Proc. West. Pharmacol. Soc. 46: 153-155 (2003) Effect of β-sitosterol as Inhibitor of 5α-reductase in Hamster Prostate Marisa Cabeza1*, Eugene Bratoeff2, Ivonne Heuze1, Elena Ramírez2, Mauricio Sánchez1 and Eugenio Flores2 Department of Biological Systems & Animal Production Metropolitan University-Xochimilco1, Mexico D. F., Mexico, Department 2 of Pharmacy, Faculty of Chemistry of National University of Mexico, Mexico D. F. *email: marisa@cueyatl.uam.mx It has been reported that β-sitosterol (U) obtained from plants inhibits the growth and migration of one type of prostate cancer cell and to slow the growth of prostate tumors in laboratory mice [1]. These data suggest that an androgenic mechanism of action could be involved, since growth of most prostate cancers is androgen-dependent. On the other hand β-sitosterol like other drugs derived from plants have a long tradition in the medical treatment of benign prostate hyperplasia (BPH) in Europe. At the present time no mechanism of action or precise classification of the active compounds for many of these drugs has been established, although substantial symptomatic improvement has been reported. Recently it has been reported that βsitosterol significantly improves the symptoms and urinary flow parameters present in BPH [2]. The purpose of this study was to determine if βsitosterol has an effect on the weight of hamster prostate. Also it was of interest to know if this steroid could act an inhibitor of 5α-reductase, the enzyme that converts testosterone (T) to his more active form, dihydrotestosterone (DHT)[3]. Furthermore it was of interest to determine if this compound binds to the androgen receptor. METHODS: Animal treatment: Adult male golden hamsters (150200 g) were obtained from Metropolitan University-Xochimilco of Mexico. Gonadectomies were performed under light ether anesthesia 30 d before the experiments. The biological activity of the pure compound (U), obtained from Sigma-Aldrich, was determined in gonadectomized male hamsters divided in several groups. Daily subcutaneous injections of 400 and 800 µg of U dissolved in 200 µl of sesame oil were administered for 6 d together with 200 µg of T. Two groups of animals were kept as a control; one was injected with 200 µl of sesame oil, the second with 200 µg of T for 6 days. After treatment, animals were sacrificed by ether anesthesia and the prostate dissected and weighed In vitro metabolic studies with prostates: Prostates from gonadectomized animals were removed, blotted and weighed prior to use. Tissues were homogenized in 3 volumes of TEDAM buffer (20 mM tris-HCl, 1.5 mM EDTA and 1 0mM sodium molybdate) at pH 7.4 (4ºC). Homogenates were centrifuged at 140,000 x g for 60 min [8] in a SW 60 Ti rotor (Beckman instruments, Palo Alto, CA). Pellets were separated, washed with 3 tissue volumes of medium A [20 mM potassium phosphate, pH 7 containing 0.32 M sucrose, 0.1 mM dithiothreitol (Sigma-Aldrich, Inc)] and centrifuged two additional times at 440 x g at 0ºC for 10 min [4]. Washed pellets were suspended in medium A and stored at -70ºC. The suspension (6.8 mg protein/ml determined by the Bradford method [5]) was used as source of 5α-reductase. Inhibitory effect of finasteride and U on hamster prostatic 5∝reductase: In order to calculate IC50 (the concentration of the steroid required to inhibit 5α-reductase activity by 50%), two series of tubes containing different concentrations of finasteride (100 pM-100 nM) or U (20 nM-10 µM) were incubated in duplicate, in the presence of: 1 mM of dithiothreitol, 40 mM sodium phosphate buffer pH of 7, 2 mM NADPH, 2 nM [1,2,6,7H3]T (specific activity 95 Ci/mmol) and 250 µg of protein in a final volume of 1 ml. The reaction was carried out in duplicate at 37ºC for 60 min and stopped by mixing with 1 ml of dichloromethane. The dichlorometane fraction was separated and the extraction was repeated x4. The extract was evaporated to dryness under a nitrogen stream and suspended in 50 µl of methanol spotted on HPTLC Keiselgel 60 F254 plates. T and DHT were used as carriers and the plate was developed in chloroform:acetone=9:1. Plates were air-dried and the chromatography was repeated x2. T standard was visualized under UV lights [254 nm] and DHT was detected using phosphomolibdic acid reagent (8% in methanol) and heating of the plate. DHT containing areas were cut out and the strips soaked in 5 ml in Ultima Gold [Packard] and the radioactivity measured by scintillation. Competition binding studies: Tubes containing 3.15 nM [H3]DHT (Specific activity 110 Ci/mmol) plus a range of increasing concentrations (10-8-10-4 M) of nonradioactive DHT and U were prepared [6]. Aliquots of 200 µl of prostatic cytosol (2.4 mg protein, determined by the Bradford method [5]) were added and incubated (by duplicate) for 18 h at 4º C. After this time, 800 µl