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Therapeutic Discovery Berberine Suppresses Androgen Receptor Signaling in Prostate Cancer Jing Li 1,4,5 , Bo Cao 2,4 , Xichun Liu 1,4 , Xueqi Fu 6 , Zhenggang Xiong 1 , Li Chen 5 , Oliver Sartor 3,4 , Yan Dong 2,4,6 , and Haitao Zhang 1,4,6 Abstract The androgen receptor (AR) is critical in the normal development and function of the prostate, as well as in prostate carcinogenesis. Androgen deprivation therapy is the mainstay in the treatment of advanced prostate cancer; however, after an initial response, the disease inevitably progresses to castration-resistant prostate cancer (CRPC). Recent evidence suggests that continued AR activation, sometimes in a ligand-independent manner, is commonly associated with the development of CRPC. Thus, novel agents targeting the AR are urgently needed as a strategic step in developing new therapies for this disease state. In this study, we investigated the effect of berberine on AR signaling in prostate cancer. We report that berberine decreased the transcriptional activity of AR. Berberine did not affect AR mRNA expression, but induced AR protein degradation. Several ligand-binding, domain-truncated AR splice variants have been identified, and these variants are believed to promote the development of CRPC in patients. Interestingly, we found that these variants were more susceptible to berberine-induced degradation than the full-length AR. Furthermore, although the growth of LNCaP xenografts in nude mice was inhibited by berberine, and AR expression was reduced in the tumors, the morphology and AR expression in normal prostates were not affected. This study is the first to show that berberine suppresses AR signaling and suggests that berberine, or its derivatives, presents a promising agent for the prevention and/or treatment of prostate cancer. Mol Cancer Ther; 10(8); 1346–56. Ó2011 AACR. Introduction Prostate cancer is the second most common cancer and a leading cause of cancer mortality among men in the United States (1). Androgen deprivation therapy (ADT), which aims to reduce the level of circulating androgens or to block the binding of androgens to their receptor, is a mainstay treatment for advanced prostate cancer. How- ever, after a moderate, short-term response, the disease eventually progresses to castration-resistant prostate can- cer (CRPC), which is a lethal and incurable. Therefore, preventing the transition to CRPC and treating CRPC effectively have become critical challenges for prostate cancer management. The androgen receptor (AR), a member of the steroid nuclear receptor family, is activated upon binding to androgens. The AR signaling axis not only plays a crucial role in the normal growth of the prostate, but also pro- motes prostate cancer initiation and progression. Recent studies have shown that AR reactivation is the driving force for the development of CRPC. Several mechanisms have been proposed to explain the sustained AR activa- tion in a low-androgen environment. First, AR amplifica- tion and AR overexpression, which were seen in 20% tumors from CRPC patients by gene expression analysis and immunohistochemistry assay (2, 3), could sensitize the receptor to low concentrations of androgens (4). Laboratory studies indicate that AR overexpression is necessary and sufficient to induce CRPC in a xenograft model (5). Second, AR mutations could alter its ligand- binding specificity and allow it to be activated by non- androgen steroids or increase its binding affinity for androgens (5–8). Third, tyrosine kinases could activate AR directly by phosphorylation (9, 10). Fourth, recent studies indicate that CRPC cells acquire the ability of intracellular synthesis of testosterone and dihydrotestos- terone (DHT) from weak adrenal androgens (11) or from cholesterol (12–15). Recently, several AR alternative spli- cing variants that lack the ligand-binding domain (LBD) Authors' Affiliations: Departments of 1 Pathology and Laboratory Med- icine, 2 Structural and Cellular Biology, and 3 Urology, and 4 Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana; 5 Department of Pharmacology, Norman Bethune School of Medicine, and 6 Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun, China Note: Supplementary data for this article are available at Molecular Cancer Therapeutic Online (http://mct.aacrjournals.org/). Corresponding Author: Haitao Zhang, Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Avenue SL-79, New Orleans, LA 70112. Phone: 504-988-7696; Fax: 504- 988-7389; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-10-0985 Ó2011 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 10(8) August 2011 1346 on June 5, 2019. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst May 25, 2011; DOI: 10.1158/1535-7163.MCT-10-0985

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Page 1: Berberine Suppresses Androgen Receptor Signaling in ...mct.aacrjournals.org/content/molcanther/10/8/1346.full.pdf · Berberine Suppresses Androgen Receptor Signaling in Prostate Cancer

Therapeutic Discovery

Berberine Suppresses Androgen Receptor Signalingin Prostate Cancer

Jing Li1,4,5, Bo Cao2,4, Xichun Liu1,4, Xueqi Fu6, Zhenggang Xiong1, Li Chen5, Oliver Sartor3,4,Yan Dong2,4,6, and Haitao Zhang1,4,6

