Antiandrogenic Effects of Novel Androgen Synthesis Inhibitors on Hormone-Dependent Prostate Cancer1

[CANCER RESEARCH 60, 6630–6640, December 1, 2000] Antiandrogenic Effects of Novel Androgen Synthesis Inhibitors on Hormone-dependent Prostate Cancer1

Brian J. Long, Dmitry N. Grigoryev, Ivo P. Nnane, Yang Liu, Yang-Zhi Ling2, and Angela M. Brodie3 Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT BPH4 and prostatic carcinoma (3). The two most important androgens in prostate cancer etiology are testosterone and DHT. Testosterone is ␣ We have found that in addition to being potent inhibitors of 17 - synthesized primarily in the testes and also, to a lesser extent, in the hydroxylase/C -lyase and/or 5␣-reductase, some of our novel androgen 17,20 adrenals. Testosterone is further converted to the more potent andro- synthesis inhibitors also interact with the mutated androgen receptor ␣ (AR) expressed in LNCaP prostate cancer cells and the wild-type AR gen DHT by the enzyme 5 -reductase, which is localized primarily in expressed in hormone-dependent prostatic carcinomas. The effects of the prostate (4). Although testosterone and DHT both stimulate the these compounds on the proliferation of hormone-dependent human pros- growth of normal and malignant prostate tissue, DHT is believed to be tatic cancer cells were determined in vitro and in vivo. L-2 and L-10 are the more important androgen (5, 6). ⌬4-3-one-pregnane derivatives. L-35 and L-37 are ⌬5-3␤-ol-androstane Androgen ablation therapy has been shown to produce the most derivatives, and L-36 and L-39 are ⌬4-3-one-androstane-derived com- beneficial responses in patients with hormone-responsive prostatic pounds. L-2, L-10, and L-36 (L-36 at low concentrations) stimulated the tumors. Orchidectomy (castration, either surgical or medical with a growth of LNCaP cells, indicating that they were interacting agonistically luteinizing hormone-releasing hormone analogue) remains the stand- with the mutated AR expressed in LNCaP cells. L-35, L-37, and L-39 ard treatment option for most patients. However, both methods, which acted as LNCaP AR antagonists. To determine whether the growth mod- ulatory effects of our novel compounds were specific for the mutated result in reduced androgen production by the testes, fail to alter LNCaP AR, competitive binding studies were performed with LNCaP androgen production by the adrenal glands. Studies in the United cells and PC-3 cells stably transfected with the wild-type AR (designated States and Europe have reported that although androgen ablation PC-3AR). Regardless of AR receptor type, all of our novel compounds therapy alone is an effective treatment, a combination therapy of were effective at preventing binding of the synthetic androgen methyl- orchidectomy with antiandrogens, to inhibit the action of adrenal trienolone[17␣-methyl-(3H)-R1881 to both the LNCaP AR and the wild- androgens, significantly prolongs the survival of prostate cancer pa- type AR. L-36, L-37, and L-39 (5.0 ␮M) prevented binding by >90%, tients (7–9). Given that efforts to block the production or effects of whereas L-35 inhibited binding by 30%. To determine whether the com- adrenal androgens result in worthwhile therapeutic gains, this labora- pounds were acting as agonists or antagonists, LNCaP cells and PC-3AR tory has been designing and evaluating novel compounds that inhibit cells were transfected with the pMAMneoLUC reporter gene. When luciferase activity was induced by dihydrotestosterone, all of the com- androgen production from all sites in the body. The compounds pounds were found to be potent inhibitors of transcriptional activity, and developed are steroidal antagonists of the steroidogenic enzymes ␣ ␣ the pattern of inhibition was similar for both receptor types. However, C17,20-lyase and 5 -reductase. C17,20-lyase catalyzes both the 17 -

L-2, L-10, and L-36 were determined to be AR agonists, and L-35, L-37, hydroxylation and the cleavage of the C17,20-side chain during the and L-39 were wild-type AR antagonists. When tested in vivo, L-39 was conversion of the 21-carbon steroids pregnenolone and progesterone the only AR antagonist that proved to be effective at inhibiting the growth to the 19-carbon androgens dehydroepiandrosterone and andro- of LNCaP prostate tumor growth. L-39 slowed tumor growth rate in stenedione, respectively (10). The enzyme has an identical amino acid LNCaP tumors grown in male SCID mice to the same level as orchidec- sequence in both testicular and adrenal tissue (11), indicating that tomy, significantly reduced tumor weights (P 0.05), significantly low- < inhibitors of this enzyme would be equally effective at both sites. Two ered serum levels of prostate-specific antigen (P < 0.02), and significantly isoforms of 5␣-reductase occur in the body (type I and type II), and lowered serum levels of testosterone (P < 0.05). L-39 also proved to be effective when tested against the PC-82 prostate cancer xenograft that the type II enzyme is the predominant form in the human prostate. expresses wild-type AR. These results show that some of our compounds Inhibitors of this isoenzyme would essentially prevent prostatic accu- initially developed to be inhibitors of androgen synthesis also interact with mulation of the potent androgen DHT. the human AR and modulate the proliferation of hormone-dependent A number of C17,20-lyase-inhibitors have been described. However, prostatic cancer cells. Therefore, compounds such as L-39, which have most are not specific, and only the imidazole antifungal agent keto- multifunctional activities, hold promise for the treatment of androgen- conazole has been used clinically to reduce testosterone levels in dependent prostate tumors. patients with advanced prostate cancer (12–14). The major drawback with ketoconazole is that it is not very potent or specific. It is only a

INTRODUCTION moderate inhibitor of C17,20-lyase, inhibits cortisol production, and has a number of significant side effects. Nevertheless, recent studies Prostatic carcinoma is the most commonly diagnosed malignancy have reported that ketoconazole was effective in reducing PSA levels in men in the United States and is second only to lung cancer in in 55% (15) and 62.5% (16) of patients who had progressed after cancer-related deaths (1, 2). Androgens play an important role in antiandrogen (flutamide) withdrawal. These results indicate that com- controlling the growth of the normal prostate gland, and in promoting pounds more specific and selective than ketoconazole may be more effective for the treatment of prostate cancer. Several inhibitors of Received 5/5/00; accepted 9/29/00. 5␣-reductase have also been described and finasteride, which is a 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 more potent inhibitor of the type II than the type I isoenzyme (17), has 18 U.S.C. Section 1734 solely to indicate this fact. been approved for the treatment of BPH (18). Although effective at 1 Supported by NIH Grant CA-27440 and a grant from Paramount Capital, Inc., New York, New York. 2 Present address: Department of Medicinal Chemistry, School of Pharmacy, Beijing 4 The abbreviations used are: BPH, benign prostatic hyperplasia; DHT, dihydrotest- ␣ Medical University, Beijing 100083, China. osterone; C17,20-lyase, cytochrome P450 17 -hydroxylase/C17,20-lyase; PSA, prostate- 3 To whom requests for reprints should be addressed, at University of Maryland, specific antigen; AR, androgen receptor; G418, geniticin; FBS, fetal bovine serum; DPBS, Department of Pharmacology and Experimental Therapeutics, 655 West Baltimore Street, Dulbecco’s PBS; [3H]R1881, methyltrienolone [17␣-methyl-[3H]]-R1881; IMEM, im- Baltimore, MD 21201-1559. proved minimum essential medium. 6630

