20(s)-protopanaxadiol-aglycone downregulation of the full-length and splice variants of androgen...

20(S)-Protopanaxadiol-aglycone downregulation of thefull-length and splice variants of androgen receptor

Bo Cao1,2, Xichun Liu3, Jing Li3,4, Shuang Liu1, Yanfeng Qi1, Zhenggang Xiong3, Allen Zhang5, Thomas Wiese6, Xueqi Fu2,

Jingkai Gu2, Paul S. Rennie7, Oliver Sartor8, Benjamin R. Lee8, Clement Ip9, Lijuan Zhao4, Haitao Zhang2,3

and Yan Dong1,2,10

1 Department of Structural and Cellular Biology, Tulane University School of Medicine, Tulane Cancer Center, New Orleans, LA2 College of Life Sciences, Jilin University, Changchun, China3 Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, Tulane Cancer Center, New Orleans, LA4 Department of Pathophysiology, School of Basic Medicine, Jilin University, Changchun, China5 Benjamin Franklin High School, New Orleans, LA6 Department of Biochemistry, College of Pharmacy, Xavier University of Louisiana, New Orleans, LA7 The Vancouver Prostate Center, Vancouver, BC, Canada8 Department of Urology, Tulane University School of Medicine, Tulane Cancer Center, New Orleans, LA9 Department of Cancer Prevention and Control, Roswell Park Cancer Institute, Buffalo, NY10 National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun, China

As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective

chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and

progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone

has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate

cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of

androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR-Vs) lacking the ligand-

binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the

present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and

activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins

followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the

transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP

xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD

suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further

developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.

Prostate carcinogenesis is characterized by a latency of 20–40years.1 Most prostate cancers remain asymptomatic for years,even though they are malignant histologically.1 The long la-

tency of prostate carcinogenesis presents an ideal opportunityfor chemoprevention to inhibit or delay the clinical symp-toms or to reverse the progression of the disease. Although

Key words: 20(S)-protopanaxadiol-aglycone, androgen receptor, prostate cancer

Abbreviations: AR: androgen receptor; AR-Vs: androgen receptor splice variants; ARE: androgen-responsive element; DHT:

dihydrotestosterone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H & E: hematoxylin and eosin; LBD: ligand-binding domain;

PPAR: peroxisome proliferator-activated receptor; PPD: 20(S)-protopanaxadiol-aglycone; PSA: prostate-specific antigen; qRT-PCR:

quantitative reverse transcription-PCR; SRB: sulforhodamine B

Additional Supporting Information may be found in the online version of this article.

Grant sponsor: National Cancer Institute; Grant number: K01CA114252; Grant sponsor: American Cancer Society; Grant number: RSG-

07-218-01-TBE; Grant sponsor: National Major Special Project for Science and Technology Development of Ministry of Science and

Technology of China; Grant number: 2009ZX09304-003; Grant sponsors: Chinese Scholarship Council Fellowship; Louisiana Cancer

Research Consortium Start-up Fund; Tulane Cancer Center Developmental Fund; Tulane University School of Medicine Pilot Fund

DOI: 10.1002/ijc.27754

History: Received 19 May 2012; Accepted 6 Jul 2012; Online 31 Jul 2012

Correspondence to: Yan Dong, Department of Structural and Cellular Biology, Tulane University School of Medicine, 1430 Tulane Avenue

SL-49, New Orleans, LA 70112, E-mail: [email protected] or Lijuan Zhao, School of Basic Medicine, Jilin University, Changchun 130021,

China, E-mail: [email protected]

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Int. J. Cancer: 000, 000–000 (2012) VC 2012 UICC

International Journal of Cancer

IJC

the etiology of prostate cancer remains poorly understood, thevital role of androgen signaling in the development and pro-gression of prostate cancer has been documented extensively.2

Finasteride and dutasteride are inhibitors of 5a-reductases,which are responsible for the conversion of testosterone to themore potent androgen dihydrotestosterone (DHT). So far, theyare the only chemopreventive agents that have been showndefinitively in large-scale randomized trials to decrease prostatecancer incidence.3,4 However, for both agents, the decreaseappears to be limited to low-grade tumors with a Gleason scoreof 6 or lower, and significant increase in the incidence of high-grade prostate cancers has been observed in the treatmentgroups.3–5 As a consequence, 5a-reductase inhibitors are notapproved by FDA for the prevention of prostate cancer.5

The mechanism underlying the increased incidence ofhigh-grade prostate cancers after finasteride and dutasteridetreatments is still unknown. A recent study reported significantupregulation of androgen receptor (AR) in prostate tissue byfinasteride treatment in patients with benign prostatic hyperpla-sia.6 Increase in AR expression has been shown to be sufficientto convert prostate tumor growth from an androgen-sensitive tocastration-resistant stage,7 and therefore may represent a mecha-nism underlying disease progression after finasteride treatment.Furthermore, the levels of constitutively active, ligand-independ-ent AR splice variants (AR-Vs) have been reported to be upreg-ulated during prostate cancer progression.8–11 These AR-Vs lackthe ligand-binding domain (LBD), and thus may not be respon-sive to treatment with finasteride or dutasteride. Therefore, itbecomes imperative to develop new chemopreventive agentsthat could disrupt AR signaling through both the full-length andsplice variants of AR.

