Antiandrogen effects on androgen-responsive mammary tumour cells in culture

Journal Printed

Biochrmarrrr Vol Great Britain

pp. 819

822, 1981

0022.4731/81/090819-04802.00/O Press Lid

ANTIANDROGEN EFFECTS ON ANDROGEN-RESPONSIVE MAMMARY TUMOUR CELLS IN CULTURE JEANYATESand R. J. B. KING Hormone Biochemistry Department, Imperial Cancer Research Fund, P.O. Box 123, Lincoln’s Inn Fields, London WCZA 3PX, England (Received 26 November 1980) SUMMARY The effect of three antiandrogens, flutamide, hydroxyflutamide and cyproterone acetate on the growth of an androgen responsive mouse mammary tumour cell line has been investigated. These effects have been correlated with binding of the compounds to the androgen receptor. The growth inhibitory activities of hydroxyflutamide and cyproterone acetate are clearly related to their ability to inhibit androgen binding to cytoplasmic and nuclear receptors. Flutamide, however, inhibits cell growth in both the presence and absence of testosterone but does not inhibit DHT binding to the receptor.

INTRODUCTION

carcinoma. S115 cells can be maintained in culture for long periods of time whilst retaining their androgenMany antiandrogens have been developed and have responsiveness. When cultured in the presence of achieved widespread clinical use in prostatic hypertestosterone the cells exhibit a two to threefold stimuplasia and carcinoma, hirsutism and acne. However, lation of growth by testosterone or Sa-dihydrotestosmany of these compounds, such as cyproterone terone [9, lo]. It was thus felt that S115 cells would be acetate (6cc-chloro-17x-hydroxy-lcr,2a-methylene-4,6an ideal experimental system in which to test antianpregnanediene-3,20-dione-17-acetate), which have a drogenic compounds. In addition it was hoped that steroidal structure, possess progestational and other further insight would be gained into the mechanism hormonal activities which can cause unwanted side by which steroid effects on cell proliferation are effects. mediated by hormone receptors. Two non-steroidal The non-steroidal compound flutamide (a, CL, compounds, flutamide and hydroxyhutamide and the z-trifluoro-2-methyl-4’-nitro-m-propionotoluidide) is steroidal antiandrogen cyproterone acetate have been devoid of hormonal activity and is a potent antianexamined. drogen in orchidectomized androgen-treated rats [l]. Studies in vivo have shown that flutamide antagonizes the biological activity of androgens in a manner simiEXPERIMENTAL lar to that of cyproterone acetate. It inhibits the cytoCell culture plasmic and nuclear binding of Sa-dihydrotestosterStock cells were routinely cultured in a humidified one (DHT) in the rat sex accessory glands and eliminates the testosterone-induced stimulation of RNA atmosphere of 5% carbon dioxide in air at 37°C in polymerase activity [24]. However, in all of these Dulbecco’s modified Eagle’s medium supplemented investigations flutamide was found to be less active in with 2% foetal calf serum (Gibco Bio-Cult, Glasgow, vitro and it was concluded that a metabolite of flutaScotland), 40 nM N-2-hydroxyethylpiperazine-N’-2ethane-sulphonic acid (Sigma Chemical Co.) and mide might be the active agent. When administered to human males as a single 35 nM (0.01 &ml) testosterone (Steraloids Ltd, Croydose, flutamide was indeed found to be rapidly don, England). Cells were subcultured twice weekly. metabolized. The major product isolated from plasma Cell growth experiments was a hydroxylated derivative a, a, a-trifluoro-Z methyl-4’-nitro-m-lactotoluidide which was suggested Cells from stock plates were suspended in 0.05% to be the active antiandrogen [S]. This compound is trypsin buffered with 0.02% EDTA (pH 7.3), counted 1.5 times as antiandrogenic in vivo as flutamide and is on a haemocytometer and added to prepared growth also devoid of hormonal activity [6]. It is far more medium with or without 35 nM testosterone at the effective than flutamide in inhibiting cytoplasmic and required cell concentration. Aliquots (5 ml) of cell suspension were placed in 50mm plastic tissue culture nuclear binding of DHT [7,8]. As a means of investigating the role of steroid hordishes. For growth curves cells were seeded at 2 x lo4 cells per dish, the medium was changed to mones in regulating the growth of mammary tumours we have been using a cell line derived from the androinclude the relevant antiandrogen at a concentration of 1 pg/ml after 24 h and on every third day theregen-responsive Shionogi 115 (S115) mouse mammary 819