reducing DHT levels in patients with prostate cancer, finasteride also fectamine-Plus were from Life Technologies, Inc. (Grand Island, NY). Phenol increases the bioavailable levels of testosterone (19), which can stim- red-free IMEM and phenol red-free trypsin/EDTA were from Biofluids Inc. ulate tumor growth (20). Therefore, compounds designed to inhibit (Rockville, MD). FBS was supplied by Summit Biotechnology, Inc. (Fort ␣ Collins, CO), and the PSA ELISA kit was from DSL, Inc. (Webster, TX). both C17,20-lyase and 5 -reductase would be expected to be more clinically beneficial to prostate cancer patients than compounds which Hydroxypropyl cellulose was from Sigma Chemical Co. (St. Louis, MO). The inhibit only one of the enzymes. luciferase reporter construct pMAMneoLUC was purchased from Clontech (Palo Alto, CA). The luciferase assay kit was from Promega (Wisconsin, MI). The growth effects of testosterone and DHT on prostate cancer cells Wild-type human AR-expressing vector pCMV5-hAR and the LNCaP AR- are mediated by the androgens binding to their cognitive nuclear expressing vector pCMV5-LNCaPAR were generous gifts from Dr. Elizabeth receptors, which in turn bind to specific response elements in the Wilson (University of North Carolina, Chapel Hill, NC). Matrigel was kindly promoter regions of androgen-regulated genes. The products of these supplied by Dr. Hynda Kleinman (NIH, Bethesda, MD). genes modulate cellular proliferation (21). Antiandrogens such as Steroids and Chemical Inhibitors. Testosterone, DHT, flutamide, keto- flutamide have also been used clinically for the treatment of prostate conazole, and the antiprogestin triamcinolone acetonitride were purchased cancer. However, the results were disappointing. Some patients from Sigma Chemical Co. (St. Louis, MO). Finasteride was a gift from Merck tended to improve after flutamide was withdrawn after relapse (22, Research Laboratories (Rahway, NJ). L-2, L-10, L-35, L-36, L-37, and L-39 23). Nonetheless, as described above, clinical trials which combined were synthesized in our laboratory according to the procedures described by the therapies of orchidectomy with the antiandrogen flutamide re- Ling et al. (27). [3H]R1881 (specific activity 70–87 Ci/mMol) was obtained ported a significantly longer survival period than orchidectomy alone from DuPont NEN (Boston, MA). Testosterone RIA kits were purchased from (7–9). This implies that a treatment regimen involving total androgen DSL, Inc. ablation in combination with an antiandrogen may be a more effective Cell Culture. LNCaP, PC-3, and CV-1 cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin treatment option for patients with androgen-dependent prostate tu- solution. To determine the effect of steroids and novel compounds on cell mors. proliferation, hormone-dependent LNCaP and hormone-independent PC-3 Previously we have reported the synthesis and testing of several cells were transferred into steroid-free medium 3 days prior to the start of ␣ steroidal inhibitors of C17,20-lyase and 5 -reductase (24–28). These experiments. Steroid-free medium consisted of phenol red-free IMEM compounds were shown to be effective inhibitors of human testicular supplemented with 5% dextran-coated, charcoal-treated serum, and 1% ␣ C17,20-lyase and prostatic 5 -reductase in vitro (See Table 1). In the penicillin/streptomycin solution. Serum was depleted of steroids as de- present study we report that some of these compounds are also very scribed previously (38). Growth studies were then performed by plating potent antiandrogens, and that this property contributes, at least in cells (1.5 ϫ 104) in 24-place multiwell dishes (Corning, Inc., Corning, part, to their growth-inhibitory effects on androgen-dependent LNCaP NY). After a 24-h attachment period, the medium was aspirated and prostate cancer cells in vitro and in vivo. LNCaP cells are the most replaced with steroid-free medium containing vehicle or the indicated frequently studied AR-positive prostate cancer cell line that can be concentrations of androgens and novel compounds. This medium was readily grown in tissue culture (29–31). LNCaP cells are not only changed every 3 days and the cells were counted 9 days later using a Coulter Counter model Z-1 (Coulter Electronics, Hialeah, FL). Cell num- androgen-responsive but also androgen-dependent. They respond to bers were expressed as a percentage of vehicle-treated cells. androgens with increased cellular proliferation and elevated expres- Wild-type LNCaP cells and CV-1 cells were transfected with the sion of PSA (32). Although LNCaP cells have a mutated AR (33, 34), pMAMneoLUC plasmid as we have described previously (39). Briefly, they have been used extensively for research on the causes, treatment, 2 ϫ 105 cells in a 35-mm2 dish were exposed to 3 ml of Opti-MEM and prevention of prostate cancer (35). We have used these cells in containing Lipofectamine (30 ␮l) and 6 ␮g of the pMAMneoLUC plasmid

culture and as tumors in nude mice to compare the efficacy of our for5hat37°C in a 5% CO2 incubator. The medium was then changed to ␣ compounds with the known inhibitors of C17,20-lyase and 5 -reduc- routine culture medium for 72 h. Cells were then grown in medium tase, ketoconazole, and finasteride, respectively. The antiandrogenic supplemented with 750 ␮g/ml G418. The surviving colonies were picked properties of the compounds were compared with flutamide. and grown in selective media. Stable selectants were tested twice per month for luciferase activity, as described in the later section. After 3 months (ϳ23 passages), the transfectants with the highest luciferase activity were MATERIALS AND METHODS selected and designated LNCaP-LUC and CV-1LUC, respectively. These Materials. The wild-type LNCaP human prostate cancer cell line and CV-1 cell lines and the PC-3AR cell line were routinely cultured in RPMI 1640 green monkey kidney cells were obtained from the American Type Culture medium supplemented with 10% FBS, 1% penicillin/streptomycin solution, ␮ Collection (Rockville, MD). PC-3 prostate cancer cells and PC-3 cells stably and 750 g/ml G418. 3 transfected with the human wild-type androgen receptor (designated PC-3AR) Competitive Binding of [ H]R1881 to the LNCaP AR and Wild-Type were kindly provided by Dr. Marco Marcelli (Baylor College of Medicine, AR in the Presence of Novel Compounds. Competitive binding studies with Houston, TX; Refs. 36 and 37). RPMI 1640 medium, penicillin/streptomycin the synthetic androgen R1881 were performed essentially as described by solution, trypsin/EDTA solution, G418, Opti-MEM, Lipofectamine, and Lipo- Wong et al. (40) and Yarbrough et al. (41). Wells in 24-place multiwell dishes were coated with poly-L-lysine (0.05 mg/ml) for 30 min and dried. To determine the kinetics of R1881-binding to the LNCaP AR and the wild-type AR, LNCaP cells and PC-3AR cells (2–3 ϫ 105) were plated in steroid-free Table 1 IC50 values (nM) of the steroidal compounds against human testicular ␣ medium and allowed to attach. The following day, the medium was replaced C17,20-lyase and prostatic 5 -reductase from human BPH microsomes with serum-free, steroid-free IMEM supplemented with 0.1% BSA and con- C -lyase 5␣-Reductase 17,20 taining [3H]R1881 (0.01–10 nM) in the presence or absence of a 200-fold IC50 (nM) IC50 (nM) excess of cold R1881 to determine nonspecific binding and 1 ␮M triamcin- Ϯ a Ϯ L-2 43 1.2 75 1.3 olone acetonitride to saturate progesterone and glucocorticoid receptors. Fol- L-10 NI 19 Ϯ 1.5 L-35 21 Ϯ 1.7 NI lowing a 2-h incubation period at 37°C, cells were washed twice with ice-cold L-36 39 Ϯ 1.0 31 Ϯ 2.0 DPBS and solubilized in DPBS containing 0.5% SDS and 20% glycerol. L-37 42 Ϯ 1.9 1277 Ϯ 39 Extracts were removed and the cell-associated radioactivity counted in a L-39 67 Ϯ 8.5 33 Ϯ 2.5 Finasteride NI 33 Ϯ 1.1 scintillation counter. The data were analyzed using RADLIG 40 software Ketoconazole 76 Ϯ 2.0 NI (Biosoft, Ferguson, MO), and Kd and Bmax determined by Scatchard plot a Values show the mean Ϯ SE of three experiments. NI, No Inhibition. These results transformation. When the concentration of R1881 required to almost saturate have been previously reported in Ref. 27. AR in both cell lines was established, the ability of the test compounds (5.0 6631

