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and Prostatic Diseases (2007) 10, 378–383 & 2007 Nature Publishing Group All rights reserved 1365-7852/07 $30.00 www.nature.com/pcan ORIGINAL ARTICLE

Suppressive effects of , finasteride and on development of prostatic lesions in a transgenic rat model

Y-M Cho1, S Takahashi, M Asamoto, S Suzuki, M Tang and T Shirai Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences, Mizuho-cho, Mizuho-ku, Nagoya, Japan

Transgenic (TG) rats bearing a probasin promoter/simian virus 40 T antigen (SV40 Tag) construct were treated with antiandrogens to examine their ability to suppress prostate carcinogenesis. Finasteride and flutamide were administered to 10-week-old TG rats five times a week for 2, 5 and 7 weeks. -treated exhibited atrophic glandular structures with almost no expression of SV40 Tag and only weak signals for receptors. Furthermore, quantitative data for ventral prostate adenocarcinomas showed significant decrease with antiandrogen treatment. Both finasteride and flutamide had the ability to suppress SV40 Tag-driven carcinogenesis through their different antiandrogenic mechanisms, suggesting that this TG model is suitable for exploring the potential of agents to inhibit development. Prostate Cancer and Prostatic Diseases (2007) 10, 378–383; doi:10.1038/sj.pcan.4500971; published online 24 April 2007

