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Experimental Therapeutics, Preclinical Pharmacology |
-induced Apoptosis in Prostate Cancer Cells through Inhibition of Nuclear Factor-
B by an IB "Super-Repressor"1
Department of Internal Medicine, Division of Hematology/Oncology [H. J. M., M. A. W., K. J. P.], Department of Animal Medicine, Division of Pathology, [D-L. L., E. T. K.], and Department of Surgery, Division of Urology [K. J. P.], University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) levels. Many
androgen-insensitive prostate cancer cells are TNF- insensitive
because of the expression of antiapoptotic genes as part of the nuclear
factor-
B (NF-B) family of transcription factors. NF-B
stimulates gene transcription when expressed in the nucleus;
however, in resting cells, this nuclear import is prevented by
association with the cytoplasmic inhibitor IB. This cytoplasmic
retention of NF-B is uncoupled by many extracellular signals
including low levels of TNF-. During normal cell activation, nuclear
translocation of NF-B is preceded by phosphorylation and degradation
of IB. When phosphorylation is blocked, IB remains intact,
thereby blocking NF-B translocation to the nucleus and subsequent
activation of antiapoptotic genes that cause TNF- insensitivity. We
tested whether a "super-repressor" of NF-B activity could be
transfected into prostate cancer cells and make them TNF- sensitive.
PC-3 and LNCaP cells were stimulated with TNF- (10 ng/ml) for
24 h in the presence or absence of the IB
"super-repressor" (p6R-IBS32A + S36A). NF-B
activity was measured by electrophoretic mobility shift assay and the
steady state levels of the cytoplasmic IB protein were measured
by Western blot. Secretory IL-6 and IL-6 mRNA were measured by ELISA.
p6R-IBS32A + S36A blocked the stimulation of NF-B
activity by TNF- in prostate cancer cells. It also subsequently
decreased IL-6 production by TNF-. We conclude that these data
demonstrate that inhibition of NF-B selectively sensitizes
previously insensitive prostate cancer cells to TNF-. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3
is
known to possess potent antitumor activity both in vivo
(1)
and in vitro (2
, 3)
. TNF- is a polypeptide mediator of a variety of cellular
responses, including apoptotic or necrotic cell lysis and proliferation
(4
, 5)
. It is predominately released from macrophages in
response to foreign microbial components and has been established as an
important mediator of tumor cell death, as demonstrated in previous
experiments by others as well as the principal investigator and
colleagues (3
, 6, 7, 8)
.
TNF- is not released from prostate cancer cells themselves but from
associated macrophages after a relapse in disease (9)
.
Studies have shown that patients with hormone-refractory prostate
cancer demonstrate high serum TNF- levels as compared with untreated
patients (9, 10, 11, 12)
. The relationship between TNF-
sensitivity and hormone responsiveness has not yet been explored.
However, androgen-insensitive prostate cancer cells, PC-3 and JCA-1,
have proven to be TNF- insensitive, whereas androgen-sensitive
prostate cancer cells, LNCaP, are TNF- sensitive (13)
.
TNF- triggers a number of signal transduction processes, which lead
to either apoptosis or proliferation based on the TNF- threshold of
a cell. Most cells do not undergo apoptosis when exposed to low levels
of TNF- (Fig. 1A)
. This is
likely a protective mechanism by the cell based on the induction by
TNF- of antiapoptotic genes such as A20, the Bcl-2 family member A1,
manganese superoxide dismutase, and cellular inhibitor of apoptosis-2,
all targets of the NF-B family of transcription factors
(14)
. It is this activation of antiapoptotic genes that
makes cells insensitive to TNF--induced apoptosis. Apoptosis is
induced by high levels of TNF- binding to its receptor, TNF-RI, and
activating members of the caspase family of proteases (Fig. 1B
; Ref. 14
). TNF--insensitive cells do not
undergo TNF--induced apoptosis, regardless of the amount of TNF-
present (Fig. 1C)
. Instead, TNF- causes constitutive
expression of antiapoptotic genes protecting the cells from
TNF--induced apoptosis.
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B is a well-established inducible transcription factor
of great importance in cytokine-mediated inflammation
(15)
. It is responsible for the rapid induction of many
cytokines and adhesion molecules involved in the inflammatory and
immune response systems (15
, 16)
. Specifically, NF-B
participates in the activation of TNF--dependent expression of IL-6,
intercellular adhesion molecule-1, and matrix metalloproteinase-9,
among others, in various cell systems (17
, 18)
. IL-6, an
inflammatory cytokine, is a known autocrine and paracrine growth factor
in androgen-insensitive human prostate carcinomas (17)
.
