J Cancer Res Clin Oncol (2007) 133:493–500

DOI 10.1007/s00432-007-0193-1

ORIGINAL PAPER

Ursolic acid, a naturally occurring triterpenoid, demonstrates anticancer activity on human prostate cancer cells

E. Kassi · Z. Papoutsi · H. Pratsinis · N. Aligiannis · M. Manoussakis · P. Moutsatsou

Received: 3 August 2005 / Accepted: 15 January 2007 / Published online: 15 February 2007© Springer-Verlag 2007

AbstractPurpose Glucocorticoids are widely used as adjuvanttherapy in hormonal refractory prostate cancer; theirtherapeutic role, however, remains unclear. Ursolicacid, a natural triterpene, structurally similar to dexa-methasone, exhibits antitumor eVects in various celltypes. Our main objective was to investigate the eVectsof ursolic acid on cell viability, apoptosis and bcl-2 pro-tein, in human hormone refractory and androgen-sen-sitive prostate cancer cells.Methods The ursolic acid-induced changes in cellviability, apoptosis and bcl-2 protein were examined

in human hormone refractory prostate cancer PC-3cells and androgen-sensitive LNCaP cells, by MTTassay, Xow cytometry and western blot analysis,respectively.Results Ursolic acid inhibited signiWcantly the cellviability and induced apoptosis in PC-3 cells at 55 �Mand in LNCaP cells at 45 �M associated with a downre-gulation of bcl-2 protein.Conclusions The antiproliferative and apoptoticeVects of ursolic acid in PC-3 and LNCaP cells impli-cate its potential therapeutic use for the treatment ofhormone refractory and androgen-sensitive prostatecancer. The downregulation of bcl-2 may be one of themolecular mechanisms via which it induces apoptosisin PC-3 and LNCaP cells.

Keywords LNCaP cells · Dexamethasone · RU486 · Ursolic acid · Prostate cancer

Introduction

Carcinoma of the prostate is a major health problem.In the United States, of the more than 700,000 newcases of cancer occurring in men each year, approxi-mately one third or 230,000 new cases were expectedto be cancer of the prostate in 2005 (Chodak 2006).As most prostate tumors are androgen-dependent,the common strategy for therapy at advanced diseasestages is the androgen ablation manipulations, whichinclude castration or luteinizing hormone-releasinghormone analogs (LHRH-A) with or without the useof anti-androgens. Prostate-speciWc antigen (PSA)analysis is the most useful tumor marker for the fol-low-up of patients with androgen-sensitive prostate

Financial support by a grant PAVE from the General Secretariat of Research and Technology in cooperation with the company ELPEN.

E. Kassi · Z. Papoutsi · P. Moutsatsou (&)Department of Biological Chemistry, Medical School, University of Athens, 75 Mikras Asias Street, 115 27 Goudi, Athens, Greecee-mail: pmoutsatsou@med.uoa.gr

H. PratsinisLaboratory of Cell Proliferation and Ageing, Institute of Biology, NCSR Demokritos, 153 10 Athens, Greece

N. AligiannisLaboratory of Pharmacognosy, Department of Pharmacy, University of Athens, Panepistimiopolis, 157 71 Zografou-Athens, Greece

M. ManoussakisLaboratory of Pathophysiology, Medical School, University of Athens, 75 M.Asias, 115 27 Athens, Greece

123

494 J Cancer Res Clin Oncol (2007) 133:493–500

cancer since PSA gene is androgen-regulated and itspromoter contains an androgen response element(Cleutjens et al. 1996). However, ultimately advancedstage prostate cancer becomes eventually refractoryto androgen ablation (Isaacs 1999).

The use of dexamethasone as adjuvant to chemo-therapy has resulted clinical beneWts in patients withhormone refractory prostate cancer (HRPC) and clin-ical trials showed that PSA decline can serve as anintermediate endpoint of clinical response in HRPC(Sartor et al. 1998; Nishimura et al. 2000; Koutsilieriset al. 2001, 2004). However, a subset of HRPCpatients failed to respond to dexamethasone and PSAlevels did not decline after dexamethasone but ratherincreased (Storlie et al. 1995). DiVerent glucocortic-oids such as prednisone, hydrocortisone or dexameth-asone resulted in variable declines in PSA levels ofpatients with HRPC, possibly due, in part, to the typeand dose of diVerent glucocorticoids (Sartor et al.1998; Kelly et al. 1995; Storlie et al. 1995). In supportto clinical studies, in vitro studies regarding the eVectof dexamethasone on growth and apoptosis of andro-gen-sensitive and androgen-insensitive prostate can-cer cells (LNCaP and PC-3, respectively) giveconXicting results, supporting either growth arrest orno eVect at all (Nishimura et al. 2001; Reyes-Morenoet al. 1995; Lin et al. 1995; Carollo et al. 1998; Xinget al. 2001; Fakih et al. 2002). Taken together, despitethe multiple studies (in vivo and in vitro) on glucocor-ticoid use in androgen-independent prostate cancer,their exact therapeutic role remains unclear (Fakihet al. 2002).

