Comparative Antitumor Effects of Hormonal Ablation, Estrogen

Home , Aromatase inhibitor, Estramustine, Trioxifene

[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 andrÃ²- 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 mÃ©tastasesof 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

CICH2CHj Nâ€”¿Câ€”¿O / CICH2CH2

a.) Estracyt'M (EMP)

Nâ€”Câ€” 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 37Â°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 Â±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 cancer, the effects of castration, hypophysectomy, es pounds were added to the plates at a concentration of 10.0 Mg/ml for trogen agonist, estrogen cytotoxic derivative, and estrogen an 60-min exposure. The PAIII cells were then centrifuged at 1000 x g for tagonist were assessed using in vitro culture and 28-day in vivo 5 min at room temperature and washed once in 10.0 ml Hanks' bal treatment protocols. anced salt solution containing 10% heat-inactivated horse serum and plated as described above. PAIII cells in suspension were also incubated MATERIALS AND METHODS with 10.0 Mg/mlof the test compounds for 14 days. PAIII colony count determinations in test groups were compared to untreated control plate Cell Culture and Clonogenic Assay. PAIII cells were kindly provided values. Criterion of in vitro sensitivity to a given agent was defined as a by Dr. Morris Pollard (Lobund Laboratory, University of Notre Dame). treatment colony count value <30% of control level. PAIII cells were grown as monolayer cultures passed twice weekly using PAIII Cell Culture for in Vivo Studies. A stock culture of PAIII rat trypsinicitrate solution to dissociate cells. Trypsin:citrate solution is prostatic adenocarcinoma cells at passage 107 was supplied by Dr. composed of 0.25% trypsin, 46.7 ITIMsodium citrate, 94.8 IHMNaCI, 6.8 Morris Pollard. This original stock culture was expanded through 2 ITIMKC1,and 0.002% phenol red. PAIII cells were grown in CM at 37Â°C passages and stored in liquid nitrogen using 10% dimethyl sulfoxide as under 5% CO2 in air. CM is composed of RPMI 1640 supplemented a cryoprotectant. The cells were kept in liquid nitrogen as 1.0-ml ali- with 10% PCS, 2 mm L-glutamine, \x MEM vitamins, Ix MEM es quots at a minimal concentration of 1.0 x 106 cells/ml. PAIII cells were sential amino acids, \x MEM nonessential amino acids, 2 x IO-5 M grown in antibiotic-free, minimal essential medium with Earle's salts 2-mercaptoethanol, 50 units penicillin/ml, and 5 mg streptomycin/ml. (Gibco, Grand Island, NY) supplemented with 10% PCS (Hyclone 4664

