effect of permixon on human prostate cell growth: lack of apoptotic action

The Prostate 61:73 ^ 80 (2004)

Effectof PermixononHumanProstateCell Growth:LackofApoptoticAction

Brian Hill1 and Natasha Kyprianou2*1DivisionofUrology,UniversityofMaryland SchoolofMedicine, Baltimore,Maryland

2DivisionofUrology,Departmentof SurgeryUniversityof KentuckyCollege ofMedicine, Lexington,Kentucky

BACKGROUND. Permixon, a phytotherapeutic agent derived from the saw palmetto orSerenoa repens plant, is a lipid/sterol extract that is believed to interfere with 5a-reductaseactivity, thus inhibiting prostate growth. In this study, we investigated the magnitude andspecificity of the effect of Permixon on cell proliferation and apoptosis in humanprostate cancercells.METHODS. The effect of Permixon was examined in androgen-independent PC-3 prostatecancer cells, androgen-sensitive LNCaP prostate cancer cells, and MCF-7 breast cancer cellsin vitro. Cell growth, apoptosis induction, and cell proliferation was studied after exposure toPermixon at two concentrations (10 and 100mg/ml). Cell proliferation and cell cycle progressionwere determined after 24 hr on the basis of 3[H]-thymidine incorporation assay andflowcytometric analysis, respectively. Apoptosis induction was evaluated in treated anduntreated cultures using the Hoescht staining and caspase-3 activation.RESULTS. Exposure of prostate and breast cancer cells to a high dose of Permixon (100 mg/ml)resulted in a significant decrease in the rate of cell growth; an effect thatwas not time-dependentand was not associated with cell cycle arrest. Permixon treatment (at either high or low dose)had no effect on apoptosis induction in prostate cancer cell lines (P> 0.6). Furthermore, in vitroPermixon was a weak inhibitor of 5a-reductase activity type 2 in prostatic homogenates.CONCLUSIONS. The results indicate the ability of Permixon to affect prostate cancer cellgrowthwithout inducing apoptosis or cell cycle arrest. This effect was not prostate-specific andwas onlymanifested at high concentrations of Permixon. Furthermore ourfindings indicate thatPermixon is weak inhibitor of 5a-reductase compared to finasteride. This study challengesprevious evidence on the anti-growth effect of Permixon in the prostate and its ability to inhibit5a-reductase activity, while questioning apoptosis as a mechanism of action of thisphytotherapeutic against prostate growth, a concept that may have therapeutic significance.Prostate 61: 73–80, 2004. # 2004 Wiley-Liss, Inc.

KEY WORDS: apoptosis; permixon; prostate cancer; cell proliferation; 5a-reductase

INTRODUCTION

Current therapy for prostate cancer is limited by thepropensity of the disease to progress from androgen-dependent to an androgen-independent state [1]. In thenormal prostate, organ homeostasis is maintained by adynamic balance between the rate of cell proliferationand the rate of apoptosis [2]. Disruption of themolecular mechanisms that regulate apoptosis and cellproliferation among the stroma and epithelial cellpopulations, may underlie the abnormal growth ofthe gland that characterizes neoplastic developmentof the prostate [3,4]. Thus apoptosis induction

provides a relevant endpoint for testing existing ornew drugs for therapeutic efficacy against prostate

Grant sponsor: Merck and Co. (an educational grant and studymaterial).

*Correspondence to: Dr. Natasha Kyprianou, Division of Urology,MS 283, 800, Rose Street, University of Kentucky Medical Center,Lexington, KY 40536. E-mail: [email protected] 4 September 2003; Accepted 26 January 2004DOI 10.1002/pros.20088Published online 16 March 2004 in Wiley InterScience(www.interscience.wiley.com).

� 2004 Wiley-Liss, Inc.

growth disorders, benign prostatic hyperplasia (BPH),and prostate cancer [3].