The ginseng root is one of the most commonly used me-dicinal herbs in the United States.12 It is used as a tonic forenhancing well-being and promoting longevity, and also as acomplementary therapy particularly for cancer intervention.12

It has been used for centuries by humans and reported tohave minimal side effects. Ginsenosides are considered themain active ingredients responsible for the pharmaceuticalfunctions of the ginseng root.13 There are two major groupsof ginsenosides, the protopanaxadiol and the protopanaxatriolgroup. Orally ingested ginsenosides are mainly metabolizedby intestinal bacteria through deglycosylation.13 The sugarmoieties of the ginsenosides in the protopanaxadiol and theprotopanaxatriol group are cleaved to give 20(S)-protopanaxa-diol-aglycone (PPD) and 20(S)-protopanaxatriol-aglycone,respectively.13 PPD has been reported to inhibit the growth of

prostate cancer cells both in vitro and in vivo.14,15 The growthinhibition has been attributed to PPD suppression of cell pro-liferation and induction of apoptosis, which are accompaniedby downregulation of cyclins and cyclin-dependent kinases aswell as induction of p21 expression and PARP cleavage.14,15

Beyond these, very little is known about the mechanismunderlying the action of PPD in prostate cancer.

While taking a close look at the structure of PPD, wefound that it shares the four trans-ring rigid steroid skeletonwith steroid hormones, including androgens (Fig. 1a).Accordingly, it is reasonable to believe that PPD might beable to modulate AR signaling. In fact, PPD inhibition of thegrowth of LNCaP prostate cancer cells has been reported tolead to reduced secretion of prostate-specific antigen (PSA), awell-known target of AR, although it is unclear as to whetherthe reduction in PSA secretion is a direct consequence ofPPD inhibition of AR signaling.15 In this report, we describea series of experiments that were designed to characterize theeffect of PPD on AR signaling pathway.

Materials and MethodsCell lines and reagents

LNCaP, 22Rv1, and PC-3 cells were obtained from AmericanType Culture Collection at Passage 4. LAPC-416 and C4-217

were provided by Drs. Charles Sawyers and Shahriar Koo-chekpour, respectively, and cultured as described.17,18 Cellsused in this study were within 20 passages (�3 months ofnoncontinuous culturing). Cell growth was determined byusing the sulforhodamine B (SRB) assay as described.19 PPDwas obtained from the Organic Chemistry Laboratory at JilinUniversity, Changchun, China. Compound of purity of>98%, as determined by high-performance liquid chromato-graph, was used in cell culture studies, and that of >95% wasused in animal studies.

Construction of AR-promoter-luciferase reporter

gene plasmids

An 8 kb (�6,885 to þ1,115) and a 1.7 kb (�600 to þ1115)fragment of the 50-flanking region of the human AR genewere PCR amplified from an AR-containing BAC clone,RP11-807F19 (Roswell Park Cancer Institute). These two frag-ments were then separately cloned into the pCRTM2.1-TOPOVR

vector (Invitrogen), and subcloned into the pGL4.19[luc2CP/Neo] rapid response luciferase expression vector (Promega).The authenticity of the constructs, pGL4-ARpro8 and pGL4-ARpro1.7, was confirmed by DNA sequencing.

What’s new?

This is the first report on 20(S)-protopanaxadiol-aglycone as an effective agent to downregulate the expression and activity of

the full-length androgen receptor and its constitutively-active splice variants in prostate cancer. It is also the first to show the

in vivo efficacy of 20(S)-protopanaxadiol-aglycone against androgen-receptor-expressing prostate tumors. Considering the

critical roles of androgen receptor and its splice variants in disease progression, our findings suggest that 20(S)-

protopanaxadiol-aglycone is a promising agent for prostate cancer intervention.

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2 PPD downregulates androgen receptor


Reporter gene assay

The androgen-responsive element (ARE)-luciferase reporterplasmid, containing three repeats of the ARE region ligatedin tandem to the luciferase reporter,20 or the pGL4-ARpro8or pGL4-ARpro1.7 AR-promoter-luciferase reporter con-struct, was transiently transfected into cells as described.21 Tocontrol for uniform transfection efficiency, transfection wasconducted in a 10-cm dish, and the transfected cells weresubsequently replated in triplicate onto 24-well plates andallowed to recover overnight before treatments. Luciferase ac-tivity was normalized by the protein concentration of thesample.

Quantitative reverse transcription-PCR (qRT-PCR)

RNA was isolated with the PerfectPure RNA Cultured Cell Kit(Fisher Scientific). The TaqManV

R

PCR primers and probes forb-actin, AR, and PSA were from Applied Biosystems, and theqRT-PCR analysis was performed as described.22 The primersequences for AR isoforms and the qRT-PCR analysis of ARisoforms were as described in Ref. 9.

