The Prostate 57:14 ^23 (2003)

Perillyl AlcoholMediatedRadiosensitizationviaAugmentationof the Fas Pathway

in ProstateCancerCells

Deepika Rajesh and Steven P. Howard*

DepartmentofHumanOncology,Medical School,UniversityofWisconsin,Madison,Wisconsin

BACKGROUND. The management of hormone-insensitive locally advanced prostate canceris difficult and complex and there is an urgent need for the development of effective chemo-therapeutic agents intended for combination with currently available treatment modalities.METHODS. The present paper demonstrates the effectiveness of the monoterpene perillylalcohol (POH) as potent radiosensitizer on DU145 and PC3 cell lines by performing clonogenicsurvival assays, cycle analysis, andassays todetect viability, apoptosis, andFas receptor/ligandby flow cytometry.RESULTS. POH pretreatment resulted in a dose dependent sensitization to kill cell byradiation. Furthermore, POH treatment induced a transient G2/M arrest, enhanced the expres-sion of the membrane bound form of the Fas ligand and sensitized the cells to Fas mediatedapoptosis.CONCLUSIONS. The unique manner of radiosensitization in addition to its low toxicityprofile makes POH a promising new agent for preclinical evaluation as a potential radio-sensitizer in the treatment of prostate cancer. Prostate 57: 14–23, 2003. # 2003 Wiley-Liss, Inc.

KEY WORDS: monoterpenes; radiation; apoptosis; cell cycle and Fas

INTRODUCTION

Prostate cancer is the most common cancer amongmen and accounts for 43% of all male cancers. The5-year survival rate for men diagnosed with localizedprostate tumors discovered at this stage is 99%;63% survive beyond 10 years; 15% survive beyond15 years [1]. The management of locally advancedprostate cancer is difficult and challenging, as thesetumors often become hormone-insensitive and unre-sponsive to standard chemotherapeutic agents [2].

Research strategies have been directed towards thedevelopment of new agents directed against novelcellular targets to be used either as single agents or incombination with currently available treatment mod-alities. The management of locally advanced prostatecancer is clinically challenging, as these tumors oftenbecome hormone-insensitive and unresponsive tocurrent chemotherapeutic agents [3]. The use of radio-therapy and hormonal therapies are important treat-ment options that usually precede chemotherapy. Inspite of escalating doses of radiotherapy, 20–25% of

prostate cancer patients with non-invasive disease(stages T1–T2) ultimately relapse. A major reason forfailure to control local disease is the intrinsic radioresistance of these tumors. Therapeutic options forpatients with advanced disease, once limited to theuse of androgen deprivation and external beam radio-therapyhaveexpanded to includeanumberof systemicinterventions, including secondary hormonal manip-ulations, chemotherapy [4] using alkylating agents

Grant sponsor: The University of Wisconsin Comprehensive CancerCenter; Grant sponsor: The Robert Draper Technology InnovationGrant (Funded by the Wisconsin Alumni Research Foundation);Grant number: 135-8439.

*Correspondence to: Steven P. Howard, MD, PhD, Department ofHuman Oncology, K4/354, CSC, 600 Highland Avenue, Universityof Wisconsin Comprehensive Cancer Center, Madison, WI 53792.E-mail: howard@mail.humonc.wisc.eduReceived 8 November 2002; Accepted 4 February 2003DOI 10.1002/pros.10269

� 2003 Wiley-Liss, Inc.

(hydroxymethylacylfulvene (HMAF) [5–7], Taxanes(BMS-275183) [8], docetaxel [9], estramustine [10,11],mitoxantrone [12,13], pyrimidine analogs etanidazole(ETA) [14,15], and systemic radionuclide therapy [16]for the treatment of prostate cancer. The poor clinicalresponse of this tumor type has prompted researchstrategies towards the development of more effectivechemotherapeutic agents for use in the treatment ofadvanced hormone-refractory prostate cancer.