Keywords: chemoprevention; transgenic rat; finasteride; flutamide

Introduction Approaches to influence androgen levels in prostatic tissue include (a) inhibition of the pituitary secretion of Among US men, prostate cancer is the most common luteinizing hormone (LH) by LH-releasing hormone non-cutaneous neoplasm and the second leading cause (LHRH) analogs, (b) prevention of the conversion of of cancer death. It has been estimated that there will be to (DHT) by 5a-reduc- approximately 234 460 new cases of prostate cancer and tase in the prostate and (c) blocking prostatic androgen 27 350 associated deaths in the United States in 2006.1 receptors (ARs) using -like antagonists with no Over 60% of these newly diagnosed cases of prostate intrinsic activity, to reduce potentially unacceptable cancer will be pathologically advanced; at this stage, systemic toxicity.6 One such is the azasteroid there is no cure and the prognosis is dismal. Since the finasteride.7 Another potential suppressive agent is frequency of latent prostatic tumors has been shown to the nonsteroidal antiandrogen flutamide, which exerts increase with each decade of life from the 50s (5.3–14%) to effects by interfering with the binding of DHT or the 90s (40–80%)2 and relatively long time periods are testosterone to the AR.5 necessary for progression to actual prostate cancer, the The study of prostate cancer chemoprevention and concept of chemoprevention has attracted much attention. treatment has been hindered by the lack of appropriate The majority of prostate initially respond to animal models. Recently, we have established transgenic androgen ablation therapy because of hormone-depen- (TG) rats, designated TRAP (Transgenic Rat for Adeno- dent growth,3 but relapse with generation of hormone- carcinoma of the Prostate), bearing a probasin promoter/ independent cancer cell occurs and eventually leads to a simian virus 40 T antigen (SV40 Tag) construct in which fatal outcome in many cases.4,5 Although androgen androgen-dependent prostate cancers develop rapidly.8–10 ablation at this hormone refractory stage is ineffective, In our TRAP model, high-grade prostatic intraepithe- androgen deprivation strategy as an early intervention lial neoplasia (PINs) are found from 4 weeks of age, and may delay the promotion and/or progression of prostate prostate adenocarcinomas in the ventral, dorsolateral cancer, resulting in reduced morbidity and mortality. and anterior lobes are observed at 100% incidences before 15 weeks of age. Prostate adenocarcinomas Correspondence: Dr S Takahashi, Department of Experimental developing in TRAP are strictly androgen dependent in Pathology and Tumor Biology, Nagoya City University Graduate a pattern similar to that of human primary prostate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, cancer. TRAP lesions have many advantages, for Nagoya 467-8601, Japan. example, their large size allows adequate materials to E-mail: [email protected] be obtained and development of prostate cancer occurs 1Current address: Division of Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan in a shorter experimental period and more efficiently Received 20 December 2006; revised and accepted 26 March 2007; compared with several other rat prostate carcinogenesis 11–19 published online 24 April 2007 models. These characteristics of TRAP may make it Suppressive effects of antiandrogens, finasteride and flutamide Y-M Cho et al particularly suitable for evaluation of strategies for were demonstrated with diaminobenzidine as the 379 chemoprevention and treatment. The present study was substrate. conducted to assess whether finasteride and flutamide have the ability to suppress prostate carcinogenesis in our TRAP model. Statistical analysis Statistical analysis of differences between means and incidences was carried out using analysis of variance and Materials and methods the Kruskal–Wallis test, respectively. When positive results were obtained, the Mann–Whitney U-test with Animal and treatment Bonferroni correction was applied to evaluate the Male heterozygous TRAP rats established in our labora- statistical significance between treatment groups. tory with a Sprague–Dawley genetic background were used in the present study.8,9 They were housed three/ cage on wood-chip bedding in an air-conditioned animal Results room at 23721C and 50710% humidity, with food (Oriental MF, Oriental Yeast Co., Tokyo, Japan) and tap TRAP rats treated with finasteride or the high dose of water available ad libitum. Finasteride (Merck KGaA, flutamide for 5 and 7 weeks showed significantly Darmstadt, Germany) and flutamide (Nippon Kayaku (Po0.001) lower prostate weights relative to body Co. Ltd, Tokyo, Japan) were suspended in 0.5% methyl- weights as compared to controls (Table 1). Macroscopi- cellulose 400 cP (Wako Pure Chemical Industries Ltd, cally, prostates of TRAP in the non-treated group showed Osaka, Japan) and administrated to 10-week-old TG rats slight enlargement and irregular surfaces but no appar- five times a week via intragastric route at concentrations ent nodule or mass formation. In contrast, antiandrogen of 10 and 5 or 20 mg/kg, respectively. Animals were treatment was associated with obvious prostate and then killed at weeks 2, 5 and 7, and lesions in the prostate seminal vesicle . Histopathological findings for were histopathologically classified as described pre- prostatic lesions are summarized in Table 2. Antiandro- viously into PIN and adenocarcinoma categories.8 The gen treatment for 2 weeks caused destruction of areas of lesions in each prostate sample were quantita- glandular structure with inflammatory cell infiltration tively measured with an Image Processor for Analytical (Figure 1a). After treatment for 7 weeks, atrophic glands Pathology (Sumika Technos Co., Osaka). Testosterone were frequently observed with minimal nuclear atypia levels in serum were also analyzed by radioimmuno- (Figures 1b and 2). PINs and/or adenocarcinomas were assay at a commercial laboratory (SRL, Tokyo). detected in all lobes. There were no statistically sig- The present experiment was performed under proto- nificant differences in the incidences of PINs or cols approved by the Institutional Animal Care and Use adenocarcinomas among the groups. However, the areas Committee of Nagoya City University Graduate School of lesions were significantly reduced in finasteride- and of Medical Sciences. high dose of flutamide-treated groups in the ventral and lateral lobes, but not the dorsal lobe. The lesions also tended to be suppressed without significance by low- Immunohistochemistry analysis dose flutamide in all lobes (Table 3). The avidin–biotin–peroxidase complex (ABC) method Almost no expression of the SV40 Tag and only weak was used to determine the expression of the SV40 signals for AR were detected in involuted glands of rats Tag and the AR in prostate epithelial cells. Prostate treated with antiandrogens (Figure 3). Serum testoster- sections were treated with mouse anti-SV40 Tag (1:250, one levels were not influenced by finasteride treatment. Pharmingen, San Diego, CA, USA), or rabbit anti-AR On the other hand, the levels of testosterone were (1:200, Affinity Bioreagents, Golden, CO, USA) and significantly (Po0.001) increased in the flutamide-trea- then sequentially with secondary antibody and ABC ted groups in a dose-dependent manner at week 5. At (Vectastain ABC Elite kit, Vector Laboratories, week 7, however, levels had returned to normal level Burlingame, CA, USA). The sites of peroxidase binding (Figure 4).