Laboratories. Logan, UT). The cells were grown to confluency and noi. Pleural lesions on the lung surface were counted using a dissecting harvested using 0.06 units/cm2 trypsin (2x recrystallized; Worthington microscope by methods published earlier (21). Biochemical Corp., Freehold. NJ). Histopathological Analysis. After weighing lymph nodal tissues at Preparation of Reagents. E2B, testosterone, and estradici were pur necropsy, specimens were collected for histology from representative chased from Sigma Chemical Company (St. Louis, MO). EMP (Fig. PAIII control and treatment groups. Tissues were placed in 10% neu la) was a gift from Hoffmann-LaRoche (Nutley, NJ). Antiestrogen, tral buffered formalin. Histological preparations of paraffin-embedded LY117018 (Fig. lÃ²), aromatase inhibitor, LY183648 (Fig. le), and samples were sectioned at 6 MM.stained with hematoxylin and eosin, LY186564, a derivative of estromustine phosphate (Fig. Id), were syn and examined by light microscopy. thesized at the Lilly Research Laboratories (Indianapolis, IN). E2B, Statistical Methods. Bartlett's test determined that there were sig EMP, LY 117018, LY 183648, and LY 186564 were dissolved in ethanol nificant (P < 0.001) differences among the group variances for the tail, and diluted to a final ethanohpeanut oil ratio of 1:10. All compounds gluteal and iliac lymph nodal, and pulmonary focal variables. There for injection were stored in lightproof containers at -20Â°C before for fore, Dunnett's test was performed on the rank transformed data, pro mulation and in lightproof bottles at room temperature during com viding a nonparametric test (25). Monotonie decreases in tumor pa pound administration. rameter mean responses were assessed in treated animals for dose Experimental Animals. Breeding stock of LW rats was a gift from dependency using linear regression of the logio of the test agent dose Dr. Morris Pollard. LW rats were maintained as a closed colony at against the appropriate experimental parameter (26). HarÃanIndustries (Cumberland, IN). Male rats that weighed between 110 and 125 g were used. Two or 3 rats were housed in screen-bottomed cages in a light-controlled environment (lights on, 6 a.m.; lights off, 8 RESULTS p.m.). Water and powdered Rodent Laboratory Chow 5001 (Ralston- Purina, St. Louis, MO) were supplied ad libitum. These investigations Lack of in Vivo Antitumor Effects. Administration of the were conducted under practices outlined for the care and use of labo EMP analogue LY 183648 and the aromatase inhibitor LY- ratory animals set forth by the NIH and the American Association for 186564 had no effect on PAIII growth or metastasis (data not Laboratory Animal Care. shown). Surgical Procedures. All surgical procedures were done under Meto- Clonogenic Assay. The addition of E2, T, EMP, or LY- fane anesthesia (methoxyflurane; Pittman-Moore, Washington Cross 117018 (10.0 Mg/ml) to media for either 60 min or continuously ing, NJ). Incision sites were shaved and washed with povidone-iodine for 14 days had no effect upon the colony-forming ability of (PVP-I Prep Solution: Baxter Healthcare Corp., McGaw Park, IL) PAIII cells (Table 1). before surgical manipulation. Surgical instruments were sterilized by In Vivo Body Weight Effects. There was no difference in the autoclaving. Between procedures on different animals, surgical instru weight gains of PAIII-bearing and tumor-free rats. Castration, ments were washed in a solution of 70% ethanol and dried with sterile gauze sponges. Sterile gloves were worn for all surgical procedures. hypophysectomy, and administration of E2B, EMP, and Castrations and removal of the epididymal fat pad were done through LY117018 for 28 days significantly inhibited body weight gains the scrotal route 2 days before injection of PAIII cells. Hypophysecto- (P < 0.05). Inhibition of final to initial body weight ratios in mies were performed on rats weighing 75 to 90 g through the nasopha- PAIII-bearing hypophysectomized and castrated rats was 47 ryngeal route 10 days before the start of the studies. After hypophysec- and 20% from the PAIII plus vehicle control body weight tomy, rats were weighed every third day and animals in ill health or means (Table 2). E2B treatment of PAIII-bearing rats produced those that had gained >5 g body weight were not used for the studies. significant (P < 0.05) decreases in final to initial body weight In Vivo Dose-Response Studies. On day 0, animals were randomized ratios (Table 3). Relative to PAIII-bearing control rats, maxi into 5 or 6 groups (8 to 10 rats/group). All except one of these groups mal E2B-induced inhibition of body weight gain was 28% at a were given injections of PAIII cells. Injections of tumor cells and sur daily dose of 0.005 mg/kg. EMP treatment of PAIII-bearing gical procedures were performed under light metofane anesthesia. Us ing a 25-gauge needle. IO6PAIII cells in a volume of SUM'were injected rats produced significant (P < 0.05) dose-dependent decreases (r2 = 0.998, P < 0.031) in final to initial body weight ratios s.c. into the dorsal surface halfway between the base and tip of the tail. Control animals were given injections of saline. Rats not receiving (Table 4). Maximal inhibition of body weight gain was 28% at injections of PAIII cells were designated the non-PAIII-bearing control a daily dose of 0.005 mg/kg EMP. group. All studies were conducted for 28-29 days. In the dose-response LY117018 administration to PAIII-bearing rats also pro studies, PAIII-bearing experimental groups were administered duced significant (P < 0.05) dose-dependent decreases (r2 = LY186564 (2.0, 10.0, and 20.0 mg/kg), LY183648 (2.0, 10.0, and 20.0 0.968, P < 0.115) in final to initial body weight ratios (Table 5). mg/kg), EMP (0.1, 1.0, and 10.0 mg/kg), E2B (0.001, 0.005, 0.01, and Maximal LY117018-induced inhibition of body weight gain 0.05 mg/kg), or LY117018 (2.0, 10.0, and 20.0 mg/kg) as 0.2-ml single was 31% at a daily dose of 20.0 mg/kg. daily s.c. injections. Daily injections were given in the morning (8 to 9 a.m.). Control groups received equivolumetric vehicle injections. In the In Vivo Accessory Sex Organ Regression. Hormonal abla combination study, E2B (0.005 and 0.1 mg/kg/day) and LY117018 tion procedures and administration of E2B, EMP, and (20.0 mg/kg/day) were administered as 2 separate daily 0.1-ml injec LY117018 for 28 days produced significant (P < 0.05) de tions. E2B and LY117018 treatment and control groups received single creases in normalized accessory sex organ weights. Castration 0.2-ml vehicle injections. Compounds for injection were formulated for 2-week treatment in tervals on a body weight basis. Initial dose formulations were calculated Table 1 Effects of 10 i^g/ml eslradiol, testosterone, estramustine phosphate, and using previously observed 2-week body weight gains in untreated rats. LYI 17018 on the in vitro clonogenic activity of PAIII cells" After 2 weeks, doses in each group were adjusted relative to change in control60-min of body weight. One-half of the rats in each treatment group were sacrificed on day CompoundEstradiol exposure106.7exposure Continuous 28. The remaining animals were sacrificed the following day. Rats were Â±16.6Â» 98.2 Â±9.8 sacrificed by CO2 asphyxiation, body weights were recorded, and tails Testosterone 104.8 Â±2.9 97.3 Â±29.2 Estramustine phosphate 79.6 Â±10.6 83.3 Â±4.0 were amputated 1 in. from the base and weighed. The gluteal and iliac LYI 17018% 78.5 Â±13.7 96.4 Â±0.5 lymph nodes were also excised and weighed. The lungs were removed " Control growth = 171.7 Â±31.1 colonies (n = 6). For experimental details, see and inflated through the trachea with about 5.0 ml Bouin's fixative. The "Materials and Methods." lungs were placed in Bouin's fixative for 24 h and stored in 70% etha- Â»Experimental values represent the mean Â±SD of triplicate observations. 4665