AbstractThe androgen receptor (AR) is critical in the normal development and function of the prostate, as well as in

prostate carcinogenesis. Androgen deprivation therapy is the mainstay in the treatment of advanced prostate

cancer; however, after an initial response, the disease inevitably progresses to castration-resistant prostate

cancer (CRPC). Recent evidence suggests that continued AR activation, sometimes in a ligand-independent

manner, is commonly associated with the development of CRPC. Thus, novel agents targeting the AR are

urgently needed as a strategic step in developing new therapies for this disease state. In this study, we

investigated the effect of berberine on AR signaling in prostate cancer. We report that berberine decreased the

transcriptional activity of AR. Berberine did not affect AR mRNA expression, but induced AR protein

degradation. Several ligand-binding, domain-truncated AR splice variants have been identified, and these

variants are believed to promote the development of CRPC in patients. Interestingly, we found that these

variants were more susceptible to berberine-induced degradation than the full-length AR. Furthermore,

although the growth of LNCaP xenografts in nude mice was inhibited by berberine, and AR expression was

reduced in the tumors, themorphology andAR expression in normal prostates were not affected. This study is

the first to show that berberine suppresses AR signaling and suggests that berberine, or its derivatives,

presents a promising agent for the prevention and/or treatment of prostate cancer. Mol Cancer Ther; 10(8);

1346–56. �2011 AACR.

Introduction

Prostate cancer is the second most common cancer anda leading cause of cancer mortality among men in theUnited States (1). Androgen deprivation therapy (ADT),which aims to reduce the level of circulating androgens orto block the binding of androgens to their receptor, is amainstay treatment for advanced prostate cancer. How-ever, after a moderate, short-term response, the diseaseeventually progresses to castration-resistant prostate can-cer (CRPC), which is a lethal and incurable. Therefore,preventing the transition to CRPC and treating CRPC

effectively have become critical challenges for prostatecancer management.

The androgen receptor (AR), a member of the steroidnuclear receptor family, is activated upon binding toandrogens. The AR signaling axis not only plays a crucialrole in the normal growth of the prostate, but also pro-motes prostate cancer initiation and progression. Recentstudies have shown that AR reactivation is the drivingforce for the development of CRPC. Several mechanismshave been proposed to explain the sustained AR activa-tion in a low-androgen environment. First, AR amplifica-tion and AR overexpression, which were seen in 20%tumors from CRPC patients by gene expression analysisand immunohistochemistry assay (2, 3), could sensitizethe receptor to low concentrations of androgens (4).Laboratory studies indicate that AR overexpression isnecessary and sufficient to induce CRPC in a xenograftmodel (5). Second, AR mutations could alter its ligand-binding specificity and allow it to be activated by non-androgen steroids or increase its binding affinity forandrogens (5–8). Third, tyrosine kinases could activateAR directly by phosphorylation (9, 10). Fourth, recentstudies indicate that CRPC cells acquire the ability ofintracellular synthesis of testosterone and dihydrotestos-terone (DHT) from weak adrenal androgens (11) or fromcholesterol (12–15). Recently, several AR alternative spli-cing variants that lack the ligand-binding domain (LBD)

Authors' Affiliations: Departments of 1Pathology and Laboratory Med-icine, 2Structural and Cellular Biology, and 3Urology, and 4Tulane CancerCenter, Tulane University School of Medicine, New Orleans, Louisiana;5Department of Pharmacology, Norman Bethune School of Medicine, and6Edmond H. Fischer Signal Transduction Laboratory, College of LifeSciences, Jilin University, Changchun, China

Note: Supplementary data for this article are available at Molecular CancerTherapeutic Online (http://mct.aacrjournals.org/).

Corresponding Author: Haitao Zhang, Department of Pathology andLaboratory Medicine, Tulane University School of Medicine, 1430 TulaneAvenue SL-79, New Orleans, LA 70112. Phone: 504-988-7696; Fax: 504-988-7389; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-0985

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(8) August 20111346

on June 5, 2019. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 25, 2011; DOI: 10.1158/1535-7163.MCT-10-0985

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but retain the DNA-binding domain have been identifiedin both xenografts and human tissues (16–19). Thesevariants, referred to as ARDLBDs hereafter, have beenshown to be transcriptionally active in the absence ofandrogens and drive androgen-independent growth ofprostate cancer cells (17–19). Collectively, these studiesprovide new insights into the development of CRPC andunderscore the importance of AR as a direct therapeutictarget in all stages of prostate cancer.Berberine (2,3-methylenedioxy-9,10-dimenthoxyproto-

berberine chloride; BBR) is an isoquinoline alkaloid(Fig. 1A)with a long history of use in traditionalmedicine.It can be isolated from several plants of the genera Berberis

and Coptis, including goldenseal, Oregon grape, and bar-berry. Recent studies have reported that BBR has antic-ancer activities against several types of tumors (20, 21),including prostate cancer (22, 23). In this study, we testedthe hypothesis that BBR suppresses the AR signalingpathway, which has not been reported previously.

Materials and Methods

ReagentsThe reagents MTT, sulforhodamine B, cycloheximide,

and 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) were purchased from Sigma-Aldrich. R1881and dihydrotestosterone (DHT) were obtained fromSteraloids, Lipofectamine and Plus from Invitrogen,MG132 from the American Peptide Company, and thepan-caspase inhibitor z-VAD-fmk from Becton Dickin-son Pharmingen. Berberine (purity >99%) was acquiredfrom the Northeast Pharmaceutical Group and fromSigma-Aldrich.

