Estrogen Receptor Binding Affinity Research Articles

Synthesis Studies and the Evaluation of C6 Raloxifene Derivatives.

Methodology is described for the synthesis of C6 derivatives of raloxifene, a prescribed drug for the treatment and prevention of osteoporosis. Studies have explored the incorporation of electron-withdrawing substituents at C6 of the benzothiophene core. Efficient processes are also examined to introduce hydrogen bond donor and acceptor functionality. Raloxifene derivatives are evaluated with in vitro testing to determine estrogen receptor (ER) binding affinity and gene expression in MC3T3 cells.

Synthesis and biological evaluation of novel D-ring fused steroidal N(2)-substituted-1,2,3-triazoles.

In this study, a series of 13 new D-ring fused steroidal N(2)-substituted-1,2,3-triazoles were synthesized, characterized and evaluated for their biological activities. The relative binding affinities of the synthesized compounds for the ligand-binding domains of estrogen receptors α and β, androgen receptor and glucocorticoid receptor demonstrated that androstane derivatives 3a and 3h and estratriene derivative 4e showed highly specific and strong binding affinity for estrogen receptor β, while 3b, 3e, 4a and 4b displayed high binding affinity for the glucocorticoid receptor. The synthesized compounds were tested for their ability to inhibit aldo-keto reductases 1C3 and 1C4 in vitro by monitoring NADPH consumption using fluorescence spectroscopy. The most potent aldo-keto reductase 1C3 inhibitors were compounds 3h (71.17%) and 3f (69.9%). Moreover, a molecular docking study was carried out for compounds 3f and 3h against aldo-keto reductase 1C3 and results showed that compounds 3h and 3f could bind in the same site and orientation as EM1404. However, polar atoms in the triazole group enable additional hydrogen bonding deeper in SP1 with Tyr319, Tyr216 and the NADP+ cofactor, which are not visible in the AKR1C3-EM1404 crystal structure. The synthesized compounds were screened for their anticancer activity against four cancer cell lines. Compound 3f demonstrated moderate toxic effects across various cancer types, while displaying lower toxicity towards the healthy cell line. In summary, our findings indicate that N(2)-substituted-1,2,3-triazoles are high-affinity ligands for estrogen receptor β and glucocorticoid receptor, inhibitors of aldo-keto reductase 1C3 enzyme, and exhibit antiproliferative effects against cancer cells, suggesting that they could serve as scaffolds for anticancer drug development.

The obesogenic side of Genistein.

Genistein (GN) has been highly recommended for its medicinal properties like anticancer, antidiabetic, antihyperlipidemic, antiviral, and antioxidant activities among others. Recently, scientists realized that Genistein is an endocrine disruptor. It is an obesogen that interferes with the endocrine system causing obesity through many mechanisms like inducing adipocyte differentiation, lipid accumulation, and transformation of some stem cells into adipocytes (bone marrow mesenchymal stem cells for example) in vitro. Animal studies show that GN upregulates genes associated with adipogenesis like CCAAT/enhancer binding protein alpha (Cebpα), CCAAT/enhancer binding protein beta (Cebpβ), and PPARγ. In silico studies reveal a strong binding affinity for estrogen receptors. All these findings were contingent on concentration and tissues. It is beyond dispute that obesity is one of the most frustrating medical conditions under the sun. The pathophysiology of this disease was first attributed to a high-calorie diet and lack of physical activity. However, studies proved that these two factors are not enough to account for obesity in both children and adults. This mini review highlights how Genistein interaction with the peroxisome proliferator-activated receptor gamma protein can cause obesity.

Exploring aryl hydrocarbon receptor (AhR) as a target for Bisphenol-A (BPA)-induced pancreatic islet toxicity and impaired glucose homeostasis: Protective efficacy of ethanol extract of Centella asiatica

