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

Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201

	ABSTRACT

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We have found that in addition to being potent inhibitors of 17-hydroxylase/C_17,20-lyase and/or 5-reductase, some of our novel androgen synthesis inhibitors also interact with the mutated androgen receptor (AR) expressed in LNCaP prostate cancer cells and the wild-type AR expressed in hormone-dependent prostatic carcinomas. The effects of these compounds on the proliferation of hormone-dependent human prostatic cancer cells were determined in vitro and in vivo. L-2 and L-10 are ⁴-3-one-pregnane derivatives. L-35 and L-37 are ⁵-3ß-ol-androstane derivatives, and L-36 and L-39 are ⁴-3-one-androstane-derived compounds. L-2, L-10, and L-36 (L-36 at low concentrations) stimulated the growth of LNCaP cells, indicating that they were interacting agonistically with the mutated AR expressed in LNCaP cells. L-35, L-37, and L-39 acted as LNCaP AR antagonists. To determine whether the growth modulatory effects of our novel compounds were specific for the mutated LNCaP AR, competitive binding studies were performed with LNCaP cells and PC-3 cells stably transfected with the wild-type AR (designated PC-3AR). Regardless of AR receptor type, all of our novel compounds were effective at preventing binding of the synthetic androgen methyltrienolone[17-methyl-(³H)-R1881 to both the LNCaP AR and the wild-type AR. L-36, L-37, and L-39 (5.0 µM) prevented binding by >90%, whereas L-35 inhibited binding by 30%. To determine whether the compounds were acting as agonists or antagonists, LNCaP cells and PC-3AR cells were transfected with the pMAMneoLUC reporter gene. When luciferase activity was induced by dihydrotestosterone, all of the compounds were found to be potent inhibitors of transcriptional activity, and the pattern of inhibition was similar for both receptor types. However, L-2, L-10, and L-36 were determined to be AR agonists, and L-35, L-37, and L-39 were wild-type AR antagonists. When tested in vivo, L-39 was the only AR antagonist that proved to be effective at inhibiting the growth of LNCaP prostate tumor growth. L-39 slowed tumor growth rate in LNCaP tumors grown in male SCID mice to the same level as orchidectomy, significantly reduced tumor weights (P < 0.05), significantly lowered serum levels of prostate-specific antigen (P < 0.02), and significantly 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 expresses wild-type AR. These results show that some of our compounds initially developed to be inhibitors of androgen synthesis also interact with the human AR and modulate the proliferation of hormone-dependent prostatic cancer cells. Therefore, compounds such as L-39, which have multifunctional activities, hold promise for the treatment of androgen-dependent prostate tumors.

	INTRODUCTION

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Prostatic carcinoma is the most commonly diagnosed malignancy in men in the United States and is second only to lung cancer in cancer-related deaths (1 , 2) . Androgens play an important role in controlling the growth of the normal prostate gland, and in promoting BPH⁴ and prostatic carcinoma (3) . The two most important androgens in prostate cancer etiology are testosterone and DHT. Testosterone is synthesized primarily in the testes and also, to a lesser extent, in the adrenals. Testosterone is further converted to the more potent androgen DHT by the enzyme 5-reductase, which is localized primarily in the prostate (4) . Although testosterone and DHT both stimulate the growth of normal and malignant prostate tissue, DHT is believed to be the more important androgen (5 , 6) .

Androgen ablation therapy has been shown to produce the most beneficial responses in patients with hormone-responsive prostatic tumors. Orchidectomy (castration, either surgical or medical with a luteinizing hormone-releasing hormone analogue) remains the standard treatment option for most patients. However, both methods, which result in reduced androgen production by the testes, fail to alter androgen production by the adrenal glands. Studies in the United States and Europe have reported that although androgen ablation therapy alone is an effective treatment, a combination therapy of orchidectomy with antiandrogens, to inhibit the action of adrenal androgens, significantly prolongs the survival of prostate cancer patients (7, 8, 9) . Given that efforts to block the production or effects of adrenal androgens result in worthwhile therapeutic gains, this laboratory has been designing and evaluating novel compounds that inhibit androgen production from all sites in the body. The compounds developed are steroidal antagonists of the steroidogenic enzymes C_17,20-lyase and 5-reductase. C_17,20-lyase catalyzes both the 17-hydroxylation and the cleavage of the C_17,20-side chain during the conversion of the 21-carbon steroids pregnenolone and progesterone to the 19-carbon androgens dehydroepiandrosterone and androstenedione, respectively (10) . The enzyme has an identical amino acid sequence in both testicular and adrenal tissue (11) , indicating that inhibitors of this enzyme would be equally effective at both sites. Two isoforms of 5-reductase occur in the body (type I and type II), and the type II enzyme is the predominant form in the human prostate. Inhibitors of this isoenzyme would essentially prevent prostatic accumulation of the potent androgen DHT.

