ICI 164,384, a Pure Antagonist of Estrogen-Stimulated MCF-7 Cell Proliferation and Invasiveness1
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[CANCER RESEARCH 49. 6929-6934, December 15. I989| ICI 164,384, a Pure Antagonist of Estrogen-stimulated MCF-7 Cell Proliferation and Invasiveness1 Erik W. Thompson,2 Deborah Katz, Thomas B. Shima, Alan E. Wakeling, Marc E. Lippman, and Robert B. Dickson Laboratory of Developmental Biology and Anomalies, NI DR, NI H, Belhesda, Maryland 20892 [E. W. T., T. B. S.]; Vincent T. Lombardi Cancer Center, Georgetown University Medical Center, 3800 Reservoir Rd., NW Washington, DC 20007 ¡D. K., M. E. L., R. B. D.J; and ICI Pharmaceuticals. Alderly Park, Macclesfield, Cheshire, England [A. E. W.I ABSTRACT ment and testing of new antiestrogens with the objective of achieving drugs with pure antagonist properties. Some success Estrogen is known to stimulate the proliferation and basement mem was seen with the benzothiophene antiestrogens, particularly brane ¡nvasivenessofthe MCF-7 human breast cancer cell line. We have compared the new steroidal antiestrogen ICI 164,384, the triphenyleth- LY 117018, which exhibits similar activities to OHT in block ylene 4-hydroxytamoxifen (OHT), and the benzothiophene LY 117018, ing estrogen-induced proliferation of MCF-7 cells (22), and for their effects on the proliferation and invasiveness of the MCF-7 cell show less partial agonism of estrogen effects on MCF-7 cells line and its antiestrogen-resistant variant LY-2. While all three anties- (4, 23), or on progesterone receptor expression in, or growth trogens blocked the proliferative effects of 17/J-estradiol on MCF-7 cells, of, the immature rat uterus (24). Although this molecule pro OHT and LY 117018, but not ICI 164,384 stimulated their proliferation vides a good tool for the study of estrogen action in vitro, its in the absence of estrogen. The proliferative effects of OHT and LY use in the clinic is severely limited by poor pharmacokinetics 117018 were blocked by ICI 164,384. Basement membrane ¡nvasiveness (25, 26). ICI 164,384 is a new steroidal antiestrogen which of MCF-7 cells was stimulated by 17/3-estradiol and OHT, but not LY exhibits no estrogenic effect on uterine growth in immature rats 117018 or ICI 164,384. Both ICI 164,384 and LY 117018 were able to and mice, and reverses estrogen-induced proliferation of cul block the invasiveness induced by either 17/9-estradiol or OHT. The LY- tured MCF-7 cells (27-29). It binds to partially purified estro 2 antiestrogen-resistant variant of the MCF-7 cell line showed increased basal proliferation, and responded only slightly to estrogen. ICI 164,384, gen receptor, but unlike estrogen, does not stimulate the affinity but not OHT or LY 117018 antagonized the effects of 17/3-estradiol, but of the receptor complex for DNA cellulose (30). did not reduce proliferation below control levels. The LY-2 line was not Recently, we have demonstrated that invasion of a reconsti resistant to the antiestrogenic effects of LY 117018 or ICI 164,384 on tuted basement membrane by MCF-7 cells in vitro is estrogen invasiveness, and was stimulated by LY 117018 for this parameter. Thus, stimulated (31), and that this activity is also stimulated by ICI 164,384 is a pure antiestrogen for MCF-7 cell proliferation and tamoxifen and OHT, presumably due to estrogen agonist prop invasiveness, and may offer clinical advantage over nonsteroidal anties- erties (32). Basement membranes surround epithelial tissues, trogens which can stimulate these activities in tumor models in vitro. nerves, and muscles, providing physical support to these tissues and segregating parenchymal and mesenchymal elements (33). INTRODUCTION Their major components, collagen IV, laminin, and heparan sulfate proteoglycan are common to all known basement mem Appreciation of the mechanism of action of estrogen in branes (34). The interaction of tumor cells with basement cellular systems, and of its importance in human breast cancer membranes is a critical step in metastasis, since the cells en growth, has led to the development and clinical application of counter and pass numerous basement membranes as they dis antiestrogens. Nonsteroidal antiestrogens like nafoxidine and seminate (35). Basement membrane-invasion initiates with tu clomifene have been studied in clinical trials, and tamoxifen, a mor cell attachment to laminin (36), which induces a protease substituted triphenylethylene, has been used successfully in the cascade terminating with active collagenase IV, specific for clinical management of advanced breast cancer for many years basement membrane collagen degradation (37, 38, 44). The (1, 2). Although clearly of clinical value, tamoxifen and its importance of these events to tumor cell metastasis has been active metabolite OHT' show evidence of partial estrogen agon- demonstrated with the dramatic reduction