Note: Descriptions are shown in the official language in which they were submitted.
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USE OF PHOSPHATASES TO TREAT TUMORS OVEREXPRESSING N-COR
Certain embodiments of this invention were created in the
performance of a Cooperative Research and Development
Agreement with the National Institute of Health, U.S.
Department of Health and Human Services. Consequently, the
Government of the United States has certain rights in the
invention.
Throughout this application, certain publications are
referenced. Full citations for these publications may be found
immediately preceding the claims. The disclosures of these
publications in their entireties are hereby incorporated by
reference into this application in order to more fully
describe the state of the art to which this invention relates.
Background of the Invention
Despite the medical advances of the past few decades, cancer
continues to plague people of all ages. The prevalence of
various forms of cancer and the lack of effective treatments
for many forms is a testament to the problems these diseases
present.
Of the many cancers still lacking an effective treatment,
glioblastoma multiforme (GBM) is one of the most lethal.
Patients diagnosed with GBM have a grim prognosis. Patients
may be treated with surgery, radiotherapy and chemotherapy,
but the median survival is still less than one year. (Stupp et
al. (2005)) This short survival time has remained virtually
unchanged over the past 30 years due to the lack of an
effective treatment.
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GBM is the most common primary brain tumor. GBM is also the
most malignant primary brain tumor. (Stupp et al. (2005)) It
grows rapidly within the brain and may reach a large size
before any symptoms occur and a diagnosis is made. GBM's
malignancy typically remains in the cerebral hemispheres of
the brain; however, glioblastomas can form in the brainstem,
the cerebellum and the spinal cord. GBM does not usually
spread to other parts of the body.
GBM tumors form from the supportive or glial tissue of the
brain. GBM tumor cells look very different from normal brain
cells. GBM cells are poorly differentiated, neoplastic
astrocytes. GBM tumors are characterized by molecular lesions,
cellular pleomorphism, mitotic figures, and multinucleated
giant cells. (U.S. Patent Publication No. 2005/0203082, Hsu et
al.) The World Health Organization classifies GBM as having 3
or 4 of the following histologic criteria: (1) nuclear atypia,
(2) mitoses, =(3) endothelial proliferation, and (4) necrosis.
The cause of GBM is unknown. GBM tumors may develop from much
less malignant precursor tumors, called astrocytomas
(secondary GBMs), or it may form de novo, with no evidence of
a precursor tumor (primary GBM).
Due to the lethality of GBM and the sensitive nature of its
location within the human body, it is imperative that new
treatments and better modes of diagnosis be developed.
The subject invention provides novel methods of treating GBM.
It also provides novel methods of diagnosing'and screening for
this deadly disease.
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Summary of the Invention
The invention provides a method of treating a patient
suffering from a tumor overexpressing N-CoR comprising
administering to the patient one or more phosphatase ligand,
alone or in combination with one or more retinoid receptor
ligand, or one or more histone deacetylase ligand, or both, in
each case in an amount effective to treat the patient.
This invention also provides a method of inhibiting growth of
a tumor overexpressing N-CoR in a patient, comprising
administering to the patient one or more phosphatase ligand,
alone or in combination with one or more retinoid receptor
ligand, or one or more histone deacetylase ligand, or both, in
each case in amounts effective to affect N-CoR so as to induce
differentiation of cells of the tumor overexpressing N-CoR and
inhibit growth of the tumor in the patient.
This invention further provides a method of identifying a
compound or a mixture of compounds capable of inducing
differentiation or inhibiting proliferation of cells of a
tumor overexpressing N-CoR, comprising the steps of (a)
culturing a first population of the specified human cells in
the absence of the compound or the mixture of compounds in
both serum and serum free conclitions; (b) separately culturing
a second population of such human cells in the presence of the
compound or the mixture of compounds; (c) comparing the rate
of growth of the cultured human cells in step (a) with the
rate of growth of the cultured human cells in step (b); (d)
identifying the compound or the mixture of compounds which
inhibited, or reduced the rate of, growth of the cultured
human cells in step (b) as compared to the rate of growth of
the cultured human cells in step(a); and (e) measuring the
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level of N-CoR in the cytoplasm and in the nucleus of the
cultured human cells from step (b) whose growth was inhibited
or whose rate of growth was reduced in the presence of the
compound or the mixture of compounds with the =levels of N-CoR
in the cultured human cells from step (a), wherein the
presence in the sample of decreased levels of N-CoR indicates
that the compound or the mixture of compounds is capable of
inducing differentiation of cells of tumors overexpressing N-
CoR, so as to thereby identify the compound or the mixture of
compounds.
This invention also provides a method of determining the
likelihood of successfully treating a subject suffering from a
tumor overexpressing N-CaR, comprising the steps of (a)
obtaining a sample from the subject containing cells of a
tumor overexpressing N-CoR; and (b) measuring the level of N-
CoR in the cytoplasm and in the nucleus of cells in the sample
so obtained, wherein the presence in the sample of an
increased level of N-CoR in the nucleus of the cells indicates
that there is a greater likelihood of successfully treating
the subject.
This invention further provides a method of assessing the
likelihood that a patient is suffering from a tumor
overexpressing N-CoR, comprising the steps of (a) obtaining a
serum sample from the subject; and (b) measuring the level of
N-CoR in the serum sample so obtained, wherein the presence in
the serum sample of increased levels of N-CoR relative -to a
normal reference standard indicates that the patient is likely
suffering from a tumor overexpressing N-CoR.
This invention still further provides a method of assessing
the likelihood that a patient previously suffering from and
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treated for a tumor overexpressing N-CoR has suffered a
recurrence of such tumor, comprising the steps of (a)
obtaining a serum sample from the subject; and (b) measuring
the level of N-CoR in the serum sample so obtained, wherein
the presence in the serum sample of increased levels of N-CoR
relative to a previous' level of N-CoR indicates that the
patient is likely suffering from a recurrence of a tumor
overexpressing N-CoR.
This invention provides a method of treating a patient
suffering from glioblastoma multiforme, comprising
administering to the patient one or more phosphatase ligand,
alone or in combination with one or more retinoid receptor
ligand, or one or more histone deacetylase ligand, or both, in
each case in amounts effective to treat the patient.
This invention also provides a method of inhibiting growth of
a tumor in a patient suffering from glioblastoma multiforme,
comprising administering to the patient one or more
phosphatase ligand, alone or in combination with one or more
retinoid receptor ligand, or one or more histone deacetylase
ligand, or both, in each case in amounts effective to affect
N-CoR so as to induce differentiation of glioblastoma
multiforme tumor cells and inhibit growth of the tumor in the
patient.
This invention further provides a method of identifying a
compound or a mixture of compounds capable of inducing
differentiation or inhibiting proliferation of glioblastoma
multiforme tumor cells, comprising the steps of (a) culturing
a first population of human brain cells in the absence of the
compound or the mixture of compounds in both serum and serum
free conditions; (b) separately culturing a second population
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of such human brain cells in the presence of the compound or
the mixture of compounds; (c) comparing the rate of growth of
the cultured human brain cells in step (a) with the rate of
growth of the cultured human brain cells in step (b); (d)
identifying the compound or the mixture of compounds which
inhibited, or reduced the rate of, growth of the cultured
human brain cells in step (b) as compared to the rate of
growth of the cultured human brain cells in step (a) ; and (e)
measuring the level of N-CoR and the level of a glioblastoma
multiforme lineage marker in the cytoplasm and in the nucleus
of the cultured human brain cells from step (b) whose growth
was inhibited or whose rate of growth was reduced in the
presence of the compound or the mixture of compounds with the
levels of N-CoR and the glioblastoma multiforme lineage marker
in the cultured human brain cells from step (a), wherein a
decrease in the level of N-CoR and an. increase in the
glioblastoma multiforme lineage marker indicate that the
compound or the mixture of compounds is capable of inducing
differentiation of glioblastoma multiforme tumor.cells, so as
to thereby identify the compound or the mixture of compounds.
This invention also provides a method of determining the
likelihood of successfully treating a subject suffering from
glioblastoma multiforme, comprising the steps of (a) obtaining
a sample from the subject containing glioblastoma multiforme
cells; and (b) measuring the level of each of N-CoR and a
glioblastoma multiforme lineage marker in the cytoplasm and in
the nucleus of cells in the sample so obtained, wherein the
presence in the sample of an increased level of N-CoR and a
low or undetectable level of glioblastoma multiforme lineage
marker in the cytoplasm of the cells indicates that there is a
greater likelihood of successfully treating the subject.