A number of C_17,20-lyase-inhibitors have been described. However, most are not specific, and only the imidazole antifungal agent ketoconazole has been used clinically to reduce testosterone levels in patients with advanced prostate cancer (12, 13, 14) . The major drawback with ketoconazole is that it is not very potent or specific. It is only a moderate inhibitor of C_17,20-lyase, inhibits cortisol production, and has a number of significant side effects. Nevertheless, recent studies have reported that ketoconazole was effective in reducing PSA levels in 55% (15) and 62.5% (16) of patients who had progressed after antiandrogen (flutamide) withdrawal. These results indicate that compounds more specific and selective than ketoconazole may be more effective for the treatment of prostate cancer. Several inhibitors of 5-reductase have also been described and finasteride, which is a more potent inhibitor of the type II than the type I isoenzyme (17) , has been approved for the treatment of BPH (18) . Although effective at reducing DHT levels in patients with prostate cancer, finasteride also increases the bioavailable levels of testosterone (19) , which can stimulate tumor growth (20) . Therefore, compounds designed to inhibit both C_17,20-lyase and 5-reductase would be expected to be more clinically beneficial to prostate cancer patients than compounds which inhibit only one of the enzymes.

The growth effects of testosterone and DHT on prostate cancer cells are mediated by the androgens binding to their cognitive nuclear receptors, which in turn bind to specific response elements in the promoter regions of androgen-regulated genes. The products of these genes modulate cellular proliferation (21) . Antiandrogens such as flutamide have also been used clinically for the treatment of prostate cancer. However, the results were disappointing. Some patients tended to improve after flutamide was withdrawn after relapse (22 , 23) . Nonetheless, as described above, clinical trials which combined the therapies of orchidectomy with the antiandrogen flutamide reported a significantly longer survival period than orchidectomy alone (7, 8, 9) . This implies that a treatment regimen involving total androgen ablation in combination with an antiandrogen may be a more effective treatment option for patients with androgen-dependent prostate tumors.

Previously we have reported the synthesis and testing of several steroidal inhibitors of C_17,20-lyase and 5-reductase (24, 25, 26, 27, 28) . These compounds were shown to be effective inhibitors of human testicular C_17,20-lyase and prostatic 5-reductase in vitro (See Table 1 ). In the present study we report that some of these compounds are also very potent antiandrogens, and that this property contributes, at least in part, to their growth-inhibitory effects on androgen-dependent LNCaP prostate cancer cells in vitro and in vivo. LNCaP cells are the most frequently studied AR-positive prostate cancer cell line that can be readily grown in tissue culture (29, 30, 31) . LNCaP cells are not only androgen-responsive but also androgen-dependent. They respond to androgens with increased cellular proliferation and elevated expression of PSA (32) . Although LNCaP cells have a mutated AR (33 , 34) , they have been used extensively for research on the causes, treatment, and prevention of prostate cancer (35) . We have used these cells in culture and as tumors in nude mice to compare the efficacy of our compounds with the known inhibitors of C_17,20-lyase and 5-reductase, ketoconazole, and finasteride, respectively. The antiandrogenic properties of the compounds were compared with flutamide.

View this table: [in this window] [in a new window]   Table 1 IC50 values (nM) of the steroidal compounds against human testicular C17,20-lyase and prostatic 5-reductase from human BPH microsomes

	MATERIALS AND METHODS

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Steroids and Chemical Inhibitors.
Testosterone, DHT, flutamide, ketoconazole, and the antiprogestin triamcinolone acetonitride were purchased from Sigma Chemical Co. (St. Louis, MO). Finasteride was a gift from Merck Research Laboratories (Rahway, NJ). L-2, L-10, L-35, L-36, L-37, and L-39 were synthesized in our laboratory according to the procedures described by Ling et al. (27) . [³H]R1881 (specific activity 70–87 Ci/mMol) was obtained from DuPont NEN (Boston, MA). Testosterone RIA kits were purchased from DSL, Inc.