in lung colonization ism both in vivo and in vitro. They stimulate uterine growth in by metastatic melanoma cells injected into the nude mouse in ovariectomized rats and mice (3-5), and stimulate progesterone the presence of peptides which block the attachment of cells to receptor expression (6-10), morphological changes (11), and basement membranes (39) or in the presence of inhibitors of elevation of estrogen-dependent mRNA species (12-14) in cul collagenase type IV (40). tured estrogen-dependent human MCF-7 breast carcinoma In this study, we compared the effects of OHT, LY 117018 cells. Proliferation of MCF-7 cells in vitro is also stimulated by and ICI 164,384 on the basement membrane invasiveness and tamoxifen and OHT (15, 16), an effect which was previously proliferation of the MCF-7 cell line. We have similarly exam masked by the estrogenic nature of phenol red, the pH indicator ined the stable, estrogen receptor-positive, but antiestrogen- dye used for culture media ( 17). Although tamoxifen is generally resistant, MCF-7 cell variant LY-2 (41). The data provide tumoristatic to MCF-7 tumors grown in the nude mouse (18. further evidence that ICI 164,384 is a pure antiestrogen lacking 19), prolonged tamoxifen exposure can lead to tamoxifen- agonist activity, and suggest that such pure antiestrogens are resistant (20) and tamoxifen-stimulable (21) tumors. good candidates for clinical use. These considerations have fostered the continuous develop- Rcceived 2/3/89; revised 8/28/89; accepted 9/21/89. MATERIALS AND METHODS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Cell Lines. MCF-7 cells were obtained from Dr. Marvin Rich (Mich 1This work. Erik W. Thompson, and Thomas B. Shima were supported in igan Cancer Foundation) and used at passage 23. MDA-MB-231 cells part by the Breast Cancer Study Group, National Cancer Institute. NIH. 2To whom requests for reprints should be addressed, at Vincent T. Lombardi were obtained from the ATCC (Rockville, MD). Both cell lines were Cancer Center. Georgetown University Medical Center. 3800 Reservoir Road. maintained in Costar T75 flasks with Richters modified Minimal NW. Washington. DC 20007. Essential Medium (IMEM; Biofluids, Rockville, MD) supplemented 'The abbreviation used is: OHT, 4-hydroxytamoxifen. with 2 HIMglutamine and 5% fetal bovine serum (GIBCO, NY; FBS/ 6929 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MCF-7 CELL INHIBITION WITH ICI I6-U84 MEM). LY-2 cells were obtained from Diane Bronzert, NCI, NIH, and for this study. Effects on MCF-7 cells were compared to the maintained under the same conditions as MCF-7 cells. To deplete cells effects on the antiestrogen-resistant LY-2 subclone of the MCF- of estrogens, they were passaged for I week in phenol red-free IMEM 7 cell line. supplemented with 5% estrogen-depleted calf serum (DCS/MEM) Proliferation Studies. Fig. 1 shows dose response data for the which was made by sequential treatment of calf serum (GIBCO, New York, NY) with sulfatase (Sigma, St. Louis, MO) and dextran-coated effect of ICI 164,384 on the proliferation of estrogen-dependent charcoal (Sigma, St. Louis, MO). NIH-3T3 cells, kindly provided by MCF-7 cells and estrogen-insensitive MDA-MB-231 cells after Dr. Stewart Aaronson, NIH, were maintained in Dulbecco's modified 9 days of treatment. This time point shows the maximum effects minimal essential medium (Biofluids, Rockville, MD) supplemented of ICI 164,384 on cell populations within the logarithmic with 5% FBS. growth phase. Although this experiment was performed in Estrogen and Antiestrogens. 17/S-Estradiol was obtained from Sigma, DCS/MEM, control cultures (ethanol alone) showed logarith St. Louis. MO, tamoxifen (TAM) and 4-hydroxytamoxifen (OHT) were mic growth for the 9-day experimental period (doubling time, obtained from ICI America, Inc., Wilmington, DE, LY 117108 was a 108 h), suggesting the presence of residual estrogens. On days gift from Eli Lilly Co., Indianapolis, IN. ICI 164,384 was synthesized 6 (not shown) and 9 (Fig. 1), MCF-7 cells were inhibited in a by Jean Bowler, ICI Pharmaceuticals, Cheshire, England. Stock solu tions of these agents in absolute ethanol were stored at —¿20°C,and dose-dependent manner by increasing concentrations of ICI 164,384, with maximal effect at 10~7M.MCF-7 cultures treated diluted lOOOx in the appropriate medium when required. Control with 10~7M ICI 164,384 remained at constant density after day cultures received the ethanol vehicle (0.1%) alone. Proliferation Studies. MCF-7 cells were carried in 5% FBS/MEM 3. Proliferation was stimulated approximately twofold by 10~9 and transferred to 5% DCS/MEM without phenol red approximately M 17/3-estradiol (not shown). Under similar conditions, the 1 week before experiments were begun. Cells were plated in six-well growth of estrogen receptor-negative MDA-MB-231 cells was dishes (Costar) at approximately 20,000 cells/well.