ERα Activity Research Articles

Androgen receptor and estrogen receptor variants in prostate and breast cancers

The androgen receptor (AR) and estrogen receptor alpha (ERα) are steroid receptor transcription factors with critical roles in the development and progression of prostate and breast cancers. Advances in the understanding of mechanisms underlying the ligand-dependent activation of these transcription factors have contributed to the development of small molecule inhibitors that block AR and ERα actions. These inhibitors include competitive antagonists and degraders that directly bind the ligand binding domains of these receptors, luteinizing hormone releasing hormone (LHRH) analogs that suppress gonadal synthesis of testosterone or estrogen, and drugs that block specific enzymes required for biosynthesis of testosterone or estrogen. However, resistance to these therapies is frequent, and is often driven by selection for tumor cells with alterations in the AR or ESR1 genes and/or alternatively spliced AR or ESR1 mRNAs that encode variant forms AR or ERα. While most investigations involving AR have been within the context of prostate cancer, and the majority of investigations involving ERα have been within the context of breast cancer, important roles for AR have been elucidated in breast cancer, and important roles for ERα have been elucidated in prostate cancer. Here, we will discuss the roles of AR and ERα in breast and prostate cancers, outline the effects of gene- and mRNA-level alterations in AR and ESR1 on progression of these diseases, and identify strategies that are being developed to target these alterations therapeutically.

PFKFB3 regulates breast cancer tumorigenesis and Fulvestrant sensitivity by affecting ERα stability

Estrogen receptor alpha (ERα) is expressed in approximately 70% of breast cancer cases and determines the sensitivity and effectiveness of endocrine therapy. 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase3 (PFKFB3) is a glycolytic enzyme that is highly expressed in a great many human tumors, and recent studies have shown that it plays a significant role in improving drug sensitivity. However, the role of PFKFB3 in regulating ERα expression and the underlying mechanism remains unclear. Here, we find by using immunohistochemistry (IHC) that PFKFB3 is elevated in ER-positive breast cancer and high expression of PFKFB3 resulted in a worse prognosis. In vitro and in vivo experiments verify that PFKFB3 promotes ER-positive breast cancer cell proliferation. The overexpression of PFKFB3 promotes the estrogen-independent ER-positive breast cancer growth. In an estrogen-free condition, RNA-sequencing data from MCF7 cells treated with siPFKFB3 showed enrichment of the estrogen signaling pathway, and a luciferase assay demonstrated that knockdown of PFKFB3 inhibited the ERα transcriptional activity. Mechanistically, down-regulation of PFKFB3 promotes STUB1 binding to ERα, which accelerates ERα degradation by K48-based ubiquitin linkage. Finally, growth of ER-positive breast cancer cells in vivo was more potently inhibited by fulvestrant combined with the PFKFB3 inhibitor PFK158 than for each drug alone. In conclusion, these data suggest that PFKFB3 is identified as an adverse prognosis factor for ER-positive breast cancer and plays a previously unrecognized role in the regulation of ERα stability and activity. Our results further explores an effective approach to improve fulvestrant sensitivity through the early combination with a PFKFB3 inhibitor.

Molecular insights into the potential effects of selective estrogen receptor β agonists in Alzheimer's and Parkinson's diseases.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common neurodegenerative disorders. Pathologically, AD and PD are characterized by the accumulation of misfolded proteins. Hence, they are also called as proteinopathy diseases. Gender is considered as one of the risk factors in both diseases. Estrogens are widely accepted to be neuroprotective in several neurodegenerative disorders. Estrogens can be produced in the central nervous system, where they are called as neurosteroids. Estrogens mediate their neuroprotective action mainly through their actions on estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). However, ERα is mainly involved in the growth and development of the primary and secondary sexual organs in females. Hence, the activation of ERα is associated with undesired side effects such as gynecomastia and increase in the risk of breast cancer, thromboembolism, and feminization. Therefore, selective activation of ERβ is often considered to be safer. In this review, we explore the role of ERβ in regulating the expression and functions of AD- and PD-associated genes. Additionally, we discuss the association of these genes with the amyloid-beta peptide (Aβ) and α-synuclein mediated toxicity. Ultimately, we established a correlation between the importance of ERβ activation and the process underlying ERβ's neuroprotective mechanisms in AD and PD.

