Green tea constituent epigallocatechin-3-gallate selectively inhibits COX-2 withoutaffecting COX-1 expression in human prostate carcinoma cellsTajamul Hussain1, Sanjay Gupta2, Vaqar M. Adhami1 and Hasan Mukhtar1*1Department of Dermatology, University of Wisconsin, Madison, WI, USA2Department of Urology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA

Overexpression of cyclooxygenase (COX)-2 has been implicated inmany pathologic conditions, including cancer. One practical infer-ence of this finding is that sustained inhibition of COX-2 couldserve as a promising target for prevention or therapy of cancer.Conventional nonsteroidal antiinflammatory drugs (NSAIDs) andrecently developed COX-2-specific inhibitors have shown consid-erable promise in prevention of some forms of human cancer;however, its application is limited due to severe toxic side effectson normal cells. Therefore, there is a need to define novel, nontoxicdietary constituents with proven chemopreventive effects throughother pathways that also possess COX-2 but not COX-1 inhibitoryactivity. Recent studies on green tea and its major polyphenolicconstituent (-)epigallocatechin-3-gallate (EGCG) have established itsremarkable cancer preventive and some cancer therapeutic effects.Here, we show that EGCG inhibits COX-2 without affecting COX-1expression at both the mRNA and protein levels, in androgen-sensi-tive LNCaP and androgen-insensitive PC-3 human prostate carci-noma cells. Based on our study, it is tempting to suggest that acombination of EGCG with chemotherapeutic drugs could be animproved strategy for prevention and treatment of prostate cancer.© 2004 Wiley-Liss, Inc.

Key words: green tea; epigallocatechin-3-gallate; cell growth; pros-taglandin E; cyclooxygenase-2; apoptosis

Cyclooxygenase (COX)-1 and -2 are key enzymes that catalyzethe rate-limiting step in prostaglandin biosynthesis.1,2 COX-1 is ahousekeeping enzyme, constitutively expressed in most mamma-lian tissue and is responsible for maintaining normal cellularphysiologic functions.1 COX-2, on the other hand, is an inducibleisoform, rapidly induced by growth factors, tumor promoters,oncogenes and carcinogens.2 Aberrant or increased expression ofCOX-2 has been implicated in many pathologic conditions, includ-ing tumor promotion and angiogenesis.3,4 Compelling evidencefrom genetic and clinical studies indicates that overexpression ofCOX-2 is associated with carcinogenesis.5–9 Deletion of theCOX-2 gene has been shown to suppress the development ofintestinal polyps in mice with the adenomatous polyposis coli(APC) gene, suggesting that COX-2 expression is essential fortumorigenesis.10 In addition, overexpression of COX-2 in themammary glands of transgenic mice results in enhanced tumori-genesis in multiparous (nonvirgin) females.11 These experimentaldata strongly suggest that inappropriate expression of COX-2 is acritical event in tumorigenesis. One practical implication of thesefindings is that sustained inhibition of COX-2 could be of signif-icant clinical importance in prevention and therapy of many humancancers. Conventional nonsteroidal antiinflammatory drugs(NSAIDs) and recently developed COX-2-specific inhibitors haveshown considerable promise in the prevention of some forms ofhuman cancer; however, their application is limited due to severetoxic side effects on normal cells.12,13 Therefore, novel nontoxicCOX-2-specific inhibitors that have the ability to spare normalcells from their cytotoxic effects are required. One approach ofdefining such agents is to assess dietary substances that haveproven cancer preventive or therapeutic effects through multiplepathways that include COX-2 but not COX-1 inhibitory activity.Such agents could be used in combination with NSAIDs or che-motherapeutic drugs.

Green tea, a popular beverage consumed worldwide, has beenwidely appreciated for its cancer chemopreventive effects.14,15 Thecancer chemopreventive effects of green tea have been attributed

to its major polyphenolic constituent, epigallocatechin-3-gallate(EGCG).14,15 Recent reports suggest that EGCG may also possesscancer therapeutic effects.16,17 Previous studies from our labora-tory have shown that EGCG, at pharmacologically attainable con-centrations, results in inhibition of cell growth, arrest of the cellcycle in the G0/G1 phase and induction of apoptosis in somehuman carcinoma cells, without affecting the normal cells.18 Insubsequent studies, many other laboratories reported similar find-ings in many other cancer cell lines.19–21 More recently, we havedemonstrated the importance of green tea polyphenols in inhibitingprostate carcinogenesis in transgenic adenocarcinoma of themouse prostate (TRAMP) model that emulates most forms ofhuman prostate cancer.22