Cell culture and MTT assayCells from LNCaP, 22Rv1, and PC-3 cell lines were

obtained from the American Type Culture Collection atPassage 4. The LAPC-4 cell line was provided by Dr. C.L.Sawyers (24). C4–2B cells were provided by Dr. S. Koo-chekpour at Louisiana State University Health SciencesCenter, New Orleans, LA. All cells used in this studywere within 20 passages after receipt or resuscitation (�3months of noncontinuous culturing). The requirementof androgen for growth was tested intermittently, andthe expression of AR was assessed by Western blotanalysis during the study. LNCaP, LAPC-4, 22Rv1, C4–2B, and PC-3 prostate cancer cells were maintained inRPMI 1640 medium supplemented with 10% FBS, 2mmol/L glutamine, 100 Units/mL penicillin, and 100mg/mL streptomycin. In experiments requiring androgenstimulation, LNCaP and 22Rv1 cells were cultured inphenol red–free RPMI 1640 medium supplemented with10% charcoal-stripped FBS (cs-FBS). MTT assay wasconducted in 96-well plates in octuplicate. Cells wereseeded at a density of 3 � 103 cells/well overnight,and treated with BBR for 24, 48, or 72 hours. IC50 valuesof BBR were calculated using the software Origin 6.0.

Apoptosis assayLNCaP cells were plated onto 96-well plates at a

density of 6 � 103 cells/well in RPMI 1640 medium with10% FBS overnight, and treated with BBR for 24 hours.Apoptosis was detected by the Cell Death DetectionELISA Kit (Roche). Cell number was determined inparallel by sulforhodamine B assay and was used tonormalize the apoptosis result. Data were expressed asfold of apoptosis induction over that of control.

Transient transfection and reporter-gene assayLNCaP and 22Rv1 cells were seeded on 10-cm dishes at

a density sufficient to become 80% to 90% confluent after

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Figure 1. Berberine inhibits prostate cancer cell growth and inducesapoptosis. A, structure of berberine. B, BBR inhibited the growth ofLNCaP. Cells were treated with various concentrations of BBR for24, 48, or 72 hours. MTT assay was conducted to determine cell viability.The viability of control cells was set at 100%. The data plotted aremean � SD; n ¼ 8. C, BBR induced apoptosis in LNCaP. Apoptosis wasquantitated by cell death ELISA in LNCaP cells treated with indicatedconcentrations of BBR for 24 hours. Data plotted are mean � SD;n ¼ 3. *, P < 0.05; **, P < 0.01.

Berberine Downregulates Androgen Receptor

www.aacrjournals.org Mol Cancer Ther; 10(8) August 2011 1347

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24 hours. Transient transfection was carried out by usingthe Lipofectamine and Plus reagents according to themanufacturer’s instructions. Cells were transfected with4 mg of the ARR3 plasmid (single luciferase assay) orcotransfected with 4 mg ARR3 plasmid and 200 ng pRL-TK plasmid (dual luciferase assay). After incubating withthe transfection mixture for 4 hours, cells were trypsi-nized and re-plated in triplicate onto 24-well plates at adensity of 6 � 104 cells/well in RPMI 1640 mediumcontaining 10% cs-FBS. Cells were allowed to recoverovernight before treatment with 0, 25, 50, or 100 mmol/LBBR in the presence or absence of 1 nmol/L DHT. Forsingle-luciferase assay, LNCaP cells were lysed with 1XReporter Lysis Buffer (Promega) at 6 or 24 hours aftertreatment, and luciferase activity was assayed by usingthe Luciferase Assay System (Promega). Protein concen-tration was determined by using the BCA Protein AssayKit (Pierce). The luciferase activity was normalized by theprotein concentration of the same sample. Dual luciferaseassay was conducted when there was a need to compareresults from different cell lines. The assay was conducted24 hours after treatment using the Dual Luciferase Repor-ter Assay System (Promega). Renilla luciferase activitywas used to normalize the activity of firefly luciferase.

Quantitative reverse-transcription PCRQuantitative reverse-transcription PCR (qRT-PCR)was

carried out as described previously (25). cDNA wassynthesized from 1.5 mg of total RNA by the SuperScriptIII Reverse Transcriptase (Invitrogen). The mRNAs forAR, prostate-specific antigen (PSA), and 24-dehydrocho-lesterol reductase (DHCR24) were analyzed by the rela-tive gene expression protocol, and b-actinwas used as theinternal control.

