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Grape-derived Chemopreventive Agent Resveratrol Decreases Prostate-Specific Antigen (PSA) Eapression in LNCaP Cells by an Androgen Receptor (AR)-Independent Mechanism Tze-chen Hsfeh, and Joseph M. Wu* *A,jfliation ojauthors: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York. Correspondence to: Joseph M. Wu, Ph.D., Room 147, Department of Biochemistry and Molecular Biology, Basic Sciences Building, New York Medical College, Valhalla, NY 10595. Telephone: (914)594-4891; Fax: (914)594-4058; E-mail: Joseph Wu@nymc.edu Key Words: Resveratrol, prostate LNCaP cells, prostate specific antigen (PSA), androgen receptor (AR)

Prostate carcinoma is the most frequently diagnosed male cancer and the second leading cause of cancer-related deaths in men in Western society (1). Risks for prostate cancer range from unmodifiable factors such as age, race, genetics, to modifiable ones such as diet and nutrition, occupational exposures, and possibly vasectomy (2). Unlike other forms of malignancy, prostate carcinoma is unique in that initially the cancer cells are relatively slow growing and sensitive to androgen ablation, and may remain subclinical for an extended period of time. When the disease progresses to an advanced stage, the cells are highly proliferative and extremely malignant, become hormone refractory and resistant to therapies, and as a result produce high fatality (3). Because of these distinct features of prostate carcinogenesis, early detection and easily compliant preventive measures should be considered as complements to formulation of new treatment strategies. Prostate-specific antigen (PSA) is a 34-kD kallikrein-like serine protease produced exclusively by the epithelial cells lining the prostatic acini and ducts. Clinically PSA is most frequently utilized as a serum marker for screening, diagnosis and staging of prostate cancer, and as a prognosticator of patients' response to treatment modalities (4-6). The PSA gene, with its five exons spanning over approximately 6 kb on the long arm of chromosome 19 (7), is known to be under the control of androgens in the androgen- responsive LNCaP cells (8,9), by a mechanism involving the binding of the androgen- activated AR to androgen responsive elements, including the steroid receptor binding consensus sequences (10,11). Several studies show the coordinated expression of AR and PSA in LNCaP cells (12-16). Dietary factors may contribute to as much as a third of potentially preventable cancers and the long-term preventive^effect of plant-based diets on turmorigenesis and other 2 2SOS-v42/S~.

chronic diseases is well-documented (17). Resveratrol (3,5,4'-trihydroxystibene), a phytoalexin with a relatively broad distribution in plants and in human foods such as grapes, peanuts and mulberries, was recently reported to exhibit chemopreventive properties (18). In model assay systems resveratrol was shown to display anti-initiation, antipromotion and antiprogression activities (18). The goal of this study was to investigate whether resveratrol may affect prostate cell proliferation and modulate the expression of AR and PSA in a coordinate manner. Accordingly we determined the growth modulatory activities of resveratrol using the androgen-responsive human prostatic LNCaP cells, as follows. LNCaP cells were treated with 2.5x10-5, 2.5x10-6 and 2.5x10'' M of resveratrol for 2 to 4 days. Cell count at each of the respective time points was determined. No inhibition of growth was seen at the lowest concentration of resveratrol. At the higher 2.5x10"' M and 2.5x10-6 M, however, a statistically significant time-dependent suppression of proliferation was observed in treated cells, as compared to controls (Table 1). Moreover, at 2.5x10'S M resveratrol, approximately 9-15% of the cells became apoptotic after 4 days of treatment (data not shown). To determine the effects of resveratrol on expression of prostate specific genes, the levels of intracellular and secreted PSA were measured using the TANDEM-E kit (assay 1), as well as Western blot analysis (assay 2). In both assays, the amount of biological specimens was varied to verify that changes in the PSA were proportional to the control and treated extracts or media added. Results of assay I show that treatment with resveratrol resulted in a progressive reduction in both intracellular and secreted PSA (Fig. 1, panels A and B). After 4-daytreatment, intracellular and secreted PSA levels decreased 3

