Prostate Androgen Receptor Research Articles

Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses.

On the basis of results of studies using high doses of estrogens, exposure to estrogen during fetal life is known to inhibit prostate development. However, it is recognized in endocrinology that low concentrations of a hormone can stimulate a tissue, while high concentrations can have the opposite effect. We report here that a 50% increase in free-serum estradiol in male mouse fetuses (released by a maternal Silastic estradiol implant) induced a 40% increase in the number of developing prostatic glands during fetal life; subsequently, in adulthood, the number of prostatic androgen receptors per cell was permanently increased by 2-fold, and the prostate was enlarged by 30% (due to hyperplasia) relative to untreated males. However, as the free serum estradiol concentration in male fetuses was increased from 2- to 8-fold, adult prostate weight decreased relative to males exposed to the 50% increase in estradiol. As a model for fetal exposure to man-made estrogens, pregnant mice were fed diethylstilbestrol (DES) from gestation days 11 to 17. Relative to controls, DES doses of 0.02, 0.2, and 2.0 ng per g of body weight per day increased adult prostate weight, whereas a 200-ng-per-g dose decreased adult prostate weight in male offspring. Our findings suggest that a small increase in estrogen may modulate the action of androgen in regulating prostate differentiation, resulting in a permanent increase in prostatic androgen receptors and prostate size. For both estradiol and DES, prostate weight first increased then decreased with dose, resulting in an inverted-U dose-response relationship.

Autoregulation of androgen receptor in rat ventral prostate: involvement of c-fos as a negative regulator

Recent work from our laboratory has focused on elucidating the mechanism of androgen regulation of the androgen receptor (AR). We have demonstrated that testosterone increases AR protein and binding within 1 h in the ventral prostate of adult rats castrated for 24 h. Cycloheximide administered with testosterone reduces AR and AR mRNA levels. AP-1/c-fos transcription factor has been shown to function as a negative repressor in many systems. c-fos mRNA levels were decreased 1 h after testosterone treatment in the ventral prostate, whereas they were increased in cycloheximide alone or cycloheximide-testosterone treated groups as compared to vehicle control. c-fos protein was also increased in the testosterone-cycloheximide treated group as compared to testosterone alone or cycloheximide alone groups at 1 h. By 3 h, the tissue recovers from the inhibitory effect of cycloheximide as evidenced by restoration of AR and an increase in AR mRNA levels. At this time c-fos protein levels were reduced after treatment with cycloheximide and testosterone and c-fos mRNA levels were comparable to the controls. These results suggest that elevation of c-fos expression is associated with a decrease in AR and AR mRNA and provide correlative data supporting negative repression by c-fos on androgen receptor levels. Between the age of 25–85 days, serum testosterone levels reached adult levels by the age of 55 days. Steady-state AR mRNA levels increased significantly by the age of 85 days while c-fos mRNA levels remained at low baseline levels at all ages. Thus, in addition to the circulating levels of serum testosterone, other age related factors are also involved in the regulation of AR mRNA levels. Furthermore, androgens appear to maintain androgen receptor levels and androgen sensitivity by continuous suppression of the repressor. c-fos.

Androgens are not major down-regulators of androgen receptor levels during growth of the immature rat penis

