J. steroid Biochem. Vol. 31, No. 5, pp. 845-852, 1988 0022-473 I j88 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright 0 1988 Pergamon Press plc ANDROGENIC ACTIVITY OF SYNTHETIC PROGESTINS AND SPIRONOLACTONE IN ANDROGEN-SENSITIVE MOUSE MAMMARY CARCINOMA (SHIONOGI) CELLS IN CULTURE I. A. LUTHY, D. J. BEGIN and F. LABRIE* MRC Group in Molecular Endocrinology, Lava1 University Medical Center, Quebec GlV 4G2, Canada
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`(Received 18 December 1987)
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`Smmmuy-A series of compounds designed to block the action of androgens in target tissues, and called antiandrogens, have been developed for the treatment of androgen-sensitive diseases, especially prostate cancer, hirsutism, precocious puberty and deviant sexual behavior. In order to further assess the androgenic activity of these compounds, we have studied their effect on the growth of an androgen-sensitive clone of the mouse mammary carcinoma Shionogi SC-l 15 cells in culture. Hydroxyflutamide did not affect the doubling time (7.40 f 0.09 vs 7.20 f 0.12 days) characteristic of these cells. However, all of the other compounds tested stimulated cell growth. Thus, in the presence of cyproterone acetate, cells had an accelerated growth rate and shorter generation time of 6.28 &- 0.06 days (P < 0.01). In the presence of 1 PM spironolactone, the generation time was 4.96 f 0.04 days (P < 0.01). With chlormadinone acetate, the doubling time was reduced to 3.79 f 0.08 days while for megestrol acetate, the doubling time was 3.63 f 0.04 days (P < 0.01). The synthetic progestin Medroxyprogesterone acetate had the most potent androgenic effect reducing the doubling time to 1.85 + 0.05 days (P < 0.01). For comparison, dihydrotestosterone gave a doubling time of 1.76 + 0.07 days. When hydroxy-flutamide (5 p M) was added simultaneously with each “progestin”, the EDSo value of action of all the compounds was increased in a competitive manner, thus indicating that the mitogenic effect on cell growth of all compounds is mediated by the androgen receptor. Of all the compounds used, only hydroxy-Flutamide was devoid of any androgenic activity and thus meets the criteria of a pure antiandrogen. INTRODUCTION Since the observation of Huggins and his colleagues in 1941 on the important role of testicular androgens in prostate cancer growth [ 11, the standard treatment of this disease has been orchiectomy and estrogens. However, orchiectomy is psychologically unaccept- able for a large number of patients while treatment with estrogens in order to block gonadotropin secre- tion causes serious cardiovascular complications leading to death in 15% of patients during the first year of treatment [2]. A recently proposed alternative is the use of agonists of luteinizing hormone-releasing hormone (LHRH) which cause a complete blockade of testicular androgen secretion without side effects other than those related to hypoandrogenism [3]. However, since the effect of these peptides is limited to the blockade of testicular androgens, one cannot expect to improve the prognosis of prostate cancer beyond the results previously achieved with or- chiectomy, namely a remission in 60-80% of cases for a limited time interval, thus leaving 2&40% of *To whom correspondence should be addressed at: MRC Group in Molecular Endocrinology, Lava1 University Medical Center, 2705, Laker Boulevard, Quebec GlV 4G2, Canada. patients without any significant effect of treatment on the cancer [4, 51. The well recognized sensitivity of prostate cancer to androgens has stimulated the development of compounds, called antiandrogens, which are aimed at blocking the action of androgens in target tissues [6,7]. The interest in these compounds is strength- ened by the recent observation that the blockade of androgens of both testicular and adrenal origins by combining a pure antiandrogen with castration at the start of treatment of advanced prostate cancer yields a higher response rate, a more prolonged disease-free period and an improved survival while maintaining a good quality of life [4,8,9]. The use of this combina- tion therapy follows the recognition that adrenal steroids contribute approx 50% of all androgens present in the prostatic cancer tissue [4]. Antiandro- gens thus offer an alternative to hypophysectomy or adrenalectomy without the complications of glucocorticoid replacement therapy. Since the aim of endocrine therapy in prostate cancer is maximal blockade of androgen action, the ideal antiandrogen should be a compound having potent antiandrogenic activity while being devoid of any androgenic, glucocorticoid, progestational, es- trogenic or any other hormonal or antihormonal 845
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`action [lo]). It thus becomes of major interest to medium was changed every third day and the cells investigate in detail the properties of available drugs subcultured as described above. in order to choose the best antiandrogen for clinical use.