JEAN YATES and R. J. B. KING

after; the cells were counted daily. For dose response curves cells were seeded at lo5 cells per dish, the medium was changed to include antiandrogens at the desired concentration after 24 h and cell counts were performed three days later. Cell numbers were estimated from triplicate Coulter Counter readings of trypsinised cell suspensions diluted with Isoton (Coulter Electronics Ltd, Harpenden, England). All cell counts were expressed as the mean of triplicate plates, standard errors were always less than 10% of the mean. Androgen receptor assays Cells were grown to a sub-confluent monolayer and were then maintained in serum- and hormone-free medium for 24 h. The cells were removed from the dishes by scraping with a rubber policeman and the pellet homogenized in 10mM Tris, 1 mM EDTA, 10 mM DTT buffer, pH 7.4 containing 10% glycerol. A cytosol was prepared by centrifuging at 100,000g for 30 min. Incubations were carried out for 4 h at

4°C using 10 nM C3H]-DHT (56 Ci/m mol) and free steroid was removed by treatment with dextran coated charcoal for 5 min at 4°C. Nuclear translocation assays Sub-confluent monolayers of cells were changed to serum- and testosterone-free medium 24 h before use. The cells were incubated with 10 nM C3H]-DHT and 1 PM cold DHT or anti-androgen in medium for 2 h at 37°C. After scraping and homogenization in 10mM Tris, 1 mM EDTA, 10mM DTT buffer containing 0.1% Triton X-100 the nuclear fraction was obtained by centrifugation at 500 g for 5 min. The nuclear pellet was resuspended in buffer and the receptor precipitated by treatment with protamine sulphate for 15 min at 0°C. Bound C3H]-DHT was extracted from the pellet using absolute alcohol. RESULTS

Effects on cell growth

In the presence of 35 nM testosterone cell growth is inhibited by one hundredfold excess of each of the

0.“~

D-r*

Fig. 2. The recovery of growth following readdition of 35 nM testosterone (T) to previously deprived cells in the presence and absence of 3.62 mM flutamide (F).

three antiandrogens tested, hydroxyflutamide and cyproterone acetate being more effective than tlutamide (Fig. 1). This inhibition only seems to act on the growth rate and not on the final cell density both of which are increased by testosterone. Flutamide also exerts an inhibitory effect in the absence of testosterone apparently throughout the whole period of growth. This effect is shown to a much smaller degree by hydroxyflutamide and cyproterone acetate. The significance of these effects can be seen by a comparison of cell numbers during the rapid growth phase and at saturation density (Table 1). If testosterone is added to cultures growing for 7 days, either in the presence or absence of flutamide, the growth rate increases (Fig. 2) and a final cell density equal to that of cells continuously in the presence of testosterone (as shown in Fig. 1) is reached by day 13.

Dose response of antiandrogen effects When tested over a wide range of doses in a short term growth assay, hydroxyflutamide and cyproterone acetate were again more effective in terms of overall inhibition than flutamide (Fig. 3). In addition they were active at lOO-fold lower concentrations than flutamide. In the absence of testosterone only flutamide showed an inhibitory effect.

D.“S

Fig. 1. The effect of antiandrogens on the growth of ,915 cells in the presence ~ and absence ----- of 35 nM testosterone (T). CA 2.40 mM cyproterone acetate. HF 3.42 mM hydroxflutamide. F 3.62 mM Butamide.