This study was undertaken to investigate the prevalent hypothesis that androgens are responsible for the organ-specific down-regulation of penile androgen receptors (ARs) and decline of penile growth in the rat during sexual maturation. Sexually immature male rats (21 days old) were castrated and treated for 3 days (“short-term”), with high doses of: (a) testosterone and the α-reductase inhibitor finasteride ( T F ); (b) dihydrotestosterone (DHT); or (c) finasteride alone (F). Intact and castrate controls received vehicle only. PolyA + RNA was analysed by Northern blot hybridization and ARs were estimated in the penis and ventral prostates by (3-H)R-1881 binding in the cytosol. Short-term castration, with or without F, increased penile AR mRNA, whereas high doses of T F and DHT reduced it considerably. Although penile cytosol AR concentration in the control castrates, with or without F, paralleled the AR mRNA rise, treatment with androgens left cytosol AR content per organ and AR concentration above those of the intact rat penis despite the drop in AR mRNA. A “long-term” treatment (10 days) on 19-day-old rats with either medium or high doses of T F and DHT also failed to down-regulate penile cytosol ARs below the intact controls. Western blot analysis of penile cytosol AR levels confirmed these results. Block of pituitary FSH and LH release by a GnRH antagonist in castrates receiving T F or DHT at high doses did not modify the response. In the case of intact rats, high doses of T F or DHT actually increased penile cytosol AR content. No difference was observed between T F and DHT effects. In contrast to what occurs during sexual maturation, the prostate ARs and growth rate responded to all treatments in a similar way to what was observed in the penis. Our results suggest that increases in serum T or DHT are not major factors in the physiological down-regulation of ARs and androgen-dependent growth in the rat corpora cavernosa.

The Development of Casodex™ (Bicalutamide): Preclinical Studies

Casodex (bicalutamide, Zeneca Limited) was developed for the treatment of prostate cancer from a series of nonsteroidal compounds related to flutamide. Casodex is a selective antiandrogen that binds to rat, dog and human prostate androgen receptors, and has approximately a 4-fold higher affinity for the rat androgen receptor than hydroxyflutamide, the active metabolite of flutamide. Casodex also binds to androgen receptors found in the LNCaP human prostate tumour and the Shionogi S115 mouse mammary tumour cell line, as well as androgen receptors transfected into CV-1 and HeLa cells. In all cases, Casodex behaves as a 'pure' antiandrogen and inhibits gene expression and cell growth stimulated by androgens. Studies in vivo show that Casodex is a potent antiandrogen in the rat. In contrast to flutamide, which produces dose-related, marked increases in serum luteinising hormone (LH) and testosterone, Casodex has little effect on serum LH and testosterone; that is, it is peripherally selective. The peripheral selectivity of Casodex has now been shown to be due to poor penetration across the blood-brain barrier. In dogs, Casodex has exquisite potency and causes dose-related atrophy of the prostate gland and epididymis; with an oral ED50 of 0.1 mg/kg, it is about 50 times as potent as flutamide in this species. Casodex is also peripherally selective in the dog. In addition, magnetic resonance imaging studies have shown that Casodex is a potent antiandrogen in the monkey. Casodex, at a daily oral dose of 25 mg/kg effected a highly significant reduction in the growth of Dunning R3327H transplantable rat prostate tumours that was equivalent to that achieved by either surgical or medical castration with the LH-releasing hormone agonist Zoladex (goserelin). In a comparative study, flutamide was shown to be both less potent and less active than Casodex. In these preclinical studies, Casodex was well tolerated. The preclinical properties of Casodex give it advantages, with respect to potency, tolerability and the maintenance of effective antiandrogen serum concentrations, over other available antiandrogens. Moreover, it has a half-life that is compatible with once-daily administration.

Distribution of Androgen Receptor‐Like Immunoreactivity in the Brains of Cynomolgus Monkeys

A polyclonal antibody, PA1, raised in a rabbit against fusion proteins containing fragments of the human prostatic androgen receptor (AR) was used to map the distribution of AR-like immunoreactivity in the brains of adult male and female cynomolgus monkeys. PA1 AR-immunoreactive (ARir) labeling occurred in the cell nuclei and, more weakly, in the cytoplasm of brain cells. The PA1 ARir labeling occurred primarily in brain regions previously shown on the basis of gonadal steroid autoradiography to contain androgen receptors. However, the distribution of PA1 ARir staining was substantially more restricted than that of autoradiographic labeling using 3H-androgens. The pattern of PA1 ARir labeling was closely similar between animals and occurred in the lateral septum, medial preoptic area, bed nucleus of stria terminalis, anterior, cortical, accessory basal and medial amygdala, several hypothalamic nuclei including the supraoptic, anterior, paraventricular, ventromedial and arcuate nuclei, and the premammillary nucleus. No significant sex differences were observed. With the exception of the supraoptic nucleus, reported not to be labeled by autoradiography, earlier autoradiographic findings and the current immunocytochemical results, although not congruent, have noteworthy similarities.