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This invention still further provides a method of assessing
the likelihood that a patient is suffering from glioblastoma
multiforme, comprising the steps of (a) obtaining a sample' of
cerebrospinal fluid and/or tumor cells or serum from the
subject; and (b) measuring the level of N-CoR in the
cerebrospinal fluid and/or the cells or serum in the sample so
obtained, wherein the presence in the sample of increased
levels of N-CoR in the cerebrospinal fluid relative to a
normal reference standard indicates that the patient is likely
suffering from glioblastoma multiforme. If N-CoR is increased
in the serum but not in the cerebral spinal fluid this would
indicate that the patient is likely suffering from a tumor
overexpressing N-CoR but not necessarily a glioblastoma
multiforme.
Finally, this invention provides a method of assessing the
likelihood that a patient previously suffering from and
treated for glioblastoma multiforme has suffered a recurrence
of glioblastoma multiforme, comprising the steps of (a)
obtaining a sample of cerebrospinal fluid and/or tumor cells
or serum from the subject; and (b) measuring the level of N-
CoR in the cerebrospinal fluid and/or in the cells or serum in
the sample so obtained, wherein the presence in the sample of
increased levels of N-CoR in the cerebrospinal fluid or serum
relative to the amount of N-CoR previously in the cerebral
spinal fluid indicates that the patient is likely suffering
from a recurrence of glioblastoma multiforme.
Brief Description of the Figures
Figure 1A. Differential expression of N-CoR in normal and GBM
brain tissue. Total proteomic analysis of microdissected
normal glial tissue (white matter) compared with GBM was
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performed by two dimensional gel electrophoresis (2-DGE). The
highlighted region which is magnified on the right panels
shows a consistent protein pattern in normal glial and GBM and
unique expression of N-CoR in GBM. Protein identification was
performed by liquid chromatography-mass spectrometry.
Figure lB. Expression of N-CoR in GBM by immunohistochemistry:
N-CoR protein is present in both nucleus and cytoplasm in GBM.
Right panel: arrows point to N-CoR staining (circled) in
nucleus (right) and cytoplasm (left). Left panel: no N-CoR is
seen in normal tissue.
Figure 1C. Expression of N-CoR in GBM by Western blot
analysis: N-CoR is present in GBMs on lanes 2-4 (molecular
weight 270 kDa). N-CoR is absent in normal white matter (lane
1). P-actin was used- as internal positive quantitative
control.
Figure 1D. Subcellular localization of N-CoR correlates with
glial differentiation: N-CoR and GFAP immunolabeling in GBM.
Cell on right demonstrating nuclear N-CoR localization shows
no cytoplasmic GFAP. Cell on left with absent N-CoR labeling
shows cytoplasmic expression of GFAP.
Figure 1E. Co-expression of nuclear localization of N-CoR and
cytoplasmic expression of CD133 in GBM primary culture:
Immunolabeling of nuclear localization of N-CoR and
cytoplasmic CD133 are present in the same GBM cells.
Figure 2A. GFAP expression by CNTF-treated BTSC: GFAP
expression was induced in glioma stem cells by treatment with
CNTF and detected by 2-DGE and LCMS. Arrow points to the GFAP
spot (circled).
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Figure 2B. Cytoplasmic N-CoR fraction increased by CNTF
treatment of BTSC. Cytoplasmic level of N-CoR expression in
BTSC shows gradual increase from day 0 to day 7 upon CNTF
treatment. P-actin is shown as quantitative internal control.
Figure 2C. Logarithmic growth curve of gliomal cell line, U343
MG-A, treated with retinoic acid (RA), okadaic acid (OA),
combination of retinoic acid and okadaic acid (RA/OA), and
control (NC) for 16 days: Individual treatment with retinoic
acid and okadaic acid show a modest inhibition of growth.
Combination of retinoic acid and okadaic acid shows
synergistic reduction in cell growth. Error bars indicate 1
SD.
Figure 3. Logarithmic curve of gliomal cell line U373 treated
with endothal (End), endothal thioanhydride (ET), nor-
cantharidin (nor-Can) and compound LB-1. Increasing dosages
demonstrate a greater inhibition of growth. Error bars
indicate SD.
Figure 4A. Logarithmic curve of gliomal cell line =U373
treated with all-trans retinoic acid (ATRA). Increasing dosage
shows a modest inhibition of growth. Error bars indicate SD.
Figure 4B. Inhibition of gliomal cell line U373 treated
with endothal (End) and compound LB-1 with and without all-
trans Retinoic acid (ATRA) for 7 days. Individual treatment
with endothal and compound LB-1 shows modest inhibition of
growth. Combination of End or compound LB-1 with ATRA shows
synergistic reduction in cell growth. Error bars indicate SD.
Figure 4C. Inhibition of growth of gliomal cell line U373
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by endothal (End) with and without 13-cis Retinoic Acid (cis-
RA). Individual treatment with End and cis-RA show a modest
inhibition of growth. Combination of End and cis-RA show a
synergistic reduction in cell growth. Error bars indicate SD.
Figure 5A. Inhibition of growth of gliomal cell line U373
with Valproic Acid (Val). Increasing doses of Val (mM) shows a
greater inhibition of cell growth. Error bars indicate SD.
Figure 5B. Inhibition of growth of gliomal cell line U373
by Trichostatin A (TSA) . Increasing doses of TSA (ug/mL) show
a greater inhibition of cell growth. Error bars indicate SD.
Figure 6A. Inhibition of growth of kidney cancer cell
line, UMRC by endothal thioanhydride (ET), endothal (End),
all-trans Retinoic Acid (ATRA), Trichostatin A (TSA) and
norcantharidin (nor-Can) for 7 days. Error bars indicate SD
Figure 6B. Inhibition of growth of gliomal cell line U373
by endothal thioanhydride (ET), endothal (End), all-trans
Retinoic Acid (ATRA), Trichostatin A (TSA) and norcantharidin*
(nor-Can) for 7 days. Individual treatment with endothal
thioanhydride showed the greatest inhibition of growth. Error
bars indicate SD.
Figure 6C. Inhibition of growth of breast cancer cell
line, MCF-7 by Inhibition of UMRC by endothal thioanhydride
(ET), endothal (End), all-trans Retinoic Acid (ATRA),
Trichostatin A (TSA) and norcantharidin (nor-Can) for 7 days.
Treatment with individual doses of endothal, ATRA and TSA
surprisingly showed an inhibition in growth. Error bars
indicate SD.
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Detailed Description of the Invention
As used in this application each of the following terms has
the meaning set forth below.
As used herein, "administering" an agent may be performed using
any of the various methods or delivery systems well known to
those skilled in the art. The administering can be performed,
for example, orally, parenterally, intraperitoneally,
intravenously, intraarterially, transdermally, sublingually,
intramuscularly, rectally, transbuccally, intranasally,
liposomally, via inhalation, vaginally, intraoccularly, via
local delivery, subcutaneously, intraadiposally,
intraarticularly, intrathecally, into a cerebral ventricle,
intraventicularly, intratumorally, into cerebral parenchyma or
intraparenchchymally.
The following delivery systems, which employ a number of
routinely used pharmaceutical carriers, may be used but are
only representative of the many possible systems envisioned
for administering compositions in accordance with the
invention.
Injectable drug delivery systems include solutions,
suspensions, gels, microspheres and polymeric injectables, and
can comprise excipients such as solubility-altering agents
(e.g., ethanol, propylene glycol and sucrose) and polymers
(e.g., polycaprylactones and PLGA's).
Implantable systems include rods and discs, and can contain
excipients such as PLGA and polycaprylactone.
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Oral delivery systems include tablets and capsules. These can
contain excipients such as binders (e.g.,
hydroxypropylmethylcellulose, polyvinyl pyrilodone, other
cellulosic materials and starch), diluents (e.g., lactose and
other sugars, starch, dicalcium phosphate and cellulosic
materials), disintegrating agents (e.g., starch polymers and
cellulosic materials) and lubricating agents (e.g., stearates
and talc).
Transmucosal delivery systems include patches, tablets,
suppositories, pessaries, gels -and creams, and can contain
excipients such as solubilizers and enhancers (e.g., propylene
glycol, bile salts and amino acids), and other vehicles (e.g.,
polyethylene glycol, fatty acid esters and derivatives, and
hydrophilic polymers such as hydroxypropylmethylcellulose and
hyaluronic acid).
Dermal delivery systems include, for example, aqueous and
nonaqueous gels, creams, multiple emulsions, microemulsions,
liposomes, ointments, aqueous and nonaqueous solutions,
lotions, aerosols, hydrocarbon bases and powders, and can
contain excipients such as solubilizers, permeation enhancers
(e.g., fatty acids, fatty acid esters, fatty alcohols and
amino acids), and hydrophilic polymers (e.g., polycarbophil
and polyvinylpyrolidone). In one embodiment, the
pharmaceutically acceptable carrier is a liposome or a
transdermal enhancer.