Cell Culture.
LNCaP, PC-3, and CV-1 cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin solution. To determine the effect of steroids and novel compounds on cell proliferation, hormone-dependent LNCaP and hormone-independent PC-3 cells were transferred into steroid-free medium 3 days prior to the start of experiments. Steroid-free medium consisted of phenol red-free IMEM supplemented with 5% dextran-coated, charcoal-treated serum, and 1% penicillin/streptomycin solution. Serum was depleted of steroids as described previously (38) . Growth studies were then performed by plating cells (1.5 x 10⁴ ) in 24-place multiwell dishes (Corning, Inc., Corning, NY). After a 24-h attachment period, the medium was aspirated and replaced with steroid-free medium containing vehicle or the indicated concentrations of androgens and novel compounds. This medium was changed every 3 days and the cells were counted 9 days later using a Coulter Counter model Z-1 (Coulter Electronics, Hialeah, FL). Cell numbers were expressed as a percentage of vehicle-treated cells.

Wild-type LNCaP cells and CV-1 cells were transfected with the pMAMneoLUC plasmid as we have described previously (39) . Briefly, 2 x 10⁵ cells in a 35-mm² dish were exposed to 3 ml of Opti-MEM containing Lipofectamine (30 µl) and 6 µg of the pMAMneoLUC plasmid for 5 h at 37°C in a 5% CO₂ incubator. The medium was then changed to routine culture medium for 72 h. Cells were then grown in medium supplemented with 750 µg/ml G418. The surviving colonies were picked and grown in selective media. Stable selectants were tested twice per month for luciferase activity, as described in the later section. After 3 months (23 passages), the transfectants with the highest luciferase activity were selected and designated LNCaP-LUC and CV-1LUC, respectively. These cell lines and the PC-3AR cell line were routinely cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin solution, and 750 µg/ml G418.

Competitive Binding of [³H]R1881 to the LNCaP AR and Wild-Type AR in the Presence of Novel Compounds.
Competitive binding studies with the synthetic androgen R1881 were performed essentially as described by 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 x 10⁵) were plated in steroid-free medium and allowed to attach. The following day, the medium was replaced with serum-free, steroid-free IMEM supplemented with 0.1% BSA and containing [³H]R1881 (0.01–10 nM) in the presence or absence of a 200-fold excess of cold R1881 to determine nonspecific binding and 1 µM triamcinolone acetonitride to saturate progesterone and glucocorticoid receptors. Following a 2-h incubation period at 37°C, cells were washed twice with ice-cold DPBS and solubilized in DPBS containing 0.5% SDS and 20% glycerol. Extracts were removed and the cell-associated radioactivity counted in a scintillation counter. The data were analyzed using RADLIG 40 software (Biosoft, Ferguson, MO), and K_d and B_max determined by Scatchard plot transformation. When the concentration of R1881 required to almost saturate AR in both cell lines was established, the ability of the test compounds (5.0 µM) to displace [³H]R1881 (5.0 nM) from the receptors was determined as described above.

Transient Transfection of PC-3AR Cells with the pMAMneoLUC Plasmid and CV-1LUC Cells with the pCMV5-hAR and pCMV5-LNCaPAR Plasmids.
Cells were grown in steroid-free IMEM for 3 days and plated (4 x 10⁴ cells/well) in 24-well plates in phenol red-free IMEM supplemented with 10% charcoal-stripped serum and no antibiotics. After a 24-h incubation period, the cells were washed twice with DPBS and each well was incubated with 250 µl of phenol red-free IMEM containing 2 µl of PLUS-reagent, 4 µl of Lipofectamine, and 4 µg of the pMAMneoLUC plasmid for PC-3AR cells and 0.5 µg of the pCMV5-hAR or pCMV5-LNCaPAR plasmids for CV-1LUC cells. After a 5-h incubation period, 250 µl of routine medium were added to each well and the cells were incubated for an additional 24 h. The resultant cells, designated PC-3AR/LUC, CV-1LUC/hAR, or CV-1LUC/LNCaPAR, were assayed for luciferase activity as described in the following section.