BPH is attributable to proliferation of the epithelialand stromal smoothmuscle components of the prostateresulting in an enlargement and causing urinaryobstructive symptoms in aging men [5]. Dihydrotes-tosterone (DHT) is the critical intracellular androgenthat controls the growth of the prostate and is causallyinvolved in the development of BPH [5]. Consequentlyeffective drugs have been developed to reduce theactivity of androgens on target cells, by inhibiting theaction of 5a-reductase isoenzymes which are respon-sible for the metabolic conversion of testosterone toDHT. Finasteride selectively inhibits the action of oneisoenzyme (type II), thus reducing volume of theprostate in men with BPH and improves urinarysymptoms [6]. Clinical studies have established thatthe use of 5a-reductase inhibitors (by inhibiting growthof the enlargedgland) in the treatmentofBPH,providessymptomatic relief of patients with lower urinary tractsymptoms (LUTS) [7]. Induction of apoptosis of bothprostate epithelial and stromal cell components hasbeen identified as a major molecular mechanismunderlying this reduction in the prostate volume byfinasteride [8,9]. Herbal remedies have also becomeparticularly popular for patients with LUTS, since theprimary goal of treatment is a subjective decrease invoiding symptoms [10,11]. The escalating popularity ofalternative medicines however, has increased thescrutiny regarding their efficacy, safety and mech-anism of action.

The saw palmetto plant or Serenoa repens, is a smalldwarf palm indigenous to the southeastern UnitedStates andWest Indies [12]. For several years now, sawpalmetto has been used to treat symptomatic BPHthroughout Europe [10,12]; its mechanism of actionhowever is not fully elucidated. Permixon, a hexanelipid/sterol extract of the fruits of this dwarf palm hasbeen shown to act as a weak, non-competitive inhibitorof both isoforms of 5a-reductase (type I and II) [13,14].Rapidly growing clinical interest in 5a-reductaseinhibitors has identified the therapeutic use of Per-mixon for the treatment of BPH with minimal site-effects [10,15]. The documented decrease in DHT andthe rise in testosterone in prostate tissue from BPHpatients treated with Permixon [14], confirmed thecapacity of this agent to inhibit in vivo 5a-reductaseactivity in human benign prostate with direct clinicalimplications. A marked increase in epidermal growthfactor (EGF) was associated with the reduction in DHTlevels [14]. These biochemical effects, similar to thoseobtained with finasteride [16], were particularly evi-dent in the periurethral region, whose enlargement isresponsible for the urinary obstruction symptom inBPH.

There is increased controversy however surround-ing the biochemical significance and theprecise cellularaction of these effects. A decrease in serum DHT wasnoted only inmen treated with finasteride in a study ofhealthy volunteers randomized to receive finasteride,Permixon, and placebo for a week [17]. At the bio-chemical level, a recent randomized, placebo controltrial failed to reveal an association saw palmetto herbalblend therapy in BPH patients with neither apoptosisinduction nor changes in the prostate tissue expressionof the androgen receptor [18]. Moreover, the morpho-logical changes in the prostate tissue characterized as‘‘atrophy,’’ were not accompanied by an increase in theapoptotic index or was there any difference in themainapoptosis regulators such as bcl-2 in the clinical study.

Several biochemical studies have analyzed the directeffect of Permixon on benign and malignant prostatecells. Using a model for BPH which exhibits many ofthe phenotypic characteristics of the human prostate,including both isoforms of 5a-reductase, PSA, andandrogen receptors, Bayne et al. [19], demonstratedthat Permixon effectively inhibits both isozymes of 5a-reductase in benign prostate cells, without interferingwith PSA expression or inducing apoptosis [19]. Amore recent report however challenges this evidenceby suggesting an apoptotic action by Permixon againstbenign and malignant prostate cells [20,21]. Consider-ing that androgens exert their growth regulatoryactivities via growth factor signal transduction, onemay speculate that in addition to targeting the andro-gen axis, targeting certain growth factor signalingpathways might dictate the prostate cellular responseto Permixon, as observed for finasteride treatment [7,8].Indeed saw palmetto has been reported to have anti-inflammatory effects in the prostate via inhibition ofarachidonic acid metabolites and reactive oxygenspecies [22], while other evidence suggests that sawpalmetto can inhibit fibroblast growth factor (FGF)induced prostatic epithelial cell proliferation [23].

In this study, we conducted an in vitro analysis toevaluate the prostate cell response to Permixon in orderto determine the ability of this phytotherapeutic agentto suppress prostate cell growth by affecting cellproliferation and apoptosis.

