Effects of Bromocriptine and Tamoxifen on Growth and Hormone Receptor Levels of 7,12-Dimethylbenz(a)anthraceneinduced Mammary Tumors in Rats

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1 Endocrinol. Japon. 1983, 30 (4), Effects of Bromocriptine and Tamoxifen on Growth and Hormone Receptor Levels of 7,12-Dimethylbenz(a)anthraceneinduced Mammary Tumors in Rats NORIO WASEDA, YUZURU KATO, NORIO MATSUSHITA AND HIROO IMURA Second Medical Clinic, Department of Medicine, Kyoto University Faculty of Medicine, Kyoto 606 Abstract The role of prolactin (PRL) and estrogen in the growth of 7,12-dimethylbenz(a)- anthracene (DMBA)-induced rat mammary tumors was investigated. Tumor growth was suppressed by both bromocriptine and tamoxifen. Combined administration of ovine PRL and tamoxifen attenuated the inhibitory action of tamoxifen on tumor growth. Serum PRL levels were decreased by both bromocriptine and tamoxifen administration. Estrogen receptors (ER) in tumor tissues were decreased in number by tamoxifen, but not by bromocriptine. Progesterone receptors (PgR) in DMBAinduced mammary tumors were abolished by tamoxifen and significantly reduced by bromocriptine. Combined administration of ovine PRL with tamoxifen attenuated the inhibition of PgR induced by tamoxifen alone, while ER remained undetectable. Specific PRL binding to the liver was significantly reduced by treatment with bromocriptine, tamoxifen and tamoxifen plus ovine PRL, whereas PRL receptors in mammary tumor were not influenced by these drugs. These results suggest that PRL may stimulate DMBA-induced tumor growth independently of ER and that a part of the inhibiting effect of tamoxifen on tumor growth may be explained by the decreased PRL secretion. It is well known that growth of rat mammary tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) is dependent mostly on the action of estrogens and prolactin (PRL) (Asselin et al., 1977; Heuson et al., 1971/72; Leung and Sasaki, 1975; Manni et al., 1977; McGuire et al., 1974; Meites, 1972; Sterental et al., 1963). The response of a hormone-dependent tumor is likely to depend not only on the circulating Received April 27, 1983 The abbreviations used are: ER, estrogen receptor; PgR, progesterone receptor; PRL, prolactin; PRL-R, prolactin receptor; hgh, human growth hormone; DMBA, 7,12-dimethylbenz(a)anthracene; R5020, 17, 21-dimethyl-19-norpregna-4, 9-diene-3, 20-dione; DCC, dextran-coated charcoal. hormone levels but also on hormone receptor levels. Specific receptors for estrogen (De- Sombre et al., 1976; Leung and Sasaki, 1975; McGuire and Julian, 1971; Terenius, 1973), progesterone (Asselin et al., 1976; Horwitz and McGuire, 1977; Terenius, 1973) and PRL (Holdaway and Friesen, 1976; Smith et al., 1976) have been demonstrated in DMBA-induced mammary tumors. Furthermore, it has been reported that estrogen receptor levels in DMBA-induced rat mammary tumors are increased by estrogen and PRL (Asselin et al., 1977; Sasaki and Leung, 1975; Vignon and Rochefort, 1976), whereas PRL receptor levels are differently influenced by estrogen and PRL (Asselin et al., 1977; Holdaway and Friesen, 1976; Smith et al.,

