Lupeol, a dietary triterpene, inhibits Wnt/-catenin - Carcinogenesis

Carcinogenesis vol.30 no.5 pp.808–817, 2009doi:10.1093/carcin/bgp044Advance Access publication February 20, 2009

Lupeol inhibits proliferation of human prostate cancer cells by targeting b-cateninsignaling

Mohammad Saleemy, Imtiyaz Murtazay, RohintonS.Tarapore1, Yewseok Suh, Vaqar Mustafa Adhami,Jeremy James Johnson2, Imtiaz Ahmad Siddiqui, NaghmaKhan, Mohammad Asim, Bilal Bin Hafeez, MohammedTalha Shekhani, Benyi Li3 and Hasan Mukhtar�

Department of Dermatology, University of Wisconsin, 1300 UniversityAvenue, MSC-25B, Madison, WI 53706, USA, 1Molecular andEnvironmental Toxicology Center and 2School of Pharmacy, University ofWisconsin, Madison, WI 53706 and 3Department of Urology, 2045 LiedBiomedical Research Facility, University of Kansas Medical Center, MailStop 3035, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA

�To whom correspondence should be addressed. Tel: þ1 608 263 3927;Fax: þ1 608 263 5223;Email: [email protected]

Lupeol, a dietary triterpene, was shown to decrease serum pros-tate-specific antigen levels and inhibit the tumorigenicity of pros-tate cancer (CaP) cells in vivo. Here, we show that Lupeol inhibitsthe proliferative potential of CaP cells and delineated its mecha-nism of action. Employing a focused microarray of human CaP-associated genes, we found that Lupeol significantly modulates theexpression level of genes such as ERBB2, tissue inhibitor of metal-loproteinases-3, cyclin D1 and matrix metalloproteinase (MMP)-2that are known to be associated with proliferation and survival. Acommon feature of these genes is that all of them are known toeither regulate or act as downstream target of b-catenin signalingthat is highly aberrant in CaP patients. Lupeol treatment signifi-cantly (1) reduced levels of b-catenin in the cytoplasmic andnuclear fractions, (2) modulated expression levels of glycogen syn-thase kinase 3 beta (GSK3b)–axin complex (regulator of b-cateninstability), (3) decreased the expression level and enzymatic activityof MMP-2 (downstream target of b-catenin), (4) reduced the tran-scriptional activation of T Cell Factor (TCF) responsive element(marker forb-catenin signaling) in pTK-TCF-Luc-transfected cellsand (5) decreased the transcriptional activation of MMP-2 gene inpGL2-MMP-2-Luc-transfected cells. Effects of Lupeol treatmenton b-catenin degradation were significantly reduced in CaP cellswhere axin is knocked down through small interfering RNA trans-fection and GSK3b activity is blocked. Collectively, these datasuggest the multitarget efficacy of Lupeol on b-catenin-signalingnetwork thus resulting in the inhibition CaP cell proliferation.We suggest that Lupeol could be developed as an agent for chemo-prevention as well as chemotherapy of human CaP.

Introduction

In recent years, there is an intense activity to identify novel therapeu-tic modalities and preventive approaches for prostate cancer (CaP).Epidemiological and laboratory studies suggest that diet-based natu-rally occurring agents, due to their ability to target multiple signalingpathways, their cost-effectiveness and most importantly their humanacceptability could be ideal candidates for the treatment and preven-tion of human CaP (1–4). At the present time many such agents arebeing investigated in preclinical settings and emerging data with someof the agents in clinical settings is encouraging (3,4). We recentlyshowed that Lupeol [Lup-20(29)-en-3b-ol], a diet-based triterpene

found in fruits such as olive, mango, strawberry, grapes, figs and inseveral medicinal plants activates apoptotic machinery (Fas signalingthat generally is impaired in CaP cells) and inhibits the tumorigenicityof human androgen-sensitive CaP cells with a concomitant decreasein serum prostate-specific antigen levels under in vivo conditions (5).We suggested that Lupeol could be developed as a potential agent forthe treatment of human CaP (5). Recently, Lee et al. (6) showed thatLupeol treatment inhibits head and neck cancer in a mouse tumorxenograft model. In the current study, we provide evidence to showthat Lupeol significantly reduces the proliferative and clonogenicpotential of androgen-sensitive as well as androgen-insensitive CaPcells by modulating b-catenin-signaling pathway.

Materials and methods

Cell culture

Human CaP cells LNCaP and DU145 and fetal bovine serum were obtainedfrom American Type Culture Collection (Manassas, VA). Cells were culturedin appropriate media containing 10% fetal bovine serum supplemented with1% penicillin–streptomycin (Cellgro Mediatech, Herndon, VA).

[3H]-thymidine incorporation assay

Stock solution of Lupeol (Sigma, St Louis, MO) was prepared as describedearlier (5). Cells grown in 24-well cluster plates were subjected to Lupeoltreatment for 48 h, the last 16 h of which was in the presence of [3H]thymidine(0.5 lCi/ml). Cells were then washed twice with cold phosphate-buffered salineand then were incubated with trichloroacetic acid solution on ice for 30 min andsubsequently, the acid-insoluble fraction was dissolved in 1 ml 1 M NaOH.Incorporated [3H]thymidine was quantified by liquid scintillation counting.

Colony formation studies

A total of 0.5% agar was prepared in RPMI containing 20% fetal calf serum(bottom layer). Cells (1 � 105 cell per 100 mm plate) in 20% fetal calf serumand 0.7% agarose (top layer) were plated and incubated at 37�C overnightbefore treatment with Lupeol. The medium was removed and replaced withfresh medium containing Lupeol every 3 days. After 21 days of incubation, thecells were stained with 0.05% Crystal Violet/methanol for 2 h and colonieswere counted in two colony grids using a microscope.

Microarray analysis

LNCaP cells were treated with subtoxic dose (20 lM) of Lupeol. After 48 h ofincubation, cells were harvested and RNA was isolated by using RNeasy kit(Qiagen, Valencia, CA). Next, 4 lg of RNA was enzymatically converted intocomplementary RNA, labeled and hybridized with the microarrays (imprintedwith 288 well-characterized CaP-associated genes) as per vendor’s protocol(Super Array, Frederick, MD) followed by detection with the chemilumines-cent reagents and X-ray film. Data were acquired and analyzed by using GEsuper array software. A cutout point of 2-fold was selected for analysis. Themicroarray experiments were conducted three times independently.

