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REVIEWEndocrine-Related Cancer (2006) 13 751–778
Mechanisms of action of novel agents forprostate cancer chemoprevention
Rana P Singh1 and Rajesh Agarwal1,2
1Department of Pharmaceutical Sciences, School of Pharmacy and 2University of Colorado Cancer Center, University of Colorado
Health Sciences Center, Denver, Colorado 80262, USA
(Requests for offprints should be addressed to R Agarwal; Email: [email protected])
Abstract
Despite advances in the understanding of prostate cancer (PCa) growth and development, it is stillthe leading incidence of cases and the second leading cause of mortality due to cancer in men.The problem of early diagnosis compounded with the emergence of androgen independenceduring commonly used anti-androgen therapy of PCa, have been discouraging for optimaltherapeutic response. Recently, many chemopreventive agents, including silibinin, inositolhexaphosphate, decursin, apigenin, acacetin, grape seed extract, curcumin, and epigalloca-techin-3 gallate have been identified in laboratory studies, which could be useful in themanagement of PCa. In vivo pre-clinical studies have indicated chemopreventive effect of manysuch agents in PCa xenograft and transgenic mouse models. The molecular targets of theseagents include cell signaling, cell-cycle regulators, and survival/apoptotic molecules, which areimplicated in uncontrolled PCa growth and progression. Furthermore, angiogenic and metastatictargets, including vascular endothelial growth factor, hypoxia-inducing factor-1a, matrixmetalloproteinase, and urokinase-type plasminogen activator are also modulated by manychemopreventive agents to suppress the growth and invasive potential of PCa. This reviewfocuses on novel PCa chemopreventive observations in laboratory studies, which could providethe rationale for the prospective use of chemopreventive agents in translational studies.
Endocrine-Related Cancer (2006) 13 751–778
Introduction
Prostate cancer (PCa) is a chronic disease, and
therefore, its chemoprevention is emerging as an
attractive additional strategy for disease control. In
recent years, considerable progress has been made in
this direction, which has led to the identification of
novel cancer chemopreventive agents and their mode
of action. Since the existing therapies for PCa have
done little in the improvement of morbidity and
mortality, the clinical application of chemoprevention
should be explored enthusiastically. Several molecular
pathways and various stages of cancer could be the
targets for chemoprevention. However, according to
most researchers, cancer chemoprevention includes
prevention, suppression and/or reversal of early and/or
late stages of cancer growth, and development by
ideally non-toxic natural or synthetic agents.
PCa behavior is mostly unpredictable; however, it
usually takes longer time for progression to malig-
nancy and metastasis and, therefore, provides a broader
Endocrine-Related Cancer (2006) 13 751–778
1351-008806/013–751 q 2006 Society for Endocrinology Printed in Great B
window for its management, including the suitability
for chemopreventive intervention. Whereas, it is likely
that chemoprevention modality of cancer management
would not necessarily eliminate the lesions, it is
expected to halt neoplastic progression of pre-
neoplastic lesions that certainly would improve
morbidity and survival time in PCa patients. As
described in detail later in the clinical studies section,
PCa certainly provides this opportunity where several
chemoprevention trials in patients start at pre-neoplas-
tic condition known as high-grade prostatic intra-
epithelial neoplasia (PIN) and the positive outcomes
are measured as reduction in clinical PCa.
Life style and dietary habits have been identified as
major risk factors in PCa growth and progression
(Whittemore et al. 1995, Potter & Steinmetz 1996,
Clinton & Giovannucci 1998, Abdulla & Gruber 2000,
Agarwal 2000). Epidemiological data indicate that
vegetables and fruits containing chemopreventive
agents could have protective effect against cancer
ritain
DOI:10.1677/erc.1.01126
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R P Singh and R Agarwal: PCa chemoprevention
(Whittemore et al. 1995, Potter & Steinmetz 1996,
Clinton & Giovannucci 1998, Abdulla & Gruber
2000). However, due to lack of sufficient pre-clinical
and clinical studies, it has been difficult to generalize
such statement for the human population. Nonetheless,
the major advantage of chemoprevention as compared
with other forms of treatment is the lack of systemic
toxicity. Another advantage of chemoprevention is
recent laboratory findings showing that when used in
combination, chemopreventive agents improve the
therapeutic response of other PCa treatment modal-
ities, such as anti-androgen, chemo- or radiotherapy in
rodent models or cell culture studies (Tyagi et al.
2002d, Deeb et al. 2003, Chendil et al. 2004,
Venkateswaran et al. 2004, Narayanan et al. 2005).
Therefore, in the present circumstances, chemopre-
ventive agents could be used as an alternative, as well
as a combination treatment option in the management
of PCa. In the following sections, we have described
the emerging PCa chemopreventive agents with their
potential molecular targets and clinical implications
where applicable.
Potential PCa chemopreventive agents
Usually, PCa patients live with the disease for many
years until it becomes metastatic and, therefore,
present an opportunity for chemopreventive interven-
tion. There is also a wide variation in PCa incidence
among different ethnic populations, wherein dietary
habit has been identified as one of the major etiologic
factors (Clinton & Giovannucci 1998, Abdulla &
Gruber 2000). Epidemiological studies suggest that
about two-thirds of cancer can be prevented by
incorporating healthy agents in, and reducing those,
which increase cancer risk from the diet (Clinton &
Giovannucci 1998, Abdulla & Gruber 2000, Singh
et al. 2002b). Naturally occuring cancer chemopre-
ventive agents, include flavonoids, isothiocyanates,
indoles, dithiolthiones, coumarins, and organosulfides
(Kelloff et al. 1999, Agarwal 2000, Singh et al. 2002b,
Stewart et al. 2003, Park & Surh 2004, Sarkar & Li
2004, Tsuda et al. 2004, Bektic et al. 2005). The
mechanism-based efficacy of some common PCa
chemopreventive agents, such as genistein (isoflavo-
noid), curcumin (polyphenol), epigallocatechin gallate
(EGCG; flavanol), indole-3-carbinol (isothiocyanates),
and resveratrol/procyanidins has been well reviewed
recently (Singh et al. 2002b, Tsuda et al. 2004), and
therefore, only a brief account of these agents is provided
here. Some of these agents are being considered for
clinical trials in PCa patients. The novel chemopreven-
tive agents discussed in detail include silibinin
752
(flavonoid), inositol hexaphosphate (IP6), decursin
(coumarin), apigenin (flavanol), and acacetin (flavone),
which have shown activity against a wide range of
cancers, including PCa. These agents, except silibinin,
are still in laboratory/pre-clinical stages; however, the
studies conducted so far have provided encouraging
results for their further investigation in pre-clinical PCa
models followed by clinical potential. Extensive studies
with silibinin in cell culture and animal models of PCa
have been described in the following sections, which
have led to its clinical trial in PCa patients at the
University of Colorado Health Sciences Center.
The anti-cancer efficacy of chemopreventive agents
has been linked to the modulation of mitogenic
signaling, cell-cycle regulation, survival/apoptotic
signaling, angiogenic, and metastatic events in cancer
cells. These agents have been shown to ultimately
inhibit PCa cell proliferation in vitro, as well as tumor
growth in vivo. Silibinin, from milk thistle, the most
extensively studied chemopreventive agent in our
laboratory, inhibits the growth of human PCa
DU145, PC3 (both hormone refractory), and LNCaP
(androgen dependent) cells, and rat PCa H-7, I-8, and I-
26 cells (Zi et al. 1998, 2000b, Zi & Agarwal 1999,
Tyagi et al. 2002c). IP6, a form of carbohydrate present
in most cereals, legumes, oil seeds, and nuts, has been
shown to suppress the growth of human, rat, and mouse
PCa cells in culture (Singh & Agarwal 2005). Further,
oral consumption of silibinin in diet or IP6 in drinking
water or grape seed extract (GSE) by gavage
suppresses the growth of DU145 tumor xenograft in
athymic nude mice (Singh et al. 2002b, 2004a,b). Our
pilot studies in transgenic adenocarcinoma of mouse
prostate (TRAMP) model have also revealed the
chemopreventive activity of these three agents against
the growth of prostate tumor (R P Singh, K Raina,
M Pollack & R Agarwal unpublished observation).
More recently, anti-proliferative effect of decursin
(from the roots of Angelica giga) was observed against
both human androgen-dependent and hormone-refrac-
tory PCa cells (Yim et al. 2005, Jiang et al. 2006).
Apigenin and acacetin, present in many fruits and
vegetables, also inhibit the growth of human PCa cells
(Shukla & Gupta 2004a,b, Singh et al. 2005a).
Recently, oral apigenin has also been reported to
inhibit prostate tumor growth in TRAMP model
(Shukla et al. 2005). It is important to mention that
none of these agents has shown any adverse health
effect in animal studies. Moreover, for many new
agents, the in vivo studies are yet to be done to estimate
their chemopreventive potential; however, many
targets have been identified for these agents in PCa
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Endocrine-Related Cancer (2006) 13 751–778
cell-culture studies. An account of such chemopreven-
tive targets in PCa has been described below.
Targets for PCa chemoprevention
The mechanism-based efficacy without any consider-
able side effect is an essential requirement for the
clinical development of a cancer chemopreventive
agent. In PCa, often, ligand-induced signaling drives
cell proliferation and survival, which are accompanied
by deregulated cell-cycle progression (Jones et al. 2001,
Fernandez et al. 2002). Many signaling pathways are
constitutively active that lead to persistent growth and
progression in PCa (Gioeli et al. 1999). In this fashion,
PCa cells keep accumulating neoplastic genetic and
epigenetic changes leading to metastatic disease.
Furthermore, neo-angiogenesis has been observed as
an obligatory requirement for the solid tumor growth,
including prostate tumors (Lara et al. 2004, Cox et al.
2005). In addition, activated endothelial cells, as well as
advanced PCa cells have been found to have intrinsic
invasive potential (Pienta&Loberg 2005, Singh&Figg
2005). A summary of these PCa chemopreventive
targets is included in Fig. 1. These neoplastic biological
events are regulated by sequential activation and
deactivation or expression and suppression of many
cellular molecules, which could be targeted by
chemopreventive agents to intervene with the tumor
Figure 1 Potential targets forPCachemoprevention.AR, androgen regrowth factor receptor; STAT, signal transducer and activator of transCDKI, CDK inhibitor; Rb, retinoblastoma; NF-kB, nuclear factor-kB; Ainhibitor of apoptosis protein; VEGF, vascular endothelial growth factuPA, urokinase-type plasminogen activator; Bcl-2, B-cell CLL/lympho
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growth and progression. It is important to briefly
mention here that apart from these mechanisms, natural
chemopreventive agents also possess anti-oxidant
activity and can inhibit inflammation and stimulate
phase II detoxification enzyme activity. Further, there
are other molecular targets, such as fatty acid synthase,
inducible nitric oxide synthase (iNOS), and cyclo-
oxygenase-2 (COX-2), as well as stromal and epithelial
cell interaction and associated paracrine regulatory
mechanisms, which could be modulated by many
chemopreventive agents in their overall cancer pre-
ventive efficacy. However, in the following sections, we
have focused on those chemopreventive targets, which
are summarized in Fig. 1.