We earlier reported that EGCG inhibits cell growth and inducesapoptosis in both androgen-sensitive LNCaP and androgen-insen-sitive DU145, human prostate carcinoma cells.23 In addition, greentea and EGCG have been shown to inhibit COX-2 activity andexpression in both cell culture systems and in vivo models of somecancer types.24–27 Based on these observations and many clinicalstudies, COX-2 inhibitors are considered effective agents for pre-vention and therapy of some forms of human cancer. Since EGCGhas been shown to cause cell growth inhibition and apoptosis ofvarious cancer cells, we considered the possibility that EGCG mayalso inhibit COX-2 expression. In the present study, we show thatconcomitant with EGCG-induced cell growth inhibitory effects, italso inhibits COX-2 but not COX-1 expression, in both androgen-sensitive and androgen-insensitive human prostate carcinomacells. These observations may contribute to EGCG-induced selec-tive cell growth inhibition and apoptosis of cancer cells andsuggest the possible role of green tea and EGCG in prevention andtherapy of prostate cancer.

Material and methodsMaterials

Androgen-sensitive LNCaP and androgen-insensitive PC-3,human prostate carcinoma cell lines, were obtained from Amer-ican Type Culture Collection (Rockville, MD). Purified prepa-ration (�98%) of EGCG was a kind gift from Dr. YukihikoHara, Mitsui Norin Co. Ltd. (Shizuoka, Japan). Arachidonicacid (AA) was purchased from Sigma Chemical Company (St.Louis, MO). FBS was obtained from Gibco BRL (Gaithersburg,

T.H. and S.G. contributed equally to this work.Abbreviations: AA, arachidonic acid; APC, adenomatous polyposis coli;

COX, cyclooxygenase; EGCG, (-)epigallocatechin-3-gallate; NSAIDs,nonsteroidal antiinflammatory drugs; PGE2, prostaglandin E2; RT-PCR,reverse transcriptase-PCR.

Grant sponsor: USPHS; Grant number: RO1CA78809; Grant sponsor:American Institute for Cancer Research; Grant number: 00A30; Grantsponsor: Department of Defense; Grant number: DAMD 17-00-1-0527;Grant sponsor: Cancer Research and Prevention Foundation; Grant spon-sor: O-CHA (Tea) Pioneer Academic Research Grant.

*Correspondence to: Hasan Mukhtar, Helfaer Professor of CancerResearch and Vice Chair, University of Wisconsin, Department of Der-matology, 1300 University Ave., Medical Sciences Center, B-25, Madison,WI 53706, USA. Fax: �608-263-5223. E-mail: hmukthar@wisc.edu

Received 17 May 2004; Accepted after revision 28 July 2004DOI 10.1002/ijc.20629Published online 28 September 2004 in Wiley InterScience (www.

interscience.wiley.com).

Int. J. Cancer: 113, 660–669 (2005)© 2004 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

MD). Monoclonal antibodies for anti-COX-2 (Clone 33, Cat.610203) and anti-COX-1 (Cat. 160110) were purchased fromBD Transduction Laboratories (Lexington, KY) and CaymanChemical (Ann Arbor, MI), respectively, while anti-actin anti-body was obtained from Santa Cruz Biotechnology, Inc. (SantaCruz, CA). A prostaglandin E2 (PGE2) detection kit was ob-tained from Cayman Chemical (Ann Arbor, MI). A cell deathdetection ELISA kit was purchased from Roche MolecularBiochemicals, USA (Indianapolis, IN).

Cell culture and treatmentsHuman prostate carcinoma cells were cultured in RPMI-1640

growth medium supplemented with 10% FBS, 1% penicillin and1% streptomycin. Cells were maintained at 37°C in a humidifiedCO2 incubator. For experiments, LNCaP and PC-3 cells wereplated at low density in RPMI-1640 complete medium and wereallowed to grow until �40% confluent. After this, the completegrowth medium was changed to serum-free RPMI-1640 mediumfor 24 hr, and thereafter cells were treated with 5 �M AA (dis-solved in DMSO to a final concentration of 0.02%), alone or incombination with 10, 25, 50, or 100 �M EGCG (dissolved inPBS), for 24 hr and were processed for determination of mRNAand protein expression. For examining the effect of EGCG on cellgrowth, incubation was continued for 48 hr and cell viability wasassessed by Trypan blue exclusion assay following standard pro-tocol. To study the effect of EGCG on PGE2 secretion, LNCaP andPC-3 cells were treated with 5 �M AA alone or in combinationwith 10, 25, 50, or 100 �M EGCG for 24 hr. Culture supernatantsfrom treated and untreated cells were collected and assayed forPGE2 levels. To determine the effect of exogenous PGE2 additionon cell proliferation and apoptosis, LNCaP and PC-3 cells weretreated with 0.01, 0.1, 1 or 10 �M PGE2 along with 5 �M AA and100 �M EGCG for 48 hr, which were evaluated by a Trypan blueexclusion assay and cell death detection ELISA kit, respectively.For examining the effect of celecoxib, a COX-2-specific inhibitoron cell growth and PGE2 levels, the cells were treated with 5 �MAA alone or in combination with 10 �M celecoxib for 6 hr andwere processed for determination of cell growth, PGE2 levels andCOX-2 protein expression.