Treatment of cells

For dose-dependent studies, the cells were treated with Lupeol (5–50 lM) for48 h in complete cell medium. To investigate the phosphorylation of b-cateninby Lupeol, CaP cells were treated with Lupeol (20–40 lM) and cells werecollected at 24 and 48 h later. After treatment with Lupeol, the cell lysates wereprepared from harvested cells and stored at �80�C for later use.

Western blot analysis

Western blot analysis was performed as described earlier (5). Antibodies usedin the immunoblotting were procured from Cell Signaling, Danvare, MA [anti-b-catenin, antiphospho-b-catenin, anti-Cdk2, anti-Cyclin D1, anti-cmyc, anti-matrix metalloproteinase (MMP)-2, anti-ERBB2, anti-insulin-like growthfactor (IGF)-1R, anti-glycogen synthase kinase 3 beta (GSK3b), antiphospho-GSK3b and anti-axin], Santa Cruz Biotechnology, Santa Cruz, CA (anti-tissueinhibitor of metalloproteinases (TIMP)-3 and anti-Lamin,) and Sigma (anti-b-actin). Densitometry measurements of the scanned bands were performedusing digitalized scientific software program UN-SCAN-IT (Silk ScientificCorporation, Orem, UT). Data were normalized to loading control.

Abbreviations: BIO, (2#Z,3#E)-6-bromoindirubin-3#-oxime; CaP, prostatecancer; GSK3b, glycogen synthase kinase 3 beta; IGFBP, insulin-like growthfactor binding protein; MMP, matrix metalloproteinase; siRNA, small interfer-ing RNA; TCF, T Cell Factor; TIMP, tissue inhibitor of metalloproteinases.

yThese authors contributed equally to this work.

� The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 808

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GSK3b inhibition studies

CaP cells were pretreated with 0.1 lM of GSK3b inhibitor, (2#Z,3#E)-6-bromoindirubin-3#-oxime (BIO) (Calbiochem, San Diego, CA) for 4 h beforeLupeol treatment (20 lM) for 24 h. Cell lysates were obtained for immunoblotand immunoprecipitation analysis.

Immunoprecipitation

Cell lysates were precleared by adding 1 lg of appropriate control immuno-globulin G (corresponding to the host species of primary antibody) togetherwith 20 ll of resuspended volume of Protein A-Agarose (Santa Cruz Bio-technology) at 4�C for 30 min. Beads were pelleted and cell lysates weretransferred to a fresh centrifuge tube. Equal amounts (200 lg) of cellularprotein from the cell lysates were incubated overnight with 10 ll of specificprimary antibodies (anti-human axin and anti-human GSK3b) at 4�C. Afterovernight incubation, 20 ll of protein A-Agarose was added and the mixturewas incubated for 3 h. Immunoprecipitates were collected by centrifugationand were washed thrice with the cell lysis buffer [50 mM N-2-hydroxyethyl-piperazine-N#-2-ethanesulfonic acid (pH 7.55), 1 mM ethylenediaminetetra-acetic acid, 1 mM dithiothreitol and protease inhibitor cocktail). The finalpellets were dissolved in 20 ll of 2� protein loading buffer. The expressionlevels of proteins (axin and GSK3b) were determined by immunoblot anal-ysis. Further, to confirm the specificity of antibodies (axin and GSK3b im-munoprecipitates) used for immunoprecipitation, a non-specific antibody(anti-cyclin E antibody) was used as a negative control. Cyclin E was im-munoprecipitated in CaP cells and loaded in parallel on sodium dodecylsulfate gels. The specificity of antibodies was confirmed by stripping immu-noblots and reprobing them for Cyclin E (negative control). Finally, 10% ofpost-cleared protein was also loaded as input control on sodium dodecylsulfate Gel. After detecting axin and GSK3b proteins, immunoblots werestripped and reprobed for b-actin to confirm the equal loading.

Small interfering RNA transfection

A total of 2 � 106 cells were transfected with 100 nM of small interferingRNA (siRNA) directed against axin (Santa Cruz Biotechnology). Control cellswere transfected with scrambled siRNA (100 nM). After overnight incubation,transfected cells were treated with Lupeol (20–40 lM) and 24 h later wereharvested and cell lysates were processed.

Transcriptional activity of TCF and MMP

The human MMP-2 promoter luciferase plasmid (pGL2-MMP-2-luc) was de-veloped as described earlier (7). pTK-TCF-Luc (TopFlash and FopFlash) wasprocured from Upstate Laboratories (Lake Placid, NY). Cells were transfectedwith the plasmids (200 ng per well) for 24 h. Renilla luciferase (20 ng per well,pRL-TK; Promega, Madison, WI) was used as an internal control. In addition,for controls, the same amount of empty vectors were transfected in cells. After12 h post-transfection, fresh media was added with Lupeol (5–10 lM) andincubated for 24 h. The cells were then harvested and transcriptional activitywas measured in terms of luciferase activity in quadruplicates by using dual-luciferase reporter assay system (Promega). Relative luciferase activity wascalculated with the values from vector alone group with or without Lupeol-treated group. To confirm the effect of Lupeol treatment on the general tran-scriptional machinery, CaP cells were transfected with 50 ng per well Renillaluciferase plasmid (pRL-TK). The transcriptional activity was measured ina dose- and time-dependent manner.

Gelatin zymography

Equal number of cells (1 � 106) were seeded in the plates and after 24 h weretreated with Lupeol. After 48 h post-treatment, the conditioned media wereharvested, concentrated and electrophoresed (10 lg protein) under non-reducing conditions. The gelatinolytic activity of MMP-2 was determined byemploying zymography kit (Invitrogen, Carlsbad, CA) as per vendor’s protocol.

Statistical analyses

Student’s t-test for independent analysis was applied to evaluate differencesbetween the treated and untreated cells with respect to the expression of var-ious proteins using S-plus Software (Insightful, Seattle, WA). A P-value of,0.05 was considered to be statistically significant.