Intercellular adhesion molecule-1 is an established adhesion molecule
involved in prostate cancer metastasis (17)
, whereas
matrix metalloproteinase-9 plays an essential role in the destruction
of the basement membrane because of its ability to proteolyze type IV
collagen (18)
.
The prototypical form of NF-B is a heterodimeric inducible complex
containing two DNA binding subunits, p50 (NF-B1) and p65 (RelA),
both of which belong to the Rel family of transcription factors
(19
, 20) . This heterodimer is the most potent gene
transactivator within the NF-B family (21)
. When
NF-B is expressed in the nucleus, it stimulates gene transcription
via the potent transactivation domain located within the COOH-terminal
half of RelA (22)
. However, in resting cells, the nuclear
import of NF-B is prevented because of a high-affinity association
of its RelA subunit with a labile cytoplasmic inhibitor called IB
(19
, 23)
. This IB-dependent mechanism for the
cytoplasmic retention of NF-B is uncoupled by many extracellular
signals including low levels of TNF- (24)
. After this
cellular stimulation, IB is phosphorylated at serines 32 and 36
by a specific kinase IKK (25)
, ubiquitinated, and
undergoes proteolysis in proteosomes, enabling NF-B to translocate
to the nucleus, where it binds to NF-B DNA binding sites and
stimulates transcription of many cytokines, chemokines, and adhesion
molecules (26)
.
A "super-repressor" form of IB with mutations at serines 32
and 36, located in the NH2-terminal part of the
polypeptide, has been shown to effectively prevent IB
phosphorylation, degradation, and NF-B activation in other systems
(Fig. 2
; Ref. 20
, 27, 28, 29, 30
).
With phosphorylation, blocked IB remains intact, thereby,
blocking NF-B translocation to the nucleus and subsequent activation
of antiapoptotic genes that cause TNF- insensitivity, thereby
allowing the cells to proceed through apoptosis. This IB
"super-repressor" has also been shown to induce apoptosis in other
systems (31
, 32)
. The "super-repressor" produces
constitutive repression of NF-B-directed transcription, despite
the presence of agonists that normally induce the degradation of
IB and the nuclear translocation of NF-B (24)
.
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B activity in cultured cells
(20)
. They inhibit IB degradation through their
ability to block the catalytic activity of the proteosome complex
(20)
. Although very effective in blocking the induction of
NF-B-activated antiapoptotic genes, proteosome inhibitors are
nonspecific and therefore potentially very toxic. However, the IB
"super-repressor," p6R-IBS32A + S36A, has
also proven to be very effective in inhibiting activation of
NF-B20. Its ability to be genetically
engineered with a PSA promoter provides specificity for prostate cancer
cells and therefore makes it an ideal candidate for hormone-refractory
prostate cancer gene therapy.
We report an efficient transduction of exogenous
"super-repressor," p6R-IBS32A + S36A,
NF-B into both TNF--insensitive PC-3 cells and TNF--sensitive
LNCaP cells. This "super-repressor" blocked IB
phosphorylation, NF-B translocation and activation, IL-6 production,
and induced apoptosis in transfected PC-3 and LNCaP cells exposed to
TNF-. Our data demonstrate that p6R-IBS32A +
S36A is a powerful tool that can sensitize
TNF--insensitive prostate cancer cells to undergo apoptosis.
Moreover, blockage of IL-6 production decreases cell proliferation of
androgen-independent prostate cancer cells.
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MATERIALS AND METHODS |
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Transfections.
The IBS32A/S36A plasmid used in this study
was described previously (21
, 33)
. Transfections were
conducted by using SuperFect (Qiagen, Valencia, CA). The procedure was
followed according to the manufacturer’s protocol. Briefly, cells were
seeded overnight at 60% confluency. The DNA plasmid (10 µg/10-cm
dish or 2 µg/6-cm dish) containing the p6R-IBS32A +
S36A "super-repressor" was mixed with 10 or 25 µl of
SuperFect, respectively. In control experiments, the empty eukaryotic
expression vector, p6R, was similarly introduced into cells. Cells were
incubated with the DNA mixture for 2 h at 37°C. Equal amounts of
additional fresh media were then added to the cells. The cultures were
incubated at 37°C for 24 h and replaced with fresh media. Cells
were then treated with TNF- (10 ng/ml) for 24 h.
Cytosolic and Nuclear Extracts.