Table 2 Inhibition by hypophyseclomy and lack of effect of castration on the growth and metastasis of the PAIII prostatic adenocarcinoma in male Lobund Wistar rats"

tumor to initial normalized tail wt lymph node lymph node TreatmentPAIII body wtratio2.089 (g/wt)2. 100 g body (mg/wt)278.7 100 g body (mg/100wt)269.8 g body focinos.41.0 9)Â»No+ vehicle (n = Â±0.010'2.207 139 Â±0.052 Â±24.95.4 Â±40.429.6 Â±5.71.0 (5.293 Â±0.077)"1.850

9)PAIIIPAIII (n = Â±0.0811.758 Â±0.027' Â±0.7'253.5 Â±2.5'166.1 Â±1.0'44.1 (4.87027)*'2.440 Â±0.1

8)PAIII+ castration (n = Â±0.012'1.176 Â±0.049 Â±38.37.9 Â±35.316.7 Â±6.223.1 (5.184 +0.099)''2.43

+ hypophysectomy (n = 15)Final Â±0.017'Primary Â±0.08' Â±1.4'Iliac Â±1.1'Pulmonary Â±3.5' (2.429 Â±0.081)*'Glutea! â€¢¿Fordetails, see "Materials and Methods." ' Numbers in parentheses, number of observations. ' Mean Â±SEM. " Mean Â±SEM of the absolute tail weight (g). ' Significantly different from PAIII plus vehicle-treated controls (P < 0.05) using Dunnett's test on ranked data.

Table 3 Lack of effects ofE2B on the growth and metastasis of the PAIII prostatic adenocarcinoma in male Lobund Wistar rats"

tumor to initial (Tail Weight) lymph node lymph node TreatmentPAIII body wtratio2.042 (g/100wt)2.377 g body (mg/100gwt)239.8 body (mg/100wt)298.2 g body focinos.73.1 + vehicle (n = 9)* Â±0.037C Â±0.076 Â±14.4 Â±60.0 Â±27.1 No PAIII (n = 9) 2.1 12Â±0.0971.865 1.825Â±0.029"2.271 2.3 Â±0.4"136.0+25.8 10.0Â±1.0"201.7 0.4 Â±0.4"65.9 PAIII +E2B0.001 mg/kg/day (n = 9) Â±0.064 Â±0.127 Â±43.3 Â±32.7 0.005 mg/kg/day (n = 8) 1.517 Â±0.045'' 2.406 Â±0.050 166.9 Â±28.9 168.1 Â±48.6 87.3 Â±46.4 0.01 mg/kg/day (n = 8) 1.648 Â±0.048" 2.481 Â±0.048 152.1 Â±31.0 244.0 Â±58.7 76.3 Â±53.5 0.05 mg/kg/day (n = 8)Final 1.569 Â±0.073"Primary 2.383 Â±0.059Gluteal 133.1 Â±24.3Iliac 166.9 Â±33.5Pulmonary 58.5 Â±47.3 â€¢¿Fordetails, see "Materials and Methods." * Numbers in parentheses, number of observations. <â€¢MeanÂ±SEM. " Significantly different from PAIII plus vehicle-treated controls (/' < 0.05) using Dunnett's test on ranked data.

Table 4 Lack of effects of EMP on the growth and metastasis of the PAIII prostatic adenocarcinoma in male Lobund Wistar rats"