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`Doubling-time experiments
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`EXPERIMENTAL
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`Culture of Shionogi cells
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`counter. Shionogi male mice bearing tumors of the
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`So far, the androgenic/antiandrogenic activity of Cells were plated in 24-well plates at a density of the various antiandrogenic compounds used has been 20,000 cells/well in MEM-2% DCC-FCS medium measured in intact animal models [7, 1 l-141. In the containing, when indicated, 1 PM of the following present study, we have taken advantage of the avail- compounds: medroxyprogesterone acetate, megestrol ability of a highly androgen-sensitive clone derived acetate, chlormadinone acetate, spironolactone, cy- from the Shionogi carcinoma cell line in order to proterone acetate and hydroxy-flutamide while the assess the androgenic activity of the compounds concentration of DHT was 100 nM. The control wells presently proposed for the treatment of prostate contained the same ethanol concentration (0. I %). cancer and/or hirsutism, namely cyproterone ace- Samples were taken daily for measurement of cell tate, medroxyprogesterone acetate, spironolactone, number. The medium was changed every second day chlormadinone acetate, megestrol acetate and Flut- and the cells were grown up to 40 days. At the end amide. Specificity of the action of each drug on the of the indicated incubation periods, cells were washed androgen receptor has been tested using competition with Dulbecco’s phosphate buffered saline (without with the pure antiandrogen Flutamide [7, 11, 151. calcium and magnesium). A pancreatin-EDTA incu- bation was then performed. The enzymatic treatment was stopped with MEM medium containing 10%
`cell line SC-l 15 were kindly provided by Dr K. The cells were incubated with increasing concen- Matsumoto, Osaka, Japan. Cells were dispersed trations of the indicated compounds in the presence from finely minced tumors by a 2-h treatment at or absence of 5 PM hydroxy-flutamide. DHT and 37°C in 25 mM Hepes buffer (137 mM NaCl; medroxyprogesterone acetate were incubated during 5 mM Na,HPO,; 10 mM glucose, pH 7.2) containing 8 days while chlormadinone acetate, spironolactone 510 U/ml collagenase (Clostridium, Boehringer), and megestrol acetate were incubated during 14 days 550 U/ml hyaluronidase II (Sigma) and 4% bovine and cyproterone acetate and hydroxy-flutamide dur- serum albumin (fraction V, Schwartz-Mann). Dis- ing 25 days. persed cells were collected by centrifugation (500g for 10 min), washed twice by suspension in Minimal
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`Measurements of ED,, values of stimulation of cell
`proliferation
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`Calculations and statistical analyses
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`Essential Medium (MEM) containing 5% dextran- The potency of each compound as stimulator of coated charcoal-treated fetal calf serum (DCC-FCS), cell proliferation was measured as ED,, value using 1% non-essential amino acids, 10 U/ml penicillin, an iterative least square method [18].
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`50
`pg/ml streptomycin and 100 nM Sa-dihydrotes- tosterone (DHT) (Steraloids).