Luciferase Activity Assay.
LNCaP-LUC cells were transferred to steroid-free medium 3 days before the start of the experiment and plated at 1 x 10⁵ cells/well in steroid-free medium. PC-3AR/LUC cells, CV-1LUC/hAR cells, and CV-1LUC/LNCaPAR cells were transiently transfected as described above. After a 24-h incubation period in steroid-free medium, each well was treated with ethanol vehicle or the selected steroids and novel compounds (5 µM) in triplicate. After a 24-h treatment period, the cells were washed twice with ice-cold DPBS and assayed using the Luciferase kit according to the manufacturer’s protocol. Briefly, the cells were lysed with 200 µl of luciferase lysing buffer, collected in a microcentrifuge tube and pelleted by centrifugation. Supernatants (100 µl aliquots) were transferred to the corresponding wells of white 96-well plates (Polyfiltronics, Inc., Boston, MA). Luciferin (50 µl) was added to each well, and the light produced during the luciferase reaction was measured in a Victor 1420 Multilabel counter (Wallac, Inc., Gaithersburg, MD). The effects of the 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 (Frederick, 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 x 10⁷ cells/ml. Each mouse received s.c. injections at one site on each flank with 100 µl of cell suspension. Tumors were measured weekly with calipers, and tumor volumes were calculated by the formula 0.5236 x r₁² x 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 µl of tumor suspension using a 18-gauge needle. Tumors were allowed to grow (3 months) and then were measured weekly. Tumor volumes were then calculated weekly according to the formula 0.5236 x r₁² x 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 3 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 decapitation and the blood was collected. Tumors were excised, weighed, and stored in liquid nitrogen for additional analysis.

Testosterone RIA Assays.
For measurement of serum testosterone levels, 50 µl of mouse serum were assayed according to the instructions provided with the ¹²⁵I-testosterone RIA kit supplied by DSL, Inc. Radioactivity was counted using a Packard Cobra II gamma counter. For measurement of tumor testosterone levels, whole tumors were homogenized in phosphate buffer (pH 7.4; 0.1 M). The homogenates were then centrifuged at 2000 x g for 20 min to remove debris. Fifty-µl aliquots of the tissue supernatant were used to determine the tumor testosterone concentration, as described above.

Measurement of Serum PSA Levels.
Serum PSA levels were determined using a PSA ELISA kit supplied by DSL, Inc. Briefly, 2.5 µl of serum diluted 1:10 in DPBS was mixed with assay buffer to a final volume of 75 µl and added to duplicate wells in the 96-well plate that had been coated with an anti-PSA antibody. Following a 1-h assay and extensive washing of the plate, the wells were treated for 30 min with a second anti-PSA antibody labeled with horseradish peroxidase. After washing, the wells were treated with the tetramethylbenzidine substrate for 10 min, and the absorbance was read at 450 nm with a Dynatech MRX plate reader.

Statistical Analysis.
One-way ANOVA on SigmaStat for Windows version 1.0 was used to compare the different treatment groups at the 95% confidence level. A P of <0.05 was considered to be statistically significant.

	RESULTS

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Growth Effects on LNCaP and PC-3 Cell Cultures in Vitro.
The structures of the compounds are shown in Fig. 1 , and their in vitro activities against C_17,20-lyase and 5-reductase are provided in Table 1 . For growth studies, androgen-responsive LNCaP cells and androgen-independent PC-3 cells were deprived of steroids for 3 days before the experiments. After plating and attachment, cells were treated with the compounds at concentrations of 0.1 µM, 1.0 µM, and 5.0 µM for 9 days (Fig. 2A and 2B) . In agreement with our previous findings (45 , 46) , DHT and testosterone (both at 1 nM) were potent stimulators of LNCaP cell growth in vitro, and increased cell number by 5.9-fold and 4.4-fold, respectively (Fig. 2A) . Neither testosterone nor DHT had any effect on the growth of hormone independent PC-3 prostate cancer cells (Fig. 2B) . At each of the concentrations tested, the antiandrogen flutamide also increased the growth rate of LNCaP cells, whereas ketoconazole had no effect on cellular proliferation. Finasteride (1 µM and 5 µM) inhibited growth by 60% compared with vehicle-treated cells. L-37 was the most potent inhibitor of cell growth and did so in a dose-dependent manner. Names of the "L" compounds are shown in Fig. 1 . L-37 inhibited cell growth by 34% at 0.1 µM, 54% at 1.0 µM, and 67% at 5 µM. L-35 inhibited cell growth by 40% at both 1-µM and 5-µM concentrations. L-10 increased cell number by approximately 2-fold at each of the concentrations tested. L-2 was found to be the most potent stimulator of cell growth and 0.1 µM, 1.0 µM, and 5.0 µM 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 µM and 1.0 µM) increased the growth rate of LNCaP cells by 2.3-fold and 2-fold, respectively. However, at a 5-µM 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 µM, 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-µM concentration inhibited the growth of PC-3 cells by approximately 30% (Fig. 2B) .