tumor to initial (Tail Weight) lymph node lymph node TreatmentPAIII body wtratio1.885 (g/100wt)2.159 g body (mg/100 g bodywt)86.7 (mg/100gbodywt)92.4 focinos.90 + vehicle (n = 9)Â» Â±0.046' + 0.026 Â±14.4 Â±33.9 Â±5752 No PAIII (n = 7) 2.000 Â±0.0541.812 1.688 +0.046"2.138Â± 2.0 Â±0.3"105.3 7.0 Â±0.9"93.8 PAIII + EMP 0.1 mg/kg/day (n = 10) Â±0.036 0.042 Â±35.6 Â±21.7 Â±15 1.0 mg/kg/day (n = 10) 1.463 Â±0.030" 2.244 Â±0.037 67.3 Â±18.4 102.9 Â±26.6 97 Â±51 10.0 mg/kg/day (n = 10)Final 1.168 Â±0.024"Primary 2. 190 Â±0.036Gluteal 45.6 Â±13.5Iliac 64.6+ 19.2Pulmonary 162 Â±94 " For details, see "Materials and Methods." * Numbers in parentheses, number of observations. c Mean Â±SEM. "Significantly different from PAIII plus vehicle-treated controls (/' - 0.05) using Dunnett's test on ranked data. and hypophysectomy produced a 94% regression of ventral pro- Testicular Responses. Twenty-eight days after hypophysec static weight (Table 6). These treatments also decreased semi tomy, there was a significant (P < 0.05) regression of normal nal vesicular weights by 87 and 97%, respectively (Table 6). E2B ized testicular weights (Table 6). E2B (Table 7) and EMP ad administration to PAIII-bearing rats produced significant (P < ministration (Table 8) to PAIII-bearing rats also produced 0.05) regression of ventral prostatic and seminal vesicular significant (P< 0.05) regression of testicular weights. Maximal weights (Fig. 2). Maximal E2B-induced regression of ventral testicular regression was 62% from PAIII-bearing control levels prostatic weight was 84% from controls at 0.01 mg/kg E2B. at the daily E2B dose of 0.01 mg/kg. BMP-induced regression This same E2B dose produced a 69% regression of seminal was 65% at the 10.0 mg/kg EMP dose level. LY117018 admin vesicular weights. EMP treatment of PAIII-bearing rats pro istration to PAIII-bearing rats produced significant (P < 0.05) duced significant (P < 0.05) dose-dependent decreases in ven increases in normalized (per 100 g body weight) testicular tral prostatic (r2 = 0.974, P < 0.102) and seminal vesicular weights (Table 5). In these animals, the absolute testicular weights (r2 = 0.896, P < 0.209) (Fig. 3). Relative to PAIII- weights were unchanged from control values. bearing controls, maximal regression with daily EMP doses of Adrenal Responses. E2B (Table 7) and EMP (Table 8) ad 10.0 mg/kg were 72% for ventral prostatic and 87% for seminal ministration to PAIII-bearing rats produced significant (P < vesicular weights. LY117018 administration to PAIII-bearing 0.05) stimulation of normalized (per 100 g body weight) adrenal rats also produced significant (P < 0.05) dose-dependent re weights. Maximal stimulation was 168% at the daily E2B dose gression (r2 = 0.947, P< 0.147) of ventral prostatic weight with of 0.01 mg/kg and 174% at 10.0 mg/kg EMP, respectively. In maximal inhibition of 44%. Maximal LY117018-induced inhi these animals, adrenal wet weights (mg) were also significantly bition of seminal vesicular weights was 30% (Fig. 4). (P < 0.05) increased over PAIII-control levels. LY 117018 4666

Table 5 Inhibitory effects of LYl 17018 on body weight gains and stimulator)' effects on testicular and adrenal organ weights in male Lobuna Wistar rats inoculated with PAIII cells"

to initial body adrenal wt lesticular wt TreatmentPAIII wtratio1.630Â±0.043f1.654 (mg/kg bodywt)21.13Â± (g/kg bodywt)44.51 10)*No+ vehicle (n = 1.10(47.50 2.20(1 Â± Â±1.85)Â«'22.55 000.90 Â±47.50)''46.04 10)PAIIIPAIII + vehicle (n = Â±0.0291.348 Â±1.39(51.20 1.01(1049.10Â± +2,94)''24.34 Â±51.20)"54. +LY1170182.0 10)10.0mg/kg/day (n = Â±0.027'1. Â±1.14(44.50 14Â±0.88'(988.50 Â±2.33)''28.05 Â±48.00)"61.32 9)20.0mg/kg/day (n = 237Â±0.041'1.142 Â±0.95'(48.00 1.73'(1031.44Â±Â± Â±4.93)''28.71 48.00)"59.12 mg/kg/day (n = 9)Final + 0.037'Normalized Â±2.34'(44.00 Â±1.74"'(945.43 Â±2.98)''Normalized Â±38.00) ' For details, see "Materials and Methods." * Numbers in parentheses, number of observations. ' Mean Â±SEM. " Mean Â±SEM of the absolute organ wet weight (mg). ' Significantly different from PAIII plus vehicle-treated controls (P < 0.05) using Dunnett's test on ranked data.