of dextran-coated charcoal in TEDAM buffer (containing dithiotreitol) was added and the mixture incubated for 40 min at 4°C. To prepare the dextran-coated charcoal mixture, dextran was agitated for 30 min before adding the charcoal to the mixture. The tubes were vortexed and immediately centrifuged at 800 g for 10 min and 200 µl aliquots counted by scintillation. RESULTS: After gonadectomy the weight of the prostate decreased significantly (p<0.005). Treatment with vehicle alone (control) did not change this whereas subcutaneous injection of 200 µg of T for 6 d significantly increased (p<0.005) the weight of the prostate in gonadectomized male hamsters (Table I). When T and U were injected together at two different doses, the weight of seminal vesicles decreased in a dose dependent manner suggesting an inhibitory effect of 5α-reductase. Duplicate experiments were carried out in quadruplicate. The mean of weights are shown in Table I. 153 Table I. Effect of β-sitosterol (U) on prostatic weight in gonadectomized hamsters treated with T. The weight of the prostate is given in mg ± standard deviation. Significant differences were observed between treated with T (p<0.009) and treated with T+U at different doses. 100 90 80 Control T T+U (400 µg) T+U (800 µg) Prostate Weight (mg) 70 47.2 ± 9.9 60 % Binding Treatment 93.3 ± 26.9 72.1 ± 14.7 60.8 ± 7.2 20 100 % of activity of 5a-reductase Compound U 75 HO IC 50 = 2.7 mM 25 0 0 2 4 6 8 40 30 5α-reductase activity inhibition: Since the weight of the prostate depends on the 5α-reduced T [7], it was important to determine the effect of U on the in vitro activity of 5α-reductase. The results (Fig. 1) obtained from two separate experiments performed in duplicate demonstrated that β-sitosterol (U) inhibited 5α-reductase activity. The concentration of U necessary to inhibit 50% of the enzymatic activity (IC50) was of 2.7 µM at pH of 7, whereas that for finasteride, a type 2 selective 5α-reductase inhibitor, at pH of 7 was 10.12 nM (see: Cabeza et al. this volume). 50 50 10 mM concentration of U Figure 1. Inhibitory effect of β-sitosterol on hamster prostatic 5αreductase activity. Results are the average of two experiments. IC50 represents the β-sitosterol concentration necessary to inhibit 50% enzymatic activity. β-sitosterol competition of androgen receptor binding. The effect of increasing concentrations of non-radioactive steroids upon [H3]DHT binding to androgen receptors from male hamster prostate in two different experiments is shown in Fig. 2. U was a poor competitor and did not exhibit any apparent affinity for the androgen receptor. 10 0 0 200 400 600 800 1000 1200 [uM] Figure 2. Binding Specificity for the androgen receptor. Compound U (β-sitosterol) was a poor competitor at the androgen receptor, whereas cold DHT competed for [H3]DHT binding to the androgen receptor. DISCUSSION: This study describes the 5α-reductase inhibitory activity of the β-sitosterol. This compound significantly inhibits growth of the gonadectomized hamster prostate treated with T at two different doses thus indicating that β-sitosterol has a pharmacological activity from subcutaneous application in this model. In humans treated orally with β-sitosterol, no relevant reduction of prostatic volume was observed [2]. This difference between humans and hamsters may be attributable to the doses chosen or the purity of the compounds utilized [2]; we employed pure β-sitosterol purchased from Sigma-Aldrich, whereas Berges et al. [2] used a mixture of phytosterols. On the other hand β-sitosterol inhibited 5α -reductase activity with a value of IC50 2.7 µM, as compared to that of finasteride of 10.12 nM. These data indicate that β-sitosterol is less potent than finasteride in the inhibition of 5α-reductase. However, U did not exhibit any affinity to the androgen receptor thus indicating that the effect observed is not due to the binding of βsitosterol to the androgen receptor. ACKNOWLEDGEMENTS: We gratefully acknowledge the financial support of CONACYT for the project G-33450-M. REFERENCES: 1. Awad AB, Fink CS, Williams H, Kim U: Eur J Cancer Prev 10:507 (2001). 2. Berges RR, Windeler J, Trampisch HJ, Senge TH and the β-sitosterol study group: LANCET 345:1529 (1995). 154 3. Bruchovski N, Sadar MD, Akakura K, Goldemberg Sl, Matsuoka K, Rennie PS: J Steroid Biochem Mol Biol. 59:397 (1996). 4. Hirosumi J, Nakayama T, Fagan K, Sawada N, Chida N, Inami M, Takahashi S, Kojo H, Notsu Y and Okuhara M: J Steroid Biochem Mol Biol 52:357 (1995). 5. Bradfod MM, Anal Biochem 72:248 (1986). 6. Cabeza M, Quiroz A, Bratoeff E, Heuze I, Ramírez E and Flores E: Proc West Pharmacol Soc 45:166 (2002). 7. Brooks JR, Baptista EM, Berman C, Ham EA, Hichens HM, Johnston DBR, Primka RL, Rasmusson GH, Reynolds GF, Schmitt SM and Arth GE: Endocrinology 109: 830 (1981). 155 View publication stats