Hisataka Shikama

Stabilization of androgen receptor protein is induced by agonist, not by antagonists ☆

The action of nuclear receptor ligands in target tissues is specified mainly by the expression levels of their cognate nuclear receptors. The expression levels of these receptors are controlled through transcriptional and post-transcriptional events. Among post-transcriptional events, the effect of ligand on nuclear receptor protein turnover still remains largely unknown. Therefore, we studied the effects of agonist and antagonists on the turnover of the human androgen receptor (hAR) protein in stably transformed Chinese hamster ovary cells expressing exogenous hAR. Western blot analysis showed that the most potent androgen, dihydrotestosterone (DHT), stabilizes hAR with the induction of the transactivation function of hAR. However, this androgen-induced stabilization of hAR protein was abrogated by well-known androgen antagonists, hydroxyflutamide and bicalutamide (BIC), with inhibition of the transactivation function of hAR. Thus, the present study suggests that androgen antagonists exert their effects through, at least in part, abrogating the agonist-induced stabilization of hAR protein as well as blocking the ligand-induced transactivation function of hAR.

Inhibitory effects of a novel aromatase inhibitor, YM511, on growth of endometrial explants and insulin-like growth factor-I gene expression in rats with experimental endometriosis

Estrogen deprivation therapy effectively prevents progress of endometriosis but the precise mechanism by which estrogen stimulates growth of endometriotic implants is still unknown. We examined effects of hypoestrogenic state induced by ovariectomy, gonadotropin-releasing hormone agonist (leuprolide) or aromatase inhibitor (YM511), on growth of experimental endometrial explant, a section of endometrium transplanted under the renal capsule, in rats. Ovariectomy gradually reduced the volume of endometrial explants for 21 days. YM511 (0.1 mg/kg) and leuprolide (1 mg/rat) completely reduced volume of endometrial explants but they differed widely in the onset of inhibitory action. YM511 prevented growth of explants on day 4 but leuprolide had no inhibitory effect until day 15. YM511 dose-dependently reduced volume of endometrial explants and its minimum effective dose was 0.04 mg/kg. Insulin-like growth factor-I (IGF-I) mRNA expression in endometrial explant and uterus was examined on day 4. YM511 decreased IGF-I expression in endometrial explant and uterus by 58% and 48%, respectively. Reductions of the extent of IGF-I expression by YM511 and ovariectomy were comparable. A significant correlation between the volume and IGF-I mRNA expression in endometrial explant suggests that local expression of this gene may play an important role in stimulating growth of endometrial explants.

The novel hypoglycemic agent YM440 normalizes hyperglycemia without changing body fat weight in diabetic db/db mice

To determine the relationship between hypoglycemic activity and body weight gain induced by insulin sensitizers, we compared the effects of thiazolidinedione analogs (troglitazone and pioglitazone) and the oxadiazolidinedione analog (Z)-1,4-bis4[(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)methyl]phen oxy¿but-2-ene (YM440) in diabetic db/db mice. Oral treatment with YM440(100 mg/kg) for 28 days decreased the blood glucose concentration (control v YM440, 418 +/- 12 v243 +/- 44 mg/dL). The hypoglycemic activity of this agent was comparable to that of troglitazone (300 mg/kg) and pioglitazone (100 mg/kg). There were no changes in food intake among the groups. Troglitazone and pioglitazone, but not YM440, significantly increased body weight gain during treatment (control, 7.2 +/- 0.5 g; YM440, 7.5 +/- 0.8 g; troglitazone, 10.9 +/- 0.8 g; and pioglitazone, 14.5 +/- 1.1 g). To further assess whether the increase in body weight by troglitazone or pioglitazone was due to adipogenesis, the weight of intraabdominal fat tissue (epididymal, retroperitoneal, and perirenal) was determined. There were no differences in the total weight of visceral fat between the control and YM440 treatment (3.53 +/- 0.23 and 3.60 +/- 0.16 g). In contrast, troglitazone and pioglitazone significantly increased the fat weight (4.31 +/- 0.13 and 4.66 +/- 0.19 g). Thiazolidinediones are known as ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor responsible for adipogenesis. Troglitazone and pioglitazone activated PPARgamma and increased triglyceride accumulation and mRNA expression of fatty acid-binding protein (FABP) in 3T3-L1 cells. However, YM440 had no effect on these indices for adipocyte differentiation. These results suggest that the mechanism is different for the hypoglycemic action of YM440 versus the thiazolidinediones. YM440 ameliorates hyperglycemia without changing PPARgamma activity, adipocyte differentiation, or fat weight. Thus, YM440 could be a useful hypoglycemic agent for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) without affecting body weight.