Western blotting

The procedure was described previously.23 Immunoreactivebands were quantitated by densitometry and normalized toglyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thefollowing antibodies were used: anti-PSA (Neomarkers), anti-GAPDH (Millipore), anti-AR (Millipore), anti-AR3 (PrecisionAntibody), anti-peroxisome proliferator-activated receptor c

(PPARc) (Santa Cruz Biotechnology), anti-p53 (Cell Signal-ing) and anti-histone H3 (Cell Signaling).

Cell-free AR ligand-binding assay

The assay was performed by using the PolarScreenTM ARCompetitor Assay Kit (Invitrogen) as per manufacturer’s pro-tocol. In essence, the relative affinity of the test compound toAR was analyzed by competing with a fluorescent androgenligand (FluormoneTM AL Green) for binding to the His- andGST-tagged AR-LBD. The FluormoneTM AL Green or differ-ent concentrations of the test compound (PPD, DHT or bica-lutamide) were incubated with AR-LBD (His-GST) in dupli-cate in dark for 4 hr, and the polarization values were thenmeasured. The fluorescence polarization values of 1 � AR-LBD (His-GST)/FluormoneTM AL Green Complex and 1 �AR-LBD (His-GST) were considered as 0 or 100% AR boundby the test compound, respectively.

Whole-cell AR ligand-binding assay

The assay was performed by using the test compound (PPD,DHT, or bicalutamide) to compete with 0.1 nM [H3]-R1881(a potent synthetic androgen) for AR binding. Cells wereincubated with different concentrations of PPD, DHT orbicalutamide in the presence of 0.1 nM [H3]-R1881 (Perki-nElmer) for 1.5 hr. At the end of the incubation, the cellswere washed three times with ice-cold PBS to remove excess[H3]-R1881. The AR-bound R1881 was extracted with etha-nol, and radioactivity measured by scintillation counting.

Figure 1. (a) Structures of PPD and DHT. (b,c) PPD inhibition of the growth of LNCaP cells (b), as well as LAPC-4, C4-2 and 22Rv1 cells (c).

Cells were treated with different doses of PPD as indicated (b) or 20 lg ml�1 PPD (c) for 24, 48 or 72 hr, and growth response determined

by the SRB assay. *, p < 0.01 from vehicle control. (d) PPD suppression of AR transactivation. LNCaP cells transfected with the ARE-

luciferase construct were treated with 20 lg ml�1 PPD for indicated time in the presence or absence of 1 nM DHT, and luciferase activity

analyzed. * and **, p < 0.01 from control or DHT-treated sample, respectively. Carcinog

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Immunoprecipitation assay

Cells were lysed with a lysis buffer containing 20 mM Tris–HCl (pH 7.5), 50 mM NaCl, 20 mM Na2MoO4, 0.5% NP-40,1 mM EDTA, 1 mM EGTA, and 2 mM DTT. Cell lysate (1.2mg) was incubated with 4 lg anti-AR antibody (Millipore) ormouse IgG (as a negative control) overnight at 4�C.DynabeadsVR Protein G (Invitrogen) was then added. After anadditional incubation for 1 hr at 4�C on a platform rocker,the beads were captured by using a magnetic separation rack,washed four times with the lysis buffer, boiled in SDS-loadingbuffer for 10 min, and subjected to Western blot analysiswith an antibody against Hsp90 (Santa Cruz Biotechnology)or AR (Millipore).

AR N-C interaction assay

The intensity of AR N–C interaction was assessed by using amammalian two-hybrid system.24 This system includes twofusion protein constructs, VP-A1 and GALD-H, as well asone reporter gene plasmid, G5E1bLuc. VP-A1 is the fusionconstruct of the N-terminal residues 1–503 of AR and theactivation domain of the herpes simplex virus VP16 protein.GALD-H is the fusion construct of the C-terminal ligand-binding domain of AR (624–919) and the GAL4 DNA-bind-ing domain. The G5E1bLuc construct contains the luciferasereporter gene downstream of five consensus GAL4 bindingsites and the minimal promoter of the adenovirus E1b gene.To control for uniform transfection efficiency, the three con-structs were cotransfected into AR-null PC-3 cells at 1:1:5 ra-tio in a 15-cm dish as described.21 Cells were subsequentlyreplated in triplicate onto six-well plates and allowed torecover overnight before treatments. Luciferase activity wasnormalized by the protein concentration of the sample. Theuse of AR-null cells in this assay was to avoid interference ofthe interaction between transfected AR N- and C-terminalfragments from the endogenous AR.25

LNCaP tumor xenograft model

Male nude mice were obtained from the NCI Animal Pro-duction Center at 5–6 weeks of age. After 1 week of adapta-tion, mice were inoculated subcutaneously with 4 � 106

LNCaP cells suspended in 50% Matrigel on both dorsalflanks. The day following inoculation, mice were randomlyassigned to two groups and received 40 mg kg�1 PPD inolive oil or olive oil as control through oral gavage 6 daysweekly. Body weights and tumor dimensions were monitoredweekly. Tumor volume was calculated as 0.524 � width2 �length.26 At the termination of the experiment, mice wereanesthetized, and blood collected for serum PSA determina-tion using quantitative ELISA (United Biotech). Tumors wereremoved, weighed, and fixed in 10% formalin for paraffinembedding and histological analyses. All animal procedureswere approved by the Tulane University Institutional AnimalCare and Use Committee.