Given the many side eVects of glucocorticoid useand due to the variable eVects of glucocorticoids onHRPC, limited only to a subgroup of patients, the dis-covery of new alternatives is highly desirable. Triterp-enes are compounds isolated from plants with chemicalstructure very similar to that of dexamethasone (Chaet al. 1998). Ursolic acid (UA), a pentacyclic triterpeneis non-toxic; it presents anti-invasive eVect in tumorcells, and it has been shown to induce growth arrestand apoptosis in a variety of tumors. Furthermore, itexhibits chemopreventive properties by preventingmalignant transformation of normal cells (Novotnyet al. 2001; Ovesna et al. 2004). Since the apoptosisinduction is a useful endpoint assay for evaluating thetherapeutic potency of a compound against prostatecancer, we conducted an in vitro investigation to evalu-ate the potency of ursolic acid to suppress PC-3 (repre-senting hormone refractory prostate cancer) andLNCaP (representing model for androgen-sensitiveprostate cancer) cellular growth by aVecting the cellviability/proliferation, apoptosis and apoptosis-related

bcl-2 protein. Its potential to induce PSA expression inLNCaP cells was also assessed.

Materials and methods

Isolation of ursolic acid

Ursolic acid was isolated from aerial parts of SalviaoYcinalis (Labiatae). Air-dried, powdered plant mate-rial (1 kg) was extracted at room temperature withCH2Cl2 (3L £ 3) and then MeOH (3L £ 3). TheCH2Cl2 soluble extract was concentrated underreduced pressure to give a residue (25 g), which waschromatographed over a silica gel 60H column andeluted with CH2Cl2–MeOH (100:0 ! 0:100) (0.5–1.5 l)to give 14 fractions. The sixth fraction (0.92 g), elutedwith CH2Cl2–MeOH 97:3, was re-chromatographedover silica gel and eluted with CH2Cl2–MeOH(99.5:0.5 ! 98:2) to aVord ursolic acid (18 mg).

Cell viability/proliferation MTT assay

We used two established prostate carcinoma cell lines,the PC-3 (androgen receptor negative, glucocorticoidreceptor positive), and LNCaP (androgen receptorpositive, glucocorticoid receptor negative) cell linesrepresentative of androgen receptor independent andandrogen receptor dependent prostate cancers, respec-tively (Horoszewicz et al. 1983; Schuurmans et al.1988). The LNCaP cells are expressing mutated andro-gen receptor and wild type estrogen receptor alpha(King et al. 2006). LNCaP cell-line was routinely cul-tured in RPMI 1640 and PC-3 in Dulbecco’s MinimalEssential Medium, supplemented both with penicillin(100 U/ml), streptomycin (100 �g/ml) and 10% FetalBovine Serum (FBS) (media and antibiotics from Bio-chrom KG, Berlin, Germany) at 37°C and 5% CO2.Adherent cells were subcultured using a trypsin 0.25%-EDTA 0.02% solution. Cytotoxicity was estimated bya modiWcation of the MTT assay (Denizot and Lang1986). BrieXy, cells were plated in 96-well Xat-bot-tomed microplates at a density of 10,000 cells/well.Twenty-four hours after the plating, ursolic acid wasadded at Wnal concentrations 100, 80, 60, 40, 20, 10, and2 �M. After a 48-h incubation, the medium wasreplaced with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma, St.Louis, MO,USA) dissolved at a Wnal concentration of 1 mg/ml inserum-free, phenol-red-free RPMI (Biochrom KG)and DMEM (Biochrom KG), respectively for a further4-h incubation. Then, the MTT-formazan was solubi-lized in isopropanol and the optical density was

123

J Cancer Res Clin Oncol (2007) 133:493–500 495

measured at a wavelength of 550 nm and a referencewavelength of 690 nm. The eVect of dexamethasone(Sigma) (10¡5–10¡8M) and RU486 (10¡6–10¡8M) wasalso examined.

