Enhanced pup retrieval behaviour in a mouse model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy to affect women of reproductive-age world-wide. Hyperandrogenism is both a hallmark feature of PCOS, and is hypothesised to be an underlying mechanism driving the development of the condition in utero. With circulating hormones known to profoundly influence maternal responses in females, we aimed to determine whether maternal behaviour is altered in a well-described prenatally androgenised (PNA) mouse model of PCOS. Mouse dams were administered with dihydrotestosterone or vehicle on days 16, 17 and 18 of pregnancy. Maternal responses were assessed in both the dihydrotestosterone-injected dams following parturition and in their adult female PNA offspring. Exposure of dams to excess androgens during late pregnancy had no detrimental effects on pregnancy outcomes, including gestation length, pup survival and gestational weight gain, or on subsequent maternal behaviour following parturition. By contrast, PNA virgin females, modelling PCOS, exhibited enhanced maternal behaviour when tested in an anxiogenic novel cage environment, with females rapidly retrieving pups and nesting with them. In comparison, most control virgin females failed to complete this retrieval task in the anxiogenic environment. Assessment of progesterone receptor and oestrogen receptor α immunoreactivity in the brains of virgin PNA and control females revealed increased numbers of oestrogen receptor α positive cells in the brains of PNA females in regions well known to be important for maternal behaviour. This suggests that increased oestrogenic signalling in the neural circuit that underlies maternal behaviour may be a possible mechanism by which maternal behaviour is enhanced in PNA female mice.

Activable Targeted Protein Degradation Platform Based on Light-triggered Singlet Oxygen.

Targeted protein degradation technologies (e.g., PROTACs) that can selectively degrade intracellular protein are an emerging class of promising therapeutic modalities. Herein, we describe the conjugation of photosensitizers and protein ligands (PS-Degrons), as an activable targeted protein degradation platform. PS-Degrons are capable of degrading protein of interest via light-triggered 1O2, which is orthogonal and complementary to existing technologies. This generalizable platform allows controllable knockdown of the target protein with high spatiotemporal precision. Our lead compound PSDalpha induces a complete degradation of human estrogen receptor α (ERα) under visible light. The high degrading ERα efficacy of PSDalpha enables an excellent anti-proliferation performance on MCF-7 cells. Our results establish a modular strategy for the controllable degradation of target proteins, which can hopefully overcome the systemic toxicity in clinical treatment of PROTACs. We anticipate that PS-Degrons would open a new chapter for biochemical research and for the therapeutics.

Design, synthesis, and biological evaluation of novel morpholinated isatin-quinoline hybrids as potent anti-breast cancer agents.

Keeping in view the emerging need for potent and safer anti-breast cancer agents as well as the pharmacological attributes of isatin, quinolone, and morpholine derivatives, novel hydrazine-linked morpholinated isatin-quinoline hybrids were designed, synthesized, and evaluated as anti-breast cancer agents. The synthesized hybrid compounds were preliminarily screened against two breast cancer cell lines (MCF-7 and MDA-MB-231). Almost all synthetics showed potent inhibitory potential against hormone-positive MCF-7 cells while being inactive against hormone-negative MDA-MB-231 cells. Potent compounds were further evaluated against the L929 (noncancerous skin fibroblast) cell line and found to be highly selective for MCF-7 cells over L929 cells. Cell cycle analysis confirmed that the most potent compound AS-4 (MCF-7: GI50 = 4.36 µM) causes mitotic arrest at the G2 /M phase. Due to higher selectivity toward estrogen receptor alpha (ERα)-dependent MCF-7 cells, various binding interactions of AS-4 with ERα are also streamlined, suggesting the capability of AS-4 to completely block ERα. Overall, the study suggests that AS-4 can act as a potential lead for further development of potent and safer anti-breast cancer agents.

Calcineurin regulates the stability and activity of estrogen receptor α.