Cloning and partial sequence of the rabbit androgen receptor: expression in fetal urogenital tissues.

Genetic studies indicate that the actions of testosterone and dihydrotestosterone are mediated by a single androgen receptor (AR). The basis for the differential effects of these hormones during development is not known. To address this problem, we have turned to the rabbit, a species in which the endocrine and metabolic events in male sexual differentiation have been studied in detail. As a first step, we have cloned a partial cDNA encoding the rabbit prostate AR and have analyzed the expression of the cDNA in tissues of rabbit embryos at the time during which male phenotypic differentiation takes place. The coding sequence of the rabbit AR cDNA reveals a high degree of conservation with the sequences of the human, mouse, and rat ARs. By Northern analysis the principal transcript expressed in the mesonephros and the anlage of the external genitalia on gestation day 18 appeared to be identical to the mRNA expressed in adult prostate and epididymis.

The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen

Brief administration of estrogen to newborn rats permanently imprints adult prostatic androgen receptor (AR) expression in a lobe-specific manner. To delineate this effect, we examined the immediate effects of early estrogen exposure on the changing AR pattern in the developing ventral, dorsal, and lateral prostate lobes. Antibodies against rat AR (PG-21) were used in combination with several antibodies to cell-specific antigens for positive cellular identification by immunocytochemistry. At birth, mesenchymal cells of the ventral prostate were strongly AR positive (AR+). Epithelial cells stained only for basal cell cytokeratins and, in contrast to earlier reports, many were AR+ on day 1. Between days 3-5, periductal mesenchymal cells differentiated into smooth muscle cells which retained strong AR+ staining, whereas interductal fibroblasts exhibited a decreased incidence of AR+ cells. Between days 5-10, luminal epithelial cells first appeared, and a striking increase in AR staining intensity was noted relative to that in the basal cells. During puberty, basal cells lost their AR immunoreactivity. Similar changes were observed in the dorsal and lateral lobes. Newborn rats were given 25 micrograms estradiol benzoate on days 1, 3, and 5 and were killed thereafter. By day 6, AR staining was markedly decreased to a weak to moderate intensity in all cell types, and by day 10, AR was virtually absent in the separate lobes. Growth and epithelial cytodifferentiation were significantly retarded. Between days 15-30, evidence of luminal cell cytodifferentiation was noted; however, this was frequently not associated with an increase in AR staining. In the ventral and dorsal lobes, a continuous peripheral layer of AR-negative basal cells surrounded the ducts in the central and proximal regions, and this was associated with a permanent inability of luminal epithelial cells to express AR. Epithelial and smooth muscle AR expression was observed only in the distal tips. In contrast, AR expression rapidly returned in all regions of the lateral lobes, except the proximal ducts. We conclude that 1) basal epithelial cells express AR as early as day 1 of life and should be considered as possible direct targets of androgen action during prostate morphogenesis; 2) differentiation into luminal cells is associated with an increase, rather than an induction, of AR expression; and 3) periductal smooth muscle cells retain strong AR expression throughout development and should be considered primary targets for androgen-mediated morphogenesis. Neonatal estrogen initially down-regulates AR expression in all cells of three lobes, which may explain the overall growth retardation.

The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen.