Investigating the effect of hesperetin on estrogen receptor alpha (ERα) expression, phosphorylation and activity in MCF-7 cells

PurposeThe most common malignancy among women worldwide is breast cancer. The estrogen receptor plays a vital role in this cancer. One of the most well-known mechanisms that affects the activity of this receptor is its phosphorylation by protein kinase pathways. Hesperetin, a flavonoid abundant in citrus species such as lemons, grapefruits, and oranges, is the aglycone form of hesperidin. It has undergone thorough evaluation for its potential anti-cancer properties, particularly in the context of breast cancer. Studies have shown that hesperetin has an effect on intracellular kinase pathways. The aim of this study was to investigate the effect of hesperetin on the expression, phosphorylation and activity of estrogen receptor alpha (ERα) in MCF-7 breast cancer cell line. Study design and methodsMCF-7 cells were cultured in RPMI-1640 phenol red-free medium supplemented with charcoal-stripped FBS and treated with hesperetin. The MTT method was used to evaluate cell survival. The levels of the ERα protein and its phosphorylated form (Ser118) were determined via western blotting. A luciferase reporter vector was used to evaluate ERE activity. ResultsThe results of this study indicated that hesperetin reduced the survival of MCF-7 cells in a dose-dependent manner. The expression and phosphorylation (at Ser118) of the ERα significantly increased and decreased, respectively, in the groups treated with hesperetin. Hesperetin increased the activity of the ERα in the absence of E2, although these differences were not statistically significant. Conversely, in the presence of E2, hesperetin caused a significant decrease in receptor activity. ConclusionBased on the results of this study, it can be concluded that hesperetin has a significant effect on ERα expression, phosphorylation and activity.

The Estrogen Receptor Alpha Regulates the Sex-dependent Expression and Pronociceptive Role of Bestrophin-1 in Neuropathic Rats

Bestrophin-1, a calcium-activated chloride channel (CaCC), is involved in neuropathic pain; however, it is unclear whether it has a dimorphic role in female and male neuropathic rats. This study investigated if 17β-estradiol and estrogen receptor alpha (ERα) activation regulate bestrophin-1 activity and expression in neuropathic rats. Neuropathic pain was induced by L5-spinal nerve transection (SNT). Intrathecal administration of CaCCinh-A01 (.1–1 µg), a CaCC blocker, reversed tactile allodynia induced by SNT in female but not male rats. In contrast, T16Ainh-A01, a selective anoctamin-1 blocker, had an equal antiallodynic effect in both sexes. SNT increased bestrophin-1 protein expression in injured L5 dorsal root ganglia (DRG) in female rats but decreased bestrophin-1 protein in L5 DRG in male rats. Ovariectomy prevented the antiallodynic effect of CaCCinh-A01, but 17β-estradiol replacement restored it. The effect of CaCCinh-A01 was prevented by intrathecal administration of MPP, a selective ERα antagonist, in rats with and without prior hormonal manipulation. In female rats with neuropathy, ovariectomy prevented the increase in bestrophin-1 and ERα protein expression, while 17β-estradiol replacement allowed for an increase in both proteins in L5 DRG. Furthermore, ERα antagonism (with MPP) prevented the increase in bestrophin-1 and ERα protein expression. Finally, ERα activation with PPT, an ERα selective activator, induced the antiallodynic effect of CaCCinh-A01 in neuropathic male rats and prevented the reduction in bestrophin-1 protein expression in L5 DRG. In summary, data suggest ERα activation is necessary for bestrophin-1’s pronociceptive action to maintain neuropathic pain in female rats. PerspectiveThe mechanisms involved in neuropathic pain differ between male and female animals. Our data suggest that ERα is necessary for expression and function of bestrophin-1 in neuropathic female but not male rats. Data support the idea that a therapeutic approach to relieving neuropathic pain must be based on patient's gender.

Estrogen signaling in the dorsal raphe regulates binge-like drinking in mice

Estrogens promote binge alcohol drinking and contribute to sex differences in alcohol use disorder. However, the mechanisms are largely unknown. This study aims to test if estrogens act on 5-hydroxytryptamine neurons in the dorsal raphe nucleus (5-HTDRN) to promote binge drinking. We found that female mice drank more alcohol than male mice in chronic drinking in the dark (DID) tests. This sex difference was associated with distinct alterations in mRNA expression of estrogen receptor α (ERα) and 5-HT-related genes in the DRN, suggesting a potential role of estrogen/ERs/5-HT signaling. In supporting this view, 5-HTDRN neurons from naïve male mice had lower baseline firing activity but higher sensitivity to alcohol-induced excitation compared to 5-HTDRN neurons from naïve female mice. Notably, this higher sensitivity was blunted by 17β-estradiol treatment in males, indicating an estrogen-dependent mechanism. We further showed that both ERα and ERβ are expressed in 5-HTDRN neurons, whereas ERα agonist depolarizes and ERβ agonist hyperpolarizes 5-HTDRN neurons. Notably, both treatments blocked the stimulatory effects of alcohol on 5-HTDRN neurons in males, even though they have antagonistic effects on the activity dynamics. These results suggest that ERs’ inhibitory effects on ethanol-induced burst firing of 5-HTDRN neurons may contribute to higher levels of binge drinking in females. Consistently, chemogenetic activation of ERα- or ERβ-expressing neurons in the DRN reduced binge alcohol drinking. These results support a model in which estrogens act on ERα/β to prevent alcohol-induced activation of 5-HTDRN neurons, which in return leads to higher binge alcohol drinking.