Western blotting and immunoprecipitationCells were washedwith ice-cold PBS and lysedwith 2�

Cell Lysis Buffer (Cell Signaling) containing a phospha-tase inhibitor and the protease inhibitor cocktail (Sigma)by incubating on ice for 30 minutes. Lysates were col-lected by centrifugation and protein concentrations weredetermined by the BCA method. For immunoprecipita-tion, cells were lysed with the lysis buffer (50 mmol/LTris-HCl, pH 8.0, 125 mmol/L NaCl, 5 mmol/L EDTA,0.5% NP-40), and 1.2 mg of total protein were incubatedwith the anti-HSP90a/b antibody or the mouse immu-noglobulin G (IgG; negative control) overnight at 4�C.Protein-G Agarose (Invitrogen) was added and the mix-ture was incubated for 1 hour at 4�C. The immunocom-plex was precipitated by a magnetic rack, washed 4 timeswith the lysis buffer, and resuspended in the SDS-loadingbuffer. The samples were separated on 10% SDS-PAGEand transferred onto polyvinylidene fluoride mem-branes. After blocking in TBS buffer (150 mmol/L NaCl,10 mmol/L Tris, pH 7.4) containing 5% nonfat milk, theblots were incubated with a primary antibody overnightat 4�C and a fluorescent-labeled secondary antibody for 1hour at room temperature. The fluorescent signals were

obtained by the Odyssey Infrared Imaging System (LI-COR Bioscience). The primary antibodies used were asfollows: anti-AR (Millipore), anti-PSA, anti-HSP90a/b(Santa Cruz Biotechnology), anti-DHCR24 (Cell Signal-ing), and anti-GAPDH (Chemicon).

Immunocytofluorescence imagingLNCaP cells were cultured on poly-D-lysine-coated

cover slides in phenol red–free RPMI 1640 medium plus10% cs-FBS overnight and treated with BBR for 2 hour.R1881, a synthetic androgen, was added to make up afinal concentration of 1 nmol/L. After 2.5 hours, cellswere fixed, permeabilized, and incubated with an anti-AR antibody and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Nuclei were counter-stained with 40, 6-diamidino-2-phenylindole (DAPI).Images were captured at�63 magnification using a LeicaTCS SP2 confocal microscope.

In vivo tumor growth in a xenograft modelLNCaP cells (4 � 106) were collected in 70 mL PBS and

mixed with 70 mL Matrigel Matrix (Becton DickinsonBiosciences). The mixture was injected subcutaneouslyto one side of the dorsal flank of 6- to 7-week-old malenu/nu mice (National Cancer Institute, Frederick, MD).When tumor volume reached 100 mm3, mice were ran-domized into 2 groups (n ¼ 6) and treated with vehicle(DMSO, 1mL/kg) or BBR (5mg/kg, dissolved inDMSO),respectively, by daily intraperitoneal (i.p.) injection.Tumor volumes were measured every 3 days and calcu-lated using the formula: r1� r2

2� 0.52, where r1 > r2. Micewere sacrificed 15 days after treatment.

Immunohistochemistry stainingTumor tissues were fixed in 10% buffered formalin and

embedded in paraffin. The sections were dewaxed andrehydrated in graded ethanol. Antigen retrieval wascarried out by soaking the sections in 10 mmol/L sodiumcitrate buffer (pH 6.0) and heating in a high-powermicrowave oven for 20 minutes. The sections were incu-bated with a mouse anti-AR monoclonal antibody (Bio-Genex) for 30 minutes at room temperature. Afterwashing, the secondary antibody was added and incu-bated for 30 minutes. Reactive products were visualizedby staining with 3,30-diaminobenzidene and counter-stained with hematoxylin for 30 seconds. Images cover-ing the entire slide were captured with the same opticalparameters. All images, excluding areas near the edges orwith necrosis, were analyzed using the ImageJ Software(NIH). Cells with strong, medium, weak, or negativestaining for AR were counted and the percentage of cellsin each category was calculated.

Statistical analysisAll results are presented as mean � SD unless other-

wise stated. Statistical significance was determined withthe Student’s t test (2-tailed). P < 0.05 was consideredstatistically significant.

Li et al.

Mol Cancer Ther; 10(8) August 2011 Molecular Cancer Therapeutics1348

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Results

BBR inhibits cell growth and induces apoptosis inprostate cancerWe first used the MTT assay to investigate the effect of

BBR on cell viability in several prostate cancer cell lines,including LNCaP, LAPC-4, 22Rv1, C4-2B, and PC-3. TheAR-expression and androgen-dependence status ofthese lines are described in Table 1. Cells were treatedwith increasing concentrations of BBR (from 1.56 to 100mmol/L in 2-fold increments) for 24, 48, or 72 hours. TheMTT results show that BBR reduced the viability of all celllines tested in a dose- and time-dependent manner(Fig. 1B for LNCaP, remaining data not shown). TheIC50 values indicate that AR-positive cell lines are moresensitive to BBR than the AR-negative, androgen-inde-pendent PC-3 cells. These results indicate that BBR mayexert its growth inhibitory effect by disrupting the ARsignaling pathway.Furthermore, we conducted apoptosis assay in LNCaP

cells after 24 hours of BBR treatment. As shown in Fig. 1C,a dose-dependent induction of apoptosis was detected,starting at 12.5 mmol/L. This result confirms previousreports showing that BBR is a potent inducer of apoptosisin prostate cancer cells (23).