Results

Effect of Lupeol on androgen-sensitive CaP cell proliferation

Recently, we showed significant growth inhibitory effects of Lupeol(5–30 lM) on androgen-sensitive CaP cells without producing anyeffect on the viability of normal prostate epithelial cells (5). It is wellknown that proliferating cells exhibit increased thymidine incorpora-tion into DNA that arises from increased growth factor expression and

activity in cancer cells (8). We investigated the effect of Lupeol treat-ment on the rate of proliferation of androgen-sensitive LNCaP cells bymeasuring the rate of uptake of [3H]-thymidine by dividing cells.Lupeol treatment caused a significant decrease of [3H]-thymidineuptake by LNCaP cells suggesting the antiproliferative potential ofLupeol (Figure 1A).

Effect of Lupeol treatment on the clonogenic potential of androgen-sensitive CaP cells

Next, we asked whether treatment with Lupeol could exert greateractivity on the formation of colonies, which allows an investigationover a longer period of time and that mimics cellular physiologyin vivo. We observed that the colony-forming ability of LNCaP cellswas inhibited by 20–50% (P , 0.05), after treatment with Lupeol(Figure 1B). Interestingly, even after 21 days, the colony formationwas significantly decreased in Lupeol-treated cells as compared withcontrol that exhibited numerous colonies (Figure 1B). These datasuggest that the sustained effect of Lupeol on growth-promotingand proliferative property of androgen-sensitive CaP cells.

Effect of Lupeol treatment on expression of CaP-associated genes

To define the mechanism through which Lupeol inhibits or reducesthe proliferation of CaP cells, we employed a focused microarray.The effect of subtoxic dose of Lupeol (20 lM) treatment on 288well-characterized human CaP-associated genes was assessed. Acutout point of 2-fold was selected for analysis. Lupeol treatmentwas observed to modulate the messenger RNA expression level ofseveral genes (Table I). Only those genes are represented in Table Ithat exhibit changes of 2- or .2-fold in their expression. We ob-served that Lupeol treatment significantly decreases the messengerRNA level of several genes associated with the growth, survival andproliferation of CaP cells. The prominent proliferation-associatedgenes whose expression was observed to be downregulated byLupeol treatment are androgen receptor, IGF-1R, Cyclin D1, myc,MMP-2, Cdk2, Jun, nuclear factor kappa B1 and ERBB2 (Table I).In addition, Lupeol treatment was observed to upregulate the ex-pression of several genes that are known to negatively regulate thesurvival and proliferation of human CaP cells (9–11). These includeTIMP-3, KLK10, six-transmembrane epithelial antigen of the pros-tate (STEAP)-1 and insulin-like growth factor binding protein(IGFBP)-6 (Table I). Lupeol was also observed to induce the ex-pression of TP53, RB1, RASSF1, MAPK12, PAR-4, PTEN, AGTR2and CD82, genes that are known to negatively regulate the prolifer-ation; however, the fold changes were ,2-fold.

To further validate the microarray data at translational level, wetreated LNCaP cells with Lupeol at the doses of 5–30 lM for 48 h.These doses were selected on the basis of our published data (5). Toinvestigate whether the modulations induced by Lupeol occurs attranslational level, we performed the immunoblot analysis of pro-liferation-associated proteins (Cdk2, c-myc, IGF-1R, ERBB2/HER2and TIMP3) selected from the Table I. We observed that Lupeoltreatment significantly decreased protein levels of in Cdk2, c-myc,IGF-1R and ERBB2 in LNCaP cells (Figure 1C). Lupeol was alsoobserved to increase the level of TIMP-3 protein in cells in a dose-dependent manner (Figure 1D). It is well known that IGF-1R be-comes activated after phosphorylation and the activated IGF-1Rrelays the tumorigenic signals in CaP cells (12,13). Next, we eval-uated the phosphorylation of IGF-1R protein and observed thatLupeol treatment significantly decreased the phosphorylation ofIGF-1R (Figure 1C). These data suggest that Lupeol-induced mod-ulations observed at messenger RNA level corroborated with trans-lational level changes in CaP cells.

Effect of Lupeol treatment on the expression level of b-cateninsignaling in androgen-sensitive CaP cells

Lupeol was observed to induce modulation in the expression level ofIGF-1R, ERBB2, TIMP3, cyclin D1 and MMP-2 (Table I) and it isnoteworthy that the common feature shared by all of these genes is

Lupeol inhibits b-catenin signaling in prostate cancer cells

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Fig. 1. Effect of Lupeol on proliferation, clonogenic potential, the expression level of proliferation-associated proteins and b-catenin signaling in LNCaP cells.(A) Histogram showing the rate of [3H]thymidine uptake in LNCaP cells treated with Lupeol. Cells were subjected to Lupeol treatment for 48 h, the last 16 h ofwhich in the presence of [3H]thymidine (0.5 lCi/ml). Incorporated [3H]thymidine was quantified by liquid scintillation counting as described under Materials andMethods. Each bar in the histogram represents mean ± standard error, ‘�’ indicates P , 0.05. All experiments were repeated three times with similar results. (B)Histogram showing number of colonies formed by LNCaP cells treated with Lupeol. Cells seeded in agarose and incubated at 37�C were treated with Lupeol asdescribed under Materials and Methods. After 21 days of incubation, the cells were stained with crystal violet–methanol and colonies were counted. Each bar in thehistogram represents mean ± standard error, � indicates P , 0.05. All experiments were repeated three times with similar results. (C–E) Immunoblots representthe effect of Lupeol treatment on the protein level of Cdk2, c-myc, ERBB2, IGF-1R, TIMP3, b-catenin (total, cytosolic and nuclear), phosphorylated-IGF-1R,GSK3b and axin in LNCaP cells. Cells were treated with vehicle (dimethyl sulfoxide þ alcohol) only or specified concentrations of Lupeol for 48 h. (F)Immunoblots represent the effect of Lupeol on the phosphorylation of b-catenin in LNCaP cells at 24 h post-treatment. Cells were treated with vehicle (dimethylsulfoxide þ alcohol) only or specified concentrations of Lupeol for 24 h and harvested (Figure 1C–F). Proteins levels in these experiments were determined bywestern blot analysis by using specific antibody. The immunoblots shown here are representative of three independent experiments with similar results. The detailsare described under Material and Methods. ‘V’ represents vehicle. Values above the immunoblots represent relative densities (in terms of fold units) of the bandsnormalized to b-actin. Equal loading was confirmed by stripping immunoblots and reprobing them for b-actin (for total and cytosolic fractions) and Lamin (fornuclear fraction).