Cell signaling targets
Androgen receptor (AR) signaling
The steroid enzyme 5a-reductase converts testosteroneinto dihydrotestosterone (DHT), an active androgen,
which binds to AR leading to its nuclear translocation
for the transcriptional activation of androgen-respon-
sive genes (Sommer & Haendler 2003; Fig. 2). In the
beginning, PCa growth is primarily regulated by
androgens, and therefore, androgen ablation therapy
is carried out as the first line of treatment; however,
PCa relapses within a few years and at this stage, the
ceptor;EGFR,epidermal growth factor receptor; IGFR, insulin-likecription; TLR, toll-like receptor; CDK, cyclin-dependent kinase;TM, ataxia telangiectasia-mutated; Chk, checkpoint kinase; IAP,or; HIF, hypoxia-inducible factor; MMP, matrix metalloproteinase;ma-2; E2F, E2-promoter binding factor.
753
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Figure 2 Androgen receptor and receptor tyrosine kinase signaling targeted by chemopreventive agents in PCa to inhibit cell growth,deregulated cell-cycle progression, and cell survival. Dotted arrow indicates that ERK1/2 and AKT signaling also enhances cell growthand survival via pathways other than CDK–cyclin. Upward arrow indicates increase in protein expression of insulin-like growth factor-binding protein-3 and cyclin-dependent kinase (CDK) inhibitors (CIP1, KIP1, and INK4 family members) or interaction betweenretinoblastoma (Rb) family proteins andE2Fs.DHT, dihydrotestosterone; AR, androgen receptor; EGF, epidermal growth factor; EGFR,EGF receptor; TGFa, transforming growth factor a; IGF, insulin-like growth factor; IGF-IR, IGF-I receptor; IRS-1, insulin receptorsubstrate-1; ERK1/2, extracellular signal-regulated kinase1/2; ppRb, hyperphosphorylated Rb.
R P Singh and R Agarwal: PCa chemoprevention
malignancy is androgen independent (Feldman &
Feldman 2001, Sommer & Haendler 2003). Recent
studies have suggested that various approaches
employed in androgen ablation therapy do not cause
a complete depletion of androgens (Scher & Sawyers
2005). In such conditions, even nanomolar concen-
trations of testosterone and DHT, most likely produced
by adrenal glands or tumor itself, are sufficient for the
transactivation of AR to support PCa cell proliferation
(Mohler et al. 2004, Titus et al. 2005). AR gene
amplification accompanied by an increased AR mRNA
and protein production had been frequently observed
during the relapse of the disease after hormone ablation
therapy (Feldman& Feldman 2001, Edwards et al. 2003,
Ford et al. 2003, Sommer & Haendler 2003). This could
be an adaptive response of the tumor cell to become
sensitive to low concentrations of the androgens.
Furthermore, AR mutations have been observed in up
to 50%of the PCa tissue samples, which aremostly in the
ligand-binding domain and are associated with the gain
of the receptor function (Buchanan et al. 2001, Scher &
754
Sawyers 2005). Overall, these observations suggest that
ARsignaling is important in the growthof PCa, aswell as
in the development of hormone resistance and relapse of
the disease, and that if AR signaling and associated
molecular events could be targeted by chemopreventive
agents, this approach should help suppress PCa growth
and progression.
Silibinin has been shown to decrease prostate specific
antigen (PSA) protein expression in LNCaP cells
together with cell growth inhibition via cell-cycle arrest
(Zi & Agarwal 1999; Fig. 2). In a follow-up study, it was
reported that silibinin and its crude form silymarin inhibit
androgen-stimulated cell proliferation together with
secretion of both PSA and human glandular kallikrein
in LNCaP cells (Zhu et al. 2001). The study also showed
that the transactivation activity of AR is diminished by
both agents together with nuclear levels of AR; however,
expression and steroid-binding ability of total AR were
not affected (Zhu et al. 2001). Decursin is also reported
recently to exert strong inhibitory effect on AR
expression and activity in androgen-dependent PCa
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Endocrine-Related Cancer (2006) 13 751–778
cells (Jiang et al. 2006). Quercetin and selenium are
reported to inhibitARactivity inLNCaPcells,whichwas
accompanied by the reduction in cell proliferation
(Morris et al. 2005). In another study, the inhibitory
effect of quercetin on both expression and function ofAR
is reported in LNCaP PCa cells (Xing et al. 2001).
Theaflavin-3,30-digallate and penta-O-galloyl-b-D-glu-cose have been shown to inhibit the activity of
5a-reductase, as well as the production of androgens
(Lee et al. 2004a). Both these compounds also suppress
the expression of AR and its activity in LNCaP cells
leading to cell-growth inhibition. Genistein, an active
component in soy diet, has also been shown to decrease
AR expression and PSA secretion in LNCaP cells at
physiological concentrations (Bektic et al. 2004).
Whereas, the role of non-steroidal anti-inflammatory
drugs (NSAIDs) has been well established in the
chemoprevention of colon cancer, with regard to PCa,
flufenamic acid, an NSAID, is found to transcriptionally
inhibit the expression ofAR andAR-promoter activity in
LNCaP cells (Zhu et al. 1999). Ornithine decarboxylase
(ODC) is an androgen-responsive gene and over-
expressed in PCa (Mohan et al. 1999). It has been
shown that green tea polyphenols inhibit testosterone-
inducedODC activity, as well as anchorage-independent
growth of LNCaP cells, and also inhibit ODC activity in
rat prostate (Gupta et al. 1999). Together, these reports
suggest that suppression ofARexpression and activityby
chemopreventive agents could have profound effect on
PCa growth.
Receptor tyrosine kinase signaling
Epidermal growth factor receptor (EGFR) and insulin-
like growth factor receptor-type I (IGF-IR) have been
identified as major tyrosine kinase membrane
receptors, which are constitutively active in PCa
(Lorenzo et al. 2003, Liao et al. 2005; Fig. 2). An
increased production of growth factors to act as ligands
and EGFR family receptors is the major contributing
factor in the progression of PCa to the advanced and
androgen-independent stages (Lorenzo et al. 2003).
Many studies have shown the high levels of EGFR and
transforming growth factor a (TGFa) in PCa that leadsto the formation of an autocrine loop for a constitu-
tively active mitogenic signaling in advanced human
PCa cells (Mendelsohn & Baselga 2000, Lorenzo et al.
2003). With regard to their tissue levels, human PIN
and primary and metastatic PCa show frequent
expression of EGFR family receptors (Myers et al.
1994, Mendelsohn & Baselga 2000, Di Lorenzo et al.
2002). EGFR itself and other members in its family are
usually activated by ligand binding and/or receptor
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homo/hetero-dimerization, and subsequently their
intrinsic tyrosine kinase activity transmits both
mitogenic and survival signals via Shc/Ras/Raf/
MAPK and/or PI3K/Akt pathways (Whitmarsh et al.
1998, Fischer et al. 2003). Consistent with the
observations regarding constitutively active EGFR
signaling pathway, constitutive activation of mitogen-
activated protein kinase (MAPK)/extracellular
signal-regulated kinase1/2 (ERK1/2) and associated
transcription factors is also observed and implicated in
PCa progression to an androgen-independent and
aggressive stage based on Gleason scores (Wasylyk
et al. 1993, Gioeli et al. 1999). The role of EGFR
activation in epithelial cancer growth is further
demonstrated and established by the studies showing
that inhibition of ligand binding to EGFR either by the
antibodies, such as IMC-C225 and ABX-EGF or by the
receptor tyrosine kinase inhibitors, such as Iressa and
Tarceva, results in promising growth inhibitory out-
comes at least in those epithelial cancers that express
high levels of EGFR and its ligands (Mendelsohn &
Baselga 2000, Lage et al. 2003). Together, these
findings convincingly suggest that downregulation of
EGFR signaling would be a vital strategy in the
chemopreventive intervention of PCa.
To our knowledge, for the first time we showed that
both silymarin and silibinin inhibit TGFa- and EGF-
caused tyrosine phosphorylation of EGFR and its
adaptor protein Shc in androgen-independent human
PCa DU145 cells, which harbor constitutively active
EGFR (Zi et al. 1998, Sharma et al. 2001). In the
follow-up of detailed mechanistic investigations,
silibinin inhibited ligand binding to EGFR and its
internalization in to the cytoplasm, EGFR dimeri-
zation, ERK1/2 activation, as well as cell proliferation
(Sharma et al. 2001). Silibinin also inhibited TGFamRNA expression and decreased both secreted and
cellular levels of TGFa in LNCaP and DU145 cells
(Sharma et al. 2001). In a more recent study, we found
that silibinin selectively exerts biological effects in 9L-
EGFR cells (9L rat glioma cells lacking EGFR
message were transfected with human EGFR for its
overexpression) via inhibition of EGF-induced EGFR
activation; the parental 9L glioma cells lacking EGFR
did not respond to silibinin-caused inhibition of cell
growth and proliferation (Hannay & Yu 2003, Qi et al.
2003). The findings of this study clearly suggest that
growth inhibitory and apoptotic effects of chemopre-
ventive agents, including silibinin could be achieved in
PCa by targeting EGFR signaling. Consistent with this
suggestion, other studies by us have shown that natural
products, such as IP6 and GSE inhibit EGFR–ERK1/2
signaling together with growth and proliferation in
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R P Singh and R Agarwal: PCa chemoprevention
DU145 cells (Zi et al. 2000a, Tyagi et al. 2003a).
Dietary genistein is also reported to inhibit EGFR and
ERK1/2 expression and incidence of poorly differ-
entiated prostatic adenocarcinomas in animal models
(Wang et al. 2004). Other chemopreventive agents
have also been shown to inhibit EGFR–ERK1/2
signaling in PCa (Bhatia & Agarwal 2001).