AR immunohistochemical analyses

Sections of paraffin-embedded blocks were deparaffinized,rehydrated, and immersed in 3% H2O2 to quench endoge-nous peroxidase activity. Antigen retrieval was achieved byheating samples in 10 mM citrate buffer (pH 6.0) to nearboiling for 20 min. The slides were cooled to room tempera-ture, and preincubated with blocking serum to prevent non-specific binding. Primary antibody incubation, with a mono-clonal AR antibody (BioGenex), was conducted at room tem-perature for 1 hr. The reaction was visualized using the ABCStaining System (Santa Cruz Biotechnology), with diamino-benzidine as the chromogen. Sections were counterstainedwith hematoxylin. As the negative control, the primary anti-body was replaced with a nonimmune IgG at the same con-centration, and no reactivity was detected.

Images were sampled sequentially throughout each sectionusing a 20� objective, with areas of necrosis, preparationartifacts, or edges avoided. Each field of view was digitized at200� magnification. All digitized images were analyzed usingthe ImageJ software (NIH). The diaminobenzidine color wasextracted from each digitized image using the color deconvo-lution function in ImageJ, and the extracted color image wastransformed into an 8-bit grayscale image. A histogram wassubsequently derived for each image using the Auto Thresh-old tool in ImageJ. The two peaks on each histogram depictthe distribution of pixels in areas stained positively (leftpeak) or negatively (right peak) for AR. The floor betweenthe two peaks defined background intensity. The relative den-sity of each immunopositive pixel was calculated as: relativedensity ¼ log (I0) – log (Ii), where I0 is the intensity of thebackground in each field, and Ii the intensity of individualpixel.27

Statistical analysis

The Student’s two-tailed t test was used to determine themean differences between treatment and control. Data arepresented as mean 6 SEM.

ResultsPPD inhibition of the growth of prostate cancer cells

We first assessed the effect of PPD on the growth of andro-gen-dependent LNCaP cells by the SRB assay. As presentedin Figure 1b, PPD inhibited the growth of LNCaP cells in adose- and time-dependent manner. LNCaP cells express amutant but functional AR.28 We also examined the effect ofPPD on the growth of androgen-dependent, wild-type-AR-expressing LAPC-4, the C4-2 castration-resistant derivative ofLNCaP, and castration-resistant 22Rv1 cells. The 22Rv1expresses both the full-length AR and the constitutively activeLBD-truncated AR-Vs, which have been correlated with pros-tate cancer progression and recurrence.8–11 As shown in Figure1c, PPD also caused a progressive inhibition of the growth ofLAPC-4, C4-2 and 22Rv1 cells as a function of time. The data

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thus confirm previous reports showing the ability of PPD toinhibit the growth of prostate cancer cells.14,15

PPD suppression of AR signaling

We then investigated the effect of PPD on AR transactivation bythe reporter gene assay. LNCaP cells transfected with the ARE-luciferase reporter construct20 were exposed to PPD for 3, 6, 12or 16 hr. As shown in Figure 1d, PPD inhibited not only DHT-induced but also basal AR trans-activating activity. Reduction ofDHT-induced AR activity appeared earlier, at 6 hr, whereasdecrease of basal AR activity was not observed until 12 hr post-treatment. We proceeded to examine the effect of PPD on anendogenous AR-target gene, PSA, by qRT-PCR and Westernblotting. As shown in Figures 2a and 2b, the PSA data were con-sistent with the reporter gene result, displaying a time-depend-ent downregulation. A similar decrease of PSA level was alsodetected in LAPC-4, C4-2 and 22Rv1 cells (Figure 2c).

Limited ability of PPD to compete with androgens for

binding to AR

Because PPD bears structural similarity to androgens, it isconceivable that PPD may inhibit AR transactivation byfunctioning as an AR antagonist. We carried out a whole-cellAR ligand-binding assay in LNCaP cells and a cell-free ARligand-binding assay to characterize the interaction betweenPPD and AR. Both assays showed that the binding affinity of

PPD to AR is �10,000–40,000-fold less than that of DHT and�80-fold less than that of the anti-androgen bicalutamide. Wethen studied whether the suppressive activity of PPD on ARsignaling differs in the presence of different concentrations ofDHT by using PSA mRNA expression as the read-out. As pre-sented in Figure 2d, the ability of PPD to downregulate PSAexpression was not weakened with increasing concentrationsof DHT. Taken together, the data indicate that PPD suppres-sion of AR transactivation is unlikely to be mediated by com-peting with androgens for binding to AR. It is of note that PPDdoes not have agonistic activity against AR either. This is evi-denced by the AR activity data presented in Figure 1d underandrogen-depleted condition.

