The Prostate 67:1641^1653 (2007)

Resveratrol SensitizesAndrogen IndependentProstateCancerCells toDeath-ReceptorMediated

ApoptosisThroughMultipleMechanisms

Catherine Gill,* Sinead E. Walsh, Colm Morrissey,John M. Fitzpatrick, and R. William G. Watson

UCDSchoolofMedicineandMedical Sciences,MaterMisericordiaeUniversityHospital,UCDConway Institute of BiomolecularandBiomedical Research,University CollegeDublin, and

DublinMolecularMedicineCentre,Dublin, Ireland

BACKGROUND. Acritical factor in prostate cancer development andprogression is the alteredexpression of apoptotic regulatory proteins which renders cells resistant to both hormone- andchemo-therapies. Resveratrol, a dietary component with chemopreventive properties has beenreported to resensitize a variety of cancer cell types to apoptosis. In the current study, the ability ofresveratrol pre-treatment to sensitize hormone refractory prostate cancer cell lines (PC-3 andDU145) to apoptosis and the mechanisms involved were investigated.METHODS. Apoptosis was assessed using several established parameters and proteinexpression was analyzed by Western blot and flow cytometry. IAP knockdown was achievedusing RNAi while inhibition of Akt phosphorylation was achieved by pre-incubation with thePI3-kinase inhibitor LY294002.RESULTS. Pre-treatment with resveratrol sensitized PC-3 and DU145 cells to agents thatspecifically target death receptors (TRAIL, Fas, TNFa) but not agents that initiate apoptosisthrough other mechanisms (Etoposide, Paclitaxel, Tunicamycin, Thapsigargin). Resveratrol pre-treatment altered the expression of IAPs and Bax, and decreased Akt phosphorylation in PC-3cells, leading to increased caspase activation and apoptosis. While knockdown of IAPs usingsiRNAdid notmimic the effects of resveratrol, inhibition ofAkt phosphorylation usingLY294002sensitized PC-3 cells to TRAIL induced apoptosis but not to etoposide or tunicamycin.CONCLUSION. Altering apoptotic susceptibility in advanced androgen independent diseaserequires manipulation of a broad signaling pathway. Use of resveratrol or inhibition ofAkt phosphorylation may represent an important therapeutic approach in combinationwith conventional therapies for the treatment of prostate cancer. Prostate 67: 1641–1653,2007. # 2007 Wiley-Liss, Inc.

KEY WORDS: resveratrol; prostate cancer; TRAIL; IAP; Akt; apoptosis

INTRODUCTION

Prostate cancer is the most frequently diagnosedcancer in men. Current treatment for localized diseaseinvolves surgery or radiation therapy and is potentiallycurative. Locally advanced disease is treated withandrogen ablation, resulting in death of androgendependent cancer cells by apoptosis, however, the cellsultimately become resistant to this treatment as theyare no longer dependent on androgen for growthand survival and therapy fails. This stage of diseaseis referred to as androgen-refractory or androgen

depletion-independent, and hormonal manipulationor chemotherapy is essentially palliative with noeffective cure for advanced prostate cancer.

Grant sponsor: Irish Cancer Society; Grant sponsor: EU (E)UROES-TROGEN(E)S; Grant number: QLK6-CT-2000-00565.

*Correspondence to: Catherine Gill, UCD School of Medicine andMedical Sciences, Conway Institute, University College Dublin,Belfield, Dublin 4, Ireland. E-mail: catherine.gill@ucd.ieReceived 2 May 2007; Accepted 20 July 2007DOI 10.1002/pros.20653Published online 6 September 2007 in Wiley InterScience(www.interscience.wiley.com).

� 2007Wiley-Liss, Inc.

Increased cellular resistance to apoptosis plays animportant role in the development of androgenindependence and failure of subsequent chemotherapy[1]. Depending on the trigger, apoptosis is initiatedthrough one of a number of different signaling path-ways. These include the ‘‘extrinsic’’ or death receptorpathway, whereby ligands such as Fas and TRAILbind to specific receptors on the cell surface leadingto caspase activation and cell death [2], and the‘‘intrinsic’’ pathway of which mitochondrial disrup-tion and cytochrome c release are central components[3]. Recent evidence points to a third pathway ofcaspase activation initiated in response to agents thatdisrupt endoplasmic reticulum (ER) homeostasis.Though the mechanism is yet to be established, agentsthat cause ER stress ultimately result in caspase-3activation and the downstream events of apoptosis [4].These pathways are not mutually exclusive andcross talk can occur, for example, in many cell types,caspase-8 can activate Bid which subsequently trans-locates to the mitochondria leading to cytochrome crelease [5,6]. Apoptotic signaling is tightly regulated byproteins, some of which are specific for particularpathways, such as cFLIP-mediated inhibition of deathreceptor signaling, and others that act at a number oflevels in the cell, such as inhibitor of apoptosis proteins(IAPs) which are reported to act both at the receptorand by inhibiting caspases downstream of mito-chondria [7–9]. Expression levels of these proteins area key determinant of cell fate and thus changes in theirexpression play an important role in promotingsurvival of cancer cells. Altered expression of Bcl-2family members is frequently reported in humancancer [10] as is deregulated expression of IAPs [11],and heat shock proteins (Hsps) [12]. In prostate cancerincreased expression of anti-apoptotic Bcl-2 is closelyassociated with development of androgen indepen-dence [13]. Hsp27 levels are also increased afterandrogen ablation and its overexpression in LNCaPcells renders them highly resistant to androgen with-drawal [14]. We and others have reported increasedexpression of IAPs in prostate cancer cell lines [15]and biopsy specimens [16]. Due to the frequency ofderegulated apoptosis in cancer cells, much of thecurrent research is aimed at finding ways to restoresusceptibility to apoptosis within the cell, therebyre-sensitizing these cells to conventional therapies.A major drawback in these efforts is that targeting asingle, overexpressedprotein in a cancer cell is unlikelyto be effective, as demonstrated previously [17]. It istherefore necessary to identify a panel of proteins thatmight be targeted simultaneously or to find singleagent with multiple effects.