Cell viabilityThe viability of cells in various treated and control groups was

determined by Trypan blue exclusion assay, and morphologicexamination of cells was done under light microscopy. Briefly,total viable cells in control and treated groups were counted underthe microscope and control group represented as 100%.

PGE2 estimationAfter various treatments, culture medium was collected and

used immediately or stored at –80°C until assayed. PGE2 levels inthe culture supernatants were estimated using a PGE2 monoclonalenzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) asdetailed by the manufacturer.

DNA fragmentation assayAfter treatment of cells as described above, the cells were

washed twice with PBS (pH 7.4), incubated with DNA lysis buffer(10 mM TRIS, pH 7.5, 400 mM NaCl, 1 mM EDTA and 1% TritonX-100) for 30 min on ice and then centrifuged at 14,000g at 4°C.The supernatant obtained was incubated overnight with RNAse(0.2 mg/ml) at room temperature and then with Proteinase K(0.1 mg/ml) for 2 hr at 37°C. DNA was extracted using phenol:chloroform (1:1) and precipitated with 95% ethanol for 2 hr at–80°C. The DNA precipitate was centrifuged at 14,000g at 4°C for15 min and the pellet was air-dried and dissolved in 20 �l ofTRIS-EDTA buffer (10 mM TRIS-HCl, pH 8.0, and 1 mMEDTA). Total amount of DNA was resolved over 1.5% agarosegel, containing 0.3 �g/ml ethidium bromide in 1� TBE buffer (pH8.3, 89 mM TRIS, 89 mM boric acid and 2 mM EDTA) (BioWittaker, Inc., Walkersville, MD). The bands were visualizedunder UV transilluminator (model TM-36, UVP, Inc., San Gabriel,

CA) followed by Polaroid photography (MP-4 Photographic Sys-tem; Fotodyne, Inc., Hartland, WI).

Apoptosis by ELISAAfter EGCG treatment, the extent of apoptosis was determined

by Cell Death Detection ELISAPLUS assay according to the man-ufacturer’s protocol (Roche Diagnostic Corporation, Indianapolis,IN). Briefly, the cells were harvested after EGCG treatment andincubated over ice for 30 min in TRIS lysis buffer (50 mMTRIS-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 20 mM NaF,0.5% NP-40, 1% Triton X-100) containing fresh protease inhibi-tors (5 �g/ml aprotinin, 10 �g/ml phenylmethylsulfonyl fluorideand 10 �g/ml sodium vanadate) and then centrifuged at 15,000gfor 10 min at 4°C. The total cell lysate was used for proteindetermination by the DC BioRad� protein assay. The lysates(30 �g of total protein) were added to lysis buffer and pipetted ona streptavidin-coated 96-well microtiter plate to which immunore-agent mix was added and incubated for 2 hr at room temperaturewith continuous shaking at 800g. The wells were then washed withwashing buffer, the substrate solution was added, and the colordeveloped (10–20 min) was read at 405 nm against the blankreference wavelength of 490 nm. The enrichment factor (totalamount of apoptosis) was calculated by dividing the absorbance ofthe sample (A405 nm) by the absorbance of the controls withouttreatment (A490 nm).

Reverse transcriptase (RT)-PCRTotal RNA (1 �g) was reverse transcribed for cDNA synthesis

using M-MLV Reverse Transcriptase and Oligo(dT)12–18 primer(Gibco BRL, Grand Island, NY). PCR was performed using gene-specific primers for COX-2 or COX-1. The GAPDH gene wascoamplified to serve as an internal control. Primer sequences usedfor COX-2 were sense 5�-GGT CTG GTG CCT GGT CTG ATGATG-3� and antisense 5�-GTC CTT TCA AGG AGA ATG GTCG-3�; for COX-1, sense 5�-GTT CAA CAC CTC CAT GTT GGTGGA C-3� (774 bp) and antisense 5�-TGG TGT TGA GGC AGACCA GCT TC-3� (494 bp); and for GAPDH, sense 5�-TGA AGGTCG GAG TCA ACG GAT TTG GT-3� and antisense 5�-CATGTG GGC CAT GAG GTC CAC CAC-3� (983 bp). PCR wasperformed under exponential conditions that allowed the amountsof PCR products to be proportional to the amounts of input RNA.PCR products were electrophoresed on 1.5% agarose gel contain-ing 0.5 �g/ml ethidium bromide as previously described.28

