The agonistic bioanalytical equivalent concentration: A novel tool for assessing the endocrine activity of environmental mixtures.

Cell-based bioassays are very sensitive and allow integrative effect screening of the whole environmental sample, which is usually composed of a mixture of agonists and antagonists. Measured toxicity is usually expressed as a bioanalytical equivalent concentration. So far, it is not possible to distinguish which part of this value is caused by the agonists and which by the antagonists. In this article, we present a simple method to analyze the dose-response curve of a mixture and to determine an agonistic bioanalytical equivalent concentration: a concentration of a reference chemical that would elicit the same effect as do only agonists in an unknown mixture. The method has been validated using several artificially prepared mixtures of agonists and competitive antagonists measured in a recombinant yeast assay. No difference was observed between the calculated equivalent concentrations and the used concentrations of the agonist in the mixture.

Single-dose pharmacokinetics and pharmacodynamics assessment of oestriol and trimegestone containing vaginal rings in healthy women with childbearing potential.

PURPOSE: To evaluate the pharmacokinetics and pharmacodynamics of oestriol (E3) and trimegestone (TMG) in healthy women after application of three different vaginal rings over 21 days. The vaginal rings had a nominal delivery rate of 0.413/0.050 mg/day (Test 1), 0.311/0.090 mg/day (Test 2) and 0.209/0.137 mg/day (Test 3) E3/TMG. METHODS: Thirty-five healthy women were randomised to receive a single application of Test 1, 2 or 3 (Clinical Trial NCT03343912). The E3 and TMG plasma concentration was determined by LC-MS/MS. Oestradiol (E2) and progesterone (PG) serum concentrations, and bleeding patern were determined as pharmacodynamic parameters. Safety was assessed by evaluation of adverse events and local tolerability. RESULTS: The total and maximum exposure of E3 and TMG increased in a proportional ratio to dose. However, not in a magnitude which was expected from the dose differences for E3. During Test 2 and 3 treatment all E2 and PG values remained on a well suppressed level until end of treatment. E2 and PG serum levels increased distinctly earlier after ring removal with Test 1 compared to Test 2 and 3. Test 3 achieved 95.24% of "no bleeding" days under treatment followed by Test 1 (91.67%), and Test 2 (86.15%). CONCLUSIONS: The Test 3 formulation presented the best dose combination of E3/TMG for contraception. Moreover, all vaginal rings were well tolerated.

Effects of androgens and estrogens on sirtuin 1 gene expression in human aortic endothelial cells.

OBJECTIVES: To investigate the effect of androgens and estrogens on surtuin 1 (SIRT1) expression in human aortic endothelial cells (HAECs). METHODS: Real-time polymerase chain reaction analysis of SIRT-1 expression over 48 hours (h) was performed in HAECs treated with various concentrations of dehydroepiandrostendione (DHEA), androstenedione and testosterone (androgens), estrone (E1), estradiol (E2), and estriol (E3) (estrogens) to investigate the dose-dependency of time courses. The influence of high glucose on SIRT1 expression induced by the androgens and estrogens was also examined. RESULTS: Dehydroepiandrostendione, androstenedione, and testosterone remarkedly produced a dose-dependent increase in SIRT1 expression in the range of 10 to 20 µg/ml. High glucose (40mM) medium had significantly inhibitory effects on 10 µg/ml DHEA-induced SIRT1 expression (p=0.024). Estrone and E2, but not E3, caused a marked dose-dependent increase in SIRT1 expression from 10 to 20 µg/ml. Treatment with 20 mM or 40 mM glucose medium did not significantly inhibit E1- and E3-induced SIRT1 expression in control medium; however, both high glucose mediums significantly emphasized E2-induced SIRT1 expression in control medium (p=0.007, p=0.005). CONCLUSION: These results suggest that DHEA, androstenedione, testosterone, E1, and E2 definitely activate SIRT1 expression in HAECs. A high glucose medium is potent to inhibit the basal gene expression; however, it could not reduce powerful androgen- and estrogen-induced SIRT1 expression in HAECs.