Cell pellets were washed in PBS, pelleted again, resuspended in buffer
A [10 mM HEPES (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 5 mM
DTT, 5 mM phenylmethylsulfonyl fluoride and protease
inhibitors (50 µg/ml antipain, 2 µg/ml aprotinin, 1 µg/ml
leupeptin, and 1 µg/ml pepstatin)] and placed on ice for 10 min. The
cells were then vortexed and centrifuged for 10 s. The supernatant
was placed in a separate tube, and 10 mM EDTA, 120
mM KCl, and 20% glycerol were added. This mixture was
designated cytosolic extracts and stored at -80°C. The pellets were
resuspended in equal volumes of buffer C [10 mM HEPES (pH
7.9), 25% glycerol, 1.5 mM MgCl2,
0.2 mM EDTA, 0.8 M KCl, and protease
inhibitors] and incubated for 20 min on ice. Samples were centrifuged
for 5 min at 10,000 x g at 4°C. Supernatants were
designated nuclear extracts and were stored at -80°C.
Western Blot Analysis.
Equal amounts of cytosolic extracts (50 µg) were analyzed by
SDS-PAGE, followed by Western blotting using a polyclonal rabbit
IB antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Immunoreactive IB was detected using the enhanced
chemiluminescence (ECL) light detecting kit (Amersham).
EMSA.
NF-B oligonucleotide probe (Santa Cruz Biotechnology) was labeled
with [
32P]ATP to 50,000 cpm/ng using
polynucleotide kinase. Nuclear extracts (5 µg) were incubated with 1
µg of poly(deoxyinosinic-deoxycytidylic acid) a 20-µl volume of gel
shift reaction buffer [10 mM Tris (pH 7.5), 50
mM NaCl, 1 mM DTT, 1 mM EDTA, and
5% glycerol] and 0.5 ng of labeled oligonucleotide probe for 20 min
at room temperature. For supershifts with p65 antibody, nuclear
extracts from untransfected PC-3 cells were preincubated with 1 µl of
RelA antibody against the COOH-terminal portion of the molecule (Santa
Cruz Biotechnology) for 15 min at room temperature before the addition
of binding buffer and probe. DNA-protein complexes were resolved by
electrophoresis through a 4% polyacrylamide gel containing 50
mM Tris (pH 7.5), 0.38 M glycine, and 2
mM EDTA. The gel was then dried and visualized by
autoradiography.
RNA Extraction and Amplification.
RNA was isolated from control, p6R, and mutant, p6R-IBS32A
+ S36A, transfected PC-3 cells using the TRIzol method
(Life Technologies). Total RNA (10 µg) was amplified and measured
using a mRNA IL-6 Quantikine ELISA (R&D Systems, Minneapolis, MN).
IL-6 ELISA.
An IL-6 ELISA of cell culture supernatants from control, p6R, and
mutant, p6R-IBS32A + S36A, transfected PC-3
cells was performed in triplicate according to the manufacturer’s
specifications (R&D Systems). Supernatants were removed at various time
points after TNF- (10 ng/ml) stimulation.
Apoptosis Detection.
Annexin V fluorescent staining was assayed using an Annexin V apoptosis
kit (Santa Cruz Biotechnology), and Apoptag fluorescent staining was
detected using an immunohistochemistry kit (Intergen, Purchase, NY),
according to manufacturer’s protocol.
Statistical Analysis.
Data are expressed as a means ± SE. Statistical significance was
performed by the two-tailed Student t test for paired data
and considered significant if Ps were <0.05.
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RESULTS |
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B in Transfected LNCaP
(LNCaPT) and PC-3 (PC-3T) Cells. induced IB degradation, we transfected
LNCaP and PC-3 cells with the IB "super-repressor,"
p6R-IBS32A + S36A, which is not susceptible to
phosphorylation at NH2-terminal serines 32 and 36
and is therefore resistant to subsequent degradation. Western blot
analysis of cells transfected with p6R-IBS32A +
S36A and treated with 10 ng/ml of TNF- for 24 h
demonstrated blockage of IB phosphorylation and degradation (Fig. 3)
. The TNF--insensitive PC-3 cells
constitutively degraded IB until its phosphorylation was blocked
(Fig. 3
, Lane 4). This phenomenon was present 24 h
after TNF- was added to the cultures. After 48 h in culture,
p6R-IBS32A + S36A alone could stop degradation
of IB (data not shown). On the basis of these data, it appears
that TNF- expedites the blocked degradation of IB. The
TNF--sensitive LNCaP cells only degraded IB in the presence of
10 ng/ml TNF- (Fig. 3
, Lanes 8 and 12), except
where phosphorylation was blocked by the IB "super-repressor"
(Fig. 3
, Lane 10).