Table 6 Effects of castration and hypophysectomy for 28 days on ventral prostatic, seminal vesicular, and testicular weights in male Lobund Wistar rats inoculated with PAIII prostatic adenocarcinoma cells"

Ventral prostate Seminal vesicle Testis (mg/100g (mg/100g (g/100g Ss (10) Treatment body wt) body wt) body wt) 5% VENTRAL PROSTATE 80.08 Â±3.43' 90.94 Â±5.22 43.26 + 0.94 gS SEMINAL VESICLE PAIII + vehicle Â°Ã¤ (n = 9)Â» x~ ul E U) O" No PAIII (n = 9) 86.35 Â±5.85 76.29 Â±6.36 42.29 + 0.61 > H 4.35 + 0.96" 11.82+ 1.35" PAIII + castration mu>S. E (Â«= 8) o o PAIII + hypophy- 4.46 + 0.97" 2.43 + 0.13" 14.15+1.39" sectomy (n = ) " For details, see "Materials and Methods." * Numbers in parentheses, number of observations. 10.0 <â€¢Mean+ SEM. EMP (MG / KG- DAY) " Significantly different from PAIII plus vehicle-treated controls (P < 0.05) using Dunnett's test. 28 DAYS Fig. 3. Regressive effects of EMP on ventral prostatic and seminal vesicular weights in male Lobund Wistar rats inoculated with PAIII prostatic adenocarci noma cells. *, Significantly different from PAIII + vehicle (P < 0.05) using Dunnett's test. Numbers in parentheses, number of observations. For details, see "Materials and Methods."

â€¢¿ VENTRAL PROSTATE (10) â€¢¿ SEMINAL VESICLE

(9)'

Fig. 2. Regressive effects of E2B on ventral prostatic and seminal vesicular PAIN + VEHICLE NO PAIII weights in male Lobund Wistar rats inoculated with PAIII prostatic adenocarci noma cells. *, Significantly different from PAIII + vehicle (P < 0.05) using LY117018 (MG/KG-DAY) Dunnett's test. Numbers in parentheses, number of observations. For details, see 28 DAYS "Materials and Methods." Fig. 4. Regressive effects of LYl 17018 on ventral prostatic and seminal vesic ular weights in male Lobund Wistar rats inoculated with PAIII prostatic adenocarcinoma cells. *, Significantly different from PAIII + vehicle (P < 0.05) using Dunnett's test. Numbers in parentheses, number of observations. For details, see administration to PAIII-bearing rats produced significant (P < "Materials and Methods." 0.05) dose-dependent (r2= 0.976, P < 0.100) increases in nor malized (per 100 g body weight) adrenal weights (Table 5) with Antitumor Effects. Twenty-eight days after hypophysec maximal stimulation of 29% over PAIII controls at a daily dose tomy, there was a significant (P < 0.05) inhibition of the growth of 20.0 mg/kg. In these animals, the absolute adrenal weights and metastasis of the PAIII adenocarcinoma in LW rats (Table were unchanged from control values. 2). While normalized tail weights (g/100 g final body weight) 4667

Table 7 Adrenal and testicular weight responses to E2B in male Lobuna Wistar rats inoculated with PAIll cells"

adrenal wt testicular wt TreatmentPAIII toratio2.042 initial body wt (mg/kgwt)17.05 body (g/kg bodywt)46.41 Â±0.037'-2.1 9)No + vehicle (n = Â±0.47(42.20 Â±0.78(1010.25 Â±3.51)"16.82 Â±16.76)''45.98 9)PAIIIPAIII (n = 12Â±0.0971.865 +0.48(43.20 0.58(1026.12Â± Â±3.28)rf18.88 Â±30.63)''46.23 +E2B0.001 =0.005mg/kg/day (n 9)= Â±0.0641.517 Â±0.40(50.00 0.40(1020.33Â± Â±3.35)rf38.56 Â±21.21)-'23.10 =0.01 mg/kg/day (n 8)8)8)Final Â±0.045'1.648 Â±1.60'(64.20 Â±2.48'(426.43 Â±2.60)*'47.47 Â±50.64)*'18.29Â± =0.05mg/kg/day (n Â±0.048'1.569 Â±1.86'(67.00 1.90'(364.90 Â±5.59)*'40.44 Â±41.07)*'28.28 mg/kg/day (n =b= Â±0.073'Normalized Â±2.49'(82.20 Â±2.18'(458.38 Â±5.13)*'Normalized Â±31.94)*' " For details, see "Materials and Methods." * Numbers in parentheses, number of observations. ' Mean Â±SEM. * Mean Â±SEM of the absolute organ wet weight (mg for adrenals and testes). ' Significantly different from PAIII plus vehicle-treated controls (P < 0.05) using Dunnett's test on ranked data.

Table 8 Adrenal and testicular weight responses to EMP in male Lobund Wistar rats inoculated with PAIII cells"

to initial body adrenal wt testicular wt TreatmentPAIII wtratio1. (mg/kgwt)19.65 body (g/kg bodywt)42.27 9)Â»No+ vehicle (n = c2.000885 Â±0.046 Â±0.60(49.60 Â±0.79(107. Â±1.69)''17.93 50 Â±11.99)*42.18 7)PAIIIPAIII + vehicle (n = Â±0.0541.812 Â±0.98(48. Â±0.84(1127.62 12Â±2.73)-*2 +48.12)-'43.67 +EMP0.1 10)1.0mg/kg/day (n= +0.0361.463 1.64Â±0.67(56.91 0.78(1056.09Â± Â±3.21)''36.30 If42.00+ 56.9 10)10.0mg/kg/da> (n = Â±0.030'1.168 Â±0.48'(70.12 Â±2.39(833.12 Â±1.48)*'51.26+ Â±72.85)*19.07Â± mg/kg/day (n = 10)Final Â±0.024'Normalized 1.61'(82.12 1.08'(311.00Â± Â±2.85)*'Normalized 16.41)*' " For details, see "Materials and Methods." * Numbers in parentheses, number of observations. ' Mean + SEM. Ã¤Mean Â±SEM of the absolute organ wet weight (mg). ' Significantly different from PAIII plus vehicle-treated controls (P < 0.05) using Dunnett's test on ranked data.