Monoterpenesarenon-nutritivedietarycomponentsfound in the essential oils of citrus fruits and otherplants. Monoterpenes produce significant tumor re-gression inboth chemically inducedand transplantableanimal tumor models [17]. For example, D-limonene,which comprises >90% of orange peel oil and Perillylalcohol (POH) from cherries have chemotherapeuticactivity against pancreatic, mammary, and prostatictumors [18] and also have chemopreventive activityagainst rodent mammary, skin, liver, lung, and forest-omach cancers [19]. POH isolated from the essential oilsof lavendin, peppermint, spearmint, and several otherplants, is the most potent monoterpene tested to date.Although its mechanism of action is unclear, POH canmodulate cellular processes that control cell growthand differentiation including: (1) G1 cell cycle arrestand induction of apoptosis [20,21] (2) inhibition ofisoprenylation of small GTP-binding proteins involvedin signal transduction [22,23], and (3) differential generegulation including over expression of themannose-6-phosphate/insulin-like growth factor II (M6P/IGF II)[17] and transforming growth factor-beta (TGF-b)type II receptor genes [24].

POH is currently being evaluated in Phase I andPhase II clinical trials [25–27]. The formulation and thepharmacokinetics of POH in advanced cancer hasbeen validated and a reliable maximum tolerated dosedetermined. POH at 1,600–2,100 mg/m2 p.o. threetimes daily is well tolerated on a 14-day on/14-day offdosing schedule. The plasma of patients under studyrevealed a dose-dependent increase in levels of thetwo main metabolites, perillic acid and dihydroperillicacid [27].

In the present paper, we evaluated the effect of POHtreatment on PC3 and DU145 prostate cancer cell lines.POH treatment resulted in a dose dependent sensiti-zation to cell kill by radiation in the clinically relevantdose range of radiation. POH induced a transient blockin the G2/M phase of the cell cycle and induced apop-tosis in both the cell lines. POH treatment enhancedthe expression of the membrane bound form of the Fasligand in and sensitized the cells to Fas mediatedapoptosis. Thus, the efficacy of POH in vivo can beenhanced by its potential to be used as a radiosensi-tizer at relatively non-toxic doses for the treatment ofprostate cancer.

MATERIALSANDMETHODS

Materials

POH, propidium iodide (PI), RNase H were pur-chased from Sigma Aldrich (St. Louis, MO), antibodiesto Cyclin B and D were purchased from Santa CruzBiotechnology (Santa Cruz, CA), annexin V stainingkit was purchased from Clonetech (Palo Alto, CA),Antibodies to Fas ligand and Fas receptor and secon-dary antibodies streptavidin PE and the isotype controlantibodies were purchased from BD Pharmingen(San Diego, CA).

Cell Lines

The DU145 and PC3 cell lines originally from ATCCwere obtained from Dr. Ajit K. Verma, Department ofHuman Oncology, University of Wisconsin-Madisonand maintained in a humidified incubator at 378C.DU145 cellswere grown inDMEMmedium containing10% fetal bovine serum, 1% penicillin, streptomycin,while PC3 cells were grown in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 1% peni-cillin, streptomycin.

Cell Treatments andClonogenic Survival Assays

PC3 and DU145 cells were treated with varyingdoses of POH (0.1–0.5 mM) for a period of 72 hr. Stocksolution of POH was made in medium. Control disheswere treated with different doses of radiation and theplating efficiencywas determined by treating cellswithmedium alone. All cells were irradiated at a dose ofapproximately 7.5Gy/minusing a 137Cs irradiator. Thedose of irradiation varied from 1 to 8.5 Gy. Followingirradiation, both POH treated and untreated cells wereharvested, washed with PBS, plated at the desired cellnumber, and incubated for 2 weeks. The colonies werestainedwith crystal violet and quantified. Survival wasdetermined as the ratio of plating efficiencies for eachirradiated group to that of the unirradiated control.

Apoptosis Assays

The percent of cells undergoing apoptosis and thedifferent phases of the cell cycle were determined bythe method described by Darzynkiewicz et al. [28].Briefly, the cells were treatedwith 0.1–1mMPOH for aperiod of 72 hr. At appropriate times after treatment,the cells were harvested by trypsin–EDTA treatmentand washed twice with ice-cold PBS and fixed in ice-cold 95% ethanol. The fixed cells were stained using aPI staining solution containing 20 mg/ml PI, 200 mg/mlRNase H, and 0.1% NP40 solution. The cells werefiltered through a 40 mMpore nylonmesh (Tetkop, Inc.)and analyzed using a Beckton-Dickinson FACStar plusflow cytometrywith excitation 488 nM. The percentage

Radiosensitization and ProstateCancer 15

of cells in sub-G0/G1 (apoptotic) peak and the distri-bution of cells in G0/G1, S, and G2/M phases weredetermined using the ModfitLT version 2.0 (VeritySoftware, Topham, Maine) and Cell Quest software(Becton Dickinson, San Jose, CA).

