BOMT (6 alpha-bromo-17 beta-hydroxy-17 alpha-methyl-4-oxa-5 alpha-androstan-3-one) is not an androgen antagonist within the central nervous system.

This study investigates the efficiency of BOMT as an androgen antagonist within the central nervous system. The efficiency of BOMT in suppressing neural receptor binding of testosterone, and the ability of this antiandrogen to block the feedback loop of testosterone onto the central nervous system, as evidenced by plasma testosterone levels, is reported. BOMT was found to be unable to open the feedback loop of testosterone onto the central nervous system, which was correlated with the low competing efficiency of this antiandrogen for receptor sites in vitro within the hypothalamic-preoptic area of the brain - a region known to be involved in gonadotrophin secretion. The observed divergence in the degree of antiandrogenicity of BOMT between peripheral and central target tissues of testosterone is discussed.     

Design of antiandrogens and their mechanisms of action: a case study (anandron)

The design of a new drug is conditioned by knowledge of the biochemical mechanisms involved in the etiology of the disease to be treated. With regard to endocrine pathologies, such knowledge can be obtained in the clinic from systematic assays of urinary and plasma hormones, enzyme activities and target tissue receptor concentrations. The present paper describes the results of our assays of plasma 3 alpha-androstanediol glucuronide, 5 alpha-reductase and androgen receptor in prostate cancer patients. The activity of the nonsteroid antiandrogen anandron is discussed in relation to these parameters: anandron may inhibit slightly adrenal androgen biosynthesis but, in particular, counters the action of these adrenal androgens on the prostate. It does not inhibit rat prostate 5 alpha-reductase activity but interacts with androgen receptor to exert an antiandrogen action.     

Cyclin E as a coactivator of the androgen receptor

Androgens play an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The enhancement of AR activity by cyclin E was resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E was shown to bind directly to the COOH terminus portion of the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute to persistent activation of AR function, even during androgen ablation therapy.     

A bioisosteric approach to the discovery of indole carbinol androgen receptor ligands

Two potential bioisosteres of the nonsteroidal antiandrogen bicalutamide, an imidazolidinone and an indole, were synthesized and tested for their androgen receptor binding. Indole was discovered to be a suitable bioisostere for the acyl anilide moiety in the parent compound. Several analogs in the indole series were found to be 10-fold better than bicalutamide in binding to the recombinant androgen receptor binding domain.     

Study of the impact of the T877A mutation on ligand‐induced helix‐12 positioning of the androgen receptor resulted in design and synthesis of novel antiandrogens

Antiandrogen flutamide, an antagonist of the wild‐type androgen receptor (AR), is used in the clinics for treating metastatic prostate cancer. However, the T877A mutated AR is paradoxically activated by hydroxyflutamide, an active form of flutamide. Despite of crystallographic studies, how the T877A mutation results in antagonist‐agonist conversion of hydroxyflutamide remains a puzzle. Here, started from a structural model of the apo form of AR ligand‐binding domain (AR‐LBD), we have investigated the impact of the T877A mutation on ligand‐induced helix‐12 positioning by replica‐exchange molecular dynamics (REMD) simulations with an unique protocol, which is capable of simulating the H12 dynamics and keeping the main body of AR‐LBD unchanged. Specifically, (i) we have computationally demonstrated that on the binding of hydroxyflutamide, the apo form of H12 rearranges into the agonistic form in the T877A mutant, but into the antagonistic forms in the wild‐type receptor, shedding light on hydroxyflutamide agonism/antagonism; (ii) By REMD simulations, we have predicted antiandrogen SC184 is a non‐agonist of the T877A mutant. This was confirmed by luciferase assays; and (iii) on the basis of the binding modes of hydroxyflutamide and SC184 from the simulations, we designed a novel flutamide derivative called SC333, which was subsequently predicted to be a pure antagonist of the T877A mutant. We then synthesized and experimentally confirmed SC333 is a pan‐antiandrogen effective against the wild‐type and the T877A and W741C mutated ARs, showing low micromolar cytotoxicity in LNCaP cells. Importantly, we demonstrated that distribution of the H12 conformations from REMD simulations is correlated with ligand agonist/antagonist activity. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens

The human prostate tumor cell line LNCaP containd an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor proteon and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induced reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.     