Similar to EGFR, IGF receptor (IGFR) signaling
promotes growth and survival of PCa, and is
constitutively active in the advanced stage of the
disease, as well as closely linked to the PCa growth and
progression in many studies (Yu & Rohan 2000, Liao
et al. 2005; Fig. 2). Epidemiological studies in
European and Western countries indicate a positive
association between PCa risk and higher circulating
levels of IGF-I and lower levels of IGF-binding protein
(IGFBP)-3 (Chan et al. 1998, Chokkalingam et al.
2001). IGFs are mitogenic ligands and overexpressed
in PCa, and their activity is firmly controlled by the
presence of IGFBPs. IGF activates tyrosine kinase
activity of IGFR and triggers downstream signaling
either via PI3K/Akt and/or Ras/Raf/MAPK (Yu &
Rohan 2000, Liao et al. 2005). IGFBPs sequester IGFs
and reduce their availability to IGFR, and thereby
downregulate IGFR activation (Grimberg & Cohen
2000). IGFs/IGFBP-3 plasma level is suggested as a
potential predictor and end-point surrogate biomarker
for PCa risk and progression (Harman et al. 2000, Chan
et al. 2002). Interestingly, PSA has protease activity for
IGFBP-3 (Cohen et al. 1994) that suggests a possible
regulatory link between AR and IGFR signaling.
Animal studies also suggest that IGFR signaling
promotes growth and metastatic potential of PCa
(DiGiovanni et al. 2000). Taken together, a well
studied and established role of IGF-I-mediated mito-
genic and survival signaling cascades in both PCa cells
and animal models and their clinical relevance,
convincingly suggest that it could be a promising
target for chemopreventive agents.
Our studies show that silibinin downregulates IGF-
IR signaling in PCa and inhibits tumor cell growth,
both in vitro and in vivo (Zi et al. 2000b, Singh et al.
2002b). The inhibitory effect of silibinin on IGF-IR
signaling was, in part, via an upregulation in both the
message and protein levels of IGFBP-3, as well as its
secreted levels. The use of IGFBP-3 antisense-
oligonucleo-tide partially reversed the effect of
silibinin on insulin receptor substrate-1 tyrosine
phosphorylation and cell proliferation in human PCa
PC3 cells (Zi et al. 2000b). In DU145 prostate tumor
xenograft study, silibinin increased IGFBP-3
expression and secretion from tumor cells (Singh
et al. 2002b, 2003b). Dietary silibinin is also observed
756
to decrease IGF-I and increase IGFBP-3 levels in
plasma of TRAMP mice (R P Singh, K Raina, M
Pollack & R Agarwal unpublished observations).
Recently, apigenin is reported to inhibit IGF-IR
signaling by decreasing IGF-I level with a concomitant
increase in IGFBP-3 level in androgen-dependent PCa
22Rv1 cells, both in vitro and in vivo (Shukla et al.
2005). Green tea polyphenols have also been shown to
increase IGFBP-3 and decrease IGF-I levels in
TRAMP model, and the authors have claimed this to
be the mechanisms of their efficacy in inhibiting
prostate tumor growth in TRAMP model (Adhami
et al. 2004). Genistein also downregulates IGF-IR in
TRAMP mice (Wang et al. 2004). Vitamin D analogs,
including EB1089 have an anti-proliferative effect in
PCa cells (Nickerson & Huynh 1999). In this study,
EB1089-induced regression in prostate tumor has been
shown to involve an alteration in the availability of
IGF-I as a result of increased (15–25-fold) production
of IGFBP-2 to IGFBP-5. The castration of rats also
leads to upregulation of IGFBPs in the ventral prostate
accompanied by its regression. These findings suggest
that chemopreventive agents could potentially down-
regulate IGF-IR signaling by modulating the
expression and interaction of IGF-I, IGF-IR, and
IGFBPs in both in vitro and in vivo PCa models, and
that these effects of phytochemicals might be important
contributing factors in their efficacy towards the
inhibition of PCa growth and progression (Fig. 2).
Signal transducers and activators of transcrip-
tion (STATs) signaling
STATs transduce mitogenic and survival signals of
many growth factors and cytokines via tyrosine and
serine phosphorylation by upstream kinases (Yu &
Jove 2004). STAT signaling is under tight and transient
regulation in normal cells, but becomes aberrant in
many types of malignancies, including PCa (Ni et al.
2000, Yu & Jove 2004). Of the seven STAT proteins,
STAT3 is persistently activated in PCa and most
frequently in the hormone-resistant stage of the disease
(Ni et al. 2000). The significance of STAT3 activation
in PCa is further established by the studies showing
that a disruption in its activation or expression results
in growth inhibition and apoptotic death of PCa cells
(Ni et al. 2000, Gao et al. 2005, Horinaga et al. 2005).
Many STAT3-responsive elements have been ident-
ified in several genes that influence cell-cycle
progression, such as cyclin D1 and angiogenesis,
such as vascular endothelial growth factor (VEGF;
Yu & Jove 2004). Interleukin (IL)-6 has been shown to
mediate its oncogenic effect in LNCaP cells via Janus
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Endocrine-Related Cancer (2006) 13 751–778
kinases (usually JAK2) and STAT3 (Chen et al. 2000,
Matsuda et al. 2001). Additionally, STATs are
activated by cytokine receptors, G-protein coupled
receptors or growth factor receptors having intrinsic
tyrosine kinase activity, or by intracellular non-
receptor tyrosine kinases (Yu & Jove 2004). Phos-
phorylation of STAT causes its dimerization, necessary
for its nuclear translocation and transcriptional
activity. STAT3 binds to human serum-inducible
element (HSIE) and interferon-gamma activated site
(GAS) sequences to mediate its anti-apoptotic and
oncogenic effects (Yu & Jove 2004). Similar to
AG490, a JAK2 tyrosine kinase inhibitor, antisense
oligonucleotide for STAT3 inhibits STAT3 DNA-
binding activity in DU145 cells and decreases the cell
survival (Mora et al. 2002). Increased expression of
STAT3 in tumors has been crucial in the processes
evading immunological surveillance. Overall, these
findings render STAT3 as a potential target for the
intervention of PCa, including hormone-resistant
phenotype of the disease.
Zyflamend, a herbal preparation and non-selective
inhibitor of cyclooxygenase activity, is reported to
inhibit STAT3 phosphorylation in LNCaP cells
(Bemis et al. 2005). Studies with chemopreventive
agents on STATs in PCa are limited. However, in
other cancer cell types, such as multiple myeloma
cells, curcumin is shown to inhibit constitutive and
IL-6-induced STAT3 phosphorylation and nuclear
translocation, as well as cell survival (Bharti et al.
2003). Curcumin has also been shown to inhibit
interferon-a-induced STAT1 phosphorylation.
Compared with AG490, curcumin is reported to be
about 16 times faster and 10 times more effective
inhibitor of STAT3 phosphorylation (Bharti et al.
2003). In other studies, curcumin is found to suppress
JAK–STAT inflammatory signaling in brain microglia
and T-cell leukemia (Kim et al. 2003, Rajasingh et al.
2006). Green tea polyphenols are shown to inhibit
STAT1 phosphorylation and its DNA-binding activity
in lung and colon epithelial cell lines for their anti-
inflammatory activity (Watson et al. 2004). We have
also observed the inhibitory effect of silibinin on
STAT3 tyrosine phosphorylation in alveolar lung
epithelial cancer A549 and human PCa DU145 cells
(M Chittezhath, R P Singh & R Agarwal unpublished
observations). Although, there is a considerable lack
of studies assessing whether chemopreventive agents
target STAT3 and/or inhibit its activation in PCa; a
suggestion could be made for their modulatory effect
on STAT signaling that most likely plays a critical
role in their overall anti-PCa efficacy.
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b-catenin signaling
b-catenin was first identified as a structural component
in adherens junction formation participating with other
proteins to regulate cell–cell adhesion. Later, cyto-
plasmic and nuclear b-catenin were described as
potential effector of wingless-type MMTV integration
site family (wnt) signaling associated with prolifer-
ation, differentiation, and metastasis, which is main-
tained at low levels in the absence of wnt stimulation
(Chesire & Isaacs 2003). Adenomatous polyposis coli
(APC) tumor suppressor and axin proteins form a
complex with b-catenin and facilitate its N-terminal
phosphorylation by glycogen synthase kinase-3bfollowed by its ubiquitination and proteosomal
degradation (Davies et al. 2001). On the other side,
enhanced b-catenin levels facilitate its nuclear translo-
cation and interaction with DNA-binding transcription
factors, more frequently with T-cell factor (TCF)/
lymphoid enhancer factor family proteins (Chesire &
Isaacs 2003, Yardy & Brewster 2005). The activated
b-catenin upregulates genes (e.g., c-myc) by transacti-
vation of TCF transcription factors, where it competes
with their co-repressors for the binding and then
recruits transactivating factors. In the nucleus, it also
faces nuclear export by APC into the cytoplasm, as
well as nuclear retention by TCF. Formation of PIN in
transgenic mice expresses constitutively active
b-catenin (Gounari et al. 2002). Alterations in APC
or axins, or activating mutations in b-catenin itself
have been observed to increase its nuclear activity in
PCa, and therefore, indicates its oncogenic nature in
tumor formation and progression (Gerstein et al. 2002,
Yardy & Brewster 2005).
Chemopreventive agents, celecoxib, and
a-difluoromethylornithine have been shown to restore
b-catenin levels in the dorso-lateral prostate of
TRAMP mice, and the authors have suggested these
findings as an anti-metastatic effect of the agents
(Gupta et al. 2000, 2004a). Since these studies did not
examine the localization of b-catenin in the cyto-
plasmic membrane, cytoplasm and/or nucleus or its
serine/threonine and tyrosine phosphorylation status,
the authors’ claim is in contrast to the basic cell
biology findings, because it is not clear how the
increased level of b-catenin could have inhibited PCa
growth and progression by these chemopreventive
agents. Despite the advances in the understanding of
basic mechanisms of wnt-b-catenin signaling, it
remains mostly unexplored in PCa chemoprevention
research. Therefore, more studies with known and new
PCa chemopreventive agents are needed in this
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R P Singh and R Agarwal: PCa chemoprevention
direction to establish the role of b-catenin signaling in
their efficacy against PCa.
Toll-like receptor (TLR) signaling
Chronic inflammation is an etiological event for many
human cancers, including PCa. TLRs are among
important mediators of inflammation as they recognize
a broad range of microbial pathogens, including
bacteria and viruses and interact with various adaptor
proteins to activate different transcription factors,
including nuclear factor-kB (NF-kB) and activator
protein-1 (AP-1), and induce innate and adaptive
immune response (Kawai & Akira 2006). Recent
studies show that sequence variations in TLR4 gene
(inherited polymorphisms) and TLR6–TLR1–TLR10
gene cluster are associated with PCa risk (Zheng et al.