␮M) to displace [3H]R1881 (5.0 nM) from the receptors was determined as decapitation and the blood was collected. Tumors were excised, weighed, and described above. stored in liquid nitrogen for additional analysis. Transient Transfection of PC-3AR Cells with the pMAMneoLUC Plas- Testosterone RIA Assays. For measurement of serum testosterone levels, mid and CV-1LUC Cells with the pCMV5-hAR and pCMV5-LNCaPAR 50 ␮l of mouse serum were assayed according to the instructions provided with Plasmids. Cells were grown in steroid-free IMEM for 3 days and plated the 125I-testosterone RIA kit supplied by DSL, Inc. Radioactivity was counted (4 ϫ 104 cells/well) in 24-well plates in phenol red-free IMEM supplemented using a Packard Cobra II gamma counter. For measurement of tumor testos- with 10% charcoal-stripped serum and no antibiotics. After a 24-h incubation terone levels, whole tumors were homogenized in phosphate buffer (pH 7.4; period, the cells were washed twice with DPBS and each well was incubated 0.1 M). The homogenates were then centrifuged at 2000 ϫ g for 20 min to with 250 ␮l of phenol red-free IMEM containing 2 ␮l of PLUS-reagent, 4 ␮l remove debris. Fifty-␮l aliquots of the tissue supernatant were used to deter- of Lipofectamine, and 4 ␮g of the pMAMneoLUC plasmid for PC-3AR cells mine the tumor testosterone concentration, as described above. and 0.5 ␮g of the pCMV5-hAR or pCMV5-LNCaPAR plasmids for CV-1LUC Measurement of Serum PSA Levels. Serum PSA levels were determined cells. After a 5-h incubation period, 250 ␮l of routine medium were added to using a PSA ELISA kit supplied by DSL, Inc. Briefly, 2.5 ␮l of serum diluted each well and the cells were incubated for an additional 24 h. The resultant 1:10 in DPBS was mixed with assay buffer to a final volume of 75 ␮l and cells, designated PC-3AR/LUC, CV-1LUC/hAR, or CV-1LUC/LNCaPAR, added to duplicate wells in the 96-well plate that had been coated with an were assayed for luciferase activity as described in the following section. anti-PSA antibody. Following a 1-h assay and extensive washing of the plate, Luciferase Activity Assay. LNCaP-LUC cells were transferred to ste- the wells were treated for 30 min with a second anti-PSA antibody labeled with roid-free medium 3 days before the start of the experiment and plated at horseradish peroxidase. After washing, the wells were treated with the tetra- 1 ϫ 105 cells/well in steroid-free medium. PC-3AR/LUC cells, CV-1LUC/ methylbenzidine substrate for 10 min, and the absorbance was read at 450 nm hAR cells, and CV-1LUC/LNCaPAR cells were transiently transfected as with a Dynatech MRX plate reader. described above. After a 24-h incubation period in steroid-free medium, Statistical Analysis. One-way ANOVA on SigmaStat for Windows ver- each well was treated with ethanol vehicle or the selected steroids and sion 1.0 was used to compare the different treatment groups at the 95% novel compounds (5 ␮M) in triplicate. After a 24-h treatment period, the confidence level. A P of Ͻ0.05 was considered to be statistically significant. cells were washed twice with ice-cold DPBS and assayed using the Lucif- erase kit according to the manufacturer’s protocol. Briefly, the cells were RESULTS lysed with 200 ␮l of luciferase lysing buffer, collected in a microcentrifuge tube and pelleted by centrifugation. Supernatants (100 ␮l aliquots) were Growth Effects on LNCaP and PC-3 Cell Cultures in Vitro. The transferred to the corresponding wells of white 96-well plates (Polyfiltron- structures of the compounds are shown in Fig. 1, and their in vitro ics, Inc., Boston, MA). Luciferin (50 ␮l) was added to each well, and the ␣ activities against C17,20-lyase and 5 -reductase are provided in light produced during the luciferase reaction was measured in a Victor 1420 Table 1. For growth studies, androgen-responsive LNCaP cells and Multilabel counter (Wallac, Inc., Gaithersburg, MD). The effects of the androgen-independent PC-3 cells were deprived of steroids for 3 steroids and novel compounds on DHT-induced luciferase transcription were determined using the same protocol. In Vivo Studies with LNCaP Tumors in SCID Mice and PC-82 Xenografts in Athymic Nude Mice. Male SCID mice and athymic nude mice 4–6 weeks of age were purchased from the National Cancer Institute (Fred- erick, MD). Animals were housed in a pathogen-free environment under controlled conditions of light and humidity and received food and water ad libitum. Inoculation of LNCaP Cells into Male SCID Mice. LNCaP tumors were grown s.c. in male SCID mice essentially as described by Sato et al. (42) with modifications based on the breast cancer model described by Yue et al. (43, 44). LNCaP cells were grown in routine culture medium (RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin) until 80% confluent. Cells were scraped into DPBS, collected by centrifugation, and resuspended in Matrigel (10 mg/ml) at 3 ϫ 107 cells/ml. Each mouse received s.c. injections at one site on each flank with 100 ␮l of cell suspension. Tumors were measured weekly with calipers, and tumor volumes were calculated by ϫ 2 ϫ Ͻ the formula 0.5236 r1 r2 (r1 r2). Inoculation of PC-82 Xenograft Tumors Into Male Athymic Nude Mice. Hormone-dependent PC-82 tumor xenografts were kindly provided by Dr. John Isaacs (John Hopkins School of Medicine, Baltimore, MD). PC-82 tumors were minced into fine pieces, washed with DPBS, filtered, and resuspended in Matrigel at a concentration of 100 mg/ml The mice were inoculated s.c. at one site on each flank with 100 ␮l of tumor suspension using a 18-gauge needle. Tumors were allowed to grow (ϳ3 months) and then were measured weekly. Tumor volumes were then calculated weekly according to the formula ϫ 2 ϫ Ͻ 0.5236 r1 r2 (r1 r2). When tumors reached a measurable size, the mice were randomized into treatment groups (five animals/group) and treated as described in the following section. Treatment. For LNCaP tumor experiments, treatments began 4–5 weeks after cell inoculation when measurable tumor volume was 500 mm3 (For PC-82 xenografts, this was ϳ3 months). For each experiment, groups of six mice (five mice for PC-82 xenografts) with comparable total tumor volumes were either castrated or treated with the novel compounds at 50 mg/kg/day. Mice were castrated under methoxyfluorane anesthesia via the abdominal approach. Compounds and reference drugs were prepared at 10 mg/ml in a 0.3% solution of hydroxypropyl cellulose in saline, and mice received s.c. injections daily. Control and castrated mice were treated with vehicle only. Treatments lasted for 28 days, after which time the animals were sacrificed by Fig. 1. Chemical structures of the novel compounds evaluated in this study. 6632