Flow cytometry

PC-3 and LNCap cells were plated at a density of75,000/well in 24-well plates and grown with DMEMand RPMI 1640 respectively containing 5% FBS. Theday after, ursolic acid was added at a concentration of55 and 45 �M (respectively) and cells were incubatedfor 24, 48 and 72 h. There was a vehicle control(absence of compound) for each incubation period.Apoptosis of PC-3 and LNCap cells was assessed byXow cytometry analyzing DNA content in Annexin V-FITC and propidium iodide (PI) stained cells (TACSAnnexin V-FITC Apoptosis Detection Kit,Cat#TA4638, R & D Systems) with a FACSCalibur(Becton Dickinson) cytometry. BrieXy, cells wereharvested with trypsin/EDTA, washed, then incubatedfor 15 min with Annexin V-FITC and PI, and Wnallyanalyzed by Xow cytometry.

Bcl-2 determination by Western Blot analysis

PC-3 cells were grown in DMEM medium andLNCaP in RPMI 1640 supplemented both with peni-cillin/streptomycin and 10% FBS, as previouslydescribed. After incubation for 24 and 48 h with vehi-cle control (in the absence of compound) or ursolicacid (at Wnal concentration 55 �M for PC-3 and 45 �Mfor LNCaP cells), cells (1 £ 107) were washed twice incold PBS and lysed with 1 ml lysis buVer (50 mMTris–HCl pH = 7,4, 150 mM NaCl, 1 mM EGTA,1 mM EDTA, 1% NP-40 with aprotinin 2 �g/ml, leu-peptin 2 �g/ml, PMSF 100 �g/ml) (Jiang et al 2004).Cells were lysed at 4°C for 30 min with occasionalvortexing followed by brief sonication. Lysates werespun at 14,000 rpm for 10 min to remove cellulardebris. Protein concentration in supernatants wasdetermined with Bradfoard assay. Sixty microgram ofeach sample were subjected to electrophoresis on12% SDS-polyacrylamide gels and transferred tonitrocellulose membrane. The blots were blocked,incubated with anti-bcl-2 antibody (N-19, Santa-Cruz) at 1:500 dilution and anti-actin antibody (MAB1501, Chemicon) at 1:2,000 dilution, washed withTBST and incubated with secondary antibody, anti-rabbit (Pierce) at 1:7,000 dilution and antimouse(Santa Cruz) at 1:2,000 dilution, respectively. Chemi-luminescence detection kit was used for visualizationof the signal. Densitometric quantiWcation of the

bands (Scion Image), normalization with actin andcalculation of the ratio bcl-2 with UA/ bcl-2 withoutUA 24 h, and bcl-2 with UA/bcl-2 without UA 48 hwas also carried out.

PSA measurement in LNCaP cells

Cultures of LNCap (ATCC Cell Bank) were grown inRPMI 1640 (Gibco BRL) and PC-3 cells (ATCC CellBank) in DMEM supplemented with 10% FBS(Gibco BRL), 50 units/ml Penicillin and 50 �g/mlStreptomycin in T-75 cm2 Xasks at 37°C and 5% CO2atmosphere. Stock cultures were subcultured every 6–7 days. Cells were plated at an initial density of170,000 cells/well in 24-mm-diameter multiwell plasticculture dishes (6-well-plate); cells were then grown inthe same medium as previously described until con-Xuence (5 days approximately). After 5 days of cellculture, the medium was changed to RPMI 1640 with-out Phenol Red supplemented with 2% FBS DCC(Dextran Coated Charcoal Slurry) treated and50 units/ml Penicillin and 50 �g/ml Streptomycin.After 2 days of cell culture, ursolic acid (10¡5–10¡7M)was added and a 48-h incubation followed. Each time,the culture Xuids were collected from the dish andcentrifuged at 12,000 rpm for 10 min. The superna-tants were used for total PSA measurement. The cellswere removed carefully from the dish using cell-scrapers, collected in approximately 200 �l PBS buVerand counted using a hemocytometer plate. The eVectof vehicle control (in the absence of compound),dexamethasone (10¡5–10¡8 M), and RU486 (10¡6–108 M) was also investigated. Total PSA was mea-sured in all samples directly and after dilution 1:30using a non-competitive direct sandwich ELISA tech-nique. BrieXy, standards and samples were incubatedtogether with biotinylated anti-PSA monoclonal anti-body in Streptavidin coated microtiter strips. Afterincubation, horseradish peroxidase (HRP) labeledanti-PSA Mab (Monoclonal Antibody) was added,incubated and then detected by substrate/Chromogenreagent. The color intensity was determined in amicrotiter plate spectrophotometer at 405 nm afterthe addition of stop solution.

Statistical analysis

All values are expressed as the mean § standard devia-tion of four measurements. Statistical analysis was per-formed using t test, two-tailed distribution, assumingtwo-sample unequal variance. A P < 0.05 value wasconsidered statistical signiWcant (*P < 0.05, **P < 0.01,***P < 0.001).

