British Journal of Urology (1977), 49, 695-700
0
The Effect of Antiandrogens and Stilboestrol on the Cytosol Receptor in Rat Prostate R. GHANADIAN, c. B. SMITH, G. WILLIAMS and G. D. CHISHOLM
Prostnfe Research Laboratory, Royal Postgraduate Medical School, London, W12 OHS
It has been demonstrated that antiandrogens can act by inhibiting the binding of androgens within the prostatic cell in both experimental animals and man (Fang and Liao, 1969; Dorfman, 1971; Hansson and Tveter, 1971; Sufrin and Coffey, 1973). Some of these compounds have been used in the management of patients with prostatic tumours and these reports have provided considerable information regarding their various actions (Scott and Wade, 1969; Chisholm and O’Donoghue, 1975; Jonsson and Nilsson, 1976). However, there is no reliable index or method for choosing a particular antiandrogen for clinical practice due to the fact that in most of these investigations the drugs have not been compared in an equimolar system. In addition, little is known about the effects of stilboestrol, the most widely used oestrogen, on the steroid receptor complex in the prostate. In the present study we have measured equimolar effects of representative compounds from 3 main classes of antiandrogens and the non-steroidal oestrogen, stilboestrol, on the dihydrotestosterone receptor complex isolated from rat prostate. These effects have been compared to the inhibitory effect of a similar concentration of testosterone. Materials and Methods Animals. Male albino Wistar rats of 300 to 400 g body weight were used in all experiments. Bilateral orchidectomy was performed, under fluothane anaesthesia, 24 hours prior to the experiment. Steroids. [I u,Za-3H]testosterone, Sp. activity, 49 Ci/mmol (Radiochemical Centre, Amersham, UK). The following unlabelled steroids were used: testosterone, 5a-dihydrotestosterone, androsterone (3a-hydroxy-5a-androstan-17one), androstanediol (5a-androstane-3P,17P-diol),androstenediol (androst-5-ene-3fl,17j%diol),androstanedione (5a-androstane-3,17-dione)and androstenedione (androst-4-ene-3J7-dione). Antiandrogens. 3 classes of antiandrogens were used : (a) 17cc-hydroxyprogesterone derivatives: Cyproterone acetate (Schering) and chlormadinone acetate (Syntex). (b) 19 nor-testosterone derivative: 17~-(2-methallyl)19 nortestosterone (SC9022) (Searle). (c) Non-steroidal antiandrogen: Flutamide (SCH 13521, Schering, USA). Other Compounds. Diethylstilboestrol. Solvents. Redistilled chloroform, acetone, diethyl ether (peroxide free), Triton X-100 and Toluene (Analar grade). Chromatographic Materials. Sephadex G-25 and G-200. Aluminium-backed silica gel plates, F254 for thin layer chromatography (T.L.C.). Scintillation Fluid. Toluene: Triton X-I00 (2 : 1) containing 0.4% diphenyloxazole (P.P.O.) was used for aqueous samples. Toluene containing 0.4% (w/v) P.P.O. was used for all other samples. Read at the 33rd Annual Meeting of the British Association of Urological Surgeons in Aberdeen, June 1977.
695
696
BRITISH JOURNAL OF UROLOGY
Buffers. Two buffers were used. Buffer A: 20 mM Tris-HC1 buffer, pH 7.4 containing 320 mM sucrose and 3 mM magnesium chloride. Buffer B: 20 mM Tris-HCI buffer, pH 7.4 containing 1.5 mM EDTA, disodium salt and 2 mM 2-mercaptoethanol.
Preparation and Labelling of Tissue Prostatic tissues were labelled either in vivo or in vitro and subsequently subjected to cell fractionation. (a) In vivo Experiments
Animals castrated 24 hours previously received an intraperitoneal injection of 1 pM/100 g body weight of the test drug in ethanol/saline (1 : 1 v/v). Control animals received an equal volume of ethanol/saline only. Each animal received 10 pCi/lOO g body weight [3H] testosterone in 20% ethanol/saline following the injection of drug. 1 hour after the administration of radioactivity the animals were killed and for each experiment the prostates from 2 animals were pooled. The tissue was minced, blotted, weighed and used for cytosol preparation.