by approximately 80%, as compared to controls. Similar results were obtained using assay 2 (Fig.1, panel C). Because expression of the PSA gene is coupled to the activation of AR by androgens (12-16), it is of interest to ask whether the PSA/AR genes may be similarly controlled, in a coordinated manner, by resveratrol. Accordingly, levels of AR were assayed by Western blot analysis, using the cognate antibody (Fig.1, panel C), or by binding with the radioactive ligand methyltrienolone [3H]R1881 (Fig. 1, panel D) (13,19). As controls, we also tested the ability of resveratrol to directly displace binding of the radioactive ligand to the androgen receptor. No competition was observed with resveratrol added at ten times the highest concentration used in these experiments. With either assay, little or no change in AR expression could be detected between control and resveratrol-treated cells. Thus, it would appear that the prostate tumor marker PSA is down regulated by resveratrol, by a mechanism independent of changes in AR. Since PSA has been suggested to regulate prostate cell growth (20), the decrease in PSA expression following treatment with resveratrol, may partly account for the resveratrol-induced growth suppression in LNCaP cells. The fact that rcsveratrol is capable of modulating the growth and specific phenotype expression in prostatic tumor cells independent of AR raise the possibility that it may be considered as an easily compliant preventive modality for prostate cancer with clinically measurable benefits. 4

References (1) Parker SL, Tong T, Bolden S, Wingo PA. Cancer statistics, 1997. CA Cancer J Clin 1997;47:5-27. (2) Bosland MC. Hormonal factors in carcinogenesis of the prostate and testis in humans and in animal models. In: Huff J, Boyd J, and Barrett JC, eds. Cellular and molecular mechanisms of hormonal carcinogenesis: Environmental influences. Wiley-Liss, Inc., 1996:309-352. (3) Isaacs JT. Prostatic cancer: an age-old problem. In: Yang SS, and Warner HP, eds. The underlying molecular, cellular, and immunological factors in cancer and (4) (5) (6) aging. Plenum Press, 1993:167-184. Shih WJ, Collins J, Mitchell B, and Wierzbinski B. Serum PSA and PAP measurements discriminating patients with prostate carcinoma from patients with nodular hyperplasia; Keetch DW, and Andriole GL. The use of tumor markers in prostate cancer. In: Dawson NA, and Vogelzang NJ, eds. Prostate cancer. Wiley- Liss, Inc., 1994: 95-112. Whittemore AS, Lele C, Friedman GD, Stamey T, Vogelman JH, Orentreich N. Prostate-specific antigen as predictor of prostate cancer in black men and white men. J Natl Cancer Inst 1995;87:354-360. Herrada J, Dieringer P, and Logothetis CJ. Characterization of patients with androgen-independent prostatic carcinoma whose serum prostate specific antigen decreased following flutamide withdrawal. J Urol 1996;155:620-623. 5

(7) Riegman PH, Vlietstra RJ, van der Korput JA, Brinkmann AO, and Trapman J. The promoter of the prostate specific antigen gene contains a functional androgen responsive element. Mol Endocrinol 1991;5:1921-1930. (8) Montgomery BT, Young CY, Bilhartz DL, Andrews PE, Prescott JL, Thompson NF, and Tindall DJ. Hormonal regulation of prostate-specific antigen (PSA) glycoprotein in the human prostatic adenocarcinoma cell line, LNCaP. The Prostate 1992;21:63-73. (9) Gau JT, Salter RD, Krill D, Grove ML, and Becich M.J. The biosynthesis and secretion of prostate-specific antigen in LNCaP cells. Canc Res 1997;57:3830- 3834. (10) Lindzey M, Kumar MV, Grossman M, Young C, and Tindall DJ. Molecular mechanisms of androgen action. Vitamins and Hormones 1994;49:383-432. (11) Wang LG, Liu XM, Kreis W, and Budman DR. Down-regulation of prostate- specific antigen expression by finasteride through inhibition of complex formation between androgen receptor and steroid receptor-binding consensus in the promoter of the PSA gene in LNCaP cells. Canc Res 1997;57:714-719. (12) Lee C, Sutkowski DM, Sensibar JA, Zelner D, Kim I, Amsel I et al. Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone- Endocrinology 1995;136:796-803. (13) Hsieh TC, Ng CY, Mallouh C, Tazaki H, and Wu JM. Regulation of growth, PSA/PAP and androgen receptor expression by 1a,25-dihydroxyvitamin D3 in the 6