123

496 J Cancer Res Clin Oncol (2007) 133:493–500

Results

EVect of UA on cell viability/proliferation

The dose response curve concerning the eVect of UAon cell viability of PC-3 and LNCaP cells is shown inFig. 1. Results are expressed as percentage of survivalof untreated cells (in the absence of test compounds).The growth of PC-3 cells was inhibited by ursolic acid(Fig. 1). Dexamethasone did not inhibit the PC-3 cellu-lar growth, whereas RU486 at 10¡4M inhibited thegrowth of cells (data not shown). Neither Dexametha-sone nor RU486 inhibited the growth of LNCaP cells(data not shown), whereas ursolic acid inhibited signiW-cantly the growth of LNCaP cells (Fig. 1). The IC50 forursolic acid was approximately 32.6 �M for PC-3 cellsand 15.7 �M for LNCaP cells.

EVects of UA on apoptosis

The combination of Annexin V-FITC and propidiumiodide allows for the diVerentiation between earlyapoptotic cells, late apoptotic or necrotic cells and via-ble cells. Ursolic acid at a concentration 55 �� inducedsigniWcantly (P < 0.01) apoptosis in PC-3 cells after 48and 72 h incubation (viable cells = 79.86 § 1.39% oftotal cells after 48 h incubation, and 48.7 § 2.45% oftotal cells after 72 h of incubation). In Fig. 2a, there isan example of the analysis of DNA content in AnnexinV-FITC and propidium iodide (PI) stained PC-3 cellsafter 24, 48, and 72-h-exposure to ursolic acid (55 �M).For each incubation period there was a vehicle control

(without compound). At a concentration 45 ��, itinduced signiWcantly (P < 0.01) apoptosis in LNCaPcells after incubation for the same periods (viablecells = 79.96 § 0.9% of total cells after 48 h incubation,and 54.48 § 2.06% of total cells after 72 h of incuba-tion). Figure 2b shows an example of the analysis ofDNA content in Annexin V-FITC and propidiumiodide (PI) stained LNCaP cells after 24, 48 and 72 hexposure to ursolic acid (45 �M). As previously men-tioned, for each incubation period there was a vehiclecontrol (without compound). In both Fig. 2a and b,controls are representative.

Bcl-2 protein levels in PC-3 and LNCaP cells

Levels of bcl-2 protein in PC-3 cells were decreasedafter incubation with ursolic acid (55 �M) (Fig. 3A1).The downregulation of bcl-2 expression (as percentageof control), after normalization with actin, was statisti-cally signiWcant (P < 0.001). As it is shown in Fig. 3A2,there was a 65 and 53% downregulation of bcl-2expression after incubation with ursolic acid for 24 and48 h, respectively. Similarly, in LNCaP cells the bcl-2protein was completely suppressed after incubationwith ursolic acid for 24 h (Fig. 3b).

EVect of Ursolic acid on PSA levels in LNCaP cells

Dexamethasone (10¡5–10¡8 M), RU486 (10¡6–10¡8 M)and ursolic acid (10¡5–10¡7 M) increased signiWcantly(P ranging from 0.05 to 0.001) PSA levels in LNCaPcells (data not shown). The percentage of increaseranged, in comparison to vehicle control, from132 § 7.2% to 149 § 12% for dexamethasone, from147 § 4.6% to 161 § 1.8% for RU486 and from124 §4% to 141.8 § 1.3% for ursolic acid. PC-3 cells did notinduce PSA expression in presence of dexamethasone,RU486 or ursolic acid (data not shown), as expected.

Discussion

Due to the high prevalence of prostate cancer amongthe common cancers in men, and because of the partialclinical responses in hormone refractory prostate can-cer even after the use of combined therapies with che-motherapy and dexamethasone, the development ofnew agents for advanced stage prostate cancer treat-ment has become a challenging issue. The main objec-tive of the present study was to investigate theantigrowth eVects of ursolic acid and its impact oninduction of apoptotis and bcl-2 protein in PC-3 pros-tate cancer cells, representing model for hormone

Fig. 1 Cytotoxicity of ursolic acid on PC-3 and LNCaP cells. Af-ter incubation of cells with the indicated concentrations of Urso-lic acid for 48 h, cytotoxicity was determined by the MTT assay.Each point of the dose response curve is the average of threewells. The error bars represent standard deviations. The resultsshown are representative of three experiments

123

J Cancer Res Clin Oncol (2007) 133:493–500 497

refractory prostate cancer and in LNCaP cells, repre-senting model for androgen-sensitive prostate cancer.The uncontrolled cellular growth is among the maincharacteristics of almost all types of cancer, includingprostate cancer, due to defects either in cell prolifera-tion and/or in apoptosis (Bruckheimer and Kyprianou