Solutions, suspensions and powders for reconstitutable
delivery systems include vehicles such as suspending agents
(e.g., gums, zanthans, cellulosics and sugars), humectants
(e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and
propylene glycol), surfactants (e.g., sodium lauryl sulfate,
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Spans, Tweens, and cetyl pyridine), preservatives and
antioxidants (e.g., parabens, vitamins E and C, and ascorbic
acid), anti-caking agents, coating agents, and chelating
agents (e.g., EDTA).
As used herein, "therapeutically effective amount" means an
amount sufficient to treat a subject afflicted with a disease
(e.g. glioblastoma multiforme) or to alleviate a symptom or a
complication associated with the disease.
As used herein, "treating" means slowing, stopping or
reversing the progression of a disease, particularly
glioblastoma multiforme.
As used herein, "overexpressing N-CoR" means that the level of
the nuclear co-receptor (N-CoR) expressed in cells of the
tissue tested are elevated in comparison to the levels of N-
CoR as measured in normal healthy cells of the same type of
tissue under analogous conditions. The nuclear receptor co-
repressor (N-CoR) of the subject invention may be any molecule
that binds to the ligand binding domain of the DNA-bound
thyroid hormone receptor (T3R) and retinoic acid receptor
(RAR) . (U.S. Patent No. 6,949,624, Liu et al.) Examples of
tumors that overexpress N-CoR may include glioblastoma
multiforme, breast cancer (Myers et al.), colorectal cancer
(Giannini and Cavallini), small cell lung cancer (Waters et
al.) and ovarian cancer (Havrilesky et al.).
The invention provides a method of treating a patient
suffering from a tumor overexpressing N-CoR comprising
administering to the patient one or more phosphatase ligand,
alone or in combination with one or more retinoid receptor
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ligand, or one or more histone deacetylase ligand, or both, in
each case in an amount effective to treat the patient.
The phosphatase ligand may be selected from the group
consisting of 1-nor-okadaone, antimonyl tartrate,
bioallethrin, calcineurin, cantharidic acid, cantharidin,
calyculin, cypermethrin, DARPP-32, deamidine, deltamethrin,
diaminopyrroloquinazolines, endothal, endothal thioanhydride,
fenvalerate,* fostriecin, imidazoles, ketoconazole, L-4-
bromotetramisole, levamisole, microcystin LA, microcystin LR,
microcystin LW, microcystin RR, molybdate salts, okadaic
acid, okadol, norcantharidin, pentamidine, pentavalent
antimonials, permethrin, phenylarsine oxide, phloridzin,
protein phosphatase inhibitor-1 (I-1), protein phosphatase
inhibitor-2 (I-2)pyrophosphate, salubrinal, sodium fluoride,
sodium orthovanadate, sodium stibogluconate, tartrate salts,
tautomycin, tetramisole, thrysiferyl-23-acetate, vanadate,
vanadium salts and antileishmaniasis compounds, including
suramin and analogues thereof.
In the method of the invention, the histone deacetylase ligand
may be an inhibitor, e.g. the histone deacetylase inhibitor
HDAC-3 (histone deacetylase-3). The histone deacetylase ligand
may also be selected from the group consisting of 2-amino-8-
oxo-9,10-epoxy-decanoyl, 3-(4-aroyl-lH-pyrrol-2-yl)-N-hydroxy-
2-propenamide, APHA Compound 8, apicidin, arginine butyrate,
butyric acid, depsipeptide, depudecin, HDAC-3, m-
carboxycinnamic acid bis-hydroxamide, N-(2-aminophenyl)-4-[N-
(pyridin-3-ylmethoxycarbonyl) aminomethyl] benzamide, MS 275,
oxamfiatin, phenylbutyrate, pyroxamide, scriptaid, sirtinol,
sodium butyrate, suberic bishydroxamic acid, suberoylanilide
hydroxamic acid, trichostatin A, trapoxin A, trapoxin B and
valproic acid.
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The subject application encompasses compounds which inhibit
the enzyme histone deacetylase (HDAC). These HDAC enzymes
posttranslationally modify histones (U.S. Patent Publication
No. 2004/0197888, Armour et al.) Histones are groups of
proteins which associate with DNA in eukaryotic cells to form
compacted structures called chromatin. This compaction allows
an enormous amount of DNA to be located within the nucleus of
a eukaryotic cell, but the compact structure of chromatin
restricts the access of transcription factors to the DNA.
Acetylation of the histones decreases the compaction of the
chromatin allowing transcription factors to bind to the DNA.
Deacetylation, catalysed by histone deacetylases (HDACs),
increases the compaction of chromatin, thereby reducing
transcription factor accessibility to DNA. Therefore,
inhibitors of histone deacetylases prevent the compaction of
chromatin, allowing transcription factors to bind to DNA and
increase expression of the genes.
This invention also provides a method of inhibiting growth of
a tumor overexpressing N-CoR in a patient, comprising
administering to the patient one or more phosphatase ligand,
alone or in combination with one or more retinoid receptor
ligand, one or more histone deacetylase ligand, or both, in
each case in amounts effective to affect N-CoR so as to
thereby induce differentiation of cells of the tumor
overexpressing N-CaR and inhibit growth of the tumor in the
patient.
This invention further provides a method of identifying a
compound or a mixture of compounds capable of inducing
differentiation or inhibiting proliferation of cells of a
tumor overexpressing N-CoR, comprising the steps of (a)
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culturing a first population of the specified human cells in
the absence of the compound or the mixture of compounds in
both serum and serum free conditions; (b) separately culturing
a second population of such human cells in the presence of the
compound or the mixture of compounds; (c) comparing the rate
of growth of the cultured human cells in step (a) with the
rate of growth of the cultured human cells in step (b); (d)
identifying the compound or the mixture of compounds which
inhibited, or reduced the rate of, growth of the cultured
human cells in step (b) as compared to the rate of growth of
the cultured human cells in step(a); and (e) measuring the
level of N-CoR in the cytoplasm and in the nucleus of the
cultured human cells from step (b) whose growth was inhibited
or whose rate of growth was reduced in the presence of the
compound or the mixture of compounds with the levels of N-CoR
in the cultured human cells from step (a), wherein the
presence in the sample of decreased levels of N-CoR indicates
that the compound or the mixture of compounds is capable of
inducing differentiation of cells of tumors overexpressing N-
CoR, so as to thereby identify the compound or the mixture of
compounds.
In the method of the invention, the level of N-CoR in the
cytoplasm and in the nucleus may be measured by either
indirect immunofluorescence microscopy, direct
immunofluorescence microscopy, FACS, or other methods for
detecting and measuring amounts of specific proteins in
tissues including assessment of the amounts of proteins in the
nucleus versus the cytoplasm and in cell lysates, or a
combination thereof.
N-CoR is expressed in the nucleus of the undifferentiated
tumor or stem cells and is only present in the cytoplasm at
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amounts detectable by immunochemistry and Western blotting
when the cell undergoes differentiation. N-CoR ' is not
detectable by immunochemistry and Western blotting in either
the nucleus or the cytoplasm of normal or fully differentiated
cells.
Thus, in the methods of the invention, an assessment of the
percentage of cells with N-CoR in the cytoplasm relative to
the percentage of cells with N-CoR in the nucleus is
representative of the ratio of more differentiated cells to
less differentiated cells in a given tissue.
In the method of the invention, tumors that overexpress N-CoR
may include glioblastoma multiforme, breast cancer, colorectal
cancer, small cell lung cancer and ovarian cancer.
This invention also provides a method of determining the
likelihood of successfully treating a subject suffering from a
tumor overexpressing N-CoR, comprising the steps of (a)
obtaining a sample from the subject containing cells of a
tumor overexpressing N-CoR; and (b) measuring the level of N-
CoR in the cytoplasm and in the nucleus of cells in the sample
so obtained, wherein the presence in the sample of an
increased level of N-CoR in the nucleus of the cells indicates
that there is a greater likelihood of successfully treating
the subject.
In the method, the level of N-CoR in the cytoplasm and in the
nucleus may be measured by either indirect immunofluorescence
microscopy, direct immunofluorescence microscopy, FACS, or
other methods for detecting and measuring amounts of specific
proteins in tissues including assessment of the amounts of
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proteins in the nucleus versus the cytoplasm and in cell
lysates, or a combination thereof.