2 530 WASEDA et al. Endocrinol. Japon. August ). There have been conflicting reports cocerning the role of ER in the action of PRL (Leung and Sasaki, 1975; Manni et al., 1977). In the present experiments, we examined the relationship between growth of DMBAinduced mammary tumors and the changes in PRL, estrogen and progesterone binding to mammary tumors when the action of circulating estrogens is blocked by tamoxifen and when the secretion of rat PRL is suppressed by bromocriptine. membrane fraction. Protein concentrations were determined by the method of Lowry et al. (1951). ER Assay Cytosol estrogen receptor (ER) was measured by a modification of the method of McGuire et al. (1977). In brief, cytosol (200ƒÊl) diluted with phosphate buffer to a protein concentration of 0.5 to 1.5 mg/ml was incubated for 15 hr at 0 Ž with increasing quantities of [3H]-estradiol (90 Ci/mmol; New England Nuclear, Boston, Mass., U. S. A., 0.03 to 0.4 pmol). An additional set of tubes with 0.4 pmol [3H]- estradiol was incubated with 80 pmol of diethylstilbestrol to determine nonspecific binding. Unbound hormone was removed by the addition of dextrancoated charcoal (DCC). Materials and Methods Animals Female Sprague-Dawley rats were maintained in a temperature-controlled room under artificial lighting (lights on at 0600 h, off at 1800 h) and fed laboratory chow (Oriental Yeast Co., Tokyo) and water ad libitum. Mammary tumors were induced by intragastric administration of 15 mg DMBA (Nakarai Chemical, Kyoto) dissolved in 1 ml sesame oil in 50-day-old rats. Tumor growth was measured twice a week with calipers, and tumor size was estimated as the product of the two largest perpendicular diameters. When the tumor size exceeded 100 sq mm, the animals were treated once a day for 2 weeks with one of the following: 1) vehicle (0.5 ml/rat, s. c.), 2) bromocriptine (600ƒÊg/100 g body wt., s. c.), 3) tamoxifen (250ƒÊg/rat, s. c.), 4) tamoxifen (250ƒÊg/rat, s. c.) plus ovine PRL (100ƒÊg/rat, s. c.). The estrous cycle was checked by smear examination throughout the experimental period. Rats were killed by decapitation 24 hr after the last injectjon on the day of estrous except for the tamoxifen-treated animals which showed a prolonged diestrous. Mammary tumors and liver tissue were excised and stored at-70 Ž until assayed. Blood samples were centrifuged and serum was kept at-20 Ž until assayed. Processing of Tissues Frozen tissues were pulverized and weighed. The following procedures were performed at 0 Ž, unless otherwise indicated. The powder was homogenized in phosphate buffer (5 mm sodium phosphate-1 mm monothioglycerol-10% glycerol, ph 7. 4) with three 10-sec bursts of a Polytron PT-10 homogenizer at the lowest setting. The homogenate was centrifuged at 800 g for 10 min, and the pellet was washed with phosphate buffer. All the supernatants were combined and centrifuged at 105,000 g for 30 min to obtain the supernatant cytosol and the precipitate PgR Assay Cytosol progesterone receptor (PgR) was measured by a modification (Waseda et al., 1981) of the method of Horwitz and McGuire (1977). A cytosol preparation (200 ƒêl) of the type described above was incubated for 15 hr at 0 Ž with different quantities of [3H] R 5020 (85.8 Ci/mmol; New England Nuclear, Boston, Mass., U. S. A.; to 0.2 pmol). An additional set of tubes with 0.2 pmol [3H] R5020 was incubated with 40 pmol of unlabeled R5020 to determine nonspecific binding. Unbound hormone was removed by the DCC method. PRL-R Assay Membrane PRL receptor (PRL-R) was measured by a modification of the method of Kato and Ohgo (1975). Membrane particles (100 pl) diluted with 25 mm Tris-HC1 (ph 7.6) to a protein concentration of 3 mg/ml and 125I-hGH (approximately 50,000 cpm, pci/pg, 100ƒÊl) were incubated for 1 hr at 37 Ž with 25 mm Tris-HCl buffer (ph 7.6) containing 10 mm CaCl2 and 1% BSA in a total volume of 500 ƒêl. An additional set of tubes was run as