In recent years the importance of dietary compo-nents such as anti-oxidants in disease prevention has

become an important area of research. Resveratrol, aphytoalexin found in redwine and foods suchas grapesand peanuts, has potent chemopreventive propertiesand has been reported to inhibit distinct phases ofcarcinogenesis in vitro [18]. Previous studies havedemonstrated that resveratrol can not only induceapoptosis directly in PC-3 and LNCaP cells [19,20], butcan also sensitize LNCaP and other cell types toincreased apoptosis induced by TRAIL and otherchemotherapeutic agents [21–23]. The mechanism ofsensitization has been reported to involve down-regulation of the expression of survivin, a memberof the IAP family [21,22] and altered expression ofBcl-2 family proteins [21,23]. Thus, apart from a rolein chemoprevention, resveratrol has the potentialto be used therapeutically to enhance the effects ofchemotherapeutic agents by regulating sensitivity toapoptosis.

In the current study, we investigated the abilityof resveratrol to sensitize androgen independentprostate cancer (AIPC) cell lines to apoptosis inductionby a range of triggers. We demonstrate that resveratrolsensitizes specifically to agents that initiate apoptosisvia a death receptor pathway. Where resveratrol wasassociated with alterations in Akt phosphorylation,cIAP-1, cIAP-2 and Bax expression, only specificinhibition of Akt phosphorylationmimicked the effectsof resveratrol. Thus resveratrol or specific inhibitors ofAkt signalingmay be useful in sensitizingAIPC cells toreceptor-mediated apoptosis

MATERIALSANDMETHODS

Cell Culture andTreatment

The humanprostate cancer cell lines PC-3 andDU145were purchased from the American Type CultureCollection (ATCC) and maintained in RPMI-1640medium supplemented with 10% FBS, 50 U/ml pen-icillin/50 mg/ml streptomycin and 2 mM L-glutamine(Invitrogen). For experiments cells were incubated withresveratrol (Sigma) for 24 hr prior to treatment withTRAIL (Peprotech), soluble Fas ligand (Alexis), TNFa(R&D Systems), Paclitaxel, Etoposide, Tunicamycin orThapsigargin (Sigma). Media was not changed prior totreatment with the apoptosis-inducing agents, that is,resveratrol was not removed from the cells during thesetreatments. LY294002 (Calbiochem) was added for 1 hrprior to treatment with TRAIL.

Quantif|cation of Apoptosis

Apoptotic events were described as a percentageof total events with hypodiploid DNA assessed bypropidium iodide incorporation. Cells were harvestedby trypsinization, permeabilized with a hypotonic

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1642 Gill et al.

fluorochrome solution (50 mg/ml PI, 3.4 mM sodiumcitrate, 1 mM Tris, 0.1 mM EDTA, and 0.1% TritonX-100) and incubated on ice for 10min prior to analysis.Samples were run on an Epics XL-MCL Coulter EliteCytometer (Coulter Cytometry). Five thousand eventswere gated on PI intensity and analyzed using Mplussoftware.

Hoechst Staining andMicroscopy

Cellswere seeded in 6-well culture dishes 24 hr priorto addition of resveratrol. Following TRAIL treatment,cells were stained live with Hoechst 33258 (Sigma) byaddition directly to the well at a final concentrationof 1 mg/ml. After incubation for 10 min, nuclearmorphology was observed using a Zeiss Axiovert200M microscope and images were acquired usingAxiovision 3.1 software.

Cell SurfaceDeath ReceptorDetection

Following treatment with resveratrol, cells werewashed twice in PBS (supplemented with 0.5% BSA)and resuspended in the same buffer to a finalconcentration of approximately 4� 106 cells/ml. Phy-coerythrin-conjugated anti-TRAIL R1, anti-TRAIL R2or isotype control (R&D Systems; 10 ml) were added to25 ml of cell suspension (approximately 100,000 cells),which was then incubated at 48C for 40 min. Followingincubation the cells were washed twice in PBS/0.5%BSA and resuspended in 400 ml of PBS prior to flowcytometric analysis. Cells were analyzed using a FACScalibur (Becton Dickinson) flow cytometer and CellQuest software with a total of 10,000 events acquiredper sample.