StainingWithAnnexinVand Propidium Iodide

POH induced cell death was confirmed by dualcolored (PI and annexin V) flow cytometry [29]. Celllines were treated with varying doses of POH, for theindicated times, harvested by using cell dissociationsolution, washed, and subsequently stained with anti-body to annexin V conjugated to FITC and with PI[10 mg/ml] using the Apoalert Kit (Clonetech) accord-ing to the manufacturer’s instructions. Viable [annexinV�/PI�] pre-apoptotic [annexin Vþ/PI�], apoptotic[annexinVþ/PIþ], and residual damaged cells [annexinV�/PIþ] were estimated using the Cell Quest software(Becton Dickinson).

Staining for Cyclins

Staining for cyclins was performed according to themethod described by Darzynkiewicz et al. [28]. Briefly,semi confluent cultures of DU145 and PC3 cells weretreated with the indicated concentrations POH, 5.5 Gyradiation or a combination of POHand 5.5Gy radiationtreatment for 72 hr. The cells were harvested and fixedin ice-cold ethanol overnight at 48C. The cells werewashed, centrifuged, and suspended in permeabiliza-tion solution (0.25%Triton X-100 in PBS pH 7.4) andkept on ice for 5 min. The cells were washed again, resuspended in 100 ml wash buffer (1% BSA in PBS); andincubated with antibodies to cyclin B1, cyclin D1, orisotype control for 60 min at room temperature (RT).The cells were washed, and stained with goat anti-mouse FITC for 30 min at RT. The stained cell pre-paration was stained with 5 mg/ml PI and 200 mg/mlDNase-free RNase A. The cells were analyzed on aFACScan flow cytometer (Becton Dickinson) and theresults were acquired using CELL quest software andthe ModfitLT version 2.0 (Verity Software). PI stainingwas used to reveal the various phases of the cell cycleand the cyclin staining in theG0/G1 andG2/Mphase ofthe cell cycle was estimated as the mean fluorescenceintensity (MFI). The MFI recorded by the isotypecontrol for each sample was subtracted from the MFIrecorded after staining using the specific antibody toeliminate non-specific binding of the fluorochorme foreach sample.

Dual Stainingof Fas Ligand and Receptor

Prostate cell lines were treated with 0.1–0.7 mMPOH, 5.5 Gy radiation, or a combination of 0.5 mMPOH and 5.5 Gy radiation simultaneously for 48 hr.

At the end of the incubation, the cells were harvestedusing cell dissociation solution. The resulting cell pelletwas stained with biotinylated antibody specific to thehuman Fas-ligand or Biotinylated IgG1 k isotype con-trol for 45 min at 48C. The cells were washed andincubatedwithAPCconjugated antibody specific to thehuman Fas (Pharmingen) and streptavidin conjugatedphycoerythrin (PE). The cells incubated with biotiny-lated IgG1 k isotype were incubated with streptavidin-conjugated PE as well as streptavidin conjugated toAPC to eliminate non-specific binding of each treatedsample. The cells were washed and before analysis PIwas added to the cells at a concentration of 20 mg/ml.This was done to gate the live cells and the staining inthis population was determined. The cells were anal-yzed on a FACScan flow cytometer (BectonDickinson).The MFI of the Fas ligand was determined on a linearscale, while the CD95 stainingwas determined on a logscale using CELL quest software (Becton Dickinson).

Sensitization toAnti-FasAntibody-MediatedApoptosis

Subconfluent cultures of DU145 and PC3 cell lineswere treated with 1 mM POH in the presence andabsence of 5.5 Gy radiation for 48 hr. A parallel set ofplates were treatedwith cycloheximide (10 mg/ml) andanti-Fas agonisitc monoclonal antibody (CH11) at aconcentration of 100 ng/ml for 48 hr. This set served asa positive control to determine the intactness of the Fassignaling cascade. To determine POH mediated sensi-tization to Fas mediated apoptosis, cells were treatedwith 1 mM POH and 5.5 Gy radiation in the presenceand absence of monoclonal antibody (CH11). For in-hibition experiments, the antagonistic antibody ZB4was added at a concentration of 500 ng/ml to cellstreated with 1 mM POH, 5.5 Gy radiation, and mono-clonal antibody (CH11). For all treatments, the cellswere treated with medium containing either POH,alone or with cycloheximide or agonisitc antibodyCH11 or antagonistic antibodies, or a combination ofall the reagents immediately after radiation treatment.All the reagentswere added to the cells at the same timeand theplateswere incubated for 48hr.At the endof theincubation, the cells were harvested, fixed in ethanol,and stainedwithPI for quantification of subG0/G1 cellsvia flow cytometric analysis as described before. All thetreatment sets were harvested at the end of 48 hr.