1.0 ␮M, and 5.0 ␮M L-2 increased cell number by 4.6-fold, 4.4- fold, and 2.9-fold, respectively. L-36 showed a biphasic effect on cell growth. The lower concentrations (0.1 ␮M and 1.0 ␮M) increased the growth rate of LNCaP cells by 2.3-fold and 2-fold, respectively. However, at a 5-␮M concentration, L-36 inhibited cell growth by 50%. L-39 was mildly growth-stimulatory to LNCaP cells at the two lower concentrations, but at the higher concentration of 5.0 ␮M, L-39 had little effect on the growth rate. When the compounds were tested against androgen-independent PC-3 cells over the same 9-day treatment period, the only compounds that modulated growth were L-37 and L-39, which at the 5.0-␮M concentration inhibited the growth of PC-3 cells by approximately 30% (Fig. 2B). To determine whether the mechanism of action of the compounds was mediated by the AR, the ability of the compounds to compete with DHT and modulate cell proliferation was investigated (Fig. 3A). Flutamide and ketoconazole had no effect on DHT-induced LNCaP cell proliferation. Surprisingly, the 5␣- reductase inhibitor finasteride reversed the growth stimulatory effect of DHT on LNCaP cells in a dose-dependent manner. L-2 and L-10 had no effect on DHT-induced LNCaP cell proliferation at concentrations of 0.1 ␮M and 1.0 ␮M. At a concentration of 5.0 ␮M, both compounds slightly reduced proliferation to levels similar to those when the compounds were tested in the absence of DHT. No synergistic or additive effects were observed when LNCaP cells were cotreated with the growth stimulatory compounds L-2 or L-10 and DHT. L-36 had no effect on DHT-induced LNCaP cell proliferation at concentrations of 0.1 ␮M and 1.0 ␮M. However at 5.0 ␮M concentration, L-36 inhibited cell proliferation by Ͼ90%. L-37 was shown to be the most potent inhibitor of DHT-induced LNCaP cell proliferation. L-37 inhibited DHT- induced growth by 73%, 88%, and 95% at concentrations of 0.1 ␮M, 1.0 ␮M, and 5.0 ␮M, respectively. L-35 and L-39 were also effective at reversing the growth effects of DHT and did so in a dose-dependent manner. At the 5.0 ␮M concentration, L-35 and L-39 inhibited DHT- induced LNCaP cell proliferation by 66% and 79%, respectively. These Fig. 2. The effect of the novel compounds and reference drugs on the growth of (A) results suggest that the compounds may be acting to block the action of hormone-dependent LNCaP prostate cancer cells and (B) hormone-independent PC-3 prostate cancer cells in steroid-free medium. Cells were grown in steroid-free medium for DHT in stimulating cell proliferation. 3 days before plating. Triplicate wells were then treated with 0.1 ␮M, 1.0 ␮M, and 5.0 ␮M The ability of the novel compounds to compete with testosterone of the reference drugs and compounds for 9 days, as described in “Materials and for the AR and modulate cell growth in vitro was also investigated Methods.” Cell numbers are expressed as a percentage of the mean number of cells in the control (vehicle-treated) wells. The results show the mean and SE from three separate (Fig. 3B). When cells were cotreated with testosterone (1 nM) and the experiments. reference drugs or novel compounds for 9 days, the results were similar to those obtained when cell growth was stimulated by DHT. L-37, L-35, and L-39 again proved to be the most effective com- days before the experiments. After plating and attachment, cells pounds at inhibiting testosterone-induced growth of LNCaP cells in were treated with the compounds at concentrations of 0.1 ␮M, 1.0 vitro. Each of these compounds inhibited growth in a dose-dependent ␮M, and 5.0 ␮M for 9 days (Fig. 2A and 2B). In agreement with our manner. previous findings (45, 46), DHT and testosterone (both at 1 nM) LNCaP and PC-3AR Androgen Receptor Binding Assays. The were potent stimulators of LNCaP cell growth in vitro, and inability of the novel compounds to modulate the growth of LNCaP creased cell number by 5.9-fold and 4.4-fold, respectively (Fig. cells and to inhibit DHT and testosterone-induced cell proliferation 2A). Neither testosterone nor DHT had any effect on the growth of implied that they may be interacting with the LNCaP AR. To deter- hormone independent PC-3 prostate cancer cells (Fig. 2B). At each mine whether this was specific for the mutated AR expressed in of the concentrations tested, the antiandrogen flutamide also in- LNCaP cells, kinetic studies were performed with the synthetic an- creased the growth rate of LNCaP cells, whereas ketoconazole had drogen methyltrienolone [3H]R1881 binding to the mutated AR ex- no effect on cellular proliferation. Finasteride (1 ␮M and 5 ␮M) pressed in LNCaP cells and the wild-type AR expressed in PC-3AR inhibited growth by 60% compared with vehicle-treated cells. L-37 cells. The data were linearized by Scatchard transformation (data not was the most potent inhibitor of cell growth and did so in a shown) and the Kd and Bmax for both types of AR were determined. dose-dependent manner. Names of the “L” compounds are shown LNCaP cells express a single class of high affinity binding sites with ␮ ϭ Ϯ ϭ ϫ 5 Ϯ ϫ 4 in Fig. 1. L-37 inhibited cell growth by 34% at 0.1 M, 54% at 1.0 Kd 0.78 0.01 nM and Bmax 2.60 10 2.27 10 ␮M, and 67% at 5 ␮M. L-35 inhibited cell growth by 40% at both receptors/cell. PC-3AR cells also express a single class of high- ␮ ␮ ϭ Ϯ 1- M and 5- M concentrations. L-10 increased cell number by affinity binding sites with Kd 0.20 0.01 nM and ϭ ϫ 4 Ϯ ϫ 3 approximately 2-fold at each of the concentrations tested. L-2 was Bmax 4.8 10 6.5 10 receptors/cell. The ability of the 3 found to be the most potent stimulator of cell growth and 0.1 ␮M, compounds to compete with 5 nM [ H]R1881 for binding to both types 6633