2004, Chen et al. 2005, Sun et al. 2005), and that
clinical PCa tissue harbor an enhanced expression of
TLR1, TLR2, and TLR3 (Konig et al. 2004). Whereas,
the functional role of TLR variants remains to be
established in the growth and development of PCa,
recently the role of TLR4 has been established in lung
inflammation and tumorigenesis in animal models
suggesting TLRs to be one of the potential targets for
cancer chemoprevention (Bauer et al. 2005).
Many chemopreventive agents have been shown to
inhibit downstream intermediate effector molecules of
TLR signaling, such as MAPKs, AP-1, and NF-kBactivation, as well as cytokine production in PCa;
however, their modulatory effects on TLR activation
remain to be studied. Helicobacter pylori infection
stimulates TLR4 glycosylation, which initiates intra-
cellular signaling in the infected host cell (Lee et al.
2004b). EGCG is shown to completely block TLR4
glycosylation and activation resulting in an inacti-
vation of ERK1/2 and NF-kB and a decreased
synthesis of the proinflammatory mediator hydroxyei-
cosatetraenoic acid in gastric mucosa (Lee et al.
2004b). Since many intermediate signaling molecules
are common to other membrane receptor signaling,
genetic manipulations, such as knockout/knockdown
need to be employed to define the effect of
chemopreventive agents specifically on TLR signaling
in PCa.
Cell-cycle regulatory targets
Cyclin-dependent kinases (CDKs) and cyclins
Deregulated cell-cycle progression is a fundamental
biological phenomenon underlying the growth and
development of cancer. Cell-cycle progression is
regulated by the activity of CDKs (serine/threonine
758
kinases) in non-covalent association with their regulat-
ory subunits, cyclins, and CDK inhibitors (CDKIs;
Sherr 2000, Vermeulen et al. 2003). Activation of
different CDKs is specific to the distinct phases of the
cell cycle, for example, CDK4 and CDK6 with D-type
cyclins are activated for early and mid-G1-phase
progression, whereas CDK2–cyclin E for late-G1 and
G1–S transition, CDK2–cyclin A for S-phase pro-
gression, and Cdc2 (or CDK1)–cyclin B for G2–M-
phase transition (Grana & Reddy 1995, Sherr &
Roberts 1999, Sherr 2000, Senderwicz & Sausville
2000). Further, Cdc25 tyrosine phosphatases regulate
G2–M checkpoint transition (Nilsson & Hoffmann
2000), where Cdc25 dephosphorylates Cdc2 facilitat-
ing a complex formation with cyclin B to drive the cell
through G2–M checkpoint transition. In genotoxic
stress, ataxia telangiectasia-mutated (ATM)/ataxia
telangiectasia and rad3 (ATR)-related kinase–check-
point kinase (Chk)1/2 cascade activation causes
inhibitory phosphorylation of Cdc25 to halt the cell-
cycle progression at G2–M transition (Kawabe 2004).
Various genetic and epigenetic changes or oncogenic
alterations enhance Cdc2 kinase activity in many
cancers, including PCa (Fu et al. 2004a, Kawabe
2004). Additional studies also show the overexpres-
sion/activation of cyclin/CDK and their activating
phosphatases (Cdc25A/B) in many cancer cells,
including PCa (Grana & Reddy 1995, Fu et al.
2004a), and that many prostate tumors carry mutations,
which deregulate at least one of the CDKs; most
frequently CDK2, CDK4, CDK6, or CDK1. Moreover,
neoplastic mitogenic signaling is linked to the
activation of CDK–cyclin complex for uncontrolled
cell proliferation and survival (Fig. 2). Taken together,
these studies suggest that a correction in the regulation
of unchecked cell-cycle progression, or alternatively a
cell-cycle arrest, could be an effective strategy to
control the growth and proliferation of cancer cells and
to induce their apoptotic death. As summarized next,
various chemopreventive agents have shown promis-
ing outcomes in these directions.
Silibinin has been reported to induce G1 arrest in
human and rat PCa cells together with an inhibition in
the kinase activity of CDK4, CDK6, and CDK2, which
are known to regulate G1 phase of the cell cycle
(reviewed recently by Singh & Agarwal 2004). The
observed inhibitory effect of silibinin on the kinase
activity of various CDKs was in part due to a strong
decrease in the protein levels of both CDKs and
cyclins, and was evidenced in both androgen-indepen-
dent (DU145) and androgen-dependent (LNCaP)
human PCa cells (Zi et al. 1998, Zi & Agarwal
1999). Similar observations have also been reported
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Endocrine-Related Cancer (2006) 13 751–778
recently with apigenin in DU145 cells (Shukla &
Gupta 2004a,b), and its analogs, such as acacetin and
linarin DU145, PC3, and LNCaP cells (Singh et al.
2005a). We have also reported recently that the G1
arrest-inducing effect of decursin in human PCa cells is
via a decrease in the levels of CDKs and cyclins and
associated kinase activity (Yim et al. 2005). Similarly,
as reviewed recently by us, EGCG, genistein, and
quercetin also induce G1 arrest in various PCa cells by
modulating CDK–cyclin activity (Agarwal 2000,
Singh et al. 2002a). Together, these studies suggest
that chemopreventive agents potentially decrease the
protein levels of CDKs and/or cyclins that in part are
responsible for an inhibition in the kinase activity of
CDKs causing a halt in deregulated cell-cycle
progression of PCa cells (Fig. 2).
More recently, silibinin has also been shown to
decrease Cdc2 and cyclin B1 expression and associated
kinase activity leading to a G2–M arrest in PCa PC3
cells (Deep et al. 2006). In this study, silibinin also
decreased Cdc25B and Cdc25C protein levels, but
increased Cdc25C(Ser216) phosphorylation leading to
its enhanced binding with 14-3-3b and cytoplasmic
retention in an inactive form. Further, silibinin
enhanced Chk2 phosphorylation at Thr68/Ser19 site,
which was responsible for Cdc25C phosphorylation as
confirmed by the use of Chk2 siRNA (Deep et al. 2006).
This study, for the first time, showed that silibinin
causes inhibitory activation of Chk2–Cdc25C–Cdc2/
cyclin B1 cascade leading to G2–M arrest and
associated apoptosis in human PCa cells. Other
chemopreventive agents have also been shown to
induce G2–M arrest in PCa cells by inhibiting
Cdc2–cyclin B1 activity (Singh et al. 2002a).
CDK inhibitors
CDKIs (such as Cip1/p21, Kip1/p27, and Kip2/p57)
physically interact with CDK–cyclin complexes to
negatively regulate CDK activity and cell-cycle
progression (Sherr & Roberts 1999, Dai & Grant
2003). Whereas, Cip1/p21 is involved in all phases of
the cell cycle and therefore known as a universal
inhibitor of various CDKs’ kinase activity, Kip1/p27
usually regulates cell-cycle progression through G1–S
checkpoint transition (Xiong et al. 1993, Toyoshima &
Hunter 1994, Dai & Grant 2003). These CDKIs bind at
ATP-binding sites in cyclin–CDK complex and inhibit
CDK activity. Cip1/p21 is regulated by many agents in
p53-dependent or -independent manner, and is altered
at both transcriptional and post-transcriptional levels
(Zi et al. 1998, Gartel & Tyner 2002). The down-
modulation of CDKIs is facilitated by ubiquitination
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and proteosomal degradation involving Skp2 (Bloom
& Pagano 2003). Another family of CDKIs includes
INK4 (INK4b/p15, INK4a/p16, INK4c/p18, and
INK4d/p19); the members in this family specifically
bind to and inhibit CDK4 and CDK6 activity (Hirai
et al. 1995, Ortega et al. 2002). CDKI/INK genes are
often mutated and/or have very low levels of
expression in cancer cells that lead to a loss in their
control on cell-cycle progression (Tsihlias et al. 1999,
Ortega et al. 2002). Interestingly, mitogenic signals
also decrease the levels of CDKIs, and anti-mitogenic
signals induce their expression that inhibits cell
proliferation; this approach has provided a potential
strategy for cancer control (Liu et al. 1996). The
importance of INK has been shown in CDK4 knockin
mice, in which normal CDK4 gene was replaced by a
CDK4 R24C (Arg24 replaced with cysteine making it
insensitive to INK4) mutant (Sotillo et al. 2001). These
mice develop a wide spectrum of spontaneous tumors
and are highly susceptible to carcinogens. Recent
reports also suggest the prognostic significance of these
CDKIs in PCa (Tsihlias et al. 1999). Overall, these
studies convincingly suggest that chemopreventive
agents targeting CDKIs for their upregulation could be
a valuable strategy against PCa growth and progression
(Fig. 2).
Studies by us have shown that silibinin induces protein
expression of Cip1/p21 and Kip1/p27 followed by their
increased interactionwithCDK2,CDK4, andCDK6, and
subsequent inhibition of CDKs’ activity in human PCa
cells (Zi et al. 1998, Zi & Agarwal 1999, Deep et al.
2006). Other agents, such as IP6 also increases Kip1/p27
andCip1/p21 protein levels in a dose-dependent but p53-
independent manner, and induces G1 arrest in human
PCa cells (Singh et al. 2003a, Agarwal et al. 2004a). IP6-
induced increase in Kip1/p27 and Cip1/p21 was also
associated with their increased binding with CDKs and
cyclins, and subsequent decrease in the kinase activity of
G1-phase CDKs. In our ongoing study with siRNA for
Kip1/p27 and Cip1/p21, these functional genes were
observed decisive in causing both silibinin and IP6-
induced G1 arrest in DU145 cells (R P Singh, S Roy &
R Agarwal unpublished observations). Grape seed
extract (GSE) has also been shown to induce Kip1/p27
and Cip1/p21 protein levels and associated G1 arrest in
DU145 cells (Agarwal et al. 2000). Yet again, decursin-
causedG1 arrest inDU145 cells has been associatedwith
an increased expression of Cip1/p21 and Kip1/p27
protein levels (Yim et al. 2005). Apigenin has also
been shown to induce Cip1/p21, Kip1/p27, INK4a/p16,
and INK4c/p18 protein levels and cell-cycle arrest in
DU145 cells (Shukla & Gupta 2004a,b). Similarly,
EGCG is shown to induce G1 arrest in DU145 PCa cells
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R P Singh and R Agarwal: PCa chemoprevention
via induction of INK4c/p18 and INK4a/p16 with a
concomitant decrease in the CDKs’ activity and has been
reviewed recently (Singh et al. 2002a). Since increased
rate of cancer progression, aggressive phenotype, and
decreased response to therapy are linked to the loss and/
or low expression of CDKIs in PCa (Cheng et al. 2000),
these findings suggest that upregulation of CDKIs by
chemopreventive agents could inhibit PCa growth and
progression.