The estrogenic impact of Bisphenol-A (BPA), a widely recognized endocrine disruptor, causes disruption of pancreatic β-cell function through estrogen receptors (ERs). While BPA's binding affinity for ERs is significantly lower than that of its natural counterpart, estrogen, recent observations of BPA's affinity for aryl hydrocarbon receptor (AhR) in specific cellular contexts have sparked a specific question: does AhR play a role in BPA's toxicological effects within the endocrine pancreas? To explore this question, we investigated BPA's (10 and 100 μg/ kg body weight/day for 21 days) potential to activate AhR within pancreatic islets and assessed the protective role of ethanol extract of Centella asiatica (CA) (200 and 400 mg/kg body weight/day for 21 days) against BPA-mediated toxicity in mouse model. Our results indicate that BPA effectively triggers the activation of AhR and modulates its target genes within pancreatic islets. In contrast, CA activates AhR but directs downstream pathways differentially and activates Nrf2. Additionally, CA was observed to counteract the disruption caused by BPA in glucose homeostasis and insulin sensitivity. Furthermore, BPA-induced oxidative stress and exaggerated production of proinflammatory cytokines were effectively counteracted by CA supplementation. In summary, our study suggests that CA influenced AhR signaling to mitigate the disrupted pancreatic endocrine function in BPA exposed mice. By shedding light on how BPA interacts with AhR, our research provides valuable insights into the mechanisms involved in the diabetogenic actions of BPA.

Challenging Approach to the Development of Novel Estrogen Receptor Modulators Based on the Chemical Properties of Guaiazulene.

Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of N,N-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the N,N-dialkylaminoethyl substituent as a privileged functional group for SERMs.

Hibiscus sabdariffa anthocyanins are potential modulators of estrogen receptor alpha activity with favourable toxicology: a computational analysis using molecular docking, ADME/Tox prediction, 2D/3D QSAR and molecular dynamics simulation

The estrogen hormone receptor (ER) mediated gene expression mainly regulate the development and physiology of the primary and secondary reproductive system alongside bone-forming, metabolism and behaviour. Over-expressed ER is associated with several pathological conditions and play a crucial role in breast cancer occurrence, progression and metastasis. Hibiscus sabdariffa L. or roselle is a rich source of naturally occurring polyphenolic compounds that reportedly have robust estrogenic activity. However, the estrogen-like ingredient of the plant remains ambiguous. This study has screened a library of already recorded and less-explored compounds of Hibiscus sabdariffa for their estrogen receptor binding affinity and safety using a suite of computational methods that include protein-ligand docking, ADME and Toxicity prediction, and 2D/3D QSAR. The study revealed that the estrogen-receptor binding potential of Pelargonidin, Delphinidin, Cyanidin, and Hibiscetin are more efficient than popular breast cancer drugs, Tamoxifen and Raloxifene. Besides, the compounds exhibited favourable toxicological parameters with potent bioactivity towards binding ER-α subunit. Thus, these compounds can serve as prototypes for designing novel Selective Estrogen Receptor Modulator molecules with a few structural modifications. This is the first report exploring the phytochemical basis of estrogenic activity of Hibiscus sabdariffa L. Communicated by Ramaswamy H. Sarma

Safety effect of fermented oyster extract on the endocrine disruptor assay in vitro and in vivo

Oyster (Crassostrea gigas) is a marine bivalve mollusk widely distributed in coastal areas, and have been long widely used in industrial resources. Several studies demonstrated that fermented oyster (FO) extract attribute to bone health, but whether administration of FO play as an endocrine disruptor has not been studied. Therefore, in the present study, we investigated the effect of FO on the endocrine system in vitro and in vivo. As the results of the competitive estrogen receptor (ER) and androgen receptor (AR) binding affinities, FO was not combined with ER-α, ER-β, and AR. However, 17β-estradiol and testosterone, used as positive control, were interacted with ER and AR, respectively. Meanwhile, oral administration of 100 mg/kg and 200 mg/kg of FO doesn’t have any harmful effect on the body weight, androgen-dependent sex accessory organs, estrogen-dependent-sex accessory organs, kidney, and liver in immature rats. In addition, FO supplementation has no effect on the serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and 17β-estradiol. However, the relative weight of androgen- and estrogen-dependent organs were significantly increased by subcutaneously injection of 4.0 mg/kg of testosterone propionate (TP) and by orally administration of 1.0 μg of 17α-ethynyl estradiol (EE) in immature male and female rats, respectively. Furthermore, TP and EE administration markedly decreased the serum LH and FSH levels, which are similar those of mature Sprague-Dawley (SD) rat. Furthermore, the testosterone and 17β-estradiol levels were significantly enhanced in TP and EE-treated immature rats. Taken together, our findings showed that FO does not interact with ER and AR, suggesting consequentially FO does not play as a ligand for ER and AR. Furthermore, oral administration of FO did not act as an endocrine disruptor including androgenic activity, estrogenic activity, and abnormal levels of sex hormone, indicating FO may ensure the safety on endocrine system to develop dietary supplement for bone health.