Cancer-associated fibroblasts rewire the estrogen receptor response in luminal breast cancer, enabling estrogen independence.

Advanced breast cancers represent a major therapeutic challenge due to their refractoriness to treatment. Cancer-associated fibroblasts (CAFs) are the most abundant constituents of the tumor microenvironment and have been linked to most hallmarks of cancer. However, the influence of CAFs on therapeutic outcome remains largely unchartered. Here, we reveal that spatial coincidence of abundant CAF infiltration with malignant cells was associated with reduced estrogen receptor (ER)-α expression and activity in luminal breast tumors. Notably, CAFs mediated estrogen-independent tumor growth by selectively regulating ER-α signaling. Whereas most prototypical estrogen-responsive genes were suppressed, CAFs maintained gene expression related to therapeutic resistance, basal-like differentiation, and invasion. A functional drug screen in co-cultures identified effector pathways involved in the CAF-induced regulation of ER-α signaling. Among these, the Transforming Growth Factor-β and the Janus kinase signaling cascades were validated as actionable targets to counteract the CAF-induced modulation of ER-α activity. Finally, genes that were downregulated in cancer cells by CAFs were predictive of poor response to endocrine treatment. In conclusion, our work reveals that CAFs directly control the luminal breast cancer phenotype by selectively modulating ER-α expression and transcriptional function, and further proposes novel targets to disrupt the crosstalk between CAFs and tumor cells to reinstate treatment response to endocrine therapy in patients.

Effect of Per and Poly-Fluoroalkyl Substances on Pregnancy and Child Development.

Childhood obesity is significantly influenced by maternal exposure to Per- and Poly-Fluoroalkyl Substances (PFAS) during pregnancy. PFAS exposure occurs through the Peroxisome Proliferator-Activated Receptor (PPAR-γ) receptor, leading to increased fat deposition and profound health effects in child growth and development. Despite ongoing investigations, the relationship between maternal serum PFAS concentration and child obesity requires further exploration. This study aimedto review the possible effects of Per and poly-fluoroalkyl substances exposure and their mechanism in overweight/obese children from pregnant ladies. A detailed literature survey was conducted using online databases, including Science Direct, Google Scholar, Scopus, Cochrane, and PubMed. The study focused on the diverse effects of PFAS on maternal and child health, with particular emphasis on neurological complications. Child growth development depends upon breastfeeding and placenta health, which is disrupted by PFAS exposure, ultimately destroying the body mass index of the child. Neurotoxicity testing utilized the SH-SY5Y human-derived cell line as an in vitro model, revealing PFAS-induced increases in adipocyte number, reduced cell size, altered lipid conglomeration, increased adiposity, and changes in liver function. in vivo studies in mice and human cell lines indicated PPAR-γ and ER-α activation, leading to adiposity and weight gain through Estrogen signaling and Lipid metabolism. PFAS concentrations positively correlated in maternal sera, analyzed by liquid chromatography/quadrupole mass spectrometry. PFAS, with a long half-life of 3.5-8.5 years, is commonly found in the serum of pregnant women, crossing the placenta barrier. This exposure disrupts placental homeostasis, negatively impacting mechanisms of action and potentially leading to deterioration in pregnancy and child health. Further research is needed to comprehensively understand the complex interplay between PFAS exposure and its implications for maternal and child well-being.

Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

Rationale: 17β-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors.

New generation estrogen receptor-targeted agents in breast cancer: present situation and future prospectives.

Endocrine therapy which blocking the signaling of estrogen receptor, has long been effective for decades as a primary treatment choice for breast cancer patients expressing ER. However, the issue of drug resistance poses a significant clinical challenge. It's critically important to create new therapeutic agents that can suppress ERα activity, particularly in cases of ESR1 mutations. This review highlights recent efforts in drug development of next generation ER-targeted agents, including oral selective ER degraders (SERDs), proteolysis targeting chimera (PROTAC) ER degraders, other innovative molecules such as complete estrogen receptor antagonists (CERANs) and selective estrogen receptor covalent antagonists (SERCAs). The drug design, efficacy and clinical trials for each compound were detailed.