Western BlotAnalysis

Cells were harvested, washed once in PBS and lysedinwhole cell lysis buffer (20mMHEPESpH7.5, 350mMNaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1%NP-40, 1 mM NaF and 1 mM Na3VO4, plus cocktail ofprotease inhibitors). For PARPwesterns cellswere lysedinUrea lysis buffer (7MUrea, 2MThiourea, 2%CHAPS,1% DTT, 0.8% Pharmalyte, protease inhibitors). Totalcellular protein was determined by means of theBradford method. Equal amounts of protein (20–30 mg)were subjected to SDS–polyacrylamide gel electro-phoresis on 8–12% gels before being transblotted ontoImmobilin P (Millipore) membranes. Western blottingwasperformedusingantibodies to cIAP-1, cIAP-2,XIAP(R&D Systems), survivin (Abcam), caspases 3, -7, -8, -9,Akt and pAkt (serine 473) (Cell Signalling Technology),Bcl-2 and Bcl-x (BD Transduction Laboratories),Bax (Upstate Biotechnology), PARP (Biomol), b-Actin(Sigma) and GAPDH (Chemicon), followed by incuba-

tion with the appropriate horseradish peroxidase-conjugated secondary antibodies. Signals were detectedusing ECLTM (Amersham Biosciences).

CaspaseActivityAssays

Cells were harvested and pelleted by centrifugationat 200g for 5 min, resuspended in ice-cold PBS andcentrifuged for a further 5 min at 200g. The pellet wasthen resuspended in a 25 ml volume, transferred to amicrotitre plate and snap-frozen. DEVDase activitywas determined fluorometrically using a modifiedversion of the method developed by Nicholson et al.[24]. Briefly, assay buffer (100mMHEPES, 10%sucrose,0.1% CHAPS, 5 mM DTT, 0.0001% NP-40, pH 7.25)containing 50 mM substrate (DEVD-AMC, PeptideInstitute, Inc., Japan) was added to samples. Cleavageof the fluorogenic substrate was measured using aWallac 1420 Multilabel counter (PerkinElmer LifeSciences) with 355 nm excitation and 460 nm emissionwavelengths. Protein concentration of samples wasdetermined by the Bradford method and activity wasexpressed as change in fluorescence/min/mg protein.

RNAInterference

PC-3 cells were seeded in antibiotic-free media for24 hr prior to transfection with siRNA targeting cIAP-1(Ambion) and cIAP-2 (Dharmacon SMART pool)and corresponding non-targeting controls (Ambionand Dharmacon). Transfections were carried out inOptiMem medium (Gibco) using Lipofectamine 2000transfection reagent (Invitrogen). Twenty-four hourspost-transfection cells were harvested and assayed forRNA and protein expression levels of the target ofinterest. At the same time corresponding samples weretreated as described in the text.

Statistical Analysis

Statistical analysis was carried out using GraphpadInStat 3 software (GraphPad Software, Inc., San Diego,CA). Results were considered statistically significant atP< 0.01.

RESULTS

Resveratrol SensitizesAndrogen IndependentProstate Cancer Cell Lines toApoptosisMediated

ThroughDeath Receptor Signaling

In order to investigate whether resveratrol couldsensitize AIPC cell lines to apoptosis, PC-3 and DU145cellswerepre-treated for 24 hrwith resveratrol followedby treatment with a range of apoptosis inducing agents.The optimal resveratrol dose of 50 mM was chosenfollowing dose response experiments using 10–50 mM

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Resveratrol Sensitizes Prostate CancerCells toApoptosis 1643

resveratrol (data not shown). As shown in Figure 1A–C,PC-3 cells were highly resistant to apoptosis induced byall agents investigated. Paclitaxel did induce someapoptosis at 24 hr (Fig. 1B), however prolonged treat-ment with paclitaxel did not increase apoptosis levels(data not shown). Pre-treatment with resveratrol sensi-tized PC-3 cells to apoptosis induced by Fas, TNFa, andTRAIL, all of which act by binding death receptorson the cell surface (Fig. 1A). In contrast, apoptosis ratesdid not increase in response to agents thought tosignal primarily through a mitochondrial pathway ofapoptosis (Paclitaxel, etoposide; Fig. 1B) or through ERdisruption (thapsigargin, tunicamycin; Fig. 1C) despitepre-treatment with resveratrol. DU145 cells, while moresensitive to resveratrol alone, were also highly resistanttoFas, TNFa, andTRAIL. Pre-treatmentwith resveratrolled to a significant increase in death receptor mediatedapoptosis compared with treatment with resveratrol oreither agent alone (Fig. 1D). In the case of mitochondrial

or ER triggers no significant increase was observedat 24 hr of treatment (Fig. 1E,F). Thus in AIPC celllines PC-3 and DU145, pre-treatment with resveratrolsensitizes to death receptor-mediated apoptosis but notto apoptosis initiated through other pathways.