(b) In vitro Experiments 24-hour castrated rats were sacrificed and the ventral prostates dissected out. For each experiment prostatic tissue was pooled from 3 animals. The tissue was minced and washed 3 times in buffer A prior to incubation with 0.25 pCi of [3H] testosterone per 2 ml of buffer A with or without tested drug at 0.1 pM. The tissue was incubated at 30°C
Table I Comparison of the Effect of Antiandrogens and Testosterone on the in vitro uptake of [3H] Testosterone by Rat Ventral Prostate. Values are Mean f S.E.M. Compound (Number of experiments)
Uptake of [3H] testosterone
dpm/mg Tissue
~ _ _ _
Testosterone (10) Cyproterone acetate __ (7) Chlormadinone acetate (7) sc 9022 (13) Flutamide (22)
~~
~~~
Control 1807.2+ 116.2
With compound 834.3k 131.4
53.3 k 6.9
1781.9k 138.3
1015.81102.7
42.5k4.2
1595.6 152.7
+
994.2 k 68.7
36.3 14.2
1850,Ok 115.0
991.8k82.5
46.2 2.7
1305.5k61.5
1319.5567.0
No inhibition
Table II In vivo and in vitro Metabolites of [3H] Testosterone, recovered from the Steroid Receptor Complex, isolated from the Cytosol of Rat Ventral Prostate
% Steroid recovered (mean+ S.E.M.) Steroids recovered Dihydrotestosterone Testosterone Androstanediol Androstenediol Androstendione Androsterone Androstanedione Others
]
% Inhibition
_____
In vivo
In vitro
79.07k2.52 1 0 6 7 11.64
77.40k4.74 7-46? 1.71
2.87 k 1.07
6.92 & 1.65
2.97+ 0.35 1.47+ 1.32 < 0.57 2.43 k 0.26
3.02k 0.58 0.58 f025 2 4 0 k 0.64 1.82k0.45
+
697
ANTIANDROGENS AND STILBOESTROL IN RAT PROSTATE
for 1 hour and subsequently was washed 3 times with buffer A, blotted, weighed and used for the preparation of cytosol. Uptake Studies
In vitro uptake of radioactivity by prostatic tissue was carried out as described previously (Ghanadian and Fotherby, 1970). The incubation media contained 100 pg of test drug and 0.25 pCi of [3H] testosterone per 2 ml media. Preparation of Cytosol Labelled tissue from in vivo or in vitro experiments was homogenised with a Teflon-glass motor-driven homogeniser in 3 volumes of buffer B using 5 strokes each of 10 sec duration and 30 sec cooling intervals. The homogenate was centrifuged at 105,000 g for 1 hour at 2°C. The cytosol was removed. Aliquots (0.05 ml) were taken for the measurement of protein and the remainder stored at - 20°C for analysis. Analysis of the Cytoplasmic Extract (a) Characterisation of the receptor protein was performed by sucrose gradient centrifugation (Ghanadian, Smith and Chisholm, 1977). ( b) Binding analysis. Cytoplasmic extracts (1.0 ml) were subjected to gel-filtration on either Sephadex G-200 or G-25 columns, eluted with buffer B at a flow-rate of 50 cm3/hour and fractions of 2 ml were collected. Aliquots (1 ml) from each fraction were transferred to scintillation vials together with 10 ml of scintillation fluid and the radioactivity was counted in an Intertechnique SL40 scintillation spectrometer. Analysis of Radiometabolites Radiometabolites were identified as previously described (Ghanadian, Smith and Chisholm, 1977). Other Procedures Protein was measured in 0.05 ml aliquots of cytosol using bovine serum albumin as described by Lowry et al. (1951).
Results
1. Uptake Studies The in vitro effects o f antiandrogens on the uptake of tritiated testosterone by the whole tissue are shown in Table I. In this study, no effect was observed with flutamide while the remaining 3 antiandrogens produced similar inhibitory effects on the uptake of tritiated testosterone. Table LII
In vivo and in vitro Inhibition o f [W] Dihydrotestosterone binding to the Cytoplasmic Receptor Proteins from the Rat VentraI Prostate by Testosterone and Antiandrogens % Inhibition In vitro
In vivo
Compound (Number of experiments) Testosterone (5)
Cyproterone acetate (5)
Chlormadinone acetate (5)
SC 9022 (5)
Flutamide (5) Stilboestrol (5)
4917-1
Mean k S.E.M.
Range
MeankS.E.M.
Range
89.55 k 2.60
81'94-93.74
76.88 f2.68
67.82-83'26
49.46k 3.94
40.21-60.59
47.01 k 2.47
40.75-55.16
50.89k 8.28
2010-66.70
48.55k 1.77
43.92-54'89
56.68 k 5.69
36.35-67.96
44.33 k 5.45
2462-55'68
44.39k 5.25
25.95-51.24
No inhibition
-
N o inhibition
-
2.46k 1.62
0-8.0
q
,698 12
Void volume
10
2 " 2
-
I
/,!'
8-
I:
2E:
*. E
BRITISH JOURNAL OF UROLOGY
6-
I '
5
I:
I' I'
4-
I: 2-
10 20 Fraction no. (2 ml)
30
Fig. 1. A typical example of the inhibition of [ZH] dihydrotestosterone binding to the receptor complex by tested compounds. The samples were analysed o n Sephadex G-25. Radioactivity elution profiles of the cytosol are shown for (a) untreated (-), (b) cyproterone acetate (0.1 P M )(- - -), (c) testosterone (0.1 P M )(- - - -). Fig. 2. Analysis of prostatic cytosol on sucrose density gradients in the presence of 0.4 M KCl. Labelled cytosol from 24-hour castrated rats was centrifuged at 60,000 rpm for 16 hours at 2°C. Sedimentation was from right to left. Bovine serum albumin with a sedimentation coefficient of 4.6 sec was used as a standard.