Antiandrogen action in cultured tumour cells

821

Table 1. Antiandrogen effects on cell growth (day 7) and saturation density (day 10). Data from Fig. 1. Antiandrogen concentrations as in Fig. 1 Cells x 105/plate Antiandrogen Cyproterone acetate Cyproterone acetate Hydroxyflutamide Hydroxyflutamide Flutamide

Testosterone. + + + + + + -

Flutamide

Day 7 18.25 f 10.00f 3.87 f 3.16 f 18.25 + 14.26 k 3.87 k 3.26 k 13.99 + 10.31 k 4.84 + 2.61 It

Day 10

0.56 18.28 + 0.12 0.12*** 18.79 + 0.02 0.17 7.04 f 0.34 0.15* 6.24 f 0.45 0.56 18.28 f 0.12 0.39; 21.79 + 0.17 0.17 7.04 f 0.34 0.03 5.94 f 0.23+ 0.71 18.41 + 0.68 0.08, 20.10 + 0.25 0.32 7.50 f 0.54 0.08’ 3.95 + 0.10**

Significance levels are * < 0.05; ** < 0.01; *** < 0.001. Remainder no significant effect Competition for androgen receptor Specific binding to the cytoplasmic androgen receptor is eliminated by a lOO-fold excess of cold DHT (Fig. 4). Hydroxyflutamide and cyproterone acetate also inhibit cytoplasmic binding of C3H]-DHT although with a lower affinity. Flutamide shows no inhibition. Similarly, while DHT, hydroxyflutamide and cyproterone acetate all inhibit nuclear uptake of @f]-DHT, flutamide has no effect (Table 2). DISCUSSION

The stimulatory effect of testosterone on S115 cell proliferation rate can be inhibited, although not completely eliminated, by these antiandrogens. The effect

Fig. 3. The dose-dependent inhibition of S115 cell growth by flutamide, hydroxyflutamide and cyproterone acetate in the presence and absence of testosterone during a three. day growth period. Each point represents the mean of triplicate culture plates and results are expressed as the percentage reducficwri5~.~_epakc~arittr~

Table 2. Inhibition of specific nuclear uptake of [“HI-DHT (10 nM) by antiandrogens (1 PM)

Inhibitor DHT Flutamide Hydroxyflutamide Cyproterone acetate

Inhibition of [“HI-DHT nuclear uptake (%) 100 0 60 76

of testosterone on the final cell density is not, how-’ ever, affected. Thus the S115 cell system provides an ideal model in which to investigate potential antiandrogens with regard to both inhibition of growth and cytoplasmic and nuclear receptor binding. Whereas receptor binding alone cannot distinguish between androgens and antiandrogens, the effect of the compounds on the growth rate.of cells in the presence or absence of testosterone yields information about antagonist or agonist activity respectively. In the case of hydroxyflutamide and cyproterone acetate there is a,

Fig. 4. The inhibition of specific cytoplasmic receptor binding of r3H]-DHT by antiandrogens. Each point represents tht Lana&of &+ate determinations.

JEAN YATES and R. I. B. KING

822

close parallel between inhibition of binding and of growth. The same effect has previously been described for another antiandrogen BOMT (6a-bromo-17/3-hydroxy-17a-methyl-4-oxa-Sa-androstan-3-one) and for oestradiol both of which bind to the androgen receptor and inhibit the stimulation of growth. Diethylstilboestrol which does not bind to the receptor has no effect on growth at physiological concentrations [l 11. The lower concentrations apparently required to inhibit growth probably reflect the different methods used in their determination; it is assumed that the concentration of the compounds in the cells is substantially higher than that in the medium. The effects of flutamide, however, are more difficult to interpret. It is possible that flutamide binding to the androgen receptor is very weak and that, in these assay conditions, inhibition is masked by the high concentration of C3H]-DHT under equilibrium conditions. Such weak binding may only be detected by shorter incubation times. The fact that hydroxyhutamide, but not flutamide, binds to the androgen receptor whilst both are active antiandrogens might indicate that flutamide exerts its effect through conversion to the more active metabolite as suggested earlier [S]. However, the fact that flutamide, but not hydroxyflutamide, exerts an inhibitory effect in the absence of testosterone suggests that this cannot be the complete explanation. The effect of flutamide on the growth of S115 cells may be analogous to that of the antioestrogen Tamoxifen on the MCF-7 human breast cancer cell line with the notable difference that Tamoxifen binds strongly to the oestrogen receptor [12]. The growth of S115 cells in the absence of testosterone shows that they are androgen responsive rather than androgen dependent (Fig. 1) and indicates that receptor-mediated proliferation can proceed in the absence of hormone. There is, therefore, a possibility that flutamide exerts its effect by depressing the activity of the receptor or altering it to a less efficient form without significant binding. REFERENCES 1. Neri R. 0.. Florance K., Koziol P. and Van Cleave S.:

