Tokai J Exp Clin Med., Vol. 26, No. 2 , pp.71-75, 2001

Relationship between Prostatic Atrophy and Apoptosis in the Canine Spontaneous Benign Prostatic Hyperplasia (BPH) following Chlormadinone Acetate (CMA)

Masanori MURAKOSHI, Rie IKEDA, Norio FUKUI and Takaharu NAKAYAMA

＊ Safety Research Department, Teikoku Hormone Mfg. Co., Ltd., Kawasaki

(Received April 9, 2001; Accepted May 22, 2001)

The effect of a synthetic steroidal antiandrogen, chlormadinone acetate (CMA), on sponta- neous benign prostatic hyperplasia (BPH) in dogs was investigated. Male beagle dogs (5 -8 years old) were divided into four experimental groups. Group 1 consisted of untreated con- trols. Groups 2 to 4 received CMA 0.03, 0.1, and 0.3 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro-mus- cular stroma was prominent. To evaluate the frequency of apoptosis, we counted the posi- tive cells stained by the nick end labeling method. In group 1, the apoptotic index was 0.76±0.03％. In groups 2 to 4, apoptotic index were 15.41±1.26％, 2.63±0.98％ and 1.45± 0.85％, respectively. Apoptotic cell death was mainly observed in the glandular epithelial cells. Based on our data, regression of BPH after treatment with CMA may be apoptotic cell death.

Key words : Chlormadinone acetate (CMA), Benign prostatic hyperplasia (BPH), Canine, Apoptosis, Atrophy

INTRODUCTION DNA fragmentation is sequently followed by irreversible morphological changes Biochemical and morphologic studies have termed apoptosis [13, 33] which characteris- demonstrated that the involution of normal tically involves chromatic condensation, prostate after castration is not the result of nuclear disintegration, cell surface blebbing, necrotic cell death, but is an active process and eventually cellular fragmentation, into a brought about by the initiation of a series of cluster of membrane-bound apoptotic bodies specific biochemical steps that lead to the within the prostate [5, 14, 27]. programmed cell death (apoptosis) of andro- Both man and dogs spontaneously devel- gen-dependent glandular epithelial cells op benign prostatic hyperplasia (BPH) with within the prostate [5, 14, 25]. Associated age [1, 10, 18, 30, 34]. Canine BPH is with this programmed cell death is the believed by many investigators to be an enhanced expression of series of genes with- appropriate model for the study of human in the prostate [2, 16, 17, 26]. BPH, although there are important differ- Like other systems in which programmed ences between the conditions in the two cell death occurs [3, 29, 31, 32], androgen species [1, 10, 18, 30, 34]. Thus, the human ablation induced prostatic cell death initially disease is often a multinodular process involved fragmentation of genomic DNA. thought to arise a priurethal stromal nodule, This DNA fragmentation occurs via activa- which is then secondarily invaded by glan- tion of endonucleases within the prostatic dular elements [8]. In contrast, canine BPH glandular cell nucleus which degradates the is a diffuse epithelial or glandular process genomic DNA into nucleosomal oligomers with less stromal involvement [4]. Although (i.e. multiples of a 180-base pair subunit) some differences exist between human and lacking intranucleosomal breaks in the DNA canine BPH, the dog is considered to be a [14, 15]. good animal model of BPH to test the effi-