2. Separation of the Receptor and Identijcation of Radiometabolites The steroid receptor complex was isolated by column chromatography. The binding patterns on Sephadex G-25 (Fig. 1) and G-200 were found to be similar. This binding protein was then analysed by gradient centrifugation. When high salt solution (0.4 M KCl) was used the sedimentation coefficient was found to be 3.6 sec (Fig. 2). Analysis of [3H] steroids associated with the binding protein was carried out by thin layer chromatography. In both in vivo and in vitro studies 77 to 79 % of the radioactive steroid recovered was 5a-dihydrotestosterone. Testosterone was only 7 to 10% of the radioactivity recovered. Other androgens were present at a much lower level (Table 11). 3. Effects of Test Compounds on the Cytoplasmic DHT Receptor For the routine measurement of the effect of antiandrogens and stilboestrol on the binding of dihydrotestosterone to the receptor complex, samples were sequentially analysed on a column of Sephadex G-25 (Fig. 1). The total counts associated with bound radioactivity (fractions 5 to 10) were adjusted for variations in the protein content of the applied cytosol and the percentage inhibition compared to the control (untreated) was calculated. The inhibitory effect of 1 pmo1/100 g body weight of the 4 test antiandrogens on the dihydrotesterone receptor complex in vivo ranged from 44.39 to 56.6%. Apart from the 19-nor-testosterone derivative (SC 9022) which produced a somewhat higher inhibition than the non-steroidal compound (flutamide) no other significant differences were observed. The two 17a-hydroxyprogesterone derivatives tested in this experiment (cyproterone acetate and chlormadinone acetate) produced an almost identical effect on the cytosol receptor protein both in vivo and in
ANTIANDROGENS AND STILBOESTROL IN RAT PROSTATE
699
vitro. Flutamide produced no in vifro effect. The non-steroidal oestrogen (stilboestrol) had negligible in vitro and no in vivo effect on the cytoplasmic D HT receptor complex (Table 111).
Discussion When the inhibitory effects of equimolar doses of antiandrogens and stilboestrol were compared to that of testosterone, a significantly higher inhibition was produced by testosterone. This applied to both in vivo and in vitro experiments. There was no significant difference between the in vivo inhibitory effects amongst the 3 classes of antiandrogens tested. Stilboestrol did not produce any in vivo effect. With the exception of flutamide, similar results were observed in vitro. Previous studies have demonstrated the equipotency of cyproterone acetate and flutamide, in vivo (Neri et al., 1972; Liao, Howell and Chang, 1974). In the present study, the potency of 17a-hydroxyprogesterone derivatives (cyproterone acetate and chlormadinone acetate) and the non-steroidal compound (flutamide) was found to be similar in vivo. However, no in vitro effect was observed with flutamide. Katchen and Buxbaum (1975) found that following the injection of tritiated flutamide in man more than 97 % of the compound was converted to several radiometabolites. They suggested that the side-chain hydroxylated metabolite could be the active form of this compound. This may explain the inability of flutamide to act in vifro. Our results also suggest that the 19-nor-testosterone derivative (SC 9022) is somewhat more active than flutamide. Skinner et al. (1975) found that 19-nor-dihydrotestosterone derivatives were slightly more potent than cyproterone acetate. It appears that the addition of a methylene group to 17a-hydroxyprogesterone derivatives does not alter the potency of the compound and that cyproterone acetate produces similar effects to chlormadinone acetate. Stilboestrol was included in this study because of its wide use in patients with prostatic carcinoma. We did not consider stilboestrol an antiandrogen in the strict sense since this drug does not compete with testosterone at the target tissue level (Dorfman, 1971). This study clearly demonstrates that stilboestrol does not have an inhibitory effect on the cytoplasmic binding of Sa-DHT to the receptor. Fang, Anderson and Liao (1969) reported only slight effect of stilboestrol on the nuclear retention of 5a-dihydrotestosterone using 500 times higher concentration than used in this study. It has been well documented that stilboestrol substantially reduces the level of plasma testosterone by inhibiting gonadotrophin production as well as stimulating sex hormone binding globulin (SHBG) in man (Jonsson and Nilsson, 1976). Some effect of stilboestrol on the 5a reductase has also been reported. In an in vitro study, Lee et al. (1973) measured the inhibitory effects of 6 oestrogens on the testosterone reduction in rat prostate and found that only those oestrogens with a complete steroid structure and a free phenolic hydroxyl group at position 3 of the steroid nucleus inhibited testosterone 5a reduction. However, in liver this structural requirement was not essential. They concluded that there was a negligible effect of stilboestrol on the conversion of testosterone to 5a-dihydrotestosterone in the prostate. Similarly, Belham and Neal (1971) found little effect on this enzyme in rat prostate. Other research groups (Farnsworth, 1969) have reported more effect of stilboestrol on this enzyme in the prostate. In the present study the existence of a receptor protein which binds preferentially to dihydrotestosterone was confirmed. In the presence of a high salt solution the steroid receptor complex had a sedimentation coefficient of 3.6 sec and this is in agreement with previous reports characterising receptor proteins (Ghanadian, 1976). Analysis of the [3H] steroids associated with the receptor complex showed that 77 to 79 % of the radioactivity recovered was dihydrotestosterone. Other workers have reported that 69% (Rennie and Bruchovsky, 1972) and 8.5% (Fang et al., 1969) of the steroid bound to the receptor was dihydrotestosterone. Summary The effects of 4 antiandrogens and stilboestrol on the cytosol receptor of the rat ventral prostate have been compared.
