[CANCER RESEARCH 33, 849-855, April 1973] Suppression of Pituitary Tumor Growth and Function by Ergot Alkaloids1' 2 Robert M. MacLeod3 and Joyce E. Lehmeyer Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22901 SUMMARY mammary tumorigenesis. Among the drugs that are of considerable interest in control of prolactin release are the The in vivo and in vitro effects of ergot derivatives on ergot alkaloids. prolactin and growth hormone biosynthesis in the rat have Nagasawa and Meites (9) and Wuttke et al. ( 14) have shown been studied. Injection with 0.05 or 0.2 mg ergotamine that ergocornine in vivo decreases serum and pituitary tartrate had no effect on in vitro prolactin synthesis by the prolactin levels. When added in vitro, it decreases the release of pituitary gland. Ergocornine and ergocryptine, however, prolactin by the pituitary gland (3). It has also been shown inhibited both synthesis and release of prolactin. Incubation of that both ergocornine and ergocryptine inhibit the growth of glands with 4 or 40 /nM ergotamine greatly decreased prolactin 7,12-dimethylbenzanthracene-induced mammary tumors (1,9) synthesis and release but had no effect on growth hormone. and the growth of spontaneous mammary tumors as well (12). Ergocornine, 10 /¿M,and ergocryptine, 10 juM, almost Yanai and Nagasawa (16) demonstrated that ergocornine completely blocked prolactin release and decreased synthesis inhibited the appearance of spontaneous mammary tumors and release of growth hormone as well. and suppressed prolactin secretion in mice. Recently, we Daily administration of 0.05 mg ergotamine for 13 days to reported our finding that ergotamine, ergocryptine, and rats bearing the prolactin- and adrenocorticotropic hormone- ergocornine were all effective in suppressing growth of several secreting pituitary Tumor 7315a dramatically inhibited tumor pituitary tumors (7). Similarly, Quadri et al. (11) found that growth and reversed the adrenal hypertrophy caused by the injection of ergocornine or ergonovine induced regression of tumor adrenocorticotropic hormone. Ergotamine, ergo the growth of the prolactin-secreting pituitary Tumor cryptine, and ergocornine were all effective in suppressing the MtTWIS. growth of pituitary tumors and reversed the spleno- The present study is in agreement with these latter reports hepatomegalia caused by growth hormone-secreting tumors. In on the effects of ergot alkaloids on pituitary tumors. addition, the ergots decreased the high circulating prolactin Additionally, the work presented here demonstrates that the levels found in tumor-bearing animals. Ergotamine alone was primary effect of ergotamine on animals bearing pituitary able to overcome the atrophy of the pituitary glands of tumors is a direct, suppressive effect on the tumor itself, rather tumor-bearing animals and allow gland function to return than on the pituitary gland of the host. toward normal. Ergocryptine and ergocornine tended further to suppress gland function. These data demonstrate that MATERIALS AND METHODS hormone synthesis and release by the pituitary gland and pituitary tumors can be inhibited by derivatives of the ergot Animals. Mature Wistar-Furth rats (obtained from A. R. alkaloids. Schmidt Co., Inc., Madison, Wis.) were inoculated with pituitary Tumors MtTWS, MtTWIS, and SMtTWS as pre viously described (5). Pituitary Tumor 7315a was transplanted INTRODUCTION into mature female Buffalo rats (obtained from Simonsen Laboratories, Gilroy, Calif.). Mature female Sprague-Dawley For the past several years, investigations in this laboratory rats were obtained from Flow Research Animals, Dublin, Va. have been directed toward the mechanisms governing the All rats were routinely housed 4 to 5/cage at 22—23°and function of the pituitary gland and the function of pituitary allowed water and Purina laboratory chow ad libitum. tumors. More specifically, we have been interested in the Materials. Tissue Culture Medium 199 was obtained from endocrine mechanisms involved in prolactin synthesis and Microbiological Associates, Bethesda, Md. Leucine-4,5-3H, release by the pituitary gland and have been ultimately 29.8 Ci/mmole, was a product of International Chemical and concerned with the role of this hormone in supporting Nuclear Corp., Irvine, Calif. Ergotamine tartrate, ergocryptine, and ergocornine maléatewere gifts from Sandoz Pharmaceuti 1This investigation was supported by USPHS Grant CA-07535 from cals, Hanover, N. J. In some cases, the ergotamine tartrate the National Cancer Institute. injected was Gynergen (Sandoz). The drugs were all injected 'Portions of this work were presented at the Annual Meeting of the s.c. American Association for Cancer Research, 1972. Organ Culture. Incorporation of leucine-4,5-3H into prolac 'Investigation conducted while a recipient of Research Career Development Award CA-07665 from the National Cancer Institute. tin and growth hormone was studied by incubating bisected Received September 5, 1972; accepted January 8, 1973. pituitary glands in 0.5 or 1 ml of Tissue Culture Medium 199 APRIL 