PPD downregulation of AR protein levels

To understand the mechanism by which PPD inhibits ARtransactivation, we assessed PPD modulation of AR protein.As shown in Figure 3a, in all the four cell lines examined, adecrease of AR protein was already evident at 6 hr after PPDtreatment, and the decrease became more significant withtime. In contrast, no decrease of another nuclear receptor,PPARc, was observed (Supporting Information Figure), indi-cating the specificity of the effect. It is important to note thatPPD downregulated not only the full-length AR but also theconstitutively active truncated AR-Vs (the 22Rv1 panel ofFigure 3a). The magnitude of downregulation of the AR-Vswas similar to that of the full-length AR. The downregulationof AR-Vs also resulted in a decrease in the activity of AR-Vs.This is indicated by the ability of PPD to inhibit PSA expres-sion even after specific knockdown of the full-length AR(Figure 3b).

To further determine whether PPD downregulation of ARrequires the presence of androgen, we cultured the LNCaPand 22Rv1 cells in androgen-deprived condition, and treatedthe cells with PPD in the presence or absence of DHT. Thedata are presented in Figure 3c. In accordance with the abil-ity of androgen to stabilize the full-length AR protein andthe lack of the LBD of the AR-Vs, an increase of the full-length AR protein, but not the AR-Vs, was evident after ex-posure of cells to DHT. PPD downregulated basal and andro-gen-stabilized AR protein to a similar extent, indicating thatPPD downregulation of AR does not require the presence ofandrogen. The data thus lend further support to our conclu-sion that PPD suppression of AR signaling is unlikely to bemediated by competing with androgens for binding to AR.

PPD suppression of AR transcription

We then examined the changes of AR transcripts by qRT-PCR. As shown in Figures 4a and 4b, PPD treatment alsodownregulated the mRNA levels of the full-length AR andthe AR-Vs, namely AR3, AR4 and AR5. To determinewhether the reduced transcript levels were contributed byPPD inhibition of AR promoter activity, we constructed tworeporter gene plasmids of the human AR promoter, pGL4-ARpro8 and pGL4-ARpro1.7. The 8-kb fragment (�6,885 to

Figure 2. (a) PPD downregulation of PSA mRNA in LNCaP cells.

Cells were treated with 20 lg ml�1 PPD for indicated time in the

presence or absence of 1 nM DHT, and PSA mRNA determined by

qRT-PCR. * and **, p < 0.01 from control or DHT-treated sample,

respectively. (b) PPD downregulation of PSA protein in LNCaP cells.

Cells were treated with 20 lg ml�1 PPD for indicated time, and

PSA protein detected by Western blotting. (c) PPD downregulation

of PSA mRNA in LAPC-4, C4-2 and 22Rv1 cells. Cells were treated

with 20 lg ml�1 PPD for 12 hr, and PSA mRNA determined. *, p <

0.01 from vehicle control. (d) No weakening of PPD effect on PSA

expression by increasing concentrations of DHT. LNCaP cells were

treated with 20 lg ml�1 PPD and indicated concentration of DHT

for 6 hr, and PSA mRNA determined by qRT-PCR. *, p < 0.01 from

respective DHT-treated sample.

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Cao et al. 5


þ1,115) in pGL4-ARpro8 contains �6.9 kb of the 50-flank-ing region upstream of the entire 50-untranslated region, andthe 1.7-kb fragment (�600 to þ1,115) in pGL4-ARpro1.7contains 600 bp of the 50-flanking region upstream of theentire 50-untranslated region. LNCaP cells transfected withpGL4-ARpro8 or pGL4-ARpro1.7 were exposed to PPD for12 hr. As shown in Figure 4c, PPD treatment led to a �80%reduction of the activity of both promoters. The data there-fore demonstrate the ability of PPD to suppress the tran-scription of the AR gene. However, suppression of transcrip-tion may not contribute to the initial decline of AR protein.This is because PPD downregulation of AR transcripts wasnot observed until 12 hr after treatment (Figures 4a and 4b),lagging behind the drop in protein (evident already at 6 hr,Fig. 3a). The initial rapid decline of AR protein, instead,may be the result of PPD modulation of AR proteindegradation.

PPD induction of proteasome degradation of AR

Proteasome-mediated pathway has been described as themain machinery regulating AR protein degradation.29–31 Todetermine the involvement of proteasome in PPD-mediateddecrease of AR protein, we assessed the effect of the MG132proteasome inhibitor on PPD downregulation of AR inLNCaP cells and AR3 (the only AR-V for which a specificantibody is available) in 22Rv1 cells. Cells were pretreatedwith PPD for 6 hr prior to the addition of MG132, and thetreatment continued for an additional 6 hr. The reason thatMG132 was only present during the last 6 hr of treatmentwas because of the cytotoxicity associated with longer treat-ment with PPD and MG132. As shown in Figure 4d, treat-ment with MG132 alone did not cause a significant changein AR or AR3 protein level (comparing lanes 1 and 2). Thisis consistent with previous reports.32,33 However, MG132treatment was able to reverse the PPD effect, bringing PPD-suppressed AR or AR3 level respectively from 38 or 48% ofcontrol (Lane 4) back to 64 or 68% of control (Lane 5), thesame as the pre-MG132 level (Lane 3). The data thus indi-cated that promoting AR degradation through the protea-some pathway contributes significantly to PPD downregula-tion of both the full-length and truncated AR. Todemonstrate the specificity of PPD-induced AR degradation,we assessed the effect of PPD on the level of the p53 protein,which is known to be regulated by the proteasome-mediatedpathway.34 No change in p53 protein level was detected (Sup-porting Information Fig. 1).