Table 1 Relative prostate weights in TRAP rats treated with finasteride or flutamide for 5 and 7 weeks Duration Treatment No. of rats Final body weight (g) Relative prostate weight (%)

Dorsolateral lobe Anterior lobe and

5 weeks Finasteride 10 mg/kg 5 429.4715.9 0.1570.02 0.2470.03*** Flutamide 20 mg/kg 5 397.7753.2 0.1170.02** 0.2170.07*** Flutamide 5 mg/kg 5 424.8743.5 0.1570.02 0.3570.05* Control 5 407.8756.1 0.1770.06 0.4070.16

7 weeks Finasteride 10 mg/kg 5 478.4754.9 0.1270.01*** 0.1870.02*** Flutamide 20 mg/kg 5 449.4725.8 0.1170.02*** 0.2270.09*** Flutamide 5 mg/kg 5 469.3748.3 0.1970.01 0.3870.07 Control 5 462.7727.1 0.2270.03 0.4370.06

Values are mean7s.d. *, **, ***: significantly different from the control group at Po0.05, 0.01 and 0.001, respectively.

Prostate Cancer and Prostatic Diseases Suppressive effects of antiandrogens, finasteride and flutamide Y-M Cho et al 380 Table 2 Histopathological findings of neoplastic lesions in TRAP rats Duration Treatment No. of rats No. of rats with prostatic lesion

Ventral Dorsal Lateral Anterior

PIN Ca PIN Ca PIN Ca Ca

2 weeks Finasteride 10 mg/kg 5 0 5 3 2 1 4 5 Flutamide 20 mg/kg 4 0 4 3 1 0 4 4 Flutamide 5 mg/kg 5 1 4 3 2 1 4 5 Control 5 0 5 3 2 3 2 5

5 weeks Finasteride 10 mg/kg 5 1 4 1 4 1 4 5 Flutamide 20 mg/kg 5 1 4 2 3 0 5 5 Flutamide 5 mg/kg 5 0 5 3 2 0 5 5 Control 5 1 4 2 3 1 4 5

7 weeks Finasteride 10 mg/kg 5 0 5 3 2 0 5 5 Flutamide 20 mg/kg 5 0 5 4 1 2 3 5 Flutamide 5 mg/kg 5 0 5 1 4 0 5 5 Control 5 1 4 2 3 1 4 5

Abbreviations: Ca, carcinoma; PIN, prostatic intraepithelial neoplasia; TRAP, TG rat for adenocarcinoma of the prostate.

Figure 1 Representative findings for involuted prostate cancer lesions in TRAP rats treated with antiandrogen flutamide for 2 (a) and 7 (b) weeks. Original magnification, Â 400. TRAP, TG rat for adenocarcinoma of the prostate.