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B Translocation and Activation.B DNA binding activity using a radiolabeled binding site as a
B probe. TNF- strongly induced NF-B activity in untransfected
and control cells (Fig. 4A)
.
Transfection with p6R-IBS32A + S36A and
stimulation with TNF- (10 ng/ml) for 24 h significantly reduced
the cytokine-mediated NF-B nuclear translocation (Fig. 4A
, Lanes 4 and 10) In PC-3 cells without the IB
"super-repressor" and TNF- stimulation, the constitutive
degradation of IB causes NF-B to be constitutively expressed
in the nucleus, where it transcribes antiapoptotic genes that cause
TNF- insensitivity. However, LNCaP cells only degrade IB in
the presence of TNF-
, Lanes 8 and
12), except where the "super-repressor" is present (Fig. 4
, Lane 10), and therefore have significantly less NF-B
in the nucleus. Again, after 48 h in culture
p6R-IBS32A + S36A alone could decrease NF-B
translocation to the nucleus (data not shown). A supershift assay, with
lysate obtained from untransfected PC-3 cells (Fig. 4B)
, was
performed with a p65 antibody to demonstrate specificity to the NF-B
complex p65. Both a negative control without nuclear extracts as well
as a competitive control using a 100-fold excess of unlabeled consensus
mutant NF-B were used to establish specificity of the reaction (data
not shown).
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BS32A + S36A.B activity by p6R-IBS32A +
S36A in prostate cancer cells suggested that TNF--mediated,
NF-B-dependent gene induction could be down-regulated by this
reagent. As shown in Fig. 5
, TNF-
induction of IL-6 mRNA expression is inhibited in
p6R-IBS32A + S36A transfected PC-3 cells,
when compared with untransfected and control cells, reaching a maximum
of only 60 amol/ml.
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BS32A + S36A
would be able to prevent IL-6 secretion resulting from TNF- and
other stimuli. Fig. 6
demonstrates that
PC-3 cells transfected with p6R-IBS32A + S36A
and treated with TNF- (10 ng/ml) have significantly decreased levels
of IL-6 secretion when compared with untransfected and control cells.
This is particularly apparent at 24 h after TNF- stimulation,
where IL-6 levels are decreased to 480 (pg/ml).
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B Significantly Sensitizes Prostate Cancer Cells to
Apoptosis Induced by TNF-. (10 ng/ml) for 24 h induces apoptosis as assessed by morphological examination, propidium
iodide staining, Annexin V staining, and Apoptag staining. Annexin V
staining of cells transfected with p6R-IBS32A +
S36A and sensitized with TNF- demonstrate an increase in
apoptotic activity (Fig. 7A)
when compared with untransfected and control cells (Fig. 7, B and 7C)
. Fig. 8
shows columns illustrating the intensity of Apoptag staining in PC-3
and LNCaP cells, respectively. Again, when cells are transfected with
the IB mutant and sensitized with TNF-, there is a significant
increase in apoptotic cell death.
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DISCUSSION |
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levels when compared with
untreated patients or patients in remission (9)
. These
patients also experience a higher early mortality rate, and it seems
likely that serum TNF- levels increase in prostate cancer patients
with end-stage disease (9)
. The prostate cancer cells
found in relapsing patients are primarily androgen insensitive because
of prior androgen ablation therapy. Studies have shown that these
androgen-insensitive prostate cancer cells are also TNF- insensitive
(13)
. We proposed that this was attributable to NF-B
activation of antiapoptotic genes, such as those that cause TNF-
insensitivity. There are some cancer cells that are particularly
sensitive to the cytotoxic effect of TNF- (8)
. However,
TNF- has also been implicated in tissue remodeling and proliferation
in some cell types (8)
. This paradox is because of two
separate and distinct TNF--mediated pathways present in cells. One
pathway leads to apoptosis and the other to activation of protective
antiapoptotic genes through NF-B. Which pathway is activated depends
on the TNF- threshold of that particular cell. Typically, lower or
normal levels of TNF- induce the NF-B protective pathway to keep
the cell from undergoing unnecessary apoptosis, where higher or toxic
levels induce apoptosis. TNF--insensitive cells, such as
androgen-insensitive prostate cancer cells, do not experience
TNF--induced apoptosis, even at the LD10 dose
of 10 ng/ml. This is attributable to the constitutive degradation of
IB and therefore NF-B activation of antiapoptotic genes. Even
TNF- levels of 100 ng/ml could not force the TNF--insensitive
cells into apoptosis. The fact that the androgen-insensitive prostate
cancer cells were also TNF- insensitive, coupled with the increased
serum TNF- levels of relapsing prostate cancer patients, lead us to
believe that these cells were thriving in a toxic TNF- environment
attributable to the induction of NF-B antiapoptotic genes.