Natural epiestriol-16 act as potential lead molecule against prospective molecular targets of multidrug resistant Acinetobacter baumannii-Insight from in silico modelling and in vitro investigations.

The current study aimed to identify putative drug targets of multidrug resistant Acinetobacter baumannii (MDRAb) and study the therapeutic potential of natural epiestriol-16 by computer aided virtual screening and in vitro studies. The clinical isolates (n = 5) showed extreme dug resistance to carbapenems and colistins (p ≤ .05). Computational screening suggested that out of 236 natural molecules selected, 06 leads were qualified for drug likeliness, pharmacokinetic features and one potential molecule namely natural epiestriol-16 (16b-Hydroxy-17a-estradiol) exhibited significant binding potential towards four prioritised drug targets in comparison with the binding of faropenem to their usual target. Natural epiestriol demonstrated profound binding to the outer membrane protein (Omp38), protein RecA (RecA), orotate phosphoribosyltransferase (PyrE) and orotidine 5'-phosphate decarboxylase (PyrF) with binding energy of -6.0, -7.3, -7.3 and -8.0 kcal/mol respectively. MD simulations suggested that 16-epiestriol-receptor complexes demonstrated stability throughout the simulation. The growth curve and time kill assays revealed that MDRAb showed resistance to faropenem and polymyxin-B and the pure epiestriol-16 showed significant inhibitory properties at a concentration of 200 µg/mL (p ≤ .5). Thus, natural epiestriol-16 can be used as potential inhibitor against the prioritised targets of MDRAb and this study provide insight for drug development against carbapenem and colistin resistant A. baumannii.

Estrogen induces indoleamine 2,3-dioxygenase expression via suppressors of cytokine signaling 3 in the chorionic villi and decidua of women in early pregnancy.

PROBLEM: Indoleamine 2,3-dioxygenase (IDO) is a key protein that participates in the protection of embryos against the mother's immune system during pregnancy. How the expression of this protein is regulated at the maternal-fetal interface remains largely unknown. METHOD OF STUDY: The chorionic villi and decidua of women in early pregnancy were collected. Tissue explants of the chorionic villi and decidua were cultured in media containing varying concentrations of 17ß-estradiol and estriol with or without fulvestrant. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the expression of IDO and the suppressors of cytokine signaling 3 (SOCS3) in the cultured tissues from chorionic villi and decidua. RESULTS: Both IDO and SOCS3 were expressed in chorionic villi and decidua. The expression of IDO was increased in tissue explants from chorionic villi and decidua cultured in medium containing different concentrations of 17ß-estradiol or estriol, and this increase was reversed when fulvestrant was added to the medium. The expression of IDO was upregulated, and SOCS3 expression was downregulated the most in tissue explants from chorionic villi and decidua that were cultured in medium containing 17ß-estradiol at a concentration of 10 ng/mL or estriol at a concentration of 1 µg/mL. This increase in IDO and decrease in SOCS3 were reversed when fulvestrant was added to the medium at a concentration of 10 µg/mL. CONCLUSION: At a concentration similar to that present during pregnancy, estrogen may upregulate the expression of IDO via downregulating SOCS3, which implies that estrogen may contribute to the prevention of allogeneic fetal rejection, and further studies may strengthen the possibility of using estrogen as an immune modulator.

Species differences in ligand interaction and activation of estrogen receptors in fish and human.

Estrogen receptor (ER) sequences vary between species and this suggests that there are differences in the ligand-specificity, leading to species-specific effects. This would indicate that it is not possible to generalize effects across species. In this study, we investigated the differences in activation potencies and binding affinities of ER´s alpha (α) and beta (ß) in human, zebrafish and sea bream to elucidate species differences in response to estradiol, estrone, estriol and methyltestosterone. In vitro analysis showed that estradiol had the highest activity for all the ER´s except for human ERß and seabream ERß2. Alignment of the ligand binding domain and ligand binding pocket (LBP) residues of the three species showed that different residues were involved in the LBPs which led to differences in pocket volume, affected binding affinity and orientation of the ligands. By combining in silico and in vitro results, it was possible to identify the ligand specificities of ER´s. The results demonstrated that the human ER´s show lower resolution in ligand-dependent activation, suggesting higher promiscuity, than the zebrafish and seabream ER´s. These results show species-specificity of ER´s and suggest that species-specific differences must be taken into consideration when studying different exposure scenarios.