Uncovering a novel mechanism: Butyrate induces estrogen receptor alpha activation independent of estrogen stimulation in MCF-7 breast cancer cells.

Butyrate is a promising candidate for an antitumoral drug, as it promotes cancer cell apoptosis and reduces hormone receptor activity, while promoting differentiation and proliferation in normal cells. However, the effects of low-dose butyrate on breast cancer cell cultures are unclear. We explored the impact of sub-therapeutic doses of butyrate on estrogen receptor alpha (ERα) transcriptional activity in MCF-7 cells, using RT-qPCR, Western blot, wound-healing assays, and chromatin immunoprecipitation. Our results showed that sub-therapeutic doses of sodium butyrate (0.1 - 0.2 mM) increased the transcription of ESR1, TFF1, and CSTD genes, but did not affect ERα protein levels. Moreover, we observed an increase in cell migration in wound-healing assays. ChIP assays revealed that treatment with 0.1 mM of sodium butyrate resulted in estrogen-independent recruitment of ERα at the pS2 promoter and loss of NCoR. Appropriate therapeutic dosage of butyrate is essential to avoid potential adverse effects on patients' health, especially in the case of estrogen receptor-positive breast tumors. Sub-therapeutic doses of butyrate may induce undesirable cell processes, such as migration due to low-dose butyrate-mediated ERα activation. These findings shed light on the complex effects of butyrate in breast cancer and provide insights for research in the development of antitumoral drugs.

Stimulation of estrogen receptor-alpha by hydroxyanilide fungicide, fenhexamid promotes lipid accumulation in 3 T3-L1 adipocyte

Fenhexamid are fungicides that act against plant pathogens by inhibiting sterol biosynthesis. Nonetheless, it can trigger endocrine disruption and promote breast cancer cell growth. In a recent study, we investigated the mechanism underlying the lipid accumulation induced by fenhexamid hydroxyanilide fungicides in 3 T3-L1 adipocytes. To examine the estrogen receptor alpha (ERα)-agonistic effect, ER transactivation assay using the ERα-HeLa-9903 cell line was applied, and fenhexamid-induced ERα agonist effect was confirmed.Further confirmation that ERα-dependent lipid accumulation occurred was provided by treating 3 T3-L1 adipocytes with Methyl-piperidino-pyrazole hydrate (MPP), an ERα-selective antagonist. Fenhexamid mimicked the actions of ERα agonists and impacted lipid metabolism, and its mechanism involves upregulation of the expression of transcription factors that facilitate adipogenesis and lipogenesis. Additionally, it stimulated the expression of peroxisome proliferator-activated receptor (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), and sterol regulatory element-binding protein 1 (SREBP1) and significantly elevated the expression of fatty acid-binding protein 4 (FABP4). In contrast, in combination with an ERα-selective antagonist, fenhexamid suppressed the expression of adipogenic/lipogenic transcription factors.These results suggest that fenhexamid affects the endocrine system and leads to lipid accumulation by interfering with processes influenced by ERα activation.

MYSM1 acts as a novel co-activator of ERα to confer antiestrogen resistance in breast cancer

Endocrine resistance is a crucial challenge in estrogen receptor alpha (ERα)-positive breast cancer (BCa). Aberrant alteration in modulation of E2/ERα signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Herein, we demonstrate that MYSM1 as a deubiquitinase participates in modulating ERα action via histone and non-histone deubiquitination. MYSM1 is involved in maintenance of ERα stability via ERα deubiquitination. MYSM1 regulates relevant histone modifications on cis regulatory elements of ERα-regulated genes, facilitating chromatin decondensation. MYSM1 is highly expressed in clinical BCa samples. MYSM1 depletion attenuates BCa-derived cell growth in xenograft models and increases the sensitivity of antiestrogen agents in BCa cells. A virtual screen shows that the small molecule Imatinib could potentially interact with catalytic MPN domain of MYSM1 to inhibit BCa cell growth via MYSM1-ERα axis. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ERα action and provide a potential therapeutic target for endocrine resistance in BCa.

