject a second therapeutic agent. The second therapeutic
agent can be administered before, during or after the
administration of the compound of Formula I.
[0080] Second therapeutic agents for treatment of breast cancer
include, but are not limited to, taxanes, e.g., paclitaxel (TAXOL,
BRISTOL-MYERS SQUIBB Oncology, Princeton, N.J.) and doxetaxel
(TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil,
vincristine, vinblastine, gemcitabine, ixabepilone, doxorubicin,
anti-estrogens such as tamoxifen and raloxifene, aromatase
inhibitors such as 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene,
keoxifene, LY117018, onapristone, and toremifene (Fareston), and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin, and immuno-modulating agents.
[0081] Therapeutic Compositions and Formulations
[0082] The subject invention further provides therapeutic
compositions that contain a therapeutically effective amount of the
therapeutic agent of the subject invention and a pharmaceutically
acceptable carrier or adjuvant.
[0083] The therapeutic agent used in these therapies can be in a
variety of forms. These include for example, solid, semi-solid, and
liquid dosage forms, such as tablets, pills, powders, liquid
solutions or suspensions, suppositories, and injectable and
infusible solutions. The preferred form depends on the intended
mode of administration and therapeutic application.
[0084] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for local injection administration to human beings.
Typically, compositions for local injection administration are
solutions in sterile isotonic aqueous buffer. Generally, the
ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is administered by injection, an ampoule of sterile
water for injection or saline can be provided so that the
ingredients may be mixed prior to administration.
[0085] The subject invention also provides for a therapeutic method
by administering therapeutic or pharmaceutical compositions in a
form that can be combined with a pharmaceutically acceptable
carrier. In this context, the compound may be, for example,
isolated or substantially pure. The term "carrier" refers to a
diluent, adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum oil such as
mineral oil; vegetable oil such as peanut oil, soybean oil, and
sesame oil; animal oil; or oil of synthetic origin.
[0086] Suitable carriers also include ethanol, dimethyl sulfoxide,
glycerol, silica, alumina, starch, sorbitol, inosital, xylitol,
D-xylose, manniol, powdered cellulose, microcrystalline cellulose,
talc, colloidal silicon dioxide, calcium carbonate, magnesium
cabonate, calcium phosphate, calcium aluminium silicate, aluminium
hydroxide, sodium starch phosphate, lecithin, and equivalent
carriers and diluents. Saline solutions and aqueous dextrose and
glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions.
[0087] Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol, and
the like. The therapeutic composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents.
[0088] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary,
depending such as the type of the condition and the subject to be
treated. The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary,
depending such as the type of the condition and the subject to be
treated. In general, a therapeutic composition contains from about
5% to about 95% active ingredient (w/w). More specifically, a
therapeutic composition contains from about 20% (w/w) to about 80%
or about 30% to about 70% active ingredient (w/w).
[0089] The therapeutic agent of the subject invention can be
formulated according to known methods for preparing
pharmaceutically useful compositions. Formulations are described in
detail in a number of sources which are well known and readily
available to those skilled in the art. For example, Remington's
Pharmaceutical Science by E. W. Martin describes formulations which
can be used in connection with the subject invention. In general,
the compositions of the subject invention will be formulated such
that an effective amount of the bioactive compound(s) is combined
with a suitable carrier in order to facilitate effective
administration of the composition.
[0090] The therapeutic or pharmaceutical compositions of the
subject invention can also be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free
amino groups such as those derived from hydrochloric, phosphoric,
acetic, oxalic, or tartaric acids, etc., and those formed with free
carboxyl groups such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0091] Routes of Administration
[0092] The compounds and compositions of the subject invention can
be administered to the subject being treated by standard routes,
including oral, or parenteral administration including intravenous,
subcutaneous, topical, transdermal, intradermal, transmucosal,
intraperitoneal, intramuscular, intracapsular, intraorbital,
intracardiac, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, and
epidural injection, infusion, and electroporation, as well as
co-administration as a component of any medical device or object to
be inserted (temporarily or permanently) into a subject.
[0093] The amount of the therapeutic or pharmaceutical composition
of the subject invention effective in the treatment of breast
cancer will depend on a variety of factors, such as the route of
administration and the seriousness of the condition, and should be
decided according to the judgment of the practitioner and each
patient's circumstances. In general, the dosage ranges from about
0.01 .mu.g/kg to about 10 mg/kg, about 0.01 .mu.g/kg to about 1
mg/kg, about 0.01 .mu.g/kg to about 100 .mu.g/kg, about 0.01
.mu.g/kg to about 10 .mu.g /kg, or about 0.01 .mu.g/kg to about 1
.mu.g/kg. Such a unit dose may be administered once to several
times (e.g. two, three and four times) every two weeks, every week,
or every day.