Evidence of increased estrogenicity upon metabolism of Bisphenol F - Elucidation of the key metabolites

The increasing concern over bisphenol A (BPA) has directed much attention toward bisphenol F (BPF) and bisphenol S (BPS) as BPA alternatives for the development of “BPA-free” products. Consequently, BPS and BPF were frequently detected in surface water, sediment, sewage effluent, indoor dust, and even in food and biological fluids in humans. Thus, environmental researches start to focus on the potential environmental risks of BPA alternatives. While the estrogenically active metabolites and the specific estrogenically active structure are still unknown. In this study, the MTT assay on acute cytotoxicity and the recombinant transactivation assay were carried out to determine whether BPF and BPS are suitable alternatives to BPA. Our results show that the cytotoxic and estrogenic activities of BPS and BPF are lower than those of BPA. However, after the addition of a rat liver homogenate to simulate mammal metabolism, BPF exhibited higher estrogenic activity than BPA. To identify the chemical structures and estrogen receptor binding affinities of active estrogenic metabolites, LC-MS, MetaPrint2D(-React), and VirtualToxLab were integrated. The observed results indicated that the para-hydroxylated BPF and BPF-OCH3 might have strong ER binding affinities. These results demonstrate that metabolization is important to consider upon investigating endocrine disruption of chemicals getting into contact with humans, such as in dental sealing or food packaging. Alternatives to potentially hazardous substances should be thoroughly tested prior to use.

Binding Assays Using a Benzofurazan-Labeled Fluorescent Probe for Estrogen Receptor-Ligand Interactions.

Binding assays are widely used to study the estrogenic activity of compounds targeting the estrogen receptor (ER). The fluorescence properties of benzofurazan (BD), an environmentally sensitive fluorophore, are affected by solvent polarity. In this study, we synthesized BD-labeled estradiol (E2) derivatives hoping to develop a fluorescent ligand to be used in ER binding assays, without the separation of free- from bound-ligand. Three fluorescent ligands with a BD skeleton were obtained and their fluorescence properties were investigated. Analysis of the fluorescent ligands and human recombinant ERα (hr-ERα) interactions revealed that the fluorescence intensity increased in hydrophobic environments, such as the receptor-binding site. In saturation binding assays, ABD-E2 derivative 2c showed positive cooperative binding, and its dissociation constant (Kd) and Hill coefficient were 23.4 nM and 1.34, respectively. The estrogenic compounds affinity, assessed by competitive binding assays was well correlated with the results obtained by conventional studies, using the fluorescence polarization method. Overall, the developed assay using BD-labeled ligands was a simple, rapid, and reliable method for the evaluation of ER binding affinity.

Alternaria toxins as casein kinase 2 inhibitors and possible consequences for estrogenicity: a hybrid in silico/in vitro study

Emerging mycotoxins produced by Alternaria spp. were previously reported to exert cytotoxic, genotoxic, but also estrogenic effects in human cells. The involved mechanisms are very complex and not fully elucidated yet. Thus, we followed an in silico target fishing approach to extend knowledge on the possible biological targets underlying the activity of alternariol, taken as the signature compound of Alternaria toxins. Combining ligand-based screening and structure-based modeling, the ubiquitous casein kinase 2 (CK2) was identified as a potential target for the compound. This result was validated in a cell-free in vitro CK2 activity assay, where alternariol inhibited CK2 with an IC50 of 707 nM. As CK2 was recently discussed to influence estrogen receptor (ER) transcription and DNA-binding affinity, we assessed a potential impact on the mRNA levels of ERα or ERβ by qRT-PCR and on nuclear localization of the receptors by confocal microscopy, using estrogen-sensitive Ishikawa cells as a model. While AOH did not affect the transcription of ERα or ERβ, an increase in nuclear localization of ERα after incubation with 10 µM AOH was observed. However, this effect might be due to ER binding affinity and therefore estrogenicity of AOH. Furthermore, in silico docking simulation revealed not only AOH, but also a number of other Alternaria toxins as potential inhibitors of CK2, including alternariol monomethyl ether and the perylene quinone derivative altertoxin II (ATX-II). These findings were representatively confirmed in vitro for the perylene quinone derivative altertoxin II, which was found to inhibit the kinase with an IC50 of 5.1 µM. Taken together, we propose CK2 inhibition as an additional mechanism to consider in future studies for alternariol and several other Alternaria toxins.