700
BRITISH JOURNAL OF UROLOGY
Equimolar doses of cyproterone acetate, chlormadinone acetate, SC 9022 and flutamide inhibited the binding of dihydrotestosterone to the receptor complex in vivo by 44 to 50 % whereas stilboestrol had no effect. In the in vitvo studies an equimolar concentration produced similar effects to those observed in vivo with the exception of flutamide which had no effect. This technique provides a method for assessing and comparing the effectivenessof antiandrogens on the androgen receptor complex of the prostate. The authors wish to thank the Medical Research Council for financial support.
References BELHAM, J. E. and NEAL,G. E. (1971). Testosterone action in the rat ventral prostate. Biochemical Journal, 125, 81-91. (CHISHOLM, G. D.and O’DONOGHUE, E. P. N. (1975). The non-surgical treatment of prostatic carcinoma. Vitamins and Hormones, 33,377-397. DORFMAN, R. I. (1971). Antiandrogens. In Proceedings of 3rd International Congress on Hormonal Steroids, eds V. James and L. Martini. Amsterdam: Excerpta Medica, pp. 995. K. M. and LIAO,S. (1969). Receptor proteins for androgens. Journal of Biological Chemistry, FANG,S., ANDERSON, 24, 6584-6595. FANG,S. and LIAO,S.(1969). Antagonistic action of anti-androgens on the formation of a specific dihydrotestosterone-receptor protein complex in rat ventral prostate. Molecular Pharmacology, 5,420-431. FARNSWORTH, W. (1969).A direct effect of oestrogens on prostatic metabolism of testosterone. Investigative Urofogy, 6,423-427. GHANADIAN, R. and FOTHERBY, K. (1970). Uptake of 3H testosterone by tissues of the male rat. Steroidologia, 1, 193-200. GHANADIAN, R. (1976). Endocrine control of the prostate: mechanism of action of androgens. In Scientific Foundations of Urology, eds. D. I. Williams and G. D. Chisholm. London: Heinemann, Vol. 11, pp. 138-146. GHANADIAN, R., SMITH,C. B. and CHISHOLM, G.D. (1977). Identification of an androgen receptor in the cytosol of the female Mastomys prostate. Molecular and Cellubr Endocrinology, 8,147-15 5 . HANSSON, V. and TVETER, K. J. (1971). Effect of anti-androgens on the uptake and binding of androgen by human benign nodular prostatic hyperplasia in vitro. Acta Endocrinologica, 68, 69-78. JONSSON, G.and NILSSON,T.(1976). Pharmacology of drug therapy. In Scientific Foundations of Urology, eds. D. I. Williams and G. D. Chisholm. London: Heinemann, vol. 11, pp. 347-353. KATCHEN, B. and B ~ B A U S. M (1975). , Disposition of a new, non-steroid, antiandrogen, a,a,cr-trifluoro 2-methyl4’-nitro-m-propionotoluidide(flutamide), in men following a single, oral 200 mg dose. Journal of Clinical Endocrinology and Metabolism, 41, 373-379. LEE,D.K.H., YOUNG,J. C., TAMURA, Y., PATTERSON, D. C., BIRD,C. E. and CLARK,A. F. (1973). Effects of oestrogens on the A 4 reduction of testosterone by rat prostate and liver preparation. Canadian Journal of Biochemistry, 51,735-740. LIAO,S., HOWELL, D. K. and CHANG,I. M. (1974). Action of a non-steroidal antiandrogen, flutamide, on the receptor binding and nuclear retention of 5a-dihydrotestosterone in rat ventral prostate. Endocrinology, 94, 1205-1209. LOWRY, 0 .H., ROSEBROUGH, M. J., FARR,A. L. and RANDALL, R. J. (1951). Protein measurement with the folinphenol reagent. Journal of Biological Chemistry, 193,265-276. NERI,R., FLORANCE, K., KOZIOL, P. and VAN-CLEAVE, S.(1972). A biological profile of a non-steroidal antiandrogen, SCH 13521 (4’-nitro-3‘-trifluoromethylisolbutyranilide). Endocrinology, 91,427-437. RENNIE,P.and BRUCHOVSKY, N. (1972). I n vitro and in vivo studies on the functional significance of androgen receptors in rat prostate. Journal of Biological Chemistry, 247, 1546-1554. SCOTT, W. W. and WADE,J. C. (1969). Medical treatment of benign nodular prostatic hyperplasia with cyproterone acetate. British Journal of Urology, 101, 81-85. SKINNER, R. W. S., POZDERAC, R. V., COUNSELL, R. E. and WEINHOLD, P. A. (1975).The inhibitive effects of steroid analogues in the binding of tritiated 5a-dihydrotestosterone to receptor proteins. Steroids, 25, 189-202. SLJFRIN,G. and COFFEY, D. S. (1973). A new model for studying the effect of drugs on prostatic growth. Antiandrogens and DNA synthesis. Znveztigative Urology, 11, 45-54.
The Authors R. Ghanadian, PhD, Lecturer and Research Fellow. C . B. Smith, MA(Cantab), Research Assistant. G . Williams, FRCS, Senior Urological Registrar. C . D. Chisholm, ChM, FRCS, Consultant Urological Surgeon and Senior Lecturer.