[0094] In one embodiment, the compounds and compositions of the
subject invention and any second therapeutic agent are administered
simultaneously or sequentially to the patient, with the second
therapeutic agent being administered before, after, or both before
and after treatment with the compounds of the subject invention.
Sequential administration may involve treatment with the second
therapeutic agent on the same day (within 24 hours) of treatment
with the subject compound. Sequential administration may also
involve continued treatment with the second therapeutic agent on
days that the subject compound is not administered.
[0095] In addition, in vitro assays may optionally be employed to
help identify optimal dosage ranges. The precise dose to be
employed in the formulation will also depend on the route of
administration, and the seriousness of the disease, condition or
disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances. Effective doses may
be extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0096] 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
Over-Expression of PCK- in Benign and Malignant Breast Biopsies
[0097] This Example reveals that PKC- is present in high levels in
benign and malignant breast tumor tissue, but is absent in normal
breast tissue (FIG. 1). Briefly, breast tissue biopsy specimens
were obtained from three normal subjects, a patient with
fibroadenoma (benign), a patient with invasive ductual carcinoma,
and a patient with invasive lobular carcinoma. 50 .mu.g of each
biopsy specimen was subjected to gel electrophoresis and Western
blots with monoclonal antibodies against PKC- (cat. # 610176, BD
Transduction, San Diego, Calif.) at a 1:2000 dilution (5 .mu.g).
Secondary antibodies obtained from Accurate JOM035146, Westbury,
NY) were used at a 1.5:10000 dilution (48 .mu.g). To control equal
loading of the proteins, .beta.-actin was also probed with goat
polyclonal antibodies (SC-1616) at a 2.5:2000 dilution (10 .mu.g)
and secondary antibodies SC-2350 at a 1:2000 dilution (8 .mu.g,
Santa Cruz Biotechnology). Du-145 cell lysates (50 .mu.g)
containing PKC- were used as positive control for PKC-
immuno-reactivity (not shown).
[0098] FIG. 1A shows that PKC- was abundantly present in
fibroadenoma and invasive carcinoma tissue, but was absent in
normal breast tissue. PKC- was identified by Western blots as a 67
kD band, which is consistent with the immuno-reactive signal
obtained from Du-145 prostate cancer cells expressing PKC- (data
not shown). Control .beta.-actin Western blots showed .beta.-actin
immuno-reactive bands at a molecular weight of 42 kD. The
.beta.-actin immuno-reactive bands were of equal intensity,
indicating that equal amount of protein was loaded into each lane.
In addition, PKC- proteins detected with Western blots were
quantified, and the mean plus and minus of the standard error (SE)
value was calculated.
[0099] FIG. 1B shows Western blots probing for PKC- in normal
breast tissue specimens (n=5), benign breast tissue specimens (n=9;
i.e., fibroadenoma), and malignant breast tissue specimens (n=11;
i.e., invasive and non-invasive ductual and lobular carcinoma). The
results revealed that PKC- was present in low levels in normal and
benign breast tissue (FIG. 1B). In comparison, there was a 435%
increase in PKC- expression in malignant tissue as compared to
normal tissue (P=0.008). Additionally, there was a 251% increase in
PKC- expression in malignant tissue as compared to benign tissue
(P=0.05). The levels of PKC- expression between normal and benign
breast tissue were not significantly different. Control
.beta.-actin Western blots showed equal density of .beta.-actin
immuno-reactive bands at a molecular weight of 42 kD, indicating
that equal amount of protein was loaded into each lane (Western
blots not shown due to space limitations).
EXAMPLE 2
Association of PCK- with the Proliferation and Survival of Breast
Cancer Cells
[0100] To investigate whether PKC- plays any role in the
proliferation/survival of breast cancer cells, MDA-MB-468 human
breast cancer cells were plated. Cell lysates were subject to
Western blot analysis when cells reached 100% and 50% confluence.
Western blot results showed that PKC- was present in large
quantities in rapidly proliferating, 50% confluent cells (FIG. 2A).
In comparison, PKC- protein content in 100% confluent cells was 43%
lower than that in 50% confluent cells. Differences between PKC-
protein content in 100% confluent and rapidly proliferating, 50%
confluent cells were significant at P=0.04 (n=3).
[0101] To investigate whether PKC isozymes play any role in cell
proliferation/survival, Western blots were performed to detect the
levels of PKC-.alpha., PKC-.delta., and PKC-.epsilon. in 100%
confluent and proliferating 50% confluent cells. Westerns blot
results showed invariant levels of PKC-.alpha., PKC-.delta. and
PKC-.epsilon. in 100% confluent versus proliferating 50% confluent
cells. The results showed that PKC- was up-regulated during the
course of cell proliferation, while other PKCs such as PKC-.alpha.,
PKC-.delta. and PKC-.epsilon. were not involved in the cell
proliferation process.