Estrogen receptor α (ER-α) mediates estrogen-dependent cancer progression and is expressed in most breast cancer cells. However, the molecular mechanisms underlying the regulation of the cellular abundance and activity of ER-α remain unclear. We here show that the protein phosphatase calcineurin regulates both ER-α stability and activity in human breast cancer cells. Calcineurin depletion or inhibition down-regulated the abundance of ER-α by promoting its polyubiquitination and degradation. Calcineurin inhibition also promoted the binding of ER-α to the E3 ubiquitin ligase E6AP, and calcineurin mediated the dephosphorylation of ER-α at Ser294 in vitro. Moreover, the ER-α (S294A) mutant was more stable and activated the expression of ER-α target genes to a greater extent compared with the wild-type protein, whereas the extents of its interaction with E6AP and polyubiquitination were attenuated. These results suggest that the phosphorylation of ER-α at Ser294 promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser118 by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to ß-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser294 and Ser118 Finally, the expression of the calcineurin A-α gene (PPP3CA) was associated with poor prognosis in ER-α-positive breast cancer patients treated with tamoxifen or other endocrine therapeutic agents. Calcineurin is thus a promising target for the development of therapies for ER-α-positive breast cancer.

BPA-induced prostatic hyperplasia in vitro is correlated with the unbalanced gene expression of AR and ER in the epithelium and stroma.

As a typical environmental endocrine disruptor (EED), bisphenol A (BPA) can induce pathological hyperplasia of the prostatic epithelium and stroma. This study concentrates mainly on the effect and underlying mechanisms of BPA on prostatic hyperplasia, which is based on the culture of primary human prostate epithelial cells (HPEpiC) and human prostate fibroblasts (HPrF). In an effect to screen the optimal pro-survival BPA levels, HPEpiC and HPrF were, respectively, exposed to concentration gradients of BPA (10-12 M-10-4 M) solution diluted with two corresponding medium and incubated for 72 h at 37°C. CCK-8 assay showed that 10-9 M-10-5 M BPA could facilitate the proliferation of HPEpiC, while similar proliferative effect of HPrF only needed 10-11 M-10-7 M BPA. HPrF were more sensitive to BPA than HPEpiC. The qualification of PCNA gene expression measured using quantitative real-time polymerase chain reaction (qRT-PCR) also mirrored the BPA-induced cell proliferation. Additionally, our results considered that androgen receptor (AR), estrogen receptor (ERα, ERß), and NFKB1 gene expressions exhibited up-regulation in HPEpiC treated with 10-9 M BPA for 72 h. However, in HPrF, the identical BPA treatment could activate ERα, ERß, and NFKB1 gene expressions and down-regulated the expression of AR levels. It is further confirmed that low-dose BPA can indeed promote the proliferation of human prostate cells in vitro, and the mechanisms of BPA for prostatic epithelial and stromal hyperplasia may not be consistent.

A new α-pyrone from Arthrinium pseudosinense culture medium and its estrogenic activity in MCF-7 cells.

A new α-pyrone analog, arthrifuranone A (1) was isolated from an EtOAc-extract of Arthrinium pseudosinense culture medium. The isolation workflow was guided by a Molecular Networking-based dereplication strategy. The chemical structure of the new compound was elucidated using MS and NMR spectroscopic techniques, and the absolute configuration was established by the Mosher's method and gauge-including atomic orbital NMR chemical shift calculations, followed by DP4 + analysis. The isolated compound was evaluated for its estrogenic activity using the MCF-7 estrogen responsive human breast cancer cells. Compound 1 showed estrogenic activity by increasing the proliferation of MCF-7 cells at the concentration of 3.125 µM via phosphorylation of estrogen receptor-α.

Doxazosin and erlotinib have anticancer effects in the endometrial cancer cell and important roles in ERα and Wnt/ß-catenin signaling pathways.