PPD disruption of AR N–C interaction

The stability of the AR protein has been reported to be regu-lated by Hsp90 and the interaction between the N- and C-termini of AR. Inhibiting Hsp90 activity or disruptingHsp90-AR association leads to reduced stability andincreased proteasomal degradation of AR.32,35 AR N–C inter-action stabilizes AR by preventing ligand dissociation and ARdegradation.36 We therefore characterized the effect of PPDon Hsp90-AR association and AR N-C interaction. The asso-ciation of Hsp90 and AR was evaluated by immunoprecipita-tion. No change in Hsp90-AR association was observed (Fig.4e). We then assessed AR N–C interaction using a mamma-lian two-hybrid system24 in AR-null PC-3 cells to avoid in-terference from the endogenous AR.25 Cells transfected withthe mammalian two-hybrid system were exposed to DHTwith or without PPD for 3, 6, or 12 hr. As shown in Figure4f, exposure to DHT resulted in AR N–C interaction, whichwas markedly inhibited by PPD.

PPD suppression of tumor growth and AR expression

in vivo

Male nude mice implanted with LNCaP cells on both dorsalflanks were divided into two groups receiving either olive oilas control (n ¼ 8 mice) or 40 mg kg�1 PPD (n ¼ 11 mice)through oral gavage 6 days per week. As shown in Figure 5a,

Figure 3. (a) PPD downregulation of AR protein. Western blot

analysis of AR protein in cells treated with 20 lg ml�1 PPD. (b)

Specific knockdown of the full-length AR does not affect PPD

downregulation of PSA mRNA. The 22Rv1 cells infected with

lentivirus encoding the control shRNA or a full-length-AR-specific

shRNA9 were treated with 20 lg ml�1 PPD in androgen-depleted

condition, and the cell lysates subjected to qRT-PCR analysis of

PSA mRNA or Western blot analysis of AR proteins. * and **, p <

0.01 from untreated control-shRNA-transfected or AR-shRNA-

transfected cells, respectively. (c) PPD downregulation of AR

protein in the presence or absence of DHT. Western blot analysis

of AR protein in LNCaP or 22Rv1 cells treated with 20 lg ml�1 PPD

in the presence or absence of 1 nM DHT for 6 or 12 hr. The

numbers in the tables denote relative normalized intensities of the

AR protein bands compared to the untreated control value of 100.

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palpable tumors appeared in both groups on day 16. How-ever, the average tumor size in the PPD group was signifi-cantly smaller than that in the control group. At the conclu-sion of the study (Day 65), the tumor incidence was 87.5%(14/16) in the control group, but 59% (13/22) in the PPDgroup, representing a 32.6% decrease of tumor take rate byPPD. Additionally, the average weight of the tumors at theend of the study was 0.43 6 0.07 g in the control group, but0.22 6 0.04 g in the PPD group, indicating an �49% inhibi-tion of tumor growth by PPD (Fig. 5c). Each individual tu-mor was considered as independent in the above data analy-sis. We also calculated tumor volumes and weightsconsidering each individual mouse as independent, and tu-mor volumes and weights remain statistically differentbetween control and PPD treatment groups (Supporting In-formation Fig. 2). PPD supplementation did not appear tocause severe toxicity since mice in the treatment group hadsimilar body weights as the control mice (Fig. 5b).

We then measured PSA levels by ELISA in mouse serumand AR protein expression by immunohistochemistry in for-malin-fixed tissues. Mean serum PSA concentration afternormalization by tumor weight was reduced by one third byPPD (Fig. 5d). The result of quantitative analysis of AR im-

munostaining of the xenograft tumors is shown in the toppanel of Figure 6a. The quantitation was performed accord-ing to the method developed by Kim et al. for optimized im-munohistochemical quantitation of AR.27 Images weresampled sequentially throughout sections from four tumorsin the control group and five tumors in the PPD group usinga 20� objective, with areas of necrosis, preparation artifactsor edges avoided. Depending on the size of the tumors, 10–25 image fields were captured for each tumor section, anddigitized and quantitated as described in Materials and Meth-ods. The data are presented as percentage of pixels with vary-ing AR immunostaining intensity. PPD treatment resulted ina shift in the distribution of AR intensity, from the highertwo tertiles to the lowest tertile. The mean pixel intensity inthe PPD group was decreased to 73% of the control. Thedata therefore corroborated our in vitro results, showing theeffectiveness of PPD in downregulating AR level and activityin cancer cells in vivo. On the other hand, no change in ARexpression was observed in the normal prostate glands ofmice (Fig. 6b). Hematoxylin and eosin (H & E) staining ofthe tissues showed no apparent histopathological change ofthe tumors or the prostate glands after PPD treatment (Figs.6c and 6d).