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that these are directly or indirectly associated with b-catenin-signal-ing pathway (14–16). These genes are known to either regulate or actas downstream targets of b-catenin signaling (14–16). For example,IGF-1R is known to regulate the location, stability and transcriptionalactivity of b-catenin in cancer cells (17). Similarly, ERBB2/HER2 isreported to induce the expression of cyclin D1 whose expression isprimarily known to be regulated by b-catenin (18). Further, b-cateninsignaling is also reported to be increased in cells lacking TIMP-3suggesting the regulatory action of TIMP-3 on b-catenin signaling(15). Compared with normal prostate epithelial cells, CaP cells havebeen reported to express high b-catenin protein levels (19). Stabilizedb-catenin accumulates in the cytoplasm and translocates to the nu-cleus to induce transcriptional activation of proliferation-associatedgenes leading to increased proliferation of epithelial cells (20,21).Abnormal cytoplasmic/nuclear b-catenin expression has been re-ported to be associated with high Gleason scores in CaP patients(22). Since Lupeol was observed to modulate the expression of pro-liferation-associated genes (having direct or indirect associationwith b-catenin signaling), we next evaluated the effect of Lupeol onb-catenin signaling in CaP cells. Lupeol treatment was observed tosignificantly decrease the protein level of whole and cytoplasmicb-catenin protein in LNCaP cells in a dose-dependent manner (Figure1E). We also observed that Lupeol treatment significantly decreasesnuclear b-catenin level in LNCaP cells (Figure 1E). As evident fromthe densitometric analysis of immunoblots, Lupeol-induced b-catenindegradation was significant in LNCaP cells (Figure 1E).

Degradation of b-catenin in cells is known to be preceded by itsphosphorylation. We next investigated the effect of Lupeol treatmenton the phosphorylation of b-catenin in a time-dependent manner. Asevident from the densitometric analysis, Lupeol treatment was ob-served to increase the phosphorylation of b-catenin at 24 h post-treatment (Figure 1F). At 48 h time point, Lupeol was observed todecrease the total pool of b-catenin (including phosphorylated, total,

cytosolic and nuclear b-catenin) in CaP cells (Figure 1E). These datasuggest that Lupeol treatment initiates the molecular events very early(24 h post-treatment) that ultimately result in the loss of b-cateninlevels (Figure 1E).

GSK3b and axin are known to phosphorylate b-catenin proteinleading to its degradation by proteasomes; thus, act as upstream reg-ulators of b-catenin signaling (23,24). Increased phosphorylation ofGSK3b is known to render b-catenin molecule defective and GSK3band axin expressions are reported to be defective in CaP cells (23–25).Next, we evaluated the effect of Lupeol treatment on GSK3b and axinprotein levels. Lupeol treatment was observed to increase the GSK3bprotein level in cells; however, marginal change in the phosphorylationof GSK3b was observed at the dose of 30 lM (Figure 2A). Further, todetermine the role of GSK3b in Lupeol-induced b-catenin degradation,LNCaP cells were pretreated with the GSK3b inhibitor, BIO (0.1 lM),for 4 h prior to Lupeol treatment and cells were analyzed for b-cateninprotein level. Inhibition of GSK3b by BIO prevented Lupeol-inducedphosphorylation of b-catenin protein in LNCaP cells (Figure 2B).These findings further support the role of GSK3b activation inLupeol-induced b-catenin degradation in CaP cells.

The degradation of b-catenin depends on b-catenin phosphoryla-tion, which occurs in a multiprotein complex containing axin, GSK3band b-catenin. It is believed that in this complex, GSK3b phosphor-ylates the b-catenin primarily when it is bound to axin. Lupeol treat-ment was observed to increase the expression level of axin protein incells (Figure 2A). To determine the effect of Lupeol treatment on thelevel of axin and GSK3b when in complex form, immunoprecipitationand immunoblot analyses were performed. As shown in Figure 2C,treatment of LNCaP cells with Lupeol induced the levels of axin andGSK3b in their complex thus increasing the possibility of b-catenindegradation in LNCaP cells.

To confirm that Lupeol-induced degradation of b-catenin is throughthe induction of axin protein, we investigated the effect of Lupeol

Table I. List of selected genes modulated by Lupeol treatment of CaP cells

Gene symbol Fold change Function Potential implications in CaP pathogenesis

DownregulatedAIG1 30.0 Integral to membrane Cell proliferationCyclin D1 2.5 Cell cycle, cell growth and/or maintenance Cell proliferationCDK2 22.0 Cell cycle; G2/M transition, kinase activity Cell proliferationCDK4 9.0 Regulation of cell cycle; protein kinase activity Cell proliferationCDK5 37.0 Transferase and kinase activity; protein phosphorylation kinase activity; cell cycle Cell proliferationCDK9 14.0 Transcription elongation factor Cell proliferationCLDN 3 90.0 Transmembrane receptor activity; tight junction InvasionERBB2 20.0 Kinase activity, phosphorylation; receptor activity Cell proliferation, tumorigenesisEGR3 12.0 Transcription factor activity, regulation of transcription, Cell proliferationESR1 30.0 Signal transduction; transcription factor and receptor activity Cell survival and proliferationHIP1 2.0 Cytoskeleton; actin binding; phospholipid binding Cell proliferationIGF2 2.0 Cell cycle; growth factor and activity; signaling cascade Cell proliferation, invasionIGFR1 30.0 Phosphorylation; signal transduction; activity Antiapoptosis; cell proliferationIL12A 2.5 Inflammatory response; G-protein signaling: chemokine receptor activity Inflammation, cell growthILK 58.0 Phosphorylation; transferase activity; cell-matrix adhesion; signaling cascade Cell proliferation, invasionJUN 15.0 Transcription factor activity, RNA pol II transcription factor activity Cell survival, tumorigenesisKLK1 2.6 Proteolysis; chymotrypsin activity; trypsin activity; tissue kallikrein activity Survival, invasion, antiapoptosis,MMP2 3.0 Hydrolase activity; extracellular matrix; gelatinase A activity MetastasisMYC 15.0 Transcription factor activity; regulation of transcription from Pol II promoter Cell proliferation; tumorigenesisNFjB1 45.0 Signal transduction; nucleus; protein binding; transcription factor activity Survival, invasion, chemoresistanceNR1H2 8.5 Transcription factor activity; steriod hormone receptor activity Survival, invasion, chemoresistanceNRIH3 2.0 Transcription factor and coactivator activity; steroid hormone receptor activity Survival, invasion, chemoresistanceNR2C2 2.0 Transcription factor and coactivator activity; steroid hormone receptor activity Survival, invasion, chemoresistancePCNA 8.0 Regulation of cell cycle; DNA replication; DNA repair Cell proliferationPI3KCG 2.0 Transferase activity; signaling transduction; kinase activity Cell survival, chemoresistance