ERα and Wnt/ß-catenin pathways are critical for the progression of most endometrial cancers. We aimed to investigate the cytotoxic and apoptotic effects of tamoxifen and quinazoline derivative drugs of doxazosin and erlotinib, and their roles in ERα and Wnt/ß-catenin signaling pathways in human endometrial cancer RL 95-2 cell. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay and xCELLigence systems were performed to evaluate cytotoxicity. Furthermore, apoptotic induction was tested by Annexin V analysis. Caspase-3 and -9 activity and changes in the mitochondrial membrane potential were evaluated. The level of reactive oxygen species was measured by incubating with dichlorofluorescein diacetate. Protein ratios of p-ERα/ERα, GSK3ß/p-GSK3ß, and p-ß-catenin/ß-catenin and expression levels of ESR1, EGFR, c-Myc genes were evaluated to elucidate mechanisms in signaling pathways. We found that the tested drugs showed cytotoxic and apoptotic effects in the cells. Doxazosin significantly reduced ESR1 expression, slightly reduced the p-ß-catenin/ß-catenin ratio and c-Myc expression. Erlotinib significantly increased c-Myc expression while significantly decreasing the p-ß-catenin/ß-catenin and p-ERα/ERα ratio, and ESR1 expression. However, we observed that the cells develop resistance to erlotinib over a certain concentration, suggesting that ERα, ESR1, EGFR, and c-Myc may be a new target for overcoming drug resistance in the treatment of endometrial cancer. We also observed that erlotinib and doxazosin play an important role in the ERα signaling pathway and can act as potent inhibitors of PKA and/or tyrosine kinase in the Wnt/ß-catenin signaling pathway in RL 95-2 cell. In conclusion, doxazosin and erlotinib may have a possible therapeutic potential in human endometrial cancer.

USP35, regulated by estrogen and AKT, promotes breast tumorigenesis by stabilizing and enhancing transcriptional activity of estrogen receptor α.

Although endocrine therapies targeting estrogen receptor α (ERα) are effective in managing ER positive (+) breast cancer, many patients have primary resistance or develop resistance to endocrine therapies. In addition, ER+ breast cancer with PIK3CA activating mutations and 11q13-14 amplification have poor survival with unclear mechanism. We uncovered that higher expression of deubiquitinase USP35, located in 11q14.1, was associated with ER+ breast cancer and poor survival. Estrogen enhanced USP35 protein levels by downregulating USP35-targeting miRNA-140-3p and miRNA-26a-5p. USP35 promoted the growth of ER+ breast cancer in vitro and in vivo, and reduced the sensitivity of ER+ breast cancer cells to endocrine therapies such as tamoxifen and fulvestrant. Mechanistically, USP35 enhanced ERα stability by interacting and deubiquitinating ERα, and transcriptional activity of ERα by interacting with ERα in DNA regions containing estrogen response element. In addition, AKT, a key effector of PI3K, phosphorylated USP35 at Serine613, which promoted USP35 nuclear translocation, ERα transcriptional activity, and the growth of ER+ breast cancer cells. Our data indicate that USP35 and ERα form a positive feedback loop in promoting the growth of ER+ breast cancer. USP35 may be a treatment target for ER+ breast cancer with endocrine resistance or with PIK3CA mutations or hyperactivation of the PI3K pathway.

Maternal exposure of mice to sodium p-perfluorous nonenoxybenzene sulfonate causes endocrine disruption in both dams and offspring.

The toxicity of certain novel perfluoroalkyl substances (PFCs) has attracted increasing attention. However, the toxic effects of sodium p-perfluorous nonenoxybenzene sulfonate (OBS) on the endocrine system have not been elucidated. In this study, OBS was added to the drinking water during the pregnancy and lactation of the healthy female mice at dietary levels of 0.0 mg/L (CON), 0.5 mg/L (OBS-L), and 5.0 mg/L (OBS-H). OBS exposure during the pregnancy and lactation resulted in the presence of OBS residues in the placenta and fetus. We also analyzed physiological and biochemical parameters and gene expression levels in mice of the F0 and F1 generations after maternal OBS exposure. The total serum cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels were significantly increased in female mice of the F0 generation. The androgen levels in the serum and the ovarian mRNA levels of androgen receptor (AR) also tended to increase after maternal OBS exposure in the F0 generation mice. Moreover, maternal OBS exposure altered the mRNA expression of endocrine-related genes in male mice of F1 generation. Notably, the serum TC and LDL-C levels were significantly increased in 8-weeks-old male mice of the F1 generation, and the serum high-density lipoprotein cholesterol (HDL-C) levels were decreased in 24-week-old male mice of the F1 generation. These results indicated that maternal OBS exposure can interfere with endocrine homeostasis in the F0 and F1 generations. Therefore, exposure to OBS during pregnancy and lactation has the potential toxic effects on the dams and male offspring, which cannot be overlooked.