RESULTS

POHInduced Radiosensitizationin Prostate CancerCell Lines

The clonogenic survival assay was carried out todetermine the effect of different doses of POH radiation

16 Rajesh andHoward

and a combination of both in PC3 and DU145 cell lines.The results in Figures 1 and 2 reveal the effect of POHalone and in combination with various doses of radi-ation onDU145 and PC3 cell lines. Treatmentwith 0.1–0.3 mM of POH appeared to be relatively non-toxic tothe cells. Higher concentrations between 0.7 and 1 mMresulted in a drastic reduction of the surviving fractionin both the cell lines. The clinically achievable range ofPOH was up to 0.5 mM, and hence, radiosensitizationexperimentswereperformed in thedose rangebetween0.1 and 0.5 mM POH. Pretreatment of DU145 (Fig. 1)and PC3 (Fig. 2) cell lines with POH revealed a dosedependent sensitization of these cells to radiation in-duced cell death. This effect was seen with as low as0.3 mMPOH. The next set of experiments were design-ed to characterize themode of cell death in POH treatedcells.

POHInducesApoptosis inProstate CancerCell Lines

DU145 and PC3 cells were treated with POH (0.1–1 mM) in the presence and absence of 5.5 Gy radiationfor a period of 72 hr and the cells in the G0/G1, G2/M,and sub G0/G1 phase of the cell cycle were determinedafter staining the cells with PI. The results revealed adose dependent increase in the sub G0/G1 population

in both the cell lines. PC3 cells (Fig. 3) were more sus-ceptible to POH than DU145 cells (Fig. 4).

POH induced cell death was confirmed by stainingcells treated with varying doses of POH and stainingfor annexin V on the cell membrane via dual colored(PI and annexin V-FITC) flow cytometry. POH treatedcells showed an increase in the early apoptotic [annexinVþ/PI�] and apoptotic [annexin Vþ/PIþ] population ofcells indicating the onset of apoptosis, in both PC3(Fig. 5A) and DU145 (Fig. 5B) cell lines.

POH-Treatment InducedChangesin Cell Cycle Progression

Treatment with DU145 and PC3 cells with varyingconcentrations of POH in the presence and absence ofradiation revealed an increase in theG2/Mphase of thecell cycle, and a decrease in the G0/G1 phase of the cellcycle (Figs. 3 and 4). The increase in the G2/M phase ofthe cell cycle was more predominant at higher doses ofPOH and in the presence of radiation. The increase inthe cells in the G2/M phase of the cell cycle appearedto precede the onset of apoptosis in both the cell lines.To correlate these findings, further experiments wereperformed to determine the effects of POH and radi-ation on cyclin B1 and cyclinD1 levels in theG0/G1 andG2/M phases of the cell cycle.

Figs. 1and2. SubconfluentculturesofPC3cellsorDU145cellswere treatedwith0.1^ 0.5mMPOHfor72hrandsubsequently toincreasingdosesofradiationfrom1to5.5Gy forPC3and1to8.5Gy forDU145cells.ControlcellsweretreatedwiththeindicateddosesofPOH,radiation,ormediumalone.Thecellswereharvestedandanoptimumnumberofcellswereplated.Theresultingcolonieswerestainedandcounted.Survivalwas determinedas theratio ofplatingefficiencies for each irradiatedgroup to thatof theunirradiatedcontrol.Eachgraphedpointrepresentsmeanvalues� SEvaluesof triplicatedishes.

Radiosensitization and Prostate Cancer 17

POH-Treatment Increased theLevels of Cyclins B andD

Sub confluent cells of DU145 and PC3 cells weretreated with varying doses of POH in the presence andabsence of radiation and dual staining was performedwith FlTC conjugated cyclin B1 and cyclin D1 anti-bodies and PI. The percentage of cells in the G0/G1 andG2/M phases of the cell cycle and amount of cyclin B1and D1 levels in the respective compartments weredetermined by flow cytometry. The results in Figure 6revealed an increase in the levels of cyclin B1 and inthe G0/G1 and G2/M phase of the cell cycle in DU145cells while an increase of cyclin B1 predominantly inthe G2/M phase of the cell cycle was seen in PC3 cells.Since cyclin B1 promotes G2/M transition, the increasein the levels of cyclin B1 in the G2/M compartmentcould be correlated to the transient block in the G2/Mphase of the cell cycle.