MATERIALSANDMETHODS

Cell Culture

Four different human prostate cell lines were used:the androgen-sensitive, (mutant) androgen-receptorpositive LNCaP human prostate cancer cells, theandrogen-independent prostate cancer cells PC-3, andDU-145 cells and the benign prostate epithelial cells,BPH-1. TheMCF-7 human breast cancer cells were alsoused for the control experiments (ATCC, Rockville,

74 Hill andKyprianou

MD). Prostate cell lines were maintained in RPMI-1640medium (Life Technologies Inc., Gaithersburg, MD)supplemented with 10% fetal calf serum (FCS; Colla-borative Biomedical Products, Bedford, MA) andantibiotics, penicillin/streptomycin (M.A. Biopro-ducts, Walkersville, MD). MCF-7 cells are maintainedin DMEM (Life Technologies Inc.), supplemented with10%FCS and antibiotics. Cell culturesweremaintainedat 378C in a humidified atmosphere of 5% CO2. A stocksolution of Permixon was prepared in ethanol at aconcentration of 10 mg/ml and appropriate workingdilutions were prepared in culture media. Parallelcontrol cultureswere supplementedwith an equivalentvolume of ethanol.

Cell ViabilityAssay

Cellswere plated at a density of 1� 105 cells perwellin 6-well plates and at subconfluency cells wereexposed to two different doses (10 and 100 mg/ml) ofPermixon for 1, 2, 3, 4, and 5 days. After treatment, cellviability was assessed by trypan blue exclusion assay.Mean values from triplicate wells were determined foreach treatment and expressed as% of untreated controlcultures.

Cell ProliferationAssay

Cells were plated in 6-well plates at a density of1� 105 cells/well and after 24 hr cell cultures wereexposed to increasing concentrations of Permixonwithin the pharmacological dose range (10, 50, and100 mg/ml) for 24 hr, and the rate ofDNAsynthesiswasdetermined using the [3H]-thymidine uptake assay[24]. Mean values from triplicate wells for each treat-ment were determined.

Cell Cycle Analysis

To examine the effect of Permixon on cell cycledistribution of asynchronous populations of LNCaPand PC-3 prostate cancer cells, replicative DNAsynthesis and DNA content were analyzed usingbivariate flowcytometric analysis. Asynchronous cul-tures of LNCaP and PC-3 cells were treated withPermixon (10 and 100 mg/ml) for 24 hr, and cells werepulsed-labeled with 5-bromo-2-deoxy [1,2-3H]-uridine(Amersham Intern., IL). After staining with fluores-cein-conjugated anti-Brd antibodies (BectonDickinson;PC-Lysis Program) and counterstaining with propi-dium iodide, sampleswere analyzed by flowcytometryusing the FACScan (Becton Dickinson).

Apoptosis Evaluation

Apoptosis assayswere performedusing theHoechststaining [25]. PC-3 and LNCaP cells were cultured in

the presence or absence of increasing concentrations ofPermixon and apoptosis induction were evaluatedafter 24 and 48 hr. Quantitative evaluation of the cellsexhibiting apoptotic morphology in treated anduntreated control cultures was performed by countingthe fluorescent-positive apoptotic cells over the totalnumber of cells [25]. A time course of apoptosisinduction was assessed after treatment of cells with100 mg/ml for 1, 2, 3, 4, and 5 days. Parallel cultures ofPC-3, DU-145, and BPH-1 prostate epithelial cells wereused as negative controls since these cells lack theandrogen receptor and should be unresponsive tothe effects of Permixon [13].

Western Blot Analysis

Cells were treated with Permixon (100 mg/ml) for 6,9, and 24 hr and after treatment, total cellular proteinwas extracted from cell pellets by homogenization inRIPA buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1%Nonidet p40, 0.5% deoxycholate sodium salt, 1 mMphenylmethysulphonyl fluoride, and 2 mg/ml aproti-nin). Aliquots of protein (30 mg) in Tris-buffered salinewere subjected to electophoretic analysis through12.5% sodium dodecyl sulfate polyacrylamide gel.Proteins were subsequently transferred onto Immobi-lon-P membranes; membranes were exposed to anti-body against caspase-3 from Pharmigen (San Diego,CA) and the antibody against m-actin was fromCalbiochem (La Jolla, CA) as previously described[25,26]. Protein expression was detected using thechemiluminescence system (ECL) (ECL, Amersham,Life Sciences) and autoradiography using X-ray film(Amersham).