2000). Agents, which show antiproliferative action maybe able to eliminate the number of cancer cells andmay be used in cancer treatment. Herein, we demon-strated that ursolic acid inhibited signiWcantly the cellviability of PC-3 and LNCaP cells at low concentra-tions, 32.6 and 15.7 �M, respectively, implicating that it

Fig. 2 a An example of the analysis of DNA content in AnnexinV-FITC and propidium iodide (PI) stained PC-3 cells after 24, 48,72 h exposure to ursolic acid (55 �M). For each incubation periodthere was a diVerent vehicle control (without compound). Ursolicacid induce statistically signiWcant apoptosis (viablecells = 48.7 § 2.45% of total cells) after 72 h exposure (P < 0.01)[Early Apoptotic (% of total cells): Control (representativephoto) = 3.95 § 0.268; ursolic acid 24 h = 3.96 § 0.667; ursolicacid 48 h = 5.30 § 0.65; ursolic acid 72 h = 13.66 § 1.71 average §SD; triplicate determinations] [Late apoptotic or necrotic (% oftotal cells): Control = 4.67 § 0.64; ursolic acid 24 h = 6.2 § 0.8;ursolic acid 48 h = 14.8 § 0.9; ursolic acid 72 h = 36.26 § 2.25;average § SD; triplicate determinations]. b An example of the

analysis of DNA content in Annexin V-FITC and propidiumiodide (PI) stained LNCaP cells after 24, 48, and 72 h exposure toursolic acid (45 �M). For each incubation period there was adiVerent vehicle control (without compound). Ursolic acid inducestatistically signiWcant apoptosis (viable cells = 54.48 § 2.06% oftotal cells) after 72 h exposure (P < 0.01) [Early Apoptotic (% oftotal cells): control (representative photo) = 0.34 § 0.025; ursolicacid 24 h = 0.54 § 0.04; ursolic acid 48 h = 0.87 § 0.11; ursolicacid 72 h = 21.24 § 1.88 average § SD; triplicate determinations][Late apoptotic or necrotic (% of total cells): control = 13.2 §1.09; ursolic acid 24 h = 17.65 § 0.8; ursolic acid 48 h = 19.16 § 1;ursolic acid 72 h = 24.27 § 1.67; average § SD; triplicate determi-nations]

123

498 J Cancer Res Clin Oncol (2007) 133:493–500

may be useful as prostate anticancer agent. In ourstudy, dexamethasone as well as RU486, a widelyknown antiandrogen/antiglucocorticoid compound,showed variable eVects on the cellular proliferation ofPC-3 and LNCaP prostate cancer cells. In the same linewith our Wndings, in vitro studies regarding the eVect ofdexamethasone and RU486 on growth of PC-3 andLNCaP prostate cancer cells give conXicting results,supporting either growth arrest or no eVect at all

(Nishimura et al. 2001; Reyes-Moreno et al. 1995; Linet al. 1995; Carollo et al. 1998; El Etreby et al. 2000;Xing et al. 2001; Liang et al. 2002; Fakih et al. 2002).The above implicate that UA may be a better alterna-tive to glucocorticoids in combined chemotherapies forthe treatment of prostate cancer.

Apoptosis is widely accepted as an ideal mechanismfor reduction of prostate cancer cells. Dysregulation ofapoptosis is associated with resistance to chemother-apy while agents, which can induce apoptosis may beuseful in the management and therapy of prostate can-cer (Bruckheimer and Kyprianou 2000). We observeda signiWcant increase in apoptosis of PC-3 cells by treat-ment with ursolic acid at low concentrations (55 ��),implicating that the mechanism by which UA inhibitedthe growth of prostate cancer cells was through apop-tosis. The UA-associated induction of apoptotic pro-cess may be accomplished by various pathways, theantiapoptotic bcl-2 protein being among the majorapoptosis-related mediators. Given that recent datasuggest that bcl-2 is an attractive molecular target inthe treatment of HRPC and plays an important role inthe response to chemotherapy, we further investigatedthe bcl-2 responses to the presence of UA in the hor-mone refractory PC-3 cells (Sonpavde and Hutson2006). Our data demonstrated a decreasing eVect ofUA at 55 �M on bcl-2 protein content, implicating thatthe UA-induced apoptosis in PC-3 cells is mediated, atleast in part, via triggering the mitochondrial pathwayand that ursolic acid may be a promising therapeuticagent for HRPC. Interestingly, Choi et al. (2000) havealso shown that ursolic acid induced apoptosis inhuman prostate epithelial cells. However, their datasuggested that the apoptotic signals evoked by UAtreatment might converge caspases (caspase-1, -3, -8,and -9) activation through downregulation of c-IAPsfamily proteins and without alterations in bcl-2 and baxexpression. In our study, UA triggered apoptosis alsoin the androgen-sensitive LNCaP cells at a low concen-tration (45 �M), via triggering the mitochondrial path-way as demonstrated by its decreasing eVect on bcl-2protein content. Taking into consideration that othercurrently used agents for prostate cancer as monother-apy or combined chemotherapy have been shown toinduce apoptosis at a concentration range 1–100 ��,which is similar to the apoptosis induced concentrationof ursolic acid (45–55 �M), present data suggest furtherthat UA has the potential to overcome the androgenand chemotherapy refractoriness of prostate cancer asmonotherapy or combined therapy (Budman et al.2002).