POH treatment increased the expression of cyclinD1 in the G2/M compartment in PC3 cells and in theG0/G1 andG2/Mcompartments (Fig. 7) inDU145 cells.

Figs. 3 and 4. The effects of various concentrations of POH on the induction of apoptosis in prostate cancer cell lines. Subconfluent cul-tures of PC3 (Fig. 3) and DU145 (Fig. 4) cells were treated with the indicated concentrations of POH in the presence and absence of 5.5 Gyradiationfor72hr.Attheendof theincubation,thecellswereharvested,fixedinethanol,andstainedwithpropidiumiodide(PI).Thehistogramsreveal the percentage of cells in sub G0/G1,G0/G1, and G2/M phases of the cell cycle. Each graphed point representsmean values� SE valuesof triplicatedishes.

Fig. 5. A,B: POH-induced apoptosis ofprostate cancer cell linesas determined by dual colored (PI and annexinV) flow cytometricanalysis. Subconfluentcultures of PC3 (A) andDU145 (B) cellsweretreated with or without the indicated concentrations of POH for72 hr.The cellswere harvested and subsequently stainedwith anti-body to annexinV conjugated to FITC andwith PI [10 mg/ml].Viable[annexin V�/PI�] pre-apoptotic [annexin Vþ/PI�], apoptotic[annexinVþ/PIþ], and the residual damaged [annexinV�/PIþ] cellswere quantitated by flow cytometric analysis. Each point is anaverageofduplicatedishes (variation<5%).

18 Rajesh andHoward

The D type cyclins (cyclin D1, D2, and D3) promotecell cycle progression from G1 to S phase by bindingto and activating the cyclin dependent kinase (cdk)4 and 6. A family of inhibitors of cdks (cdkIs) p15,p16, p18, p27, and p21 inactivate cdks and arrest cellsat checkpoints by inhibiting cyclin/cdks complexes.Although there was a decrease in the percentage ofcells in the G0/G1 phase of the cell cycle the expressionof cyclin D1 increased in POH treated cells. Furtherexperiments are needed to explain the unscheduledcyclin production in prostate cancer cell lines and todetermine the role of cdks and cdkIs in POHmediatedradiosensitization.

Perillyl Alcohol Pretreatment Enhancedthe Expression of theMembraneBound Fromof the Fas Ligand

The next series of experiments were designed toidentify the mechanism of cell death induced by POHand radiation in prostate cancer cells. Several labora-tories have reported on the apoptotic potentials ofhuman prostate cancer (PC) cell lines in response tocross-linking of Fas (CD95/APO-1) with agonistic anti-Fas antibodies [30,31]. The Fas (CD95) and Fas ligand(Fas-L) are an interacting receptor ligand pair thatelicit apoptosis in many cell types [32] and faultyregulation of the Fas system has been described in a

variety of human tumors with different histogeneticorigin [33]. Prostate cancer cell lines as well as prostatetissue specimens from benign andmalignant epithelialcells have been reported to express the normal trans-membrane anddeath domains of Fas antigen by immu-nohistological and PCR analysis [34–37].

Simultaneous staining of Fas ligand andFas receptorwas performed on PC3 and DU145 cells treated withPOH in the presence and absence of radiation. Theresults showed a dose dependent up-regulation of themembrane bound form of the Fas ligand in PC3 (Fig. 8)and DU145 (Fig. 9) cell lines. POH treatment did notalter the level of expression of the Fas receptor in boththe cell lines.

Perillyl Alcohol Pretreatment InducedSensitization to FasMediatedApoptosis

Since POH induced up-regulation of the membranebound form of the Fas-ligand, it was of interest todetermine if POH could sensitize and glioma cells toFasmediated apoptosis by an agonistic antibodyCH11.PC3 andDU145 cell lines were treatedwith 1mMPOHin the presence and absence of 5.5Gy radiation for 48 hrto check for augmentation of POHmediated cell deathin the presence radiation. To determine POHmediatedsensitization to Fasmediated cell death, anti-Fasmono-clonal antibody (CH11) was added to the cultures at a