Retinoblastoma protein and E2F
Retinoblastoma (Rb/p110) and Rb-family proteins,
such as Rb/p107 and Rb2/p130 have been reported as
important players in cell-cycle regulation and are
mostly inactivated in cancer cells, including PCa
(Paggi et al. 1996, Chau &Wang 2003). These nuclear
phosphoproteins are phosphorylated by CDKs (CDK4/
6–cyclin D1 and CDK2–cyclin E complexes) and
subsequently become unable to sequester E2F family
of transcription factors (Chau & Wang 2003, Seville
et al. 2005). Thus free E2Fs, in response to mitogenic
signal, induce gene transcription required for the cell-
cycle progression (Chau & Wang 2003). Conversely,
hypophosphorylated Rb and Rb-related proteins
sequester E2Fs and inhibit cell proliferation. Rb-family
genes are critically targeted for inactivation by
transforming oncoproteins, and Rb is mutated in
many cancers, including PCa (Chau & Wang 2003,
Seville et al. 2005). High levels of E2Fs have also been
observed in neoplastic cells. It is also important to
emphasize here that E2Fs are downstream targets for
the tumor suppressor p53, as well as Rb proteins
(Slansky & Farnham 1996), and that most of the
advanced PCa tissue samples and established prostate
carcinoma cells show increased E2F transcriptional
activity as a consequence of loss of p53 and/or Rb
(Chau & Wang 2003). Rb/p107 and Rb2/p130
co-operate to regulate cell-cycle progression through
G1 phase with a common goal of G1–S checkpoint
regulation (Sage et al. 2000). These studies suggest
that upregulation of Rb-family proteins, as well as their
increased hypophosphorylation by chemopreventive
agents could check the deregulated cell-cycle pro-
gression in PCa (Fig. 2).
Consistent with the above suggestion, studies by us
have shown that silibinin increases hypophosphory-
lated levels of Rb and Rb-related proteins together with
their increased binding with E2Fs, and that this effect
of silibinin was associated with growth inhibition via
G1 arrest in human PCa cells (Tyagi et al. 2002a,b).
Consistent with a decrease in CDK–cyclin kinase
activity in LNCaP cells, silibinin increased
760
hypophosphorylated levels of Rb/p110 with a specific
decrease in Rb/p110 phosphorylation at Ser780,
Ser795, Ser807/811, and Ser249/Thr252 sites (Tyagi
et al. 2002a). Silibinin also caused a moderate to strong
decrease in E2F1, E2F2, and E2F3 levels in LNCaP
cells (Tyagi et al. 2002a), and E2F4 and E2F5 levels in
DU145 cells (Tyagi et al. 2002b). Overall, these effects
of silibinin were suggestive of suppression in E2F
transcriptional activity leading to a G1 arrest in PCa
cell-cycle progression. We have also observed that IP6
modulates Rb-related protein–E2F complex in causing
G1 arrest in human PCa cells. IP6 increases hypopho-
sphorylated levels of Rb/p110 in LNCaP cells, and
Rb/p107 and Rb2/p130 in DU145 cells (Singh et al.
2003a,b, Agarwal et al. 2004a), together with an
increase in pRb/p107 and pRb2/p130 interaction with
E2F4 and a decrease in the level of transcriptionally
active-free E2Fs leading to growth inhibition of PCa
cells via G1 arrest (Singh et al. 2003a). The decreased
phosphorylation of Rb family proteins is a conse-
quence of the inhibitory effect of IP6 on CDK–cyclin
kinase activity (Singh et al. 2003b, Agarwal et al.
2004a). Recently, a novel flavonoid licochalcone,
isolated from the roots of licorice, is reported to inhibit
Rb phosphorylation at Ser780 together with a decrease
in the levels of different E2Fs in PC3 cells and this
effect was associated with G2–M cell-cycle arrest (Fu
et al. 2004b). Equiguard, a polyherbal supplement,
inhibits Rb phosphorylation, as well as exhibits anti-
tumor activity against LNCaP cells (Lu et al. 2004).
Indole-3-carbinol also decreases hyperphosphorylated
levels of Rb in PCa cells (Sarkar & Li 2004). Overall,
these reports suggest potential anti-cancer activity of
chemopreventive agents via targeting Rb–E2F
complexes most likely via their modulatory effects on
CDK–cyclin–CDKI axis in PCa.
Telomerase
The telomerase (a reverse transcriptase enzyme) activity
is essential to maintain a specialized heterochromatin
structure, a tandem repeats of the hexanucleotide
sequence with a terminal 30 G-rich overhang called
telomere, which protects the ends of chromosomes
(Neumann & Reddel 2002, Biroccio & Leonetti 2004).
Telomeres shorten during each cell division, and normal
cells stop replicating once telomeres become critically
short; however, almost all human tumor cells acquire
telomerase activity to overcome this limitation (Kim et
al. 1994, Mathon & Lloyd 2001, Orlando et al. 2001).
Telomerase activity is absent in normal prostate;
however, primary prostate tumors show elevated
telomerase activity that is also correlated with their
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Endocrine-Related Cancer (2006) 13 751–778
aggressiveness (Lin et al. 1997, Kim & Hruszkewycz
2001, Orlando et al. 2001). Furthermore, androgen
depletion has been shown to activate telomerase in the
prostate of monkeys (Ravindranath et al. 2001), and
therefore, could contribute to PCa progression during
anti-androgen therapy. These studies suggest that
telomerase activity could be another potential target for
chemopreventive agents in their efficacy against PCa
growth and progression (Fig. 2).
By using telomeric repeat amplification protocol
assay, it has been shown that androgen-sensitive
LNCaP cells possess DHT-dependent telomerase
activity, and silibinin has been shown to decrease basal,
aswell asDHT-inducedmRNAexpressionof telomerase
catalytic subunit (Thelen et al. 2004). It has been
suggested that the inhibitory effect of silibinin on DHT-
caused increase in telomerase activity via AR possibly
involves the downregulation of telomerase and other
genes, including PSA. Though not in PCa, EGCG has
been reported to downregulate telomerase activity in
human breast carcinoma MCF-7 cells, which has been
associated with the suppression of cell viability (Mittal
et al. 2004). Nevertheless, more studies are needed to
establish telomerase as a potential target for chemopre-
ventive agents in their efficacy against PCa.
Cell survival/apoptotic targets
NF-kB pathway
NF-kB has been implicated in the growth and survival
of many types of cancer cells, including PCa (Karin
et al. 2002). NF-kB is a family of homo- or
heterodimeric transcription factors, including p50,
p52, p65, RelB, and c-Rel (Schmitz & Baeuerle
1995, Karin et al. 2002). NF-kB is sequestered by
inhibitory-kB (IkB) proteins in cytoplasm, which are
phosphorylated at serine sites by IkB kinases (IKKs)
followed by their ubiquitination and degradation in
response to anti-apoptotic/survival signals (Baeuerle
1998, Karin et al. 2002). Thus, free NF-kB translocates
to the nucleus and binds to a common DNA sequence
motif, known as kB site, in a broad spectrum of genes
that include inflammatory cytokines, chemokines, cell
adhesion molecules, and growth and survival genes
(Giuliani et al. 2001, Karin et al. 2002). ERK1/2 and
Akt pathways are also known to activate NF-kB(Agarwal et al. 2005, Hsiung et al. 2005, Jeong et al.
2005). Both constitutive and induced activation of NF-
kB play a crucial role in the development of
chemotherapy resistance in various cancer cells,
including PCa (Flynn et al. 2003). NF-kB is
constitutively active in androgen-independent PCa,
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where it causes the activation of many genes, including
those involved in apoptosis resistance, angiogenesis,
invasion, and metastasis (Sumitomo et al. 1999,
Lindholm et al. 2000, Karin et al. 2002, Suh & Rabson
2004). Together, these studies suggest that targeting
NF-kB activation by chemopreventive agents could be
a logical strategy to control PCa growth, tumor
angiogenesis, metastasis, and chemoresistance.
Our studies have shown that silibinin inhibits IKKakinase activity accompanied by an inhibition in IkBaphosphorylation and NF-kB transcriptional activity in
androgen-independent PCa DU145 cells (Dhana-
lakshmi et al. 2002). Silibinin also showed a decrease
in nuclear translocation of p65 and p50 with a
concomitant increase in their cytoplasmic levels. In
this study, we also found that silibinin strongly
increases the sensitivity of DU145 cells to otherwise
resistant TNFa-induced apoptosis, and that this effect
of silibinin was via an inhibition of TNFa-induced NF-kB activation (Dhanalakshmi et al. 2002). The
inhibitory effect of silibinin on NF-kB signaling
could be an important mechanism of its anti-tumor
efficacy observed in many studies. IP6 has also been
shown to inhibit NF-kB activation and cell survival in
human PCa DU145 cells (Agarwal et al. 2003).
Similarly, indole-3-carbinol is shown to inhibit NF-
kB activation in advanced human PCa cells and
sensitizes them for chemotherapy (Sarkar & Li
2004). It has been reported recently that curcumin
and phenethyl isothiocyanate inhibit the growth of PC3
prostate tumor xenografts in nude mice, and that they
inhibit NF-kB activation; the effect of these two agents
was stronger when combined together than each agent
alone (Khor et al. 2006). Many other chemopreventive
agents, such as genistein, EGCG, and selenium
compounds have been shown to inhibit NF-kBactivation in different PCa cells as one of their
mechanisms of chemopreventive efficacy (Davis
et al. 1999, Gasparian et al. 2002, Gupta et al.
2004b). Overall, these studies clearly suggest that NF-
kB signaling is an important target for chemopreven-
tive agents to check the growth, survival, and drug
resistance in PCa, and that the effective inhibitors of
this pathway have potential application in clinic.