UpregulatedSTEAP1 6.0 Intercellular junction; transporter activity; vesicular fraction Invasion inhibitionKLK10 2.0 Hydrolase activity; chymotrypsin activity Inhibition of turmor growth and invasionIGFBP6 2.0 Signal transduction Tumor growth inhibitionPLG 6.3 Hydrolase activity; plasmin activity Tumor growth inhibitionTGFB1I1 41.0 Adhesion; transcription coactivator: AR binding Tumor growth inhibitionTIMP3 2.0 Extracellular matrix; metalloendopeptidase inhibitor activity Induces apoptosis, inhibits invasion


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Fig. 2. Effect of Lupeol treatment on the upstream regulators and downstream targets of b-catenin in CaP cells. (A) Immunoblots represent the effect of Lupeoltreatment on the expression level of GSK3b, axin and the phosphorylation of GSK3b protein in LNCaP cells. Cells were treated with vehicle (dimethyl sulfoxide þalcohol) only or specified concentrations of Lupeol for 48 h. The expression level of GSK3b, axin and phosphorylated GSK3b were determined by western blotanalysis. Equal loading was confirmed by stripping immunoblots and reprobing them for b-actin. (B) Immunoblots represent the effect of Lupeol on thephosphorylation of b-catenin in LNCaP cells at 24 h post-treatment in presence of GSK3b inhibitor (BIO). CaP cells were pretreated with 0.1 lM of BIO for 4 hbefore Lupeol treatment (20 lM) for 24 h. The expression of phosphorylated b-catenin was determined by western blot analysis in total cell lysates. Equal loadingwas confirmed by stripping immunoblots and reprobing them for b-actin. The immunoblots shown here (Figure 2A and B) are representative of three independentexperiments with similar results. The details are described under Materials and Methods. ‘V’ represents vehicle. Values above the immunoblots represent relativedensities (in terms of fold units) of the bands normalized to b-actin. (C) Immunoblots represent the effect of Lupeol on the GSK3b and axin complex in LNCaPcells. LNCaP cells were treated with the specified concentrations of Lupeol (20 lM) for 48 h. Cells were harvested and total cell lysates were processed for theimmunoprecipitation analysis as described under Materials and Methods. V represents vehicle. (D) Immunoblots represent the effect of Lupeol on the GSK3b andaxin complex in axin-silenced LNCaP cells. Cells transfected with 100 nM of siRNA directed against axin and scrambled siRNA (100 nM) were treated withLupeol and vehicle alone. Cells were harvested after 24 h post-Lupeol treatment and cell lysates were processed for immunoprecipitation analysis as describedunder Materials and Methods. The expression of GSK3b and axin proteins in the immunocomplex was determined by western blot analysis. Input control consistedof 10% of post-cleared protein. After detecting axin protein in input samples, immunoblots were stripped and reprobed for b-actin to confirm the equal loading.The immunoblots shown here are representative of three independent experiments with similar results. V represents vehicle-treated cells. (E) Histogram representsthe effect of Lupeol treatment on the transcriptional activation of TCF (marker of b-catenin activity) in LNCaP cells. pTK-TCF-Luc (pTopFlash)-transfectedLNCaP cells were treated with Lupeol and the transcriptional activity was measured as described under Materials and Methods. pFopFlash was used as a negativecontrol and Renilla luciferase was used as an internal control. Relative luciferase activity was calculated with the values from vector alone group with or withoutLupeol-treated group. (F) Immunoblots represent the effect of Lupeol treatment on the expression level of Cyclin D1 and MMP-2 proteins. Cells were treated withvehicle (dimethyl sulfoxide þ alcohol) only or specified concentrations of Lupeol and the expression of Cyclin D1 and MMP-2 were determined in whole-celllysates. Equal loading was confirmed by stripping immunoblots and reprobing them for b-actin. The immunoblots shown here are representative of threeindependent experiments with similar results. ‘C’ represents untreated cells and V represents vehicle-treated cells. The values above the immunoblots representrelative densities (in terms of fold units) of the bands normalized to b-actin. (G) Histogram represents the effect of Lupeol treatment on the transcriptionalactivation of MMP-2 in LNCaP cells. pGL2-MMP-2-luc-transfected LNCaP cells were treated with Lupeol and the transcriptional activity was measured in termsof luciferase activity as described under Materials and Methods. Relative luciferase activity was calculated with the values from vector alone group with or withoutLupeol-treated group. (H) Histogram represents the effect of Lupeol treatment on Renilla luciferase activity in LNCaP cells. LNCaP cells were transfected withpRLTK-luc plasmid (50 ng) and treated with Lupeol. After 24 h of Lupeol treatment, the transcriptional activity was measured as described under Materials andMethods. Data are presented as percent luciferase activity. Each bar in the histogram represents mean ± standard error. (I) Image represents the gelatinolytic effectof LNCaP cells treated with Lupeol. The gelatinolytic activity of MMP-2 was determined in conditioned media harvested from treated cells by employingzymography as described under Materials and Methods.