PSA is a glycoprotein, which is highly and speciWcallyexpressed in prostate tissue. It is an androgen-dependent

Fig. 3 a EVect of ursolic acid (Wnal concentration 55 �M) on bcl-2 expression in PC-3 cells. 1 Western blot analysis shows less in-tense bands in PC-3 cells treated with UA for 24 and 48 h in com-parison to untreated cells (Control). 2 DensitometricquantiWcation of the bands, normalization with actin and calcula-tion of the % downregulation of bcl-2 expression after incubationwith UA for 24 h (65%) and 48 h (53%) compared with untreatedcells (Control). b EVect of ursolic acid (Wnal concentration45 ��) on bcl-2 expression in LNCaP cells. Western blot analysisshows that bcl-2 protein levels were completely suppressed afterincubation with UA for 24 h in comparison to untreated cells(Control)

123

J Cancer Res Clin Oncol (2007) 133:493–500 499

protein, the gene of which carries androgen responseelement in its promoter, therefore its speciWc regula-tion by androgen (Cleutjens et al. 1996). PSA is themost widely accepted marker in the detection of earlycancer whereas the serial blood sample analysis andfollow up of PSA levels in prostate cancer patientsunder treatment, is a standard employed procedure toassess the response to therapy as well as the cancerprogression in androgen-sensitive prostate cancer.EVective anticancer agents are expected to result in adecline of PSA levels. However, in the presence ofmutated androgen receptors, other steroids, not onlyandrogens, have been shown to induce PSA expression(Cleutjens et al. 1997). Similar to previous reports, wefound that dexamethasone as well as RU486 inducedPSA expression in LNCaP cells, which are known tocontain a mutant form of androgen receptor (Krishnanet al. 2002; Cleutjens et al. 1996; Song et al. 2004). Inlight of the abovementioned, many concerns arisewhether the presence of various androgen receptor(AR) mutant forms in prostate cancer may lead tovarying responses to dexamethasone treatment whenthis type of prostate cancer progress from androgen-dependent to an androgen-independent state (Isaacs1999; Buchanan et al. 2001; Koutsilieris et al. 2004).Evidently, it is a necessity to develop new agents,which may be eVective against androgen-sensitiveprostate cancer carrying a mutant type AR, which mayprogress to androgen-independent prostate cancer.

Our study revealed that ursolic acid increased PSAlevels in LNCaP cells, however, it induced antigrowtheVects and apoptosis in this cell type, implicating thatPSA is not the factor based on which one could supportany eVect on prostate cancer cell growth. The diver-gence of ursolic acid action on LNCaP cell growth andapoptosis vis a vis PSA expression may be partlyexplained by the fact that ligand-mediated regulationof a single target gene, such as PSA, may diVer from itsgeneralized eVects on growth or apoptosis which arethe reXection of much more complex transcriptionalfunctions. It is known that modulation of multiplepathways are required for the treatment of prostatecancer, such as the growth factor signaling, inhibitionof NFkB signaling, modulation of coactivators/core-pressors, suppression of cell cycle genes, or inductionof apoptosis process accomplished by either caspase-3and caspase-9 activation or alteration of the Baxprotein or increased intracellular Ca2+ levels (Ca2+

inXux) or even by changes in mitochondrial permeabil-ity transition (MPT) (Schuurmans et al 1988; Songet al. 2004; Xing et al. 2001; Bruckheimer and Kypria-nou 2000; Tenta et al. 2004; Sonpavde and Hutson2006; Nishimura et al. 2001). In this regard, one may

speculate further that the diVerences among ursolicacid and dexamethasone on LNCaP cell growth incomparison with PSA levels may be due to diVerencesin the underlying cellular mechanisms triggered bythese diVerent compounds.