Effect of Estriol, Chorionic Gonadotropin, and Oncostatin M on the Expression of Recombinase RAG-1 in Regulatory T Lymphocyte Subpopulations.

We studied the effect of estriol, chorionic gonadotropin, oncostatin M, and hormone-cytokine combinations on the expression of recombinase RAG-1 in T-regulatory (Treg) and T helper 17 (Th17) lymphocytes. It was found that estriol in a concentration corresponding to the first trimester of pregnancy increased the level of Treg (CD4+FoxP3+) cells and suppressed the formation of Th17 (CD4+RORC+) lymphocytes. This effect was nor observed after individual administration of chorionic gonadotropin and oncostatin M, but some combinations of the studied hormones with oncostatin M increased the percentage of CD4+FOXP3+ cells. In the presence of oncostatin M, the studied hormones enhanced the expression of RAG-1 in CD4+FoxP3+ cells, but not in CD4+RORC+ cells, thereby initiating of Treg T-cell receptor (TCR) revision. The mechanisms of hormone cytokine control of induction of the immune tolerance during pregnancy are discussed.

Hormonal Regulation of Dendritic Cell Differentiation in the Thymus.

We studied the effect of hormones estriol, ghrelin, kisspeptin, and chorionic gonadotropin in concentrations corresponding to their content in the peripheral blood in each trimester of pregnancy on the expression of membrane molecules on myeloid and plasmacytoid dendritic cells of the thymus. It was found that thymic myeloid dendritic cells are sensitive to the action of estriol and kisspeptin. Estriol in a concentration of the first trimester of pregnancy reduces the number of myeloid dendritic cells expressing receptor for thymic stromal lymphopoietin (CD11c+TSLP-R+) and inhibitory molecule B7-H3 (CD11c+CD276+). In contrast to estriol, kisspeptin regulates the processes of differentiation of thymic myeloid dendritic cells in concentrations typical of the second-third trimesters and reduced their total number (CD11c+) and the number of cells expressing TSLP-R (CD11c+TSLP-R+). Estriol and kisspeptin do not affect the total number of plasmacytoid dendritic cells (CD303+) and expression of TSLP-R and CD276 by these cells. Ghrelin and chorionic gonadotropin in the studied concentrations had no significant effect on the total number of thymic myeloid and plasmacytoid dendritic cells and on the expression of membrane molecules of TSLP-R and CD276.

Estriol Inhibits Dermcidin Isoform-2 Induced Inflammatory Cytokine Expression Via Nitric Oxide Synthesis in Human Neutrophil.

BACKGROUND: An increase in the level of cytokines like TNF-α and IL-6 causes the inflammatory surge in acute ischemic heart disease (AIHD). OBJECTIVE: A high-level dermcidin isoform-2 (DCN-2) occurrence in AIHD was subjected to determine a possible regulation of cytokines expression. The effect of estrogen to counteract the inflammatory response was determined. METHODS: Blood was collected from AIHD patients and normal volunteers with consent. Nitric oxide (NO) synthesis was done with methemoglobin method.TNF-α and IL-6 expression were determined by ELISA and Western blot. RESULTS: (DCN-2) incubation with 120nM to the normal neutrophil solution for 2h resulted in the increase of TNF-α from 3.82±1.53pg/ml to 20.7±6.9pg/ml and IL-6 from 3.27±1.52pg/ml to 47.07±3.4pg/ml. In AIHD patients, the cytokine level was18.3- 27.3pg/ml, with a median value 21.86pg/ml (TNF-α) and IL-6 level was 23.54- 52.73pg/ml, with a median value 42.16pg/ml. Treatment with 0.6nM estriol, a kind of female steroid hormone estrogen for 45min decreased the elevated cytokine level in 120nM DCN-2 treated normal neutrophils. DCN-2 induced TNF-α synthesis in neutrophils was further determined by Western blot technique with a thickened band intensity of TNF-α. Estriol (0.6nM) treatment also influenced the DCN-2 induced inhibition of nitric oxide (NO) synthesis from 0nmol NO/ml to 0.56nmol/ml. The subsequent reduction of TNF-α level correlates the increase of NO level. CONCLUSION: In conclusion, the stress-induced DCN-2 production in AIHD propagates the inflammatory response. Steroid molecule like estriol plays a protective role by reducing DCN-2 responses in the NO synthesis.