Western blot analysisCell lysates (25 �g) were resolved on 12% SDS-polyacrylamide

gels and electroblotted on a nitrocellulose membrane. Membranewas blocked for 2 hr at room temperature in 5% nonfat dry milkdissolved in TBS buffer (10 mM TRIS, pH 7.5, 100 mM NaCl,0.1% Tween-20). Blot was incubated with anti-COX-2- and anti-COX-1-specific monoclonal antibodies for 2 hr at room tempera-ture. Incubation with appropriate HRP-conjugated secondary an-tibody (Amersham Life Sciences, Inc., Arlington Heights, IL) wascarried out for 90 min at room temperature and signals weredetected using a chemiluminescence system (ECL; AmershamPharmacia Biotech, Piscataway, NJ) and autoradiography. To en-sure equal loading of protein samples, blot was stripped andincubated with anti-�-actin antibody and processed as previouslydescribed.28

Statistical analysisDensitometric measurements of the bands in Western blot anal-

ysis were performed using the digitalized scientific software pro-gram UN-SCAN-IT purchased from Silk Scientific Corporation(Orem, UT). Student’s 2-tailed t-test was employed to assess thestatistical significance between the control and treated groups, andp-values less than 0.05 were considered significant.

661SELECTIVE INHIBITION OF COX-2 BY EGCG

ResultsAA stimulates cell growth and PGE2 levels in human prostatecarcinoma cells

COX-2 catalyzes the rate-limiting step in prostaglandin synthe-sis from AA; therefore, in the first experiment we incubated bothandrogen-sensitive LNCaP and androgen-insensitive PC-3 humanprostate carcinoma cells with AA to observe cell growth and PGE2levels. As shown in Figure 1a, treatment of LNCaP cells with AAexhibited a significant increase in cell growth and PGE2 levels ina dose-dependent fashion. Stimulation of LNCaP cells with AAresulted in a significant growth of cells at 2 �M (88%), 5 �M(102%) concentration of AA and PGE2 levels (243% at 2 �M and329% at 5 �M), with a dose-dependent increase observed up to10 �M concentration of AA. Similar effects were observed inPC-3 cells with AA stimulation in both cell growth (125% at 2 �Mand 152% at 5 �M) and PGE2 levels (257% at 2 �M and 346% at5 �M) (Fig. 1b). Based on this data, we selected a dose concen-tration of 5 �M AA for further studies.

Celecoxib overcomes the effect of AA in inhibiting cell growth,PGE2 levels and COX-2 expression in human prostatecarcinoma cells

Next we asked whether treatment of cells with celecoxib, aspecific COX-2 inhibitor, inhibits AA-induced cell proliferationand PGE2 levels. To accomplish this goal, the cells were incubatedwith 5 �M concentration of AA alone or with 10 �M celecoxib for6 hr. Treatment of LNCaP cells with celecoxib was found to resultin a significant decrease in cell growth (19%; p � 0.05) and PGE2levels (30%; p � 0.001) stimulated by AA. Further, these effectswere found to correlate with a modest decrease in COX-2 proteinexpression after celecoxib treatment (Fig. 2a). Similar inhibitoryeffects were observed on AA-stimulated PC-3 cell growth (24%;p � 0.05), PGE2 levels (36%; p � 0.001) and COX-2 protein

expression after treatment of cells with a 10 �M concentration ofcelecoxib (Fig. 2b).

EGCG overcomes the effect of AA in inhibiting cell growth andPGE2 levels in human prostate carcinoma cells

Next we evaluated the efficacy of EGCG for its ability toovercome the growth-stimulating effects of AA on both LNCaPand PC-3 cells. Treatment of LNCaP cells with a 5 �M concen-tration of AA exhibited almost 200% increase in cell growth and300% increase in PGE2 levels in the cell supernatants. Further,treatment of LNCaP cells with EGCG resulted in a dose-dependentdecrease in cell growth to 20% at 10 �M, 37% at 25 �M, 62% at50 �M and 78% at 100 �M EGCG. A significant decrease in PGE2expression was observed at 10 �M (15%), 25 �M (36%), 50 �M(59%) and 100 �M (85%) concentrations of EGCG (Fig. 3a). Asimilar effect was observed with PC-3 cells where treatment ofcells with EGCG resulted in a dose-dependent decrease in AA-induced cell growth (19% at 10 �M, 36% at 25 �M, 60% at50 �M and 71% at 100 �M) and PGE2 levels (17% at 10 �M, 34%at 25 �M, 57% at 50 �M and 83% at 100 �M) (Fig. 3b).

EGCG affects cell morphology and induces apoptosis in humanprostate carcinoma cells

Next we observed morphologic changes in LNCaP and PC-3cells treated with EGCG. Treatment of LNCaP and PC-3 cells withAA alone resulted in increased cell survival and proliferation.Previous studies have shown that AA is metabolized to a widerange of ecosanoids that affect survival and proliferation of pros-tate carcinoma cells.29 A marked difference in cell morphologywas observed with EGCG at 100 �M concentration in LNCaPcells compared to nontreated cells and cells treated with a 5 �Mconcentration of AA. EGCG treatment showed pronounced alter-ations of membrane morphology characterized by the formation ofmembrane blebs, which were consistent with apoptosis (Fig. 4a).