androgen-dependent LNCaP cells. Biochem Biophys Res Commun 1996;223:141- 146. (14) Hsieh TC, Chen SS, Wang W, and Wu JM. Regulation of androgen receptor (AR) and prostate specific antigen (PSA) expression in the androgen-responsive human prostate LNCaP cells by ethanolic extracts of the Chinese herbal preparation, PC- SPES. Biochem Mol Biol Int 1997;42:535-544. (15) Hsieh TC, and Wu JM. Induction of apoptosis and altered nuclear/cytoplasmic distribution of the androgen receptor and prostate-specific antigen by 1a,25- dihydroxyvitamin D3 in androgen-dependent LNCaP cells. Biochem Biophys Res Commun 1997;235:539-544. (16) Konno S, Mordente JA, Chen Y, Wu JM, Tazaki H, and Mallouh C. Effects of brefeldin A on androgen receptor-mediated cellular responses in human prostatic carcinoma LNCaP cells. Mol Urol 1998;2:7-11. (17) Miller AB. Diet and cancer: a review. Rev Oncol 1990;3:87-95. (18) Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CWW, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997;275:218-220. (19) Turcotte G, Chapdelaine A, Roberts KD, and Chevalier S. Androgen binding as evidenced by a whole cell assay system using cultured canine prostate epithelial cells, J Steroid Biochem1988;29:69-76. (20) Wang LG, Liu XM, Kreis W, and Butman DR. Involvement of prostate specific antigen in the stimulation of LNCaP cell growth. Oncol Rep 1996;3:911-917. 7

Table 1. Cell culture and treatment with resveratrol. LNCaP cells were maintained and cultured as previous publications from this laboratory (13-15). Cells were treated with different concentrations of resveratrol (2.5x10"5, 2.5x10-6 and 2.5x10"' M). A stock solution of resveratrol (Sigma Chemical Co.) was prepared at a concentration of 12.5 mM in dimethyl sulfoxide (DMSO) and stored at -20°C. The resveratrol was diluted in RPMI1640 medium to the desired final concentrations. The carrier solvent (0.2 % DMSO) was added into control cultures. On days 2-4, cells were harvested to check cell number and cell viability. Media from control and treated cells were saved for PSA analysis. Resveratrol Percent Control Cell Growth concentration (M) 2 day 4 day 2.5x10'' 105.7 ± 6.4 2.5x10' 88.2 *±11.1 (p<0.05) 36.3 *±12.4(p<0.01) 2.5x10"5 70.7 *± 15.7 (p < 0.01) 49.8 * ± 15.9 (p < 0.01) The results shown represent the mean ± SD calculated from 2 experiments in triplicate. 8

Fig. 1. Panels A and B. Measurement of intracellular and secreted PSA expression. The TANDEM-E PSA kit used to measure the intracellular and secreted levels of PSA was based on the quantitative binding of PSA by the cognate antibody, followed by the retention of PSA:antibody complex on alkaline phosphatase-tagged IgG-coated beads, and the cleavage of the substrate p-nitrophenyl phosphate by the IgG-tagged alkaline phosphatase. The colored products were quantified by measuring absorbency at 405- and 450-nm (13-15). Panel C. Western blot analyses of AR, PSA and actin were performed as follows. PSA, AR and actin antibodies were obtained from commercial sources. Postmitochondrial extracts were prepared from control and treated cells using buffers supplemented with multiple protease inhibitors as previously descrcibed (13-17). Extracts were separated on 10 % SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with the respective primary and secondary antibodies. Specific immunoreactive bands were visualized with enhanced chemiluminescence system (ECL) or by color reaction, as described (13-15). Reprobing of blots was done after stripping with a buffer containing 62.5 mM Tris-HCI, pH 6.7, 100 mM 2-mercaptoethanol, 2 % SDS, at 50°C for 30 min. Panel D. For measurement of AR expression by binding to ['H]R1881, control and resveratrol-treated-cells were incubated with plain media or with media supplemented with 1.25 µM unlabeled R1881, for 15 min. They were labeled with 5 nM [3H]-R1881 (87 Ci/mmol, NEN) for 2 h at 37°C. Specific binding to labeled R1881 was assayed as described (13) using 2.5x10'3 M resveratrol-treated cells. The ability of resveratrol to compete for the binding of R1881 was determined in separate, control experiments. At a concentration ten times higher than the range used in these studies, resveratrol did not compete for binding of R1881 ^ 9

Notes Supported in part by the Vivian Wu-Au Memorial Cancer Research Fund. We thank Dr. Zhou Bao-sen for his statistical advice. 10