Brief administration of estrogen to newborn rats permanently imprints adult prostatic androgen receptor (AR) expression in a lobe-specific manner. To delineate this effect, we examined the immediate effects of early estrogen exposure on the changing AR pattern in the developing ventral, dorsal, and lateral prostate lobes. Antibodies against rat AR (PG-21) were used in combination with several antibodies to cell-specific antigens for positive cellular identification by immunocytochemistry. At birth, mesenchymal cells of the ventral prostate were strongly AR positive (AR+). Epithelial cells stained only for basal cell cytokeratins and, in contrast to earlier reports, many were AR+ on day 1. Between days 3-5, periductal mesenchymal cells differentiated into smooth muscle cells which retained strong AR+ staining, whereas interductal fibroblasts exhibited a decreased incidence of AR+ cells. Between days 5-10, luminal epithelial cells first appeared, and a striking increase in AR staining intensity was noted relative to that in the basal cells. During puberty, basal cells lost their AR immunoreactivity. Similar changes were observed in the dorsal and lateral lobes. Newborn rats were given 25 micrograms estradiol benzoate on days 1, 3, and 5 and were killed thereafter. By day 6, AR staining was markedly decreased to a weak to moderate intensity in all cell types, and by day 10, AR was virtually absent in the separate lobes. Growth and epithelial cytodifferentiation were significantly retarded. Between days 15-30, evidence of luminal cell cytodifferentiation was noted; however, this was frequently not associated with an increase in AR staining. In the ventral and dorsal lobes, a continuous peripheral layer of AR-negative basal cells surrounded the ducts in the central and proximal regions, and this was associated with a permanent inability of luminal epithelial cells to express AR. Epithelial and smooth muscle AR expression was observed only in the distal tips. In contrast, AR expression rapidly returned in all regions of the lateral lobes, except the proximal ducts. We conclude that 1) basal epithelial cells express AR as early as day 1 of life and should be considered as possible direct targets of androgen action during prostate morphogenesis; 2) differentiation into luminal cells is associated with an increase, rather than an induction, of AR expression; and 3) periductal smooth muscle cells retain strong AR expression throughout development and should be considered primary targets for androgen-mediated morphogenesis. Neonatal estrogen initially down-regulates AR expression in all cells of three lobes, which may explain the overall growth retardation.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen receptor affinity of 5'-acyl furanosteroids.

Syntheses of 5'-acyl furanosteroids are described from the corresponding unsubstituted [3,2-b]furanosteroids using acid anhydrides and acid chlorides in the presence or absence of Lewis acids. New methods have been developed to prepare 5'-acetyl derivatives: reduction of a 5'-trichloroacetyl intermediate either by sodium formaldehyde sulfoxylate or with 10% Pd/C. Most of these 5'-acyl derivatives bind to the rat ventral prostate androgen receptor. However the antiandrogenic activity was diminished when compared with 4,5'-methylsulfonyl furanosteroid. Biological studies revealed that 5'-acyl furanosteroids were either androgens or modest antiandrogens. The electrostatic potential maps of the substructures of 3, 4, and 5'-acetyl syn- and anti-furanosteroids showed striking differences which may explain, to some extent, the lack of significant antiandrogenic activity of 5'-acyl furanosteroids.

The Correlation of Serum Luteinizing Hormone Levels with the Induction of Leydig Cell Tumors in Rats by Oxolinic Acid

Studies were performed to examine the mechanism by which testicular Leydig cell tumors are induced in rats by administration of the antimicrobial agent oxolinic acid (1-ethyl-1,4-dihydro-6,7-methylenedioxy-4-oxo-3-quinolinecarboxylic acid). In these studies, the effects of oxolinic acid on serum levels of luteinizing hormone (LH), testosterone, and prolactin and the binding of testosterone to prostatic androgen receptors were examined. In a long-term hormonal study, male Wistar rats were fed a diet containing oxolinic acid at 0, 100, 1000, or 3000 ppm for 104 weeks. A statistically significant increase in serum LH levels was observed at 1000 and 3000 ppm, but no dose of oxolinic acid had a significant effect on serum testosterone levels. Serum LH levels were no longer elevated above control levels within 2 weeks of cessation of the administration of oxolinic acid. Oxolinic acid was found to have no effect on the rate of clearance of exogenous LH from the circulation. Serum prolactin levels were decreased by the administration of oxolinic acid. The increase in serum LH induced by oxolinic acid was completely blocked by the intraperitoneal injection of the dopamine antagonist haloperidol (2 mg/kg). In addition, no significant affinity of oxolinic acid for androgen receptors was found in an in vitro study. These findings suggest that: (1) oxolinic acid induces Leydig cell tumors in rats by chronically stimulating the release of LH from the pituitary, (2) the mechanism of stimulating the release of LH involves facilitation of the dopaminergic systems in the hypothalamus-pituitary axis, and (3) oxolinic acid has no effect on androgen-mediated feedback inhibition.