were significantly (P < 0.05) increased to 114%, absolute tail 140 1 weights were significantly (P < 0.05) regressed by 54% from PAIII-bearing controls. The PAIII prostatic adenocarcinoma metastasizes through lymphatic channels from the site of tumor implantation. There is a reproducible, time-dependent spread GLUTEAL of the tumor from the tail to the gluteal and iliac lymph nodes ILIAC and subsequently to the lungs. Gluteal and iliac lymph nodes in hypophysectomized rats were significantly (P < 0.05) smaller than PAIII-bearing controls. Inhibition of PAIII lymphatic me 01 tastasis to the gluteal and iliac lymph nodes after hypophysectomy was 97 and 94%, respectively. Metastatic foci in the lungs were reduced significantly (P< 0.05) by hypophysectomy. Max imal inhibition of pulmonary metastasis was 97%. Castration (Table 2), E2B (Table 3), and EMP treatment (Table 4) had no significant effects on PAIII growth and metastasis. PAIII + VEHICLE NO PAIII 10.0 20.0 LY117018 administration had no significant cytoreductive LY117018(MG/KG-DAY) effects against primary PAIII tumor growth in the tail (data not 28 DAYS shown). LY117018-treated groups exhibited significant (P < Fig. 5. Inhibitory effects of LY117018 on the gluteal and iliac lymph nodal metastasis of the PAIII prostatic adenocarcinoma in male Lobund Wistar rats. 0.05) dose-dependent regression of gluteal (r2 = 0.978, P < *, Significantly different from PAIII + vehicle (P< 0.05) using Dunnelt's test on 0.094) lymph node weights (Fig. 5) relative to PAIII-bearing ranked data. Numbers in parentheses, number of observations. For details, see controls. Iliac lymph node weights were also significantly (P < "Materials and Methods." 0.05) decreased by LY117018 treatment (Fig. 5). Maximal in hibition of PAIII lymphatic metastasis to the gluteal and iliac In a separate series of studies, LY117018 (20.0 mg/kg) and lymph nodes was 97 and 88%, respectively. Lung colony num E2B (0.005 and 0.1 mg/kg) were administered alone and in bers were reduced significantly (P < 0.05) in a dose-dependent combination daily for 28 days as described for the previous manner (r2 = 0.995, P< 0.046). Maximal inhibition of pulmo experiments. LY117018 was unable to significantly antagonize nary metastasis was 86% with a daily LY117018 dose of 20.0 the regressive effects on testicular weights of a daily dose of mg/kg (Fig. 6). 0.005 mg/kg E2B (P = 0.062, data not shown). As described 4668

80 rats at doses sufficient to inhibit prolactin to nondetectable levels (28) had no effect on PAIII growth or metastasis.4 Like 70 . wise, treatment of PAIII-bearing rats with the prolonged bio logically active somatostatin analogue (29) dPhe-Cys-Tyr- tO 60 OC LU dTrp-Lys-Val-Cys-Thr-NH2 produced no antitumor effects in o 50 this model.3 Continuing investigations into the potential role of ZI 2 pituitary-derived and related peptides in tumor growth and me 40 tastasis may elucidate new regulatory factors of proliferation (10) CL 30 and metastasis and identify new antitumor therapies. While the doses of E2B used in these studies produced sig O 20 i nificant inhibition of ventral prostatic, seminal vesicular, and 3 o. testicular weights, no in vivo antitumor responses were pro 10 duced. These findings are consistent with the lack of estrogen (10) ' responsiveness of the PAIII in vitro. PAIII cells do not possess