N-CoR is expressed in the nucleus of the undifferentiated
tumor or stem cells and is only present in the cytoplasm at
amounts detectable by immunochemistry and Western blotting
when the cell undergoes differentiation. N-CoR is not
detectable by immunochemistry and Western blotting in either
the nucleus or the cytoplasm of normal or fully differentiated
cells.
Thus, in the methods of the invention, an assessment of the
percentage of cells with N-CoR in the cytoplasm relative to
the percentage of cells with N-CoR in the nucleus is
representative of the ratio of more differentiated cells to
less differentiated cells in a given tissue.
In the method of the invention, tumors that overexpress N-CoR
may include glioblastoma multiforme, breast cancer, colorectal
cancer, small cell lung cancer and ovarian cancer.
This invention further provides a method of assessing the
likelihood that a patient is suffering from a tumor
overexpressing N-CoR, comprising the steps of (a) obtaining a
serum sample from the subject; and (b) measuring the level of
N-CoR in the serum sample so obtained, wherein the presence in
the serum sample of increased levels of N-CoR relative to a
normal reference standard indicates that the patient is likely
suffering from a tumor overexpressing N-CoR.
In the method of the invention, tumors that overexpress N-CoR
may include glioblastoma multiforme, breast cancer, colorectal
cancer; small cell lung cancer and ovarian cancer.
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This invention still further provides a method of assessing
the likelihood that a patient previously suffering from and
treated for a tumor overexpressing N-CoR has suffered a
recurrence of such tumor, comprising the steps of (a)
obtaining a serum sample from the subject; and (b)measuring
the level of N-CoR in the serum sample so obtained, wherein
the presence in the serum sample of increased levels of N-CoR
relative to a previously lower level indicates that the
patient is likely suffering from a recurrence of a tumor
overexpressing N-CoR.
In the method of the invention, tumors that overexpress N-CoR
may include glioblastoma multiforme, breast cancer, colorectal
cancer, small cell lung cancer and ovarian cancer.
This invention provides a method of treating a patient
suffering from glioblastoma multiforme, comprising
administering to the patient one or more phosphatase ligand,
alone or in combination with one or more retinoid receptor
ligand, or one or more histone deacetylase ligand, or both, in
each case in amounts effective to treat the patient.
The phosphatase ligand may be selected from the group
consisting of 1-nor-okadaone, antimonyl tartrate,
bioallethrin, calcineurin, cantharidic acid, cantharidin,
calyculin, cypermethrin, DARPP-32, deamidine, deltamethrin,
diaminopyrroloquinazolines, endothal, endothal thioanhydride,
fenvalerate, fostriecin, imidazoles, ketoconazole, L-4-
bromotetramisole, levamisole, 1-p-bromotetramisole, d-p-
bromotetramisole, p-hydroxylevamisole, microcystin LA,
microcystin LR, microcystin LW, microcystin RR, molybdate
salts, okadaic acid, okadol, norcantharidin, pentamidine,
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pentavalent antimonials, permethrin, phenylarsine oxide,
phloridzin, protein phosphatase inhibitor-1 (I-1), protein
phosphatase inhibitor-2 (I-2)pyrophosphate, salubrinal, sodium
fluoride, sodium orthovanadate, sodium stibogluconate,
tartrate salts, tautomycin, tetramisole, thrysiferyl-23-
acetate, vanadate, vanadium salts and antileishmaniasis
compounds, including suramin and analogues thereof.
In a presently preferred embodiment of the invention, the
phosphatase ligand is a protein phosphatase inhibitor, such as
endothal thioanhydride, endothal, norcantharidin or okadaic
acid.
The protein phosphatases of the subject application can be
tyrosine-specific, serine/threonine-specific, dual-specificity
phosphatases, alkaline phosphatases such as levamisole, and
acid phosphatases.
In the method of the invention, the retinoid receptor ligand
may be a retinoid, such as a retinoic acid, e.g. cis retinoic
acid or trans retinoic acid. The cis retinoic acid may be 13-
cis retinoic acid and the trans retinoic acid may be all-trans
retinoic acid.
In the practice of the method of the invention, the retinoid
receptor ligand may affect retinoid receptor activity but not
thyroid hormone receptor activity; alternatively or
additionally the retinoid receptor ligand may inhibit N-CoR
binding to the retinoid receptor but not N-CoR binding to the
thyroid hormone receptor.
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Retinoid receptor ligands used in the method of the invention
include vitamin A (retinol) and all its natural and synthetic
derivatives (retinoids).
In the method of the invention, the retinoid receptor ligand
may be selected from the group consisting of b,g-selective 6-
(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-
naph-thalenecarboxylic acid (TTNN), Z-oxime of 6-(5,6,7,8-
tetrahydro-5,5,8,8-tetramethyl-2-naphthalenylcarbonyl)-2-
naphthalenecarboxylic acid (SR11254), 4-(5,6,7,8-tetrahydro-
5,5,8,8-tetramethyl-2-anthracenyl) benzoic acid (TTAB), 4-[1-
(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-
cyclopropyl] benzoic acid (SR11246), 4-[l-(5,6,7,8-tetrahydro-
3,5,5,8,8-pentamethyl-2-naphthalenyl)-2 -methylpropenyl)benzoic
acid (SR11345), and 2-(6-carboxy-2-naphthalenyl)-2-(5,6,7,8-
tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1,3-dithiolane
(SR11253).
In the method of the invention, the histone deacetylase ligand
may be an inhibitor, e.g. the histone deacetylase inhibitor
HDAC-3 (histone deacetylase-3). The histone deacetylase ligand
may also be selected from the group consisting of 2-amino-8-
oxo-9,10-epoxy-decanoyl, 3-(4-aroyl-lH-pyrrol-2-yl)-N-hydroxy-
2-propenamide, APHA Compound 8, apicidin, arginine butyrate,
butyric acid, depsipeptide, depudecin, HDAC-3, m-
carboxycinnamic acid bis-hydroxamide, N-(2-aminophenyl)-4-[N-
(pyridin-3-ylmethoxycarbonyl) aminomethyl] benzamide, MS 275,
oxamfiatin, phenylbutyrate, pyroxamide, scriptaid, sirtinol,
sodium butyrate, suberic bishydroxamic acid, suberoylanilide
hydroxamic acid, trichostatin A, trapoxin A, trapoxin B and
valproic acid. The subject application encompasses compounds
which inhibit the enzyme histone deacetylase (HDAC) These
HDAC enzymes posttranslationally modify histones (U.S. Patent
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Publication No. 2004/0197888, Armour et al.) Histones are
groups of proteins which associate with DNA in eukaryotic
cells to form compacted structures called chromatin. This
compaction allows an enormous amount of DNA to be located
within the nucleus of a eukaryotic cell, but the compact
structure of chromatin restricts the access of transcription
factors to the DNA. Acetylation of the histones decreases the
compaction of the chromatin allowing transcription factors to
bind to the DNA. Deacetylation, catalysed by histone
deacetylases (HDACs), increases the compaction of chromatin,
thereby reducing transcription factor accessibility to DNA.
Therefore, inhibitors of histone deacetylases prevent the
compaction of chromatin, allowing transcription factors to
bind to DNA and increase expression of the genes.
This invention also provides a method of inhibiting growth of
a tumor in a patient suffering from glioblastoma multiforme,
comprising administering to the patient one or more
phosphatase ligand, alone or in combination with one or more
retinoid receptor ligand, one or more histone deacetylase
ligand, or both, in each case in amounts effective to affect
N-CoR so as to thereby induce differentiation of glioblastoma
multiforme tumor cells and inhibit growth of the tumor in the
patient.
The nuclear receptor co-repressor (N-CoR) of the subject
invention may be any molecule that binds to the ligand binding
domain of the DNA-bound thyroid hormone receptor (T3R) and
retinoic acid receptor (RAR). (U.S. Patent No. 6;949,624, Liu
et al.)
This invention further provides a method of identifying a
compound or a mixture of compounds capable of inducing
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differentiation or inhibiting proliferation of glioblastoma
multiforme tumor cells, comprising the steps of (a) culturing
a first population of human brain cells in the absence of the
compound or the mixture of compounds in both serum and serum
free conditions; (b) separately culturing a second population
of such human brain cells in the presence of the compound or
the mixture of compounds; (c) comparing the rate of growth of
the cultured human brain cells in step (a) with the rate of
growth of the cultured human brain cells in step (b); (d)
identifying the compound or the mixture of compounds which
inhibited, or reduced the rate of, growth of the cultured
human brain cells in step (b) as compared to the rate of
growth of the cultured human brain cells in step(a); and (e)
measuring the level of N-CoR and the level of a glioblastoma
multiforme lineage marker in the cytoplasm and in the nucleus
of the cultured human brain cells from step (b) whose growth
was inhibited or. whose rate of growth was reduced in the
presence of the compound or the mixture of compounds with the
levels of N-CoR and the glioblastoma multiforme lineage marker
in the cultured human brain cells from step (a), wherein a
decrease in the level of N-Cor and an increase in glioblastoma
multiforme lineage marker indicate that the compound or the
mixture of compounds is capable of inducing differentiation of
glioblastoma multiforme tumor cells, so as to thereby identify
the compound or the mixture of compounds.