This study provides important new insights into the effects of
inhibiting NF-B in TNF--insensitive prostate cancer cells. A wide
range of intracellular components are implicated in TNF--induced
cell killing, including pertussis toxin-sensitive guanine nucleotide
binding protein (34)
, phospholipase
A2 (35)
, phospholipase D activation
(36)
, and DNA damage (37)
. Despite which
receptor sets off the cytotoxic effect, the various intracellular
signals activated by TNF- lead to activation of NF-B
(4)
. Prostate cancer cells up-regulate multiple
NF-B-responsive genes in response to TNF- stimulation. These
molecules may be involved in cell proliferation and metastasis, where
modulation of their expression could be clinically beneficial. We have
targeted our intervention at the IB/NF-B pathway because most of
these genes are predominately regulated at the level of transcription.
After TNF- stimulation, NF-B maintains a balance between an
inactive and active state that relies mostly on IB
(21)
. NF-B can be shifted to an inactive or active
state by overexpression or degradation of IB (21)
.
Previous approaches to inhibit NF-B activity have focused on
endothelial or mononuclear hemopoietic cells (21)
. Most
strategies target IB through proteosome blockades,
phosphorylation inhibition, and protein overexpression (38
, 39)
. The method we used to block the NF-B pathway in
TNF--insensitive prostate cancer cells was transfection with an
IB "super-repressor" that resists TNF- induced
phosphorylation and degradation. The IB protein used was a
S32A/S36A mutant form of IB that mutated the inducible amino acid
phosphoreceptor and therefore abolished the degradation process
(21)
. Our approach was successful in maintaining IB
levels, which selectively inhibited NF-B p65 subunit nuclear
translocation, NF-B DNA binding activity, down-regulated the
induction of NF-B responsive gene IL-6, and induced
apoptosis.
Androgen-independent prostate cancer cells spontaneously release
high levels of IL-6 into the cell supernatant without exogenous
stimulation (13)
. IL-6 is a cytokine with pleiotropic
activities and has been shown to play a central role in immune
host-defense mechanisms (40)
. This cytokine has been
implicated in growth differentiation, inhibition, and proliferation,
depending upon the nature of the responsive target cells
(41)
. IL-6 has been shown to promote cell proliferation in
androgen-independent prostate cancer cells, PC-3 (42)
.
When these cells were transfected with thep6R-IBS32A +
S36A "super-repressor" and stimulated with TNF-,
IL-6 secretion and IL-6 mRNA production was decreased. Although our
results demonstrate only partial inhibition of IL-6 production, we
believe that this IL-6 is residual, because of the fact that the
"super-repressor" only inhibits new gene transcription, and that we
have demonstrated that p6R-IBS32A + S36A
significantly suppresses new IL-6 production by TNF--stimulated PC-3
cells.
Transfected prostate cancer cells stimulated with TNF- were induced
to proceed through apoptosis. With the NF-B pathway blocked by the
IB "super-repressor," the cells were forced to proceed though
TNF--induced apoptosis. Our results indicate that the previously
TNF--insensitive prostate cancer cells were made sensitive because
of transfection with p6R-IBS32A + S36A. Once
made sensitive, the cells experienced apoptotic cell death upon
stimulation with TNF-.
The complex signal transduction pathway, beginning from the
binding of a cytokine to its receptor and leading to NF-B
transcriptional activity, provides many opportunities for therapeutic
intervention (20)
. Other studies have demonstrated that
p6R-IBS32A + S36A can be successfully
incorporated within the recombinant replication-deficient adenovirus,
giving rise to possible clinical use (20
, 21)
. In future
studies, we propose using a PSA-promoter, pPSA-630 (43)
,
added to p6R-IBS32A + S36A, that would
specifically target only PSA-secreting cells such as those found in
prostate cancer relapsed patients. This PSA promoter can only be
activated in the presence of specific transcription factors present
only in PSA-producing cells. One such transcription factor is the
androgen receptor present in prostate cancer cells. This gene therapy
might be beneficial to advanced prostate cancer patients and would
avoid the severe toxicities associated with other IB inhibitors,
such as ALLN and MG-132, by specifically targeting prostate cancer
cells. With NF-B activation blocked, prostate cancer cells could
proceed through apoptosis using the elevated serum TNF- levels
already present in patients with relapsed prostate cancer.