Measurementof 5a -ReductaseActivityinHuman Prostate

The enzymatic activity of 5a-reductase was mea-sured in homogenates of human prostate as follows:Human prostate tissue from a patient who underwentprostatectomy for BPH was pulverized in Freezer–mil(Spex 6700) at the highest frequency and fractions weresubsequently homogenized in seven volumes ofsucrose buffer (0.25 M sucrose, 5 mM MgSO4, 20 mMKP, pH 6.5, 20 mM KCl, 1 mM PMSF, 1 mM dithio-threitol, and 5 mM NADPH. The prostate homogenatewas subsequently filtered and centrifuged at 1,500g (for15 min at 48C). The pellet was washed (3�) in cold0.25M sucrose buffer and it was finally resuspended infive volumes of cold 0.25 M sucrose buffer containing20% glycerol. The prepared 5a-reductase-prostatehomogenates were stored at �808C as individualaliquots.

For assay of the prostate 5a-reductase activity,different volumes of the prostate homogenate were

Lackof Apoptotic Effect by Permixon in Prostate Cells 75

added (5, 10, and 20 ml) were added to 75 ml of reactioncocktail and sampleswere incubated for 30min at 308C.The reaction was terminated by adding cyclohexane:ethyl acetate (65:35 v/v) and high speed centrifugation(14,000 rpm for 3min). The organic layer was subjectedto HPLC analysis in an HPLC system (2Waters HPLCpumps model 510, Waters Automated Gradient Con-troller, Hitachi Intelligent autosampler AS4000, Rdio-matic Flo-One beta counter model A100, Whatmanpartisil 5 column 10 cm). The retention times were11.1 min for testosterone and 6.3 min for dihy drotes-tosterone. For the 5a-reductase enzymatic assay,Permixon was added to the reaction mixture to a finalconcentration ranging from 10�4–103 mg/ml and incu-bated for 20 min at 378C. The IC50 represents theconcentration of Permixon that inhibits 50% of theconversion of testosterone to DHT.

Effect of Permixonon Prostate Specif|cAntigen (PSA) Expression

LNCaP prostate cancer cells were treated withPermixon (100 mg/ml) for 24 hr and supernatants fromtreated and untreated cells were assayed for PSAexpression using an ELISA assay and a PSA mono-clonal antibody (Fitzerald Industries, Concord, MA) aspreviously described [26]. PSA protein was detectedusing p-nitrophenyl phosphate (Sigma Chemical Co.)according to the manufacturer’s instructions. Valueswere determined as percentage of PSA expressionrelative to the untreated controls.

Statistical Analysis

All numerical data were represented as meanvalues� SEM (standard error of mean). Statisticalanalysis of the data was performed using the two-tailed Student’s t-test in the Microsoft Excel program.

RESULTS

The effect of Permixon on cell viability is shown inFigure 1. Exposure of prostate cancer cells to low-dosePermixon (10 mg/ml) had no significant effect (P> 0.3)on cell growth, while high dose Permixon (100 mg/ml)resulted in a significant loss of cell viability in both theandrogen-independent cells PC-3 and DU-145, as wellas the androgen-sensitive cells LNCaP (P< 0.001).Permixon at a high dose (100 mg/ml) had an effect onMCF-7 cell viability comparable to the one observed forthe prostate cancer cells. A time course of cell viabilityin prostate cancer cells PC-3 revealed no furtherreduction in cell growth after incubation with Per-mixon for 5 days, than the one initially observed after24 hr of treatment (Fig. 2).

The effect of Permixon on cell proliferation wassubsequently investigated. Permixon at a low dose

(10 mg/ml) had no significant effect on the proliferativerate of prostate cancer cells, while a significantreduction in the rate of [3H]-thymidine uptake wasdetected after 24 hr of treatment with high dosePermixon (Fig. 3). A similar effect was observed forthe benign prostate cells BPH-1 (data not shown). Todetermine whether a potential anti-growth effect ofPermixon was prostate cell-specific, the effect ofPermixon against the human breast cancer cells MCF-7was comparatively examined.A comparabledecreasein the rate of cell proliferation was observed for theMCF-7 breast cancer cells following treatment withPermixon (100 mg/ml).

The effect of Permixon prostate cancer cell cycleprogression was determined by flow cytometry. Thedata summarized on Table I revealed that after 24 hr ofincubationwith either a lowor ahighdose of Permixon,the percentage of the relative distribution of prostatecancer cells in each phase of the cell cycle remained

Fig. 1. Effectofpermixononcellviabilityofprostate cancercells.SubconfluentculturesofPC-3,LNCaP,andMCF-7cellsweretreatedwithtwodifferentconcentrationsofPermixon(10and100mg/ml) for24 hr. After treatment, cell viability was determinedon thebasis oftrypanblueexclusionassay.Datarepresenttheaveragevalues(intri-plicatewells) fromfour independentexperiments.