View larger version (19K): [in this window] [in a new window]   Fig. 1. Chemical structures of the novel compounds evaluated in this study.

View larger version (37K): [in this window] [in a new window]   Fig. 2. The effect of the novel compounds and reference drugs on the growth of (A) 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 3 days before plating. Triplicate wells were then treated with 0.1 µM, 1.0 µM, and 5.0 µM of the reference drugs and compounds for 9 days, as described in "Materials and 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 experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 3. The effect of novel compounds and reference drugs on LNCaP prostate cancer cell growth stimulated by (A) 1 nM DHT and (B) 1 nM testosterone. Cells were grown in steroid-free medium before plating. Triplicate wells were then cotreated with 0.1 µM, 1.0 µM, and 5.0 µM 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 of cells in the androgen-treated wells. The results show the mean and SE from three separate experiments.

LNCaP and PC-3AR Androgen Receptor Binding Assays.
The ability of the novel compounds to modulate the growth of LNCaP cells and to inhibit DHT and testosterone-induced cell proliferation implied that they may be interacting with the LNCaP AR. To determine whether this was specific for the mutated AR expressed in LNCaP cells, kinetic studies were performed with the synthetic androgen methyltrienolone [³H]R1881 binding to the mutated AR expressed in LNCaP cells and the wild-type AR expressed in PC-3AR cells. The data were linearized by Scatchard transformation (data not shown) and the K_d and B_max for both types of AR were determined. LNCaP cells express a single class of high affinity binding sites with K_d = 0.78 ± 0.01 nM and B_max = 2.60 x 10⁵ ± 2.27 x 10⁴ receptors/cell. PC-3AR cells also express a single class of high-affinity binding sites with K_d = 0.20 ± 0.01 nM and B_max = 4.8 x 10⁴ ± 6.5 x 10³ receptors/cell. The ability of the compounds to compete with 5 nM [³H]R1881 for binding to both types of AR was determined (Fig. 4) . Ketoconazole was ineffective at preventing [³H]R1881 from binding to the AR in either cell line. The antiandrogen flutamide prevented 52% of the [³H]R1881 from binding to the LNCaP AR, and 51% of the labeled androgen from binding to the wild-type AR in PC-3AR cells. Interestingly, L-35, which was one of the most effective compounds at inhibiting the growth of LNCaP cells in vitro, was the least effective compound at preventing [³H]R1881from binding to either type of AR. In LNCaP cell cultures, L-35 prevented 34% of the [³H]R1881 from binding to the AR, and in PC-3AR cells, the inhibition was 31%. Each of the other 5 novel compounds were very effective at preventing [³H]R1881 from binding to the cellular AR. The pattern was similar regardless of whether they were tested against the mutated LNCaP AR or the wild-type AR. Moreover, they prevented [³H]R1881from binding to the ARs with higher efficiencies than flutamide. In the presence of L-2, L-10, L-36, L-37, and L-39, the amount of [³H]R1881 bound to the LNCaP cells was 30%, 5.2%, 0.7%, 15.2%, and 3.3%, respectively. In PC-3AR cells, the amount of [³H]R1881 bound in the presence of L-2, L-10, L-36, L-37, and L-39 was 13.5%, 2.6%, 5.1%, 11.7%, and 2.5%, respectively.

View larger version (32K): [in this window] [in a new window]   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 and reference drugs. LNCaP and PC-3AR cells were incubated with 5 nM [3H]R1881 and 5 µM of the compounds and reference drugs for 2 h at 37°C in serum-free medium, as described in "Materials and Methods." After washing and lysing, cell-associated radioactivity was determined by liquid scintillation counting. The amount of [3H]R1881 bound to the cells in the presence of compound or reference drug, is expressed as a percentage of the amount of [3H]R1881 that bound in the presence of vehicle. The results show the mean and SE from three separate experiments. *, P < 0.001 versus control; **, P < 0.01 versus control; , P < 0.05 versus control.

View larger version (17K): [in this window] [in a new window]   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 µM) 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. Values are expressed as mean and SE. A, *P < 0.001 versus control; **P < 0.01. B, *P < 0.01; **P < 0.05 versus control.