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`RESULTS
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`Cells were plated in the same medium at a density of 75,000 cells/ml in 75 cm2 flasks under an atmo- The stimulatory activity of the various “anti- sphere of 5% carbon dioxide in air at 37°C. The androgens” on cell proliferation was first assessed by medium was changed weekly. Sa-dihydrotestosterone measurement of cell growth kinetics in the presence (DHT) was dissolved in ethanol and the final concen- of each compound alone. As illustrated in Fig. I, a tration of ethanol did not exceed 0.01% in the culture wide range of stimulatory activities was observed medium. Cells were subcultured at near-confluence when each compound was incubated with clone by gentle digestion in a solution of 0.1% pancreatin SEM-1 at a concentration of 1 PM (except for DHT (Flow Laboratories) in Hepes buffer containing which was used at a concentration of 100 nM). The 3 mM EDTA (pH 7.2). Cells were pelleted by centrif- cell number doubling-time for each compound was ugation, resuspended in culture medium, counted in as follows: hydroxy-flutamide had no effect on cell a Coulter counter and replated as described above. growth, the doubling time being superimposable
`to that of cells incubated with control medium (7.40 k 0.09 vs 7.20 + 0.12 days) while all the other Soft agar cloning was performed as described compounds stimulated cell growth: cyproterone ace- [16, 171. A single clone (SEM-I) showing DHT- tate reduced the generation time to 6.28 _+ 0.06 days sensitive growth was selected for cell growth experi- (P < 0.01 vs control and Flutamide), spironolactone ments. The cloned cells were maintained in the MEM to 4.96 &- 0.04 days (P c O.Ol), chlormadinone ace- medium described above except that the DCC-FCS tate to 3.79 + 0.08 days (P c O.Ol), megestrol acetate concentration was 2% and DHT was 100 nM. The to 3.63 + 0.04 days (P < 0.01) and medroxypro-
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`Cloning of SC- 115 cells
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`FCS and the cells were counted with a Couher
`androgen-sensitive mouse mammary carcinoma
`I. A. Lurnv et
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`Androgenic activity of progestins 847 H WA +C
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`MEG
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`HCMA TIME (DAYS, Fig. 1. Effect of a series of “antiandrogens” on the growth of an androgen-sensitive clone @EM-l) of mouse mammary carcinoma Shionogi cells in culture. The cells were incubated up to 40 days in medium (MEM + 2% dextran-coated charcoal extracted fetal calf serum) contain- ing the indicated compounds at a concentration of 1 PM (except 0.1 PM for DHT). Media were changed every second day. The compounds were: DHT: dihydrotes- tosterone; MPA: medroxyprogesterone acetate; MEG: megestrol acetate; CMA: chlormadinone acetate; SPIR: spironolactone; CPA: Cyproterone acetate; FLU-OH: hydroxy-flutamide. The control medium contained the same concentration of ethanol (0.1%). gesterone acetate to 1.85 + 0.05 days (P < 0.01). DHT included for comparison as the standard an- drogen showed an accelerated doubling-time of 1.76 + 0.07 days (P -C 0.01 vs all groups except medroxyprogesterone acetate). Following this assessment of the relative stimu- latory effect of each compound on cell growth at a concentration of 1 PM, we next examined the po- tency of each compound by measurements of ED,, values and have assessed the specificity of their action on the androgen receptor by simultaneous incubation o-3 DHT H DHT+FLU-OH W MPA
`MPA+FLU-OH 011 : : : : : I -1, -10 -9 -8 -7 -6 -5 CONC (LOG M) Fig. 2. Effect of increasing concentrations of dihydro- testosterone or medroxyprogesterone acetate in the presence or absence of hydroxy-Flutamide (5 p M) on the number of androgen-sensitive Shionogi SC-l 15 cells (clone SEM-1). The cells were incubated as described in Fig. 1, except for the concentrations of the indicated compounds and an incubation time of 8 days. Fig. 3. Effect of increasing concentrations of spironol- actone (SPIR) in the presence or absence of hydroxy- flutamide (5 PM) on the growth of androgen-sensitive Shionogi SC- 115 cells in culture (clone SEM- 1). The condi- tions were those specified in Fig. 1. The incubation was performed during 14 days. with the pure antiandrogen hydroxy-flutamide. Fig- ure 2 compares the ED,, values of DHT and me- droxyprogesterone acetate action measured after 8 days of incubation with increasing concentrations of the two compounds. The ED, value of DHT action was calculated at 0.073 nM while that of me- droxyprogesterone acetate is 42.5 nM. When hydroxy-flutamide (5 PM) was added to the incu- bation medium, the ED, values of DHT and MPA action increased to 5.9 and 222 nM, respectively. It can be seen in Fig. 3 that incubation with increasing concentrations of spironolactone for 14 days showed an ED5, value of action of the com- pound at 344 nM while, in the presence of hydroxy- flutamide, it was increased in a competitive manner ,J, , ( , , , , ) -,I -10 -9 -9 -7 -9 -5 -4 CONC (LOG M) Fig. 4. Effect of increasing concentrations of chlormadinone acetate (CMA) in the presence or absence of hydroxy- flutamide (5 PM) on androgen-sensitive Shionogi (clone SEM-1) cell growth. The conditions were as described in Fig. 1. The experiment was performed for 14 days.