ResveratrolMediated Sensitization toApoptosisInvolves Increased CaspaseActivity,PARPProcessing, andNuclear Condensation

In order to confirm that cell death resulting fromresveratrol pre-treatment was indeed apoptotic innature, a number of other biochemical and morpholog-ical markers of apoptosis were investigated. PC-3 cellswere pre-treated with resveratrol for 24 hr and caspaseactivitywasdeterminedfluorometrically after 3 and 6hrof TRAIL treatment. While TRAIL alone induced somecaspase activity, resveratrol pre-treatment lead to asignificant increase in caspase activity in response to

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Fig. 1. Resveratrol sensitizes prostate cancer cell lines to death receptor mediated apoptosis. PC-3 (A^C) and DU145 (D^F) cells werepre-treatedwith(blackbars)orwithout(graybars)50mmresveratrol for24hr followedbya further24hr treatmentwithFas (25ng/ml),TNFa(25ng/ml),TRAIL(25ng/ml),Paclitaxel(Pax;10ng/ml),Etoposide(Etop;10mm),Thapsigargin(Tg;2mm),orTunicamycin(Tm;2mg/ml).Apoptosiswas assessedbypropidiumiodideDNA stainingand flowcytometry.TRAIL treatmentwasincludedin all experiments as apositive control forresveratrolmediated sensitization.Values are expressed asmean� SD. *Significantly different fromboth resveratrol alone or single agent atP< 0.01.

1644 Gill et al.

TRAIL (Fig. 2A), corresponding to the increased levelsofapoptosis (Fig. 1). Cell lysates were also assayed for thepresence of cleaved PARP, a caspase-3 substrate whichserves as a marker of apoptosis. As shown in Figure 2B,little or no cleaved PARP was detected up to 24 hrwhen cells were treated with TRAIL alone, however,resveratrol pre-treatment led to a significant increase inthe levels of cleavedPARP following addition of TRAIL.Similarly, on a morphological level, Hoechst stainingrevealed that resveratrol pre-treatment resulted inan increase in the number of cells demonstratingcondensed or fragmented nuclei typical of apoptosis(Fig. 2C). Taken together, these data demonstrate thatsensitization to death receptor apoptosis followingresveratrol pre-treatment involves classical caspase-mediated apoptosis.

Resveratrol Increases ProcessingofCaspases 8, -9, -3, and -7

To further investigate the role of caspases, and todetermine at what level resveratrol might be exerting

its effect, the expression levels of individual caspasesand their cleaved products were analyzed by Westernblotting. As expected from the caspase assay datawhich predominantly measures activity of caspases3 and -7 (Fig. 2A), increased processing of both ofthese proteins was observed in samples treated withresveratrol prior to TRAIL as opposed to those treatedwith TRAIL alone (Fig. 3). No processing of theseproteins was observed in the presence of resveratrolalone. A similar result was found for caspase-8 whereTRAIL alone induced some caspase-8 cleavage but itseffect was significantly increased by resveratrol pre-treatment. In addition, increased levels of caspase-9cleavage products were observed in resveratrol pre-treated samples, suggesting that TRAIL mediated celldeath in these cells also encompasses themitochondrialpathway. The finding that all caspases investigatedshowed increased processing in response to resveratrolsuggests that the pro-apoptotic effect of resveratroloccurs upstream of caspase activation, possibly at thelevel of the receptor. It is also possible, however,

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Fig. 2. Resveratrol pre-treatment increases caspase activity, PARP processing and nuclear condensation followingTRAIL treatment.Cellswere pre-treated with 50 mm resveratrol for 24 hr followed by treatment withTRAIL (25 ng/ml) for the indicated times. A: DEVDaseactivity wasmeasuredusing a fluorometric assay as described inMaterials andMethods Section,DEVDase activity is expressed as change influorescence/min/mgprotein.Data representmean� SD. *Significantlydifferent from treatmentwithTRAIL alone at P< 0.001.B: Cell lysateswere assessed for PARP processing by Western blot.C: Hoechst staining of apoptotic nuclei (magnification 10�) visualized using a ZeissAxiovert200MmicroscopewithAxiovision3.1software.

Resveratrol Sensitizes Prostate CancerCells toApoptosis 1645

that the pro-apoptotic effects of resveratrol couldoccur downstream, at the level of caspase-3, and thatincreased processing of upstream caspases is as a resultof a feedback amplification loop.