6′-Methoxy Raloxifene-analog enhances mouse bone properties with reduced estrogen receptor binding

Raloxifene (RAL) is an FDA-approved drug used to treat osteoporosis in postmenopausal women. RAL suppresses bone loss primarily through its role as a selective estrogen receptor modulator (SERM). This hormonal estrogen therapy promotes unintended side effects, such as hot flashes and increased thrombosis risk, and prevents the drug from being used in some patient populations at-risk for fracture, including children with bone disorders. It has recently been demonstrated that RAL can have significant positive effects on overall bone mechanical properties by binding to collagen and increasing bone tissue hydration in a cell-independent manner. A Raloxifene-Analog (RAL-A) was synthesized by replacing the 6-hydroxyl substituent with 6-methoxy in effort to reduce the compound's binding affinity for estrogen receptors (ER) while maintaining its collagen-binding ability. It was hypothesized that RAL-A would improve the mechanical integrity of bone in a manner similar to RAL, but with reduced estrogen receptor binding. Molecular assessment showed that while RAL-A did reduce ER binding, downstream ER signaling was not completely abolished. In-vitro, RAL-A performed similarly to RAL and had an identical concentration threshold on osteocyte cell proliferation, differentiation, and function. To assess treatment effect in-vivo, wildtype (WT) and heterozygous (OIM+/−) female mice from the Osteogenesis Imperfecta (OI) murine model were treated with either RAL or RAL-A from 8 weeks to 16 weeks of age. There was an untreated control group for each genotype as well. Bone microarchitecture was assessed using microCT, and mechanical behavior was assessed using 3-point bending. Results indicate that both compounds produced analogous gains in tibial trabecular and cortical microarchitecture. While WT mechanical properties were not drastically altered with either treatment, OIM+/− mechanical properties were significantly enhanced, most notably, in post-yield properties including bone toughness. This proof-of-concept study shows promising results and warrants the exploration of additional analog iterations to further reduce ER binding and improve fracture resistance.

Estrogenic properties of Prunus cerasoides extract and its constituents in MCF-7 cell and evaluation in estrogen-deprived rodent models.

Prunus cerasoides (PC) products contain relatively high levels of flavones and isoflavones and may be potential sources of phytoestrogens for postmenopausal symptom relief. We assessed the PC extract (PCE) and its representative constituents in vitro with assays for estrogen receptor alpha binding, estrogen response element transcriptional activity, cell proliferation, and gene expression changes for pS2 in MCF-7 cells. PCE and its compounds showed strong estrogen receptor binding affinities and estrogen response element induction. A previously undescribed compound (designated as compound 18), now identified as being gentisic acid, 5-O-β-D-(6'-O-trans-4-coumaroyl)-glucopyranoside, also showed potent estrogenic properties and induced proliferation of MCF-7 cells. PCE was evaluated for its in vivo uterotrophic effects in immature female rats as well as for its lipid lowering effects in estrogen-deprived animals. For ovariectomized rats and aged female mice, PCE-treated groups had lower plasma triglyceride levels compared with control and, for the same comparison, had reduced serum levels of liver stress/damage markers. Our results point to strong estrogenic activities and beneficial metabolic effects for PCE, with properties that put PC and its extracts as promising sources of phytoestrogens for symptom relief in menopausal and postmenopausal cases.

In Silico Modeling Method for Computational Aquatic Toxicology of Endocrine Disruptors: A Software-Based Approach Using QSAR Toolbox.