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`848 I. A. Lurnv et al. Fig. 5. Effect of increasing concentrations of megestrol acetate (MEG) in the presence or absence of hydroxy- flutamide (5 PM) on androgen-sensitive Shionogi (clone SEM-1) cell growth. The conditions are as described in Fig. 1. The experiment lasted 11 days. to 1.88 PM. Figure 4 shows that the effect of chlor- madinone acetate on cell growth during the same incubation period of 14 days was exerted at ED,, values of 15 1 and 896 nM in the absence and presence of 5 PM hydroxy-flutamide, respectively. Megestrol acetate (Fig. 5) had an ED, value of action of 161 nM, this value being shifted to 146 nM in the presence of hydroxy-flutamide (5 p M). It can be seen in Fig. 6 that after 25 days of incubation, cyproterone acetate stimulated cell growth at an ED, value of 65 nM, this value being increased to 739 nM in the presence of 5 PM hydroxy-flutamide. Hydroxy- flutamide alone, on the other hand, showed no significant stimulation of cell growth during a 25-day incubation period (Fig. 7). Fig. 6. Effect of increasing concentrations of cyproterone acetate (CPA) in the presence or absence of hydroxy- flutamide (5 FM) on androgen-sensitive Shionogi cell growth. The experiment was performed as described in Fig. 1 and lasted 25 days. 6 I O-.0 FLU-OH
`(LOG M) Fig. 7. Effect of increasing concentrations of hydroxy- flutamide on androgen-sensitive Shionogi cell growth of the androgen-sensitive clone SEM-1 of Shionogi cells. The conditions are as described in Fig. 1. The experiment was performed for 25 days. DISCUSSION Antiandrogens must be strictly defined as com- pounds which inhibit androgen action at the target tissue level and do not act through inhibition of gonadotropin secretion [lo]). To be maximally effec- tive, it is clear that an antiandrogen should counter- act the effect of androgens at the receptor level without exerting any androgenic activity by itself. Compounds having mixed agonistic-antagonistic activity (such as cyproterone acetate and spirono- lactone, 11, 12, 19) cannot reach the complete in- hibition achieved with pure antiandrogens [ 11, 191. Any intrinsic agonistic activity stimulates the an- drogen receptor, such as stimulatory androgenic ac- tivity being more important in cancer cells where the stimulatory action is amplified by rapid cell division. The present data clearly show that all the compounds tested, except hydroxy-flutamide, exert significant and sometimes marked agonistic androgenic activity as assessed by stimulation of proliferation of an androgen-sensitive clone of cancer cells. Although an androgenic activity has been previously suggested for most of these compounds using androgen-sensitive parameters of response in normal tissues, it is likely that the present data obtained with cancer cells are even more relevant to human cancer. Previous studies using androgen-sensitive para- meters in normal tissues have demonstrated the androgenic activity of cyproterone acetate [7, 11, 13, 19-221. In fact, when cyproterone acetate was administered to pregnant guinea pigs [13], and rabbits [21], all female fetuses showed signs of vir- ilization of the secondary sex organs. Recently, a growth-promoting effect of cyproterone acetate has been described on the growth of Shionogi tumors in castrated mice [22]. The present data, using a simpler system, namely a clone of the same androgen-
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`Androgenic activity of progestins 849 Table I. Effect of synthetic progcstins, spironolactone and hydroxy-Flutamide on the doubling time (expressed in days) and the ED, value of stimulation of growth (expressed in nM in the absence or presence of 5gM hydroxy-Flutamide) of Shionogi carcinoma cells Compound Control Hydroxy-flutamide Cyproterone acetate Spironolactone Chlormadinone acetate Megestrol acetate Doubling time (days) 7.20*0.12 7.40 * 0.09 6.28 + 0.06. 4.96 f 0.04’ 3.79 * 0.08* 3.63 + 0.04* ED, value of growth response No hydroxy + hydroxy- Flutamidc Flutamide (nM) - - - - 65 139 344 1880 151 896 161 746 Medroxyprogesterone 1.85 :0.05* 42.5 222 DHT 1.76 f 0.07* 0.073 5.9
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`l
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`P <O.Ol. For the doubling time, all comoounds were present at a concentration of 1 pM except DHT<O.lO~M). . sensitive Shionogi cancer cells maintained
`show that cyproterone acetate exerts a direct stimu- latory effect on cell proliferation. The androgenic nature of the stimulatory effect of cyproterone ace- tate is well demonstrated by the inhibition of its effect by the pure antiandrogen hydroxy-flutamide [ 151, the active metabolite of flutamide [23]. The presence of megestrol acetate on the prostatic androgen receptor following