Androgen Receptor Function During Undernutrition

Hypothalamo-pituitary inhibition of reproductive function during undernutrition is well known, however, the physiological mechanisms leading to suppression of gonadotrophin secretion are not clear. A variety of studies have indicated that testicular negative feedback on LH secretion is enhanced during food restriction. To evaluate directly the suppression by endogenous androgens on hypothalamic GnRH pulse generator activity during food restriction and examine the mechanism underlying the increased testicular steroidal feedback, we examined (1) circulating bioactive LH (bLH) levels in response to selective cerebral androgen blockade by intraventricular administration of an androgen receptor antagonist (hydroxyflutamide, SCH 16423) and (2) the binding capacity and affinity of androgen receptors in medio-basal hypothalamus, pituitary and prostate during undernutrition of intact mature male rats. Hydroxyflutamide (20 micrograms in 10 microliters vehicle), but not vehicle alone, markedly increased bLH levels in both food restricted and ad-lib fed rats. However, the faster (geometric mean 11.4 vs 27.7 min) and greater (47.2 vs 21.9 ng/ml) increase in bLH level in food restricted compared with ad-lib fed controls demonstrates an enhanced sensitivity to blockade of androgenic negative feedback during undernutrition. Food restriction increased androgen receptor binding capacity in pituitary (3.36 vs 0.77 fmol/mg protein) but not in medio-basal hypothalamus or prostate while binding affinity was unchanged by undernutrition in all 3 tissues. These studies reveal that undernutrition both enhances tonic, androgen receptor-mediated feedback suppression of GnRH secretion and increases in pituitary (but not hypothalamic) androgen receptor numbers to cause inhibition of LH secretion.

Effects of antiandrogen TZP-4238 on the prostatic androgen receptor and prostatic androgen in rat

TZP-4238, a new steroidal antiandrogen, is an orally active drug in rats and dogs. The effects of TZP-4238 on the androgen receptor and androgen content were examined in comparison with other antiandrogens in rats. The prostate DHT levels decreased markedly within 4-8 hr after a single oral administration of TZP-4238 8mg/kg. The prostatic testosterone concentrations fell below the detection limit (5pg/g of tissue) at 4-12 hr after the initiation of treatment. The plasma testosterone level also fell to 60% of the control level after 4-8 hr and then returned to the normal range. Eight percent of DHT present in normal prostatic tissue was located in the nuclear fraction. TZP-4238 reduced the concentration of DHT in nuclei to 50% of the normal level. The concentration of the plasma drug which induced a 50% decrease in the prostatic DHT, the IC50, was about 10ng/ml, while the IC50 value for plasma testosterone was 30-50ng/ml. After oral administration of 15-OH TZP-4238, the main metabolite, the level of plasma testosterone was significantly elevated above the control level. The androgen receptor level was markedly reduced at 24 hr following castration and returned to the normal range within 5 hr of a single injection of testosterone. TZP-4238 reduced the nuclear androgen receptor level to 60% at 24 hr after a single oral dose and then, the receptor content returned to its original level. Both 15-OH TZP-4238 and cyproterone acetate also reduced the androgen receptor and DHT contents to 50%. A series of in vivo studies demonstrated that TZP-4238 inhibited the uptake of testosterone and the decrease of DHT and testosterone, and decreased the nuclear androgen receptor in the rat prostate.

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work, the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nM of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease S1 analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcription 96 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to cells results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

Effects of neonatal estrogen exposure on prostatic secretory genes and their correlation with androgen receptor expression in the separate prostate lobes of the adult rat.

