Inhibition of T-tropic HIV strains by selective antagonization of the chemokine receptor CXCR4

Mutations in the androgen receptor (AR), that alter steroid hormone specificity have been identified in a series of androgen-independent prostate cancers. To address the functional properties of these mutant ARs that may have contributed to their selection in vivo, responses to a series of steroid hormones and antiandrogens were assessed. CV-1 cells were cotransfected with wild-type or mutant ARs and a luciferase reporter plasmid regulated by an androgen-responsive element. Dose-response curves were analyzed for 5alpha-dihydrotestosterone, the most active androgen in normal prostate, and androstenedione, a major androgen derived from the adrenals. Although the mutant ARs responded to both of these steroids, the responses were equivalent to or less than the wild-type AR. In contrast, responses to flutamide, a competitive antagonist of the wild-type AR, were markedly increased by three of the mutations. Similar responses were observed with a second antiandrogen, nilutamide. Bicalutamide, another antiandrogen related to flutamide, remained an antagonist for these mutant ARs. Finally, flutamide was observed to be a weak partial agonist of the wild-type AR in this system. These results indicate that flutamide used in conjunction with androgen ablation therapy for prostate cancer may select for tumor cells with flutamide-inducible ARs.     

Treatment of metastatic prostate cancer. Lessons from the androgen receptor

The exquisite hormonal dependence of prostate cancer continues to provide an opportunity and a challenge for oncologists. It is clear that future efforts in the laboratory should include determining the frequency and spectrum of AR mutations in AI prostate cancer, the development of more effective antiandrogens, and understanding in greater detail how the AR stimulates the growth of prostate cancers. These efforts may eventually lead to treatments that greatly reduce any stimulatory effects of the AR on prostate cells, possibly resulting in a significant improvement in disease-free survival and, perhaps in conjunction with other modalities, cure of some earlier stages of disease. And even for patients with advanced disease, because hormonal therapy is generally fairly well tolerated even in the typically older prostate cancer patient, defining the contribution of AR-mediated growth to AI disease will be critically important.     

Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines

Androgen plays a critical role in regulating the growth and differentiation of normal prostate epithelia, as well as the initial growth of prostate cancer cells. Nevertheless, prostate carcinomas eventually become androgen-unresponsive, and the cancer is refractory to hormonal therapy. To gain insight into the mechanism involved in this hormone-refractory phenomenon, we have examined the potential role of the androgen receptor (AR) in that process. We have investigated the expression of AR and two prostate-specific androgen-responsive antigens, prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), for the functional activity of AR in LNCaP and PC-3 human prostate carcinoma cells. Our results are as follows. (i) Clone 33 LNCaP cells express AR, PAcP, and PSA, and cell growth is stimulated by 5alpha-dihydrotestosterone (DHT). Stimulation of cell growth correlates with decreased cellular PAcP activity. (ii) In clone 81 LNCaP cells, the expression of PAcP decreases with a concurrent decrease in the degree of androgen stimulation of cell growth, whereas the expression of PSA mRNA level is up-regulated by DHT, as in clone 33 cells. Conversely, in PAcP cDNA-transfected clone 81 cells, an additional expression of cellular PAcP correlates with an increased stimulation by androgen, higher than the corresponding control cells. (iii) PC-3 cells express a low level of functional AR with no detectable PAcP or PSA, and the growth of PC-3 cells is not affected by DHT treatment. Nevertheless, in two PAcP cDNA-transfected PC-3 sublines, the expression of exogenous cellular PAcP correlates with androgen stimulation. This androgen stimulation of cell growth concurs with an increased tyrosine phosphorylation of a phosphoprotein of 185 kDa. In summary, the data indicate that the expression of AR alone is not sufficient for androgen stimulation of cell growth. Furthermore, in AR-expressing prostate cancer cells, the expression of cellular PAcP correlates with androgen stimulation of cell proliferation.     

Determinants of plasma homocysteine

Prostate cancer remains the most commonly diagnosed cancer in American males and is the second leading cause of cancer death in this group. Hydrazine sulfate, an inhibitor of gluconeogenesis, has been proposed as a means to improve nutritional status and improve survival in patients with solid tumors. We investigated the effects of hydrazine sulfate on both in vitro and in vivo models of prostate cancer. We examined the cytotoxicity of hydrazine sulfate in both human (LNCaP and PC-3) and animal (MAT-LyLu) prostate cancer cell lines. No growth inhibition was observed. In vivo, hydrazine sulfate did not suppress the growth of implanted Dunning rat prostate MAT-LyLu cells. Hydrazine sulfate does not have activity in these models of prostate cancer and may not be an appropriate therapy for patients with prostate cancer.     