Perinatal activation of ERα and ERß but not GPER-1 masculinizes female rat caudate-putamen medium spiny neuron electrophysiological properties.

Exposure to steroid sex hormones such as 17ß-estradiol (estradiol) during early life potentially permanently masculinize neuron electrophysiological phenotype. In rodents, one crucial component of this developmental process occurs in males, with estradiol aromatized in the brain from testes-sourced testosterone. However, it is unknown whether most neuron electrophysiological phenotypes are altered by this early masculinization process, including medium spiny neurons (MSNs) of the rat caudate-putamen. MSNs are the predominant and primary output neurons of the caudate-putamen and exhibit increased intrinsic excitability in females compared to males. Here, we hypothesize that since perinatal estradiol exposure occurs in males, then a comparable exposure in females to estradiol or its receptor agonists would be sufficient to induce masculinization. To test this hypothesis, we injected perinatal female rats with estradiol or its receptor agonists and then later assessed MSN electrophysiology. Female and male rats on postnatal day 0 and 1 were systemically injected with either vehicle, estradiol, the estrogen receptor (ER)α agonist PPT, the ERß agonist DPN, or the G-protein-coupled receptor 1 (GPER-1) agonist G1. On postnatal days 19 ± 2, MSN electrophysiological properties were assessed using whole cell patch clamp recordings. Estradiol exposure abolished increased intrinsic excitability in female compared to male MSNs. Exposure to either an ERα or ERß agonist masculinized female MSN evoked action potential firing rate properties, whereas exposure to an ERß agonist masculinized female MSN inward rectification properties. Exposure to ER agonists minimally impacted male MSN electrophysiological properties. These findings indicate that perinatal estradiol exposure masculinizes MSN electrophysiological phenotype via activation of ERα and ERß.NEW & NOTEWORTHY This study is the first to demonstrate that estradiol and estrogen receptor α and ß stimulation during early development sexually differentiates the electrophysiological properties of caudate-putamen medium spiny neurons, the primary output neuron of the striatal regions. Overall, this evidence provides new insight into the neuroendocrine mechanism by which caudate-putamen neuron electrophysiology is sexually differentiated and demonstrates the powerful action of early hormone exposure upon individual neuron electrophysiology.

Daidzein Induces Intrinsic Pathway of Apoptosis along with ER α/ß Ratio Alteration and ROS Production.

BACKGROUND: Low risk of breast cancer is observed among females consuming a moderate quantity of soy throughout their life. The present study was conducted to evaluate the anticancer potential of Daidzein, one of the major Isoflavones in soy using Human breast cancer cells MCF-7. METHODS: MCF-7 were subjected to various doses of Daidzein treatment to determine the IC50 value. Onset of apoptosis was ascertained by AnnexinV assay and caspase 3/7 activity post treatment. Expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 was also assessed to further confirm apoptotic mode of cell death. ROS production post treatment with Daidzein was assessed to ascertain the apoptosis via intrinsic pathway. Expression of ER α and ER ß was evaluated by western blot analysis. RESULTS: Human breast cancer cells MCF-7 were found to be sensitive to Daidzein treatment, with an IC50 value of 50µM. Increased percentage of treated cells stained with Annexin V confirmed apoptosis mediated cell death. Activity of Caspase 3/7 activity was found to be 1.4-fold higher in Daidzein treated cells than control cells, confirming apoptosis. Daidzein caused over expression of Bax and down-regulated expression of Bcl2. There has been an outburst of ROS in Daidzein treated cells indicating that Daidzein induces apoptosis via intrinsic pathway. A decrease in the expression of ER α and increase in levels of ER ß has been observed which are conducive indicator of apoptosis. CONCLUSIONS: In conclusion, the present study suggests that Daidzein induces apoptosis in MCF-7 cells by mitochondrial pathway along with lowering the ratio of ER α/ß and an outburst of Reactive Oxygen Species(ROS).