Fig. 2. Time course of the effect of Permixon on cell viabilityof PC-3 prostate cancer cells.Cells were exposed to a high dose ofPermixon (100mg/ml) for variousperiods of time (1^5days) andcellviabilitywasassessedasdescribedforFigure1.


similar. A comparable cell cycle profile was obtainedfor the PC-3 and the LNCaP cells (Table I).

Apoptosis evaluation in treated and untreatedprostate cancer cells was performed using the Hoechststaining. The results from thequantitative evaluation ofapoptosis induction in response toPermixon are shownin Figure 4. Treatment of LNCaP prostate cancer cellswith Permixon (at high dose) for 24 hr had no effect onapoptosis induction (P> 0.6). A similar profile wasobtained for the PC-3 cells following treatment withPermixon. Longer treatment periods did not result inany changes in the apoptotic potential of prostatecancer cells (data not shown).

Execution of apoptosis occurs through activation ofthe caspase proteases. Caspases are a family of cysteineproteases that are expressed as inactive pro-enzymes innormal cells and upon activation, they are capable ofcleaving structural and functional proteins involved inkey cellular processes [27]. The lack of apoptotic actionwas confirmed by the inability of Permixon to inducecaspase-3 activation. Figure 5 reveals a Western blot

analysis of caspase-3 expression inPC-3prostate cancercells before and after treatment with high dosePermixon.Only the pro-formof caspase-3was detectedthroughout any of the time periods of exposure tothe drug (without the release of the active fragment).This result is representative of two independentexperiments.

Treatment of LNCaP prostate cancer cells withPermixon (100 mg/ml) for 24 hrs did not result insignificant changes in PSA expression compared to theuntreated control cells (96% PSA expression).

We finally examined the effect of Permixon on 5a-reductase activity in human prostate homegenates.The results from the HPLC analysis are shown inFigure 6, revealing an IC50 value forPermixonof 8.7mg/ml which would correspond to 25 mM (if one assumesan average molecular weight of the mixture of 350).These data suggest that Permixon is a very weakinhibitor compared to finasteride.

DISCUSSION

It has become increasingly accepted that sawpalmetto does something to the prostate. Reports onthe use of saw palmetto to treat a variety of prostaticconditions date to the 1800s, however, many miscon-ceptions and uncertainties remain regarding its ther-apeutic use in prostatic symptoms in men with BPH[10]. Although many potential mechanisms via whichsaw palmetto may contribute to the relief of urinarysymptoms in men with BPH have been suggested, themost widely held belief is that this herbal product is anaturally occurring 5a-reductase inhibitor [22].

This study provides in vitro evidence to suggest thatPermixon fails to induce apoptosis in a manner similarto finasteride by interfering with the androgen axis inbenign and malignant human prostate cells in vitro.Low concentrations of Permixon (10 mg/ml), appar-ently a predicted physiologically relevant concentra-tion assuming distribution in total body fluid

Fig. 3. EffectofPermixonon therateofcellproliferationofpros-tate cancer cells. Prostate cancer cells PC-3 and LNCaP cells weretreatedwith Permixon (at two differentdoses10 and100 mg/ml) for24 hr and the rate of [3H[-thymidine-uptake was determined asdescribed in ‘‘Materials and Methods.’’ Data represent meanvalues fromthreeindependentexperimentsperformedin triplicate(�SEM).

TABLE I. Effectof PermixononCell Cycle Progression of Prostate CancerCells

PC-3 LNCaP

Treatment %G1 %G2 %S %G1 %G2 %SControl 46.9 29.8 23.3 49.2 29.6 21.2Permixon (10 mg/ml) 41.2 31.0 27.8 45.7 30.9 23.4Permixon (100 mg/ml) 36.3 32.1 31.6 41.5 33.2 25.3

Asynchronous populations of PC-3 and LNCaP cells were exposed to Permixon andwere fixed at24 hr following treatment. DNAwas identifiedwith a flourescein labeled anti-BrdU antibody andthen stainedwithpropidium iodide. FACSanalysiswasperformedasdescribed in ‘‘Materials andMethods.’’ G1, S, and G2 populations were quantitated using the Multicycle software program(Phoenix Flow Systems) and results were expressed as percentage of cells in each phase of the cellcycle.