Figs. 6 and 7. Bivariate analysis of DNA content and cyclins following POH treatment: Subconfluent cultures of DU145 and PC3 cellswere treatedwith the indicated concentration of POHin thepresence and absence ofradiation for 72 hr.Following treatment, the cellswereharvested fixed in ethanol, and permeabilized.The cells were stained using specific antibodies for cyclin D1or cyclin B1or the relevant iso-type control and with goat anti-mouse FITC.The cells were stained with PI solution and analyzed on a FACScan flow cytometer (BectonDickinson, San Jose,CA).PI staining was used toviewdifferentphases of the cell cycle and the cyclin staining in theG0/G1andG2/M phase ofthe cell cyclewas estimatedusing the cell quest program.The graph represents themean fluorescence intensity (MFI) recorded for cyclin B1and cyclin D1staining after subtracting theMFI of the isotype control of each treated sample.Figure 6 reveals the effect of POHon cyclin B1levels,whileFigure7reveals theeffectofPOHonthelevelsofcyclinD11inDU145andPC3cellsin theG0/G1andG2/Mphaseof thecellcycle.

Radiosensitization and ProstateCancer 19

concentration of 100 ng/ml in the presence of POH inthe presence or absence of radiation. For inhibitionexperiments, the antagonistic antibody ZB4was addedat a concentration of 500 ng/ml to the cells treated withPOH, radiation, and CH11. At the end of the incuba-tion, cells were harvested, and the quantitative analysisof apoptosiswasperformedbyflowcytometric analysisof fixed cells stained with PI.

As seen in Figure 10, PC3 and DU145 cell lineswere sensitive to cell death mediated by the anti-Fas

antibody CH11 in the presence of cycloheximide(CHX) confirming the intactness of the Fas signalingcascade. POH pretreatment (1 mM) sensitized both thecell lines to Fas mediated apoptosis in the absenceof CHX. The Fas sensitive cell line PC3 was moresusceptible to Fas as well as POH mediated apoptosis.The highest amount of cell death was induced by acombination of POH, radiation, and anti-Fas antibodyCH11. Pretreatment with the antagonistic antibodyZB4 abrogated the activation of the Fas cascade byPOH

Figs. 8 and 9. Dual stainingof Fas ligandandreceptoronprostate cancercells.DU145andPC3cell lineswere treatedwithvaryingdoses ofPOHfor48hr.Thecellswereharvestedandtheresultingpelletwasincubatedwithbiotinylatedanti-humanantibodytoFas-L(PharmMingen)orwithanisotypematchedanti-humanantibody.Following theprimaryantibodyincubationthecellswerewashedandsubsequentlystainedwithsecondaryantibodyconjugated tophycoerythrin (PE) (PharMingen) andantibody toCD95directlyconjugated to allophycocyanin (APC).ThestainedcellswereanalyzedusingaBecton-DickinsonFACStarplus, andtheresultswereacquiredusingCELLquest software.Thegraphrepre-sents theMFIrecordedforFas-ligandandFas stainingafter subtracting theMFIof theisotypecontrolofeach treatedsampleinPC3(Fig.8)andDU145(Fig.9)cell lines.Eachpointin thegraphis anaverageof threeindependentexperiments� SEvalues.

Fig. 10. PC3 andDU145 cell lineswere treatedwith1mMPOHandin thepresence or absence of the anti-CD95 antibodyCH11 (100 ng/ml),radiation (5.5 Gy),10mg/m1cycloheximide (CHX), antagonistic antibody ZB4 (500 ng/ml), or a combination of all for 48 hr. At the end of theincubation, the cells were harvested, fixed in ethanol and stainedwith PI solution.Each point in the graph represents the sub G0/G1fractionofstainedcells.

20 Rajesh andHoward

andCH11. The abovefindings confirm the involvementof the Fas cascade in POH mediated cell death andradiosensitization.

DISCUSSION

Managing hormone-refractory prostate carcinomaremains a difficult challenge for the clinician and hencethe development of an effective radiosensitizer shouldbe particularly relevant in the management of prostatecancer. The present paper highlights the usefulness ofthe monoterpene POH as a potent radiosensitizer inprostate cancer cells using clinically relevant dosesof POH and radiation. POH pretreatment induced atransient increase in the G2/M phase of the cell cyclewithaconcomitant increase incyclinB1 levels.Althoughthere was a decrease in the percentage of cells in theG0/G1 phase of the cell cycle, an increase in cyclin D1levels in the G2/M andG0/G1 compartments of the cellcycle was observed.