Molecular determinants of resistance to antiandrogen therapy

Using microarray-based profiling of isogenic prostate cancer xenograft models, we found that a modest increase in androgen receptor mRNA was the only change consistently associated with the development of resistance to antiandrogen therapy. This increase in androgen receptor mRNA and protein was both necessary and sufficient to convert prostate cancer growth from a hormone-sensitive to a hormone-refractory stage, and was dependent on a functional ligand-binding domain. Androgen receptor antagonists showed agonistic activity in cells with increased androgen receptor levels; this antagonist-agonist conversion was associated with alterations in the recruitment of coactivators and corepressors to the promoters of androgen receptor target genes. Increased levels of androgen receptor confer resistance to antiandrogens by amplifying signal output from low levels of residual ligand, and by altering the normal response to antagonists. These findings provide insight toward the design of new antiandrogens.     

The effects of various steroids on testosterone metabolism by the sexually mature rabbit epididymis

We examined the influences of steroids present in the epididymis on androgen metabolism by epididymal tissue and on the binding of androgen metabolites to the epididymal androgen receptor in castrated adult rabbit epididymides under in vitro conditions. The conversion of [3H]testosterone to [3H]17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT) and to [3H]5 alpha-androstane-3 alpha (beta), 17 beta-diol was inhibited by unlabeled steroids in the following manner progesterone greater than testosterone greater than estradiol. Unlabeled 5 alpha-DHT did not inhibit [3H]testosterone metabolism indicating that product inhibition is not an important regulatory event. The antiandrogen cyproterone acetate did not inhibit the formation of 5 alpha-reduced metabolites of [3H]testosterone. All of the compounds used inhibited androgen binding to the classically defined cytoplasmic and nuclear androgen receptor.     

Androgen deprivation therapy in the treatment of advanced prostate cancer

This article reviews the issues and controversies relevant to the treatment of advanced prostate cancer with androgen deprivation therapy. Initially, diethylstilbestrol was used for achieving androgen deprivation, but was replaced by luteinizing hormone-releasing hormone (LHRH). Adverse events associated with LHRH agonists include the flare phenomenon, hot flashes, loss of libido, erectile dysfunction, depression, muscle wasting, anemia, and osteoporosis. Intermittent therapy has been advocated to reduce morbidity of treatment. The addition of an antiandrogen provides maximum androgen blockade. There remains controversy regarding the timing of the addition of an antiandrogen. Secondary hormonal therapies include antiandrogens, adrenal androgen inhibitors, and estrogens.     

Androgen receptor antagonist suppresses exercise-induced hypertrophy of skeletal muscle

The physiological importance of the increase in androgen receptors in exercise-induced muscle hypertrophy was investigated in rats. Together with training rat gastrocnemius muscles by electrical stimulation every other day for 2 weeks, male rats were administered the androgen receptor antagonist, oxendolone. The androgen receptor antagonist effectively decreased the wet mass of the prostate, an androgen target organ, and did not significantly affect body mass. The increase in muscle mass induced by electrical stimulation was effectively suppressed by the androgen receptor blockade. The mean degree of muscle hypertrophy in the antagonist-treated group was significantly lower than that in the control group (102.30% vs 107.41%, respectively;P=0.006). This result suggests that the androgen pathway has a significant effect in exercise-induced muscle hypertrophy and emphasizes the importance of the increase in the number of androgen receptors in exercised muscle.     

A role for the androgen receptor in the endometrial antiproliferative effects of progesterone antagonists

In women and nonhuman primates, treatment with progesterone antagonists suppresses estrogen-dependent mitotic activity in the endometrial glands. This antiproliferative effect is paradoxical, because progesterone antagonists do not bind to the estrogen receptor (ER). While this phenomenon has been termed a "functional noncompetitive antiestrogenic effect," it does not occur in all species or in all regions of the primate reproductive tract, so is best referred to as an "endometrial antiproliferative effect." Recent studies of ours in both women and macaques revealed that the endometrial androgen receptor (AR) was increased by progesterone antagonist treatment. Because androgens are known to suppress estrogen-dependent endometrial proliferation, we hypothesized that the AR was involved in the antiproliferative effects induced by progesterone antagonists. In a test of this hypothesis, we administered the antiandrogen, flutamide, along with progesterone antagonists to ovariectomized, estrogen-treated macaques. Flutamide counteracted the suppressive effects of the progesterone antagonists on endometrial wet weight, thickness, stromal compaction, and mitotic index. Hyaline degeneration of the spiral arteries was also blocked by flutamide. These data implicate the AR as a functional component of the mechanism through which progesterone antagonists induce endometrial antiproliferative effects in the presence of estrogens.     