PAIII + VEHICLE NO PANI 2.0 10.0 20.0 estrogen receptors (30), and E2 had no direct effect upon the in vitro colony-forming ability of PAIII cells, supporting the con LY117018(MG/KG- DAY) tention that estrogens do not play a direct role in mediating in 28 DAYS vivo PAIII cellular proliferation or tumor spread. Additional Fig. 6. Inhibitor) effects of LY117018 on the metastasis of the PAIII prostatic adenocarcinoma to the lungs of male Lobund Wistar rats. *. Significantly differ pharmacological evidence for a lack of estrogen influence on ent from PAIII + vehicle (P< 0.05) using Dunnett's test on ranked data. Numbers PAIII growth and metastasis is provided by the absence of in parentheses, number of observations. For details, see "Materials and Methods." antitumor responses to the potent aromatase inhibitor LY183648 (Fig. le) (31) in the 28-day in vivo PAIII assay. previously, LY117018 produced significant (P< 0.05) antime- Contrasting the results described here, estrogens have been tastatic responses by inhibiting gluteal. iliac, and pulmonary demonstrated to inhibit the growth of the androgen-sensitive metastasis of the PAIII tumor (Table 9). E2B administration R3327 prostatic adenocarcinoma (32) through suppression of did not produce antitumor effects (Table 9). E2B co-adminis circulating LH and T and by possible direct cytoreductive ac tration did not antagonize the antitumor responses produced by tivity (33). While high doses of DES effectively suppressed LY 117018. High-dose co-administration of E2B (0.1 mg/kg) growth of the androgen-resistant R3327AI subline (34), DES produced significantly (P < 0.05) greater regression of the glu was less effective at inhibiting the growth and metastasis of teal and iliac lymph node weights than observed with R3327 MAT-LyLu variant (35). An antimetastatic response to LY 117018 PETÂ«'(Table 9). single high-dose DES has been reported for the PAIII tumor Histological examination of PAIII tumors in the lymph (36). In this study, no cytoreductive effect was observed against nodes of different treatment group animals exhibited a great the primary tumor, nor was a dose-response antitumor relation diversity of morphological characteristics. It was not possible to ship defined. Both the PAIII and R3327 MAT-LyLu tumors distinguish the various treatment groups on a histopathological metastasize through lymphatic channels to form lung colonies basis. in vivo, and both tumors respond modestly to high-dose DES. The antitumor responses observed in these 2 studies resulted DISCUSSION from using DES doses in excess of those needed to produce chemical castration and probably reflect direct or indirect cy The PAIII prostatic adenocarcinoma in LW rats is a useful toreductive pharmacological activities of DES. model to assess the efficacy of cytotoxic and antimetastatic As seen with in vitro and in vivo responses to estrogen, EMP agents. Using this system, the cytoreductive and antimetastatic treatment had no effect on the in vitro growth or in vivo growth effects of warfarin (22), the diarylsulfonylurea cytotoxic agent and metastasis of the PAIII tumor. Lack of in vitro activity seen LY181984 (23), and hirudin (27) have been evaluated. How with EMP may be attributable to the 170-phosphate substitu ever, the endocrine sensitivity of this tumor has not been sys ent. The 170-phosphate presumably exists in an anionic state in tematically defined. These studies describe the in vitro and in the culture media, inhibiting entry of EMP into the PAIII cells. vivo growth and metastatic responses of PAIII to clinically used Significant in vivo regressive responses in accessory sex tissues hormonal ablative, hormonally derived cytoreductive, and hor were produced by EMP treatment of PAIII-bearing rats. Sim mone antagonist therapies. Neither T nor E2 had effects on the ilarly, LY 186564, a derivative of estromustine phosphate (Fig. in vitro clonogenic activity of PAIII cells. While producing Id) administered for 28 days in the in vivo PAIII assay, pro marked regression of male accessory sex organs, neither castra duced significant (P < 0.05) regression of the prostate and tion nor E2B administration had effects on the growth or me seminal vesicles but had no antitumor activity. While EMP is tastasis of the PAIII tumor in LW rats. Therefore, unlike the structurally related to estrogens and alkylating agents, the in R3327 Dunning prostate adenocarcinoma, the PAIII tumor is not sensitive to surgical or estrogen-induced androgen ablation. vitro antitumor activity of the compound results from disrup tion of cellular microtubules (8). In humans, selectivity for pro- Hypophysectomy produced marked inhibition of PAIII static EMP uptake has been proposed to result from high af growth and metastasis, supporting the hypothesis that a factor finity binding to an intracellular protein, designated EMBP from the pituitary is stimulatory to PAIII spread. Presently, (10). Accumulation of estramustine in the rat ventral prostate this pituitary factor is unknown. Aside from PRL or the effects results from high affinity binding to a homologous protein (9). of LH mediated through effects of testicular steroidogenesis, the role of pituitary-derived factors on prostatic tumor growth 3 B. L. Neubauer, K. L. Best. R. L. Goode, M. L. Heiman. D. M. Hoover, D. W. has received little attention. Administration of the potent Robertson. M. F. Sarosdy. C. J. Shaar. L. R. Tanzer, and R. L. Merriman, unpub dopamine agonist, pergolide mesylate, to PAIII-bearing LW lished observations. 4669

