[CANCER RESEARCH 52, 4663-4671. September 1992| Comparative Antitumor Effects of Hormonal Ablation, Estrogen Agonist, Estrogen Cytotoxic Derivative, and Antiestrogen in the PAIH Rat Prostatic Adenocarcinoma Blake Lee Neubauer,1 Kevin L. Best, Robin L. Goode, Mark L. Heiman, Dennis M. Hoover, David W. Robertson, Michael F. Sarosdy, Carl J. Shaar, Lee R. Tanzer, and Ronald L. Merriman The Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 [B. L. N., K. L. B., R. L. G., M. L. H., D. M. H., D. W. R., C. J. S., L. K. T., K. L. M.J, and Department of Surgery/Urology, University of Texas Health Sciences Center, San Antonio, Texas 78284 {M. F. SJ ABSTRACT gans. An effective therapy to treat prostatic cancer is 1.0 mg/ day DES; however, this DES dose does not always reduce cir The effects of hormonal ablation, estrogen, estrogen-derived cyto- culating T to castrate levels (3). Similarly, Prout et al. (4) toxic agent, and estrogen antagonist therapies used clinically were eval uated on in vitro colony formation, in vivo growth, and lymphatic and showed that 1.0 mg/day DES inhibited tumor growth without pulmonary metastasis of the PAIH tumor. Ventral prostatic and semi suppressing circulating T. Chlorotrianesene (TACE), which has nal vesicle weights were evaluated in the same animals to assess andrò- clinical antitumor efficacy against prostatic cancer, is also par gen-related responses. Estradici, estramustine phosphate, and testos tially effective in reducing circulating T (5). Clinical responses terone had no effects on PAIH colony formation in vitro. Castration, to estrogenic compounds in the presence of partial reductions in hypophysectomy, estradici benzoate, and estramustine phosphate treat circulating T may result from direct inhibitory effects on neo- ment of PAIII-bearing Lobund Wistar rats produced significant (P < plastic urogenital epithelia. 0.05) regression of male accessory sex organs. Of these treatments, only hypophysectomy had significant (/' < 0.05) inhibitory effects EMP (Estracyt; Fig. la) is an effective chemotherapeutic on primary PAIH growth and lymphatic and pulmonary metastasis. agent for the treatment of advanced prostatic cancer (6, 7). The LY117018 [6-hydroxy-2-(p-hydroxyphenyl)benzo(b)thien-3-yl-p-2-(l- cytotoxicity of this nitrogen mustard derivative of 170-estradiol pyrrolidinyl)ethoxy phenyl ketone] has antiestrogenic activity but pro is mediated through antimicrotubular activity (8). Estramustine duces no significant agonist responses. I.Yl 17018 had no effect upon and estromustine, 2 major metabolites of EMP, demonstrate PAIH colony formation in vitro. Following s.c. implantation of PAIH specific, high affinity binding to abundant secretory proteins of cells, LY117018 (2.0, 10.0, or 20.0 mg/kg s.c.) had no effect on primary rat (9) and human prostate (10). EMP organ selectivity result tumor growth in the tail, in vitro LY117018 administration produced ing from these ligand-protein interactions has been claimed marked antimetastatic effects. In a dose-dependent manner, LY117018 (Fig. 1). inhibited PAIH metastasis to the gluteal (97%) and iliac lymph nodes (88%) ( /' < 0.05 for both). LY117018 also maximally inhibited pulmo The antiestrogen tamoxifen has been assessed in clinical tri nary metastasis by 86% (P < 0.05). Maximal regression of 42% for als with Stage D prostatic cancer patients (11, 12). These stud ventral prostatic and 35% for seminal vesicle weights were also seen ies have shown tamoxifen to be of some effect in the palliative after LY117018 administration (/' < 0.05 for both). Co-administration treatment of the disease that could be attributable to the inher of estradiol benzoate had no antagonistic effect upon the antitumor ent estrogenicity of the compound. In addition to its estrogen- responses produced by LY117018. The mechanism of action of related activity, the antiproliferative effects of tamoxifen may LY117018 is not known. The failure of estradiol benzoate to affect involve interactions with other cellular mediators, producing PAIH growth and metastasis supports the contention that the responses stimulation of inhibitory factors or inhibition of growth factor to LY117018 are not attributable to simple antagonism of estrogen action. LY117018 may be exerting its antitumor effects through auto production (13). LYI 17018 (Fig. 1) is a potent in vitro estrogen crine, paracrine, or endocrine mechanisms. LY117018 represents a receptor binding competitor (14) and is an in vivo antagonist of class of agents with potential utility in treating metastatic cancer of the estrogen action. Unlike tamoxifen, LYI 17018 has minimal ag prostate. onist efficacy in