In this method, the glioblastoma multiforme lineage marker may
be selected from the group consisting of GFAP, nestin, tujl,
and CNPase.
A glial fibrillary acidic protein (GFAP) useful in the subject
invention is a 55 kDa cytosolic protein, a major structural
component of astroglial filaments and the major intermediate
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filament protein in astrocytes. (U.S. Patent Publication No.
2004/0253637, Buechler et al.) GFAP is specific to astrocytes
of the brain.
In the method of the invention, the level of N-CoR and the
level of the glioblastoma multiforme lineage marker in the
cytoplasm and in the nucleus may be measured by either
indirect immunofluorescence microscopy, direct
immunofluorescence microscopy, FACS, or other methods for
detecting and measuring amounts of specific proteins in
tissues including assessment of the amounts of proteins in the
nucleus versus the cytoplasm and in cell lysates, or a
combination thereof.
N-CoR is expressed in the nucleus of the undifferentiated
tumor or stem cells and is only present in the cytoplasm at
amounts detectable by immunochemistry and Western blotting
when the cell undergoes differentiation. N-CoR is not
detectable by immunochemistry and Western blotting in either
the nucleus or the cytoplasm of normal or fully differentiated
cells.
Thus, in the methods of the invention, an assessment of the
percentage of cells with N-CoR in the cytoplasm relative to
the percentage of cells with N-CoR in the nucleus is
representative of the ratio of more differentiated cells to
less differentiated cells in a given tissue. In the method,
the first population of human brain cells and the second
population of human brain cells is selected from the group
consisting of primary normal human brain cells, primary human
brain stem cells, and primary glioblastoma multiforme stem
cells. For example, the first population of human brain cells
and the second population of human brain cells may be the same
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or different, preferably the same and may be cells derived
from any of the following cell lines: U343 MG-A, U251, U373,
U87, A-172, LN-18, LN-229, M059J, M059K, and HS683.
Cell line U343 MG-A is available from the University of
California at San Francisco (UCSF) Brain Tumor Research Center
Tissue Bank. (University of California, San Francisco, Health
Sciences West building, San Francisco, California 94143-0520.)
In addition, cell lines U343 and U87 are commercially
available from EPO-GmbH, Robert-Rossle-Str.10, 13092 Berlin-
Buch, Germany.
Cell line U251 is available from Division of Cancer Treatment
and Diagnosis at National Cancer Institute Tumor Repository,
The National Cancer Institute at Frederick Bldg. 1073,
Frederick, Maryland 21702-1201.
Cell lines A-172, LN-18, LN-229, M059J, M059K, and HS683 are
available from the American Type Culture Collection (ATCC),
P.O. Box 1549, Manassas, Virginia, 20108, as ATCC No. CRL-
1620, ATCC No. CRL-2610, ATCC No. CRL-229, ATCC No. CRL-2365
and ATCC No. HTB-138, respectively.
Cell line U373 is available from the National Institute of
Neurological Disease and Stroke, Building 31, 31 Center Drive,
Bethesda, MD, 20892 and the National Institute of Health,
Building 1, 1 Center Drive, Bethesda, Maryland, 20892.
This invention also provides a method of determining the
likelihood of successfully treating a subject suffering from
glioblastoma multiforme, comprising the steps of (a) obtaining
a sample from the subject containing glioblastoma multiforme
cells; and (b) measuring the level of each of N-CoR and a
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glioblastoma multiforme lineage marker in the cytoplasm and in
the nucleus of cells in the sample so obtained, wherein the
presence in the sample of an increased level of N-CoR in the
nucleus indicated that there is a greater likelihood of
successfully treating the subject.
In the preceding method, the glioblastoma multiforme lineage
marker may be selected from the group consisting of GFAP,
nestin, tujl, and CNPase.
In the method, the level of N-CoR and the level of the
glioblastoma multiforme lineage marker in the cytoplasm and in
the nucleus may be measured by either indirect
immunofluorescence microscopy, direct immunofluorescence
microscopy, FACS, or other methods for detecting and measuring
amounts of specific proteins in tissues including assessment
of the amounts of proteins in the nucleus versus the cytoplasm
and in cell lysates, or a combination thereof.
N-CoR is expressed in the nucleus of the undifferentiated
tumor or stem cells and is only present in the cytoplasm at
amounts detectable by immunochemistry and Western blotting
when the cell undergoes differentiation. N-CoR is not
detectable by immunochemistry and Western blotting in either
the nucleus or the cytoplasm of normal or fully differentiated
cells.
Thus, in the methods of the invention, an assessment of the
percentage of cells with N-CoR in the cytoplasm relative to
the percentage of cells with N-CoR in the nucleus is
representative of the ratio of more differentiated cells to
less differentiated cells in a given tissue.
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This invention also provides a method of assessing the
likelihood that a patient is suffering from glioblastoma
multiforme, comprising the steps of (a) obtaining a sample of
cerebrospinal fluid and/or tumor cells from the subject; and
(b) measuring the level of N-CoR in the cerebrospinal fluid
and/or the cells in the sample so obtained, wherein the
presence in the sample of increased levels of N-CoR in the
cerebrospinal fluid relative to a normal reference standard
indicates that the patient is likely suffering from
glioblastoma multiforme. If N-CoR is increased in the serum
but not in the cerebral spinal fluid this would indicate that
the patient is likely suffering from a tumor overexpressing N-
CoR but not necessarily a glioblastoma multiforme.
This invention also provides a method of assessing the
likelihood that a patient previously suffering from and
treated for glioblastoma multiforme has suffered a recurrence
of glioblastoma multiforme, comprising the steps of (a)
obtaining a sample of cerebrospinal fluid and/or tumor cells
from the subject; and (b) measuring the level of N-CoR in the
cerebrospinal fluid and/or the cells in the sample so
obtained, wherein the presence in the sample of increased
levels of N-CoR in the cerebrospinal fluid relative to the
previous levels of N-CoR post-treatment indicates that the
patient is likely suffering from a recurrence of glioblastoma
multiforme.
This invention further provides a method of assessing the
likelihood that a patient is suffering from a tumor
overexpressing N-CoR, comprising the steps of (a) obtaining a
serum sample from the subject; and (b) measuring the level of
N-CoR in the serum sample so obtained, wherein the presence in
the serum sample of increased levels of N-CoR relative to a
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normal reference standard indicates that the patient is likely
suffering from a tumor overexpressing N-CoR.
This invention still further provides a method of assessing
the likelihood that a patient- previously suffering from and
treated for a tumor overexpressing N-CoR has suffered a
recurrence of a tumor overexpressing N-CoR, comprising the
steps of (a) obtaining a serum sample from the subject; and
(b) measuring the level of N-CoR in the serum sample so
obtained, wherein the presence in the serum sample of
increased levels of N-CoR relative to a previous lower levels
of N-CoR post treatment indicates that the patient is likely
suffering from a recurrence of a tumor overexpressing N-CoR.
The invention provides a use of one or more phosphatase
ligand, in an amount effective to treat a patient, alone or in
combination with one or more retinoid receptor ligand, or one
or more histone deacetylase ligand, or both, in each case in
an amount effective to treat the patient, for the preparation
of a medicament. In an embodiment of the invention, the
medicament comprises one or more phosphatase ligand alone, or
for use with, one or more retinoid receptor ligand, or one or
more histone deacetylase receptor ligand, or both.
This invention also provides the use of a phosphatase ligand,
in an amount effective to induce differentiation of cells of a
tumor overexpressing N-CoR=and to inhibit the growth of the
tumor in a patient, alone or in combination with one or more
retinoid receptor ligand, one or more histone deacetylase
ligand, or both, for the preparation of a medicament. In an
embodiment of the invention, the medicament comprises one or
more phosphatase ligand alone, or for use with, one or more
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retinoid receptor ligand, or one or more histone deacetylase
receptor ligand, or both.
The uses of the invention herein encompass the enumerated
phosphatase ligands, retinoid receptors and histone
deacetylase receptor ligands enumerated above.