Cyclic depsipeptide mycotoxin exposure may cause human endocrine disruption: Evidence from OECD in vitro stably transfected transcriptional activation assays.

The presence of cyclic depsipeptide mycotoxins in foods and feedstuffs could potentially cause endocrine disrupting effects on humans and wildlife by their inhibition of active steroidogenesis. Therefore, we attempted to assess the human estrogen receptor (ER) and androgen receptor (AR) agonistic/antagonistic effects of representative cyclic depsipeptide mycotoxins, enniatin A1 (ENN A1), and enniatin B1 (ENN B1), by OECD Performand Based Test Guideline (PBTG) No.455, VM7Luc ER transcriptional activation (TA) assay and OECD TG No. 458, 22Rv1/MMTV_GR-KO AR TA assay. No tested cyclic depsipeptide mycotoxins were found to be ER and AR agonists in VM7Luc ER TA and 22Rv1/MMTV_GR-KO AR TA assays. On the other hand, ENN A1, and ENN B1 exhibited the ER and AR antagonistic effects with IC30 and IC50 values in both TA assays. These two cyclic depsipeptide mycotoxins, which were determined as ER and AR antagonists by two in vitro assays, bound to ERα, and AR. Then ENN A1, and ENN B1 inhibited the dimerization of ERα, and AR. These results, for the first time indicated that ENN A1, and ENN B1 could have potential endocrine disrupting effects mediated by interaction of ERα and AR using international standard testing methods to determine the potential endocrine disrupting chemical.

Narasin inhibits tumor metastasis and growth of ERα­positive breast cancer cells by inactivation of the TGF­ß/SMAD3 and IL­6/STAT3 signaling pathways.

Treatment of human estrogen receptor (ER)­positive breast cancer (ER+ BC) using conventional chemotherapy remains a challenge and is often ineffective as a result of tumor metastasis. The present study aimed to investigate the ability of narasin, an ionophore antibiotic, to potentially inhibit tumor metastasis and growth in human ER+ BC. Narasin was found to have significant inhibitory abilities on cell proliferation, migration and invasion in ER+ BC cell lines MCF­7 and T47D compared with the triple­negative BC cell MDA­MB­231. For the in vivo studies, narasin effectively decreased the number of tumor metastasis nodules, tumor volume and weight without apparent toxicity in human MCF­7 nude mouse left ventricle injection tumor metastasis and xenograft models. Mechanistically, it demonstrated that exposure to TGF­ß or IL­6 induced the expression of epithelial­mesenchymal transition (EMT) markers in ER+ BC cell lines. On the contrary, narasin dose­dependently reversed EMT by increasing the expression of E­cadherin and decreasing the expression of N­cadherin, vimentin, ß­catenin and zinc finger E­box­binding homeobox 1 at the protein and gene expression levels. Gene microarray, molecular docking and western blotting were performed to demonstrate that those protein and gene expression levels are regulated by the inactivation of the TGF­ß/phosphorylated (p)­SMAD3 and IL­6/p­STAT3 signaling pathways. Taken together, these findings indicated that narasin may be a promising candidate that can be further optimized for the treatment of human ER+ BC.

Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle Cells.