described above with the addition of 1ƒÊg unlabeled ovine PRL to determine nonspecific binding. Membrane-bound and free 125I-hGH were separated by centrifugation at 1,600 g for 30 min. The pellet was washed with ice-cold 25 mm Tris-HCl-10 mm CaCl2-1% BSA buffer. Radioimmunoassay of serum PRL Serum rat PRL levels were determined by a specific radioimmunoassay (Matsushita et al., 1981) using a kit supplied by the NIAMDD Rat Pituitary Hormone Distribution Program. The minimum detectable quantity of serum PRL was 1.0 ng/ml, and the coefficient of variation between assays averaged 12%. Ovine PRL was not crossed with anti-rat PRL serum in the assay. Analysis of data For both steroids, binding data were calculated

3 Vol.30, No.4 HORMONE RECEPTORS IN DMBA-INDUCED MAMMARY TUMORS 531 and analyzed according to the method of Scatchard (1949) with a computer assisted system. In PRL-R assays, specific PRL binding was defined as the difference between the radioactivity bound in the presence and in the absence of unlabeled ovine PRL. 125I-ovine PRL showed a similar displacement curve with ovine PRL to 125I-human GH. Statistical evaluation was conducted by one-way analysis of variance in combination with Ducan's new multiple range test. Drugs Tamoxifen and bromocriptine were kindly supplied by I. C. I. Japan (Osaka), and Sandoz (Basel, Switzerland), respectively. Ovine prolactin and human growth hormone were obtained from the National Hormone and Pituitary Program (Baltimore, Maryland, U. S. A.). Tamoxifen was dissolved in physiological saline containing 30% ethanol and bromocriptine was dissolved in physiological saline containing 0.5% tartaric acid. Results Tumor Growth (Fig. 1) DMBA-induced rat mammary tumors grew steadily throughout the observation period. The administration of bromocriptine resulted in a significant reduction in the mean ( }SE) tumor size at 2 weeks when compared with the initial size and control group tumors (61.6 }10.6% vs control 197 p<0.01). However, the tumor continued to grow despite bromocriptine treatment in 2 out of 12 rats. The administration of tamoxifen also inhibited growth in 13 of 15 rat mammary tumors. Combined administration of ovine PRL with tamoxifen attenuated the regression of tumor size induced by tamoxifen (tamoxifen+ovine PRL 91 }14% vs tamoxifen 44 }7, p<0.05). Changes in ER and PgR (Fig. 2 and 3) Cytosol ER and PgR were detectable in all of the DMBA-induced mammary tumors in the control group and the means ( }SE) of the maximum binding sites of each receptor were 27.1 }5.4 fmol/mg protein and 88.9 } 21.3 fmol/mg protein, respectively. Cytosol PgR levels in DMBA-induced mammary Fig. 1. Relative sizes of DMBA-induced rat mammary tumors 2 weeks after treatment with bromocriptine, tamoxifen and tamoxifen plus ovine PRL. Tumor size is expressed as a percentage of that at the time of the first injection (taken as 100%). In the control group, vehicle was injected. Each value is the mean+se. The number of rats is shown in parenthesis. Statistical difference is shown by asterisks (*: p<0.05 vs tamoxifen, **: p<0.01 vs control). tumors were reduced in the bromocriptine treated group (28.8 }6.7 fmol/mg protein, p<0.05 vs control), whereas ER levels were not significantly changed by bromocriptine. Treatment with tamoxifen eliminated both ER and PgR from DMBA-induced mammary tumors. Combined administration of ovine PRL with tamoxifen slightly attenuated the suppressive effect of tamoxifen on PgR, while ER levels inhibited by tamoxifen were not affected by ovine PRL treatment.