0
The Effect of Antiandrogens and Stilboestrol on the Cytosol Receptor in Rat Prostate R. GHANADIAN, c. B. SMITH, G. WILLIAMS and G. D. CHISHOLM
Prostnfe Research Laboratory, Royal Postgraduate Medical School, London, W12 OHS
It has been demonstrated that antiandrogens can act by inhibiting the binding of androgens within the prostatic cell in both experimental animals and man (Fang and Liao, 1969; Dorfman, 1971; Hansson and Tveter, 1971; Sufrin and Coffey, 1973). Some of these compounds have been used in the management of patients with prostatic tumours and these reports have provided considerable information regarding their various actions (Scott and Wade, 1969; Chisholm and O’Donoghue, 1975; Jonsson and Nilsson, 1976). However, there is no reliable index or method for choosing a particular antiandrogen for clinical practice due to the fact that in most of these investigations the drugs have not been compared in an equimolar system. In addition, little is known about the effects of stilboestrol, the most widely used oestrogen, on the steroid receptor complex in the prostate. In the present study we have measured equimolar effects of representative compounds from 3 main classes of antiandrogens and the non-steroidal oestrogen, stilboestrol, on the dihydrotestosterone receptor complex isolated from rat prostate. These effects have been compared to the inhibitory effect of a similar concentration of testosterone. Materials and Methods Animals. Male albino Wistar rats of 300 to 400 g body weight were used in all experiments. Bilateral orchidectomy was performed, under fluothane anaesthesia, 24 hours prior to the experiment. Steroids. [I u,Za-3H]testosterone, Sp. activity, 49 Ci/mmol (Radiochemical Centre, Amersham, UK). The following unlabelled steroids were used: testosterone, 5a-dihydrotestosterone, androsterone (3a-hydroxy-5a-androstan-17one), androstanediol (5a-androstane-3P,17P-diol),androstenediol (androst-5-ene-3fl,17j%diol),androstanedione (5a-androstane-3,17-dione)and androstenedione (androst-4-ene-3J7-dione). Antiandrogens. 3 classes of antiandrogens were used : (a) 17cc-hydroxyprogesterone derivatives: Cyproterone acetate (Schering) and chlormadinone acetate (Syntex). (b) 19 nor-testosterone derivative: 17~-(2-methallyl)19 nortestosterone (SC9022) (Searle). (c) Non-steroidal antiandrogen: Flutamide (SCH 13521, Schering, USA). Other Compounds. Diethylstilboestrol. Solvents. Redistilled chloroform, acetone, diethyl ether (peroxide free), Triton X-100 and Toluene (Analar grade). Chromatographic Materials. Sephadex G-25 and G-200. Aluminium-backed silica gel plates, F254 for thin layer chromatography (T.L.C.). Scintillation Fluid. Toluene: Triton X-I00 (2 : 1) containing 0.4% diphenyloxazole (P.P.O.) was used for aqueous samples. Toluene containing 0.4% (w/v) P.P.O. was used for all other samples. Read at the 33rd Annual Meeting of the British Association of Urological Surgeons in Aberdeen, June 1977.
695
696
BRITISH JOURNAL OF UROLOGY
Buffers. Two buffers were used. Buffer A: 20 mM Tris-HC1 buffer, pH 7.4 containing 320 mM sucrose and 3 mM magnesium chloride. Buffer B: 20 mM Tris-HCI buffer, pH 7.4 containing 1.5 mM EDTA, disodium salt and 2 mM 2-mercaptoethanol.
Preparation and Labelling of Tissue Prostatic tissues were labelled either in vivo or in vitro and subsequently subjected to cell fractionation. (a) In vivo Experiments
Animals castrated 24 hours previously received an intraperitoneal injection of 1 pM/100 g body weight of the test drug in ethanol/saline (1 : 1 v/v). Control animals received an equal volume of ethanol/saline only. Each animal received 10 pCi/lOO g body weight [3H] testosterone in 20% ethanol/saline following the injection of drug. 1 hour after the administration of radioactivity the animals were killed and for each experiment the prostates from 2 animals were pooled. The tissue was minced, blotted, weighed and used for cytosol preparation.