1973 849 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1973 American Association for Cancer Research. Robert M. MacLeod and Joyce E. Lehmeyer containing 5 to 10 juCi of leucine-4,5-3H. Flasks were protocol supplied by the Hormone Distribution Program, incubated for 6 to 7 hr at 37°in a Dubnoff shaker and were National Institute of Arthritis and Metabolic Diseases. The gassed with 95% O2 -5% C02 . Pituitary glands were homoge prolactin provided was iodinated either with '2 51 or '3 ' I, nized in 0.5 to 1 ml of 50 mM phosphate (pH 7.2) with a glass obtained from New England Nuclear, Boston, Mass. Sheep homogenizer fitted with a Teflon pestle. Homogenates were anti-rabbit y-globulin was obtained from Dr. Ann J. Johanson frozen and thawed 3 times to lyse all granules. Duplicate of the University of Virginia Medical School. aliquots (50 //I) were then subjected to polyacrylamide gel Statistical Analysis of Experimental Data. When appro electrophoresis (4), with the sample, stacking, and separating priate, results are expressed as the mean ± S.E. The gel system of Jones et al. (2). The incubation medium proteins significance of the difference between the means was were separated on gels as well, but the 50-/J.I aliquots were determined by Student's t test. "top-loaded" according to the system described by Reisfeld et al. (13). The proteins on the gels were stained with Amido RESULTS black and band quantitated by means of a Canalco micro- densitometer with an integrator. The hormone bands on the The effects of ergot alkaloids on pituitary gland function in gels were identified by comparing their mobility with purified normal female rats are illustrated in Chart 1. The open bars in preparations of prolactin and growth hormone. Our results are the chart represent the labeled prolactin or growth hormone found in the incubation medium at the termination of the in excellent agreement with those of Jones et al. (2), who incubation. The open bars, therefore, indicate "release" of eluted these proteins from the polyacrylamide gels and established their identity by bioassay. After the identity of the newly synthesized hormone into the medium during the hormone bands was established, the bands were cut, placed in incubation period. The shaded bars represent the labeled counting vials, and dissolved in 0.5 ml 30% HjC^ at 100°. prolactin or growth hormone found in the pituitary tissue at After addition of a Triton-toluene counting solution, the vials termination of incubation. The total height of both the open were placed in a scintillation counter to determine the level of and shaded bars is the sum of the newly synthesized hormone released into the medium and that remaining within the tissue radioactivity. and is termed "total synthesis." Daily injection of 0.05 or 0.2 Prolactin Radioimmunoassay. The immunoassay of sera and incubation media was carried out using the reagents and mg ergotamine tartrate for 7 days had no effect on the release of prolactin by the pituitary gland or on total in vitro PROLACTIN synthesis of prolactin. The injection of ergocornine, however, resulted in a decrease in release of newly synthesized prolactin into the incubation medium (open bar) and a subsequent 10 accumulation of the labeled hormone within the pituitary gland (shaded bar). There was no significant effect on total rii prolactin synthesis. Ergocryptine injection almost completely * blocked prolactin release. Newly synthesized prolactin accu mulated within the tissue (shaded bar), but total synthesis was decreased compared with the control. Injection of 0.2 mg of o the drugs resulted in a slight increase in total growth hormone I synthesis. This increase in growth hormone synthesis may be an example of the reciprocal relationship between prolactin t^•I—i-i-Controi1Hh»HW1T1M n u Km U N and growth hormone, a preliminary report on which has recently appeared (8). The in vitro effects of the ergots are shown in Chart 2. a Although ergotamine had no effect on in vitro pituitary *•Tj'-I-_!>_-I-Ergotamin« hormone synthesis when injected (Chart 1), incubation of pituitary glands with a 4 ¿/Mdrug concentration resulted in a greatly decreased release of prolactin into the medium (open bar) and a subsequent accumulation of labeled prolactin within the tissue (shaded bar). Total in vitro synthesis of prolactin was also significantly decreased with respect to the Ergocty0.05 Control Ergotomin« Control Ergocor mg doily 0.2 mg daily ninitini0.2 control. At 40 /^M, the effect of ergotamine on release and mg 0.2mgdoily daily total synthesis of prolactin was even more pronounced. No effect on growth hormone production was observed. Ergocor Chart 1. Effect of administration of ergot derivatives on in vitro nine and ergocryptine, 10 juM, almost completely blocked synthesis and release of prolactin and growth hormone. Bisected female rat pituitary glands were incubated with leucine-4,5-3H for 6 to 7 hr. release of prolactin (open bar) and greatly decreased total The prolactin and growth hormone in the incubation medium and in synthesis as well.