POH has been shown to induce apoptosis in breast[24,38], pancreatic [39], and leukemic cell lines [20].This is the first report of its effect on prostate cancer celllines. Our results are in contrast with the finding ofSahin [20] and Ariazi [24] et al., where the authorsreported a block in theG0/G1phase of the cell cycle andan up regulation of the expression of cyclin D levels inbreast cancer and leukemic cell lines treatedwith POH.Increased expression of cyclinD1 [40], low endogenouslevel of cyclin B1, and the absence of the mismatchrepair system [41] has been reported in prostate cancercell lines. POH treatment in the presence or absence ofradiation could alter cell cycle regulation by accumu-lating cells in the G2/M phase of the cell cycle andcausing cell death. Further studies are needed to pre-cisely determine the role of cell cycle regulatory pro-teins in POH induced cell death and radiosensitizationin prostate cancer cells.

POH treatment caused a dose-dependent up re-gulation of the Fas ligand and sensitized cells to Fasmediated apoptosis in both Fas-resistant (DU145)and Fas-sensitive (PC3) cell lines, which was inhi-bited in the presence of antagonistic antibody. Fas isa widely expressed protein found on the plasmamembrane in most tissues including prostate. Fasexpression has been detected on the surface of anumber of different human prostate cell lines in vitro[42], [36], [32,33], [30] and on the surface of both benign[32] andmalignant human prostate tissue samples [35].Liu et al. [43] detected soluble Fas-L (sFasL) in theculturemedia of PC-3, DU145, LNCaP cells, andwithinthe intraluminal secretions of normal prostate epithe-lial cells.

For reasons unknown, prostate cancer cells co-expressCD95 and CD95L in vitro, without undergoing suicide

or fratricide [35,43]. Several reports have demonstrat-ed that a functional Fas-mediated apoptotic pathwayexists in the prostate. This evidence comes both fromin vitro and in vivo studies. Some prostate cancer celllines (PPC-1, ALVA-31, JCA-1 [36], PC-3 [44]) are sensi-tive to Fas-mediated apoptosis when challengedwith aFas agonist, i.e., anti-Fas antibody or Fas-L expressingeffector cells [42], [44], [36], while other prostate cancercell lines (DU145, ND1, JCA-1 [32,33], [30]) were foundto be resistant when challenged with a Fas agonist.This resistance, however, is overcome by pretreatmentusing sub-toxic concentrations of adriamycin (ADR),cis-diamminedichloroplatinum(II) (CDDP),VP-16, cam-ptothecin [31], [33], [30] [45]. These chemotherapeuticdrugs and have different mechanisms of action, butpresumably function to remove a block in the Fas-mediatedpathwayandallow thedeath signal to proceed.Of special interest is the finding that Fas-resistant celllines can be converted to the Fas-sensitive phenotypeby treating cells with the protein synthesis inhibitorcycloheximide treatment. Thus, the resistance towardsFas-mediated apoptosis could be due to the presenceof regulatory, labile protein(s) that act as resistance-factors, and indicates that the intrinsic propensity toundergo Fas-mediated apoptosis is intact in Fas-resis-tant prostate cancer cell lines [46], and could be a targetfor therapeutic intervention in androgen-independentmetastatic prostate cancer [32,44].

POH treatment did not change the levels of the Fasreceptor. Fas up regulation following treatment withionizing radiation and DNA damaging agents [47,48]has been shown to be dependent on wild-type p53.The presence of mutated or null p53 in the cell linesunder study explains the lack of up regulation theCD95 receptor by POH and also implies the activationof a p53 independent pathway in POH-induced radiosensitization.

In summary, our results present POHas a promisingnew agent for preclinical evaluation as a potentialradiosensitizer in the treatment of prostate cancer viaactivationof theCD95 system.POHpretreatment couldsensitizeFas resistant tumors toFasmediatedapoptosisand hence it would be worthwhile to conduct com-bined studies with POH and chemotherapeutic agents,(Mitoxantrone) or radionuclides (strontium-89 andsamarium 153) in the treatment of prostate cancer.

ACKNOWLEDGMENTS

This work was supported by start up fundsallotted to Steven P. Howard by the University ofWisconsin Comprehensive Cancer Center and theRobert Draper Technology Innovation grant fundedby the Wisconsin Alumni Research Foundation grantnumber 135-8439.

Radiosensitization and Prostate Cancer 21

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