Masanori MURAKOSHI, Safety Research Department, Teikoku Hormone Mfg. Co., Ltd., 1604 Shimosakunobe, Takatsu-ku, Kawasaki 213-0033, Japan 71 72 ― M. MURAKOSHI et al. cacy of drugs that cause shrinkage of the under pentobarbital anesthesia at the end of hyperplastic gland [19, 24, 28]. the experimental period. Chlormadinone acetate (CMA) is a Organ weight steroidal antiandrogen that is widely used in The organ weights of the prostates were the medical management of human BPH or recorded (absolute weight). Weights relative prostatic carcinoma [8, 11]. The atrophic to body weight (relative weight) were calcu- effect of CMA on the human prostatic hyper- lated. plasia has been reported by several authors Histopathological examination [8, 11]. The antiandrogenic mechanisms of Prostates were removed immediately, CMA has been evaluated biochemically and fixed in 0.1M phosphate-buffered 10％ for- immunohistochemically, such as androgen malin, and embedded in paraffin. Cut sec- receptor content, and steroid 5α-reductase tions were mounted and stained with hema- type II activity in the prostate [11, 20-23]. toxylin and eosin (HE). However, a report concerning apoptotic cell Apoptosis staining death and histopathological evaluation of Apoptosis detection by labeling of 3’OH canine BPH treated with CMA has not ends of DNA breaks using terminal appeared. deoxynucleotidyl transferase was done using In the present study, in order to confirm the ApopTag detection Kit (Oncor, the relationship between BPH regression and Gaitherburg, MD). Formalin fixed and atrophic effect of CMA, we made histopatho- paraffin sections were used. After deparaf- logical and immunohistochemical analysis of finization and hydration, the sections were the prostates of spontaneous BPH dogs incubated with 20μg/ml proteinase K administered CMA for 6 months. (Sigma Chemical Co., St. Louis, MO) at room temperature for 15 min, and then inactivat- MATERIALS AND METHODS ed by covering the sections with absolute Animals methanol containing 0.3％ hydrogen perox- Seventeen male beagle dogs were pur- ide. The sections were washed with 0.01M chased from Hazelton Research Product, Inc. PBS, equibrated, and then incubated with (Denver, PA). The animals were housed indi- terminal deoxynucleotidyl transferase in a vidually in stainless steel cages in a semi-bar- reaction buffer containing digoxigenin rier system maintained at a room tempera- dUTP at 37℃ for 10 min. The reaction was ture of 22±3℃, and relative humidity of stopped, and sections were washed for 30 60±20％, with 12 h of light (7:00-19:00). min, after which the sections were incubated The animals were given 300 g of a standard with anti-digoxigenin antibody coupled to diet (CD-1, CLEA Japan, Inc.) daily and tap peroxidase for 30 min at room temperature. water ad libitum. They were 5-8 years old, After the incubation was completed, the sec- and BPH was confirmed by digital rectal tions were treated in Graham-Karnovsky’s examination, transrectal ultrasonography, reaction medium [7], which contained 20 and transrectal prostatic biopsy. The mg％ 3,3’-diaminobenzidine (DAB, Wako prostates of all the animals showed glandu- Pure Chemical Industries, Osaka) and lar hyperplasia and hypertrophy by light 0.005％ hydrogen peroxide in 0.05M Tris- microscope (histological criteria of DeKlerk HCL buffer, pH 7.6, for 5 to 10 min at room et al. [4]). Furthermore, measured prostatic temperature. Then the sections were counter- weights were 13 g and more (Group 1: 14.8 stained for nuclei with 1％ methyl green dis- -24.3 g, Group 2: 15.2-33.8 g, Group 3: 13.0 solved in veronal acetate buffer, pH 4.2. - 14.8 g, Group 4: 13.5- 35.3 g, data not Apoptotic index was calculated as the posi- shown). tive cell number in one acinus/total cell Experiments number in the acinus ×100. Four animals served as BPH untreated Statistical analysis controls (group 1). Groups 2 to 4 were The data were expressed as mean±SD. administered orally 0.03 (group 2, n ＝ 4), Homogeneity of variance was tested by 0.1 (group 3, n＝4), and 0.3 (group 4, n＝ Bartlett’s methods, and when the assumption 5) mg/kg/day of CMA as a crystalline pow- of homogeneity of variance was met, one- der in gelatin capsules for 6 months. All ani- way layout analysis of variance was per- mals were sacrificed by exsanguination formed. When a significant difference was CMA induces apoptosis in BPH ― 73

Table 1 Effect of Chlormadinone Acetate (CMA) on Prostatic Weight

Dose Initial age Body weight Prostatic weight (g) Group N (mg/kg) (years) (kg) Absolute Relative 1 4 0 5.0±1.8 14.3±1.9 26.58±1.15 1.67±0.45 2 4 0.03 6.0±0.9 15.0±0.7 15.56±2.25a 1.02±0.36 3 4 0.1 3.8±1.0 12.3±0.9 10.01±1.40a 0.71±0.21a 4 5 0.3 6.2±1.0 14.4±1.1 8.90±0.15a 0.60±0.11a Values are the mean±S.D. a P＜0.05, significant difference from BPH control (Group 1)

Table 2 Histopathological Findings CMA (mg/kg) Organ Findings 0 (n＝4) 0.03 (n＝4) 0.1 (n＝4) 0.3 (n＝5) Prostate Glandular hypertrophy/hyperplasia 4 0 0 0 Glandular atrophy mild 0 3 2 0 moderate 0 1 1 2 severe 0 0 1 3 Prominence of fibro-muscular stroma mild 0 3 2 0 moderate 0 1 1 2 severe 0 0 1 3 observed, Dunnett’s multiple comparative atrophic and the acini became completely test was performed between the BPH control atrophic. Thus, histological features of glan- group and the other experimental groups. dular atrophy were evident in this group. In contrast, the interacinar fibro-muscular stro- RESULTS ma was prominent (Table 2). 1. Organ weight b. Apoptosis As shown in Table 1, absolute and rela- To evaluate the frequency of apoptosis, tive mean prostatic weights of groups 3 and we counted the positive cells stained by the 4, and absolute mean prostatic weights of nick end labeling method (Table 3). In group 2 were statistically significantly (p＜ group 1, the apoptotic index was about 0.05) reduced, compared to the BPH control 0.76±0.03％. In groups 2 to 4, apoptotic values. index were 15.41 ± 1.26％ (p＜ 0.01), 2. Light microscopic findings 2.63±0.98％ (p＜0.05) and 1.45±0.85％ a. HE staining (p＜0.05), respectively. In addition, positive In group 1, glandular epithelial cells were cells were mainly observed in the glandular prominent and showed papillary projections epithelial cells. extending into the acini. Thus, histological DISCUSSION features of glandular hypertrophy and/or hyperplasia were evident in this group Histologically, CMA produced marked (Table 2). The amount of interacinar stroma atrophy of the glandular epithelium. It is a was variable but not extensive. In CMA-treat- well documented fact that CMA inhibits the ed animals (groups 2 to 4), the glandular uptake of testosterone in the prostate and is epithelial cells were dose-dependently selectively incorporated into prostate cells, 74 ― M. MURAKOSHI et al.