Effects of Novel Compounds on LNCaP AR- and Wild-Type AR-Mediated Transcription. To determine whether the compounds are acting as AR agonists or antagonists, luciferase activity assays were performed using LNCaP-LUC and PC-3AR/LUC cells. DHT (5.0 nM) significantly (P Ͻ 0 0.001) stimulated luciferase production mediated by the LNCaP AR in LNCaP-LUC cells (Fig. 5A). Keto- conazole (5.0 ␮M) and flutamide (5.0 ␮M) also activated transcription of the luciferase gene in LNCaP-LUC cells by approximately 2-fold each. Finasteride had no effect on luciferase activity. L-2, L-10, and L-36 (all at 5 ␮M) stimulated luciferase production in LNCaP-LUC cells by 6-fold, 4.5-fold, and 8-fold, respectively, confirming that each of these compounds are agonists of the mutated LNCaP AR. L-39 also, surprisingly, stimulated luciferase activity in LNCaP-LUC cells by 2.5-fold, which suggested that it also has some agonistic properties. L-35 and L-37 (5 ␮M) significantly (P Ͻ 0.001) lowered luciferase transcriptional activity in LNCaP-LUC cells to barely undetectable levels, indicating that they are potent antagonists of the LNCaP AR. These studies were also performed using PC-3AR/LUC cells, which express wild-type AR (Fig. 5B). In these cells, DHT (5 nM) significantly stimulated (P Ͻ 0.01) transcription of the luciferase gene by 3.3-fold. This was considerably less than with LNCaP-LUC cells, and was attributed to the cells reacting negatively to the double transfection of foreign genes. Neither ketoconazole nor flutamide had any appreciable effects on luciferase activity levels mediated by the wild- type AR, suggesting that they are only agonistic with the mutated AR. Finasteride (5 ␮M) did not modulate wild-type AR-mediated luciferase activity levels. When the novel compounds were tested, L-2, L-10, and L-36 (all at 5 ␮M) significantly stimulated (P Ͻ 0.05) luciferase production in PC-3AR/LUC cells by 2.1-fold, 2.8-fold, and 1.7-fold, respectively, confirming that each of these compounds are also agonists of the wild-type AR. L-35 and L-37 (5 ␮M) significantly lowered (P Ͻ 0.05) luciferase transcriptional activity in PC-3AR/LUC cells, again indicating that these compounds are antagonists of both the wild-type and mutant AR. In contrast to the results obtained with

Fig. 3. The effect of novel compounds and reference drugs on LNCaP prostate cancer LNCaP-LUC cells, L-39 inhibited wild-type AR-mediated luciferase cell growth stimulated by (A)1nM DHT and (B)1nM testosterone. Cells were grown in transcriptional activity in PC-3AR/LUC cells by 35%, suggesting that steroid-free medium before plating. Triplicate wells were then cotreated with 0.1 ␮M, 1.0 this compound is an antagonist of the wild-type AR and a weak ␮M, and 5.0 ␮M of the reference drugs and compounds and the androgens, as described in “Materials and Methods.” Cell numbers are expressed as a percentage of the mean number agonist of the mutated LNCaP AR. The results indicate that L-2, L-10, of cells in the androgen-treated wells. The results show the mean and SE from three and L-36 function as agonists of both receptor types, L-35 and L-37 separate experiments. are antagonists of both receptor types, and L-39 has mixed agonist/ antagonistic activity depending on receptor type. The ability of the compounds to modulate DHT-induced luciferase of AR was determined (Fig. 4). Ketoconazole was ineffective at activity was determined in both cell types. In each of the experiments preventing [3H]R1881 from binding to the AR in either cell line. The the cells were cotreated with DHT (5 nM) and the reference drug or antiandrogen flutamide prevented 52% of the [3H]R1881 from bind- novel compound (5 ␮M). DHT (5.0 nM) significantly (P Ͻ 0 0.001) ing to the LNCaP AR, and 51% of the labeled androgen from binding stimulated luciferase production mediated by the LNCaP AR in to the wild-type AR in PC-3AR cells. Interestingly, L-35, which was LNCaP-LUC cells (Fig. 6A). Ketoconazole had no effect on DHT- one of the most effective compounds at inhibiting the growth of induced luciferase activity. In this cell line, flutamide and finasteride LNCaP cells in vitro, was the least effective compound at preventing significantly lowered DHT-induced transcriptional activity suggesting [3H]R1881from binding to either type of AR. In LNCaP cell cultures, that they compete for the LNCaP AR and prevent DHT from binding. All of the novel compounds significantly inhibited DHT-induced L-35 prevented 34% of the [3H]R1881 from binding to the AR, and in LNCaP AR-mediated luciferase transcription. The order of potency of PC-3AR cells, the inhibition was 31%. Each of the other 5 novel the compounds was L-37 Ͼ L-39 Ͼ L-2 Ͼ L-35 Ͼ L-10 Ͼ L-36, and compounds were very effective at preventing [3H]R1881 from bind- DHT-induced luciferase activity was inhibited by 99.1%, 86.8%, ing to the cellular AR. The pattern was similar regardless of whether 73.8%, 72.5%, 69.8%, and 42.1%, respectively. No synergistic or they were tested against the mutated LNCaP AR or the wild-type AR. additive effects were observed on luciferase activity levels when 3 Moreover, they prevented [ H]R1881from binding to the ARs with LNCaP-LUC cells were cotreated with the agonistic compounds, L-2, higher efficiencies than flutamide. In the presence of L-2, L-10, L-36, L-10, or L-36 in combination with DHT. These compounds compete 3 L-37, and L-39, the amount of [ H]R1881 bound to the LNCaP cells with DHT for binding to the mutated LNCaP AR and mediate tran- was 30%, 5.2%, 0.7%, 15.2%, and 3.3%, respectively. In PC-3AR scription to the same level as in the absence of DHT. When the cells, the amount of [3H]R1881 bound in the presence of L-2, L-10, compounds were tested against the wild-type receptor in PC-3AR/ L-36, L-37, and L-39 was 13.5%, 2.6%, 5.1%, 11.7%, and 2.5%, LUC cells, a similar pattern was obtained (Fig. 6B). Ketoconazole and respectively. finasteride had no effect on DHT-induced transcriptional activity and 6634