A biological profile of a nonsteroidal antiandrogen. SCH 13521 (4’-nitro-3’-trifluoromethylisobutyranilide). Endocrinology 91 (1972) 427437. 2. Mainwaring W. I. P.. Mangan F. R., Feherty P. A. and Freifeld M.: An investigation into the anti-androgenic properties of the non-steroidal compound SCH 13521 (4’-nitro-3’-trifluoromethyl-isobutyranilide). M&c. cell. Endocr. l(1974) 113-128. 3. Ghanadian R., Smith C. B., Williams G. and Chisholm G. D.: The effect of antiandrogens and stilboestrol on the cytosol receptor in rat prostate. Br. J. Ural. 49 (1977) 691-700. 4. Smith C. B., Ghanadian R. and Chisholm G. D.: Inhibition of the nuclear dihydrotestosterone receptor complex from rat ventral prostate by antiandrogens and stilboestrol. Molrc. cell. Endow. 10 (1978) 13-20. 5. Katchen B. and Buxbaum S.: Disposition of a new, nonsteroid antiandrogen, X, Z, r-trifluoro-2-methyl-4’nitro-m-propionotoluidide (Flutamide), in men following a single oral 200 mg dose. J. clin. Endocr. Metab. 41 (1975) 373--379. 6. Neri R. 0.. Koziol P. and Kung T. T.: Sch 16423 (a, a, a-trifluoro-2-methyl-4’-nitro-m-lactotoluidide) a potent antiandrogen. Endocrine Sot. Program. 57 (1975) 298. 7. Peets E.. Henson F. and Neri R. 0.: Effect of nonsteroidal antiandrogen Sch 16423 (a, a, a-trifluoro-2methyl-4’-nitro-m-lactotoluidide) on androgen disposition in rats. Endocrine Sot. Program. 47 (1975) 306. 8. Svmes E. K.. Milrov E. J. G. and Mainwarine W. 1. P.: The nuclear uptake of androgen by hum&r benign prostate in vitro: action of antiandrogens. J. Ural. 120 (1978) 180-183. 9. King R. J. B., Cambray G. J., Jagus-Smith R., Robinson, J. H. and Smith J. A.: Steroid hormones and the control of tumor growth: studies on androgen-responsive tumor cells in culture. In Receptors and Mechanism of Action oJ Steroid Hormones, Part 1 (Edited by J. R. Pasqualini). Marcel Dekker Inc., New York (1976) pp. 41-84. IO. Yates J., Couchman J. R. and King R. J. B.: Androgen effects on growth, morphology and sensitivity of S115 mammary tumor cells in culture. In Hormones and Cancer (Edited by S. Iacobelli, R. J. B. King, H. Lindner and M. E. Lippman). Raven Press, New York (1980) pp. 31-39. Il. King R. J. B., Cambray G. J. and Robinson J. H.: The role of receptors in the steroidal regulation of tumour cell proliferation. J. steroid Biochem. 7 (1976) 869-873. 12. Osborn C. K. and Lippman M. E.: Human breast cancer in tissue culture: the effects of hormones. In Breast

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