Resveratrol DoesnotAlter Cell SurfaceExpression of TRAILReceptors

An important determinant of cell sensitivity to deathligands is the cell surface expression levels of thecorresponding death receptors [25,26]. As resveratrolprimes specifically for agents that bind death receptors,and appears to act at or upstream of caspase-8activation, it was hypothesized that resveratrol pre-treatment may lead to increased expression of TRAILreceptors at the cell surface. TRAIL receptors include

DR4/TRAILR1, DR5/TRAILR2, DcR1, DcR2, andOPG. Only DR4 and DR5 transmit the TRAIL deathsignal, while the other receptors act as decoys, quench-ing the signal [27]. Cell surface expression levels ofDR4and DR5 were assessed using flow cytometry follow-ing addition of resveratrol. As shown in Figure 4, noincrease in DR4 or DR5 expression was observedcompared to isotype control. This is in keepingwith the observations of Delmas et al., who found noincrease in cell surface expression of death receptors,but rather an increased clustering of existing receptorsin lipid rafts. This, however, only partially contributedto resveratrol-mediated sensitization to apoptosis incolon cancer cells [28].

ResveratrolModulates Expression of IAPsbutManipulation of These Proteins Is Insuff|cientto

Sensitize Cells toTRAIL-InducedApoptosis

Increased caspase processing and activity inresponse to pre-treatment with resveratrol suggeststhat an inhibitor(s) may be present in these cells which,upon TRAIL treatment, limits caspase activity andinhibits cell death. We hypothesized that resveratrolrelieves this inhibition leading to the observed increasein caspase activation and cell death. The specificsensitization to death ligands together with increasedprocessing of caspase-8 suggest that such an inhibitormay act at the level of the receptor. Previous studies inneuroblastoma cells have reported that resveratroldecreases the expression of survivin, an IAP familyprotein, leading to increased sensitivity to apoptosis[21,22],while decreased expressionof cIAP-1 and cIAP-2 have been observed in resveratrol treated U937 cells[29]. cIAP-1 and cIAP-2 are reported to exert their anti-apoptotic effect, in part, at the level of the receptor[9,30,31]. Thus the expression levels of a number ofIAPs in PC-3 cellsweremeasured following resveratroltreatment. In keeping with the observations in U937cells [29], XIAP and survivin levels were unchanged,while cIAP-1 expression was decreased in response toresveratrol (Fig. 5A). Interestingly, cIAP-2 expressionwas increased in PC-3 cells. As cIAP-1 is down-regulated it was postulated that this may contributeto the increased sensitivity of cells to death receptorsignals. However, knockdown experiments usingsiRNA targeting cIAP-1 demonstrated that despitedecreasing cIAP-1 levels similar to that induced byresveratrol (Fig. 5B, inset), there was no increase in thesensitivity of the cells to TRAIL (Fig. 5B). Thus althoughaltered cIAP-1 expression may contribute to increasedsensitivity, it is likely that a number of other alterationsare also required. Given the known anti-apoptoticrole of cIAP-2 [9], it was hypothesized that decreasingcIAP-2 expression in conjunction with resveratrol

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Fig. 3. Resveratrolpre-treatmentincreasesprocessingofcaspases8, -9, -3, and -7.Cells were pre-treated with 50 mm resveratrol for24 hr followed by treatment with TRAIL (25 ng/ml) for theindicated times. The expression levels of a number of caspasesand their cleaved products were detected by Western blotting.Data shown are representative of a minimum of three independentexperiments.

1646 Gill et al.

pre-treatment should further enhance sensitivity toapoptosis. To investigate this, PC-3 cells were trans-fected with siRNA targeting cIAP-2 24 hr prior toresveratrol treatment. As shown in Figure 5C, cIAP-2knockdown was maintained following resveratroltreatment, however, this did not enhance sensitivityto subsequent treatment with TRAIL as compared topre-treatment with resveratrol alone (Fig. 5D).

Resveratrol Increases Bax Protein Expression

The finding thatmanipulation of IAP expressionwasinsufficient to increase sensitivity to apoptosis supportsthe concept that resveratrol sensitizes to apoptosisthrough modulation of multiple pathways. In order toidentify other important targets, the expression levels ofapoptosis regulatory proteins were investigated. Anal-ysis of the expressionofHsp27andHsp70,bothofwhichinhibit apoptosis and are overexpressed in cancer cells,revealed no alteration in response to resveratrol (datanot shown). Analysis of the expression of Bcl-2 familyproteins, however, revealed an increase in expressionof pro-apoptotic Bax (Fig. 6). Although expression ofanti-apoptotic Bcl-2 and BclxL appear unchanged, the

increase in Bax may be sufficient to tip the Bax/Bcl-2,Bax/BclxL ratio in favor of apoptosis.

Resveratrol Regulates Akt Phosphorylation andUse of a PI3-Kinase InhibitorDecreases AktPhosphorylation and Sensitizes toTRAIL

In order to find a potential common mediator ofchanges such as altered IAP and Bax protein expres-sion, the PI3-kinase/Akt signaling pathway wasinvestigated. Treatment of PC-3 cells for 24 hr withincreasing concentrations of resveratrol induced asignificant decrease in the levels of phosphorylatedAkt (Fig. 7A). In order to investigate the contribution ofdecreased Akt phosphorylation to resveratrol-medi-ated sensitization to TRAIL, cells were incubated withthe PI3-kinase inhibitor LY294002. Pre-treatment withLY294002 decreased phosphorylation of Akt (Fig. 7B)and significantly enhanced sensitivity of PC-3 cells toTRAIL induced apoptosis (Fig. 7C), to levels similarto those observed with resveratrol pre-treatment(Fig. 1A). LY294002 did not enhance apoptosis induc-tion by etoposide or tunicamycin however (Fig. 7C)evenwith prolonged exposure to these agents (data not

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Fig. 4. Flowcytometricanalysisofdeathreceptorexpressiononcellsurface.Following treatmentwithresveratrol,cellswereincubatedwithPE-conjugatedanti-TRAILR1,anti-TRAILR2orisotypecontrolandthenanalyzedby flowcytometry.RepresentativehistogramsaredepictedinA,whileaveragemeanfluorescence fromatleast four independentexperimentsispresentedinB.Datarepresentmean� S.D.