GPR30 mediates estrone, estriol, and estradiol to suppress gonadotropin-releasing hormone-induced luteinizing hormone secretion in the anterior pituitary of heifers.

Recent studies demonstrated that G-protein-coupled receptor 30 (GPR30) on the plasma membrane of gonadotroph cells mediates picomolar, but not nanomolar, levels of estradiol (E2) to rapidly suppress gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion in the anterior pituitary (AP). While estrone (E1) and estriol (E3) are considered "weak" estrogens that exert suppressive effects through estrogen receptors α and ß, it is conceivable that they also strongly suppress GnRH-induced LH secretion via GPR30. Both E1 and E3 are likely present within the blood at picomolar or nanomolar concentrations, indicating that such concentrations are sufficient to suppress GnRH-induced LH secretion. To evaluate this possibility, bovine AP cells were cultured under steroid-free conditions and then incubated with various concentrations (0.01 pM to 10 nM) of E2, E1, or E3, prior to stimulation with GnRH. Notably, GnRH-induced LH secretion from AP cells was inhibited by 1-100 pM E2, 1-10 pM E1, and 1-100 pM E3. GnRH-induced LH secretion from AP cells was not inhibited by lower (0.01-0.1 pM) or higher (1-10 nM) concentrations of E2, E1, and E3. These suppressive effects were inhibited by pre-treatment of AP cells with the GPR30 antagonist G36, but not with the estrogen receptor alpha antagonist. Treatment with E1 or E3 also yielded decreased cytoplasmic cAMP levels in cultured AP cells pre-treated with dopamine and phosphodiesterase inhibitors. Therefore, these results suggest that GPR30 mediates the suppressive effects of E1, E3, and E2 on GnRH-induced LH secretion from bovine AP.

A novel mechanism of non-feminizing estrogens in neuroprotection.

Estrogens are potent and efficacious neuroprotectants both in vitro and in vivo in a variety of models of neurotoxicity. We determined the structural requirements for neuroprotection in an in vitro assay using a panel of >70 novel estratrienes, synthesized to reduce or eliminate estrogen receptor (ER) binding. We observed that neuroprotection could be enhanced by as much as 200-fold through modifications that positioned a large bulky group at the C2 or C4 position of the phenolic A ring of the estratriene. Further, substitutions on the B, C or D rings either reduced or did not markedly change neuroprotection. Collectively, there was a negative correlation between binding to ERs and neuroprotection with the more potent compounds showing no ER binding. In an in vivo model for neuroprotection, transient cerebral ischemia, efficacious compounds were active in protection of brain tissue from this pro-oxidant insult. We demonstrated that these non-feminizing estrogens engage in a redox cycle with glutathione, using the hexose monophosphate shunt to apply cytosolic reducing potential to cellular membranes. Together, these results demonstrate that non-feminizing estrogens are neuroprotective and protect brain from the induction of ischemic- and Alzheimer's disease (AD)-like neuropathology in an animal model. These features of non-feminizing estrogens make them attractive compounds for assessment of efficacy in AD and stroke, as they are not expected to show the side effects of chronic estrogen therapy that are mediated by ER actions in the liver, uterus and breast.

Estrogenic compound attenuates angiotensin II-induced vascular smooth muscle cell proliferation through interaction between LKB1 and estrogen receptor α.