FIGURE 1 – Effect of AA on cell growth and PGE2 levels in human prostate carcinoma LNCaP (a) and PC-3 (b) cells. The cells were treatedwith the indicated concentrations of AA and were processed for determination of cell growth and PGE2 levels as described in the Material andMethods section. A significant increase in cell growth and PGE2 levels was observed in both cell lines after AA treatment. Each data pointrepresents mean SE of 3 independent experiments. Significance compared to control: *, p � 0.05; **, p � 0.001.

662 HUSSAIN ET AL.

FIGURE 2 – Effect of celecoxib on cell growth and PGE2 levels in AA-stimulated human prostate carcinoma LNCaP (a) and PC-3 (b) cells.The cells were treated with 5 �M AA alone or in combination with 10 �M celecoxib for 6 hr and were processed for determination of cellgrowth, PGE2 levels and COX-2 protein expression as described in the Material and Methods section. Each data point represents mean SEof 3 independent experiments. Quantitation of bands was done by densitometric analysis and is shown as fold change compared to vehicle controlat the bottom of the bands. Significance compared to control: *, p � 0.05; **, p � 0.001.

FIGURE 3 – Effect of EGCG on cell growth and PGE2 levels in AA-stimulated human prostate carcinoma LNCaP (a) and PC-3 (b) cells. Thecells were treated with 5 �M AA alone or in combination with indicated doses of EGCG and processed for determination of cell growth andPGE2 levels as described in the Material and Methods section. Each data point represents mean SE of 3 independent experiments. Significancecompared to control: *, p � 0.05; **, p � 0.001.

663SELECTIVE INHIBITION OF COX-2 BY EGCG

Similar effects were observed with PC-3 cells treated with EGCGand AA (Fig. 4b).

As cellular growth inhibition of cancer cells further results inapoptosis, we examined the effect of EGCG on apoptosis inboth cell lines. Treatment of LNCaP cells with AA and EGCGresulted in a dose-dependent increase in apoptosis, as evidentby ELISA assay, while DNA fragmentation was observed at 50and 100 �M concentrations of EGCG (Fig. 5a). Similar apo-ptotic effects were observed with treatment of PC-3 cells withAA and EGCG (Fig. 5b).

EGCG downmodulates COX-2 but not COX-1 mRNA expressionin human prostate carcinoma cells

In order to understand the molecular mechanism responsiblefor the antiproliferative effects of EGCG, we treated LNCaPand PC-3 cells with indicated concentrations of EGCG andstudied the effect on COX-1 and COX-2 mRNA expression.Status of COX-2 expression in LNCaP and PC-3 cells remainedlargely unresolved, with several studies reporting the constitu-tive levels,30,31 while others suggested the stimulus-dependentexpression.32,33 In an attempt to achieve detectable levels ofCOX-2 mRNA and protein levels, we treated these cells withAA as a stimulating agent. In the absence of AA, both LNCaPand PC-3 cells expressed detectable levels of COX-2 mRNA,which was further increased by stimuli. Subsequent treatmentwith EGCG resulted in a dose-dependent decrease in COX-2mRNA levels in LNCaP cells. Compared to AA stimulatedcells, a significant decrease in COX-2 mRNA expression wasobserved at 50 and 100 �M concentrations of EGCG. Treatmentof cells with EGCG did not affect COX-1 mRNA expression atall of the doses studied or in response to cell stimuli (Fig. 6a).

Similar results were observed after treatment of PC-3 cells withEGCG in significantly reducing COX-2 mRNA expression at25, 50, and 100 �M concentrations of EGCG. COX-1 levels inPC-3 cells did not change in response to cell stimuli or afterEGCG treatment (Fig. 6b). Importantly, COX-2 mRNA levelsin LNCaP cells were lower than those found in PC-3 cellsbefore or after cell stimuli.

EGCG downmodulates COX-2 but not COX-1 proteinexpression in human prostate carcinoma cells

Since we observed that EGCG significantly downmodulatedCOX-2 mRNA in both LNCaP and PC-3 cells, next we askedwhether inhibition of mRNA levels correlates with the corre-sponding decrease in the protein expression. Consistent withdetectable mRNA levels, PC-3 cells expressed COX-2 protein,which was detectable even in the absence of a stimulatingagent. In contrast, weak COX-2 protein expression was ob-served in LNCaP cells, which further increased after treatmentwith stimulating agent (Fig. 7a). Treatment of LNCaP cellswith indicated concentrations of EGCG significantly decreasedCOX-2 protein levels in a dose-dependent fashion, while nosignificant alteration in COX-1 protein expression was observedin these cells (Fig. 7a). A similar effect was observed in PC-3cells, were EGCG treatment resulted in a significant decrease inCOX-2 protein expression, consistent with reduced COX-2mRNA levels. In agreement with unaltered COX-1 mRNAlevels in PC-3 cells, no significant alteration in COX-1 proteinwas observed before or after cell stimulation or after EGCGtreatment in these cells (Fig. 7b).