Effects of the Antiandrogen Flutamide on the Expression of Protein Kinase C Isoenzymes in LNCaP and PC3 Human Prostate Cancer Cells

Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (alpha, beta1, epsilon, zeta) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 microM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of alpha, beta1 and zeta, but not epsilon, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCbeta1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSS-cultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR.     

Androgen binding to cytosol prepared from epididymides of sexually mature castrated rabbits: evidence for a cytoplasmic receptor.

The presence of androgen-binding activity in cytosol prepared from the major anatomical segments (caput, corpus, and cauda) of the epididymis of castrated sexually mature rabbits has been demonstrated. A portion of this binding activity is likely to be the epididymal androgen receptor. When epididymal cytosol from adult castrated rabbits is analyzed on low-ionic strength (0.01 MKCl) sucrose gradients, two peaks of macromolecular binding could be detected, one congruent to 4.6S and one congruent to 8S. On gradients containing 1.0 M KCl, only one sedimenting form congruent to 4.6S could be demonstrated, suggesting that the 8S component is composed of aggregates. If cytosol was preincubated with labeled androgen, followed by an incubation with unlabeled androgen, and subsequently analyzed for binding on low-ionic strength gradients, only the congruent to 8S peak could be detected, indicating that most of the binding in the congruent to 4.6S region was rapidly dissociable. This suggests that binding in this region was to moieties other than receptor. Since androgen binding proteins (ABP) of testicular origin would have been cleared from the epididymis at the timepoints that we concentrated on for most of these studies, the 4.6S binding probably represents the association of androgen with plasma testosterone binding globulin (TeBG). The binding of androgen to the receptor can be inhibited by cyproterone, while this antiandrogen does not inhibit binding to either ABP or TeBG at the concentration used.     

A differential ligand-mediated response of green fluorescent protein-tagged androgen receptor in living prostate cancer and non-prostate cancer cell lines.

Androgen has been shown to promote the proliferation of prostate cancer through the action of the androgen receptor (AR). Mutation (T877A) of the AR gene found in an androgen-sensitive prostate cancer cell line, LNCaP, has been postulated to be involved in hypersensitivity and loss of specificity for androgen. In the present study, trafficking of AR and AR (T877A) in living prostate and non-prostate cancer cell lines under high and low concentrations of androgen and antiandrogen was investigated by tagging green fluorescent protein (GFP) to the receptors. In the presence of a high concentration of androgen, AR-GFP localized in the nucleus by forming discrete clusters in all cell lines. AR (T877A)-GFP was also translocated to the nucleus in LNCaP and COS-1 cells by the addition of a high concentration of androgen. In contrast, in the presence of a low concentration of androgen, the translocation of AR-GFP and AR (T877A)-GFP was observed in LNCaP cells, but not in COS-1 cells. Upon the addition of antiandrogen, AR-GFP was translocated to the nucleus but did not form subnuclear foci in both COS-1 and LNCaP cells, whereas AR (T877A)-GFP in both cells was translocated to the nucleus with subnuclear foci. The present study demonstrates the differential response of nuclear trafficking of AR and its mutant in prostate cancer cell lines and COS cells, and the subcellular and subnuclear compartmentalization provide important information on the sensitivity of the AR mutation.     

Studies on the mechanism of action of Win 49596: a steroidal androgen receptor antagonist

Win 49596 is an orally active antiandrogen in the rat. This report describes a series of in vitro and in vivo studies which were performed to characterize the mechanism of action of this compound. In vitro competition and Lineweaver-Burk analyses indicate that Win 49596 binds competitively to the rat ventral prostate androgen receptor with a Ki of 2.2 +/- 0.4 microM. Similar to other androgen antagonists, the relative binding affinity (RBA) of Win 49596 was greater after 1 h of incubation with androgen receptor than after an 18 h incubation (RBA of 2.2 versus 0.05, respectively). Win 49596 did not bind to rat cytosolic uterine estrogen or progesterone receptors or thymus glucocorticoid receptors. Furthermore, Win 49596 did not inhibit rat ventral prostate 5 alpha-reductase or 3 alpha-oxidoreductase, rat adrenal 3 beta-hydroxysteroid dehydrogenase or human placental aromatase activity in vitro at concentrations as high as 10 microM. A series of in vivo studies demonstrated that Win 49596 inhibited the uptake of [3H]testosterone as well as testosterone-induced nuclear accumulation of androgen receptor in the rat ventral prostate. Collectively, these results support direct androgen receptor antagonism as the mechanism for the antiandrogenic effects of Win 49596.