The prevalence rate of cardiovascular disease is higher for males than females, and estradiol (E2) induces AMP-activated protein kinase (AMPK) activation, which is known to regulate proliferation of VSMC. We identified the estrogenic properties of nordihydroguaiaretic acid (NDGA, a lignan phytoestrogen) that inhibit VSMC proliferation and explored the underlying mechanisms. Both the phosphorylation and expression of LKB1 were increased by NDGA. In addition, NDGA significantly attenuated angiotensin II (Ang II)-induced VSMC proliferation. To elucidate the estrogenic effects, we confirmed that NDGA increased estrogen receptor α (ERα) expression, similar to treatment with E2 and estriol (E3). Furthermore, tamoxifen and ERα siRNA obstructed the effects of NDGA including ERα expression, AMPK phosphorylation and both LKB1 phosphorylation and expression. VSMC proliferation was restored by tamoxifen and ERα siRNA. LKB1 siRNA also reversed the NDGA-mediated inhibition of VSMC proliferation. The estrogenic activity of NDGA induced LKB1 translocation from nucleus to cytosol, and tamoxifen obstructed LKB1 translocation. The absence of LKB1 completely abolished the increase of ERα expression induced by NDGA. Taken together, the beneficial effects of estrogenic compound (E2 and NDGA) on inhibition of VSMC proliferation are mediated by interaction between LKB1 and ERα, suggesting a potential mechanism for females having less cardiovascular disease.

Production and Analysis of Biological Properties of Recombinant Human Apolipoprotein A-I.

Production of recombinant human apolipoprotein A-I (apoA-I) in E. coli cells is described and its biological properties are compared with those of natural protein. Recombinant apoA-I was isolated as a chimeric polypeptide and then processed to a mature form apoA-I (rapo-I). We studied the ability of the resulting protein to penetrate into hepatocyte nuclei and regulate the rate of DNA biosynthesis in complex with estriol. Penetration of rapoA-I conjugated with FITC into hepatocyte nuclei was demonstrated. rapoA-I-estriol and apoA-I-estriol complexes induced similar increase in DNA biosynthesis rate in isolated hepatocytes, which confi rms functional similarity of the obtained recombinant mature protein (rapoA-I) and native human apoA-I.

Estrone - a partial estradiol antagonist in the normal breast.

Oral hormone replacement therapy (HRT) based on estradiol-17ß (E2) greatly increases circulating estrone (E1) levels. E1 is an estrogen receptor agonist but may also be a partial E2 antagonist. We investigated the effects of circulating E1 on the association between circulating E2 and the increase in mammographic density (∂MD) in 46 healthy post-menopausal women treated with E2 2 mg and norethisterone acetate 1 mg daily. MD and serum E1 and E2 were measured before and after 6 months of treatment. At high E1 levels, ∂MD showed significant positive correlations leading to increase (∂-values) in both E1 and E2. Lowering the upper serum E1 limit strengthened the correlations to ∂E2 while the significant correlations to ∂E1 disappeared. E1 at high concentrations may act as a partial E2 antagonist also in the normal breast in vivo and disturb relationships between circulating E2 and biological estrogen effects. When investigating the relations between circulating steroids and their effects, structurally related compounds, which may act as partial antagonists, have to be considered, at least when they are present in higher concentrations.

Effects of OsteoKing on osteoporotic rabbits.