View larger version (18K): [in this window] [in a new window]   Fig. 6. The ability of the novel compounds and reference drugs to modulate DHT-induced luciferase activity mediated through the (A) 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 alone (5 nM), or a combination of DHT (5 nM) and the reference drugs or novel compounds (5 µM) 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. Values are expressed as mean and SE. A, *, P < 0.001 versus DHT; **, P < 0.01 versus DHT; , P < 0.05 versus DHT. B, *, P < 0.01 versus DHT; **, P < 0.05 versus DHT.

Growth Effects on LNCaP Tumors Grown in Male SCID Mice.
In the first experiment, the effects of L-2, L-35, and L-36 on tumor growth were determined, and orchidectomy and ketoconazole were used as the reference treatments. The mice were grouped 28 days after cell inoculation when measurable tumor volumes were approximately 500 mm³ (Fig. 7A) . After 21 days of treatment tumor volumes in the mice treated with L-2, L-36, and L-35 were similar to the castration group. However, by 28 days tumor volume in the control mice increased 4-fold over the 28 days of treatment, and tumor volume in the castrated mice increased by only 2-fold (52% reduction). None of the treatments, including ketoconazole, had any appreciable effect on the growth of the tumors after 28 days of treatment (Fig. 7A) . The reductions in tumor volumes in the mice treated with ketoconazole, L-2, L-35, and L-36 were 23%, 28%, 8%, and 12% respectively. These results were also reflected in the weights of the tumors. The only treatment that resulted in significantly (P < 0.01) smaller tumors was orchidectomy (Fig. 7B) . Tumor weight in the castrated mice was reduced by 62%. Mice that had been castrated also had significantly (P < 0.02) lower serum levels of PSA when compared with vehicle-treated animals (Table 2) . In this experiment, serum and tumor levels of testosterone were significantly reduced in the mice that were castrated. Although, serum testosterone levels in the mice treated with L-2, L-35, and L-36 were significantly lower compared with the vehicle-treated mice, tumor testosterone levels were unaffected.

View larger version (13K): [in this window] [in a new window]   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 removed and weighed. Castration significantly reduced tumor weights. *, P < 0.01 versus control.

View larger version (15K): [in this window] [in a new window]   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. 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 removed and weighed. Castration and L-39 significantly reduced tumor weights. *, P < 0.05 versus control.

View larger version (14K): [in this window] [in a new window]   Fig. 9. The effects of orchidectomy, finasteride, flutamide, and the novel compound 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 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 removed and weighed. Castration, flutamide, and L-39 significantly reduced tumor weights. *, P < 0.02 versus control.

	DISCUSSION

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As an initial approach, the effects of the compounds on the growth of hormone-dependent LNCaP cells were determined. Of the six novel compounds tested, three stimulated LNCaP cell growth (L-2, L-10, and low concentrations of L-36), two inhibited cell proliferation (L-35 and L-37), and one had no effect (L-39). The mitogenic effects of L-2 and L-10 were surprising because both of these compounds are pregnane derivatives and would not expected to be recognized as ligands by the LNCaP AR. However, the LNCaP AR contains a 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, 51, 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 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 C_17,20-lyase and 5-reductase and exhibits antiandrogenic properties, may be a suitable treatment option for such patients.

Prostate cancer cells with mutated AR respond to flutamide and hydroxyflutamide, the active metabolite of flutamide, with growth stimulation rather than growth inhibition. Our studies, in vitro and in 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 flutamide 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 investigators 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-µM 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 mediating these effects. L-35, L-37, and L-39 were also determined to be antagonists of both the wild-type AR and the LNCaP AR, indicating that they are more comparable with the second-generation antiandrogen bicalutamide than with flutamide.

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 experiment (data not shown), the growth of LNCaP prostate cancer xenografts in male SCID mice was determined to be hormone-dependent, and tumor weights were significantly lower in the animals that were castrated or treated with the 5-reductase-inhibitor finasteride. Flutamide was found to stimulate the growth of LNCaP tumor xenografts, but not to the same extent as DHT. Additionally, the maximum time of treatment was determined to be 28 days. After this time, tumors in the castrated group appeared to have acquired hormone-independence based on a determination of weekly serum PSA levels (data not shown).