(b) In vitro Experiments 24-hour castrated rats were sacrificed and the ventral prostates dissected out. For each experiment prostatic tissue was pooled from 3 animals. The tissue was minced and washed 3 times in buffer A prior to incubation with 0.25 pCi of [3H] testosterone per 2 ml of buffer A with or without tested drug at 0.1 pM. The tissue was incubated at 30°C
Table I Comparison of the Effect of Antiandrogens and Testosterone on the in vitro uptake of [3H] Testosterone by Rat Ventral Prostate. Values are Mean f S.E.M. Compound (Number of experiments)
Uptake of [3H] testosterone
dpm/mg Tissue
~ _ _ _
Testosterone (10) Cyproterone acetate __ (7) Chlormadinone acetate (7) sc 9022 (13) Flutamide (22)
~~
~~~
Control 1807.2+ 116.2
With compound 834.3k 131.4
53.3 k 6.9
1781.9k 138.3
1015.81102.7
42.5k4.2
1595.6 152.7
+
994.2 k 68.7
36.3 14.2
1850,Ok 115.0
991.8k82.5
46.2 2.7
1305.5k61.5
1319.5567.0
No inhibition
Table II In vivo and in vitro Metabolites of [3H] Testosterone, recovered from the Steroid Receptor Complex, isolated from the Cytosol of Rat Ventral Prostate
% Steroid recovered (mean+ S.E.M.) Steroids recovered Dihydrotestosterone Testosterone Androstanediol Androstenediol Androstendione Androsterone Androstanedione Others
]
% Inhibition
_____
In vivo
In vitro
79.07k2.52 1 0 6 7 11.64
77.40k4.74 7-46? 1.71
2.87 k 1.07
6.92 & 1.65
2.97+ 0.35 1.47+ 1.32 < 0.57 2.43 k 0.26
3.02k 0.58 0.58 f025 2 4 0 k 0.64 1.82k0.45
+
697
ANTIANDROGENS AND STILBOESTROL IN RAT PROSTATE
for 1 hour and subsequently was washed 3 times with buffer A, blotted, weighed and used for the preparation of cytosol. Uptake Studies
In vitro uptake of radioactivity by prostatic tissue was carried out as described previously (Ghanadian and Fotherby, 1970). The incubation media contained 100 pg of test drug and 0.25 pCi of [3H] testosterone per 2 ml media. Preparation of Cytosol Labelled tissue from in vivo or in vitro experiments was homogenised with a Teflon-glass motor-driven homogeniser in 3 volumes of buffer B using 5 strokes each of 10 sec duration and 30 sec cooling intervals. The homogenate was centrifuged at 105,000 g for 1 hour at 2°C. The cytosol was removed. Aliquots (0.05 ml) were taken for the measurement of protein and the remainder stored at - 20°C for analysis. Analysis of the Cytoplasmic Extract (a) Characterisation of the receptor protein was performed by sucrose gradient centrifugation (Ghanadian, Smith and Chisholm, 1977). ( b) Binding analysis. Cytoplasmic extracts (1.0 ml) were subjected to gel-filtration on either Sephadex G-200 or G-25 columns, eluted with buffer B at a flow-rate of 50 cm3/hour and fractions of 2 ml were collected. Aliquots (1 ml) from each fraction were transferred to scintillation vials together with 10 ml of scintillation fluid and the radioactivity was counted in an Intertechnique SL40 scintillation spectrometer. Analysis of Radiometabolites Radiometabolites were identified as previously described (Ghanadian, Smith and Chisholm, 1977). Other Procedures Protein was measured in 0.05 ml aliquots of cytosol using bovine serum albumin as described by Lowry et al. (1951).
Results
1. Uptake Studies The in vitro effects o f antiandrogens on the uptake of tritiated testosterone by the whole tissue are shown in Table I. In this study, no effect was observed with flutamide while the remaining 3 antiandrogens produced similar inhibitory effects on the uptake of tritiated testosterone. Table LII
In vivo and in vitro Inhibition o f [W] Dihydrotestosterone binding to the Cytoplasmic Receptor Proteins from the Rat VentraI Prostate by Testosterone and Antiandrogens % Inhibition In vitro
In vivo
Compound (Number of experiments) Testosterone (5)
Cyproterone acetate (5)
Chlormadinone acetate (5)
SC 9022 (5)
Flutamide (5) Stilboestrol (5)
4917-1
Mean k S.E.M.
Range
MeankS.E.M.
Range
89.55 k 2.60
81'94-93.74
76.88 f2.68
67.82-83'26
49.46k 3.94
40.21-60.59
47.01 k 2.47
40.75-55.16
50.89k 8.28
2010-66.70
48.55k 1.77
43.92-54'89
56.68 k 5.69
36.35-67.96
44.33 k 5.45
2462-55'68
44.39k 5.25
25.95-51.24
No inhibition
-
N o inhibition
-
2.46k 1.62
0-8.0
q
,698 12
Void volume
10
2 " 2
-
I
/,!'
8-
I:
2E:
*. E
BRITISH JOURNAL OF UROLOGY
6-
I '
5
I:
I' I'
4-
I: 2-
10 20 Fraction no. (2 ml)
30
Fig. 1. A typical example of the inhibition of [ZH] dihydrotestosterone binding to the receptor complex by tested compounds. The samples were analysed o n Sephadex G-25. Radioactivity elution profiles of the cytosol are shown for (a) untreated (-), (b) cyproterone acetate (0.1 P M )(- - -), (c) testosterone (0.1 P M )(- - - -). Fig. 2. Analysis of prostatic cytosol on sucrose density gradients in the presence of 0.4 M KCl. Labelled cytosol from 24-hour castrated rats was centrifuged at 60,000 rpm for 16 hours at 2°C. Sedimentation was from right to left. Bovine serum albumin with a sedimentation coefficient of 4.6 sec was used as a standard.