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological systems. In particular, quantitative structure-activity relationship (QSAR) models have been increasingly applied to assess the environmental effects of a plethora of toxic materials. In recent years, some more highlighted types of toxicants are endocrine disruptors (EDs, which are chemicals that can interfere with any hormone-related metabolism). Because EDs may significantly affect animal development and reproduction, rapidly predicting the adverse effects of EDs using in silico techniques is required. This study presents an in silico method to generate prediction data on the effects of representative EDs in aquatic vertebrates, particularly fish species. The protocol describes an example utilizing the automated workflow of the QSAR Toolbox software developed by the Organization for Economic Co-operation and Development (OECD) to enable acute ecotoxicity predictions of EDs. As a result, the following are determined: (1) calculation of the numerical correlations between the concentration for 50% of lethality (LC50) and octanol-water partition coefficient (Kow), (2) output performances in which the LC50 values determined in experiments are compared to those generated by computations, and (3) the dependence of estrogen receptor binding affinity on the relationship between Kow and LC50.

Biological evaluation of isoflavonoids from Genista halacsyi using estrogen-target cells: Activities of glucosides compared to aglycones.

The purpose of this study was to evaluate the response of estrogen target cells to a series of isoflavone glucosides and aglycones from Genista halacsyi Heldr. The methanolic extract of aerial parts of this plant was processed using fast centrifugal partition chromatography, resulting in isolation of four archetypal isoflavones (genistein, daidzein, isoprunetin, 8-C-β-D-glucopyranosyl-genistein) and ten derivatives thereof. 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein were among the most abundant constituents of the isolate. All fourteen, except genistein, displayed low binding affinity for estrogen receptors (ER). Models of binding to ERα could account for the low binding affinity of monoglucosides. Genistein and its glucosides displayed full efficacy in inducing alkaline phosphatase (AlkP) in Ishikawa cells, proliferation of MCF-7 cells and ER-dependent gene expression in reporter cells at low concentrations (around 0.3 μM). ICI182,780 fully antagonized these effects. The AlkP-inducing efficacy of the fourteen isoflavonoids was more strongly correlated with their transcriptional efficacy through ERα. O-monoglucosides displayed higher area under the dose-response curve (AUC) of AlkP response relative to the AUC of ERα-transcriptional response compared to the respective aglycones. In addition, 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein displayed estradiol-like efficacy in promoting differentiation of MC3T3-E1 cells to osteoblasts, while genistein was not convincingly effective in this respect. Moreover, 7,4΄-di-O-β-D-glucopyranosyl-genistein suppressed lipopolysaccharide-induced tumor necrosis factor mRNA expression in RAW 264.7 cells, while 7-O-β-D-glucopyranosyl-genistein was not convincingly effective and genistein was ineffective. However, genistein and its O-glucosides were ineffective in inhibiting differentiation of RAW 264.7 cells to osteoclasts and in protecting glutamate-challenged HT22 hippocampal neurons from oxidative stress-induced cell death. These findings suggest that 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein display higher estrogen-like and/or anti-inflammatory activity compared to the aglycone. The possibility of using preparations rich in O-β-D-glucopyranosides of genistein to substitute for low-dose estrogen in formulations for menopausal symptoms is discussed.

Abstract 1808: The anti-estrogen endoxifen altered bone morphology and reduced muscle function in mice