Heng-Gu-Gu-Shang-Yu-He-Ji, also known as OsteoKing, is used as a herbal Traditional Chinese Medicine for the treatment of bone disease, including femoral head necrosis and osteoarthritis. However, whether OsteoKing has anti-osteoporotic properties has remained to be elucidated. The purpose of the present study was therefore to investigate the effects of OsteoKing on ovariectomy-induced osteoporosis in rabbits. Female New Zealand white rabbits were randomly divided into an ovariectomized (OVX) group and a sham-surgery group. The rabbits in the OVX group were subjected to an ovariectomy, while the rabbits in the sham group were subjected to the removal of an area of fat near the two ovaries. Bone mineral density, mechanical properties, serum biochemical parameters and micro-architecture were examined at 150 days post-OVX to characterize the experimental animal model. Once the osteoporotic rabbit model had been established, the rabbits in the OVX group were divided into the following groups: Model group, nilestriol group and 300 and 600 mg/kg OsteoKing groups, containing 16 rabbits in each group. OsteoKing and nilestriol were administered orally. The bone mineral density, mechanical properties, serum biochemical parameters, histology and micro-architecture were examined using dual-energy X-ray absorptiometric analysis, mechanical assessments, enzyme-linked immunosorbent assays, histopathological evaluation and micro-computerized tomography examination following 60 days and 120 days of treatment, respectively. Treatment with OsteoKing led to an elevation in the bone mineral density of the vertebra and serum phosphorus levels, reduced serum concentrations of osteocalcin, procollagen type I N-terminal peptide, tartrate-resistant acid phosphatase 5b and cross-linked N-telopeptide of type I collagen, improved mechanical properties (maximum load, stiffness and energy absorption capacity), and micro-architecture of the lumbar vertebra in the OVX osteoporotic rabbit model following treatment for 120 days. In conclusion, it was demonstrated that OsteoKing is effective in the prevention of estrogen deficiency-associated bone loss and may be a promising drug for the treatment of post-menopausal osteoporosis.

Oestetrol stimulates proliferation and oestrogen receptor expression in breast cancer cell lines: comparison of four oestrogens.

OBJECTIVES: Oestetrol (15-alpha-hydroxyoestriol, E4) is an endogenous oestradiol metabolite mainly produced at high concentrations in the fetal liver. In earlier studies E4 was investigated for its use as marker for pregnancies at risk, especially with vascular problems. Some current investigations suggest that the use of E4 in hormone therapy or contraception may have advantages in terms of breast cancer risk when compared to other oestrogens. METHODS: Proliferation of two oestrogen receptor-positive breast cancer cell lines (ZR 75-1 and HCC 1500) was investigated after incubation with oestrone (E1), oestradiol (E2), oestriol (E3), and oestetrol (E4). Receptor expression of oestrogen receptor-alpha (ERα) and -beta (ERß) was determined by Western-Blot. RESULTS: All four oestrogens elicited a significant proliferative stimulation at concentrations of 10(-10) und 10(-9) M as compared to controls. Oestrone displayed a significantly weaker effect than E2. Oestetrol was significantly less effective than E2 at the lower concentration. Expression of ERα and ERß was significantly upregulated by all oestrogens tested, without differences between the latter. CONCLUSIONS: Our results indicate a slightly lower proliferative effect of E4, but only at low concentrations. However, no difference was found regarding receptor expression. Breast cancer risk associated with use of oestetrol should be tested in clinical trials.

Estriol blunts postprandial blood glucose rise in male rats through regulating intestinal glucose transporters.

Despite increased total food intake in healthy, late-stage pregnant women, their peak postprandial blood sugar levels are normally much lower than the levels seen in healthy nonpregnant women. In this study, we sought to determine whether estriol (E3), an endogenous estrogen predominantly produced during human pregnancy, contributes to the regulation of the postprandial blood glucose level in healthy normal rats. In vivo studies using rats showed that E3 blunted the speed and magnitude of the blood glucose rise following oral glucose administration, but it did not appear to affect the total amount of glucose absorbed. E3 also did not affect insulin secretion, but it significantly reduced the rate of intestinal glucose transport compared with vehicle-treated animals. Consistent with this finding, expression of the sodium-dependent glucose transporter 1 and 2 was significantly downregulated by E3 treatment in the brush-border membrane and basolateral membrane, respectively, of enterocytes. Most of the observed in vivo effects were noticeably stronger with E3 than with 17ß-estradiol. Using differentiated human Caco-2 enterocyte monolayer culture as an in vitro model, we confirmed that E3 at physiologically relevant concentrations could directly inhibit glucose uptake via suppression of glucose transporter 2 expression, whereas 17ß-estradiol did not have a similar effect. Collectively, these data showed that E3 can blunt the postprandial glycemic surge in rats through modulating the level of intestinal glucose transporters.