The invention provides a product containing a phosphatase
ligand in combination with one or more retinoid receptor
ligand, one or more histone deacetylase ligand, or both, as a
combined preparation for simultaneous, separate or sequential
use in treating a tumor overexpressing N-CoR.
This invention is illustrated in the Experimental Details
section which follows. This section is set forth to aid in an
understanding of the invention but is not intended to, and
should not be construed to limit in any way the invention as
set forth in the claims which follow thereafter.
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Experimental Deta3.ls
Materials and Methods
Example 1:
To identify novel therapeutic targets for the treatment of
glioblastoma multiforme, the proteomes of 7 GBM tissues and 7
normal brain tissues (white matter) were compared using
selective microdissection, two dimensional gel electrophoresis
(2-DGE) and liquid chromatography-mass spectroscopy (LCMS).
GBM tissue was further tested by immunohistochemistry and
Western blotting for the expression of nuclear receptor co-
repressor (N-CoR). (3-actin was used as internal positive
quantitative control for the Western blotting.
Expression of glial fibrillary acidic protein (GFAP), an
established marker of astroglial differentiation and the
subcellular localization of N-CoR was assessed by indirect
immunofluorescence microscopy was on primary cell cultures,
established cell lines (A-172, HS683, U87, U251, and U343 MG-
A), and frozen and paraffin-embedded tissue sections of GBM.
The GBM cell lines were all cultured in DMEM with 10% FCS and
high glucose DMEM/F12 with N2 supplement (serum-free) on poly-
1-ornithine and fibronectin coated plate/dish/flask.
To examine the role of N-CoR in GBM development and
differentiation, cultured brain tumor stem cells (BTSC)
isolated from GBM were treated with ciliary neurotrophic
factor (CNTF), an agent which has previously been shown to
induce the astrocytic differentiation of neural stem cells
(NSC) in vitro. (Hughes et al. (1988)) The BTSCs cells were
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then assessed for GFAP expression along with subcellular N-CoR
localization.
Finally, the GBM cell line U343 MG-A was treated with 5OpM of
retinoic acid (RA) and/or lOnM of okadaic acid (OA), a protein
phosphatase-1 inhibitor.
Three cultures for each of four treatments were grown for 16
days with cell counts obtained at baseline and for each of the
eight even numbered days. Log transformations were applied to
the cell counts, and repeated measures of analysis of variance
model used to evaluate the treatment differences over time.
Pair wise treatment comparisons used Sidak's statistic to
account for multiple testing.
Results
Comparative proteomic analysis of glioblastoma multiforme
(GBM) tissue and matched normal glial tissue demonstrated
increased expression of the nuclear receptor co-repressor (N-
CoR) in GBM. GBM tumor cells with nuclear localization of N-
CoR were relatively undifferentiated, but subject to
differentiation upon exposure to agents promoting
phosphorylation of N-CoR and its translocation to the
cytoplasm.
As shown in Figure 1A, total proteomic analysis of
microdissected normal glial tissue (white matter) compared
with GBM shows a consistent protein pattern in normal white
matter and GBM, and a unique expression of N-CoR in GBM. This
expression of N-CoR in GBM tissue was confirmed by
immunohistochemistry and Western blotting as shown in Figures
1B and 1C. In contrast, N-CoR was not detectable in normal
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brain topographically matched to the location of the tumor
specimen.
As shown in Figure 1D, Nuclear expression of N-CoR correlated
with the absence of GFAP expression in the cells of primary
cultures and tissue sections, whereas cytoplasmic expression
of N-CoR correlated with positive expression of GFAP.
Subcellular localization of N-CoR correlates with glial
differentiation. Some of the tumor cells with nuclear
expression of N-CoR also expressed CD133 as shown in Figure
1E.
As shown in Figure 2A, brain tumor stem cells (BTSCs) cultured
with CNTF began to express GFAP. Western blot analysis of the
cytoplasmic fraction of CNTF-treated BTSCs demonstrates the
translocation of N-CoR to the cytoplasm (See Figure 2B). Both
cytoplasmic N-CoR and GFAP expression peaked at day 7
following CNTF stimulation.
Figure 2C shows the logarithmic growth curve of gliomal cell
line, U343 MG-A, treated with retinoic acid (RA), okadaic acid
(OA), combination of retinoic acid and okadaic acid (RA/OA),
and control (NC) for 16 days. Curve fitting indicated
exponential cell count growth for each treatment except for
the RA/OA treatment group. Analysis of variance models were
used to examine differences across the three cultures for each
treatment. For each treatment group the cultures were similar.
~~.
Model based F-statistics indicated significant differences
across time (p=0.006) and for time by treatment interactions
(p=0.001). The differences in log cell counts among the four
treatments were significant with an F3,8=163.2 and the
resulting p-value less than 0.0001. All pair wise treatment
differences (Sidak's test) were significant with p-values less
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than 0.0001, except the RA vs OA difference (p=0.079). Cell
counts monotonically increased for OA, RA, and controls
through day 16. The number of cells with the combination
treatment (OA+RA) increased until day 10 and then decreased
for the remaining duration of the study. Retinoic acid and
low-dose okadaic acid each had a modest effect on cell growth.
Combination of retinoic acid and okadaic acid shows
synergistic reduction in cell growth.
Example 2: Effect of cantharidin analogs on GBM cells
To identify novel therapeutic targets for the treatment of
glioblastoma multiforme (GBM), cantharidin analogs were
evaluated for their ability to inhibit growth of glioblastoma
multiforme cells. Specifically, GBM cell line U373 was used
in evaluations.
The cantharidin homologs that were evaluated were
norcantharidin (nor-Can), which is a demethylated cantharidin;
endothal (End), which is a dicarboxylic acid derivative of
norcantharidin; endothal thioanhydride (ET); and the compound
LB-1, which was obtained from Lixte Biotechnology, Inc., 248
Route 25A, No. 2, East Setauket, New York, which has the
structure:
C02
TC CO N N CH3
H
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Cells were plated in triplicate on day one with and without
different amounts of each drug dissolved in media (compound
LB-1 and endothal) or in dimethylsulfoxide (endothal
thioanhydride and norcantharidin). The total number of cells
is counted in the triplicate cultures at each dose and in
controls after 7 days and the average number of cells and the
standard deviation is determined.
The amount of inhibition of GBM cell growth is expressed as
the proportion of the number of cells in the experimental
dishes compared to the number of cells in control dishes
containing only the drug vehicle and culture medium. The
average percent of control is plotted and bracketed by one
standard deviation calculated from the triplicate
measurements.
Results
Each of the norcantharidin analogs inhibited the growth of
GBMs in a dose dependent manner in vivo as shown in Figure 3.
From graphic plots of the GBM cell line U373 as a function of
exposure to different doses of drug for 7 days, the
concentration of each compound that inhibited brain tumor cell
proliferation by 50% (IC50) was estimated. The IC50s
expressed in micro-molarity (uM), were: 2.5, 3.0, 12.0, and
15.0 for endothal thioanhydride, compound LB-1,
norcantharidin, and endothal respectively as seen in figure 3.
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Example 3: Effect of selected canthari.din analogs combined
with Retinoic Acid
To identify the effect of combinations of PP2A anti-
phosphatases and retinoids affecting nuclear complexes, we
focused on water soluble cantharidin derivatives that have
been shown to be active against human GBMs in vitro, endothal
and compound LB-1.
To observe the effects of endothal in combination with
retinoic acids, endothal was combined with all-trans retinoic
acid and 13-cis retinoic acid.
Cells were plated in triplicate on day one with and without
different amounts of each drug dissolved in media (compound
LB-1 and endothal). The total number of cells is counted in
the triplicate cultures at each dose and in controls after 7
days and the average number of cells and the standard
deviation is'determined.
The amount of inhibition of GBM cell growth is expressed as
the proportion of the number of cells in the experimental
dishes compared to the number of cells in control dishes
containing only the drug vehicle and culture medium. The
average percent of control is plotted and bracketed by one
standard deviation calculated from the triplicate
measurements.
Results
Figure 4A demonstrates the effect of all-trans retinoic acid
(ATRA) when used individually. The IC50 of ATRA alone was
greater than 50 pM. Endothal and compound LB-1, each in
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combination with ATRA, synergistically inhibited proliferation
of GBM cell line U373 as seen in figure 4B. Synergism
(potentiation), of the inhibitory activity of two drugs in
combination is said to be present when the percent survival in
the presence of two drugs is less than the product of the
percent survivals of the two drugs used alone at the same
doses in the combination. The extent of synergism of compound
LB-1 and endothal (end) in combination with ATRA is quantified
below in Table 1:
Table 1. Endothal and compound LB-1 +/- ATRA Inhibition of
U373 Cells.