In conclusion, our study provides evidence that urso-lic acid initiates a cell death command in hormonerefractory PC-3 prostate cancer cells and in androgen-sensitive LNCaP cells, mediated, at least in part, via themechanism of apoptosis, involving downregulation ofbcl-2 protein. The fact that UA is also an eVectiveinducer of apoptosis in LNCaP cells emphasizes furtherthat UA has strong potential for development as anagent for the management of various subsets of prostatecancer patients such as hormone-refractory as well asandrogen-sensitive carrying mutant type AR forms. Weunderstand there are gaps between in vitro studies andclinical trials and that cell lines may not reXect the het-erogeneity of clinical malignant disease; however, ourdata may be useful to establish clinical protocols investi-gating ursolic acid in hormone and chemotherapyrefractoriness of prostate cancer. Further mechanism-based studies are needed as well as its eVects in vivobefore any recommendations can be made.

References

Bruckheimer EM, Kyprianou N (2000) Apoptosis in prostatecarcinogenesis. A growth regulator and a therapeutic target.Cell Tissue Res 301(1):153–162

Buchanan G, Greenberg NM, Scher HI, Harris JM, Marshall VR,Tilley WD (2001) Collocation of androgen receptor genemutations in prostate cancer. Clin Cancer Res 7:1273–1281

Budman DR, Calabro A, Kreis W (2002) Synergistic and antago-nistic combinations of drugs in human prostate cancer celllines in vitro. Anticancer Drugs. 13(10):1011–1016

Carollo M, Parente L, D’Alessandro N (1998) Dexamethasone-induced cytotoxic activity and drug resistance eVects inandrogen-independent prostate tumor PC-3 cells are medi-ated by lipocortin 1. Oncol Res 10(5):245–254

Cha HJ, Park MT, Chung HY, Kim ND, Sato H, Seiki M, KimKW (1998) Ursolic acid-induced down-regulation of MMP-9gene is mediated through the nuclear translocation of gluco-corticoid receptor in HT1080 human Wbrosarcoma cells.Oncogene 16:771–778

Chodak G (2006) Prostate cancer: epidemiology, screening, andbiomarkers. Rev Urol 8(suppl 2):S3–S8

Choi YH, Baek JH, Yoo MA, Chung HY, Kim ND, Kim KW(2000) Induction of apoptosis by ursolic acid through activa-tion of caspases and down-regulation of c-IAPs in humanprostate epithelial cells. Int J Oncol 17(3):565–571

Cleutjens KB, van Eekelen CC, van der Korput HA, van RooijHC, Faber PW, Trapman J (1996) Two androgen responseregions cooperate in steroid hormone regulated activity ofthe prostate-speciWc antigen promoter. J Biol Chem271(11):6379–6388

Cleutjens CB, Steketee K, van Eekelen CC, van der Korput JA,Brinkmann AO, Trapman J (1997) Both androgen receptor

123

500 J Cancer Res Clin Oncol (2007) 133:493–500

and glucocorticoid receptor are able to induce prostate-spe-ciWc antigen expression, but diVer in their growth-stimulatingproperties of LNCaP cells. Endocrinology 138(12):5293

Denizot F, Lang R (1986) Rapid colorimetric assay for cellgrowth and survival: modiWcations to the tetrazolium dyeprocedure giving improved sensitivity and reliability. JImmunol Meth 89:271–277

El Etreby MF, Liang Y, Lewis RW (2000) Induction of apoptosisby mifepristone and tamoxifen human LNCaP prostate can-cer cells in culture. Prostate 43(1):31–42

Fakih M, Johnson CS, Trump DL (2002) Glucocorticoids andtreatment of prostate cancer: a preclinical and clinical re-view. Urology 60(4):553–561

Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H,Chu TM, Mirand EA, Murphy GP (1983) LNCaP model ofhuman prostatic carcinoma. Cancer Res 43(4):1809–1818

Isaacs JT (1999) The biology of hormone refractory prostate can-cer. Why does it develop? Urol Clin North Am 26:263–273

Jiang J, Slivova V, Valachovicova T, Harvey K, Sliva D (2004)Ganoderma lucidum inhibits proliferation and induces apop-tosis in human prostate cancer cells PC-3. Int J Oncol24(5):1093–1099

Kelly WK, Scher HI, Mazumdar M, PWster D, Curley T, LeibertzC, Cohen L, Vlamis V, Dnistrian A, Schwartz M (1995) Sur-amin and hydrocortisone: determining drug eYcacy inandrogen-independent prostate cancer. J Clin Oncol13(9):2214–2222