BBR inhibits ligand-dependent and -independentAR transactivationOn the basis of the MTT results, we set out to examine

the influence of BBR on the transcriptional activity of AR.LNCaP cells were transiently transfected with an andro-gen response element (ARE)-luciferase reporter constructand cells were cultured in the presence or absence ofandrogen. Thebasal activity ofARwasvery low inLNCaPwithout androgen stimulation, but the activity wasinduced significantly (>120-fold) by adding 1 nmol/LDHT (Fig. 2A). After 6 hours of treatment, BBRinhibited androgen-stimulated AR transactivation in adose-dependent manner. AR activity was furtherinduced by DHT at 24 hours, and BBR inhibited ARactivity in a similar manner (data not shown). To assessthe effect of BBR on ligand-independent AR activity,22Rv1 and LNCaP cells were transfected with the repor-

ter construct, cultured in an androgen-depleted condi-tion, and treated with BBR for 24 hours. Consistent withbeing an androgen-independent cell line, the basal activ-ity of AR was 10 times higher in 22Rv1 than that inLNCaP cells (Fig. 2B). BBR suppressed AR activity bygreater than 90% in 22Rv1 cells, suggesting that BBRsuppresses the constitutive AR activity.

In addition to theARE-luciferase assay,weanalyzed theinfluence of BBRon the expression ofAR-regulated genes.PSA is a well-characterized target gene of AR. DHCR24has been recently identified as an AR-regulated gene (25,26). Figure 2C and D showed that, at the 24-hour timepoint, the mRNA and protein levels of both genes werereduced byBBR inLNCaP cells. This is consistentwith theresults from theARE-luciferase assay. BBR also decreasedPSA expression in C4-2B cells (Supplementary Fig. S1).

BBR downregulates AR protein expressionTo investigate howBBRexerts its inhibitory effect onAR

transactivation, we investigated whether BBR modulatedthe expression of AR. The qRT-PCR results showed thatthe ARmRNA level was not decreased by BBR after a 24-hourtreatment(Fig.3A). Instead,ARmRNAwasincreasedby the 25 and 50 mmol/L concentrations. In contrast, ARprotein was downregulated by BBR in a concentration-and time-dependent manner (Fig. 3B). A reduction of ARprotein was first detected after 16 hours of treatment with100 mmol/L BBR. After 24 hours, all 3 concentrations ofBBR decreased expression of AR protein. These resultsindicate that BBR exerts an inhibitory effect on AR ex-pression through mechanisms beyond the mRNA level.

In addition to LNCaP, we found that BBR decreasedAR expression in other AR-positive lines, including22Rv1 (Fig. 3C), LAPC-4, and C4-2B (SupplementaryFig. S2). This shows that BBR downregulation of ARprotein is universal in prostate cancer cells. It is note-worthy that we detected a high abundance of AR iso-forms in the range of 75 to 80 kDa in 22RV1 cells. TheseAR isoforms have been shown to be alternative splicingproducts and lack the LBD (16–19). These variants havebeen found to be enriched in CRPCs (27) and stronglyimplicated in promoting the progression to CRPC (17, 19).Interestingly, we found that these ARDLBDs were more

Table 1. Characteristics of the cell lines and IC50 values of berberine

IC50 mM

Cell lines Androgen dependence AR status 48 h 72 h

LNCaP Dependent Mutant and functional AR 24.4 3.4LAPC-4 Dependent Wild-type AR 37.8 7.922Rvl Independent Full-length (mutant) and truncated AR 21.9 12.7C4-2B Independent Mutant and functional AR 49.4 11.2PC-3 Independent AR null 70.2 21.5

Abbreviation: AR, androgen receptor.

Berberine Downregulates Androgen Receptor

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susceptible to BBR than the full-length AR in 22Rv1 cells(Fig. 3C).

BBR induces AR degradationThe afore-described data led us to speculate that BBR

mayaffectARprotein levels by reducing thehalf-life ofAR.To test this possibility, LNCaPcellswerepretreatedwith 50mg/mL cycloheximide for 30 minutes to stop proteinsynthesis and then treated with 100 mmol/L BBR. Cellswere lysed at different intervals for Western blotting ana-lysis. Normalized AR protein levels were analyzed bylinear regression to determine their half-life. As shownin Fig. 4A, AR half-life was 19.2 hours in control cells ascompared with 10.8 hours in cells treated with BBR,suggesting that BBR induces AR protein degradation.

It has been shown that AR could be degraded by 2pathways, one is mediated by the proteasome (28) andthe other by caspase-3 (29). To test the involvement of theproteasome pathway, LNCaP cells were treated with 100mmol/L BBR for 8 hours before the proteasome inhibitorMG132 was added. Consistent with a previous report(30), MG132 increased AR level in the absence of BBR(Fig. 4B, lane 2). In cells treated with BBR, the presence ofMG132 prevented further AR degradation (Fig. 4B, lanes4 and 5).

