Selective ligands of estrogen receptor ß discovered using pharmacophore mapping and structure-based virtual screening.

AIM: To discover novel ligands of estrogen receptor (ER) ß using pharmacophore mapping and structure-based screening. METHODS: A computer-aided strategy combining pharmacophore mapping and structure-based screening was used to screen the Maybridge and Enamine databases. Yeast two-hybrid (Y2H) assay was used to detect the activity and selectivity of the chosen compounds. The transcriptional activities of the chosen compounds were demonstrated with luciferase reporter assays. The anti-proliferative effects of ER antagonists against MCF-7 and MDA-MB-231 breast cancer cells were examined using MTT assay, and the mechanisms of action were analyzed with flow cytometry analysis and Western blotting. RESULTS: Through in silico screen, 95 compounds were chosen for testing in Y2H assay, which led to 20 potent ligands, including 10 agonists, 8 antagonists and 2 partial agonists with EC50 or IC50 values at µmol/L. Furthermore, 6 agonists exhibited absolute selectivity for ERß, and 3 agonists showed higher selectivity for ERß. The agonists 1g and 1h (10, 25, and 50 µmol/L) dose-dependently increased ER transcriptional activities, whereas the antagonists 2a and 2d (10, 25, and 50 µmol/L) caused dose-dependent inhibition on the activities. The antagonists and partial agonists at 100 µmol/L suppressed the proliferation of ERα positive MCF-7 cells and ERß positive MDA-MB-231 cells, but were more effective against MDA-MB-231 cells. Treatment of MDA-MB-231 cells with antagonists 2a and 2d (25 and 50 µmol/L) dose-dependently increased the population of cells in the S phase. Both 2a and 2d treatment dose-dependently decreased the expression levels of cyclin A and CDK2. Meanwhile, the downregulation of cyclin E was only caused by 2d, while 2a treatment did not cause significant changes in the protein levels of cyclin E. CONCLUSION: The selective ligands discovered in this study are promising drug candidates to be used as molecular probes to explore the differences between ERα and ERß.

[Study on relationship between endogenous androgens and insulin resistance at the different stages of postmenopause].

OBJECTIVE: To investigate the relationship between insulin resistance and endogenous androgens at early and late phase of postmenopause. METHODS: A total of 105 women with early postmenopause ( ≤ 5 years since menopause) and 107 women with late postmenopause ( ≥ 10 years since menopause) were enrolled in this study.In the mean time, those women were classified into normal weight[body mass index (BMI), BMI <24 kg/m(2)] group and overweight (BMI ≥ 24 kg/m(2)) group.Sex hormone-binding globulin (SHBG), testosterone (T) , dehydroepiandrosterone-sulfate (DHEA-S) , fasting blood glucose(FBG), fasting insulin (FINS) levels were measured and then calculated free androgen index (FAI) and homeostatic model assessment of insulin resistance (HOMA-IR) . The relationship between sex hormones and insulin resistance was analyzed by partial correlation and multiple linear regression analyses. RESULTS: Compared to early postmenopausal women, late postmenopausal women had higher FINS [(7.9 ± 6.6) mU/L versus (6.6 ± 4.0) mU/L] and HOMA-IR(2.1 ± 1.9 versus 1.7 ± 1.1), but they had lower DHEA-S[(0.9 ± 0.5) mg/L versus (1.1 ± 0.5) mg/L, all P < 0.05) ]. Both in early postmenopausal and late postmenopausal groups, overweight women had higher HOMA-IR (early group, 2.2 ± 1.0 versus 1.2 ± 0.9;late group, 2.8 ± 2.6 versus 1.6 ± 1.1) and FINS early group[(6.9 ± 2.9) mU/L versus (4.6 ± 2.0) mU/L];late group[(10.2 ± 9.3) mU/L versus (6.4 ± 3.6) mU/L] than those at women with normal weight group (all P < 0.05) .In early postmenopausal group, overweight women had lower SHBG[ (52 ± 37) nmol/L versus (71 ± 37) nmol/L] and higher FAI (2.5 ± 2.1) versus (1.3 ± 1.1) than those at normal weight women group (all P < 0.05) .In late postmenopausal group, overweight women had higher DHEA-S (1.0 ± 0.5) mg/L versus (0.8 ± 0.4) mg/L (P < 0.05) . The analyses suggested that in early postmenopausal group, SHBG was correlated negatively with FINS and HOMA-IR (ß = -0.386, P < 0.05;ß = -0.553, P < 0.05) , DHEA-S was correlated positively with FBG (ß = 0.348, P < 0.05) in early postmenopausal group.FAI was correlated positively with FBG in late postmenopausal group (ß = 0.505, P < 0.05) . CONCLUSIONS: The increased androgenic activities are associated with insulin resistance after of menopause. These correlations are different at different stages of postmenopause, which SHBG levels correlate with high risk of insulin resistance and DHEA-S levels correlates with high blood glucose levels at early postmenopause and FAI correlates with high blood glucose levels at late postmenopause.