RATIONALE: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an ER (estrogen receptor) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17ß-estradiol in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear. OBJECTIVE: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms. METHODS AND RESULTS: Using 3 complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with 17ß-estradiol and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα subfunctions (membrane/extranuclear versus genomic/transcriptional) demonstrated that 17ß-estradiol-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells. CONCLUSIONS: Whereas tamoxifen acts as an antiestrogen and ERα antagonist in breast cancer but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.

Inhibition of brain 17ß-estradiol synthesis by letrozole induces cognitive decline in male and female rats.

BACKGROUND: Hippocampal aromatase is responsible for local synthesis of 17ß-estradiol (E2) that has much higher concentrations than serum levels in males and females. Letrozole, an aromatase inhibitor, passes through the brain barriers, distributes to the brain, and affects local E2 synthesis. Here, the effects of intra-cerebroventricular (ICV) letrozole administration in the presence and absence of gonads were examined on the cognitive abilities of male and female rats. METHOD: Animals received intra-ICV injection of letrozole or vehicle for 14 consecutive days. Spatial working memory, novel object recognition memory, and anxiety-related behavior, were evaluated using Y-maze, object recognition test, and elevated plus maze, respectively. The E2 levels in the serum and hippocampal tissue were measured by the ELISA technique. RT-PCR was performed to assess the hippocampal estrogen receptors (ER) expression. Moreover, letrozole effect on neuronal activity of CA1 pyramidal neurons was studied by in vivo single-unit recording. RESULTS: Letrozole (0.2, 0.4, and 0.8 µg) significantly decreased the hippocampal E2 levels compared to the vehicle group. Letrozole caused cognitive impairments in a dose-dependent manner in male and female rats in the presence or absence of gonads. Dose-response analysis revealed that the minimum effective dose of letrozole on the behavioral measures was 0.4 µg. Letrozole also caused an up-regulation of ERα and ERß and a down-regulation of GPR30 gene expression. The firing rate of pyramidal neurons was reduced by letrozole in gonadal-intact animals. CONCLUSION: The detrimental effects of letrozole treatment on cognitive abilities in the presence and absence of gonads indicate that local E2 synthesis in the hippocampus is a crucial factor in normal cognitive performance. The suppressive effect of letrozole on hippocampal neuronal firing might alter synaptic plasticity that is critical for memory formation. These data potentially suggest that memory deficits following letrozole administration should be monitored.

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

Interactions of bisphenol diglycidyl ethers with estrogen receptors α: Fluorescence polarization, reporter gene, and molecular modeling investigations.

Based on human estrogen receptor α ligand binding domain (hERα-LBD) as recognition element, a fluorescence polarization assay was developed for the determination of bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE), and their derivatives. Fluorescence polarization assay showed that BADGE, BFDGE and their derivatives exhibited dose-dependent binding to the receptor protein. The results of reporter gene assay indicated that all the tested bisphenol diglycidyl ethers show no agonistic activities, but some of them exhibit anti-estrogenic activities toward ERα. All the tested bisphenol diglycidyl ethers fitted into the hydrophobic binding pocket and adopted the conformation that resembled 4-hydroxytamoxifen, a selective antagonist of ERα. Quantitative structure-activity relationship analysis showed that the binding potencies of bisphenol diglycidyl ethers with hERα-LBD might be structure-dependent. This work may provide insight into the in silico screening of ER ligands from unsuspected chemicals.

8-prenylgenistein exerts osteogenic effects via ER α and Wnt-dependent signaling pathway.