Abstract Endoxifen, the predominant CYP2D6 metabolite of the selective estrogen receptor modulator (SERM) tamoxifen, is currently being developed as a novel anti-estrogen therapy for the treatment of estrogen receptor (ER)+ breast cancer. Genetic polymorphism of CYP2D6 is a predictor of response to tamoxifen in patients, implicating endoxifen as one of the most active and relevant tamoxifen metabolites. Breast cancer patients treated with adjuvant endocrine therapies including aromatase inhibitors (AIs) and SERMs often report unmanageable musculoskeletal toxicities that can result in treatment discontinuation. While ER binding affinity and anti-tumor effects have been established in preclinical models, the effects of endoxifen on the musculoskeletal system are not fully known. Twenty-week female C57BL/6 mice underwent sham surgery or ovariectomy (OVX) and were treated daily with vehicle, the AI letrozole, or the SERM endoxifen. Body composition was assessed prospectively by DXA. Bone indices and marrow adipose tissue volume were measured by μCT and muscle contractility of the extensor digitorum longus (EDL) was measured ex vivo. After eight weeks, trabecular bone volume fraction (BV/TV) decreased by 50% in OVX-vehicle and OVX-letrozole mice, whereas BV/TV increased threefold in endoxifen mice relative to sham-vehicle. Despite the presence of significantly more trabecular bone following endoxifen treatment, cortical bone analyses revealed impaired periosteal and endosteal expansion of bone, resulting in reduced polar moment of inertia, which is a measure of resistance to fracture. Uterine weight increased significantly in endoxifen-treated mice relative to all OVX groups, similar to what is observed with tamoxifen treatment. Peripheral body fat, bone marrow adipose tissue, and circulating leptin were reduced by endoxifen, suggesting that the drug may elicit positive systemic metabolic effects. At the termination of the study, muscle-specific force was reduced in OVX-endoxifen mice relative to sham-vehicle, OVX-vehicle, and OVX-AI mice, despite no change in muscle mass. While endoxifen shows promise as a potent anti-estrogen therapy for the treatment of ER+ breast cancer, it may be important to monitor patients for endometrial proliferation, morphological changes in bone that increase fracture risk, and for musculoskeletal side effects that could reduce drug compliance. Citation Format: Laura Wright, Khalid Mohammad, Theresa Guise. The anti-estrogen endoxifen altered bone morphology and reduced muscle function in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1808.

Estrogenic activity of constituents from the rhizomes of Rheum undulatum Linné

Stilbenes have been reported to be phytoestrogen compounds owing to its structural similarity to the estrogenic agent diethylstilbestrol. To find new stilbene-derivative phytoestrogens, isolation of stilbene-rich R. undulatum was performed and led to identify six new compounds (1–5 and 28), one newly determined absolute configurations compound (27) together with 21 previously reported compounds (6–26). The structures of compounds were determined on the basis of extensive spectroscopic methods including 1D and 2D NMR and CD spectra data. All the isolated compounds were tested for their estrogenic activities in HepG2 cells transiently transfected with ERα, ERβ and ERE-reporter plasmid. Among them, stilbene-derivatives, piceatannol 3′-O-β-d-xylopyranoside (12), cis-rhaponticin (16) and rhapontigenin 3′-O-β-d-glucopyranoside (17), showed the more potent binding affinity for estrogen receptors than 17β-estrodiol.

A Cell-Permeable Stapled Peptide Inhibitor of the Estrogen Receptor/Coactivator Interaction.

We and others have proposed that coactivator binding inhibitors, which block the interaction of estrogen receptor and steroid receptor coactivators, may represent a potential class of new breast cancer therapeutics. The development of coactivator binding inhibitors has been limited, however, because many of the current molecules which are active in in vitro and biochemical assays are not active in cell-based assays. Our goal in this work was to prepare a coactivator binding inhibitor active in cellular models of breast cancer. To accomplish this, we used molecular dynamics simulations to convert a high-affinity stapled peptide with poor cell permeability into R4K1, a cell-penetrating stapled peptide. R4K1 displays high binding affinity for estrogen receptor α, inhibits the formation of estrogen receptor/coactivator complexes, and distributes throughout the cell with a high percentage of nuclear localization. R4K1 represses native gene transcription mediated by estrogen receptor α and inhibits proliferation of estradiol-stimulated MCF-7 cells. Using RNA-Seq, we demonstrate that almost all of the effects of R4K1 on global gene transcription are estrogen-receptor-associated. This chemical probe provides a significant proof-of-concept for preparing cell-permeable stapled peptide inhibitors of the estrogen receptor/coactivator interaction.

Oxidative Stress and Neuronal Damages: Estrogenic Compounds, Anti-Apoptotic Factors, and Amyloidogenesis