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treatment on the axin–GSK3b complex in LNCaP cells transfectedwith axin-targeted siRNA. As evident from immunoprecipitationanalysis in axin–GSK3b complex in axin-suppressed cells, the levelof axin protein was significantly reduced (Figure 2D). However, it isnoteworthy that such cells when were treated with Lupeol exhibitedincreased axin protein levels in axin–GSK3b complex (Figure 2D).These data suggest that Lupeol destruction of b-catenin in human CaPcells is through the induction of axin protein level in axin–GSK3bcomplex.b-Catenin acts to regulate the transcription of genes through the

binding of a complex of b-catenin and T Cell Factor (Tcf) family oftranscription factors to specific promoter elements (21,26). The de-crease of nuclear b-catenin by Lupeol treatment suggested thatb-catenin nuclear signaling might have been attenuated (Figure 1B).Next, we evaluated the effect of Lupeol treatment on the transcrip-tional activity of Tcf by transiently transfecting LNCaP cells withreporter plasmids bearing Tcf-4-binding sequences. At 24 h, Lupeolat 5 and 10 lM inhibited Tcf transcriptional activity by 50% (Figure2E). The specificity of the Lupeol effect on Tcf reporters was con-firmed by the fact that pFopflash (containing a ‘far from optimal’ Tcfbinding site) was not influenced by Lupeol (Figure 2E).b-Catenin is known to relieve the inhibition of TCF/lymphoid

enhancer factor by repressors leading to transcriptional activationof target genes, such as c-myc, MMP-7, MMP-2 and cyclin D1(21,26,27). Overexpression of b-catenin has been implicated in celltransformation and correlated with increased levels of Cyclin D1,MMP-2 and c-myc in human CaP cells (20,28–30). Next, we de-termined the effect of Lupeol treatment to cells on the protein levelsof Cyclin D1 and MMP-2. Lupeol treatment caused a significantdecrease in the Cyclin D1 and MMP-2 protein levels in LNCaP cells(Figure 2F). We also determined the effect of Lupeol treatment onthe transcriptional activation of MMP-2 gene. Lupeol (5 lM) sig-nificantly decreased MMP-2 transcriptional activation at 24 h post-treatment in cells (Figure 2G). To rule out the possibility that theeffect of Lupeol is devoid of non-specific effect on general transcrip-tion, we transfected cells with 50 ng of pRLTK-luc plasmid andinvestigated its effect on Renilla in time-dependent manner inLNCaP cells. Lupeol exhibited no significant effect on Renillaluciferase activity (Figure 2H). MMP-2 is known to degrade theextracellular matrix when secreted by CaP cells in tissues. MMP-2protein secreted by cells is known to exhibit gelatinolytic activityunder in vitro conditions when analyzed by standard zymography.We observed that Lupeol treatment significantly decreases the gelat-inolytic (enzymatic) activity of MMP-2 protein in LNCaP cells(Figure 2I).

Effect of Lupeol on b-catenin-signaling network in androgen-insensitive CaP cells

CaP disease in humans at the time of diagnosis is known to exhibita heterogeneous system comprising of epithelial cells with differenttypes of androgen status such as androgen-sensitive and androgen-insensitive cells. Since Lupeol was observed to inhibit growth andproliferation of androgen-sensitive CaP cells, we asked whether thiseffect of Lupeol is universal for all types of CaP cells. For this pur-pose, androgen-insensitive CaP cells DU145 were treated withLupeol. These doses were selected on the basis of cell viability assay,where Lupeol (10–50 lM) was observed to inhibit the growth ofandrogen-insensitive cells (DU145 and PC-3) at 48 h without anyadverse effect of normal prostate epithelial cells at these doses (datanot shown). Lupeol treatment was observed to decrease the rate ofproliferation of DU145 cells in a dose-dependent manner as wasassessed by thymidine incorporation assay (Figure 3A). In addition,prolonged Lupeol treatment (for 21 days) was also observed to sig-nificantly inhibit the colony formation ability of DU145 (Figure 3B).

Lupeol was observed to decrease the level of Cdk2, c-myc andERBB2 proteins in DU145 cells (Figure 3C). Interestingly, Lupeoldid not induce any modulation on total protein and phosphorylationlevel of IGF-1R in these cells (data not shown). Furthermore, Lupeoltreatment caused a significant increase in the expression level of

TIMP-3 protein in cells (Figure 3C). Next, we determined the effectof Lupeol treatment on b-catenin-signaling pathway in DU145cells. Lupeol treatment was observed to cause a decrease in total,cytoplasmic and nuclear b-catenin levels in DU145 cells (Figure3D). Lupeol was observed to significantly induce the phosphorylationof b-catenin in DU145 cells at 24 h time point in a dose-dependentmanner (Figure 4A).

Further, Lupeol treatment was observed to cause an increase in theexpression level of axin protein with a peak effect at 10–20 lM(Figure 4B). Lupeol treatment caused a moderate decrease in thephosphorylation of GSK3b protein; however, no effect was observedon the total GSK3b protein levels in DU145 cells (Figure 4A). Effectsof Lupeol treatment on the phosphorylation and degradation ofb-catenin were minimal in BIO-pretreated DU145 cells suggestingthe involvement of GSK3b in the Lupeol-induced effects (Figure 4C).

Next, we determined whether Lupeol has similar effect on GSK3band axin levels in complex in DU145 cells as was observed in andro-gen-sensitive LNCaP cells. Treatment of DU145 cells with Lupeolinduced the levels of axin–GSK3b complex formation (Figure 4D). Incontrast, effect of Lupeol on GSK3b–axin complex in axin-silencedDU145 cells was only marginal (Figure 4E). Next, we determinedeffect of Lupeol treatment on the downstream targets of b-catenin.Lupeol treatment decreased the TCF promoter activity in DU145cells (Figure 5A). Lupeol treatment also caused a decrease in thecyclin D1 and MMP-2 protein levels in cells (Figure 5B). Finally,Lupeol treatment resulted in decreased enzymatic activity of MMP-2 protein and transcriptional activation of MMP-2 gene in DU145cells (Figure 5C and D).

Discussion

One difficulty in successfully treating CaP is that lesions responddifferently to the treatment due to presence of a mixture of CaP cellscomprising androgen-dependent and androgen-independent cellsand the key to control of CaP seems to lie in the elimination of bothtypes of CaP cells (without affecting the normal cells) (31). Re-cently, we showed that Lupeol induces apoptosis of LNCaP cellswithout any adverse effects on the viability of normal prostate epi-thelial cells (5). One major finding of this study is that Lupeol, a diet-based agent, caused inhibition in the growth and proliferation ofhuman CaP cells irrespective of their androgen status. It is believedthat the tumor cells bear the activation of proliferation-associatedgenes that directly or indirectly cross talk with b-catenin signalingforming a signaling network allowing CaP cells to survive, prolif-erate and acquire highly aggressive androgen-independent charac-teristics even after surgery and androgen ablation therapy (32).There exists a possibility that impediment of CaP may be addressedby targeting the b-catenin-associated signaling network that is im-paired in CaP cells (33,34). Since lesions exhibit the activation ofmultiple signaling pathways, agents that target a single signalingpathway do not seem to be adequate in combating CaP. Based onour previous published data and the current study, we suggest thatLupeol could be such a multitarget agent. Lupeol is reported to in-hibit the growth and proliferation of highly aggressive pancreaticcancer and melanoma cells and inhibit the skin tumorigenesis ina mouse model through the modulation of signaling pathways suchas PI3K/Akt, nuclear factor kappa B1 and Ras/PKCa (35–37).Lupeol is reported to exhibit various beneficial pharmacologicalactivities under in vitro and in vivo conditions (5,35–37). In thecurrent study, we provide evidence that Lupeol adopts a strategyto target b-catenin-signaling network thus inhibiting the prolifera-tion of human CaP cells of both androgen-dependent and androgen-independent nature.