Inhibition of GPR30 by estriol prevents growth stimulation of triple-negative breast cancer cells by 17ß-estradiol.

BACKGROUND: Due to the lack of ERα, triple negative breast cancers (TNBCs) are not susceptible to endocrine therapy using antiestrogens. However, the majority of TNBCs express the membrane bound estrogen receptor GPR30. We have recently shown that knock-down of GPR30 expression prevented growth stimulation of TNBC cell lines by 17ß-estradiol. Now we analyzed whether specific inhibition of GPR30 represents a new option for therapy of TNBC. METHODS: Growth of TNBC cells was assessed using Alamar-blue colorimetric assay. Activation of c-Src and EGF-receptor was assessed using Western blots. Expression of c-fos, cyclin D1 and aromatase was quantified by RT-PCR. Gα-specific signaling of GPR30 was analyzed by electrophoretic mobility shift assay. RESULTS: HCC1806 cells showed the highest GPR30 expression, in HCC70 cells it was clearly lower, in MDA-MB-231 cells it was lowest. 10-8 M 17ß-estradiol significantly increased proliferation of HCC1806 cells to 134 ± 12% of control (p < 0.01). Proliferation of HCC70 cells was slightly increased to 116 ± 8% of control. Estriol significantly reduced cell number of HCC1806 cells to 16 ± 12% (p < 0.01). Cell number of HCC70 cells and of MDA-MB-231 cells was reduced to 68 ± 25% and to 61 ± 10%, respectively.Activity of Src kinase increased to 150 ± 10% (p < 0.05) by 10-8 M 17ß-estradiol treatment in HCC1806 and to 220 ± 20% in HCC70 cells (p < 0.01). Estriol treatment completely inhibited 17ß-estradiol-induced p-src activation. Transactivation of EGF-receptor increased by estradiol treatment to 350% in HCC1806 and to 280% in HCC70 cells. Estriol completely suppressed EGF-receptor transactivation. c-fos expression increased to 260% and to 190%, respectively. Estriol reduced this induction to 160% (HCC1806) and below control in HCC70 cells. Cyclin D1 was induced to 290% (HCC1806) and 170% (HCC70) and completely inhibited by estriol. 17ß-estradiol increased CREB-phosphorylation to 400%. Binding of phospho-CREB to a CRE of cyclin D1 was enhanced to 320%. CONCLUSION: Specific pharmacological inhibition of GPR30 might become a promising targeted therapy for TNBC in future.

Progesterone induces apoptosis of insulin-secreting cells: insights into the molecular mechanism.

Progesterone has been associated with the development of gestational diabetes (GD) due to the enhancement of insulin resistance. As ß-cell apoptosis participates in type 1 and type 2 diabetes pathophysiology, we proposed the hypothesis that progesterone might contribute to the development of GD through a mechanism that also involves ß-cell death. To address this question, RINm5F insulin-producing cells were incubated with progesterone (25-100 µM), in the presence or absence of α-tocopherol (40 µM). After 24 or 48 h, membrane integrity and DNA fragmentation were analyzed by flow cytometry. Caspase activity was used to identify the mode of cell death. The involvement of endoplasmic reticulum stress in the action of progesterone was investigated by western blotting. Oxidative stress was measured by 2',7'-dichlorofluorescein diacetate (DCFDA) oxidation. Isolated rat islets were used in similar experiments in order to confirm the effect of progesterone in primary ß-cells. Incubation of RINm5F cells with progesterone increased the number of cells with loss of membrane integrity and DNA fragmentation. Progesterone induced generation of reactive species. Pre-incubation with α-tocopherol attenuated progesterone-induced apoptosis. Western blot analyses revealed increased expression of CREB2 and CHOP in progesterone-treated cells. Progesterone caused apoptotic death of rat islet cells and enhanced generation of reactive species. Our results show that progesterone can be toxic to pancreatic ß-cells through an oxidative-stress-dependent mechanism that induces apoptosis. This effect may contribute to the development of GD during pregnancy, particularly under conditions that require administration of pharmacological doses of this hormone.