In conclusion, this study extends known antiapoptotic roles of NF-B
to prostate cancer cells and emphasizes that the blockage of NF-B
can selectively sensitize previously insensitive cells to apoptosis by
TNF-. These findings suggest a potential new therapeutic tool for
prostate cancer gene therapy.
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FOOTNOTES |
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1 Supported by CaPCURE and SPORE Grant P50
CA69568. 
2 To whom requests for reprints should be
addressed, at University of Michigan Medical School, Department of
Internal Medicine, 1500 East Medical Center Drive, 7303 CCGC, Ann
Arbor, MI 48109-0946. Phone (734) 647-3411; Fax: (734) 647-9480;
E-mail: muenchen{at}umich.edu
3 The abbreviations used are: TNF-, tumor
necrosis factor-; NF-B, nuclear factor-B; IL, interleukin;
EMSA, electrophoretic mobility shift assay; PSA, prostate-specific
antigen.
Received 1/ 7/00; revised 2/15/00; accepted 2/16/00.
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J. S. Ross, B. V. S. Kallakury, C. E. Sheehan, H. A. G. Fisher, R. P. Kaufman Jr., P. Kaur, K. Gray, and B. Stringer Expression of Nuclear Factor-{kappa}B and I{kappa}B{alpha} Proteins in Prostatic Adenocarcinomas: Correlation of Nuclear Factor-{kappa}B Immunoreactivity with Disease Recurrence Clin. Cancer Res., April 1, 2004; 10(7): 2466 - 2472. [Abstract] [Full Text] [PDF]
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J. An, Y.-P. Sun, J. Adams, M. Fisher, A. Belldegrun, and M. B. Rettig Drug Interactions between the Proteasome Inhibitor Bortezomib and Cytotoxic Chemotherapy, Tumor Necrosis Factor (TNF) {alpha}, and TNF-Related Apoptosis-Inducing Ligand in Prostate Cancer Clin. Cancer Res., October 1, 2003; 9(12): 4537 - 4545. [Abstract] [Full Text] [PDF]
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 A. Parcellier, E. Schmitt, S. Gurbuxani, D. Seigneurin-Berny, A. Pance, A. Chantome, S. Plenchette, S. Khochbin, E. Solary, and C. Garrido HSP27 Is a Ubiquitin-Binding Protein Involved in I-{kappa}B{alpha} Proteasomal Degradation Mol. Cell. Biol., August 15, 2003; 23(16): 5790 - 5802. [Abstract] [Full Text] [PDF]
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E. Kikuchi, Y. Horiguchi, J. Nakashima, K. Kuroda, M. Oya, T. Ohigashi, N. Takahashi, Y. Shima, K. Umezawa, and M. Murai Suppression of Hormone-refractory Prostate Cancer by a Novel Nuclear Factor {kappa}B Inhibitor in Nude Mice Cancer Res., January 1, 2003; 63(1): 107 - 110. [Abstract] [Full Text] [PDF]
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R. G. Uzzo, P. Leavis, W. Hatch, V. L. Gabai, N. Dulin, N. Zvartau, and V. M. Kolenko Zinc Inhibits Nuclear Factor-{kappa}B Activation and Sensitizes Prostate Cancer Cells to Cytotoxic Agents Clin. Cancer Res., November 1, 2002; 8(11): 3579 - 3583. [Abstract] [Full Text] [PDF]
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C. D. Chen and C. L. Sawyers NF-{kappa}B Activates Prostate-Specific Antigen Expression and Is Upregulated in Androgen-Independent Prostate Cancer Mol. Cell. Biol., April 15, 2002; 22(8): 2862 - 2870. [Abstract] [Full Text] [PDF]
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P. Schotte, G. Van Loo, I. Carpentier, P. Vandenabeele, and R. Beyaert Lithium Sensitizes Tumor Cells in an NF-kappa B-independent Way to Caspase Activation and Apoptosis Induced by Tumor Necrosis Factor (TNF). EVIDENCE FOR A ROLE OF THE TNF RECEPTOR-ASSOCIATED DEATH DOMAIN PROTEIN J. Biol. Chem., July 6, 2001; 276(28): 25939 - 25945. [Abstract] [Full Text] [PDF]
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