Resveratrol Sensitizes ProstateCancer Cells toApoptosis 1647

shown). Thus, like resveratrol, specific inhibition ofAktphosphorylation preferentially sensitizes to deathreceptor-mediated apoptosis in PC-3 cells. To inves-tigate whether inhibition to of Akt phosphorylationalso plays a role in sensitizingDU145 cells to apoptosis,cells were pre-treated with LY294002, followed byincubation with TRAIL, etoposide or tunicamycin. Incontrast to PC-3 cells, LY294002 alone induced a smallbut significant increase in apoptosis in DU145 cells,

and increased sensitivity to all three agents investi-gated (Fig. 7D).

DISCUSSION

Dietary components are being increasingly recog-nized as important chemopreventive agents in prostatecancer. A number of key studies are currently under-way including the Selenium and Vitamin E Cancer

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Fig. 5. Altered IAPexpression followingresveratrolpre-treatment.A:Cellswere treatedwithincreasingconcentrationsofresveratrol for24hr followedbyWesternblot analysis ofexpressionlevels of IAP familymembers.B: Cellswere transfectedwith siRNAtargetingcIAP-1andcorrespondingnon-targetingcontrol siRNA.Twenty-fourhourspost-transfectioncellswere treatedwithTRAIL (25ng/ml) for a further 24hrandapoptosisratesweremeasuredbyPI-DNAincorporationandflowcytometry.KnockdownofcIAP-1proteinwasconfirmedbyWesternblot(inset).C^D: Cells were transfectedwith siRNA targeting cIAP-2 and corresponding non-targeting control siRNA. 24 hr post-transfectionresveratrolwas added to samples for a further 24hr, afterwhich time cIAP-2proteinexpression levelswere determinedbyWesternblot (C).TRAILwas addedto thecells for24hr andapoptosiswasmeasuredbyPI-DNAincorporationandflowcytometry (D).

1648 Gill et al.

Prevention Trial (SELECT), which is investigating apotential protective role of these agents against prostatecancer in men [32]. Resveratrol is also recognized asa cancer chemopreventive, and although humantrials have yet to be carried out, there is muchin vitro and in vivo evidence in support of this concept[18,33–35], together with the fact that red wineconsumption is associated with decreased prostatecancer risk [36]. Interestingly in the case of resveratrol,there have been several recent reports that in additionto its chemopreventive properties, this agent can alsosensitize cancer cells to a number of chemotherapeuticagents, both conventional and novel [21–23]. Amongstthose novel chemotherapeutics are agents which bindcell surface death receptors.

Death receptor mediated apoptosis plays an impor-tant role in normal immune system homeostasis andhas been extensively studied with a view to exploita-tion for chemotherapy. While the use of TNFa and Fashas not been highly successful due to side effects suchas hepatotoxicity, TRAIL is a very promising agentfor therapy [37]. Unlike other death ligands, TRAILpotently induces death of cancer cells while sparingmany normal cell types [38]. As such, it is thoughtthat by targeting TRAIL receptors, it may be possibleto specifically eliminate cancer cells without toxicityto normal tissue. However, many cancer cell typesdevelop resistance to TRAIL, and agents that increasesensitivity of cells to death receptormediated apoptosiswill be required to compliment TRAIL treatment inthese cells.

In contrast to previous studies, we have shownthat resveratrol-mediated sensitization to apoptosis isspecific for agents that bind death receptors, includingTRAIL, in the prostate cancer cell lines PC-3 andDU145. There are a number of possible explanations forthis preferential sensitization to death ligands, includ-ing regulation of cell surface death receptor expressionby resveratrol. The current study revealed no increasedexpression of DR4 or DR5 at the cell surface, however,TRAIL decoy receptors have also been implicatedin regulating chemosensitivity [25,39] and it is possiblethat resveratrol alters their expression.Alternatively, asobserved in colon cancer cells, resveratrol may inducethe redistribution of receptors into lipid rafts therebyfacilitating death receptor signaling [28] rather thanenhancing the number of death receptors at the surfaceof tumor cells.