Our data suggest that Lupeol modulates several signaling pathwaysin CaP cells. All the genes whose expression is modulated by Lupeoltreatment (as listed in Table I) are known to be associated with theproliferation and/or survival of cancer cells. Our data is significantbecause Lupeol treatment decreases the expression of nuclear factorkappa B1 and tumor necrosis factor suggesting its potential against


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the inflammatory processes that is a common feature in CaP patients(38). ERBB2/HER2 is reported to be overexpressed in CaP patientsand preclinical and clinical data show that the activation of theHER2-kinase axis is important for the progression of CaP to andro-gen-independent disease (39). Multiple pharmaceutical agents thatblock the ERBB2/HER2-kinase axis are currently being evaluated inpatients with CaP (39). ERBB2 stimulation has been shown to acti-vate androgen receptor in a ligand-independent fashion in CaP cells(40). It is noteworthy that Lupeol was observed to significantly de-crease the expression level of ERBB2 in CaP cells (Table I andFigures 1 and 3). IGF-1R has been suggested to play an importantrole in the early androgen-dependent stages of CaP (12,13,41). Ourdata are significant as Lupeol was also observed to decrease theexpression level and phosphorylation of IGF-1R in LNCaP cells thatrepresents the androgen-dependent phase of CaP (Figure 1C).

IGFBP6 has been reported to play an important role during the pro-gression of CaP (11). IGFBP6 is an inhibitor of IGF-II and excessIGFBP6 has been reported to displace IGF-II from IGFBP2; thus,preventing it from potentiating the mitogenic action of IGF-II (42).Various studies have shown that increasing IGFBP6 level results inthe decreased viability of human CaP cells; hence, IGFB6 has beensuggested as a potential therapeutic target for human CaP treatment(43). Our data are significant because the transcriptional level ofIGFBP6 was observed to be sharply increased by Lupeol treatmentin CaP cells (Table I).

MMP-2 and myc proteins are highly expressed in CaP patientsand are reported to be associated with survival, androgen indepen-dence, angiogenesis and invasion of CaP cells (22,29,30). A posi-tive correlation has been shown to exist between MMP expressionlevels and Gleason score in CaP patients and c-myc was the first

Fig. 3. Effect of Lupeol on (A) proliferation of androgen-insensitive DU145 cells, (B) clonogenic potential of DU145 cells, (C) the expression level of prominentproliferation-associated proteins and (D) on b-catenin signaling in DU145 cells. (A) Histogram showing the rate of [3H]thymidine uptake in DU145 cells treatedwith Lupeol. DU145 cells were treated with Lupeol in the presence of [3H]thymidine and [3H]thymidine incorporation was quantified as described under Materialsand Methods. Each bar in the histogram represents mean ± standard error, ‘�’ indicates P , 0.05. All experiments were repeated three times with similar results.(B) Histogram showing number of colonies formed by DU145 cells treated with Lupeol. Cells seeded in agarose and incubated at 37�C were treated with Lupeol asdescribed under Materials and Methods. After 21 days of incubation, the cells were stained with crystal violet–methanol and colonies were counted. Each bar in thehistogram represents mean ± standard error, � indicates P , 0.05. All experiments were repeated three times with similar results. (C and D) Immunoblotsrepresent the effect of Lupeol treatment on the expression level of Cdk2, c-myc, ERBB2, TIMP3, b-catenin (total, cytosolic and nuclear), GSK3b and axin proteinsin DU145 cells. Cells were treated with vehicle (dimethyl sulfoxide þ alcohol) only or specified concentrations of Lupeol and the expression levels of proteinswere determined by western blot analysis. Equal loading was confirmed by probing the immunoblots for b-actin and Lamin. The immunoblots shown here arerepresentative of three independent experiments with similar results. ‘C’ represents untreated control and V represents vehicle. The values above the immunoblotsrepresent relative densities (in terms of fold units) of the bands normalized to b-actin.

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oncogene to be recognized as being overexpressed in human CaP(29,30,44). The proliferation pathway is known to be mediated bythe ability of c-myc to activate several cyclins, including cyclin Eand cyclin D2 (44). Interestingly, Lupeol treatment was observed todecrease the expression level of myc both at transcriptional as wellas translational level (Figures 1 and 3). We also found that Lupeol

significantly decreased the expression of MMP-2 both at transcrip-tional and translational level in CaP cells (Figures 2 and 5). MMPproteins possessing proteolytic properties when secreted in the ex-tracellular vicinity are known to exert angiogenic and metastaticproperties to the CaP cells, and Lupeol treatment was observed todecrease the proteolytic activity of secreted MMP-2 protein