FIGURE 4 – Effect of EGCG on cell morphology in AA-stimulated human prostate carcinoma LNCaP (a) and PC-3 (b) cells. The cells weretreated with 5 �M AA alone or in combination with 100 �M EGCG for 24 hr and were observed under light microscope. A significant changein cell morphology characterized by the formation of membrane blebs, consistent with induction of apoptosis, was observed after EGCGtreatment. Data are from a typical experiment repeated 4 times with similar results.

664 HUSSAIN ET AL.

PGE2 restores EGCG-inhibited cell growth and decreasesapoptosis in human prostate carcinoma cells

Because EGCG-induced cell growth inhibition and apoptosiswere associated with decreased PGE2 secretion, we asked whethersupplementing PGE2 to both LNCaP and PC-3 cells restoresEGCG-mediated effects on these cells. Treatment of LNCaP cells

with exogenous PGE2 at 0.01, 0.1, 1, and 10 �M concentrationsreversed EGCG-mediated effects and significantly restored cellgrowth in a dose-dependent fashion (Fig. 8a). Interestingly, LN-CaP cells exhibiting almost undetected levels of COX-2 proteinwere responsive to exogenous PGE2, suggesting its sensitivity toPGE2-induced cell proliferation. Similar effects were observed

FIGURE 5 – Effect of EGCG on inductionof apoptosis in AA-stimulated human pros-tate carcinoma LNCaP (a) and PC-3 (b) cells.The cells were treated with 5 �M AA aloneor in combination with indicated doses ofEGCG for 48 hr and were processed for de-termination of apoptosis by ELISA and DNAfragmentation assay as described in the Ma-terial and Methods section. The data in eachbar graph represent mean SE of 3 indepen-dent experiments. Significance compared toAA treated group: **, p � 0.001; M denotesDNA marker.

FIGURE 6 – Effect of EGCG on COX-2 and COX-1 mRNA expression in AA-stimulated human prostate carcinoma LNCaP (a) and PC-3 (b)cells. The cells were stimulated with 5 �M AA alone or in combination with indicated doses of EGCG for 24 hr; total RNA was isolated andanalyzed by RT-PCR for COX-1 and COX-2 expression. The GAPDH gene was coamplified as an internal control as described in the Materialand Methods section. A dose-dependent decrease in COX-2 mRNA expression was observed in both cell lines after EGCG treatment, whereasCOX-1 mRNA expression was unaffected by EGCG treatment.

665SELECTIVE INHIBITION OF COX-2 BY EGCG

with PC-3 cells where exogenous treatment of PGE2 resulted inrestoration of EGCG-induced cell growth inhibition (Fig. 8b).

Since apoptosis is one of the effects of cell growth inhibition, weexamined whether growth stimulation by PGE2 was mediated byinhibition of apoptosis in both LNCaP and PC-3 cells. As observedby cell death detection ELISA assay, apoptosis was significantlyinhibited by supplementation of exogenous PGE2 levels in a dose-dependent fashion in LNCaP cells (Fig. 9a). A similar effect wasobserved with PC-3 cells where exogenous PGE2 supplementationresulted in significant inhibition of apoptosis in these cells(Fig. 9b). These results reveal that cell growth inhibition andapoptosis in both androgen-sensitive LNCaP and androgen-insen-sitive PC-3 cells are regulated in part by COX-2 expression andPGE2 levels.

Discussion

There is a reasonable agreement that dietary fat plays an impor-tant role in the development and progression of prostate can-cer.34,35 AA, a member of -6 polyunsaturated fatty acids, wasfound to be an effective stimulator of human prostate cancer cellgrowth.36,37 AA can be metabolized either by the lipoxygenase(LOX) pathway leading to synthesis of hydroxy derivatives andleukotriens36 or by the COX pathway, which synthesizes prosta-glandins.37 The 2 known COX isoforms are referred to as COX-1and COX-2.1,2 COX-1 is expressed constitutively in many tissuesand cell types, while COX-2 is induced by a variety of factors suchas cytokines, growth factors, and tumor promoters.1,2 Inappropri-ate COX-2 expression has been observed in many types of humancancers, including prostate that contributes to tumorigenesis.5–9,38

Accumulating evidence suggests that increased synthesis of pros-taglandins, the metabolites of COX, possibly arises due to over-expression of COX-2 and plays an important role in cancer pro-gression.39,40 This evidence suggests that COX-2 could be animportant molecular target for prevention and intervention ofcancer.

