Combination of Resveratrol and Antiandrogen Flutamide Has Synergistic Effect on Androgen Receptor Inhibition in Prostate Cancer Cells
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ANTICANCER RESEARCH 31: 3323-3330 (2011) Combination of Resveratrol and Antiandrogen Flutamide Has Synergistic Effect on Androgen Receptor Inhibition in Prostate Cancer Cells LI KAI† and ANAIT S. LEVENSON Cancer Institute and Department of Pathology, University of Mississippi Medical Center, Jackson, MS, U.S.A. Abstract. Agents targeting the androgen receptor (AR) axis are signaling pathways (1-3). Therefore, agents other than critical for chemoprevention and treatment of prostate cancer traditional antiandrogens with the ability to inhibit AR (PCa) at all stages of the disease. Combination molecular production and block AR signaling are of great interest for both targeted therapy may improve overall efficacy. The combination chemopreventive and therapeutic strategies for all stages of PCa. of dietary compound resveratrol with known therapeutic agents, Diet-derived polyphenols are attractive clinical candidates such as the antiandrogen flutamide, may be particularly for cancer prevention and treatment because they are attractive due to the pharmacological safety of resveratrol. pharmacologically safe. Resveratrol (3,5,4’-trihydroxystilbene) Materials and Methods: Resveratrol, 5α-dihydrotestosterone is a natural phytoalexin that is synthesized in several plants as and flutamide were used in various experiments using mostly a defensive response against fungal infection and LNCaP cell line. Quantitative reverse transcription polymerase environmental stress. It is produced in the skin and seeds of chain reaction (qRT-PCR), Western blots, and luciferase assay grapes, with its further accumulation in red wine during the were utilized to examine the levels of AR mRNA, and protein fermentation process. Recently, epidemiological studies have and transcriptional activity in response to treatments. Growth demonstrated a reduced relative risk for PCa associated with proliferation assays were performed in three cell lines (LNCaP, an increased level of red wine consumption (4), which was PC3 and Du145). Results: Treatment of LNCaP cells with partly attributed to resveratrol content. resveratrol (1-100 μM) resulted in the inhibition of androgen- Resveratrol, also well known as an antioxidant, has promoted growth, inhibition of AR transcriptional activity and received increasing attention during the last fifteen years for decrease in the AR and prostate-specific antigen protein levels its potential chemopreventive and antitumor effects. From through degradation pathways. The combination of resveratrol accumulated data, it is clear that resveratrol exhibits with flutamide had a synergistic effect on down-regulation of numerous biological activities, such as inhibition of cancer AR. Conclusion: Resveratrol works in concert with cell proliferation, migration, invasion and angiogenesis and antiandrogen flutamide to reduce the amount and activity of AR, induction of apoptosis (5-11) by acting through multiple suggesting new therapeutic strategies for the treatment of PCa. signaling pathways. The goal of the current study was to examine the effects of The central role of androgen receptor (AR) signaling in prostate resveratrol on AR and its signaling (androgen-inducible cancer (PCa) is defined by the fact that PCa develops and target gene prostate-specific antigen, PSA) in PCa cells and progresses under the influence of androgens. Remarkably, AR to assess if combined treatment with the conventional continues to be expressed and active in hormone-refractory endocrine agent flutamide would result in superior effects. androgen-independent tumors ultimately using alternative Here, we report that resveratrol inhibits AR production and blocks AR signaling and acts as a 5α-dihydrotestosterone (DHT) antagonist in concert with pure antiandrogen flutamide. This makes resveratrol a very attractive and †Current address: Northwestern University, Chicago, IL, U.S.A. promising pharmacologically safe compound to be used in combination settings for improved therapeutic outcomes. Correspondence to: Anait S Levenson, Cancer Institute, University of Mississippi Medical Center, 2500 North State Street, Jackson, Materials and Methods MS 39216-4505, U.S.A. Tel: +1 6018156072, Fax: +1 6018156806, e-mail: [email protected] Materials. Resveratrol, DHT, flutamide, proteasome inhibitor N- [(phenylmethoxy)carbonyl]-L-leucyl-N-[(1R)-1-formyl-3-methylbutyl]- Key Words: Androgen receptor, combination strategy, flutamide, L-leucinamideN-benzoyloxy-carbonyl (MG132) and cycloheximide prostate cancer, prostate-specific antigen, resveratrol. (CHX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 0250-7005/2011 $2.00+.40 3323 ANTICANCER RESEARCH 31: 3323-3330 (2011) Caspase-3 inhibitor Benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val- activity. Cells were then treated with different compounds for 24 h Asp(OMe)-fluoromethylketone (Z-DEVD-FMK) was purchased from and harvested for the Dual-Luciferase Reporter Assay (Promega, Imgenex (San Diego, CA, USA). Compounds used in the experiments Madison, WI, USA). Luminometer Bio-TEK Synergy HT (Bio were dissolved in 100% ethanol or high purity dimethyl sulfoxide. TEK, Winooski, VT, USA) was used for measuring light emission. Resveratrol was stored in the dark at –20˚C to ensure its stability. Renilla luciferase activity was used for background normalization. Cell culture. LNCaP, PC3 and DU145 cells (ATCC, Manassas, VA, RNA isolation and quantitative RT-PCR (qRT-PCR). RNA was USA) were grown in RPMI- 1640 containing 10% fetal bovine isolated using RNeasy Kit (Qiagen, Valencia, CA, USA) from the serum and 1× penicillin-streptomycin-amphotericin B at 37˚C/5% resveratrol-treated cells. We performed qRT-PCR on a MiniOpticon CO2 as previously described (8, 12). For experiments involving Real-Time PCR System (Bio-Rad Laboratories) using the protocol treatment with resveratrol, DHT and flutamide, media were replaced of the SuperScript III platinum two-step qRT-PCR kit with SYBR with phenol red-free RPMI-1640 containing 5% charcoal-stripped green (Invitrogen, Carlsbad, CA, USA). Briefly, first-strand cDNA serum 16 hours prior to treatment to eliminate steroidal background was synthesized using 1 μg total RNA from treated cells. The PCR that can interfere with their action. RPMI-1640 was obtained from reaction was conducted in a reaction mix of 25 μl Platinum SYBR Gibco BRL (Gaithersburg, MD, USA). Green qPCR SuperMix-UDG with 1 μl cDNA template and 0.5 μl primers (10 μM each). The PCR reaction was run for 40 cycles at Western blot analysis. Western blot was carried out as described 95˚C for 30s, 60˚C for 30s and 72˚C for 30s. Primers were purchased previously. Briefly, LNCaP cells were treated with different from IDT (Coralville, IA, USA). The gene-specific primers for AR concentrations of resveratrol or other compounds for 24h. the cells and PSA were: forward: 5’-GGA CTT CAC CGC ACC TGA TG -3; were lysed by radioimmunoprecipitation assay (RIPA) buffer reverse: 5’-CTG GCA GTC TCC AAA CGC AT -3’ and forward: 5’- containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCI, 1% Nonidet- GCC TCT CGT GGC AGG GCA GT -3’; reverse: 5’- CTG AGG P40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate and GTG AAC TTG GGC AC -3’, respectively. β-Actin was amplified supplemented with protease and phosphatase inhibitors in parallel as the internal control. The analysis was performed using (Phosphatase Arrest II and Mammalian Protease Arrest, G- Opticon Monitor software version 3.1. The quantitation was carried Biosciences, St Louis, MO, USA).The protein concentration was out using the ΔΔCt method (14). Briefly, ΔCt for AR or PSA was measured using the Bio-Rad protein assay reagent (Bio-Rad calculated by subtracting the Ct of β-Actin mRNA from the Ct of Laboratories, Hercules, CA, USA). Equal amounts of protein (40- AR or PSA mRNA. ΔΔCt was then calculated by subtracting the ΔCt 70 μg) were resolved in 7-10% Tris-HCl Ready gels (Bio-Rad of the ethanol-treated control from the ΔCt of the resveratrol-treated Laboratories) and transferred to a nitrocellulose membrane by Mini samples. Fold change of the expressions of AR and PSA mRNA Trans-Blot Electrophoresis Transfer System (Bio-Rad were calculated by the equation 2–ΔΔCt. Laboratories). The membranes were then blocked and subsequently probed with AR (Thermo Fisher Scientific, Fremont, CA, USA) Statistical analysis. Each experiment was reproduced at least three and PSA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) times. Values are expressed as the mean±SEM of triplicate antibodies. The blots were also probed with β-Actin antibodies measurements unless otherwise stated. Student’s two tailed paired (Sigma-Aldrich) as a loading control. Signals were visualized using t-test was used to analyze differences between treated and control enhanced chemiluminescence. Images were quantified using Image cells. Combination index (CI) values (quantitative definition for J software, a public domain program at http://rsb.info.nih.gov/nih- additive effect (CI=1), synergism (CI<1), and antagonism (CI>1) in image/ . Nuclear extracts from LNCaP cells were isolated using drug combinations) for combination experiments were calculated by NE-PER Nuclear and Cytoplasmic Extraction Reagent (Pierce, Chou-Talalay method using CompuSyn software (ComboSyn, Inc. Rockford, IL, USA). Paramus, NY, USA). Cell proliferation assay. Du145 and PC3 cells were seeded at 5×105 Results cells/well density in 24-well plates using RPMI-1640 media. LNCaP cells were seeded at 1×106 cells/well density in 24-well plates using Resveratrol inhibits cell proliferation of both