Brief administration of estrogen to newborn rats permanently restricts prostatic growth and testosterone sensitivity in adulthood. Previous work demonstrated that neonatal exposure to estradiol benzoate produced lobe-specific imprints in prostatic androgen receptor (AR) expression. Epithelial cell AR was markedly reduced or absent in the adult ventral and dorsal lobes, which correlated with a lack of epithelial differentiation and responsiveness. While the lateral lobe also showed reduced growth and testosterone responsiveness after neonatal estradiol benzoate, normal cell differentiation and AR levels were observed within the adult epithelium. To determine the impact that these receptor imprints have on the functional capacity of adult tissue, we herein examined the expression of lobe-specific, androgen-dependent, or androgen-responsive secretory genes in prostates of rats given neonatal estradiol benzoate and directly compared this with epithelial cell AR using histological techniques. Sprague-Dawley rat pups were given 25 micrograms estradiol benzoate or oil on days 1, 3, and 5 and killed on day 90. Prostatic mRNA was analyzed using Northern blots and in situ hybridization. Ventral lobe mRNA was hybridized with a prostate binding protein (PBP) cDNA probe, while lateral and dorsal mRNA were hybridized with RWB (seminal vesicle secretory protein or SVS-II), probasin, and DP1 cDNA probes. Sections adjacent to those used for in situ hybridization were stained for AR by immunocytochemistry. Neonatal estradiol benzoate significantly reduced ventral lobe PBP message on Northern blots, and this was not restored with adult testosterone administration. There was a direct correlation between epithelial cell AR and PBP expression, in that PBP message and protein were only present in epithelial AR-positive cells and were absent in all AR-negative epithelium. In the lateral prostate, probasin expression was unaffected by neonatal estradiol benzoate, whereas RWB was slightly reduced using Northern analysis. By in situ hybridization, these messages were observed at normal levels in lateral lobe epithelial cells of estrogenized rats, which directly correlated with the presence of AR in those cells. In the dorsal prostate, different response patterns to neonatal estradiol benzoate were found for the three secretory genes analyzed. On Northern blots, DP1 message significantly declined, probasin mRNA was modestly suppressed, and RWB expression was significantly elevated compared to those in control tissue. In situ hybridization revealed that RWB expression in estrogenized dorsal lobes was amplified in AR-positive epithelial cells, whereas AR-negative cells appeared unaltered. In summary, prostatic functional activity after neonatal estradiol benzoate exposure is affected in a lobe-specific manner, which correlates with the AR imprints in the separate lobes.

LY207320 (6‐methylene‐4‐pregnene‐3,20‐dione) inhibits testosterone biosynthesis, androgen uptake, 5α‐reductase, and produces prostatic regression in male rats

LY207320 is an in vitro inhibitor (estimated IC50 = 0.06 microM) of steroid 5 alpha-reductase that catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT). In contrast, LY207320 was only moderately active against rat prostatic 5 alpha-reductase in vivo (32% inhibition at 50.0 mg/kg single dose). LY207320 did, however, inhibit the in vivo uptake of [3H]-T by the prostate. The antiprostatic and endocrine effects of this agent were evaluated following daily (21 days) administration to castrated, androgen-supplemented castrate, and intact rats. LY207320, which has modest progestational competitive binding activity, does not bind to rat prostatic androgen or uterine estrogen cytosolic receptors. In the castrated male rat, subcutaneously (s.c.) administered LY207320 had no androgen agonist activity, as evidenced by a lack of accessory sex organ weight gains. Administration of s.c. LY207320 to intact rats for 21 days at doses greater than 5.0 mg/kg-day produced significant (P < 0.05) reductions of seminal vesicle and ventral prostatic weights (maximal regression = -65% and -40% from control values, respectively at 50.0 mg/kg-day). The compound had no regressive activity on male accessory sex organs when administered orally. LY207320 did not alter circulating prolactin, LH, or corticosterone levels, but at high doses (> or = 50.0 mg/kg-day), lowered circulating T[-67% from intact control levels (P < 0.05)]. Histological analysis of the rat ventral prostates (RVPs) in LY207320-treated rats was consistent with an androgen-deprived state. Decreased circulating androgens and prostatic regression are associated with inhibition of testicular 17 alpha-hydroxy/C17,20-lyase enzyme activity (IC50 = 0.06 microM). These findings support the contention that LY207320 is a physiological antagonist of androgen action in male rats, and that its effects are mediated primarily through inhibition of testicular androgen production rather than accessory sex organ 5 alpha-reductase.