Green tea, a popular beverage consumed worldwide, is greatlyappreciated for its cancer chemoprevention and possible therapeu-tic effects.14,15 We recently showed that infusion of green teapolyphenols inhibits tumor progression in a transgenic mouse

model of prostate cancer that recapitulates human disease.22 Muchof the biologic effects of green tea appear to be mediated by itsmajor polyphenolic constituent EGCG.41–43 Several recent reportsdemonstrated the effect of green tea polyphenols and EGCG onCOX-2 activity and expression in cell culture system and variousanimal model systems.24–27 Recently, EGCG has been shown todownmodulate IL-1�-induced COX-2 and PGE2 levels in humanchondrocytes.24 Supplementation of green tea extract has beenshown to suppress azoxymethane-induced preneoplastic lesionsand COX-2 activity in the colonic mucosa of rats.25 Similar effectshave been observed with green tea polyphenols in inhibiting AA-stimulated COX-2 and PGE2 levels in human colon cancer.26 Morerecently, EGCG has been shown to inhibit phorbol-induced skincarcinogenesis and COX-2 expression in mouse skin and in cul-tured mammary epithelial cells.27 These experimental data suggestthat COX-2 inhibition could be one of the molecular mechanismsresponsible for the chemopreventive effects of EGCG. In ourstudies, EGCG significantly downmodulated COX-2 mRNA andprotein expression in both androgen-sensitive LNCaP and andro-gen-insensitive PC-3 cells, without affecting COX-1 levels. Thisselective effect of COX-2 inhibition in both cell lines was accom-panied with significant cell growth inhibition. These observationssuggest the involvement of COX-2 during EGCG-mediatedgrowth inhibitory effects of carcinoma cells.

It has been accepted that the antitumor activities of NSAIDs andselective COX-2 inhibitors are due to their ability to sensitizecancer cells to apoptosis and inhibit COX-2 expression.44,45 Morerecently, it has been shown that the apoptosis-inducing effects ofNSAIDs and selective COX-2 inhibitors are mediated by COX-2-independent mechanisms.46 This has been convincingly demon-strated by the use of celecoxib in COX-2-deficient tumors im-planted in a nude mouse model capable of inducing apoptosis incells that do not express COX-2.46,47 Furthermore, some NSAIDderivatives that do not inhibit COX activity retain their chemopre-ventive effect.45–47 These observations support the hypothesis thatsome of the antiproliferative effects of selective COX-2 inhibitorsare independent of COX-2 inhibition. We have recently demon-strated that EGCG inhibits cell growth and induces apoptosis inprostate cancer cell lines LNCaP and DU145.23 In the presentstudy we selected 2 different human prostate carcinoma cells:

FIGURE 7 – Effect of EGCG on COX-2 and COX-1 protein expression in AA-stimulated human prostate carcinoma LNCaP (a) and PC-3 (b)cells. The cells were stimulated with 5 �M AA alone or in combination with indicated doses of EGCG for 24 hr and processed for total celllysate for protein expression by Western blotting. Actin protein expression was used as a loading control as described in the Material andMethods section. A dose-dependent decrease in COX-2 protein expression was observed in PC-3 cells after EGCG treatment, whereas COX-1mRNA expression was unaffected in both cell lines by EGCG treatment.

666 HUSSAIN ET AL.

androgen-insensitive human prostate carcinoma PC-3 cells exhib-iting appreciable levels of COX-2 protein expression and andro-gen-sensitive LNCaP cells, which barely show detectable levels ofCOX-2 protein. In our studies, downmodulation of COX-2 proteinin PC-3 cells was accompanied with significant cell growth inhi-bition, which suggests that growth inhibitory effects of EGCGinvolved downmodulation of COX-2 protein. Interestingly, LN-CaP cells, despite barely detectable levels of COX-2 protein,showed similar sensitivity to growth inhibition by EGCG. Theseobservations suggest that growth inhibitory effects of EGCG didnot entirely depend on COX-2 inhibition in LNCaP cells andsupport the possibility of COX-2-independent mechanisms as well.It may, however, be possible that immunoblot analysis barelydetected COX-2 protein in LNCaP and this diminutive level ofprotein affected cell growth. This possibility also gains supportfrom the observation that EGCG downmodulated COX-2 mRNAin LNCaP cells. In either case, it could be argued that growthinhibition in LNCaP cells by EGCG may be due to COX-2-independent mechanisms. Similar studies are reported where cele-

coxib and the metabolites of sulindac have been shown to inhibitthe growth of LNCaP and PC-3 cells and induce apoptosis byCOX-2-independent mechanisms.30,45–47