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. INHIBITION OF HORMONE-DEPENDENT PROSTATE CANCER CELL GROWTH flutamide significantly inhibited (P Ͻ 0.01) luciferase activity in- castrated. Although, serum testosterone levels in the mice treated with duced by DHT. As was observed with the mutated LNCaP AR, all of L-2, L-35, and L-36 were significantly lower compared with the the novel compounds were effective at inhibiting DHT-induced tran- vehicle-treated mice, tumor testosterone levels were unaffected. scriptional activity mediated by the wild-type AR. The order In the second experiment, the effects of L-10, L-37, and L-39 on of potency of the compounds was L-37 Ͼ L-39 Ͼ L-35 Ͼ L-2 Ն L- tumor growth were determined and orchidectomy, finasteride, and 36 Ն L-10, which inhibited luciferase activity levels by 89.1%, ketoconazole were used as the reference treatments (Fig. 8A). The 82.2%, 69.8%, 62.9%, 59.5%, and 58.9%, respectively. mice were grouped 35 days after cell inoculation when measurable To ensure that the results obtained with these cell lines reflected the tumor volume was approximately 500 mm3 (Fig. 8A). Total tumor ability of the compounds to bind to the AR and not another steroid volume in the control mice increased 4.3-fold over the 28 days of hormone receptor, these assays were performed in steroid receptor- treatment and tumor volume in the castrated mice increased by only negative CV-1LUC cells that were transiently transfected with either 2.6-fold (38% reduction compared with control). Tumor volumes the wild-type AR or the mutated LNCaP AR constructs. The results increased 3.2-fold in the mice treated with finasteride and, as in the obtained were in agreement with the results described above, indicat- previous experiment, ketoconazole had no appreciable effect on tumor ing that the effects of the compounds were specific for the AR (data growth. Tumor volumes in the mice treated with L-10 increased by not shown). 3.3-fold (22% reduction compared with control) over the 28 days of Growth Effects on LNCaP Tumors Grown in Male SCID Mice. treatment. This compound was as effective as finasteride in this In the first experiment, the effects of L-2, L-35, and L-36 on tumor model. In contrast to the growth inhibition observed with L-37 in vitro growth were determined, and orchidectomy and ketoconazole were (Fig. 2–6), tumor volumes in the mice treated with this compound used as the reference treatments. The mice were grouped 28 days after increased 5.2-fold. In the mice treated with L-39, tumor volume cell inoculation when measurable tumor volumes were approximately increased by 2.4-fold, which was a 44% reduction versus control 500 mm3 (Fig. 7A). After 21 days of treatment tumor volumes in the mice. In this experiment, L-39 was as effective as castration at mice treated with L-2, L-36, and L-35 were similar to the castration inhibiting tumor growth. Tumor weights in the castrated and L-39- group. However, by 28 days tumor volume in the control mice treated mice were significantly (P Ͻ 0.05) lower than those in the increased 4-fold over the 28 days of treatment, and tumor volume in control mice (Fig. 8B). Compared with vehicle-treated mice, tumors in the castrated mice increased by only 2-fold (52% reduction). None of the castrated and L-39-treated mice weighed 75% and 72% less, the treatments, including ketoconazole, had any appreciable effect on respectively. As shown in Table 3, serum PSA levels were signifi- the growth of the tumors after 28 days of treatment (Fig. 7A). The cantly reduced in the mice that were castrated or treated with finas- reductions in tumor volumes in the mice treated with ketoconazole, teride or L-39 (all P Ͻ 0.02), and L-10 (P Ͻ 0.05). Serum testosterone L-2, L-35, and L-36 were 23%, 28%, 8%, and 12% respectively. levels were significantly reduced in the groups treated with castration These results were also reflected in the weights of the tumors. The (P Ͻ 0.001), L-37 (P Ͻ 0.02), and L-39 (P Ͻ 0.05). Tumor testos- only treatment that resulted in significantly (P Ͻ 0.01) smaller tumors terone was significantly reduced (P Ͻ 0.001) in the mice treated by was orchidectomy (Fig. 7B). Tumor weight in the castrated mice was castration, and of the novel compounds tested, only L-37 significantly reduced by 62%. Mice that had been castrated also had significantly reduced (P Ͻ 0.02) tumor levels of testosterone. (P Ͻ 0.02) lower serum levels of PSA when compared with vehicle- Growth Effects of L-39 on PC-82 Xenografts Grown in Male treated animals (Table 2). In this experiment, serum and tumor levels Athymic Nude Mice. The effects of L-39 on the growth of hormone- of testosterone were significantly reduced in the mice that were dependent PC-82 tumor xenografts were determined because these tumors express wild-type AR. Castration, flutamide, and finasteride were used as the reference treatments. The mice were grouped approximately 21 weeks after tumor inoculation, when measurable tumor volume was approximately 500 mm3 (Fig. 9A). Tumor volume in the control mice increased 2.2-fold over the 28 days of treatment, and tumors in the castrated mice actually regressed by 13% over the duration of the experiment. Compared with the control mice, finasteride slowed tumor growth but not to the same extent as the antiandrogen flutamide. Tumor volume in the mice treated with flutamide increased by only 1.1-fold over the 28 days of treatment. L-39 was almost as effective as flutamide at slowing the growth rate of the PC-82 tumors. Tumor volume in the mice treated with L-39 increased by 1.3-fold during the course of the experiment. These results were also reflected in the weights of the tumors. Compared with the control group, castration, flutamide, and L-39 all significantly lowered (P Ͻ 0.02) PC-82 tumor weight (Fig. 9B) and serum levels of PSA (Table 4). Castration also significantly lowered (P Ͻ 0.01) serum and tumor levels of testosterone. Flutamide had no effect on the serum levels of testosterone. Serum and tumor testosterone levels were significantly lower (P Ͻ 0.01) in the mice treat with L-39. Fig. 4. Competitive binding of the synthetic androgen [3H]R1881 to the LNCaP AR, and the wild-type AR expressed in PC-3AR cells in the presence of the novel compounds 3 and reference drugs. LNCaP and PC-3AR cells were incubated with 5 nM [ H]R1881 and 5 ␮M of the compounds and reference drugs for2hat37°C in serum-free medium, as DISCUSSION described in “Materials and Methods.” After washing and lysing, cell-associated radioactivity was determined by liquid scintillation counting. The amount of [3H]R1881 bound In the present study, we evaluated the ability of novel inhibitors of to the cells in the presence of compound or reference drug, is expressed as a percentage ␣ either C17,20-lyase and/or 5 -reductase to inhibit the growth of hor- of the amount of [3H]R1881 that bound in the presence of vehicle. The results show the P Ͻ 0.01 mone-dependent prostatic cancer cells in vitro and in vivo.Wehad ,ءء ;P Ͻ 0.001 versus control ,ء .mean and SE from three separate experiments versus control; ⌽, P Ͻ 0.05 versus control. reported previously that several of these novel compounds showed 6635

mutation in the ligand-binding domain (threonine to alanine at residue 877) that is responsible for it recognizing other steroidal compounds as either agonists or antagonists (33, 34). It has recently been reported that residue 877 contacts the ligand directly, and the mutation alters the stereochemistry of the binding pocket (49). The mutation broadens the specificity of ligand recognition and the LNCaP AR recognizes estrogens, progestins, and antiandrogens such as flutamide as androgens. The cells respond to these compounds with increased proliferation (34, 50–52). Consistent with previously published reports from this laboratory, flutamide was found to be growth stimulatory to LNCaP cells and finasteride was growth inhibitory (Fig. 2; Refs. 45 and 46). Our results show that the effects of flutamide and finasteride are specific for the LNCaP AR and not for the wild-type AR. This is in contrast to the results obtained with our novel compounds, which