[0102] FIG. 2B demonstrates that the significant increase in PKC-
levels in 50% confluent serum-cultured cells is due to rapid cell
proliferation, not serum stimulation. Specifically, 50% confluent
cells were serum-starved for 24 hours, serum-starved for 48 hours,
and serum-cultured for 24 hours, respectively, and the levels of
PKC- were measured. The results showed that similar amounts of PKC-
were present in serum-starved v. serum-cultured cells (FIG. 2B).
Thus, the increase in PKC- in 50% confluent cells is not due to
serum stimulation, but is due to the involvement of PKC- in cell
proliferation/survival.
EXAMPLE 3
PKC- as an Essential Factor for the Proliferation of Breast Cancer
Cells
[0103] This Example reveals that PKC- is required for the
proliferation and/or survival of breast cancer cells. Briefly,
MDA-MB-468 breast cells were plated on 75 cm.sup.2 flasks at a
density of 3.75.times.10.sup.5 cells/flask. Twenty-four hours post
plating, cells were incubated with either siRNA-A (120 nM;
vehicle-control) or PKC- siRNA (120 nM) according to manufacture's
instruction (Santa Cruz Biotechnology). During a 3-day incubation
period, viable cells were counted by trypan blue dye exclusion
assay. Cell viability and cell count were determined 24-72 hours
following addition of either control short interfering RNAs
(siRNA-A, vehicle control; 120 nM) or PKC- siRNA (120 nM) according
to manufacturer's instruction (Santa Cruz Biotechnology).
[0104] The results showed that exposure of MDA-MB-468 breast cancer
cells to PKC- siRNA significantly reduced cell proliferation by 57%
at 24 h (P=0.04), 75% at 48 h (P=0.04) and 75% at 72 h (P=0.02)
post treatment (FIG. 3A). Densitometry scanning of Western blots
revealed that exposure to PKC- siRNA decreased PKC- protein content
by 74% (24 hours), 68% (48 hours), and 68% (72 hours) (FIGS. 3B and
3C; n=3 different experiments). Differences between PKC- protein
content in control siRNA-A and PKC- siRNA treated cells were
significant (P=0.002 at 24 hours; P=0.008 at 48 hours and P=0.036
at 72 hours) for all time points. Control .beta.-actin Western
blots showed .beta.-actin immuno-reactive bands at a molecular
weight of 42 kD. The .beta.-actin immuno-reactive bands were of
equal intensity, indicating that equal amounts of proteins were
loaded into each lane. These results demonstrate that PKC- is
required for cell proliferation/survival.
EXAMPLE 4
Anti-Proliferative Effects of ICA-1 on Breast Cancer Cells
[0105] This Example demonstrates that ICA-1 inhibits the
proliferation of breast cancer cells. Briefly, MDA-MB-468 breast
cancer cells were treated with 0.1 .mu.M or 0.5 .mu.M ICA-1. Cell
viability and cell count were determined 24 hours following
addition of either vehicle control or ICA-1. As shown in FIG. 4,
ICA-1 potently reduced the proliferation of MDA-MB-468 cells by 77%
(0.1 .mu.M) and 50% (0.5 .mu.M; P=0.05). ICA-1 at 0.1 .mu.M was
more effective in inhibiting the proliferation of MDA-MB-468 cells
than ICA-1 at 0.5 .mu.M. This indicates that ICA-1 applied at 0.5
.mu.M in vitro may induce multiple drug resistance (MDR).
[0106] 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.
[0107] 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. 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.