Discussion orchiectomy at 5 or 20 weeks of age causes severe involution or regression, and inhibition of prostate TG animal models are powerful tools for screening carcinoma development, in clear contrast to the case chemopreventive or therapeutic agents against cancer with TRAMP mice that demonstrated the development because cancer lesions develop uniformly and rapidly in of poorly differentiated prostate cancer with 80% TG animals. To date, a number TG animals for prostate incidence at 12 weeks after castration.8,23 Oral adminis- cancer research have been established and all of them are tration of finasteride for 2 months to methylnitrosourea/ mice except our TRAP rats.20,21 Among them, TG -inoculated male Wistar rats adenocarcinoma mouse prostate (TRAMP) model, har- reduced the tumor incidence to 10%, as compared with boring rat probasin promoter/SV40 Tag gene construct, the 64.3% of the control group.24 Recently, the National is widely used. TRAMP mice demonstrated the devel- Cancer Institute-sponsored Prostate Cancer Prevention opment of well-differentiated adenocarcinomas in pros- Trial designed to investigate the efficacy of a variety of tate by 10 weeks of age and of poorly differentiated agents ranging from dietary supplements to laboratory metastatic, androgen-independent tumors by 28 weeks manufactured drugs for the primary chemoprevention of of age.22,23 In our TRAP rats, well-moderately differ- prostate cancer demonstrated that finasteride can pre- entiated adenocarcinomas similarly developed in pros- vent prostate cancer.25 The observed 25% reduction in tate by 10–15 weeks of age. However, all the 7-year period prevalence of prostate cancer in men adenocarcinomas in TRAP rats were completely andro- over age 55 years and few side effects25 provides strong gen dependent and did not metastasize.9 support for the importance of hormonal control for Hormonal factors play important roles in carcinogen- primary prostate cancer. esis as well as the homeostasis in the prostate gland. In the present study, there were massive or partial Because promote prostate carcinogenesis, pathologic responses by flutamide or finasteride treat- inhibition of androgenic functions is a fundamental ment as demonstrated by the reduction of prostatic approach to control prostate cancer. Our previous studies neoplastic lesions in TRAP rats. However, these re- on the initiation and progression of prostate cancer using sponses were not complete, that is, small or tiny foci of TRAP rats demonstrated that androgen depletion by carcinoma were still remaining. Reflecting these findings,

Prostate Cancer and Prostatic Diseases Suppressive effects of antiandrogens, finasteride and flutamide Y-M Cho et al 381

Figure 2 Representative findings for involuted prostate cancer lesions in TRAP rats treated with finasteride 10 mg/kg/day (a), flutamide 20 mg/kg/day (b), flutamide 5 mg/kg/day (c) or vehicle (d) five times a week for 7 weeks. Original magnification, Â 40. TRAP, TG rat for adenocarcinoma of the prostate.

Table 3 Quantitative data for proliferative lesions in prostate of TRAP rats treated with finasteride or flutamide for 7 weeks Treatment No. of rats Epithelial area/acinus (%) examined Ventral Dorsal Lateral

Finasteride 10 mg/kg 5 80.173.2* 60.273.9 56.973.5** Flutamide 20 mg/kg 5 77.879.1* 63.273.6 65.274.7** Flutamide 5 mg/kg 5 87.772.0 59.773.9 71.4710.0 Control 4 93.374.0 66.071.9 81.576.4

Abbreviation: TG rat for adenocarcinoma of the prostate. Values are mean7s.d. *, **: significantly different from the control group at Po0.05 and 0.01, respectively.

Figure 3 Representative findings for AR and SV40Tag protein expression in serial sections of partially involuted prostate glands in TRAP rats treated with antiandrogen finasteride for 7 weeks. Similar expression was evident in rats treated with flutamide. (a) H&E. (b) AR. (c) SV40 Tag. Original magnification, Â 400. AR, ; H&E, hematoxylin and eosin; TRAP, TG Rat for adenocarcinoma of the prostate. there were no significant differences in the incidences of tumors although well-differentiated tumors were ob- PIN or adenocarcinoma in the prostate of TRAP rats. In served more frequently,6 suggesting that it is difficult to TRAMP mice, high-dose flutamide significantly de- exert complete pathologic responses by flutamide alone creased the incidence of only poorly differentiated against well-differentiated prostate tumors induced by

Prostate Cancer and Prostatic Diseases Suppressive effects of antiandrogens, finasteride and flutamide Y-M Cho et al

382 Welfare of Japan, and a grant from the Society for Promotion of Toxicological Pathology in Nagoya, Japan.

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