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`administration was incorrectly interpreted as an antiandrogenic action [24]. In the light of the present and ,previous [ 11, 14, 191 results, binding of megestrol acetate to the androgen receptor induces an activation rather than an inactivation of the receptor. Since a major argu- ment put forward to support the use of megestrol acetate for the treatment of prostate cancer is the potential blockade of intracellular androgen ac- tion in the prostatic cancer tissue, the discovery of an androgenic action of the compound in normal [l 1,14, 191 and cancer (present study) tissues should seriously limit the potential usefulness of this com- .pound for the treatment of prostate cancer. Spironolactone, in addition to being an aldos- terone antagonist (251, has been shown to have some antiandrogenic activity [26,27]. Based on these anti- androgenic properties, spironolactone has been used for the treatment of hirsutism [26-301. The andro- genie activity of spironolactone has recently been described on rat ventral prostate growth and pros- tatic ornithine decarboxylase activity in the rat [I91 and is clearly confirmed in the present study. In fact, spironolactone is a weak and only partial androgen antagonist possessing a relatively important intrinsic androgenic activity. progestin has been shown to stimulate the weight of the preputial glands [12,33]. Moreover, when injected into female mice, medroxyprogesterone acetate, as well as megestrol acetate, induced a marked stimu- lation of kidney /3-glucuronidase activity [12]. When the two 17-acetoxyprogesterone derivatives were in- jected in the androgen-insensitive (tfm/y) mouse, there was no increase in kidney B-glucuronidase activity, thus further indicating that medroxypro- gesterone acetate and megestrol acetate exert their action through interaction with the androgen receptor. In agreement with the findings of a potent an- drogenic activity of medroxyprogesterone acetate, [‘Hlmedroxyprogesterone acetate has been found to bind to the mouse kidney androgen receptor directly without transformation [34]. In fact, medroxypro- gesterone acetate binds to the androgen receptor with high affinity, its
`value of interaction being com- parable to that of DHT itself [34]. As illustrated in Fig. 2, medroxyprogesterone is also a potent stimu- lator of androgen-sensitive cell growth, a half- maximal effect being exerted at 43 nM. The androgenic activity of medroxyprogesterone acetate has previously been reported in normal tissues. These data pertain to the masculinization of female fetuses when the compound was administered to pregnant animals [3 1,321. In addition to its mascu- linizing effects on the external genitalia and re- In previous studies performed with rat anterior pituitary cells in culture, medroxyprogesterone ace- tate was found to inhibit LHRH-induced LH release to 20% of control at an ED, value of 4.6 f 0.7 nM [35]. The potency of this effect was similar to that obtained with testosterone or DHT [36], thus indicating that MPA also exerts full androgenic activity on LH release. Under the same conditions, progesterone had no effect. In agreement with the present data on androgen-sensitive cell growth, me- gestrol acetate and chlormadinone acetate had higher ED,, values on LHRH-induced LH release measured at 200 and 100 nM, respectively. Another important finding reported with me- droxyprogesterone acetate, megestrol acetate and cy- proterone acetate is their potentiating effect on the stimulatory action of androgens on kidney B-glu- productive tract of female fetuses, this synthetic curonidase activity. This marked enhancement of the
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`action of androgens is seen at low doses of the “progestins” and is called synandrogenic activity [12]. Thus, these compounds, in addition to their intrinsic androgenic action, can potentiate the effect of an- drogens on kidney 8-glucuronidase. Similarly, pre- treatment for 24 h with cyproterone acetate has been found to potentiate the induction by testosterone of alcohol dehydrogenase in the mouse kidney [33]. In agreement with the present fundamental data, it has recently been found in a study performed by the Genitourinary Tract Cooperative Group of the Euro- pean Organization for Research on the Treatment of Cancer (EORTC) that the time of progression as well as survival were significantly reduced in the group of patients who received medroxyprogesterone acetate compared to those who received diethylstilbestrol 1381. In fact, medroxyprogesterone acetate had a dramatic harmful effect on survival which was illus- trated by the finding that 50% of the patients treated with MPA died within 1 year compared with an average of 2 year survival in all the studies reported so-far using standard hormonal therapies [S, 61. In other words, patients