Novel nonsteroidal inhibitor of cytochrome P45017α (17α-hydroxylase/C17-20 lyase), YM116, decreased prostatic weights by reducing serum concentrations of testosterone and adrenal androgens in rats

The purpose of this study was to determine the effects of a nonsteroidal C17–20 lyase inhibitor, 2‐(1H‐imidazol‐4‐ylmethyl)‐9H‐carbazole (YM116), on serum concentrations of androgens and ventral prostatic weight in rats.

Inhibitory effect of a novel non-steroidal aromatase inhibitor, YM511 on the proliferation of MCF-7 human breast cancer cell

The proliferation of MCF-7, human breast cancer cell line, was stimulated by testosterone and estradiol. The aromatase activity in MCF-7 cells, which catalysed the conversion of testosterone to estradiol, was inhibited by a novel non-steroidal aromatase inhibitor, YM5111, with the IC50 of 0.2 nM, indicating that its inhibitory activity was 5.5 times more potent than that of CGS 16949A. YM511 inhibited the proliferation of MCF-7 stimulated by testosterone but did not inhibit the cell proliferation stimulated by estradiol. The IC50 values of YM511 for cell growth and DNA synthesis were 0.13 nM and 0.18 nM, respectively, demonstrating that YM511 was about 3-5 times more potent than CGS 16949A and had no anti-estrogenic or cytotoxic activity. YM511 significantly inhibited testosterone-stimulated transcriptional activation of estrogen-responsive element (ERE) in MCF-7 cells transfected transiently with ERE-luciferase reporter plasmid. The IC50 of YM511 for transactivation was 0.36 nM, suggesting that its inhibitory potency was comparable to the inhibition of aromatase activity of MCF-7 cells. These data may indicate that the inhibition by YM511 of cell proliferation of MCF-7 is attributed to the decreased production of estrogen due to the inhibition of aromatase activity. YM511 may be useful in the treatment of estrogen-dependent cancers.

The potent and selective inhibition of estrogen production by non-steroidal aromatase inhibitor, YM511

YM511 inhibited aromatase activities in microsomes from rat ovary and human placenta competitively (IC50s: 0.4 and 0.12 nM, respectively). YM511 was about 3 times more potent than other aromatase inhibitors, such as CGS 16949A, CGS 20267 and R 76713. YM511 decreased the contents of estradiol stimulated by pregnant mares serum gonadotropin in rat ovary with an ED50 of 0.002 mg/kg, indicating that YM511 was equipotent to CGS 20267 and 3 times more potent than the other two inhibitors. Serum estradiol levels in female rats were reduced by YM511 at 0.01 mg/kg into the ovariectomized range. YM511 at 1 mg/kg for 2 weeks decreased rat uterine weight to levels comparable to ovariectomy, showing it was 10 times more potent than other inhibitors. But the maximal inhibitory effect of tamoxifen failed to reach ovariectomized level. YM511 slightly inhibited production of other steroid hormones in vitro and in vivo. The IC50s of YM511 for aldosterone and cortisol production from adrenal cells were from 5500 to 9800 times higher than that for rat ovarian aromatase and 130,000 times higher for testosterone production, indicating that YM511 is a highly specific aromatase inhibitor. The data suggest that YM511 may be a potent and selective agent for suppressing estrogen-dependent action without affecting serum levels of other steroid hormones.