Table 9 Lack of antagonism by E2B of the antimetastalic effects of LY117018 on the growth and metastasis of the PAIII prostatic adenocarcinoma in male Lobuna Wistar rats"

tumor to initial (tail wt) lymph node lymph node TreatmentPAIII body wtratio2.011 (gm/100wt)2.432 g body (mg/100gwt)207.8 body (mg/100gwt)17 body nos.71.0 foci + vehicle (n = 8)* Â±0.030r Â±0.084 Â±16.7 1.3 Â±34.0.0 Â±19.2 No PAIII (n = 8) 1.858 Â±0.097 1.994 Â±0.048' 2.2 Â±0.5' 8.8 Â±1.4' 0.5 Â±0.5' LY1mg/kg/day)E2B0.005 17018 (20.0 1.208 +0.023'1.550 2.378 Â±0.0502.580 75.7 Â±17.9'158.5 67.6 Â±20.2'186.6 4.4+1.6'25.0

mg/kg/day (n = 8) Â±0.058 Â±0.038 Â±37.3 + 57.9 Â±9.4 O.I mg/kg/day (n = 8) 1.495 + 0.050' 2.527 Â±0.050 177.5 Â±27.8 172.3 Â±45.1 70.4 + 45.5 LYI 17018 + E2BFinal 1.255 Â±0.033'Primary 2.477 Â±0.050Gluteal 61.5 Â±15.5'Iliac 33.9 Â±10.6'Pulmonary 17.6 Â±11.9' 0.01 mg/kg/dav (n = 8) E2B 0.05 mg/kg/day (n = 8) 1.158 Â±0.073' 2.472 Â±0.037 11.4 Â±3.5'-' 15.4 Â±3.8'-' 8.6 Â±6.8' " For details, see "Materials and Methods." * Numbers in parentheses, number of observations. <â€¢MeanÂ±SEM. ' Significantly different from PAIII plus vehicle-treated controls (P < 0.05) using Dunnett's test on ranked data. ' Significantly different from PAIII plus LY117018 (20.0 mg/kg/day) group (P < 0.05) using Dunnett's test on ranked data.

Expression of EMBP has also been described in 2 androgen- LY117018 administration had no activity against the in vitro responsive sublines (G and H) of the R3327 rat prostatic ade clonogenic activity of PAIII cells, nor did it inhibit tumor nocarcinoma (37). Estramustine phosphate administration has growth in the tail. These observations support the contention been shown to retard the in vivo growth of the R3327H tumor that LY117018 had no direct cytoreductive activity. PAIII me (38). Low or nondetectable levels of EMBP are characteristic of tastasis through lymphatic channels was significantly reduced, the androgen-independent sublines of the R3327 tumor (37). and spread of the tumor to the lungs was inhibited by admin Two-dimensional gel electrophoresis of endogenous proteins istration of LY 117018. The mechanism for the antimetastatic failed to detect the protein corresponding to the isoelectric action of LY117018 is unknown; however co-administration of point and molecular weight of EMBP in tissue homogenates E2B did not antagonize the effects of LY117018. This lack of taken from untreated PAIII-bearing lymph nodal tissues.4 If the antagonism supports the contention that the observed antitu- presence of EMBP is a prerequisite for localization of EMP in mor activity was not attributable to estrogen receptor-mediated prostatic cells, then the lack of in vitro and in vivo PAIII tumor events. The statistically significant decrease in gluteal and lym responsiveness presumably results from the absence of EMBP phatic lymph node weights by combination daily LY 117018 in PAIII cells. (20.0 mg/kg)-E2B (0.1 mg/kg) over single-dose LY 117018 Administration of tamoxifen, an estrogen antagonist with treatment was seen in animals exhibiting borderline pathophys- partial estrogen agonist activity, has been shown to suppress the ical presentation (unkempt fur, lethargy) and probably does not growth of the R3327 androgen-sensitive tumor (39) by decreas represent a meaningful antitumor response. ing circulating androgens (40). In the studies described herein, Comparative studies with the PAIII model are in progress LY117018, a compound with antiestrogenic activity, produced with LY117018, tamoxifen, and antiestrogens of the ben- regression of the ventral prostate and seminal vesicles and stim zothiophene class (44) to assess the relative antitumor activities ulation of the testes and adrenals. Tamoxifen, a triphenyleth- of these agents and potential interaction with the pituitary, and ylene estrogen antagonist, regresses male accessory sex organs to determine the antitumor mechanism of LY 117018. Since the through partial estrogen agonist activity (41). Unlike the de metastatic processes in the rat PAIII model may be similar in creased testicular weights produced by E2B and EMP treat human urogenital cancers, understanding the antimetastatic ment, testicular weights were unaffected by LY117018 admin mechanism of LY117018 action may provide a new approach istration. Apparent increases in normalized (per body weight) to the treatment of androgen-insensitive prostatic cancer. testicular and adrenal weights in LY117018-treated rats were attributable to unchanged organ weights in the presence of an REFERENCES inhibition of body weight gain. Increases in adrenal weights 1. Waxman, J. Hormonal aspects of prostatic cancer: a review. J. R. Soc. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1992 American Association for Cancer Research. Comparative Antitumor Effects of Hormonal Ablation, Estrogen Agonist, Estrogen Cytotoxic Derivative, and Antiestrogen in the PAIII Rat Prostatic Adenocarcinoma

Blake Lee Neubauer, Kevin L. Best, Robin L. Goode, et al.

Cancer Res 1992;52:4663-4671.

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