The altered expression of apoptosis regulatoryproteins also plays an important role in mediatingresveratrol sensitization. Here we demonstrate thatTRAIL induces a minimal level of caspase activationwhich is insufficient to kill the cells, but resveratrolenhances caspase activity above a critical threshold,suggesting that resveratrol targets inhibitory proteinsor pathways, allowing adequate activation of caspasesand cell death. Several studies have highlighted theimportance of IAPs in prostate cancer [15,16], anddecreased expression of specific IAPs in response toresveratrol treatment has been reported in neuro-blastoma [21] and leukemic cell lines [29].We observedalterations in the expression of both cIAP-1 and cIAP-2in response to resveratrol, which is in keeping with theconcept of resveratrol targeting upstream inhibitors.While XIAP is a potent caspase inhibitor at the post-mitochondrial level recent evidence indicates thatcIAP-1 and -2 cannot directly inhibit caspases [40,41].There are a number of reports suggesting that cIAP-1and -2 can prevent caspase-8 cleavage and activationindirectly at the level of the receptor, particularlywith regard to TNFa-induced apoptosis [9,30,31].Here, cIAP-1 expression decreased in association withincreased sensitivity to cell death, in keeping withobservations in U937 cells, but an increase in cIAP-2expression was unexpected, based on its putative pro-survival role [9]. The increased expression of cIAP-2in response to resveratrol may occur as part of astress response mechanism, whereby it is upregulatedto counteract the pro-apoptotic effects of resveratrol.Alternatively, the increase in cIAP-2 expression mayoccur as a direct result of the decrease in cIAP-1, as ithas previously been reported that cells from cIAP-1deficient mice express increased levels of cIAP-2[42]. Transfection of these cells with cIAP-1 revealedthat cIAP-2 is a direct target for cIAP-1-mediatedubiquitination and subsequent degradation. In keeping

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Fig. 6. Altered expression of Bcl-2 family members followingresveratrol pre-treatment. Cells were treated with increasingconcentrations of resveratrol for 24 hr followed by Westernblot analysis of expression levels of Bcl-2 family members. Datashown are representative of a minimum of three independentexperiments.

Resveratrol Sensitizes ProstateCancer Cells toApoptosis 1649

with this we have found that following siRNAmediated knockdown of cIAP-1 in PC-3 cells, cIAP-2expression is increased (data not shown). Regardless ofthe reason for increased cIAP-2 expression, wehypothesized that inhibition of cIAP-2 may furtherenhance the effect of resveratrol, however, knockdownexperiments using RNAi did not demonstrate suchan effect. Similarly knockdown of cIAP-1 alone, orcombined knockdown of cIAP-1 and -2 (data not

shown) did not enhance TRAIL mediated apoptosis.Although the significance of altered expression ofcIAP-1 and -2 following resveratrol treatment remainsunclear it is of interest to elucidate their role inapoptosis inhibition, particularly as XIAP is thoughtto be the main IAP involved in regulation of apoptosis,at least in terms of caspase inhibition [40,41]. Given themultiple changes in signaling pathways that occur incancer cells, it is likely, in the case of resveratrol that

The Prostate DOI 10.1002/pros

Fig. 7. Resveratrol decreases Akt phosphorylation, and LY294002 decreases Akt phosphorylation and sensitizes PC-3 cells specifically toTRAIL.A: Cellswere treatedwith increasing concentrations ofresveratrol for 24 hr followedbyWesternblot analysis of expression levels ofphosphorylated Akt.B,C: Cells were treated for1hr with increasing concentrations of LY94002, after which time, proteinwas isolated andanalyzedbyWesternblotting forexpressionlevelsofpAkt(B)or treatedfor24hrwithTRAIL(25ng/ml),Etoposide(Etop;10mM)orTunicamycin(Tm; 2mg/ml) followedby flowcytometric analysis of apoptosis levels (C).D:DU145 cellswere treated for1hrwith LY94002, before additionof TRAIL, Etoposide, orTunicamycin, for 24 hr followed by flow cytometric analysis of apoptosis levels.Values are expressed as mean� SD.*Significantlydifferentfromthe sample treatedwith thecorrespondingconcentrationofLY294002alone, atP< 0.005.#Significantlydifferentfromcontrol (no treatment, i.e., firstbar), atP< 0.005.

1650 Gill et al.

increased sensitivity to apoptosis results from simulta-neous targeting of a number of pathways includingthose regulated by the IAPs.

Such pathwaysmay include those regulated byBcl-2familymembers as alterations in the expression of theseproteins have also been observed in cells treated withresveratrol [19,21,23]. In the current study, it was foundthat the expression ofBaxwas increased following 24hrincubation with resveratrol while Bcl-2 and BclxL wereunchanged. This is in keeping with previous observa-tions of increased Bax expression following resveratroltreatmentwithout any alteration in Bcl-2 andBclxL [21].The increase in Bax may promote a pro-apoptoticenvironment in the cell and together with alterations inIAP expression contributes to the increased sensitivityto cell death. In vivo evidence for the importance of Baxin mediating the effects of resveratrol is demonstratedby increased Bax expression in association with aprotective role of resveratrol against colon carcino-genesis [35].