Figure 4. (a,b) PPD downregulation of AR transcripts. LNCaP, LAPC-4, C4-2 cells (a) or 22Rv1 cells (b) were treated with 20 lg ml�1 PPD,

and subjected to qRT-PCR analysis of the full-length (AR) and truncated isoforms (AR3, AR4 and AR5) of AR mRNA. *, p < 0.01 from control.

(c) PPD suppression of AR transcription. LNCaP cells transfected with the AR-promoter-luciferase construct, pGL4-ARpro8 or pGL4-ARpro1.7,

were treated with 20 lg ml�1 PPD for 12 hr, and luciferase activity analyzed. *, p < 0.01 from untreated control. (d) PPD induction of

proteasome-mediated AR degradation. Western blot analysis of AR or AR3 protein in LNCaP or 22Rv1 cells, respectively. Cells were

pretreated with 20 lg ml�1 PPD for 6 hr prior to the combined treatment with PPD and 10 lM MG132 for an additional 6 hr. The bar graph

represents densitometry analysis of the Western blot data from three independent experiments. * and D, p < 0.01 from control or cells

treated with PPD alone for 12 hr, respectively. (e) No effect of PPD on Hsp90-AR association. LNCaP cells cultured in hormone-deprived

condition were treated with 20 lg ml�1 PPD for 2 or 4 hr, and immunoprecipitation was conducted with anti-AR antibody or mouse IgG.

Two top panels are Hsp90 or AR Western analysis of the immunoprecipitates. Two bottom panels are Hsp90 or AR Western analysis of the

input samples. (f) PPD disruption of AR N–C interaction. AR N–C interaction assay in PC-3 cells transfected with a mammalian two-hybrid

system. Cells were treated with 1 nM DHT 6 20 lg ml�1 PPD. * and **, p < 0.01 from control or DHT-treated sample, respectively.

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Figure 5. PPD inhibition of the growth of LNCaP xenograft tumors and PSA secretion. (a) Mean tumor volumes in each group (n ¼ 14 for

the control group; n ¼ 13 for the PPD group). *, p < 0.05 from the control group. (b) Mean body weights of the mice in each group. (c)

Weights of individual tumors in each group at the conclusion of the experiment. *, p < 0.01 from the control group. (d) Mean serum PSA

levels, normalized by tumor weights, in each group at the conclusion of the study. *, p < 0.05 from the control group.

Figure 6. PPD downregulation of AR in LNCaP xenograft tumors. (a) AR immunohistochemical staining of sections of LNCaP xenograft

tumors. Upper panel: Quantitation of the results, which is presented as % of pixels in each image field with different AR intensity. The

numbers next to the distribution curves represent the mean pixel intensity of the respective group 6 SEM. *, p < 0.01 from the control

group. The vertical dashed lines segment the three tertiles of immunostaining intensity. Lower panels: Representative images from control

and PPD groups (magnification, �200). (b) AR staining of sections of mouse dorsolateral prostates. (c,d) H&E staining of sections of LNCaP

xenograft tumors or mouse dorsolateral prostates, respectively.

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DiscussionThe present study represents the first to demonstrate PPD asan effective agent to downregulate the expression and activityof both the full-length AR and the AR-Vs in prostate cancer.This report is also the first to show the in vivo efficacy ofPPD against AR-expressing prostate cancer cells, which con-stitute the majority of clinical prostate carcinomas. We fur-ther show that PPD suppression of tumor growth is accom-panied by a considerable decline in AR expression in prostatetumor xenografts and PSA levels in the serum. The declinein serum PSA levels is not simply a consequence of reductionof tumor sizes by PPD, as the effect remains pronounced af-ter normalization by tumor weight. The data instead indicatethe ability of PPD to suppress AR activity in the tumors.

Despite its structural similarity with androgens, PPD haslimited ability to compete with androgens for binding to AR.Instead, the effect of PPD on AR is manifested by an imme-diate drop in protein, followed by a reduction in mRNA. ARis known to negatively autoregulate its transcription.37 Adecline of AR protein would be expected to lead to anincrease in AR transcription. The fact that we observe adecrease in AR transcription following a drop of protein indi-cates that PPD could turn on a mechanism counteracting ARnegative autoregulation. This is significant because it couldlead to a sustained downregulation of AR.

We further demonstrate that the initial decrease in ARprotein could be attributed to induction of proteasome-medi-ated AR degradation, possibly resulting from PPD disruptionof AR N–C interaction. The intermolecular interactionbetween the AR N-terminal and C-terminal regions is knownto stabilize the full-length AR by preventing ligand dissocia-tion and AR degradation.36 Because AR3 and all the otherAR-Vs identified to date retain an intact N-terminal do-main,8–11,38,39 it is reasonable to believe that the full-lengthAR can also form N–C interaction with the AR-Vs. In fact,ARv567es, another major AR-V, has been shown by coimmu-noprecipitation assay to interact with the full-length AR.11

On the other hand, AR/AR3 complex has not been detectedby coimmunoprecipitation in cell lines expressing a relativelyhigh level of endogenous AR3, including 22Rv1,9,38 althoughthe activity of AR3 has been indicated to rely on the presenceof the full-length AR.39 We have also used an antibody rec-ognizing both AR and AR-Vs or specific for AR3 or an anti-FLAG antibody to precipitate the complex in an AR-null cellline with exogenously introduced AR3 together with non-tagged full-length AR or AR3 together with FLAG-taggedfull-length AR. However, no interaction between these twoforms of AR was observed even when both were highlyexpressed in the cells (data not shown). Thus, whether PPDinduction of AR3 protein degradation results from PPD dis-ruption of AR N–C interaction awaits to be determined.