Endocrine and antiprostatic effects of raloxifene (LY156758) in the male rat

The benzothiophene anti-estrogen, raloxifene [LY156758; (6-hydroxy-2-(4-hydroxyphenyl) benzo(b)thien-3-yl)(4-(2-1-piperidinyl)ethoxy)phenyl methanone hydrochloride] has selective estrogen pharmacological antagonist activity in female rats. The present studies were done in the male rat to assess activity of raloxifene related to inhibition of prostatic growth and effects on the hypothalamic-pituitary-gonadal axis. Raloxifene did not compete for binding of the androgen, [3H]-methyltrienolone (R1881) in cytosolic extracts of ventral prostate. Similarly, the compound did not inhibit prostatic 5 alpha-reductase or testicular 17 alpha-hydroxy/C17,20-lyase activities. Raloxifene had no effect on the ventral prostatic uptake of [3H]-R1881 in vivo. Administration of estradiol to castrated male rats stimulated fourfold increases of in vitro ventral prostatic binding of [3H]-R1881. Raloxifene was devoid of agonist activity in castrated animals, because the compound had no stimulatory effect on prostatic androgen receptor binding activity. When raloxifene was coadministered with estradiol, the compound markedly antagonized the estrogen-induced increase of prostatic [3H]-R1881 binding, confirming its antiestrogenic properties in male rats. Serum prolactin was also elevated significantly (P < 0.05) with a single injection of raloxifene (20.0 mg/kg). In these same animals, serum FSH was significantly (P < 0.05) decreased by one dose (10.0 mg/kg) of the compound. Luteinizing hormone levels in castrated male rats were unaffected by raloxifene administration. Raloxifene treatment of castrated males significantly (P < 0.05) antagonized the stimulatory response of the ventral prostate (VP) to exogenous androgens in a dose-dependent manner. Raloxifene treatment of intact male rats for 14 and 28 days produced significant (P < 0.05) dose-dependent regression of the VP and seminal vesicles (SV). The VP regressive responses to raloxifene were associated with a decline in serum testosterone levels. Histological analysis of the VPs in raloxifene-treated rats was consistent with an androgen-deprived state. These findings support the contention that raloxifene is a pure estrogen antagonist and a physiological antagonist of androgen action in male rats. These pharmacological properties provide support for further structure-activity and mechanistic investigations with benzothiophenes in the medical management of prostatic neoplasia.

Comparison of finasteride (proscar®), a 5α reductase inhibitor, and various commercial plant extracts in in vitro and in vivo 5α reductase inhibition

Human prostate was used as a source of 5 alpha reductase. Compounds were incubated with an enzyme preparation and [3H]testosterone. [3H]-dihydrotestosterone production was measured to calculate 5 alpha reductase activity. IC50 values (ng/ml) were finasteride = 1; Permixon = 5,600; Talso = 7,000; Strogen Forte = 31,000; Prostagutt = 40,000; and Tadenan = 63,000. Bazoton and Harzol had no activity at concentrations up to 500,000 ng/ml. In castrate rats stimulated with testosterone (T) or dihydrotestosterone (DHT), finasteride, but not Permixon or Bazoton, inhibited T stimulated prostate growth, while none of the three compounds inhibited DHT stimulated growth. These results demonstrate that finasteride inhibits 5 alpha reductase, while Permixon and Bazoton have neither anti-androgen nor 5 alpha reductase inhibitory activity. In addition, in a 7 day human clinical trial, finasteride, but not Permixon or placebo, decreased serum DHT in men, further confirming the lack of 5 alpha reductase inhibition by Permixon. Finasteride and the plant extracts listed above do not inhibit the binding of DHT to the rat prostatic androgen receptor (concentrations to 100 micrograms/ml). Based on these results, it is unlikely that these plant extracts would shrink the prostate by inhibiting androgen action or 5 alpha reductase.