Hypoglycemic agent YM440 suppresses hepatic glucose output via gluconeogenesis by reducing glucose-6-phosphatase activity in obese Zucker rats.

Using a glucose clamp, we had shown that YM440, (Z)-1,4-bis[4-[(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)methyl]phenoxy]but-2-ene, reduced the increased hepatic glucose output in obese Zucker rats. We further examined effects of YM440 on 14C-incorporation from [14C]bicarbonate into blood glucose via gluconeogenesis, and on gluconeogenic enzymatic activities. Fed obese Zucker rats showed a 4-fold increase of 14C-incorporation into blood glucose compared to that in lean rats. Glucose-6-phosphatase and fructose-1,6-bisphosphatase activities in obese rats were increased 1.4-fold and 1.6-fold compared with lean rats. YM440 (300 mg/kg for 2 weeks) decreased 14C-incorporation into blood glucose by 29% in obese rats. Glucose-6-phosphatase but not fructose-1,6-bisphosphatase activity was reduced by YM440 and closely correlated with 14C-incorporation into blood glucose, indicating a key role for glucose-6-phosphatase in hepatic glucose output. These results suggest that the increased gluconeogenesis in obese rats is mainly due to the increased activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase and that YM440 suppresses hepatic glucose output by reducing glucose-6-phosphatase activity.

A Role of Androgen Receptor Protein in Cell Growth of an Androgen-Independent Prostate Cancer Cell Line

Prostate cancer, which develops due to androgen and is initially responsive to androgen deprivation therapy, often comes to acquire androgen deprivation therapy resistance in short order. We investigated the role of androgen receptor (AR) protein in an androgen-independent prostate cancer cell line using AR ligands and AR siRNA. Although the androgen-independent cell line scarcely responded to AR ligands, their growth was attenuated by ablation of AR protein by siRNA.

Differential effects of YM440 a hypoglycemic agent on binding to a peroxisome proliferator-activated receptor γ and its transactivation

Peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-inducible transcription factor mediating glucose and lipid metabolism. Prior studies showed that YM440 ameliorated hyperglycemia in diabetic mice without affecting body fat weight or PPARgamma transactivation. In this study we have examined further the effects of YM440 on PPARgamma binding, transactivation and conformational change. YM440, pioglitazone and rosiglitazone displaced [3H]rosiglitazone from PPARgamma with K(i) values of 4.0, 3.1, and 0.20 microM, indicating that YM440 was comparable to pioglitazone and 20-fold less potent than rosiglitazone. Although pioglitazone and rosiglitazone increased both PPARgamma transactivation in cells expressing human full-length PPARgamma2 or GAL4-PPARgamma and mRNA expression of PPARgamma responsive genes in 3T3-L1 cells, YM440 had weak effects on PPARgamma transactivation and mRNA expression being 550- to 790-fold and 36- to 110-fold less active than rosiglitazone, respectively. YM440 and rosiglitazone induced interaction between PPARgamma and the transcriptional cofactor, p300 or SRC-1, but YM440 was 151- and 1091-fold less potent than rosiglitazone, respectively. The weak transcriptional activity of YM440 was not due to poor cell permeability. Limited trypsin digestion of the full-length human PPARgamma2 with YM440 or rosiglitazone showed distinct patterns of digestion, suggesting a difference in the conformational change of PPARgamma. When db/db mice were treated with YM440 (100mg/kg) for 28 days, YM440 increased hepatic glucokinase expression but not adipose tissue FABP and UCP1 expression, indicating a tissue selective expression of PPARgamma-related genes. Unique properties regarding the binding-transactivation of PPARgamma by YM440 may lead to the hypoglycemic activity without affecting body fat weight in diabetic mice.