As resveratrol alters the expression of a number ofdifferent regulators of apoptotic pathways we wishedtodetermine if therewas a commonupstreammediatorof these events. Previous studies have reported thatresveratrol modulates NFkB [43,44] and ERK [45]signaling pathways, while more recently it wasobserved in the LNCaP cells that resveratrolmodulatesthe PI3-kinase/Akt pathway [19]. The PI3-kinase/Aktpathway is an important signaling axis that regulatesdiverse cellular processes including cell growth andsurvival, proliferation and glucose metabolism. Akt isactivated by phosphorylation and both overexpressionand uncontrolled activation of Akt have been reportedin many human cancers [46]. Hyperactivation of Akt isfound in prostate cancer and is thought to be largelydue to loss or mutation of PTEN, a negative regulatorof Akt signaling [47]. Activated Akt confers resistanceto conventional cancer therapies in part through itsmodulation of apoptosis regulatory proteins, eitherdirectly by phosphorylation and inactivation, as in thecase of pro-apoptotic Bad, or indirectly by promotingtranscription of anti-apoptotic genes. Both Bax [48,49]and IAPs [50,51] are regulated by Akt. A numberof studies have reported regulation of Bax proteinexpression levels by the PI3-kinase/Akt pathway [52–54] while Akt also regulates Bax translocation to themitochondria [48,49]. Similarly, cIAP-1 protein expres-sion has been linked to Akt phosphorylation status in anumber ofmodels [50,51]. ThusAkt signaling is a likelycandidate for mediating at least some of the wide-ranging effects of resveratrol. In this study resveratroltreatment decreased the expression of phosphorylatedAkt, and replacing resveratrol with the PI3-kinaseinhibitor LY294002, which also decreases phosphor-ylation of Akt, significantly enhanced TRAILmediated

apoptosis. Similar findings have been reported inLNCaP cells where TRAIL alone induced only partialactivation of caspases while inhibition of Akt phos-phorylation using LY294002 or wortmannin, resultedin activation of downstream caspases and apoptosis[55]. Like resveratrol pre-treatment, incubation withLY294002 specifically increased sensitivity to TRAILbut not to etoposide or tunicamycin. This wouldsuggest that resveratrol is indeed acting at least inpart through the PI3K/Akt pathway in PC-3 cells. InDU145 cells, LY294002 treatment alone lead to a smallbut significant increase in apoptosis. This is in keepingwith the response to resveratrol, whereby resveratrolalone induced apoptosis in DU145. This suggests thatin these cells resveratrol is also partly acting through anAkt pathway. In contrast to the PC-3 cells however,LY294002 sensitized DU145 to all three agents (TRAIL,etoposide and tunicamycin), to varying degrees. Thedifferent response to resveratrol in the two cell lines isnot very surprising as there are somemajor differencesat a molecular level. For example, in contrast to PC-3cells, DU145 are Bax deficient and an alternativeprotein such as Bak, which is known to functionallycompensate for Bax in DU145 cells, may be a target ofresveratrol in this case [56]. Furthermore, while the Aktpathway is constitutively active in PC-3 cells due to lossof PTEN, this pathway is intact in DU145 cells. Thus analternative pathwaymay bemore important for DU145survival and resveratrol may target this pathway.Another possible explanation is that although DU145do not require constitutive Akt for survival, they aresensitive to inhibition of Akt phosphorylation. There-fore either LY294002 or resveratrol treatment alone issufficient to induce cell death. PC-3 on the other handconstitutively express high levels of phosphorylatedAkt and thus require reduction of Akt phosphorylationtogether with another stimulus (TRAIL) in order to killthe cells.

CONCLUSIONS

The current study demonstrates that resveratrolspecifically sensitizes PC-3 and DU145 cells to deathreceptor mediated apoptosis. In PC-3 cells, the mecha-nism involves altered expression of cIAP-1, -2 andBax, together with decreased phosphorylation ofAkt. Targeting an upstream signaling pathway such asthe PI3-kinase/Akt pathway may be more effective intreating prostate cancer, which has many differentsignaling aberrations, than targeting individual apo-ptotic molecules. In this respect, the use of resveratrolitself may be the most effective means of increasingsensitivity to chemotherapy as it clearly acts at manydifferent levels and possibly by additional pathwaysthat have yet to be elucidated. A key factor in

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Resveratrol Sensitizes Prostate Cancer Cells toApoptosis 1651

determining whether resveratrol may be used in theclinical setting is whether it can be delivered safely atsufficient concentrations to have a biological effect.However, despite concerns over the bioavailabilty ofthis compound in vivo, there is evidence that resveratrolis effective even at low concentrations in animal models[33–35]. Further researchwill revealwhether resveratrolor its targetsmaybeused therapeutically in combinationwith conventional therapies for advanced prostatecancer.

ACKNOWLEDGMENTS

This research was funded by a grant from the IrishCancer Society and an EU (E)UROESTROGEN(E)Sgrant contract no: QLK6-CT-2000-00565. Presentationof this work at the AACR, Innovations in ProstateCancerResearchmeeting (2007)was facilitated byUCDSeed Funding scheme.

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