- Email: [email protected]
6-Methylenic steroids for prostate cancer
308 1.25-DIHYDROXYVITAMIN03 RECEPTORS IN PRIMARY BREAST CANCERS : A POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) STUDY ; Guillemant, S. and Spyratos, F. Service de Biochimie Medicale, Faculte de Medecine PitieSalpetriere ; 75013 Paris, and Laboratoire de Biochimie, Centre Rene Huguenin ; 92210 SaintCloud, FRANCE. Sixty primary breast cancers were analyzed for the presence of 1,25 (OH) 2D3 receptors the pronostic value of which was evaluated. A previously published PAGE technique (in vitamin D eds A.W. Norman et al., W. DeGruyter, Berlin, 1985, pp 897-900) was used : Breast tumors obtained at surgery were inmediately frozen and stored in liquid N2. Tumor fragments for receptor assay (E 300 mg) were homogenized in ice-cold 0.05M Tris-HCl buffer pH 7.4, containing 25 mM 5mM C12Mg, 5 mM dithiothreitol, 2O/mM Mo04Na2 and aprotinin 200 kallicrein units/ml. KC1 A g i 100000 supernatant was prepared and incubated for 1Bh at 4°C with 2 nM tritiated 1,25(OH)203 with or without a 200-fold excess of unlabelled 1.25 (OH) 2D3. Samples containing 250400 ug of protein were PAGE run for 3h at lmA/gel and at 4'C in cylindrical separation gels (T = 10% and C = 2%). Afterwards the gels were sectioned transversaly into slides 1.2 nm thick which were counted for radioactivity. Specific radioactive binding was calculated by subtracting binding in presence of an excess of unlabelled 1.25 (OH) 2D3 from total tritium binding. Receptor-positive breast tumors frequently exhibited one slow-moving specific peak,but some receptor-positive cytosols exhibited 2 or 3 fast-moving specific peaks.Receptor-negativebreast tumors could be classified in two groups : The first one showing a flat pattern and the second one showing marked non-specific radioactive peaks.lnterestingly this later group of breast car cers was the only one to be associated with a significantly (p = 0.04)increased probability of recurrences.
_
309
THE ANDROGENRESPONSIVE HUMAN PROSTATE TUMOR
CELL LINE LNCaP: ANDROCEN RECEPTORS AND THE EFFECT OF ANDRCCENS ON THE RELEASE OF PROTEINS, Berns, E.; de Boer, U. and Mulder, E. Department of Biochemistry 11, Medical Faculty, Erasmus University, P.O. Box 17'38,3000 DR Rotterdam, THE NETHERLANDS. Differentation and proliferation rates of human prostate carcinoma are dependent upon androgen stimuli. The hormonal effects are mediated by intracellular receptors. In search for a possible autocrine regulatory function in androgen dependent tumors, we have studied synthesis and secretion of androgen stimulated protein(s) in LNCaP cells. The androgen receptor (AR) content in the nuclear extract of LNCaP cells was 1679 f 558 fmol/mg nuclear, extract protein, corresponding to 15,-20,000 sites/cell. Sucrose gradient (high salt) centrifugation revealed two receptor forms sedimenting at 4.5 and 3 S. Only the 4.5 S form bound to DNA-cellulose. Estrogen and progesterone receptors could not be detected in the nuclear extracts nor in the cytosol, indicering that these receptors are absent. When grown in media containing charcoal treated fetal calf serum, cells released significantly lower amounts of several proteins especially of a (35S)-methionine labeled protein with Mw of 42 kD. The release of this protein could be R1881 restored in cells cultured in the presence of dihydrotestosterone (DHT, O.l-1uM) or (O.lnM-O.lpM). The high concentrations of DHT needed to restore this protein is possibly related to extensive metabolism of DHT by the LNCaP cells. Addition of estrogens, corticoids or progesterone had no effect on the release of the 42 kD protein. Anti-androgens, which inhibit cell grohth. also exerted inhibitory effects on the release of the 42 kD protein. In conclusion, both cell growth and release of a 42 kD protein are under control of androgens in LNCaP cells. __31o
7
6-METHYLENIC STEROIDS FOR PROSTATE CANCER; Petrow, V.; Padilla, G.M., Department of Physiology./ Duke University Medical Center; Durham, NC 27710, USA: Kadohama, N. Department of Diagnostic‘! Immunology; Roswell Park Memorial Institute; Buffalo, NY 14263, USA; Kendle, K. School of Pharmacy; Robert Gordon's Institute of Technology, Aberdeen AB9 lFR, SCOTLAND, UK. We developed 5a-reductase inhibitors in order to determine whether prostate cancer (PC) depends upon testosterone (T) or dihydrotestosterone (DHT) for hormonal support. 6-Methylene-4-pregnene-3,20-dioneIrreversibly Inhibits human and rat prostatic ~CXreductase by a mechanism of the k,,t type. In vivo It inhibits rat prostate growth and has slight anti-DHT activity In the castrate rat. In the Copenhagen rat at ZOmg/k(sc) It inhibit growth of the Dunning R3327H and Noble tumors by 92% (117 days) and 49% (17 days) respectively No effects are apparent upon LH levels, liver and tumor histology and DNA content (ug/mg), and tumor-free carcase weight. T levels are lowered. It follows that the above prostate tumors and most likely human PC depend upon DHT and not T for hormonal support and that 6-methylene4-pregnene-3,20-dionemay represent new first-line therapy. inhibit 17a-Hydroxy-, 17a-acetoxy- and 17a-propionoxy-6-methylene-4-pregnene-3,2O-dione 5a-reductase in vitro but have negligible effect on prostate growth in vivo. 21-Acetoxy-6methylene-4-pregnene_3,20_dionehas weak antlprostatic activity. 17B-Acetoxy-6-methylene-4androsten-3-one is a potent androgen. 17S-Acetoxy-16,16-dimethyl-6-methylene-4-androsten-3one is strongly antiprostatic and closely resembles 6-methylene-4-pregnene-3,20_dione in _._ biological prorile. Supported by American Cancer Society Grant No. PDT 256.