To determine the involvement of a caspase-mediatedpathway, we pretreated LNCaP cells with a pan-caspaseinhibitor for 2 hours. Cells were then treated with BBRfor 24 hours before being lysed for apoptosis assayand Western blotting analysis. As shown in Fig. 4C,

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Figure 2. BBR inhibits the transcriptional activity of AR. A, BBR inhibited the ligand-dependent AR transactivation. LNCaP cells were transfected withthe pARR3-luc construct in bulk and divided into equal aliquots in a medium containing 10% cs-FBS. Cells were stimulated with 1 nmol/L DHT andtreated with 25, 50, and 100 mmol/L BBR for 6 hours. The luciferase activity was normalized by total protein concentration. B, BBR inhibited theligand-independent AR transactivation. LNCaP and 22Rv1 cells were cotransfected with the pARR3-luciferase and pRL-TK constructs. Cells werecultured in medium containing 10% cs-FBS and treated with BBR for 24 hours. Dual luciferase assay was conducted and the firefly luciferase activity wasnormalized by the Renilla luciferase activity. C and D, BBR downregulated the expression of AR-regulated genes. LNCaP cells were cultured inmedium containing 10% FBS, and the expression of PSA and DHCR24 was analyzed by qRT-PCR (C) and Western blotting (D) following a 24-hourtreatment. *, P < 0.05; **, P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Li et al.

Mol Cancer Ther; 10(8) August 2011 Molecular Cancer Therapeutics1350

on June 5, 2019. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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treatment with the inhibitor totally blocked apoptosisinduction by BBR, suggesting that apoptosis inducedby BBR is caspase-dependent. The AR protein wasreduced by BBR to similar extents in the presence orabsence of the inhibitor, indicating that BBR-induced ARdegradation was not mediated by a caspase-dependentpathway. Furthermore, this suggests that AR degradationis a direct effect of BBR, rather than a bystander effect ofapoptosis. Altogether, these results suggest that BBR-induced AR-protein degradation is mediated predomi-nantly through the proteasome pathway.

BBR disrupts AR-Hsp90 interactionSimilar to other steroid nuclear receptors, unliganded

AR resides in the cytoplasm and interacts with heat shockprotein 90 (Hsp90), a major chaperone protein in thecytoplasm (31). This interaction stabilizes AR (32) andmaintains the proper conformation of AR for high-affi-nity ligand binding (33). Upon ligand stimulation, ARundergoes conformational changes which lead to its dis-sociation with Hsp90, and subsequently translocates tothe nucleus to function as a transcription factor. Disrup-

tion of the AR–Hsp90 interaction will not only render ARsusceptible to protein degradation (32, 34) but also inter-fere with nuclear translocation (35, 36). We next exam-ined the interaction between AR and Hsp90 bycoimmunoprecipitation. LNCaP was cultured in a low-androgen condition for 48 hours, and then treated with100 mmol/L BBR for 2 hours. Whole-cell lysates wereimmunoprecipitated by an anti-Hsp90 antibody. Figure5A shows that the AR-Hsp90 association was signifi-cantly disrupted by BBR. Western blotting using inputcell lysates showed that neither AR nor Hsp90 level wasdecreased by BBR under this condition.

BBR inhibits AR nuclear translocationThe effect of BBR on subcellularly distributed AR

was examined by immunocytofluorescence staining.As shown in Fig. 5B, in the absence of androgen, ARstaining was predominantly cytoplasmic. Followingstimulation with R1881, the nuclear staining of ARwas increased dramatically. AR staining in the nucleuswas reduced in a dose-dependent manner in cellspretreated by BBR and accompanied by increased

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Figure 3. BBR downregulates AR protein expression. Cells were cultured in RPMI 1640 medium containing 10% FBS. A, qRT-PCR analysis of AR mRNAin LNCaP following BBR treatment for 24 hours. *, P < 0.05; **, P < 0.01. B and C, AR immunoblotting in LNCaP cells (B) and 22Rv1 cells (C) treated with100 mmol/L BBR.

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staining in the perinuclear cytoplasm. These resultssuggest that BBR inhibits AR nuclear translocation andprovide an explanation to our observation that BBRinhibited AR activity before AR-protein level wasreduced.

BBR inhibits tumor growth and suppresses ARin vivo

To test the in vivo efficacy of BBR, we first establishedLNCaP xenografts in nude mice. BBR treatment startedwhen tumors reached 100 mm3 at a dose of 5 mg/kg/d.Figure 6A shows that treatment with BBR significantlyinhibited the growth of the tumor xenografts, and thisresult was confirmed by the final tumor weights(Fig. 6B). No toxicity was observed in mice treated withBBR, as body weight changed similarly in both groups

(Supplementary Fig. S3). The expression of AR intumors was analyzed by immunohistochemical stainingof formalin-fixed tissues. AR staining was found pre-dominantly in the nucleus, both in the control andtreatment samples (Fig. 6C). A decrease in AR-stainingintensity was visually apparent in samples from thetreatment group. Quantitative analysis showed thatBBR reduced the percentages of cells with strong ormedium staining of AR, and increased the percentagesof cells stained weakly or negatively for AR. Theseresults confirmed our cell-culture data and showed thatBBR is effective in suppressing AR in vivo. Furthermore,the growth-inhibitory and AR-downregulating effectsof BBR seems to be limited to the tumors, as BBRtreatment does not affect the histology or AR expressionin normal prostate glands of mice (Fig. 6D).