Table 3 Effect of CMA on apoptosis of BPH dog prostate BPH control BPH＋CMA0.03mg/kg BPH＋CMA0.1mg/kg BPH＋CMA0.3mg/kg 0.76±0.03％ 15.41±1.26％＊＊ 2.63±0.98％＊ 1.45±0.85％＊ Apoptotic index is calculated as the positive cell number in one acinus/total cell number in the acinus ×100. Values are the mean ±S.D. ＊, P＜0.05; ＊＊, P＜0.01, significant difference from BPH control CMA: Chlormadinone acetate, BPH: Benign prostatic hyperplasia resulting in inhibiting testosterone binding attributed to the fact that apoptosis occurs to the cytosol 5α-dihydrotestosterone (DHT)- within a very long period on low dose-treat- receptor [11, 20-23]. In our previous reports ment with CMA followed by rather slow [20-23], the intensity of the immunostain- regress in prostatic size. The biological sig- ing of nuclear androgen receptor in both nificance of apoptosis and CMA-treatment is epithelial and fibro-muscular stromal cells to be further clarified. was dose-dependently weakened by treat- It is concluded that at least in part regres- ment with CMA. It is a well documented fact sion of BPH after treatment with CMA may that prostatic nuclear androgen receptor be apoptotic cell death. contents were decreased after treatment with gonadotropin-releasing hormone (GnRH) REFERENCES agonist [6] as well as cyproterone acetate [9], 1) Brendler CB., Berry SJ., Ewing LL., McCullough AR., an antiandrogenic agent. We postulated that, Cochran RC., Standberg JD., Zinkin BR., Coffey DS., decreased immunostaining of androgen Wheaton LG., Hiler ML., Niswender GD., Boddy MJ., Shapeer JH., Scott WW., Walsh PC.: Spontaneous receptor after treatment with CMA may be benign prostatic hyperplasia in the beagle. The rela- explained by a decrease in the number of tionship between age , serum hormone level, and the AR. In fact, CMA inhibits the binding to morphology and function of the canine prostate. J androgen and androgen receptor competi- Clin Invest 71: 1114-1121, 1983. tively (data not shown). Therefore, CMA 2) Buttyan R., Zakeri Z., Lockshin R., Wolgemuth D.: binds competitively to the androgen receptor Cascade induction of c-fos, c-myc and heat shock 70K from the prostate, and dose-dependently oral transcripts during expression of the rat ventral prostate gland. Mol Endocrinol 2: 650-657, 1988. administration causes regression of the 3) Cohen JJ., Duke RC.: Glucocorticoid activation of a hyperplastic prostatic weight. calcium-dependent endonuclease in thymocyte Changes in the BPH size reflect the bal- nuclei leads to cell death. J Immunol 132: 38-42, ance between cell proliferation and death. It 1984. has been accepted that certain antiandrogen 4) DeKlerk DP., Coffey DS., Ewing LL., McDermott IR., cause the suppression of DNA synthesis by Reiner WG., Robinson CH., Scott WW., Strandberg competitive inhibition against the forming JD., Talalay P., Walsh PC., Wheaton LG. Zirkin BR.: Comparison of spontaneous and experimentally complex of androgen and androgen recep- induced canine prostatic hyperplasia. J Clin Invest tor, possibly resulting in BPH regression 64: 842-849, 1979. [12]. In other words, antiandrogen is consid- 5) English HF., Kyprianou N., Isaacs JT.: Relationship ered to show static action against BPH. Thus, between DNA fragmentation and apoptosis in the the pharmacological effects of antiandrogen programmed cell death in the rat prostate following seem to be complex, i.e. the agent causes castration. Prostate 15: 233-251, 1989. apoptosis as an acute effect and also sup- 6) Forti G., Salenrno R., Moneti G., Zoppi S., Fiorelli G., Martini T., Natali A., Castanitini A., Serio M., Martini pression of cell proliferation as a subacute L., Motta M.: Three-month treatment with a long-act- effect. Present experiments clearly showed ing gonadotropin-releasing hormone agonist of that apoptosis of the hyperplastic prostatic patients with benign prostatic hyperplasia. J Clin cells occurred after CMA administration for Endocrinol Metab 68: 461-468, 1989. 6 months. However, apoptotic index in low 7) Graham RC., Karnovsky MJ.: The early stages dose-treatment with CMA was higher than absorption of injected horseradish peroxidase in the that of high dose-treatment with CMA. The proximal tubules of mouse kidney. J Histochem Cytochem 14: 291-301, 1966. results showed that the increase in the inci- 8) Harada M., Kinoshita Y., Moriyama M., Kondo, I., dence of apoptotic index did not correlated Nakahashi M., Kumagai H., Sasaki K., Hosaka M.: with loss of the prostatic weight. It may be Histological evaluation of benign prostatic hyperpla- CMA induces apoptosis in BPH ― 75

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