2-Azetidinone cholesterol absorption inhibitors: structure-activity relationships on the heterocyclic nucleus

A series of azetidinone cholesterol absorption inhibitors related to SCH 48461 ((-)-6) has been prepared, and compounds were evaluated for their ability to inhibit hepatic cholesteryl ester formation in a cholesterol-fed hamster model. Although originally designed as acyl CoA: cholesterol acyltransferase (ACAT) inhibitors, comparison of in vivo potency with in vitro activity in a microsomal ACAT assay indicates no correlation between activity in these two models. The molecular mechanism by which these compounds inhibit cholesterol absorption is unknown. Despite this limitation, examination of the in vivo activity of a range of compounds has revealed clear structure-activity relationships consistent with a well-defined molecular target. The details of these structure-activity relationships and their implications on the nature of the putative pharmacophore are discussed.     

Development of an androgen receptor-null model for identifying the initiation site for androgen stimulation of proliferation and suppression of programmed (apoptotic) death of PC-82 human prostate cancer cells

Whether androgen regulates the proliferation and survival of androgen-responsive prostate cancer cells directly or indirectly via a paracrine pathway initiated in androgen receptor (AR)-expressing stromal cells is unknown. To resolve this issue, female mice heterozygous for the testicular feminized male loss of function mutation in their X-linked AR genes were cross-bred to T cell-defective homozygous male nude mice. Using a PCR-based restriction enzyme digestion method, the resulting AR/tfm, Nu/nu F1 hybrid females were identified and back-crossed to homozygous male nude mice to produce AR-null male nude mice lacking both AR and T-cell function. Androgen-responsive PC-82 human prostate cancers were xenografted into these AR-null versus AR-wild-type male nude mice. In both backgrounds, the cancer cells did not grow in nonandrogenized hosts. In contrast, PC-82 prostate cancer cells grew with identical characteristics (i.e., take rate, morphology, PSA expression, growth rate, and percentage of cell proliferating or dying) in androgenized hosts of both backgrounds. Likewise, in both backgrounds, androgen ablation of mice bearing growing PC-82 cancers resulted in the inhibition of proliferation and activation of programmed (apoptotic) cell death of the cancer cells. These results demonstrate that both the androgen-stimulated proliferation and the suppression of programmed cell death of PC-82 human prostate cancer cells are initiated by the AR pathway directly within these cancer cells themselves and do not involve initiation by AR-expressing stromal cells in a paracrine manner.     

Regulation of immunoreactive androgen receptor in the adrenal gland of the adult rat

The androgen receptor (AR) was measured by an immunoblot assay in adult tissues of both male and female rats. Relatively high levels of AR were detected in tissues of the male urogenital tract and in the adrenal glands and gonads of both sexes. Another group of tissues, including the male levator ani/bulbocavernosus muscles, preputial gland, scrotal skin, and vagina, had low, but detectable, levels of AR. In a third group of tissues, including the uterus, kidney, spleen, liver, gut, heart, lung, pituitary, and hypothalamus, AR was undetectable. In some androgen target tissues, such as the penis, androgens cause an apparent disappearance of AR from the tissue, and in other tissues, such as the ventral prostate, androgen therapy increases the amount of detectable AR. We compared the effect of androgen on AR levels in the adrenal gland and ventral prostate, tissues that differ markedly in their trophic responses to androgen. Castration appeared to have no effect on the amount of detectable AR in the adrenal gland, whereas it caused a profound decrease in AR levels in the ventral prostate. By contrast, 7 days after hypophysectomy, AR levels declined in both the adrenal gland and the ventral prostate. The effects of hypophysectomy plus castration were similar to those of hypophysectomy alone. Administration of ACTH to hypophysectomized rats for 7 days did not reverse the effects of hypophysectomy on adrenal AR, nor did treatment with levothyroxine, dexamethasone, rat GH, or rat PRL. Treatment of hypophysectomized rats with 5alpha-dihydrotestosterone for 7 days caused a dramatic increase in the amount of detectable AR in both the ventral prostate and the adrenal gland, but had a trophic effect only in the ventral prostate. These findings suggest that the amount of immunoreactive AR detected in both the adrenal gland and the ventral prostate is enhanced by androgens: testicular androgens in the case of the ventral prostate and adrenal androgen in the case of the adrenal glands.     

