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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


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

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(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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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


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.

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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.


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




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


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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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




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.


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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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




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


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

759



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




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,


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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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

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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.




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


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


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.


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




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


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.

765



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 &



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



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

767



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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