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Figure 4. BBR induces AR proteindegradation. A, LNCaP wastreated with 50 mg/mLcycloheximide (CHX) for 30minutes before 100 mmol/L BBRwas added. AR protein wasanalyzed by immunoblotting anddensitometry. Normalized ARintensities (by GAPDH) wereanalyzed by linear regression todetermine AR half-life. B, effect ofMG132 on induced ARdegradation. LNCaP was treatedwith 100 mmol/L BBR for 8 hoursbefore MG-132 was added foranother 4 hours. C, apoptosisassay and AR protein expressionafter pretreatment with z-VAD-fmk(pan-caspase inhibitor) for 2 hoursand berberine for 24 hours.**, P < 0.01.

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Discussion

The development of CRPC following androgen depri-vation is the most critical challenge in the clinical man-agement of prostate cancer. In spite of the recentdevelopment of new therapeutic options, treatment ofpatients with CRPC remains a significant clinical chal-lenge. For example, sipuleucel-T (Provenge), the firsttherapeutic cancer vaccine approved by the Food andDrug Administration, has been shown by clinical trials toprolong the median survival of patients with metastaticCRPC by 4.1 months (37). Abiraterone, an inhibitor of therate-limiting enzyme CYP17 in androgen biosynthesis,has shown promises in clinical trials involving patientswith metastatic CRPC (38, 39). However, its efficacy

against CRPC driven by the newly discovered AR splicevariants has not been established. The lack of LBD couldrender these AR variants resistant to current androgen-blockade strategies. Therefore, suppressing the expres-sion of all forms of AR appears to be a provocative andplausible strategy against CRPC.

Bioactive natural products provide a rich source forpharmacologic discovery. Berberine has been shown toinduce apoptosis in prostate cancer cells by inducingreactive oxygen species (ROS) and oxidative stress (40).Our discovery that BBR induces AR degradation appearsto be a novel mechanism that is independent of theseactions of BBR. First, as shown in Fig. 4C, treatmentwith a pan-caspase inhibitor blocked apoptosis inductionby BBR but had no impact on AR degradation, suggestingthat AR degradation is not a result of destruction ofcellular proteins during apoptosis. Second, AR degrada-tion by BBR was not affected by treatment with a ROSscavenger, N-acetyl-L-cysteine (Supplementary Fig. S4).In LNCaP cells, it has been shown that BBR inducedapoptosis by activating p53 (22). Therefore, at least inLNCaP cells, BBR appears to modulate 2 importantsignaling pathways in prostate cancer in a highly coor-dinated manner, that is, suppression of the prosurvivalAR pathway and induction of the proapoptotic p53 path-way.

Our observation that BBR inhibited AR transactivationbefore the decrease in AR protein could be explained bythe disruption of AR–Hsp90 interaction and reduction ofAR nuclear translocation by BBR. These events wereobserved as early as 2 hours and 4 hours after treatment,respectively. The reduced AR–Hsp90 interaction is prob-ably a result of decreased chaperone activity of Hsp90, asthe level of Hsp90 protein was not affected by BBR at thistime point. The activity of Hsp90 is regulated by theacetylation status, which, in turn, is regulated by histonedeacetylase 6 (HDAC6). HDAC6 deacetylates the lysineresidues of Hsp90 and plays a critical role in maintainingthe chaperone function of Hsp90 (41). HDAC6 inhibitioninactivates Hsp90 by interfering with ATP binding andassociationwith client proteins (42). Themolecular effectsof BBR we have observed, including downregulation ofthe expression, transcriptional activity and nuclear loca-lization of AR, and disruption of the AR/Hsp90 interac-tion, are similar to those achieved by HDAC6 inhibition(41, 43). Therefore, it is possible that BBR exerts itsinhibitory action on AR through modulation of theHDAC6/Hsp90 pathway.

The client proteins of Hsp90 include steroid hormonereceptors, including the estrogen receptor (ER), glucocor-ticoid receptor, and progesterone receptor. The possibi-lity that BBR inhibits Hsp90 activity implies that BBRcould have similar effects on these receptors. Indeed, wefound that BBR reduced the protein level of ERa in MCF-7 breast cancer cells (Supplementary Fig. S5). Consistentwith our finding, it has been shown that BBR enhancesthe anticancer efficacies of ER antagonists in ER-positiveMCF-7 cells, but not in ER-negative MDA-MB-231 cells

A

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BBR 100 μmol/L+R1881(12.4% ± 3.1%)

IgG CON BBR 17-AAG

IP: HSP90

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IB: AR

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AR (FITC) DAPI Merged

Figure 5. BBR disrupts the AR–Hsp90 interaction and inhibits AR nucleartranslocation. A, LNCaP cells were cultured in medium containing5% cs-FBS for 48 hours. Coimmunoprecipitation (IP) was conducted withan anti-Hsp90a/b antibody following treatment with 100 mmol/L BBR or500 nmol/L 17-AAG for 2 hours. Western blot (WB) analysis was carriedout with the anti-Hsp90 and anti-AR antibodies. B, LNCaP cells werecultured in medium containing 10% cs-FBS overnight and treated withBBR for 2 hours. Immunocytofluorescence analysis for AR was carried outfollowing stimulation with 1 nmol/L R1881 for another 2.5 hours. Numbersin the parentheses indicate percentage� SD of cells that displayedgreaterAR staining in the nucleus than in the cytoplasm. IB, immunoblotting.