Sequence CWU 1
1
71587PRTHomo sapiens 1Met Ser His Thr Val Ala Gly Gly Gly Ser Gly
Asp His Ser His Gln 1 5 10 15 Val Arg Val Lys Ala Tyr Tyr Arg Gly
Asp Ile Met Ile Thr His Phe 20 25 30 Glu Pro Ser Ile Ser Phe Glu
Gly Leu Cys Asn Glu Val Arg Asp Met 35 40 45 Cys Ser Phe Asp Asn
Glu Gln Leu Phe Thr Met Lys Trp Ile Asp Glu 50 55 60 Glu Gly Asp
Pro Cys Thr Val Ser Ser Gln Leu Glu Leu Glu Glu Ala 65 70 75 80 Phe
Arg Leu Tyr Glu Leu Asn Lys Asp Ser Glu Leu Leu Ile His Val 85 90
95 Phe Pro Cys Val Pro Glu Arg Pro Gly Met Pro Cys Pro Gly Glu Asp
100 105 110 Lys Ser Ile Tyr Arg Arg Gly Ala Arg Arg Trp Arg Lys Leu
Tyr Cys 115 120 125 Ala Asn Gly His Thr Phe Gln Ala Lys Arg Phe Asn
Arg Arg Ala His 130 135 140 Cys Ala Ile Cys Thr Asp Arg Ile Trp Gly
Leu Gly Arg Gln Gly Tyr 145 150 155 160 Lys Cys Ile Asn Cys Lys Leu
Leu Val His Lys Lys Cys His Lys Leu 165 170 175 Val Thr Ile Glu Cys
Gly Arg His Ser Leu Pro Gln Glu Pro Val Met 180 185 190 Pro Met Asp
Gln Ser Ser Met His Ser Asp His Ala Gln Thr Val Ile 195 200 205 Pro
Tyr Asn Pro Ser Ser His Glu Ser Leu Asp Gln Val Gly Glu Glu 210 215
220 Lys Glu Ala Met Asn Thr Arg Glu Ser Gly Lys Ala Ser Ser Ser Leu
225 230 235 240 Gly Leu Gln Asp Phe Asp Leu Leu Arg Val Ile Gly Arg
Gly Ser Tyr 245 250 255 Ala Lys Val Leu Leu Val Arg Leu Lys Lys Thr
Asp Arg Ile Tyr Ala 260 265 270 Met Lys Val Val Lys Lys Glu Leu Val
Asn Asp Asp Glu Asp Ile Asp 275 280 285 Trp Val Gln Thr Glu Lys His
Val Phe Glu Gln Ala Ser Asn His Pro 290 295 300 Phe Leu Val Gly Leu
His Ser Cys Phe Gln Thr Glu Ser Arg Leu Phe 305 310 315 320 Phe Val
Ile Glu Tyr Val Asn Gly Gly Asp Leu Met Phe His Met Gln 325 330 335
Arg Gln Arg Lys Leu Pro Glu Glu His Ala Arg Phe Tyr Ser Ala Glu 340
345 350 Ile Ser Leu Ala Leu Asn Tyr Leu His Glu Arg Gly Ile Ile Tyr
Arg 355 360 365 Asp Leu Lys Leu Asp Asn Val Leu Leu Asp Ser Glu Gly
His Ile Lys 370 375 380 Leu Thr Asp Tyr Gly Met Cys Lys Glu Gly Leu
Arg Pro Gly Asp Thr 385 390 395 400 Thr Ser Thr Phe Cys Gly Thr Pro
Asn Tyr Ile Ala Pro Glu Ile Leu 405 410 415 Arg Gly Glu Asp Tyr Gly
Phe Ser Val Asp Trp Trp Ala Leu Gly Val 420 425 430 Leu Met Phe Glu
Met Met Ala Gly Arg Ser Pro Phe Asp Ile Val Gly 435 440 445 Ser Ser
Asp Asn Pro Asp Gln Asn Thr Glu Asp Tyr Leu Phe Gln Val 450 455 460
Ile Leu Glu Lys Gln Ile Arg Ile Pro Arg Ser Leu Ser Val Lys Ala 465
470 475 480 Ala Ser Val Leu Lys Ser Phe Leu Asn Lys Asp Pro Lys Glu
Arg Leu 485 490 495 Gly Cys His Pro Gln Thr Gly Phe Ala Asp Ile Gln
Gly His Pro Phe 500 505 510 Phe Arg Asn Val Asp Trp Asp Met Met Glu
Gln Lys Gln Val Val Pro 515 520 525 Pro Phe Lys Pro Asn Ile Ser Gly
Glu Phe Gly Leu Asp Asn Phe Asp 530 535 540 Ser Gln Phe Thr Asn Glu
Pro Val Gln Leu Thr Pro Asp Asp Asp Asp 545 550 555 560 Ile Val Arg
Lys Ile Asp Gln Ser Glu Phe Glu Gly Phe Glu Tyr Ile 565 570 575 Asn
Pro Leu Leu Met Ser Ala Glu Glu Cys Val 580 585 219DNAArtificial
Sequencesingle strand of PKC-iota siRNA 2caagccaagc guuucaaca
19319DNAArtificial Sequencea single strand of PKC-iota siRNA
3uguugaaacg cuuggcuug 19419DNAArtificial Sequencea single strand of
PKC-iota siRNA 4ggaacgauug gguugucau 19520DNAArtificial Sequencea
single strand of PKC-iota siRNA 5augacaaccc aaucguuucc
20619DNAArtificial Sequencea single strand of PKC-iota siRNA
6cccaauaucu ucucuugua 19719DNAArtificial Sequencea single strand of
PKC-iota siRNA 7uacaagagaa gauauuggg 19
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