Percent of Control
Observed Expected if
Additive
ATRA 25 uM 77% -
END 10 uM 65% -
ATRA 25 uM + END 10 uM 32% 50%
LB-1 1 uM 78% -
ATRA 25 uM + LB-1 luM 53% 60%
The expected percent survival of U373 cells exposed to the
combination of ATRA and End is 50% (77% by ATRA x 65% by End =
50%) whereas the observed survival was 32%. The expected
percent survival in the presence of the combination of ATRA
and LB-1 is 60% (77% by ATRA x 78% by LB-1 = 60%), whereas the
observed survival was 53%.
Endothal combined with 13-cis retinoic acid (cis-RA)
synergistically inhibits U373 cell growth as seen in Figure
4C. The extent of synergism is quantified below in Table 2.
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Table 2. Endothal +/- 13-Cis RA: Inhibition of U373 Cells
13-Cis Retinoic Acid
None 50uM
Endothal (pM) Percent of Control
1 }zM 100% 94%
pM 96% 80%
PM 73% 53%
The presence of 13-cis retinoic acid at 50 pM had little
5 effect when combined with endothal at 1.0 pM (96%). At higher
doses of endothal, simultaneous exposure to the same amount of
13-cis retinoic acid decreased cell survival from 96% at 5 uM
endothal alone to 80% in combination with 13-cis retinoic acid
and from 76% survival at 10 uM endothal alone to 53% in
10 combination with 13-cis retinoic acid.
Example 4. Effect of histone deacetylase ligands on U373 GBMs
Because retinoids are known to produce developmental
abnormalities in the fetus when the drug is given to pregnant
women, we studied the activity of valproic acid and
Trichostatin A, drugs with low toxicity in the adult, but
which also disrupt fetal development.
Results-
Both valproic acid (Val) (Figure 5A) and Trichostantin A (TSA)
(Figure 5B) had dose dependent activity as a single agent
against U373 cell growth. Although inhibitory doses of
valproic acid were in the mM range, the antiepileptic drug is
tolerated in humans at serum concentrations approaching 1.0 mM
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for weeks. Trichostatin A, in contrast, is active at nM
concentrations against U373.
Compound LB-1, when combined with Trichostatin A or when
combined with 13-cis retinoic acid synergistically inhibited
the growth of GBM cell line U373 as shown below in Table 3.
Table 3. LB-i +/- 13-cis Retinoic Acid (CIS-RA) and LB-1 +/-
Trichostatin A (TSA) Inhibition of GBM Cell Line U373
Percent of Control
Observed Expected If
Additive
Cis-RA 50 pM 93.3 +/- 2.2
TSA 0.033 pM (0.01 pg/ml) 71.6 +/- 0.4
LB-1 1 pM 97.9 +/- 1.0
LB-1 5 pM 52.5 +/- 2.9
Cis-RA 50 pM + LB-1 1pM 79.3 +/- 3.2 91.3
Cis-RA 50 pM + LB-1 5 pM 31.6 +/- 2.0 49.0
TSA 0.033 pM + LB-1 1 pM 65.7 +/- 2.0 70.1
TSA 0.033 pM + LB-1 5 pM 13.9 +/- 1.0 37.6
The two drugs are synergistic in their inhibition of the
growth of U373 cells. The percent survival of the cells after
exposure to two drugs in combination is less than would be
expected from the percent survival of the cells when exposed
to each of the two drugs at the same doses used in the
combination.
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Example 5: Determination of tumor type specificity.
To determine whether there is tumor type specificity of the
inhibitory properties of PP2A inhibitors, retinoic acid and
Trichostatin A, we measured their inhibitory effects as single
agents against the GBM line U373, a breast cancer line, MCF-7
(obtained from ATCC) and a kidney cancer cell line, UMRC (UMRC
obtained by Dr. Zhuang, NINDS, NIH from the Intramural
Research Support Program, SAIC, National Cancer Institute,
Frederick Cancer Research and Development Center).
Results:
The kidney cancer cell line, UMRC (Figure 6A) was less
sensitive than the brain tumor line, U373 (Figure 6B) whereas
the breast cancer line, MCF-7 (Figure 6C) was as sensitive as
U373 to all-trans retinoic acid, endothal thioanhydride,
norcantharidin, endothal, and Trichostatin A. There is some
cell type specificity of these drugs for GBMs. The activity of
the drugs against MCF-7 cells indicates that regimens being
developed for brain tumor treatment may also be useful against
breast cancer as well as other tumors that overexpress N-CoR.
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Discussion
To identify novel therapeutic targets, the proteomes of 7 GBM
tissues and 7 normal brain tissues were compared using
selective microdissection, two dimensional gel electrophoresis
(2-DGE) and liquid chromatography-mass spectroscopy (LCMS).
One protein found to have increased expression in GBM as
compared to normal brain is nuclear receptor co-repressor (N-
CoR), a regulator of the normal neural stem cell pool. (See
Figure lA) Expression of N-CoR in GBM was confirmed by
immunohistochemistry and Western blotting. (See Figures 1B and
1C) In contrast, N-CoR was not detectable in normal brain
topographically matched to the location of the tumor specimen.
N-CoR is expressed in the nucleus of neural stem cells (NSCs).
(Hermanson et al. (2002)) Following phosphatidyl-inositol-3-OH
kinase/Aktl kinase-dependent phosphorylation, N-CoR
translocates to the cytoplasm and leads to astrocytic
differentiation of NSCs. The nuclear retention of N-CoR,
therefore, is essential for the maintenance of NSCs in the
undifferentiated state (Hermanson et al.). Analagous to CD133+
NSC found within the developing brain, brain tumor stem cells
(BTSC) bearing CD133 have been identified within GBM. (Uchida
et al. (2000); and Singh et al. (2003)) BTSC are capable of
proliferation, self-renewal, and differentiation. BTSC, but
not CD133- differentiated tumor cells, are able to recapitulate
tumors upon xenograft transplantation. (Singh et al. (2004)).
To characterize a potential role for N-CoR in BTSC
differentiation, the relationship between astroglial
differentiation within GBMs and N-CoR localization was
investigated. Expression of glial fibrillary acidic protein
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(GFAP), an established marker of astroglial differentiation,
and the subcellular localization of N-CoR was assessed by
indirect immunofluorescence microscopy on primary cell
cultures, established cell lines (A-172, HS683, U87, U251, and
U343 MG-A), and frozen and paraffin-embedded tissue sections
of GBM. Nuclear expression of N-CoR correlated with the
absence of GFAP expression in the cells of primary cultures
and tissue sections (see Figure 1D), whereas cytoplasmic
expression of N-CoR correlated with positive expression of
GFAP (see Figure 1D). Some of the tumor cells with nuclear
expression of N-CoR also expressed CD133 (see Figure 1E).
To examine the role of N-CoR in GBM development and
differentiation, cultured BTSC isolated from GBM were treated
with ciliary neurotrophic factor (CNTF), an agent which has
previously been shown to induce the astrocytic differentiation
of NSC in vitro. (Hughes et a1.(1988)). The cultured BTSC were
then assessed for GFAP expression along with subcellular N-CoR
localization. Similar to NSC, BTSC cultured with CNTF began to
express GFAP (see Figure 2A). Western blot analysis of the
cytoplasmic fraction of CNTF-treated BTSC demonstrates
translocation of N-CoR to the cytoplasm (see Figure 2B). Both
cytoplasmic N-CoR and GFAP expression peaked at day 7
following CNTF stimulation.
Retinoids, metabolites of vitamin A, have been examined
therapeutically in a variety of tumors, including gliomas.
(Yung et al. (1996)) N-CoR is closely associated with the
retinoid receptor and is released upon ligand binding to the
receptor. (Bastien et al. (2004)) We hypothesized that one
effect of retinoids on malignant gliomas may be the induction
of differentiation by the binding of retinoids to the retinoid
receptor followed by dissociation of the N-CoR/retinoid
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receptor complex and translocation of N-CoR to the cytoplasm.
This idea would explain the previous observation of increase
GFAP expression in a glioma cell line (U343 MG-A) treated with
retinoids. (Rudka et al. (1988)) To test this we targeted two
different sites, individually or simultaneously, within the N-
CoR pathway by treating the GBM cell line U343 MG-A with 50 M
of retinoic acid (RA) and/or 10 nM of okadaic acid (OA), a
protein phosphatase-1 and protein phosphatase-2A inhibitor. By
preventing the action of protein phosphatase-1 and protein
phosphatase-2A, okadaic acid increases the phosphorylated form
of N-CoR and promotes its subsequent cytoplasmic
translocation. (Hermanson et al. (2002))
Cell counts monotonically increased for OA, RA, and controls
through day 16 (see Figure 2C). Retinoic acid and low-dose
okadaic acid each had a modest effect on cell growth.