ATM kinase and cell-cycle Chk signaling
Genotoxic stress is known to activate ATM and ATR
serine/threonine protein kinases, which phosphorylate
a histone variant H2AX(Ser139) marking an early
event in DNA damage/apoptosis and associated cell-
cycle arrest (Yang et al. 2003). These kinases also
phosphorylate Chk1 and Chk2 that subsequently cause
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R P Singh and R Agarwal: PCa chemoprevention
inhibitory phosphorylation of Cdc25C, which other-
wise dephosphorylates Cdc2 making it active for cell-
cycle progression; phosphorylated Cdc2 is inactive and
causes cell-cycle arrest in S or G2–M phase (Tenzer &
Pruschy 2003). The cell-cycle arrest is accompanied by
DNA repair, if the damage is moderate, or an induction
of apoptosis, if the damage is excessive and could not
be repaired (Tenzer & Pruschy 2003, Zhou et al. 2003).
Recently, it has been observed that downregulation of
ATM by antisense-ATM oligonucleotide sensitizes
androgen-dependent (LNCaP and CWR22-Rv1) and
androgen-independent (PC3 and DU145) human PCa
cells to radiation-induced apoptosis (Truman et al.
2005). In another study, ATM knockdown by siRNA in
p53-defective PC3 cells has been shown to increase the
sensitivity of the cells to doxorubicin-induced apopto-
sis (Mukhopadhyay et al. 2005). Furthermore, a higher
expression of ATM protein has been observed in the
higher grade PCa tissues as compared with that of
normal prostate (Angele et al. 2004); however, it has
also been reported that defective DNA-strand break
repair happens in PCa cells (LNCaP, DU145, and PC3)
as compared with normal prostate epithelial cells (Fan
et al. 2004). The aberrant DNA repair could be a
possible mechanism for acquiring genetic instability
during PCa progression or radiotherapy. Therefore, it is
plausible that inhibiting DNA repair by blocking ATM
signaling or its increased activation with the lack of
normal DNA repair might sensitize PCa cells for both
cell-cycle arrest and apoptosis.
Recently, we observed that silibinin activates ATM–
Chk2 pathway leading to H2AX(Ser139) phosphoryl-
ation together with a G2–M arrest and apoptosis in PC3
cells (Deep et al. 2006). In this study, silibinin-caused
ATM(Ser1981) phosphorylation resulted in cell-cycle
arrest and apoptosis through Chk2 activation, as Chk2
siRNA abrogated the biological responses of silibinin
(Deep et al. 2006). Other studies have shown
that natural chemopreventive agents induce
ATM(Ser1981) phosphorylation in many types of
cancer cells, including PCa. For example, genistein
causes ATM-dependent activation of Chk2 and G2–M
arrest in liver carcinoma HepG2 cells (Chang et al.
2004). Apigenin, luteolin, and kaempferol are also
reported to activate ATM/ATR kinases for G2–M cell-
cycle arrest (O’Prey et al. 2003). Overall, these studies
suggest that ATM signaling could be potentially
targeted by chemopreventive agents to induce G2–M
arrest and apoptosis in PCa cells. Since these findings
are against those showing that downregulation of ATM
decreases the survival of PCa cells, more studies are
needed to further investigate these discrepancies.
Nonetheless, studies are also needed to identify the
762
in vivo significance of the modulatory effect of
chemopreventive agents on ATM signaling in PCa.
Bcl-2 family proteins
The growth of malignant cells, including PCa is almost
always accompanied by the loss of apoptotic response,
and therefore, an induction of apoptosis in cancer cells
is long being used as a strategy to control PCa growth
and progression. Further, the agents with both growth
inhibitory and apoptotic efficacy could be more
effective in both prevention and intervention of PCa.
Activation of the caspase cascade is commonly
observed in the execution of apoptosis (Ho & Hawkins
2005, Watson & Fitzpatrick 2005). Caspase (a family
of cysteine proteases) ultimately cleaves many sub-
strate proteins, including poly-(ADP-ribose) poly-
merase (PARP) that marks the induction of apoptotic
cell death (Ho & Hawkins 2005). In mitochondrial
apoptosis, the damage of mitochondrial integrity
followed by the release of cytochrome c forming a
complex with Apaf-1 and pro-caspase 9 initiates the
activation of a caspase-mediated apoptotic cascade
(Reed 2002, Ho & Hawkins 2005, Watson &
Fitzpatrick 2005). Bcl-2 family members, consisting
of both anti-apoptotic (such as Bcl-2 and Mcl-1) and
pro-apoptotic (such as Bax and Bad) molecules, play
major roles in regulating the redox state of mito-
chondria (Reed 2002, Catz & Johnson 2003). The
balance and interaction between these proteins
determine the survival or apoptotic fate of the cell
(Catz & Johnson 2003). In cancer cells, including PCa,
the equilibrium in terms of the content and interaction
of these proteins is shifted away from the pro-apoptotic
end towards the survival end and, therefore, cells
become resistant to apoptotic death (Catz & Johnson
2003). Several studies have shown an overexpression
of Bcl-2 in about 30% of the PCa cases, which was also
correlated with the advanced stage of the disease and
poor prognosis (Raffo et al. 1995, Moul 1999, McCarty
2004). Moreover, advanced PCa also develops
apoptosis resistance towards the commonly used
androgen ablation therapy (Raffo et al. 1995, Moul
1999). Antisense oligonucleotides or siRNA for Bcl-2
or Bcl-xL, as well as a forced expression of Bax have
been shown to induce apoptosis in PCa cells (Lin et al.
2005, Yamanaka et al. 2005). Thus, chemopreventive
agents that shift the equilibrium from anti-apoptotic to
pro-apoptotic Bcl-2 family members leading to
apoptosis induction could be promising in controlling
the growth and survival of PCa cells.
Silibinin has been shown to induce apoptosis and
inhibit DU145 tumor xenograft growth (Singh et al.
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Endocrine-Related Cancer (2006) 13 751–778
2003b); however, its effect on Bcl-2 family members
as not yet been investigated. The growth inhibitory
effect of IP6 has been shown to be associated with an
induction of apoptotic death in human LNCaP and
DU145, as well as in mouse TRAMP-C1 PCa cells as
reviewed by us recently (Singh & Agarwal 2005). IP6
also causes in vivo apoptotic death of PCa cells in
DU145 tumor xenograft (Singh et al. 2004a). In
mechanistic studies, IP6 was found to activate caspases
and to cause PARP cleavage in human PCa cells (Singh
et al. 2003a). In mouse PCa cells, however, the use of a
pan-caspase inhibitor shows the involvement of both
caspase-dependent and -independent mechanisms in
IP6-induced apoptotic cell death (Sharma et al. 2003).
IP6 also increases the ratio of Bax:Bcl-2 in LNCaP
cells, which was associated with increased apoptosis
(Agarwal et al. 2004a). GSE also induces apoptotic
death through dissipation of mitochondrial membrane
potential and cytochrome c release causing the
activation of caspase cascade in DU145 cells (Agarwal
et al. 2002). In this study, GSE also showed an increase
in the cleavage of pro-caspases 3 and 9 together with an
increase in their protease activity. The use of caspase
inhibitors showed that GSE-induced apoptosis was
mostly via caspase activation. Recently, pomegranate
fruit extract has been shown to induce pro-apoptotic
Bax and Bak levels together with a decrease in the
levels of anti-apoptotic proteins, namely Bcl-2 and
Bcl-xL in PC3 cells; these effects were associated with
a decrease in cell survival (Malik et al. 2005).
Selenium compounds have been shown to decrease
the ratio of Bcl-2:Bax in human PCa-derived cells
(Husbeck et al. 2006). Similarly, EGCG has also been
shown to cause a change in Bcl-2:Bax ratio in LNCaP
cells that favors apoptosis (Hastak et al. 2003). Diallyl
trisulfide and phenethyl isothiocyanate from crucifer-
ous vegetables have been shown to induce Bad or Bak,
and decrease Mcl-1 or Bcl-xL in PCa cells together
with an apoptosis induction (Xiao & Singh 2005, Xiao
et al. 2005). Recently, many other chemopreventive
agents, including quercetin (Vijayababu et al. 2005),
EGCG (Hastak et al. 2005), sulforaphane (from
cruciferous vegetables; Choi & Singh 2005),
b-lapachone (from a South American tree Tabebuia
avellanedae; Lee et al. 2005), retinoic acid (Huynh
et al. 2006), neem (Azadirachta indica) leaf extract
(Kumar et al. 2005), and guggulsterone (from a
medicinal plant, Commiphora mukul; Singh et al.
2005c) have been shown to increase the ratio of pro-
apoptotic vs anti-apoptotic Bcl-2 family proteins and
induce apoptosis in different PCa cells. Overall, these
studies suggest that chemopreventive agents have
potential to target Bcl-2 family proteins, though these
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observations need to be further explored in pre-clinical
and clinical prostate tumors.
Inhibitor of apoptosis protein (IAP)
IAP family is known to have pleiotropic functions,
including regulation of cell-cycle progression and
inhibition of extrinsic and intrinsic pathways of cell
death (LaCasse et al. 1998, Altieri 2003). Survivin, the
smallest (16.5 kDa) protein in the family, shows very
rare expression in normal tissues excluding embryonic
tissues and those with a high rate of cellular turnover,
such as colonic mucosa; however, it is overexpressed
in many types of cancers, including PCa (LaCasse
et al. 1998, Altieri 2003, Krajewska et al. 2003, Kishi
et al. 2004). Survivin expression is also associated with
biologically aggressive phenotype and drug resistance
in prostate tumors (Kishi et al. 2004, Shariat et al.
2004). Survivin directly interacts with caspase-3 and
caspase-7 leading to an inhibition in their protease
activity and subsequent apoptosis. Downregulation of
survivin expression by anti-sense has been shown to
potentiate apoptosis induction by the chemotherapeutic
agents, such as docetaxel and etoposide in DU145 cells
(Hayashi et al. 2005). Together, these studies suggest
that a differential expression of survivin could make it
a selective target for chemopreventive agents in terms
of inducing apoptotic death only in cancer cells.
In a preliminary study, silibinin is observed to
moderately decrease survivin protein expression in
PCa cells (unpublished data). Our completed studies in
other cancer cell types and human endothelial cells
show that silibinin-caused apoptosis is associated with
a decrease in survivin protein expression concomitant
with an increase in caspase activity (Tyagi et al. 2003b,
Mallikarjuna et al. 2004, Singh et al. 2005b). Genistein
is shown to inhibit survivin expression in human PCa
LNCaP cells (Suzuki et al. 2002). The combination of
vitamins C and D is reported to inhibit survivin mRNA
expression in DU145 cells (Gunawardena et al. 2004).
However, the lack of sufficient studies with chemo-
preventive agents in terms of their effect on the
modulation of survivin levels in PCa, suggests that
more investigations are needed to define survivin’s role
in apoptotic cell death in PCa by these agents.