the female rat uterine bioassay (15). Taken together, the hormonal ablative and direct actions of estrogens INTRODUCTION and related compounds have been exploited to treat hormone- sensitive and hormone-resistant cancers in humans. Curative Administration of estrogens to decrease circulating andro- treatment of hormone-resistant prostatic cancer is currently gens is an effective hormonal ablative therapy for disseminated unavailable. This situation results from an inability to control prostatic cancer (1). The symptomatic benefits of estrogen ad the continued proliferation of androgen-insensitive metastatic ministration are mediated primarily through inhibition of LH2 cells (16) following hormonal ablative therapies. It is proposed secretion, which decreases circulating T. However, estrogen an- that cytotoxic chemotherapeutic agents offer the only effective titumor efficacy in prostatic cancer patients may not relate en therapy for metastatic prostate cancer (17). Given the effective tirely to decreased circulating T. Kyprianou and Issacs (2) dem ness of eliminating localized prostatic cancer, another potential onstrated that a critical threshold of intracellular androgen is required for hormonal stimulation of male accessory sex or- therapeutic approach may be the use of agents that inhibit the métastasesof tumor cells from the primary lesion. Development of effective agents to treat metastatic prostatic Received 1/24/92; accepted 6/16/92. cancer requires evaluation of potential therapeutic modalities in 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 accord animal models that accurately mimic the human disease state. ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at CNS/GI/GU Research The PAIH adenocarcinoma in LW rats is a model that is useful 0815, Lilly Research Laboratories, Lilly Corporate Center. Indianapolis. IN 46285. to evaluate agents to treat metastatic prostatic cancer. The 2 The abbreviations used are: LH. luteinizing hormone; T, testosterone; DES, tumor originated as an autochthonous neoplasm in an old LW diethylstilbestrol; EMP, estramustine phosphate; LW. Lobund Wistar; CM, com rat maintained under germ-free conditions (18). The PAIII tu plete medium: PCS, fetal bovine serum: MEM, minimal essential medium; E2B, 17fi-estradiol benzoate; EMBP, estramustine binding protein; LYI17018, 6-hy- mor can be grown in vitro as a monolayer (19). When injected droxy-2-0>-hydroxyphenyl)benzo(A)thien-3-yl-p-2-(/-pyrrolidinyl)ethox> phenyl ke s.c. into conventionally reared male or female LW rats, PAIII tone; E2, estradiol. 4663 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1992 American Association for Cancer Research. HORMONAL EFFECTS IN PAIII PROSTATIC ADENOCARCINOMA CICH2CHj N—¿C—¿O / CICH2CH2 a.) Estracyt'M (EMP) N—C— O H3C(CH2)2/ b.) LY117018 d.) LY186564 Fig. 1. Structures of (a) EMP, (b) LY117018, (c) LY183648, and (d) LY186564. cells form a primary adenocarcinoma that later metastasizes to Clonogenic testing of PAIII cells for in vitro drug sensitivity used a lymphatic and pulmonary sites. Metastasis to the lungs is in modification of a published colony formation assay technique (24). An underlying 1.0-ml layer of 5% agar in McCoy's medium containing dependent of the implantation site (20). When PAIII cells are injected s.c. into the tails of male LW rats, a reproducible, 10% PCS was placed in 35-mm-diameter plastic Petri dishes. PAIII time-dependent, sequential spread of the tumor through the cells were suspended at the required concentrations in a plating layer of 1.0-ml double enriched CM. The dishes were incubated at 37°Cin7.5% gluteal and iliac lymph nodes to the lungs is observed (21). The morphology of the PAIII tumor resembles anaplastic lesions in CO2 humidified air. Colonies were counted using a FAS 11 counter 14 humans, supporting its utility in evaluating cytotoxic and anti- days following plating. Minimal colony size was arbitrarily designated to be 58 /JMdiameter. For each test compound, the effect on plating metastatic agents (22, 23). We have developed this rodent efficiency was evaluated in triplicate at a concentration of 10 Mg/ml. model and are studying its relevance to human metastatic pros Plating efficiency was expressed as the means ±SD of the triplicate tate cancer. Characterization of endocrine effects on PAIII observations. Preliminary observations showed that IO4 PAIII cells growth and metastasis needs to be defined. To characterize the would predictably form about 125-175 colonies/plate. Therefore, IO4 utility of the PAIII system as a model for human metastatic PAIII cells were used as the initial inoculum for the assay. Test com prostate