2. Separation of the Receptor and Identijcation of Radiometabolites The steroid receptor complex was isolated by column chromatography. The binding patterns on Sephadex G-25 (Fig. 1) and G-200 were found to be similar. This binding protein was then analysed by gradient centrifugation. When high salt solution (0.4 M KCl) was used the sedimentation coefficient was found to be 3.6 sec (Fig. 2). Analysis of [3H] steroids associated with the binding protein was carried out by thin layer chromatography. In both in vivo and in vitro studies 77 to 79 % of the radioactive steroid recovered was 5a-dihydrotestosterone. Testosterone was only 7 to 10% of the radioactivity recovered. Other androgens were present at a much lower level (Table 11). 3. Effects of Test Compounds on the Cytoplasmic DHT Receptor For the routine measurement of the effect of antiandrogens and stilboestrol on the binding of dihydrotestosterone to the receptor complex, samples were sequentially analysed on a column of Sephadex G-25 (Fig. 1). The total counts associated with bound radioactivity (fractions 5 to 10) were adjusted for variations in the protein content of the applied cytosol and the percentage inhibition compared to the control (untreated) was calculated. The inhibitory effect of 1 pmo1/100 g body weight of the 4 test antiandrogens on the dihydrotesterone receptor complex in vivo ranged from 44.39 to 56.6%. Apart from the 19-nor-testosterone derivative (SC 9022) which produced a somewhat higher inhibition than the non-steroidal compound (flutamide) no other significant differences were observed. The two 17a-hydroxyprogesterone derivatives tested in this experiment (cyproterone acetate and chlormadinone acetate) produced an almost identical effect on the cytosol receptor protein both in vivo and in
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vitro. Flutamide produced no in vifro effect. The non-steroidal oestrogen (stilboestrol) had negligible in vitro and no in vivo effect on the cytoplasmic D HT receptor complex (Table 111).
Discussion When the inhibitory effects of equimolar doses of antiandrogens and stilboestrol were compared to that of testosterone, a significantly higher inhibition was produced by testosterone. This applied to both in vivo and in vitro experiments. There was no significant difference between the in vivo inhibitory effects amongst the 3 classes of antiandrogens tested. Stilboestrol did not produce any in vivo effect. With the exception of flutamide, similar results were observed in vitro. Previous studies have demonstrated the equipotency of cyproterone acetate and flutamide, in vivo (Neri et al., 1972; Liao, Howell and Chang, 1974). In the present study, the potency of 17a-hydroxyprogesterone derivatives (cyproterone acetate and chlormadinone acetate) and the non-steroidal compound (flutamide) was found to be similar in vivo. However, no in vitro effect was observed with flutamide. Katchen and Buxbaum (1975) found that following the injection of tritiated flutamide in man more than 97 % of the compound was converted to several radiometabolites. They suggested that the side-chain hydroxylated metabolite could be the active form of this compound. This may explain the inability of flutamide to act in vifro. Our results also suggest that the 19-nor-testosterone derivative (SC 9022) is somewhat more active than flutamide. Skinner et al. (1975) found that 19-nor-dihydrotestosterone derivatives were slightly more potent than cyproterone acetate. It appears that the addition of a methylene group to 17a-hydroxyprogesterone derivatives does not alter the potency of the compound and that cyproterone acetate produces similar effects to chlormadinone acetate. Stilboestrol was included in this study because of its wide use in patients with prostatic carcinoma. We did not consider stilboestrol an antiandrogen in the strict sense since this drug does not compete with testosterone at the target tissue level (Dorfman, 1971). This study clearly demonstrates that stilboestrol does not have an inhibitory effect on the cytoplasmic binding of Sa-DHT to the receptor. Fang, Anderson and Liao (1969) reported only slight effect of stilboestrol on the nuclear retention of 5a-dihydrotestosterone using 500 times higher concentration than used in this study. It has been well documented that stilboestrol substantially reduces the level of plasma testosterone by inhibiting gonadotrophin production as well as stimulating sex hormone binding globulin (SHBG) in man (Jonsson and Nilsson, 1976). Some effect of stilboestrol on the 5a reductase has also been reported. In an in vitro study, Lee et al. (1973) measured the inhibitory effects of 6 oestrogens on the testosterone reduction in rat prostate and found that only those oestrogens with a complete steroid structure and a free phenolic hydroxyl group at position 3 of the steroid nucleus inhibited testosterone 5a reduction. However, in liver this structural requirement was not essential. They concluded that there was a negligible effect of stilboestrol on the conversion of testosterone to 5a-dihydrotestosterone in the prostate. Similarly, Belham and Neal (1971) found little effect on this enzyme in rat prostate. Other research groups (Farnsworth, 1969) have reported more effect of stilboestrol on this enzyme in the prostate. In the present study the existence of a receptor protein which binds preferentially to dihydrotestosterone was confirmed. In the presence of a high salt solution the steroid receptor complex had a sedimentation coefficient of 3.6 sec and this is in agreement with previous reports characterising receptor proteins (Ghanadian, 1976). Analysis of the [3H] steroids associated with the receptor complex showed that 77 to 79 % of the radioactivity recovered was dihydrotestosterone. Other workers have reported that 69% (Rennie and Bruchovsky, 1972) and 8.5% (Fang et al., 1969) of the steroid bound to the receptor was dihydrotestosterone. Summary The effects of 4 antiandrogens and stilboestrol on the cytosol receptor of the rat ventral prostate have been compared.