King KJ, Nicholson HD, Assinder SJ (2006) EVect of increasingratio of estrogen: androgen on proliferation of normal hu-man prostate stromal and epithelial cells, and the malignantcell line LNCaP. Prostate 66(1):105–114

Koutsilieris M, Mitsiades C, Dimopoulos Th, Ioannidis A, Ntoun-is A, Lambou T (2001) A combination therapy of dexameth-asone and somatostatin analog reintroduces objectiveclinical response to LHRH analog in androgen ablation-refractory prostate cancer patients. J Clin Endocrinol Meta-bol 86:5729–5738

Koutsilieris M, Mitsiades CS, Bogdanos J, Dimopoulos T, Kar-amanolakis D, Milathianakis C, Tsintavis A (2004) Combina-tion of somatostatin analog, dexamethasone, and standardandrogen ablation therapy in stage D3 prostate cancer patientswith bone metastases. Clin Cancer Res 10(13):4398–4405

Krishnan AV, Zhao XY, Swami S, Brive L, Peehl DM, Ely KR,Feldman D (2002) A glucocorticoid-responsive mutantandrogen receptor exhibits unique ligand speciWcity thera-peutic implications for androgen-independent prostate can-cer. Endocrinology 143(5):1889–2000

Liang Y, Eid MA, El Etreby F, Lewis RW, Kumar MV (2002) Mi-fepristone-induced secretion of transforming growth factorbeta1-induced apoptosis in prostate cancer cells. Int J Oncol21(6):1259–1267

Lin MF, Kawachi MH, Stallcup MR, Grunberg SM, Lin FF (1995)Growth inhibition of androgen-insensitive human prostatecarcinoma cells by a 19-norsteroid derivative agent, mifepri-stone. Prostate 26(4):194–204

Nishimura K, Nonomura N, Yasunaga Y, Takaha N, Inoue H, Su-gao H, Yamaguchi S, Ukimura O, Miki T, Okuyama A(2000) Low doses of oral dexamethasone for hormone-refractory prostate carcinoma. Cancer 89:2570–2576

Nishimura K, Nonomura N, Satoh E, Harada Y, Nakayama M,Tokizane T, Fukui T, Ono Y, Inoue H, Shin M, Tsujimoto Y,Takayama H, Aozasa K, Okuyama A (2001) Potential mech-anism for the eVects of dexamethasone on growth of andro-gen-independent prostate cancer. J Natl Cancer Inst93(22):1739–1746

Novotny L, Vachalkova A, Biggs D (2001) Ursolic acid: an anti-tumorigenic and chemopreventive activity. Minireview.Neoplasma 48(4):241–246

Ovesna Z, Vachalkova A, Horvathova K, Tothova D (2004) Pen-tacyclic triterpenoic acids: new chemoprotective compounds.Minireview. Neoplasma 51(5):327–333

Reyes-Moreno C, Frenette G, Boulanger J, Lavergne E, Govin-dan MV, Koutsilieris M (1995) Mediation of glucocorticoidreceptor function by transforming growth factor beta Iexpression in human PC-3 prostate cancer cells. Prostate26(5):260–291

Sartor O, Weinberger M, Moore A, Li A, Figg WD (1998)EVect of prednisone on prostate-speciWc antigen in pa-tients with hormone-refractory prostate cancer. Urology52:252–256

Schuurmans AL, Bolt J, Voorhorst MM, Blankenstein RA, Mul-der E (1988) Regulation of growth and epidermal growthfactor receptor levels of LNCaP prostate tumor cells bydiVerent steroids. Int J Cancer 42:917–922

Song LN, Coghlan M, Gelmann EP (2004) Antiandrogen eVectsof mifepristone on coactivator and corepressor interactionswith the androgen receptor. Mol Endocrinol 18(1):70–85

Sonpavde G, Hutson TE (2006) New approaches in hormonerefractory prostate cancer. Am J Clin Oncol 29(2):196¡201

Storlie JA, Buckner JC, Wiseman GA, Burch PA, Hartmann LC,Richardson RL (1995) Prostate speciWc antigen level andclinical response to low dose dexamethasone for hormone-refractory metastatic prostate carcinoma. Cancer 76:96–100

Tenta R, Tiblalexi D, Sotiriou E, Lembessis P, Manoussakis M,Koutsilieris M (2004) Bone microenvironment-relatedgrowth factors modulate diVerentially the anticancer actionsof zoledronic acid and doxorubicin on PC-3 prostate cancercells. Prostate 59(2):120–131

Xing Y, Xiao Y, Zhang Q, Lu G (2001) The eVect of interleukin-6 on the proliferation of prostate cancer cells in vitro and themodulation of this procedure. J Tongji Med Univ 21(3):225–227

123