Antiandrogen withdrawal syndrome

The antiandrogen withdrawal syndrome was first reported in patients with prostate cancer who manifested disease progression after total androgen blockage therapy with medical or surgical castration and pure antiandrogen, flutamide; discontinuation of flutamide resulted in a decline in prostate specific antigen and, in some cases, with clinical response. Same phenomena have been reported after the withdrawal of casodex, chlormadinone acetate, megestrol acetate, diethylstilbestrol, and estramustine phosphate. Mutations in the androgen receptor(AR) gene were discovered in clinical specimens of human prostate cancer patients who showed this syndrome and some of these being identical to the mutation found in LNCaP prostate cancer cell line. Another mechanism otherwise the point mutation of AR would be also speculated.     

Nonsteroidal progesterone receptor antagonists based on a conformationally-restricted subseries of 6-aryl-1,2-dihydro-2,2,4-trimethylquinolines

A series of nonsteroidal human progesterone receptor (hPR) antagonists based on conformationally-restricted analogues of a 6-aryl-1,2-dihydro-2,2,4-trimethylquinoline pharmacophore were synthesized and evaluated for their ability to bind to the human progesterone receptor and inhibit progesterone-stimulated reporter gene expression in mammalian cells.     

Androgen metabolism and prostate cancer: establishing a model of genetic susceptibility

The prostate is an androgen-regulated organ, which has led to long-standing interest in the role of androgens in prostate carcinogenesis. Although evidence of a hormonal etiology for prostate cancer is strong, it is almost entirely circumstantial. Much of the problem in proving a causal relationship relates to the continued difficulties in reliably measuring human tissue-specific exposure to endogenous steroid hormones. The international and racial-ethnic variations in prostate cancer incidence, combined with the effects of migration on risk patterns, have suggested that whereas environmental factors are likely to be important, genetic factors might also play a central role in determining prostate cancer risk. We are developing a polygenic model of prostate carcinogenesis focused around a series of genes involved in androgen biosynthesis and androgen activation, transport, and metabolism in the prostate. In this developing model, we have initially targeted four genes based on three main criteria: (a) all encode products that play important roles in inducing androgen stimulation in the prostate; (b) all are polymorphic; and (c) all show substantial allelic variation in the polymorphic marker among the racial-ethnic groups of greatest interest in terms of prostate cancer risk. In addition to studying how the polymorphic markers of interest are related to prostate cancer development within and between racial-ethnic groups, we are concurrently evaluating whether genotypic variations correlate in the anticipated direction with biochemical parameters in vitro and in vivo. We summarize the development of this model and the state of knowledge related to each of the genes comprising the current model. We discuss the extent to which the current model can explain demographic variation in prostate cancer risk as well as the potential for future expansion of the model to incorporate environmental risk factors as well as additional genes. The model, when fully developed, can potentially provide a basis for targeting populations for screening interventions and/or preventive strategies aimed at the multigene products or at the genes themselves.     

Design and synthesis of novel alpha1a adrenoceptor-selective dihydropyridine antagonists for the treatment of benign prostatic hyperplasia

We report the synthesis and evaluation of novel alpha1a adrenoceptor subtype-selective antagonists. Systematic modification of the lipophilic 4,4-diphenylpiperidinyl moiety of the dihydropyridine derivatives 1 and 2 provided several highly selective and potent alpha1a antagonists. From this series, we identified the 4-(methoxycarbonyl)-4-phenylpiperidine analogue SNAP 5540 (-) [(-)-63] for further characterization. When examined in an isolated human prostate tissue assay, this compound was found to have a Ki of 2.8 nM, in agreement with the cloned human receptor binding data (Ki = 2.42 nM). Further evaluation of the compound in isolated dog prostate tissue showed a Ki of 3.6 nM and confirmed it to be a potent antagonist (Kb = 1.6 nM). In vivo, this compound effectively blocked the phenylephrine-stimulated increase in intraurethral pressure (IUP) in mongrel dogs, at doses which did not significantly affect the arterial pressure (diastolic blood pressure, DBP), with a DBP Kb/IUP Kb ratio of 16. In addition, (-)-63 also showed greater than 40 000-fold selectivity over the rat L-type calcium channel and 200-fold selectivity over several G protein-coupled receptors, including histamine and serotonin subtypes. These findings prove that alpha1a adrenoceptor-subtype selective antagonists such as (-)-63 may be developed as uroselective agents for an improved treatment of BPH over nonselective alpha1 antagonists such as prazosin and terazosin, with fewer side effects.