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Equimolar doses of cyproterone acetate, chlormadinone acetate, SC 9022 and flutamide inhibited the binding of dihydrotestosterone to the receptor complex in vivo by 44 to 50 % whereas stilboestrol had no effect. In the in vitvo studies an equimolar concentration produced similar effects to those observed in vivo with the exception of flutamide which had no effect. This technique provides a method for assessing and comparing the effectivenessof antiandrogens on the androgen receptor complex of the prostate. The authors wish to thank the Medical Research Council for financial support.
References BELHAM, J. E. and NEAL,G. E. (1971). Testosterone action in the rat ventral prostate. Biochemical Journal, 125, 81-91. (CHISHOLM, G. D.and O’DONOGHUE, E. P. N. (1975). The non-surgical treatment of prostatic carcinoma. Vitamins and Hormones, 33,377-397. DORFMAN, R. I. (1971). Antiandrogens. In Proceedings of 3rd International Congress on Hormonal Steroids, eds V. James and L. Martini. Amsterdam: Excerpta Medica, pp. 995. K. M. and LIAO,S. (1969). Receptor proteins for androgens. Journal of Biological Chemistry, FANG,S., ANDERSON, 24, 6584-6595. FANG,S. and LIAO,S.(1969). Antagonistic action of anti-androgens on the formation of a specific dihydrotestosterone-receptor protein complex in rat ventral prostate. Molecular Pharmacology, 5,420-431. FARNSWORTH, W. (1969).A direct effect of oestrogens on prostatic metabolism of testosterone. Investigative Urofogy, 6,423-427. GHANADIAN, R. and FOTHERBY, K. (1970). Uptake of 3H testosterone by tissues of the male rat. Steroidologia, 1, 193-200. GHANADIAN, R. (1976). Endocrine control of the prostate: mechanism of action of androgens. In Scientific Foundations of Urology, eds. D. I. Williams and G. D. Chisholm. London: Heinemann, Vol. 11, pp. 138-146. GHANADIAN, R., SMITH,C. B. and CHISHOLM, G.D. (1977). Identification of an androgen receptor in the cytosol of the female Mastomys prostate. Molecular and Cellubr Endocrinology, 8,147-15 5 . HANSSON, V. and TVETER, K. J. (1971). Effect of anti-androgens on the uptake and binding of androgen by human benign nodular prostatic hyperplasia in vitro. Acta Endocrinologica, 68, 69-78. JONSSON, G.and NILSSON,T.(1976). Pharmacology of drug therapy. In Scientific Foundations of Urology, eds. D. I. Williams and G. D. Chisholm. London: Heinemann, vol. 11, pp. 347-353. KATCHEN, B. and B ~ B A U S. M (1975). , Disposition of a new, non-steroid, antiandrogen, a,a,cr-trifluoro 2-methyl4’-nitro-m-propionotoluidide(flutamide), in men following a single, oral 200 mg dose. Journal of Clinical Endocrinology and Metabolism, 41, 373-379. LEE,D.K.H., YOUNG,J. C., TAMURA, Y., PATTERSON, D. C., BIRD,C. E. and CLARK,A. F. (1973). Effects of oestrogens on the A 4 reduction of testosterone by rat prostate and liver preparation. Canadian Journal of Biochemistry, 51,735-740. LIAO,S., HOWELL, D. K. and CHANG,I. M. (1974). Action of a non-steroidal antiandrogen, flutamide, on the receptor binding and nuclear retention of 5a-dihydrotestosterone in rat ventral prostate. Endocrinology, 94, 1205-1209. LOWRY, 0 .H., ROSEBROUGH, M. J., FARR,A. L. and RANDALL, R. J. (1951). Protein measurement with the folinphenol reagent. Journal of Biological Chemistry, 193,265-276. NERI,R., FLORANCE, K., KOZIOL, P. and VAN-CLEAVE, S.(1972). A biological profile of a non-steroidal antiandrogen, SCH 13521 (4’-nitro-3‘-trifluoromethylisolbutyranilide). Endocrinology, 91,427-437. RENNIE,P.and BRUCHOVSKY, N. (1972). I n vitro and in vivo studies on the functional significance of androgen receptors in rat prostate. Journal of Biological Chemistry, 247, 1546-1554. SCOTT, W. W. and WADE,J. C. (1969). Medical treatment of benign nodular prostatic hyperplasia with cyproterone acetate. British Journal of Urology, 101, 81-85. SKINNER, R. W. S., POZDERAC, R. V., COUNSELL, R. E. and WEINHOLD, P. A. (1975).The inhibitive effects of steroid analogues in the binding of tritiated 5a-dihydrotestosterone to receptor proteins. Steroids, 25, 189-202. SLJFRIN,G. and COFFEY, D. S. (1973). A new model for studying the effect of drugs on prostatic growth. Antiandrogens and DNA synthesis. Znveztigative Urology, 11, 45-54.
The Authors R. Ghanadian, PhD, Lecturer and Research Fellow. C . B. Smith, MA(Cantab), Research Assistant. G . Williams, FRCS, Senior Urological Registrar. C . D. Chisholm, ChM, FRCS, Consultant Urological Surgeon and Senior Lecturer.