Fig. 4. Effect of Lupeol on the phosphorylation of b-catenin and on GSK3b–axin complex in DU145 cells. (A) Immunoblots represent the effect of Lupeol on thephosphorylation of b-catenin in DU145 cells at 24 h post-treatment. Cells were treated with vehicle (dimethyl sulfoxide þ alcohol) only or specifiedconcentrations of Lupeol and the expression of phosphorylated b-catenin was determined by western blot analysis in whole-cell lysates. (B) Immunoblotsrepresent the effect of Lupeol treatment on the expression level of GSK3b, axin and on the phosphorylation of GSK3b protein in DU145 cells. Cells were treatedwith vehicle only or specified concentrations of Lupeol and the expression of GSK3b, axin and phosphorylated GSK3b were determined by western blot analysis.(C) Immunoblots represent the effect of Lupeol on the phosphorylation of b-catenin in DU145 cells at 24 h post-treatment in presence of GSK3b inhibitor (BIO).Cells treated with BIO and Lupeol as described under Materials and Methods and the expression of phosphorylated b-catenin was determined by western blotanalysis. The immunoblots (Figure 5A–C) are representative of three independent experiments with similar results. Equal loading of proteins was confirmed bystripping immunoblots and reprobing them for b-actin. ‘V’ represents vehicle-treated cells. The values above the immunoblots represent relative densities (in termsof fold units) of the bands normalized to b-actin. (D) Immunoblots represent the effect of Lupeol on GSK3b and axin complex in DU145 cells. DU145 cells weretreated with the specified concentrations of Lupeol for 48 h and were processed for the immunoprecipitation analysis as described under Materials and Methods.(E) Immunoblots represent the effect of Lupeol on the GSK3b and axin complex in axin-silenced DU145 cells. Axin-silenced DU145 cells were treated withLupeol and immunoprecipitation analysis was performed as described under Materials and Methods. Input control consisted of 10% of post-cleared protein. Afterdetecting axin protein in input samples, immunoblots were stripped and reprobed for b-actin to confirm the equal loading. The immunoblots shown here arerepresentative of three independent experiments with similar results. (D and E) V represents vehicle-treated cells, ‘WB’ represents western blot and ‘IP’ representsimmunoprecipitation.


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suggesting the efficacy of Lupeol against the spread of CaP cells(Figures 2 and 5).

The common feature shared by genes including MMP-2, myc,Cyclin D1, IGFBP6, IGF-1R, IGF2 and PI3K (Table I) is that theseare directly or indirectly associated with b-catenin-signaling path-way (16). Various studies have shown that alterations in theb-catenin pathway may contribute to progression of CaP to androgenindependence (32). Recent studies have shown that �20–40% ofhormone-refractory CaP samples exhibit nuclear localization ofb-catenin (ref. 47 and references therein). Activation of the b-cateninpathway has been observed in CaP patients and a recent studyshowed that 32% of CaP patients with advanced disease carriedmutations in b-catenin gene (45). Oncogenic activation of theb-catenin-signaling pathway has been reported to result in the ab-normal accumulation of b-catenin. The translocation of b-catenin–TCF-4 complex to nucleus leads to transcriptional activation oftarget genes, such as c-myc, MMP-2 and cyclin D1 (29,30,33).Various reports suggest that b-catenin signaling cross talks withand is influenced by the alterations in the signaling pattern of growthfactor receptors during the tumorigenesis process (36,37). It isnoteworthy that in the current study, Lupeol was observed torestore the levels of active GSK3b–axin protein complex in thecytoplasm thus decreasing the stabilized b-catenin at upstream levelof b-catenin signaling in both types of CaP cells (Figures 1 and 3).Lupeol treatment was also shown to decrease the level of b-cateninin nuclei of CaP cells thus inhibiting the transcription ofproliferation-associated genes in CaP cells (Figures 1 and 3). Thesedata further strengthen the suggestion that Lupeol is a potentinhibitor of b-catenin-signaling pathway in CaP cells.

To conclude, the multifaceted nature of the Lupeol against theb-catenin-signaling network that is involved in the proliferation andsurvival is probably to have numerous beneficial effects against thedevelopment, growth and progression of early (androgen dependent)as well as advanced stage (androgen independent) CaP in humans.Taken together, our present findings demonstrate the anticancerefficacy of Lupeol, with mechanistic rationale, against androgen-sensitive as well as androgen-insensitive human CaP cells. Theseobservations warrant further in vivo efficacy studies in models thatmimic progressive forms of human prostatic disease. The positiveoutcomes of such an in vivo study could form a strong basis for thedevelopment of Lupeol as a novel agent for human CaP preventionand/or intervention.

Funding

USA PHS (R03 CA130064 to Mohammad Saleem, Bhat, RO1 CA78809, RO1 CA 101039, P50DK 65303 to H.M.). Department ofBiotechnology, Ministry of Science and Technology, Government ofIndia (BT/IN/BTON/Nich/09/2007 to I.M.).

Fig. 5. Effect of Lupeol treatment on the (A) transcriptional activation TCFpromoter, (B) expression level of Cyclin D1 and matrix mettaloproteinase-2(MMP-2) proteins, (C) transcriptional activation MMP-2 gene and (D) on theenzyme activity of MMP-2 protein in DU145 cells. (A) Histogram representsthe effect of Lupeol treatment on the transcriptional activation of TCF(marker of b-catenin activity) in DU145 cells. pTK-TCF-Luc (pTopFlash)-transfected DU145 cells were treated with Lupeol as described underMaterials and Methods. pFopFlash and Renilla luciferase were used asnegative and internal control, respectively. For controls, the same amount ofempty vectors were transfected in cells. (B) Immunoblots represents theeffect of Lupeol treatment on the expression level of Cyclin D1 and MMP-2

proteins. DU145 cells were treated with vehicle (dimethyl sulfoxide þalcohol) only or specified concentrations of Lupeol and expression of CyclinD1 and MMP-2 proteins were determined by western blot analysis. Equalloading was confirmed by stripping immunoblots and reprobing them forb-actin. The immunoblots shown here are representative of threeindependent experiments with similar results. ‘C’ represents untreatedcontrol and ‘V’ represents vehicle-treated cells. The values above theimmunoblots represent relative densities (in terms of fold units) of the bandsnormalized to b-actin. (C) Image represents the gelatinolytic effect of CaPcells treated with Lupeol. The gelatinolytic activity of MMP-2 in theconditioned media harvested from treated cells was determined byemploying zymography kit as described under Materials and Methods (D)Histogram represents the effect of Lupeol treatment on the transcriptionalactivation of MMP-2 in CaP cells. pGL2-MMP-2-luc-transfected cells weretreated with Lupeol and the transcriptional activity was measured in terms ofluciferase activity as described under Materials and Methods. Relativeluciferase activity was calculated with the values from vector alone groupwith or without Lupeol-treated group.

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Acknowledgements

We thankfully acknowledge Dr Etty N.Benveniste (University of Alabama atBirmingham, AL) for providing pGL2-MMP-2-luc reporter plasmid.

Conflict of Interest Statement: None declared.

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Received December 2, 2008; revised January 28, 2009;accepted February 5, 2009


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Lupeol, a dietary triterpene, inhibits Wnt/-catenin - Carcinogenesis