8-prenylgenistein (8PG) was previously reported to exert stronger osteogenic activity than genistein, a well-known soy phytoestrogen. However, the molecular mechanism underlying the actions of 8PG on osteoblasts was far from clear. In the present study, the osteogenic effects and mechanisms of 8PG and genistein were studied using human BMSC and murine pre-osteoblast MC3T3-E1 cells. Our results indicated that the stimulatory effects of 8PG and genistein on osteoblast differentiation were abolished by co-incubation with MPP (10-6 M, an ERα antagonist), but not PHTPP (10-6 M, an ERß antagonist). Molecular docking indicated that the binding mode of 8PG toward ERα was similar to that of genistein and therefore could not account for their differential osteogenic actions. In silico target profiling identified the involvement of glycogen synthase kinase-3ß (GSK-3ß), a key mediator of Wnt/ß-catenin pathway, in the actions of 8PG. However, instead of directly inhibiting GSK-3ß enzymatic activities, 8PG and genistein were found to induce GSK-3ß phosphorylation at Serine-9 in osteoblastic MC3T3-E1 cells. 8PG exerted more potent effects than genistein in stimulating expressions of LRP5, ß-catenin, Runx2, osteocalcin, alp, opg, major protein and gene markers involved in Wnt signaling pathway in MC3T3-E1 cells. Moreover, the inhibition of Wnt signaling by DKK1 could be restored by treatment with 8PG and genistein. However, 8PG, but not genistein, stimulated ERα-dependent ß-catenin protein expression in MC3T3-E1 cells. Furthermore, the increase in ALP activity, LRP5 and phospho-Akt/Akt expression by 8PG and genistein were abolished by co-treatment with LY294002 (10-5 M, a PI3K pathway inhibitor). Collectively, our results suggested that the osteogenic activities of 8PG was mediated by GSK-3ß phosphorylation through the induction of Wnt/ß-catenin and ERα-associated PI3K/Akt signaling.

Roflumilast attenuates cognitive deficits in estrogen insufficient rats.

Estrogen replacement therapy including specific estrogen receptor alpha (ERα) agonist, 4,4',4″-(4-propyl-[1H] pyrazole-1,3,5-triyl) trisphenol (PPT), improves cognitive function in the females with estrogen insufficiency condition. It is well suggested that the cyclic nucleotides are considered as one of the downstream mediators to ERα receptor activity and they can be hypothesized as a potential target in the management of estrogen insufficiency condition. Roflumilast, a phosphodiesterase-4 inhibitor, increases the level of cyclic adenosine monophosphate (cAMP) in most of the tissues including the brain, and is reported to have procognitive activity in the experimental animals. Hence, the present study evaluated the therapeutic effect of roflumilast with or without PPT in rats with experimentally-induced estrogen insufficiency. Estrogen insufficiency was induced in female rats through bilateral ovariectomy on day-1 (D-1) of the experimental schedule. Roflumilast (0.3 and 1.0 mg/kg; p.o.) and PPT (333µg/kg; i.p.) attenuated ovariectomy-induced cognitive deficits in the rodents during behavioral tests. Roflumilast and PPT increased the cholinergic function and cAMP level in the rat hippocampus and prefrontal cortex. Further, ovariectomy-induced decrease in the extent of phosphorylation of ERα in both the brain regions was attenuated with the monotherapy of either roflumilast or PPT. Interestingly, the combination of 1.0 mg/kg roflumilast and PPT exhibited better therapeutic effectiveness than their monotherapy. In addition, roflumilast facilitated PPT-induced increase in the level of expression of phosphorylated protein kinase-B (Akt) in both the rat brain regions. Hence, it can be assumed that the combination of roflumilast and PPT could be a therapeutic option in the management of estrogen insufficiency-induced disorders.

The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination.

Myelin loss is the hallmark of the demyelinating disease multiple sclerosis (MS) and plays a significant role in multiple neurodegenerative diseases. A common factor in all neuropathologies is the central role of microglia, the intrinsic immune cells of the central nervous system (CNS). Microglia are activated in pathology and can have both pro- and anti-inflammatory functions. Here, we examined the effects of the flavonoid agathisflavone on microglia and remyelination in the cerebellar slice model following lysolecithin induced demyelination. Notably, agathisflavone enhances remyelination and alters microglial activation state, as determined by their morphology and cytokine profile. Furthermore, these effects of agathisflavone on remyelination and microglial activation were inhibited by blockade of estrogen receptor α. Thus, our results identify agathisflavone as a novel compound that may act via ER to regulate microglial activation and enhance remyelination and repair.