4 532 WASEDA et al. Endocrinol. Japon. August 1983 Fig. 2. Cytosol ER levels in DMBA-induced rat mammary tumors after treatment with bromocriptine, tamoxifen and tamoxifen plus ovine PRL. Means }SE are shown. The number of animals is indicated in parenthesis. Statistical difference is shown by asterisks (**: p<0.01 vs control and bromocriptine). Fig. 3. Cytosol PgR levels in DMBA-induced rat mammary tumors after treatment with bromocriptine, tamoxifen and tamoxifen plus ovine PRL. Means }SE are shown. The number of animals is indicated in parenthesis. Statistical difference is shown by asterisks (**: p<0.01 vs control). Binding of PRL to Liver and Mammary Tumors and Serum PRL Levels (Fig. 4) Specific PRL binding to particulate membranes of the liver and mammary tumors was detectable in all rats with DMBA-induced mammary tumors. Specific PRL binding to the liver was reduced by treatment with bromocriptine, tamoxifen and tamoxifen with ovine PRL (p<0.01 vs control, respectively). On the other hand, specific PRL binding to mammary tumors was not changed by any treatment with these drugs. Serum PRL levels in rats with DMBAinduced mammary tumors were greatly suppressed by treatment with bromocriptine (9.3 }1.3 ng/ml vs control 45.9 }11.2 ng/ml, p<0.01). Treatment with tamoxifen also resulted in a decrease in serum PRL levels, which was not changed by combined administration of ovine PRL with tamoxifen, although the biological PRL activity was expected to be increased by ovine PRL in the latter group. Discussion Estrogen and PRL are primary hormones required for the growth of rat mammary tumors induced by DMBA (Asselin et al.,

5 Vol.30, No.4 HORMONE RECEPTORS IN DMBA-INDUCED MAMMARY TUMORS 533 We have demonstrated that bromocriptine treatment suppressed serum PRL levels and the growth of DMBA-induced rat mammary tumors. These findings support other previous reports obtained with lergotrile (Smith et al., 1976; Sweeney et al., 1975). In the present study, ER levels in tumor tissues were not affected by bromocriptine (600ƒÊg/ 100 g body wt. x 2 weeks), whereas it was reported that bromocriptine (150 ƒêg/100 g body wt. ~3 weeks) reduced estrogen binding Fig. 4. Specific PRL binding to particulate membrane of the tumor and the liver and serum PRL levels in rats with DMBA-induced mammary tumors after treatment with bromocriptine, tamoxifen and tamoxifen plus ovine PRL. Data are mean }SE. The number of rats is indicated in parenthesis. Statistical difference is shown by asterisks (**: p <0.01 vs control). 1977; Leung and Sasaki, 1975; Manni et al., 1977; McGuire et al., 1974; Meites, 1972). It has been reported that PRL is the dominant hormone involved in the growth of DMBA-induced mammary tumors since estrogens are ineffective in stimulating tumor growth in rats after hypophysectomy (Sterental et al., 1963) and after the administration of lergotrile which inhibits the secretion of PRL (Manni et al., 1977). in tumors but not in the liver (Holdaway and Friesen, 1976). The reason for such a discrepancy is still unknown, but it may be explained by differences in experiment design. In the present experiment, we found that serum PRL levels were suppressed by tamoxifen treatment in these tumor bearing animals. As reported previously (Jordan, 1976), tumor growth was also prevented by tamoxifen and cytoplasmic ER was abolished and this was accompanied by decreased PgR levels in these mammary tumors. We observed a similar inhibitory effect of tamoxifen on ER and PgR in human breast cancer tissues (Waseda et al., 1981) and the inhibition by tamoxifen of basal PRL secretion has been also reported in eugonadal man (Gooren et al., 1980). ER levels in DMBA-induced tumors have been shown to increase with exogenous PRL treatment (Vignon and Rochefort, 1976). Following an oophorectomy-induced remission, however, perphenazine, which stimulates endogenous PRL release and increases tumor growth, is not able to raise ER levels (Manni et al., 1977). In this study, exogenous PRL attenuated tumor regression induced by tamoxifen, while ER remained undetectable. These results indicate that ER may not be indispensable for PRL action. PRL may stimulate tumor growth, at least in part, independently of ER, and a part of the inhibitory effect of tamoxifen on tumor growth may be explained by the decreased PRL secretion. However, it remains to be