118s
_
309
THE ANDROGENRESPONSIVE HUMAN PROSTATE TUMOR
CELL LINE LNCaP: ANDROCEN RECEPTORS AND THE EFFECT OF ANDRCCENS ON THE RELEASE OF PROTEINS, Berns, E.; de Boer, U. and Mulder, E. Department of Biochemistry 11, Medical Faculty, Erasmus University, P.O. Box 17'38,3000 DR Rotterdam, THE NETHERLANDS. Differentation and proliferation rates of human prostate carcinoma are dependent upon androgen stimuli. The hormonal effects are mediated by intracellular receptors. In search for a possible autocrine regulatory function in androgen dependent tumors, we have studied synthesis and secretion of androgen stimulated protein(s) in LNCaP cells. The androgen receptor (AR) content in the nuclear extract of LNCaP cells was 1679 f 558 fmol/mg nuclear, extract protein, corresponding to 15,-20,000 sites/cell. Sucrose gradient (high salt) centrifugation revealed two receptor forms sedimenting at 4.5 and 3 S. Only the 4.5 S form bound to DNA-cellulose. Estrogen and progesterone receptors could not be detected in the nuclear extracts nor in the cytosol, indicering that these receptors are absent. When grown in media containing charcoal treated fetal calf serum, cells released significantly lower amounts of several proteins especially of a (35S)-methionine labeled protein with Mw of 42 kD. The release of this protein could be R1881 restored in cells cultured in the presence of dihydrotestosterone (DHT, O.l-1uM) or (O.lnM-O.lpM). The high concentrations of DHT needed to restore this protein is possibly related to extensive metabolism of DHT by the LNCaP cells. Addition of estrogens, corticoids or progesterone had no effect on the release of the 42 kD protein. Anti-androgens, which inhibit cell grohth. also exerted inhibitory effects on the release of the 42 kD protein. In conclusion, both cell growth and release of a 42 kD protein are under control of androgens in LNCaP cells. __31o
7
6-METHYLENIC STEROIDS FOR PROSTATE CANCER; Petrow, V.; Padilla, G.M., Department of Physiology./ Duke University Medical Center; Durham, NC 27710, USA: Kadohama, N. Department of Diagnostic‘! Immunology; Roswell Park Memorial Institute; Buffalo, NY 14263, USA; Kendle, K. School of Pharmacy; Robert Gordon's Institute of Technology, Aberdeen AB9 lFR, SCOTLAND, UK. We developed 5a-reductase inhibitors in order to determine whether prostate cancer (PC) depends upon testosterone (T) or dihydrotestosterone (DHT) for hormonal support. 6-Methylene-4-pregnene-3,20-dioneIrreversibly Inhibits human and rat prostatic ~CXreductase by a mechanism of the k,,t type. In vivo It inhibits rat prostate growth and has slight anti-DHT activity In the castrate rat. In the Copenhagen rat at ZOmg/k(sc) It inhibit growth of the Dunning R3327H and Noble tumors by 92% (117 days) and 49% (17 days) respectively No effects are apparent upon LH levels, liver and tumor histology and DNA content (ug/mg), and tumor-free carcase weight. T levels are lowered. It follows that the above prostate tumors and most likely human PC depend upon DHT and not T for hormonal support and that 6-methylene4-pregnene-3,20-dionemay represent new first-line therapy. inhibit 17a-Hydroxy-, 17a-acetoxy- and 17a-propionoxy-6-methylene-4-pregnene-3,2O-dione 5a-reductase in vitro but have negligible effect on prostate growth in vivo. 21-Acetoxy-6methylene-4-pregnene_3,20_dionehas weak antlprostatic activity. 17B-Acetoxy-6-methylene-4androsten-3-one is a potent androgen. 17S-Acetoxy-16,16-dimethyl-6-methylene-4-androsten-3one is strongly antiprostatic and closely resembles 6-methylene-4-pregnene-3,20_dione in _._ biological prorile. Supported by American Cancer Society Grant No. PDT 256.
118s