Fig. 5. The effects of the novel compounds and reference drugs on transcriptional activity of luciferase mediated through (A) the LNCaP AR in LNCaP-LUC cells and (B) the wild-type AR expressed in PC-3AR/LUC prostate cancer cells. Cells in steroid-free medium were treated with vehicle, DHT (5 nM), testosterone (5 nM), and the reference drugs or novel compounds (5 ␮M) for 24 h. Cells were then assayed for luciferase activity, as described in “Materials and Methods.” The columns represent the mean of light units [counts per second (cps)/unit protein] in triplicate wells from three separate experiments. .P Ͻ 0.01ءء ;P Ͻ 0.001 versus controlء ,Values are expressed as mean and SE. A .P Ͻ 0.05 versus controlءء ;P Ͻ 0.01ء ,B very good biological activity by lowering androgen levels in a normal male rat model, suggesting that they may be effective treatment options for hormone-dependent prostatic carcinoma (47, 48). In this report we describe that some of our novel androgen synthesis inhibitors also interact with the AR expressed in prostate cancer cells, and that the antiandrogenic properties of some of the compounds contrib- ute, at least in part, to their ability to inhibit the growth of hormone dependent prostate cancers. As an initial approach, the effects of the compounds on the growth Fig. 6. The ability of the novel compounds and reference drugs to modulate DHT- of hormone-dependent LNCaP cells were determined. Of the six novel induced luciferase activity mediated through the (A) LNCaP AR in LNCaP-LUC cells and compounds tested, three stimulated LNCaP cell growth (L-2, L-10, (B) the wild-type AR expressed in PC-3AR/LUC prostate cancer cells. Cells in steroid- and low concentrations of L-36), two inhibited cell proliferation (L-35 free medium were treated with vehicle, DHT alone (5 nM), or a combination of DHT (5 nM) and the reference drugs or novel compounds (5 ␮M) for 24 h. Cells were then assayed and L-37), and one had no effect (L-39). The mitogenic effects of L-2 for luciferase activity, as described in “Materials and Methods.” The columns represent and L-10 were surprising because both of these compounds are the mean of light units [counts per second (cps)/unit protein] in triplicate wells from three P Ͻ 0.001 versus ,ء ,separate experiments. Values are expressed as mean and SE. A P Ͻ 0.01 versus ,ء ,P Ͻ 0.01 versus DHT; ⌽, P Ͻ 0.05 versus DHT. B ,ءء ;pregnane derivatives and would not expected to be recognized as DHT .P Ͻ 0.05 versus DHT ,ءء ;ligands by the LNCaP AR. However, the LNCaP AR contains a DHT 6636

vivo, used flutamide, because we have shown previously that LNCaP cells respond equally well to both compounds (45, 46). Findings of growth stimulation by flutamide led to the development of bicalutamide (Casodex), a second-generation, nonsteroidal antiandrogen (56, 57). Bicalutamide has a 4-fold-higher affinity for the AR than flut-

Table 2 Serum levels of PSA and testosterone and tumor levels of testosterone in male SCID mice after 28 days of treatment with novel compounds Serum Tumor

Testosterone Testosterone PSA (ng/ml) (ng/ml) (ng/g wt) Control 407.85 Ϯ 59.06a 0.348 Ϯ 0.04 1.247 Ϯ 0.147 Castration 158.96 Ϯ 33.64b 0.095 Ϯ 0.02c Ͻ0.01c Ketoconazole 310.83 Ϯ 74.32 0.509 Ϯ 0.152 1.645 Ϯ 0.040 L-2 338.49 Ϯ 33.62 0.102 Ϯ 0.005c 1.414 Ϯ 0.103 L-35 373.20 Ϯ 33.59 0.145 Ϯ 0.028b 1.228 Ϯ 0.123 L-36 368.63 Ϯ 52.40 0.099 Ϯ 0.042b 1.631 Ϯ 0.315 a Values are the means Ϯ SE from six animals. One tumor from each mouse was analyzed. b P Ͻ 0.02 versus the control group. c P Ͻ 0.001 versus the control group.

Fig. 7. The effects of orchidectomy, ketoconazole, and the novel compounds L-2, L-35, and L-36 on the growth of LNCaP prostate tumors in male SCID mice. Groups of six mice with LNCaP tumors were treated with the compounds at 50 mg/kg/day for 28 days. A, tumor volumes were measured weekly, and the percentage of change in tumor volume was determined. B, after 28 days of treatment, the mice were sacrificed and the tumors were P Ͻ 0.01 versus ,ء .removed and weighed. Castration significantly reduced tumor weights control. interacted with both AR types and maintained the same agonistic/ antagonistic properties regardless of receptor type. This indicates that some of our antiandrogenic compounds may be useful for the treatment of patients with tumors expressing either wild-type or mutated AR, or for patients with amplified AR expression. Androgen responsive cells are believed to respond to androgen ablation therapy by acquiring androgen-independence and adapting to growth in the absence mitogenic androgens. However, recent studies have suggested that in a subset of tumors, cells respond to reduced androgen levels by amplifying AR gene expression (53, 54). Therefore, AR-mediated signaling is likely to be important in the advanced-stage disease.

Compounds such as L-39, which is a potent inhibitor of C17,20-lyase and 5␣-reductase and exhibits antiandrogenic properties, may be a Fig. 8. The effects of orchidectomy, finasteride, ketoconazole, and the novel compounds L-10, L-37, and L-39 on the growth of LNCaP prostate tumors in male SCID mice. suitable treatment option for such patients. Groups of six mice with LNCaP tumors were treated with the compounds at 50 mg/kg/day Prostate cancer cells with mutated AR respond to flutamide and for 28 days. A, tumor volumes were measured weekly, and the percentage of change in tumor volume was determined. B, after 28 days of treatment, the mice were sacrificed and hydroxyflutamide, the active metabolite of flutamide, with growth the tumors were removed and weighed. Castration and L-39 significantly reduced tumor .P Ͻ 0.05 versus control ,ء .stimulation rather than growth inhibition. Our studies, in vitro and in weights 6637