In stroke, increased oxidative stress (OS), mediated by reactive oxygen species (ROS) including H2O2 release, can induce changes that lead to neural production of amyloid-β (Aβ), a hallmark protein in the brains of Alzheimer’s disease (AD) patients. Aβ peptide can also induce OS by itself or by activating microglia to release ROS. Estrogenic compounds can protect neurons against Aβ- and OS- induced cell death. Aβ- and OS-induced cell death is another hallmark of AD. We have studied OS-related cell damage by exposing rat primary hippocampal neurons and differentiated human SH-SY5Y neuroblastoma cells to H2O2 or Aβ1–42 and evaluated the neuroprotective potential of 17β-estradiol (E2), estrone (E1), tamoxifen (Tam), 4-OH-tamoxifen (4-OH-Tam), diethylstilbestrol (Des) and genistein (Gen) against OS. These compounds have differences in estrogen receptor (ER) binding affinities and their number of antioxidative –OH groups varies. The cell damage indicator was the lactate dehydrogenase release into culture medium. Treatment with 5 nM E2, Gen, or 4-OH-Tam for 24 h before and after the H2O2 insult was neuroprotective in both hippocampal and SH-SY5Y cultures. E2 and Gen were neuroprotective against Aβ1–42-mediated toxicity. Protection by E2 was partially mediated by Bcl-2, Bcl-xL, and BAG-1. Tam also increased Bcl-2 and Bcl-xL but was much less neuroprotective. Gen increased amyloid precursor protein (APP) synthesis, but γ-secretase component PS-1 was reduced, suggesting that Gen can increase the production of neurotrophic soluble APPa. Des increased Aβ production. In conclusion, Gen shows comparable neuroprotective efficacy to E2, and seems also to reduce Aβ production in our study. However, other neuroprotective mechanisms may exist, and further studies on this subject will enhance our understanding in this respect.

Synthetic Phenolic Antioxidants Cause Perturbation in Steroidogenesis in Vitro and in Vivo.

Synthetic phenolic antioxidants (SPAs) are closely correlated with human life due to their extensive usages, and increasing concerns have been raised on their biosafety. The previous controversial findings caused continuous debates on their potential endocrine disrupting effects. In the present study, four commonly used SPAs, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butyl hydroquinone (TBHQ) and 2,2'-methylenebis(6-tert-butyl-4-methylphenol) (AO2246), were investigated for their estrogenic effects, and the results from in vitro screening assays showed SPAs themselves had negligible estrogen receptor binding affinities. Nevertheless, significant increase in E2 secretion was observed in H295R cells treated with SPAs, especially for BHA. The transcriptional levels of steroidogenic enzymes, including StAR, 3βHSD, CYP11B1, and CYP11B2 were up-regulated via the mediation of protein kinase A (PKA) signaling pathway. In vivo experiment confirmed that waterborne exposure to BHA disturbed E2 and testosterone (T) levels in zebrafish gonad, thus causing potential estrogenic effects through the regulation of hypothalamic-pituitary-gonadal-liver axis (HPGL-axis). Accordingly, this study has provided new insights for SPA-induced endocrine disrupting effects. Considering the allowable maximum level of individual BHA or in combination with TBHQ and BHT in foodstuffs (200 mg kg-1), the perturbation in steroidogenesis observed for relatively low concentrations of SPAs would need more public attention.

A functional polymorphism in TFF1 promoter is associated with the risk and prognosis of gastric cancer.

Trefoil Factor 1 (TFF1, also named pS2), which serves as the gastrointestinal mucosal protector, is known as gastric-specific tumor suppressor gene. However, the genetic variants of TFF1 are still not well studied. In our study, we aim to explore the effects of tagging single nucleotide polymorphisms (tagSNPs) of TFF1 on risk and prognosis of gastric cancer. Seven tagSNPs of TFF1 gene were first analyzed in the discovery set, which was consisted of 753 cases and 950 cancer-free controls. Then, the validation set (940 cases and 1,042 controls) was used for further evaluation. Moreover, we also tested the relation between these tagSNPs and prognosis of gastric cancer (GC). A series of experiments were performed to investigate the underlying mechanisms. We found that rs3761376 AA in the promoter region of TFF1, could reduce the expression of TFF1 by affecting the binding affinity of estrogen receptor 1 (ESR1, ERα), and thereby increased the risk of GC (1.29, 1.08-1.53). Moreover, the rs3761376 AA genotype was also found associated with worse prognosis among patients receiving 5-FU based chemotherapy after surgery (1.71, 1.18-2.48). Further functional assays demonstrated that TFF1 could increase the chemosensitivity of 5-FU by modulating NF-κB targeted genes. These results identified the effect of rs3761376 on TFF1 expression, which accounted for the correlation with susceptibility and prognosis of GC; and this genetic variant may be a potential biomarker to predict the risk and survival of GC.