While PPD suppresses AR expression in the human pros-tate tumor xenografts, no change in AR expression isdetected in normal mouse prostates. Would the differentialresponse be due to differences in regulation of mouse versus

human AR expression or the existence of a cancer-specificfactor that is amenable to PPD modulation? In aligning thesequences of the human and mouse AR proteins, we foundthat the sequences are highly homologous, with 100% homol-ogy at the DNA-binding domain, LBD, dimer interface, andcoactivator recognition sites and 90% homology at the N-ter-mini. The nucleotide sequences of the 50-flanking region ofthe human and mouse AR gene are also highly conserved,and the transcription factors that have been shown to regu-late the transcription of the mouse AR gene have also beendemonstrated to control the expression of the human ARgene.37,40–42 Mechanism underlying degradation of the mouseAR protein is largely unknown. It is possible that differencesin AR protein degradation pathways exist between the twospecies, and could contribute to the differential response ofthe human tumor xenografts and normal mouse prostates toPPD. On the other hand, a cancer-associated factor(s) couldalso be involved in the differential response. Further researchis needed to address these possibilities.

Androgens play a key role in the development of prostatecancer. The ability of finasteride and dutasteride to reduceprostate cancer incidence attests to the viability of targetingandrogen signaling axis for prostate cancer chemoprevention.PPD suppresses androgen signaling through reducing recep-tor availability, and thus is at a different level from finaster-ide and dutasteride, which block the formation of DHT.Thus, combining these two types of drugs would be expectedto produce a more pronounced suppression of AR transacti-vation and thereby an increased chemopreventive efficacy.Additionally, finasteride treatment has been shown to lead toAR upregulation in prostate tissue.6 The fact that PPD down-regulates not only the full-length AR but also the AR-Vs thatlack the LBD indicates that it could be helpful for targetingthe androgen signaling that is resistant to finasteride anddutasteride.

The benefit of PPD intervention might also be extendedto the prevention of relapse after androgen deprivation ther-apy. Although recurrent castration-resistant prostate cancer isno longer responsive to androgen deprivation, the expressionand signaling of AR are maintained.43 Agents such as abira-terone are effective in lowering PSA and prolonging survivalin patients with castration-resistant prostate cancer, but eventhis irreversible CYP17 inhibitor has a relatively short timeto disease progression in these patients.44 One mechanism ofresistance to abiraterone has been ascribed to increasedexpression of the full-length AR and AR-Vs.45 Elevatedexpression of the full-length AR has been reported to be suf-ficient to convert prostate cancer growth from an androgen-dependent to castration-resistant stage.7 Prevalent upregula-tion of AR-Vs in castration-resistant prostate cancer has alsobeen shown to contribute to castration resistance.8–11 Thus,the fact that PPD can effectively reduce the abundance ofboth the full-length AR and the AR-Vs also indicates its greatpotential as an adjuvant to androgen deprivation therapy. Inaddition to PPD, several other natural or synthetic

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compounds have been shown to inhibit AR-Vs.46–50 The discov-ery of these compounds should generate excitement becausethere is no drug currently in clinical trials to target castration-re-sistant progression driven by the AR-Vs. Taken together, thefindings from the current study provide strong rationale for fur-ther developing PPD or its analogue as a promising agent forthe prevention and/or treatment of prostate cancer.

AcknowledgementsThe authors thank Dr. Charles Sawyers, now at Memorial Sloan Ket-tering Cancer Center, for LAPC-4 cells, Dr. Shahriar Koochekpournow at Roswell Park Cancer Institute for C4-2 cells, and Dr. Elizabeth

Wilson at the University of North Carolina for the AR N-C interac-tion mammalian two-hybrid system. They are very grateful to MaryPrice (The Louisiana Cancer Research Consortium FACS Core) andDina Gaupp (Tulane Center for Gene Therapy Histology Core Facil-ity) for their excellent technical assistance. This work was supportedby National Cancer Institute grant K01CA114252 (YD), AmericanCancer Society grant RSG-07-218-01-TBE (YD), Chinese ScholarshipCouncil Fellowship (BC), Louisiana Cancer Research Consortium Start-up Fund (YD and HZ), Tulane Cancer Center Developmental Fund(YD), Tulane University School of Medicine Pilot Fund (YD), andNational Major Special Project for Science and Technology Develop-ment of Ministry of Science and Technology of China 2009ZX09304-003 (JG).

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20(s)-protopanaxadiol-aglycone downregulation of the full-length and splice variants of androgen...

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