Accumulation of nerve growth factor and its receptors in the uterus and dorsal root ganglia in a mouse model of adenomyosis.

BACKGROUND: Adenomyosis is a common gynecological disease, which is accompanied by a series of immunological and neuroendocrinological changes. Nerve growth factor (NGF) plays a critical role in producing pain, neural plasticity, immunocyte aggregation and release of inflammatory factors. This study aimed to investigate the expression of NGF and its two receptors in uteri and dorsal root ganglia (DRG) in an adenomyosis mouse model, as well as their relationship with the severity of adenomyosis. METHODS: Forty newborn ICR mice were randomly divided into the adenomyosis model group and control group (n = 20 in each group). Mice in the adenomyosis model group were orally dosed with 2.7 µmol/kg tamoxifen on days 2-5 after birth. Experiments were conducted to identify the expression of NGF- beta and its receptors, tyrosine kinase receptor (trkA) and p75 neurotrophin receptor (p75NTR), in the uterus and DRG in four age groups (90+/-5 d, 140+/-5 d, 190+/-5 d and 240+/-5 d; n = 5 mice in each group) by western bolt, immunochemistry and real time reverse transcription-polymerase chain reaction. RESULTS: Adenomyosis, which became more serious as age increased, was successfully induced in dosed ICR mice. NGF-beta, trkA and p75NTR protein levels in the uterus and trkA mRNA levels in DRG were higher in the older aged adenomyosis model group than those in controls (190+/-5 d and 240+/-5 d groups, P < 0.05). The expression of NGF-beta and its receptors in the uterus increased gradually as age increased for adenomyosis mice (190+/-5 d and 240+/-5 d, P < 0.05, compared with 90+/-5 d) but it showed little change in control mice. The mRNA level of trkA in DRG also increased as age increased in the adenomyosis model group (190+/-5 d and 240+/-5 d, P < 0.05, compared with 90+/-5 d) but was unchanged in controls. The mRNA level of p75NTR in DRG was not different between the adenomyosis and control groups and was stable from young to old mice. CONCLUSIONS: NGF- beta can be used as an indicator for the severity of adenomyosis. The gradually increasing level of NGF- beta and its receptors while the disease becomes more severe suggests an effect of NGF- beta on pathogenic mechanisms of adenomyosis.

[Dose-dependent effects of daidzein in regulating bone formation through estrogen receptors and peroxisome proliferator-activated receptor γ].