achievable using the recommended therapeutic dose[19], had no effect on prostate cell proliferation. Theonly effect against prostate growth for both androgen-independent and androgen sensitive prostate cancercells was achieved at high dose of Permixon (100 mg/ml). The present results indicate a veryweak inhibitoryeffect of Permixon against 5 m-reductase activity ofhuman prostate homogenate, contrary to previouslypublished observations [17,18]. Furthermore, our find-ings challenge a recent report on the selectivity andspecificity of Permixon-mediated apoptosis in prostateepithelial cells and not in other cell types includingbreast cells [28] and raise questions regarding thetargeting of its therapeutic effect to prostate tumors.This lack of a regulatory function could be due to

differences in purity and content among herbalproducts provided by various manufacturers.

One has to also seriously consider the lack ofmorphological evidence of apoptosis induction bysaw palmetto among cell populations of BPH tissue inthe clinical studies by Marks et al. [18]. Interestinglyenough, amore recent report of the results of a 6-monthrandomized trial by this group of investigators,documented the ability of saw palmetto treatment tochange the DNA chromatin structure, shape, andspatial organization of prostate epithelial cells in menwith symptomatic BPH [29]. Thus while there is areasonable body of evidence that Permixon results inalterations in the human prostatic epithelium andtissue levels of DHT, the absence of any regulatoryeffect of Permixon on PSA expression or apoptosisinduction would support the paradoxic concept thatthis phytotherapeutic agent has little effect on otherandrogen-dependent molecular processes, but it is still(indirectly) capable of inhibiting 5a-reductase. Indeed,contrary to an earlier report that Permixon directly actsat the cytosolic androgen receptor [30], recent evidencesuggests that disruption of the membrane would leadto inactivation of the 5a-reducstase isoenzymes with-out inhibiting the androgen receptor activity [31]. Thedamage to the intracellular membrane of the cellsfollowing Permixon treatment may be due to a specificconstituent, myristoleic acid (a cytotolic component in

Fig. 4. Effect of permixon on apoptosis in prostate cancer cells.LNCaP cells were treatedwith two differentdoses of Permixon asindicatedfor24hr.After treatment,cellswerefixedandstainedwithHoechst 33342. The cells with the apoptotic morphology wereobservedby fluorescencemicroscopyusing anultraviolet filter andquantitatedasdescribedin‘‘MaterialsandMethods.’’Datarepresentthemeanvalues from two independent experiments performed induplicate (�SEM).

Fig. 5. Western blot analysis of caspase-3 expression in PC-3prostate cancer cells before and after treatment with Permixon.PC-3cellswere treated for6,9, and24hrwithPermixon (100mg/ml)and cell lysateswereprepared as described in‘‘Materials andMeth-ods.’’Aliquots of cell lysates (30mgprotein)were separated through12.5% sodium dodecyl sulfate-polyacrylamide gel, transferred tonitrocellulose, andprobedwith amonoclonal antibody againstcas-pase-3.Proteinexpressionwasdeterminedbyusing theScionimageprogramandnormalizedtoa-actinexpression.

Fig. 6. Inhibition of 5a -reductase activity type II in human pros-tate homogenates by Permixon. The experimental conditions forthe enzyme assay and separation of testosterone from DHTare in‘‘Materials andMethods.’’ Brieflyprostatic homogenateswere incu-bated with increasing concentrations of Permixon as indicated(10�4 ^103mg/ml), for 20min at 378C.The IC50 represents the con-centrationofpermixonthatinhibits50%of theconversionof testos-terone toDHT.


the extract from Serenoa repens), as suggested by Iguchiet al. [20]. Studies in a co-culturemodel of BPH supportsuch a novelmethod of enzyme inhibition, thatwithoutdisrupting the mechanism enhancing the androgen-responsive genes, would account for the inability ofPermixon to interferewith the expression of PSA [19].Adirect correlation of the 5a-reductase inhibitory effectof Permixon with a significant apoptotic effect againstprostate cells would potentially provide a molecularbasis of the therapeutic effect the drug in BPH patients.The current results howeverwould seriously challengesuch a concept.

ACKNOWLEDGMENTS

The authors acknowledge Dr. Stephen C. Jacobs,Division of Urology, University of Maryland, forhelpful discussions.

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effect of permixon on human prostate cell growth: lack of apoptotic action

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