The ecosanoids generated by increased turnover of COX havereceived considerable attention as mediators of inflammatory andphysiologic processes that play important roles in neoplasia.48 Theecosanoids have the ability to modulate DNA/RNA synthesis, cellmembrane activity, cell communication, and immune responsesthat may mediate carcinogenesis.48 Several studies have reportedincreased PGE2 production in various human cancer tissues andcarcinoma cell lines.48,49 In our study, treatment of LNCaP andPC-3 cells with different concentrations of PGE2 reversed theeffect of EGCG and restored cell growth to significant levels inboth cell types. These observations suggest—and it may be pos-sible that—EGCG-mediated prostate cancer cell growth inhibitionis COX-2 dependent, and the sensitivity of these cell lines togrowth stimulation by PGE2 underlines the important role ofCOX-2 in carcinogenesis. Further, a dose-dependent decrease inthe enrichment factor (cell death detection assay) with increasingconcentrations of PGE2 revealed that inhibition of apoptosis could

FIGURE 8 – Effect of PGE2 supplementation in AA-stimulated andEGCG-treated cell growth in human prostate carcinoma LNCaP (a)and PC-3 (b) cells. The cells were treated with 5 �M AA, 100 �MEGCG and PGE2 at indicated concentrations and combinations for48 hr and were processed for determination of cell growth by theTrypan blue exclusion assay as described in the Material and Methodssection. Each data point represents mean SE of 3 independentexperiments. A dose-dependent increase in cell growth was observedin both cell lines after PGE2 supplementation. Data shown here arerepresentative of 3 independent experiments with similar results.

FIGURE 9 – Effect of PGE2 supplementation in AA-mediated andEGCG-treated apoptosis in human prostate carcinoma LNCaP (a) andPC-3 (b) cells. The cells were treated with 5 �M AA, 100 �M EGCGand PGE2 at indicated concentrations and combinations for 48 hr andwere processed for determination of apoptosis by ELISA as describedin the Material and Methods section. Each data point representsmean SE of 3 independent experiments. A dose-dependent decreasein cell growth was observed in both cell lines after PGE2 supplemen-tation. Data shown here are representative of 3 independent experi-ments with similar results.

667SELECTIVE INHIBITION OF COX-2 BY EGCG

be one of the COX-2-independent mechanisms in the restoration ofcell growth in these cell lines.

In recent years, NSAIDs and COX-2-specific inhibitors havebeen used as a class of chemopreventive agents in various types ofhuman malignancies.50 Clinical studies and experimental evidencehave demonstrated the role of NSAIDs and COX-2-specific inhib-itors in inhibiting tumorigenesis.51 We have recently shown thatdietary supplementation of celecoxib inhibits prostate cancer pro-gression in the TRAMP model.52 In fact, some of the newlydeveloped COX-2-specific inhibitors (celecoxib and rofecoxib) arebeing evaluated for their efficacy in numerous clinical trials on avariety of human cancers, including breast, bladder, colon, skin,and esophagus cancer.53 Interestingly, randomized clinical trialshave confirmed that the prodrugs sulindac and celecoxib are ef-fective in inhibiting the growth of adenomatous polyps and causeregression of existing polyps in patients with an inherited syn-drome that predisposes them to colon cancer.54,55 Our study hasled to the approval of celecoxib for adjuvant treatment of familialadenomatous polyposis by the United States Food and Drug Ad-ministration.56 Despite these positive findings, the efficacy andsafety of long-term use of NSAIDs remain a major issue.57 Sinceconventional NSAIDs inhibit both COX-1 and COX-2, prolongeduse of such agents causes severe side effects, including plateletdysfunction, gastrointestinal ulceration, and kidney damage.12,13

Recently developed COX-2-specific inhibitors celecoxib and rofe-coxib have been shown to lower gastrointestinal prostaglandin

levels associated with mucosal protection and have reduced thesevere side effects associated with conventional NSAID use.58

However, the fundamental question about the safety, efficacy forchemoprevention, and prophylaxis needs further evaluation. Onthe other hand, green tea is a nontoxic, regularly consumed,popular beverage, and its major constituent EGCG selectivelyinhibits COX-2 without affecting COX-1 expression. Our presentstudy leads to a possible suggestion that natural nontoxic COX-2inhibitors could be developed as potential chemopreventive ortherapeutic agents. Recent combination studies of green tea com-ponents with the NSAID sulindac has shown synergistic efficacyand reduced adverse effects against colon carcinogenesis inazoxymethane-treated rats.59 Taken together, our results are sig-nificant in demonstrating the selective inhibition of COX-2 byEGCG, along with the inhibition of cancer cell growth, inductionof DNA damage, and apoptosis, further reiterating the remarkableproperties of green tea as a cancer chemopreventive agent forpotential human use.

Acknowledgements

Supported by grants from USPHS (RO1CA78809), AmericanInstitute for Cancer Research (00A30), and Department of Defense(DAMD 17-00-1-0527) to H.M. and funds from Cancer Researchand Prevention Foundation and the O-CHA (Tea) Pioneer Aca-demic Research Grant, Japan to S.G.

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