Combination of retinoic acid and okadaic acid shows
synergistic reduction in cell growth.
These observations demonstrate a role for N-CoR in BTSC
differentiation and suggest a new treatment paradigm for
glioblastoma multiforme. To our knowledge, this is the first
example of a therapeutic strategy directly targeting BTSCs.
Differentiation and growth inhibition of BTSCs is achieved by
the synergistic combination of two compounds acting at
different levels of the N-CoR pathway.
Several molecules, including okadaic acid, that have anti-PP2A
activity synergize with all-trans retinoic acid and 13-cis
retinoic acid in inhibiting the growth of GBM cells in vitro.
The most effective group of phosphatase inhibitors synergizing
with retinoic acids that have been evaluated are analogs of
the ancient therapeutic agent, mylabris, derived from the
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crushed bodies of the blister beetle, in which the principal
active agent is cantharidin, a known potent inhibitor of PP2A
(Wang, 1989; Peng et al., 2002).
Cantharidin has anti-tumor activity against human cancers of
the liver (hepatomas) and of the upper gastrointestinal tract
but is toxic to the urinary tract (Wang, 1989).
Norcantharidin, a demethylated cantharidin, maintains
antitumor activity of cantharidin against hepatomas and
cancers of the stomach and esophagus, but has little or no
urinary tract toxicity. Norcantharidin increased the life
span of 244 patients with primary hepatoma from 4.7 to 11.1
months and increased 1-year survival from 17% to 30% compared
to historical control patients treated with standard
chemotherapy. Norcantharidin also stimulates white blood cell
production in patients and mice, a phenomenon not understood
mechanistically, but a pharmacological effect of potential
benefit as an anticancer agent (Wang et al., 1986; Wang,
1989).
In the past, several cantharidin analogs had been synthesized
and evaluated for anti-phosphatase activity and for their
ability to inhibit the growth of cancer cells in culture
(Sakoff and McClusky, 2004; Hart et al.; 2004). Some of the
previously evaluated modified norcantharidin molecules
inhibited the growth of several human tumor cell lines. The
activity of norcantharidin analogs against GBMs or the
activity of norcantharidins combined with other potential
anti-tumor agents was not analyzed. Further studies included
16 "modifi.ed norcantharidins" evaluated for activity against
four human tumor cell lines including ovarian, kidney,
colorectal and lung as well as a mouse leukemia line. None
were as active as single agents as cantharidin or
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norcantharidin and none were evaluated for activity in
combination with another antitumor agent (McCluskey et al., US
Serial No. 2006/0030616, 2006).
A different series of cantharidin analogs had been previously
synthesized and evaluated as pesticides and for antitumor
activity against cancer cell lines. The dicarboxylic acid
derivative of norcantharidin, endothal, was developed as an
herbicide and defoliant. Forty-three analogs of endothal and
cantharidin have been developed and assessed for their
activity as herbicides and their lethality to mice (Matsuzawa
et al., 1987). Endothal thioanhydride was shown to be a more
potent insecticide than endothal but was toxic to the liver of
mice (Matsuzawa et al., 1987; Kawamura et al., 1990).
Endothal and endothal thioanhydride, like cantharidin, inhibit
the activity of PP2A and to some extent, the activity of PP1
(Erdodi et al., 1995). In cell lysates the order of the
potency of inhibition of PP2A was cantharidin, endothal and
endothal thioanhydride. In the liver of the intact animal, the
potency of the inhibition of PP2A was endothal thioanhydride,
followed by cantharidin and -endothal. The differences
depended on the lipid solubility of the compounds (Erdodi,
1995). Endothal thioanhydride is highly lipid soluble and
insoluble in water and is the most toxic in vivo, whereas
endothal is highly water soluble and is the least toxic, with
norcantharidin falling in between with respect to lipid
solubility and toxicity.
To identify novel therapeutic targets for the treatment of
glioblastoma multiforme (GBM), compound LB-l, norcantharidin
(nor-Can), endothal (End), and endothal thioanhydride (ET)
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were evaluated for their ability to inhibit glioblastoma
multiforme.
Each of the norcantharidin analogs inhibited the growth of
GBMs in a dose dependent manner in vivo as shown in Figure 3.
From graphic plots of the GBM cell line U373 as a function of
exposure to different doses of drug for 7 days, the
concentration of each compound that inhibited brain tumor cell
proliferation by 50% (IC50) was estimated. The IC50s
expressed in micro-molarity (pM), were: 2_5, 3.0, 12.0, and
15.0 for endothal thioanhydride, compound LB-1,
norcantharidin, and endothal respectively as seen in figure 3.
We found that, on a molar basis, of the phosphatase inhibitors
tested, endothal thioanhydride was the most potent inhibitor
of GBMs in vitro compared to norcantharidin and endothal.
In combination with anti-phosphatases, retinoids
synergistically inhibit the proliferation of glioblastoma
multiforme. Synergism (potentiation) of the inhibitory
activity of two drugs in combination is said to be present
when the percent survival in the presence of two drugs is less
than the product of the percent survivals of the two drugs
used alone at the same doses in the combination.
The inhibitory activity of retinoids was further evaluated in
combination with endothal as well as individually. As shown
in Figure 4A, increase in the dose of ATRA exhibits inhibitory
activity on glial cancer cells. However, as demonstrated in
Figure 4B, the combination of endothal with ATRA demonstrated
a synergistic reduction in cell growth..
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The expected percent survival of U373 cells exposed to the
combination of ATRA and End is 50% (77% by ATRA x 65% by End =
50%) whereas the observed survival was 32%. The expected
percent survival in the presence of the combination of ATRA
and LB-1 is 60% (77% by ATRA x 78% by LB-1 = 60%), whereas the
observed survival was 53%.
Endothal combined with 13-cis retinoic acid (cis-RA)
synergistically inhibits U373 cell growth over a period of 7
days as compared to the more modest inhibition when endothal
was used individually as seen in figure 4C.
The presence of 13-cis retinoic acid at 50 uM had little
effect when combined with endothal at 1. 0}zM ( 96 %). At higher
doses of endothal, simultaneous exposure to the same amount of
13-cis retinoic acid decreased cell survival from 96% at 5 uM
endothal alone to 80% in combination with 13-cis retinoic acid
and from 76% survival at 10 uM endothal alone to 53% in
combination with 13-cis retinoic acid.
Trichostatin A is a natural product extracted from
streptomyces, which has anti-fungal and anti-cancer activity
in vitro and in human cancer xenografts (Yoshida et al., 1990;
Sanderson et al., 2004). Valproic acid is a widely used anti-
seizure medicine that inhibits human cancer cells in vitro at
concentrations achievable in the plasma of humans (Gottlicher
et al., 2001; Blaheta et al., 2002). As demonstrated in
Figures 5A and 5C, both valproic acid (Val) and Trichostatin A
(TSA) had dose dependent activity as single agents against
, U373 cell growth. Although inhibitory doses of valproic acid
were in the mM range, the drug is tolerated in humans at serum
concentrations approaching 1.0 mM for weeks. Trichostatin A,
by contrast, is active at nM concentrations against U373.
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Given the low toxicity in non-pregnant adults, both compounds
combined with endothal could be potentially effective regimens
in the treatment of GBM in humans.
Cantharidin homologs and okadaic acid act synergistically when
administered with valproic acid or trichostatin A to inhibit
growth of GBM cells. Both valproic acid and trichostatin A
are known to have anti-histone deacetylase (HDAC) activity.
To determine whether there is tumor type specificity of the
inhibitory properties of PP2A inhibitors, retinoic acid and
Trichostatin A we measured their inhibitory effects as single
agents against the GBM line U373, a breast cancer line, MCF-7
(obtained from ATCC) and a kidney cancer cell line, UMRC (UMRC
obtained by Dr. Zhuang, NINDS, NIH from the Intramural
Research Support Program, SAIC, National Cancer Institute,
Frederick Cancer Research and Development Center).
The kidney cancer cell line, UMRC (Figure 6A) was less
sensitive than the brain tumor line, U373 (Figure 6B) whereas
the breast cancer line, MCF-7 (Figure 6C) was as sensitive as
U373 to all-trans retinoic acid, endothal thioanhydride,
norcantharidin, endothal, and Trichostatin A. There is some
cell type specificity of these drugs for GBMs. The activity of
the drugs against MCF-7 cells indicates that regimens being
developed for brain tumor treatment are likely also useful
against breast cancer and other tumors that overexpress N-CoR.
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