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(44). The effect of BBR on the ER signaling pathwaywarrants further investigation.

Consistent with a previous report (22), our animalstudy showed that BBR is very effective in inhibitingthe growth of prostate cancer in a xenograft model. The 5-mg/kg dose used in this experiment is well below theLD50 of BBR via the same route of injection, which isapproximately 50 mg/kg (45). It is important to point outthat, in spite of the fact that this dose was expected toachieve a blood concentration of BBR 2 orders of magni-tude lower than the doses used in the cell culture experi-ments, our in vitro findings that BBR reduced AR proteinand the expression of AR-regulated genes were con-firmed by the animal experiment. Similar blood concen-trations of BBR can be achieved in humans taking oraldoses of BBR (46). Therefore, the findings described inthis report are potentially clinically relevant.

In 22Rv1 cells, the basal activity of AR is 10 times thatobserved in LNCaP cells (Fig. 2B). Because 22Rv1 cellsexpress high levels of ARDLBDs (Fig. 3C), this ligand-independent AR transactivation is presumably driven bythe splice variants. The data in Fig. 2B show that BBR isparticularly effective in shutting down the ligand-inde-pendent AR activity in 22Rv1 cells, which is furthersupported by the data in Fig. 3C that BBR decreasedthe expression of ARDLBDsmore effectively than the full-length AR. In addition to BBR, several other natural orsynthetic compounds have been shown to have similarinhibitory effects on AR slice variants (46–49). The dis-covery of these compounds should generate excitementbecause there is no drug currently in clinical trials totarget CRPC progression driven by the AR splice var-iants. Our study suggests that BBR should be furthertested and developed in the treatment of CRPC.

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6d 9d 12d 15d Figure 6. BBR suppresses ARexpression in vivo. LNCaPxenografts were established innudemice and BBRwas given at adose of 5 mg/kg/d when tumorsize reached 100 mm3. Controlmice received the DMSO. A, serialmeasurements of tumor volume.B, final tumor weight. C, left,immunohistochemistry ofrepresentative sections of LNCaPxenografts stained for AR; right,quantitation of theimmunohistochemical results. D,hematoxylin & eosin (H&E) and ARstaining in fixed mousedorsolateral prostates. High-resolution images for AR stainingare available in SupplementaryFig. S6. All data presented aremean � SEM. *, P < 0.05;**, P < 0.01.

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In summary, this work is the first report that BBRsuppresses the AR signaling pathway in prostate cancer.BBR decreased AR transcriptional activity and reducedthe expression of AR-regulated genes in both androgen-dependent and CRPC cells. AR protein was degraded byBBR, which was predominantly mediated by a protea-some pathway. Moreover, the disruption of AR–Hsp90interaction and the inhibition of AR nuclear translocationby BBR could contribute to the depressed AR transcrip-tional activity and AR protein degradation. Interestingly,BBR induced degradation of not only the full-length AR,but also the ARDLBDs. The animal data showed that BBRexertedgrowth-inhibitory andAR-downregulating effectsspecifically in tumor tissues. Our study identifies a novelmechanism for the anticancer effect of BBR and providessupport for the use of BBR as a potential preventive andtherapeutic agent in prostate cancer, particularly for AR-mediated CRPC growth.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Drs. Charles L. Sawyers at the UCLA JonssonComprehensive Cancer Center for LAPC-4 cells, Shahriar Koochekpourat the Louisiana State University Health Sciences Center for providing theC4-2B cells, Guoyong Wang at Department of Structural and CellularBiology, Tulane University School of Medicine for assistance with con-focal microscopy, and Sudesh Srivastav at Department of Biostatistics,TulaneUniversity School of Public Health, and TropicalMedicine for helpwith statistical analysis.

Grant Support

This work was supported by start-up funds from the Louisiana CancerResearch Consortium, a Tulane Cancer Center Matching Fund, a TulaneUniversity School of Medicine Pilot Fund, a Jilin Provincial Scholarship fromJilin, China (grant to H. Zhang), and ACS grant No. RSG-07-218-01-TBE and NIHgrant No. K01CA114252 to Y. Dong.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 31, 2010; revised May 11, 2011; accepted May 14,2011; published OnlineFirst May 25, 2011.

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Published OnlineFirst May 25, 2011; DOI: 10.1158/1535-7163.MCT-10-0985