Angiogenic and metastatic targets
Tumor angiogenesis and angiogenic factors
Angiogenesis is a prerequisite for the growth of solid
tumors and their metastasis. Tumors remain avascular
and latent for years; however, tumor growth can be
initiated by neo-angiogenesis (Bergers & Benjamin
763
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Figure 3 Targeting of molecular pathways by chemopreventive agents in both PCa and endothelial cells to inhibit tumorangiogenesis. Chemopreventive agents inhibit the production and secretion of angiogenic factors in PCa cells, and also inhibitmitogenic and survival signaling in endothelial cells. Further, endothelial cell migration and capillary organization are inhibited via theinhibition of MMP activation. RTK, receptor tyrosine kinase; ERK1/2, extracellular signal-regulated kinase1/2; STAT, signaltransducer and activator of transcription; HIF-1a, hypoxia-inducible factor-1a; VEGF, vascular endothelial growth factor; iNOS,inducible nitric oxide synthase; COX-2, cyclooxygenase-2; IL, interleukin; MMP, matrix metalloproteinase.
R P Singh and R Agarwal: PCa chemoprevention
2003, Turner et al. 2003, Kirsch et al. 2004). Tumor
vasculature is also recognized as a prognostic marker
and predictor of malignant potential of PCa (Bostwick
& Iczkowski 1998, Bono et al. 2002, Turner et al. 2003).
Further, vascular endothelial cells are less likely to
acquire therapeutic resistance and are directly exposed
to the physiologically achievable agents, including
chemopreventive agents (Bergers & Benjamin 2003).
The critical dependence of solid tumors on angiogenesis
suggests that blocking angiogenesis could be a potential
strategy in controlling PCa growth. The potential anti-
angiogenic strategies targeted by chemopreventive
agents could include the blocking of production and
release of angiogenic factors, increase in anti-angio-
genic factors, and inhibition of endothelial cell
proliferation, migration and survival, and disruption of
the process of microvessel formation (Fig. 3). Chemo-
preventive agents have shown pleiotropic anti-angio-
genic effects in many studies as reviewed by us recently
(Singh & Agarwal 2003). It is important to highlight
764
here that many anti-angiogenic agents, including
phytochemicals, antibodies, and synthetic peptides are
already in clinical trials (Kerbel 2000).
Tumor cells synthesize and secrete angiogenic factors,
and via paracrine regulation of endothelial cells, initiate
neo-angiogenesis from pre-existing blood vessels (Fer-
rara 2002, Bergers & Benjamin 2003; Fig. 3). VEGF,
basic fibroblast growth factor (bFGF), and IGF-I secreted
from tumor cells are known to stimulate tumor
angiogenesis (Ferrara 2002, Bergers & Benjamin
2003). COX-2, nitric oxide synthase (NOS), and IL-8
are among the inflammatory angiogenic molecules
produced by tumor cells that influence neo-angiogenesis
(Chiarugi et al. 1998, Bergers & Benjamin 2003). In
tumor cells, the expressionof these angiogenicmolecules
is favored by the constitutively active mitogenic and
survival signaling, including those mediated via EGF
receptor, IGFR, and platelet-derived growth factor
receptor, ERK1/2 and Akt (Ferrara 2002, Singh &
Agarwal 2003). Since most of these signaling pathways
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Endocrine-Related Cancer (2006) 13 751–778
are inhibited by various chemopreventive agents in PCa,
a decreased expression of angiogenic factors was also
anticipated as reported in many studies.
Silibinin-caused inhibition of mitogenic signaling has
been shown to be associated with a decrease in both
expression and secretion ofVEGF in humanPCa cells, as
reviewed by us recently (Singh&Agarwal 2004). EGCG
is also shown to inhibit receptor tyrosine kinase signaling
causing a decrease in both expression and secretion of
VEGF and bFGF in many cancer cell types (Sartippour
et al. 2002, Lee et al. 2004a,b, Rodriguez et al. 2006).
Furthermore, silibinin, EGCG, genistein, resveratrol, and
several other agents have been shown to decreaseCOX-2
and/or iNOS expression in different cancer cells,
suggesting them as the putative targets for the
suppression of tumor angiogenesis. These chemopre-
ventive agents also inhibit NF-kB activity that is known
tomediate angiogenesis, invasion, and metastasis in PCa
as discussed earlier. These facts suggest that the
inhibition of oncogenic signaling in PCa by chemopre-
ventive agents could potentially decrease the production
of angiogenic factors by tumor cells and associated
angiogenesis.
VEGF receptor signaling
VEGF is the most important mitogenic and survival
factor for vascular endothelial cells (Ferrara 2002,
Turner et al. 2003). VEGF interacts with high affinity
to both fms-like tyrosine kinase-1 (FLT-1) and fetal
liver kinase-1 (Flk-1/KDR) receptors, which are
primarily expressed by vascular endothelial cells
(Ferrara 2002). However, these receptors are also
expressed in many PCa cells, suggesting a possible
autocrine loop for VEGF–VEGF receptor signaling
(Turner et al. 2003). The binding of VEGF to its
receptors stimulates receptor dimerization and acti-
vation leading to the propagation of signaling cascade
for enhanced proliferation and survival of endothelial
cells (Ferrara 2002, Turner et al. 2003). Therefore,
disrupting VEGF receptor signaling could be an
attractive target for chemopreventive agents to check
the growth and development of PCa, given that
angiogenesis is a prerequisite for tumor growth and
metastasis (Fig. 3).
Our studies show that silibinin strongly inhibits
growth and survival of endothelial cells (Singh et al.
2005b). Silibinin is also observed to inhibit VEGF- and
IGF-I-induced cell proliferation and survival in human
endothelial cells (R P Singh & R Agarwal unpublished
observations). Similarly, GSE also inhibits growth and
survival of endothelial cells (Agarwal et al. 2004b).
Genistein is shown to inhibit VEGF-induced tyrosine
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phosphorylation of receptor kinases in endothelial cells
(Guo et al. 1995). EGCG, catechin-3 gallate, and
epicatechin-3 gallate have been shown to inhibit
VEGF-induced tyrosine phosphorylation of Flk-1/
KDR and suppress in vitro angiogenesis in endothelial
cells (Lamy et al. 2002). Moreover, an endogenous
anti-angiogenic protein, endostatin is shown to block
VEGF-induced activation of Flk-1/KDR, ERK, and
p38MAPK (Kim et al. 2002). Costunolide, a sesqui-
terpene lactone isolated from Saussurea lappa, has
been shown to inhibit Flk-1/KDR signaling in human
vascular endothelial cells (HUVEC) together with a
suppression in VEGF-induced endothelial cell prolifer-
ation and neo-vascularization of mouse cornea (Jeong
et al. 2002). IP6 also inhibits bFGF-induced in vitro
angiogenesis and reduces VEGF expression (Vucenik
et al. 2004). These reports suggest that targeting VEGF
receptor signaling by chemopreventive agents could be
a potential anti-angiogenic mechanism to halt tumor
angiogenesis in PCa.
Hypoxia-inducible factor-1a
Hypoxia is known to be associated with an increased
tumor metastasis and poor survival in cancer patients
(Zhong et al. 1999, Harris 2002). Hypoxia activates
angiogenesis and stimulates vascular remodeling
(Harris 2002). Hypoxia inducible transcription factor
(HIF)-1 is comprised of two subunits, HIF-1a and
constitutively expressed HIF-1b, and controls the
expression of several genes regulated by hypoxia
(Harris 2002). Tumor suppressor Von Hippel-Lindeau
(VHL) usually binds to and causes proteosomal
degradation of HIF-1a (Pugh & Ratcliffe 2003).
Hypoxia or a mutation in VHL gene increases HIF-1aprotein expression mostly via an enhanced receptor
tyrosine kinase (RTK) signaling that induces the
transcription of VEGF, VEGF receptor, platelet-
derived growth factor (PDGF), and other angiogenic
mediators (Zhong et al. 2000, Kim & Kaelin 2004, Fu
et al. 2005). Androgens are shown to activate HIF-1avia autocrine RTK–PI3K/Akt signaling in PCa cells
(Mabjeesh et al. 2003). In view of these facts, HIF-1acould be a logical chemopreventive target to control
PCa tumor angiogenesis.
We have observed that silibinin inhibits hypoxia-
induced expression of HIF-1a and VEGF in human
PCa LNCaP and PC3 cells (unpublished data). A
literature search shows that chemopreventive studies
on HIF in PCa are limited. However, in other cancers,
such as HepG2 human hepatoblastoma cells, torilin, a
plant-derived sesquiterpene, downregulates hypoxia-
induced expression of VEGF and IGF-II (Kim et al.
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R P Singh and R Agarwal: PCa chemoprevention
2000). Flavopiridol is also shown to inhibit hypoxia-
induced expression of VEGF in human neuroblastoma
and monocytes (Melillo et al. 1999, Rapella et al.
2002). Nevertheless, more studies are needed to assess
the effect of chemopreventive agents on HIF-1a, aswell as HIF-1a-mediated expression of angiogenic
factors in the suppression of PCa.
Cell adhesion molecules and matrix metallopro-
teinases (MMPs)
In the process of angiogenesis, endothelial or tumor
cells show increased expression of avb3 and avb5integrins, the heterodimeric cell-surface receptors,
which is usually linked with the tumor grade (Fornaro
et al. 2001, Slack-Davis & Parsons 2004). In radio-
therapy, the activation of integrins causes radio-
resistance in human PCa as reported in a PC3
xenograft experiment (Abdollahi et al. 2005). Inhi-
bition of integrin expression prevents in vitro angio-
genesis, and suppression of integrin function is shown
to suppress tumor angiogenesis in mouse model
(Abdollahi et al. 2005, Belvisi et al. 2005). The
breakdown of tissue matrix to facilitate the movement
of newly formed endothelial cells for vessel formation
is required for angiogenesis. It is achieved by MMPs
secreted by activated endothelial or tumor cells
(Overall & Lopez-Otin 2002). MMP-2 and MMP-9
are overexpressed in invasive PCa and mediate the
invasion of both tumor and endothelial cells (Overall &
Lopez-Otin 2002, Mehta et al. 2003, Takaha et al.
2004). Together, these studies suggest that integrins
and MMPs could be targeted by chemopreventive
agents to check the invasive potential of PCa.