OBJECTIVE: To investigate different doses of daidzein (DAI) in regulating bone formation of osteoblasts, and the regulating mechanisms of estrogen receptors (ERs) and peroxisome proliferator-activated receptor γ (PPARγ) in bone formation. METHODS: Human fetal osteoblasts (hFOBs) incubated without any treatment were served as controls (control group). The hFOBs were exposed to DAI of 10(-9), 10(-7) and 10(-5) mol/L for 72 h, and to ß-estradiol-17-valerate (E(2)) of 10(-8) mol/L as positive control, respectively. Methyl thiazolyl tetrazolium assay was employed to determine the proliferation status of osteoblasts, and 4-nitrophenyl phosphate disodium salt (PNPP) method was employed to determine the activity of alkaline phosphatase (ALP). ER antagonist ICI 182780 (ICI), ERα-selective antagonist methyl-piperidino-pyrazole (MPP) and irreversible PPARγ antagonist GW9662 (GW) were used to block the corresponding receptor, while hFOBs were exposed to E(2) or different concentrations of DAI for 48 h. MTT assay and PNPP method were used respectively to determine the proliferation status and ALP activity of osteoblasts cultured in vitro. RESULTS: The osteoblast proliferation rate decreased progressively as the dose of DAI increased. Compared with the controls, the osteoblast proliferation rate in the DAI 10(-9) mol/L group increased significantly, while DAI 10(-5) mol/L group decreased significantly (P<0.05). ALP level decreased progressively as the dose of DAI increased, but there was no significant difference between groups (P>0.05). When ERs were blocked by ICI, proliferation rates in the E(2) group and DAI 10(-9), 10(-7) and 10(-5) mol/L groups were 88.16%, 76.30%, 81.18% and 83.19% respectively, which were all significantly lower than before (P<0.05). After ERα was blocked by MPP alone, proliferation rates in E(2) group and DAI 10(-9), 10(-7) and 10(-5) mol/L groups were 69.78%, 63.31%, 70.71% and 78.43%, respectively, which were also significantly lower than before (P<0.05). ALP level in the DAI 10(-9) mol/L group decreased significantly when ERα was blocked alone. When PPARγ inhibitor GW was added to the culture system, proliferation rates in E(2) group and DAI 10(-9), 10(-7) and 10(-5) mol/L groups were 103.14%, 96.99%, 112.88% and 122.22%, respectively. Compared with before, proliferation rates in DAI 10(-7) and 10(-5) mol/L groups increased significantly (P<0.05), and ALP level increased significantly (P<0.05) in the DAI 10(-5) mol/L group. CONCLUSION: DAI shows a biphasic effect on osteoporosis, whereby the effect is dose-dependent; a low-dose DAI stimulates proliferation of osteoblasts, while a high-dose DAI inhibits proliferation of osteoblasts. Low-dose DAI mainly acts on ERs, whereas high-dose DAI mainly acts on PPARγ to inhibit proliferation of osteoblasts and to some extent, acts on ERs to promote the proliferation of osteoblasts.

[Role of the cross-talk between estrogen receptors and peroxisome proliferator-activated receptor gamma in daidzein's prevention and treatment of osteoporosis in ovariectomized rats].

OBJECTIVE: To investigate the role of the cross-talk between estrogen receptors and peroxisome proliferator-activated receptor gamma in daidzein's prevention and treatment of osteoporosis in the ovariectomized rats. METHODS: Sixty four-month-old Sprague-Dawley (SD) female rats were divided into six groups. Four weeks after the establishment of osteoporosis model, the ovariectomized rats were administered different drugs by gavage respectively for three months. After sacrificing, the expressions of mRNA of ERalpha, ERbeta and PPARgamma in L(6) and right femur were measured by realtime-RT-PCR, while proteins were detected by immunohistochemistry. RESULTS: The expressions of ERalpha mRNA and protein were significantly lower than that of ERbeta in three dosing groups. Excepting that of rat femur of rats in H-Dai group, the expressions of ERbeta mRNA of lumbar spine and femur in M-Dai group (0.0177 +/- 0.0010, 0.0245 +/- 0.0013) and L-Dai groups (0.0276 +/- 0.0027, 0.0278 +/- 0.0044) and lumbar spine in H-Dai group (0.0163 +/- 0.0037) were significantly higher than that in OVX group (0.0016 +/- 0.0005, 0.0041 +/- 0.0014). There was no significant difference with E(2) group. At the same time, the expression of ERbeta protein showed into the similar trend as that of mRNA. And there was an rising expressions of ERbeta while daidzein dose decreased. On the contrary, the expressions of PPARgamma mRNA and protein decreased. CONCLUSIONS: There is an inverse cross-talk relationship between ERbeta and PPARgamma, it may play a role in daidzein's prevention and treatment of osteoporosis in ovariectomized rats.