Effect of testosterone deprivation on expression of the androgen receptor in rat prostate, epididymis and testis

Adult rats were treated with ethane dimethane sulphonate (EDS) to eliminate the Leydig cells. This treatment resulted in very low levels of testosterone in the blood and in the testis. Furthermore, histological evaluation of spermatogenesis showed no marked differences between control and EDS-treated animals. In the ventral prostate, 5 days after EDS-treatment, a 4.0 +/- 0.3-fold up-regulation of androgen receptor (AR) mRNA was observed, together with a 2.2 +/- 0.2-fold increase in actin mRNA. In the epididymis, a 2.0 +/- 0.5-fold increase in AR mRNA level was observed, without a change in actin mRNA level. In the testes of EDS-treated rats, the AR mRNA level was not changed (1.02 +/- 0.17-fold of controls), and there was also no change in actin mRNA level at 5 days after EDS-treatment. These results indicate that AR mRNA expression in the ventral prostate and epididymis is regulated differentially by testosterone when compared to regulation in the testis. Testicular androgen binding sites were assayed by Scatchard analysis of the binding of 3H-R1881 to a nuclear fraction, that was isolated by a method which involved the use of liquid nitrogen and high sucrose buffer. The number of specific binding sites per testis in EDS-treated rats with testosterone-implants, remained unaltered compared to control rats (9.1 +/- 1.4 pmol/testis). In these rats, 20% of the normal testicular testosterone level was sufficient to maintain the androgen receptor in a tight nuclear binding (transformed) form. In testes from EDS-treated rats without testosterone-implants, the AR did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals was close to control levels, as measured by nuclear 3H-R1881 binding after receptor transformation through injection of a high dose of testosterone (10 mg) 2 h before killing the rats (testosterone pulse). In the different experimental groups, FSH was not required to maintain the total testicular AR content (ligand binding). Immunoprecipitation and Western blotting of the testicular AR using specific monoclonal and polyclonal antibodies indicated that the total testicular amount of immunodetectable AR protein in long-term testosterone deprived rats was very low when compared to that in control rats or rats with testosterone-implants. This is in disagreement with results obtained in the ligand binding assay, and may point to a structural modification of the AR in the testis that possibly occurs in the prolonged absence of androgens.

Subcellular location of androgen receptor in rat prostate, seminal vesicle and human osteosarcoma MG-63 cells

Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.

17β-acylurea derivatives of 4-azasteroids as inhibitors of testosterone 5α-reductase

A series of 17β-acylurea-4-aza-5α-androstan-3-one derivatives has been assayed in vitro as inhibitors of testosterone 5α-reductase, using the particulate fraction of human hyperplastic prostate and rat prostate as enzyme sources. The most active derivatives were 1-[4-methyl-3-oxo-4-aza-5α-androstane-17β-carbonyl]-1,3-dicyclohexylurea (compound 1) and 1-[4-methyl-3-oxo-4-aza-5α-androstane-17β-carbonyl]-1,3-diisopropylurea (compound 3) which demonstrated IC 50 values of 41 and 55 nM for the human enzyme and of 83 and 53 nM for the rat enzyme, respectively. Neither compound showed any relevant binding affinity to the rat prostate androgen receptor (IC 50 of ≈ 100 and 84 μM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, compound 3 (laboratory code FCE 26073), at 3 mg/kg/day, significantly decreased the ventral prostate growth promoting effect of TP by 40–50%, whereas compound 1 was ineffective up to the dose of 10 mg/kg/day.