treated with medroxyprogester- one acetate died twice as fast as those treated with DES [38]. Such data illustrate the major importance of performing detailed preclinical studies of the prop- erties of the drugs to be used as antiandrogens before using them for the treatment of androgen-sensitive diseases such as prostate cancer. The present data show that all the compounds used, with the exception of hydroxy-flutamide, induce growth of the androgen-sensitive Shionogi SC- 115 cells in culture. In all cases, the presence of hydroxy flutamide in the incubation medium shifted the ED,, values in a competitive manner to higher concen- trations, thus indicating that the action of all these compounds is due to activation of the androgen receptor. On the other hand, hydroxy-flutamide was devoid of any androgenic effect. It has been shown that in patients who received a single administration of 50mg of cyproterone ace- tate, plasma concentrations of 250 ng/ml of the drug were achieved [39]. In women treated during 2 years for female hirsutism, the plasma concentration of cyproterone acetate ranged between 436 and 520 ng/ml for a daily dose of 100 mg [40]. Since this dose corresponds to the dose administered to patients suffering from prostate cancer and assuming a similar metabolism in men and women, the plasma concen- tration of cyproterone acetate in men treated for prostate cancer would be around 1 FM. In the present experiments, the EDJo value of the stimu- latory action of cyproterone acetate action was 65 nM. Similarly, measurements of serum MPA levels in women suffering from breast cancer, gave plasma levels of MPA of 100 ng/ml (0.25 PM) when only 600 mg was administered daily [41]. Assuming again a similar metabolism of the compound in men and women, the EDJo value of medroxyprogesterone ace- tate action measured at 42.5 nM clearly indicates that medroxyprogesterone acetate could exert stimulatory effects in prostate cancer patients treated with this drug. The present experiments were performed in a clone derived from the Shionogi mouse mammary car- cinoma which is recognized as a good experimental model for prostate cancer because of its demon- strated and well-characterized androgen sensitivity both
`[16, 17,421. In analogy with cyproterone acetate [ 111, spironolactone has recently been shown to be a mixed agonist-antagonist on the weight of the ventral prostate and on prostatic or- nithine decarboxylase activity in the castrated rat [19]. The androgenic activity of spironolactone just demonstrated in the normal prostate, is now confirmed on the proliferation of the androgen- sensitive carcinoma cell clone SEM-I. This intrinsic androgenic activity of spironolactone, in addition to preventing complete blockade of the action of endogenous androgens, is likely to exert significant stimulatory androgenic effects, especially in a low androgenic environment. Although the origin of tumors is believed to be monoclonal[43], it is clear that most, if not all, advanced tumors are composed of mixed populations of cells having a wide range of phenotypes, including variable levels of androgen sensitivities [ 16, 17,441. Tumors sensitive to low levels of androgens are thus likely to be more efficiently stimulated by low levels of endogenous androgens and to be more sensitive to the administration of compounds having some intrin- sic androgenic activity. It should be added that among the compounds tested, the worst charac- teristics belong to medroxyprogesterone acetate, megestrol acetate and chlormadinone acetate, since these three compounds are devoid of any true anti- androgenic activity [l 1, 19,451 while spironolactone and cyproterone acetate are partial androgen agonist-antagonists
`1, 191, possessing a significant level of androgenic activity. The present data stress the absolute requirement of a pure antiandrogen in order to prevent stimulation of cancer growth by the intrinsic androgenic action of compounds having mixed agonistic antagonistic activity. A pure antiandrogen is also required for maximal blockade of androgenic action at the an- drogen receptor level. Of the compounds available for the treatment of prostate cancer, the present data obtained with an experimental cancer model and well supported by previous data obtained in normal tissues [7, 1 I, 12, 13, 191 show that the only com- pound (among those tested) which meets the criteria of a pure antiandrogen devoid of any other antihormonal activity is hydroxy-gutamide, a nonsteroidal antiandrogen. Acknowledgements-I.A.L. is a holder of a Fellowship from the Consejo National de Investigaciones Cientificas y Tdcnicas de la Republic Argentina. D.J.B. is a holder of a Fellowship from the Fonds pour la formation de Chercheurs et 1’Aide B la Recherche du QuCbec, Canada.
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