6 534 WASEDA et al. Endocrinol. Japon. August 1983 elucidated whether each tumor cell responds to estrogen and PRL similarly or if there exists heterogeneity in response to both hormones. It is interesting that PgR levels in tumors were lowered by bromocriptine treatment and that PgR levels after combined administration of tamoxifen and ovine PRL were higher than those after tamoxifen treatment. Although estrogen can stimulate synthesis of PgR in DMBA-induced tumors (Horwitz and McGuire, 1977), our finding that exogenous PRL combined with tamoxifen increased PgR despite undetectable level of ER suggests the possibility that PRL has a direct action on tumor tissue by stimulating the synthesis of PgR. Although the induction of DMBAtumor is enhanced by progesterone (Huggins and Yang, 1962; Yoshida et al., 1980), the role of the hormone in the growth of established tumors remains to be elucidated. We did not find any relationship between PRL binding and tumor growth response to treatment. Neither bromocriptine nor tamoxifen affected PRL receptor levels significantly in rat mammary tumor tissues, whereas PRL binding to liver membrane preparations was significanly decreased by both drugs. These results may indicate regional heterogeneity of PRL receptors as previously reported (Smith et al., 1976) and a potential role of PRL in liver function which might affect mammary tumors through some growth factors (Francis and Hill, 1975) or enzyme modification (Richards, 1975). Some previous studies (Costlow et al., 1976; Holdaway and Friesen, 1976; Kelly et al., 1974) suggested that changes in the growth of DMBA-induced mammary tumors following endocrine treatment may be reflected by changes in PRL binding to tumor cells. However, our study and others (DeSombre et al., 1976; Smith et al., 1976) suggest that PRL dependence is not determined at the level of PRL receptors in tumor tissues. It must be investigated; along with other factors in the process which are linked to hormone-receptor coupling. Acknowledgements We thank Prof. S. Morii, Kansai Medical College, Osaka, for technical advice in the induction of mammary tumors. We thank Sandoz and I. C. I. for generous gifts of bromocriptine and tamoxifen, respectively. We also thank the National Hormone and Pituitary Program, NIH, for supplying pituitary hormones. References Asselin, J., P. A. Kelly, M. G. Caron and F. Labrie (1977). Control of hormone receptor levels and growth of 7, 12-dimethylbenz (a) anthracene-induced mammary tumors by estrogens, progesterone and prolactin. Endocrinology. 101, Asselin, J., F. Labrie, P. A. Kelly, D. Philibert and J. P. Raynaud (1976). Specific progesterone receptors in dimethylbenz (a) anthracene (DMBA)-induced mammary tumors. Steroids. 27, Costlow, M. E., R. A. Buschow and W. L. McGuire (1976). Prolactin receptors and androgen-induced regression of 7, 12-dimethylbenz (a) anthraceneinduced mammary carcinoma. Cancer Res. 36, DeSombre, E. R., G. Kledzik, S. Marshall and J. Meites (1976). Estrogen and prolactin receptor concentrations in rat mammary tumors and response to endocrine ablation. Cancer Res. 36, Francis, M. J. O. and D. J. Hill (1975). Prolactinstimulated production of somatomedin by rat liver. Nature. 255, Gooren, L., E. A. van der Veen and H. Kessel (1980). Modulation of prolactin secretion by gonadal steroids in men. In: Central and peripheral regulation of prolactin. (R. M. MacLeod and U. Scapagnini eds.) Raven Press, New York. pp Heuson, J. C., C. Waalbrook, C. Legros, G. Gallez, C. Robyn and N. L' Hermite (1971/72). Inhibition of DMBA-induced mammary carcinogenesis in the rat by 2-br-a ergocryptine (CB 154), an inhibitor of prolactin secretion, and by nafoxidine (U-11, 100A), an estrogen antagonist. Gynecol. Invest. 2, Holdaway I. M. and H. G. Friesen (1976). Correlation between hormone binding and growth response of rat mammary tumor. Cancer Res. 36, Horwitz, K. B. and W. L. McGuire (1977). Progesterone and progesterone receptors in experimental

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