Table 3 Serum levels of PSA and testosterone, and tumor levels of testosterone in male growth measurements. L-10 was found to be as effective as finasteride SCID mice after 28 days of treatment with novel compounds at inhibiting tumor growth, and L-2 and L-36 significantly lowered Serum Tumor serum levels of testosterone but not tumor testosterone levels. There- Testosterone Testosterone fore, it is likely that although the compounds are effective inhibitors PSA (ng/ml) (ng/ml) (ng/g wt) of androgen synthesis, this is overshadowed by their androgenic Control 828.22 Ϯ 36.03a 0.205 Ϯ 0.015 1.843 Ϯ 0.294 Castration 379.69 Ϯ 66.23b 0.054 Ϯ 0.019c 0.056 Ϯ 0.004c Finasteride 518.38 Ϯ 54.42b 0.638 Ϯ 0.453c 5.661 Ϯ 0.328d Ketoconazole 716.54 Ϯ 31.74 0.298 Ϯ 0.116 1.573 Ϯ 0.104 L-10 567.66 Ϯ 9.78d 0.193 Ϯ 0.020 1.550 Ϯ 0.442 L-37 897.65 Ϯ 62.13 0.095 Ϯ 0.012b 0.933 Ϯ 0.158b L-39 316.38 Ϯ 98.30b 0.129 Ϯ 0.034d 1.744 Ϯ 0.131 a Values are the means Ϯ SE from six animals. One tumor from each mouse was analyzed. b P Ͻ 0.02 versus the control group. c P Ͻ 0.001 versus the control group. d P Ͻ 0.05 versus the control group. amide and is recognized by the LNCaP AR as an antiandrogen. Clinically, bicalutamide is providing better responses and is better tolerated than flutamide (57). Experiments ongoing in this laboratory are presently comparing the antitumor effects of bicalutamide to some of our more potent novel compounds. Although the LNCaP prostate cancer cell line is the most characterized androgen-dependent model of prostate cancer, the mutation in the AR is problematic for inves- tigators researching the effects of antiandrogens. Furthermore, LNCaP cells also respond to androgens in a biphasic concentration-dependent response, with high concentrations of androgens inhibiting cell proliferation (58). We observed this with L-36, which was a potent stimulator of cell proliferation at the lower concentrations and a potent inhibitor of cell growth at the 5-␮M concentration. Therefore, whereas the competitive binding studies with the synthetic androgen [3H]R1881 indicate that the compounds are interacting with both the mutated LNCaP AR and the wild-type AR, they cannot discriminate whether the compounds are acting as agonists or antagonists. We used transcriptional activation assays with luciferase activity regulated by an androgen responsive promoter to overcome these difficulties. LNCaP-LUC cells were selected in this laboratory and responded to DHT and testosterone in a manner similar to the parent cells, indicating that no clonal variation had occurred during the selection process. The studies confirmed that the growth stimulatory compounds (L-2, L-10, and L-36) were functioning as pure AR antagonists whereas the growth inhibitory compounds (L-35, L-37, and L-39) were AR antagonists. It is interesting to note that the pregnane derivatives L-2 and L-10 were agonists of both receptor types. This suggests that the side chain modification at carbon 17 may play an important role in medi- Fig. 9. The effects of orchidectomy, finasteride, flutamide, and the novel compound ating these effects. L-35, L-37, and L-39 were also determined to be L-39 on the growth of PC-82 prostate tumor xenografts in male athymic nude mice. Groups of five mice with PC-82 prostate cancer tumors were treated with the compounds antagonists of both the wild-type AR and the LNCaP AR, indicating at 50 mg/kg/day for 28 days. A, tumor volumes were measured weekly, and the percentage that they are more comparable with the second-generation antiandro- of change in tumor volume was determined. B, after 28 days of treatment, the mice were gen bicalutamide than with flutamide. sacrificed and the tumors were removed and weighed. Castration, flutamide, and L-39 .P Ͻ 0.02 versus control ,ء .significantly reduced tumor weights Having established the in vitro activities of the novel compounds, the in vivo effects were determined using LNCaP prostatic cancer cells grown as tumor xenografts in male SCID mice. In a preliminary Table 4 Serum levels of PSA and testosterone and tumor levels of testosterone in male experiment (data not shown), the growth of LNCaP prostate cancer athymic nude mice with PC-82 tumors after 28 days of treatment with the novel compound L-39 xenografts in male SCID mice was determined to be hormone-dependent, and tumor weights were significantly lower in the animals that Serum Tumor were castrated or treated with the 5␣-reductase-inhibitor finasteride. Testosterone Testosterone Flutamide was found to stimulate the growth of LNCaP tumor xe- PSA (ng/ml) (ng/ml) (ng/g wt) nografts, but not to the same extent as DHT. Additionally, the max- Control 413.04 Ϯ 78.17a 0.216 Ϯ 0.140 2.987 Ϯ 0.326 Castration 25.08 Ϯ 7.95b 0.059 Ϯ 0.030c 0.217 Ϯ 0.024c imum time of treatment was determined to be 28 days. After this time, Flutamide 56.58 Ϯ 13.53b 0.209 Ϯ 0.096 0.446 Ϯ 0.059c tumors in the castrated group appeared to have acquired hormone- Finasteride NT NT NT independence based on a determination of weekly serum PSA levels L-39 83.15 Ϯ 27.22b 0.073 Ϯ 0.031c 1.075 Ϯ 0.135c (data not shown). a Values are the means Ϯ SE from five animals. One tumor from each mouse was analyzed. In agreement with the results obtained in vitro, the androgenic b P Ͻ 0.001 versus the control group. compounds L-2, L-36, and L-10 had no appreciable effect on tumor c P Ͻ 0.02 versus the control group. NT, not tested. 6638

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. INHIBITION OF HORMONE-DEPENDENT PROSTATE CANCER CELL GROWTH properties, which results in LNCaP tumor growth. The inability of 3. Cheng, E., Lee, C., and Grayhack, J. Endocrinology of the prostate. In: H. Lepor and L-35 and L-37 to inhibit LNCaP tumor growth in vivo was especially R. K. Lawson (eds.), Prostate Diseases, pp. 57–71. Philadelphia: W. B. Saunders Co., 1993. disappointing. Both of these compounds were the most effective at 4. Bruchovsky, N., and Wilson, J. D. The conversion of testosterone to 5␣-androstan- inhibiting LNCaP cell growth in vitro, and they were both determined 17␤-ol-3-one by rat prostate in vivo and in vitro. J. Biol. Chem., 243: 2012–2021, 1968. to be the most potent antiandrogens. We have recently determined that 5. Petrow, V. The dihydrotestosterone (DHT) hypothesis of prostate cancer and its L-37 is an inhibitor of the liver cytochrome P450 enzymes 3␣- therapeutic implications. Prostate, 9: 343–361, 1986. hydroxysteroid-oxidoreductase and 3␤-hydroxysteroid-oxidoreduc- 6. Wilson, J. D. Role of dihydrotestosterone action in androgen action. Prostate, 6 (Suppl.): 88s–92s, 1996. tase, which catalyze the metabolism of DHT to polar metabolites that 7. Crawford, E. D., Eisenberger, M. A., McLeod, D. G., Spaulding, J. T., Benson, R., are excreted in the urine (data not shown). Although we were unable Dorr, F. A., Blumstein, B. A., Davis, M. A., and Goodman, P. J. A controlled trial of to reliably measure serum levels of DHT in the serum of the animals leuprolide with and without flutamide in prostatic carcinoma. N. Engl. J. Med., 321: 419–424, 1989. because of cross-reactivity with testosterone and the novel com- 8. Crawford, E. D., Crawford, E. D., and Allen, J. A. 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The inability of L-35 to W. L. Cytochrome P450c17 (steroid 17 ␣-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues. inhibit LNCaP tumor growth may be attributed to its weak affinity for Proc. Natl. Acad. Sci. USA, 84: 407–411, 1987. the AR. Alternatively, it may be attributable to the compound being 12. Trachtenberg, J., and Pont, A. Ketoconazole therapy for advanced prostatic cancer. converted to an inactive metabolite, poor uptake of the compound in Lancet, 2: 433–435, 1984. 13. Williams, G., Kerle, D. J., Doble, A., Dunlop, H., Smith, C., Allen, J., Yeo, T., and the animals, or rapid clearance from the animals. Bloom, S. R. Objective responses to ketoconazole therapy in patients with relapsed The most effective compound at inhibiting LNCaP tumor growth in progressive prostatic cancer. Br. J. Urol., 58: 45–51, 1986. 14. Jubilerer, S. J., and Hogan, T. 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Brian J. Long, Dmitry N. Grigoryev, Ivo P. Nnane, et al.

Cancer Res 2000;60:6630-6640.

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