A new classification system for pregnancy prognosis of tubal factor infertility.

UNLABELLED: The aim of the present study was to assess the objectivity and accuracy of a new system to evaluate pregnancy prognosis in tubal factor infertility (TFI) patients. Retrospective study in 469 TFI patients were pre- and postoperatively scored using the new system as mild, moderate or severe TFI, based on tubal adhesions, patency, morphology and structure. Follow-up was assessed to determine pregnancy outcomes. Laparoscopic salpingoplasty and hydrotubation, hysteroscopic-laparoscopic salpingoplasty and hydrotubation, and laparoscopic hydrotubation all decreased TFI scores to a similar extent. The pre- and postoperative TFI classification was significantly associated with intrauterine pregnancy (mild: 43.6% vs. moderate: 34.0% vs. severe: 19.4%, P < 0.0001) and live births (mild: 35.9% and moderate: 31.5% vs. severe: 16.8%, P = 0.0002) rates. Multivariate analysis showed that the preoperative disease course (P = 0.02), preoperative TFI score (P < 0.0001), and postoperative TFI score (P = 0.0007) were independently associated with the rate of intrauterine pregnancy rate. Multivariate analysis also showed that the postoperative TFI score (P = 0.001), pelvic inflammatory disease (P = 0.03) and age (P = 0.03) were independently associated with the rate of live births. CONCLUSION: We devised a new classification system for TFI prognosis. Salpingoplasty improved these scores. Both pre- and postoperative TFI assessments using this new system are associated with pregnancy prognosis in TFI patients.

The incidence and characteristics of uterine bleeding during postoperative GnRH agonist treatment combined with estrogen-progestogen add-back therapy in endometriosis patients of reproductive age.

To evaluate the incidence and characteristics of uterine bleeding during postoperative gonadotropin-releasing hormone agonist (GnRHa) treatment combined with the lowest effective dose of estrogen-progestogen add-back therapy in Chinese women of reproductive age with endometriosis. Seventy Chinese women aged 18 to 50 years with stage III or IV endometriosis and treated with postoperative GnRHa after conservative surgery for endometriosis were eligible for this study. Patients were randomly divided into two equal groups, G and A. Group G (n = 35) received three 28-day cycles of postoperative GnRHa treatment by subcutaneous injection (goserelin, 3.6 mg). Group A (n = 35) received the same GnRHa treatment in addition to daily estradiol valerate (0.5 mg) and dydrogesterone (5 mg) add-back therapy. Serum E2 and FSH levels were assessed at the end of each treatment cycle, as well as incidence and patterns of uterine bleeding. After the last GnRHa treatment cycle, endometrial thickness was evaluated by ultrasonography and the recovery of menstruation was recorded. Uterine bleeding incidence was above 90% in both groups during the first treatment cycle (group G: 90.6%; group A: 93.8%), but decreased markedly in the second treatment cycle (group G: 15.6%; group A: 21.9%), and continued to decline until the end of the third treatment cycle (group G: 6.3%; group A: 12.5%). For each cycle, the incidence of uterine bleeding in group A was slightly but not statistically higher. Irregular spotting was the most common uterine bleeding pattern observed in each of the three treatment cycle. The addition of estrogen and progestogen therapy to a postoperative GnRHa regimen does not lead to an increase in the duration or amount of treatment-induced uterine bleeding.