In agreement with the results obtained in vitro, the androgenic compounds L-2, L-36, and L-10 had no appreciable effect on tumor growth measurements. L-10 was found to be as effective as finasteride at inhibiting tumor growth, and L-2 and L-36 significantly lowered serum levels of testosterone but not tumor testosterone levels. Therefore, it is likely that although the compounds are effective inhibitors of androgen synthesis, this is overshadowed by their androgenic properties, which results in LNCaP tumor growth. The inability of L-35 and L-37 to inhibit LNCaP tumor growth in vivo was especially disappointing. Both of these compounds were the most effective at inhibiting LNCaP cell growth in vitro, and they were both determined to be the most potent antiandrogens. We have recently determined that L-37 is an inhibitor of the liver cytochrome P450 enzymes 3-hydroxysteroid-oxidoreductase and 3ß-hydroxysteroid-oxidoreductase, which catalyze the metabolism of DHT to polar metabolites that are excreted in the urine (data not shown). Although we were unable to reliably measure serum levels of DHT in the serum of the animals because of cross-reactivity with testosterone and the novel compounds, the results (not shown) did suggest that there were elevated serum DHT levels in the animals treated with L-37. These elevated DHT levels may explain why L-37 had no effect on tumor growth in vivo. It should also be noted that L-37 does not inhibit the growth of PC-82 prostate cancer xenografts grown in athymic nude mice in vivo (data not shown). Currently, we do not know the reasons for the lack of growth inhibition observed with L-35. The inability of L-35 to inhibit LNCaP tumor growth may be attributed to its weak affinity for the AR. Alternatively, it may be attributable to the compound being converted to an inactive metabolite, poor uptake of the compound in the animals, or rapid clearance from the animals.

The most effective compound at inhibiting LNCaP tumor growth in vivo was L-39, which paralleled castration. Moreover, L-39 was also very effective at inhibiting the growth of hormone-dependent PC-82 prostate tumor xenografts in athymic nude mice. PC-82 prostate cancer cells expresses wild-type AR, and the cells cannot be grown in vitro. In these experiments, PC-82 tumors grew to a measurable size in approximately 12 weeks. The in vitro experiments had determined that L-39 was effective at inhibiting LNCaP cell growth in the presence of exogenous steroids, and that L-39 is extremely effective at preventing high affinity androgens from binding to the AR. However, when compared with L-35 and L-37, L-39 was not the most potent antiandrogen. We have recently reported on the pharmacokinetic profile of L-39 in normal (non-tumor-bearing) male mice (59) . L-39 (50 mg/kg s.c.) was shown to be cleared from the blood of the animals within 6 h of injection, and a nonpolar metabolite was detected in the serum. The metabolite has not yet been characterized, and it is not yet known whether it is contributing to the antitumor activities of L-39. It is also important to note that the multifunctional activities L-39 are most likely to explain the antitumor effects of this compound. L-39 is an inhibitor of androgen synthesis, and it is a potent antiandrogen when tested against both the mutated LNCaP AR and the wild-type AR. Although additional studies are required to determine the full potential of this compound, L-39 holds considerable promise as a treatment option for hormone dependent prostate cancer.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. John Isaacs (The John Hopkins School of Medicine, Baltimore, MD) for providing the doner PC-82 tumor xenografts, to Dr. Marco Marcelli (Baylor College of Medicine, Houston, TX) for providing PC-3 cells and PC-3 cells stably transfected with the human wild-type androgen receptor, to Dr. Elizabeth Wilson (University of North Carolina, Chapel Hill, NC) for providing the wild-type human AR-expressing vector and the LNCaP AR-expressing vector, to Dr. Hynda Kleinman (NIH, Bethesda, MD) for providing the Matrigel, and to Merck Research Laboratories (Rahway, NJ) for providing the finasteride.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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 Medical University, Beijing 100083, China.

³ To whom requests for reprints should be addressed, at University of Maryland, Department of Pharmacology and Experimental Therapeutics, 655 West Baltimore Street, Baltimore, MD 21201-1559.

⁴ The abbreviations used are: BPH, benign prostatic hyperplasia; DHT, dihydrotestosterone; C_17,20-lyase, cytochrome P450 17-hydroxylase/C_17,20-lyase; PSA, prostate-specific antigen; AR, androgen receptor; G418, geniticin; FBS, fetal bovine serum; DPBS, Dulbecco’s PBS; [³H]R1881, methyltrienolone [17-methyl-[³H]]-R1881; IMEM, improved minimum essential medium.

Received 5/ 5/00. Accepted 9/29/00.

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