Aspirin is reported to inhibit adhesion of PC3 cells to
fibronectin and vitronectin, which bind to integrin
receptors (Lloyd et al. 2003). To our knowledge, there
is no chemopreventive study on integrin signaling in
PCa, and therefore, potential chemopreventive agents
need to be investigated for their possible modulatory
effect on this signaling pathway and its possible
association with their efficacy against PCa. In other
studies, both silymarin and silibinin have been shown
to inhibit both secreted and cellular MMP-2 expression
and suppress capillary formation by human umbilical
vein endothelial cells (HUVEC) in Matrigel assay
(Jiang et al. 2000, Singh et al. 2005a,b,c). EGCG also
inhibits both MMP-2 and MMP-9 for its anti-
angiogenic/metastatic activity in TRAMP model
(Sartor et al. 2004). It also inhibits the PSA-induced
degradation of basement membrane and activation of
MMP-2 in cell culture (Pezzato et al. 2004). Recently,
employing DU145 cells in culture and tumor xenograft
766
models, curcumin has been shown to reduce the
expression of MMP-2 and MMP-9 together with
suppression in growth and invasive potential of
tumor cells (Hong et al. 2006). Genistein also inhibits
MMP-2 and MMP-9 and upregulates tissue inhibitor of
metalloproteinases (TIMP)-1 leading to the suppres-
sion of vascular growth, tumor cell invasion, and bone
metastasis (Li et al. 2004, Huang et al. 2005). RRR-a-tocopheryl succinate, a vitamin E derivative, inhibits
MMPs activity and suppresses Matrigel invasion of
PC3 and DU145 cells (Zhang et al. 2004). Luteolin and
quercetin are also shown to inhibit MMP-2 and MMP-9
secretion from tumor cells (Huang et al. 1999). In
addition to their effects on MMPs and TIMPs, the
effect of chemopreventive agents should also be
studied on endogenous angiogenesis inhibitors,
such as angiostatin, endostatin, and platelet factor
4, which might also play an important role in an
overall inhibitory effect of these agents on in vivo
tumor angiogenesis. Furthermore, the discovery of
specific vascular markers associated with tumor
development could provide substantial success
towards PCa chemoprevention via anti-angiogenic
intervention.
Urokinase-type plasminogen activator (uPA)
In addition to neo-vascularization, tumor invasion
involves enhanced degradation of extracellular matrix
that facilitates tumor metastasis. Recent studies indicate
that uPA, a serine protease, is causatively involved in the
metastatic phenotype of many cancers, including PCa
(Sheng 2001). Recently, valuable information has been
generated regarding the role of uPA system inmetastasis,
where gene amplification has been identified as one of its
mechanisms of overexpression in human PCa (Helenius
et al. 2001). Animal PCa models evidently show the
significance of uPA and uPA receptor (uPAR) in the
invasion and metastases of tumors. The knockdown of
uPA and uPAR expression by siRNAs in metastatic PCa
cell-line PC3 has been shown to suppress tumor cell
invasion (Pulukuri et al. 2005). In this study, the
abrogation of uPA–uPAR signaling is reported to inhibit
the activationofdownstreammolecular signaling targets,
including ERK1/2 and Stat3. These reports suggest that
chemopreventive intervention of uPA–uPAR signaling
could be an alternative strategy to control PCametastasis.
Green tea polyphenols are shown to inhibit uPA
expression in TRAMP mice (Adhami et al. 2004).
Genistein is reported to inhibit uPA production in PC3
cells (Skogseth et al. 2005). Retinoic acid inhibits the
malignant potential, including extracellular proteolysis
of PCa by inhibiting uPA expression (Webber &
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Table 1 Potential molecular targets of the novel chemopreven-
tive agent silibinin in prostate cancer (PCa)
Molecular alterations by silibinin in PCa
Suppression of androgen receptor (AR) nuclear
translocation
Decrease in prostate specific antigen expression and secretion
Down-regulation of AR co-activator, the prostate epithelium-
derived Ets transcription factor
Inhibitionof ligand (transforminggrowth factora, TGFa) binding to
epidermal growth factor receptor (EGFR) and its internalization
Inhibition of EGFR tyrosine phosphorylation
Inhibition of Shc and extracellular signal-regulated kinase 1/2
activation
Suppression of TGFa expression and secretion
Induction of insulin-like growth factor-binding protein-3 and its
secretion
Inhibition of insulin receptor substrate-1 phosphorylation
Induction of Cip1/p21 and Kip1/p27 levels
Suppression of cyclin-dependent kinases and cyclins levels,
and associated kinase activity
Increase in retinoblastoma protein (Rb/p110)
hypophosphorylation
Decrease in serine and threonine phosphorylation of Rb/p110
Increase in hypophosphorylation of Rb/p107 and Rb2/p130
Decrease in E2F levels
Decrease in telomerase activity
Inhibition of inhibitory kBaIkBa kinase (IKKa) activity and an
increase in IkBa level
Inhibition of constitutive nuclear factor-kB (NF-kB)
transcriptional activity
Inhibition of tumor necrosis factor a-induced NF-kB
transcriptional activity
Activation of ataxia telangiectasia-mutated kinase
and checkpoint kinase 2
Activation of caspases
Decrease in survivin expression (R P Singh & R Agarwal
unpublished observations)
Decrease in vascular endothelial growth factor expression
and secretion
Decrease in hypoxia-inducing factor-1a level (R P Singh &
R Agarwal unpublished observations)
Decrease in matrix metalloproteinase 2 activity in PCa and
endothelial cells
Inhibition of growth, survival, and migration of endothelial
cells
Inhibition of urokinase-type plasminogen activator expression
and activity
Biological effects of silibinin in PCa
Inhibition of cell growth and proliferation, and induction of
differentiation morphology in PCa cells
Induction of strong G1 and G2–M cell-cycle arrest,
and moderate apoptosis in PCa cells
Suppression of PCa tumor xenograft growth in athymic nude
mice
In vivo anti-proliferative and apoptotic effects in xenograft
study
Inhibition of angiogenesis both in vitro and in vivo tumor
xenograft
Inhibition of tumor growth in transgenic adenocarcinoma
of the mouse prostate animal model (unpublished)
Non-toxic and physiologically available in plasma/tissues in
animal and human studies
Endocrine-Related Cancer (2006) 13 751–778
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Waghray 1995). Aspirin suppresses uPA secretion from
PC3 cells and inhibits cell migration (Lloyd et al. 2003)).
An extract from a palm plant, Serenoa repens, has been
shown to inhibit uPA activity in PC3 cells, as well as
tumor cell invasion (Ishii et al.2001). Silibinin’s effect on
uPAhas not been studied in PCa, but it is shown to inhibit
the expression of uPA in A549 lung cancer cell line with
metastatic potential (Chu et al. 2004). Overall, these
studies suggest that chemopreventive targeting of uPA
has potential to inhibit the invasive process in PCa;
however, more detailed studies are needed for their
possible clinical implications.
Chemopreventive agents in PCa clinicaltrial
There are only few natural agents, which are under
investigation for their chemopreventive efficacy in
human PCa patients. However, epidemiological studies
suggest that natural agents might have protective effects
against PCa (Nelson et al. 2003). An epidemiological
study with green tea consumption has been conducted in
age-matched placebo and PCa patients with confirmed
adenocarcinomaof the prostate in southeastChinaduring
2001–2002 (Jian et al. 2004). In this study, tea
consumption was limited to green tea and 55% of PCa
patients were tea drinkers as compared with 80% in
placebo. A decreased risk of PCa was noted with
increasing frequency, duration, and quantity of green
tea consumption. In a very recent randomized controlled
trial, 200 mg thrice a day (total 600 mg/day) dose of a
preparation of green tea catechins (GTCs: -epigalloca-
techin, 5.5%; -epicatechin, 12.24%; -epigallocatechin-3-
gallate, 51.88%; -epicatechin-3-gallate, 6.12%; total
GTCs, 75.7%; caffeine, !1%) was given to men with
high-grade PIN for 1 year (Bettuzzi et al. 2006). At the
end of the study, there was only one tumor in the 30
GTCs-treated men, whereas nine tumors were observed
in the 30 placebo-treatedmen. This study did not find any
adverse effects of GTCs consumption. Another clinical
trial of green tea (250 mg twice daily) in hormone
refractory PCa patients (19 subjects) was found to have
minimal clinical activity against hormone refractory PCa
(Choan et al. 2005).However, this conclusion is based on
the disease progression only in 15 patients, who
completed at least 2months of the treatment requirement.
The a-tocopherol and b-carotene study, basically
designed for the lung cancer chemoprevention, has
shown that men, who received vitamin E for a 6-year
period had a 34% lower incidence of PCa (Albanes et al.
1995). This finding has inspired an ongoing large multi-
center trial, known as SELECT (selenium and vitamin E
cancer prevention trial), with selenium and vitamin E for
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R P Singh and R Agarwal: PCa chemoprevention
PCa prevention; the results of this trial are still awaited
(Klein et al. 2000). Silibinin is being investigated in
phase I clinical study to assess its bioavailability and
toxicity. The results obtained so far are promising to
evaluate its efficacy and associated biomarkers in PCa
patients in a pilot phase II clinical trial (Flaig et al. 2005).
Overall, these epidemiological and clinical studies
suggest the potential of natural agents in the chemopre-
vention of PCa; however, more studies are needed in the
near future for their possible clinical applications.
Conclusions
To date, other than the fact that many targets are
present on the membrane and inside the tumor cells, the
outcome of an agent targeting a single molecule has
been disappointing. The simplest reason could be that
tumors have many molecular targets, which function
aberrantly, and therefore, for the best results in the
chemoprevention, as well as therapy of PCa, a multi-
molecular targeting approach is essential. In this
regard, as discussed above, chemopreventive agents,
such as silibinin (Table 1), usually have many targets
for their effective action in neoplastic cells, and
therefore, these agents could be of potential use in
controlling PCa malignancy. However, the modulatory
effect of a chemopreventive agent on many cellular
targets could most likely be a sequential or correlative
effect, and therefore, knockout/in cell and animal
models are now essential to further identify and
establish critical molecular targets for the efficacy of
chemopreventive agents, not in PCa but in other
cancers as well. Such studies will advance the field of
cancer chemopreventive drug development. So far, the
recently identified chemopreventive agents, including
silibinin, IP6, decursin, apigenin, and acacetin, along
with green tea polyphenols, genistein, quercetin, and
curcumin have shown immense potential for PCa
control; however, more studies are needed for their
possible clinical use against PCa.
Acknowledgements
Original studies are supported by the NCI RO1 grants
CA102514 and CA104286. The authors declare that
there is no conflict of interest that would prejudice the
impartiality of this scientific work.
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