Phenomenon of suppression of estrogen signaling in breast cancer cells under ultraviolet irradiation: role of Snail proteins

Introduction. The study of the effect of irradiation or any other DNA-damaging agents on the sensitivity of tumors to conservative therapy, drug or hormonal, is among the most imporant tasks that determine the efficiency of combined therapy of cancer patients.Aim. To investigate the effect of irradiation on the activity of key signaling proteins and the level of hormone dependence of breast cancer cells.Materials and methods. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells. Ultraviolet (UV) irradiation in the range of 254 nm with the intensity of 25–50 J/m2 was used as an experimental model to study the response of tumor cells to DNA damage. Cell growth rate was determined using the MTT test, cell survival after irradiation was analyzed using the colony-forming test. Estrogen receptor transcriptional activity was determined by reporter assay; cellular protein expression was determined by immunoblotting.Results. Single UV irradiation of MCF-7 cells leads to a marked increase in the level of apoptotic markers: p53, cPARP, suppression of expression of growth signaling proteins: CDK4/6 and estrogen receptor α (ERα). The above changes are accompanied with an increase in phosphorylation of Akt protein kinase and a marked increase in the expression of Snail1, one of the key proteins of epithelial-mesenchymal transition. In UV-resistant MCF-7/UVR cell subline obtained under repeated irradiation cycles, the levels of apoptotic and growth signaling proteins (p53, cPARP, CDK4/6) return to control levels, except for the phosphorylated form of Akt and Snail1, whose content remains high. Transfection of Snail1-expressing plasmid into MCF-7 cells is accompanied by activation of apoptotic signaling, suppression of ERα activity, and development of partial hormone resistance; however, the sensitivity of cells to irradiation is practically unchanged. Transfection of microRNA-181a-2, one of the microRNAs associated with cell resistance, simultaneously activates Akt and Snail1 and leads to the development of cross-resistance of cells to irradiation and hormonal drugs.Conclusion. The obtained data allow us to consider irradiation-induced Snail1 activation as one of the factors involved in deregulation of estrogen signaling and formation of cell resistance to hormonal drugs, while simultaneous activation of Akt and Snail1 is accompanied by the development of cross-resistance to irradiation and hormonal drugs.

Fucoxanthin Abrogates Ionizing Radiation-Induced Inflammatory Responses by Modulating Sirtuin 1 in Macrophages.

Ionizing radiation (IR) triggers an overproduction of reactive oxygen species (ROS), disrupting the normal function of both immune and metabolic systems, leading to inflammation and metabolic disturbances. To address the pressing requirement for protection against IR, fucoxanthin (FX), a naturally occurring compound extracted from algae, was utilized as an efficient radioprotective agent in macrophages. In this study, we cultured murine RAW 264.7 macrophages and treated them with FX, along with agents influencing the activity of sirtuin 1 (SIRT1) and estrogen receptor α (ERα), to investigate their impact on IR-induced cellular responses. FX significantly attenuated IR-induced upregulation of pro-inflammatory genes (Il1b, Tnf, and Ccl2) and inhibited macrophage polarization toward the pro-inflammatory M1 phenotype. Additionally, FX regulated IR-induced metabolic genes mediating glycolysis and mitochondrial biogenesis. The ability of FX to mitigate IR-induced inflammation and glycolysis was ascribed to the expression and activity of SIRT1 and ERα in macrophages. This study not only uncovers the underlying mechanisms of FX's radioprotective properties but also highlights its potential as a protective agent against the detrimental effects of IR, thus offering new opportunities for enhancing radiation protection in the future.

TMEM97/Sigma 2 Receptor Increases Estrogen Receptor α Activity in Promoting Breast Cancer Cell Growth

Aberrant estrogen receptor (ER) signaling is a major driver of breast tumor growth and progression. Sigma 2 receptor has long been implicated in breast carcinogenesis based on pharmacological studies, but its molecular identity had been elusive until TMEM97 was identified as the receptor. Herein, we report that the TMEM97/sigma 2 receptor is highly expressed in ER-positive breast tumors and its expression is strongly correlated with ERs and progesterone receptors (PRs) but not with HER2 status. High expression levels of TMEM97 are associated with reduced overall survival of patients. Breast cancer cells with increased expression of TMEM97 had a growth advantage over the control cells under both nutrition-limiting and sufficient conditions, while the knockdown of TMEM97 expression reduced tumor cell proliferations. When compared to their vector control cells, MCF7 and T47D cells with increased TMEM97 expression presented increased resistance to tamoxifen treatment and also grew better under estrogen-depleted conditions. The TMEM97/sigma 2 receptor enhanced the ERα transcriptional activities and increased the expression of genes responsive to estrogen treatment. Increased TMEM97 also stimulated the mTOR/S6K1 signaling pathways in the MCF7 and T47D cells. The increased level of active, phosphorylated ERα, and the enhanced resistance to tamoxifen treatment with increased TMEM97, could be blocked by an mTOR inhibitor. The knockdown of TMEM97 expression reduced the ERα and mTOR/S6K1 signaling activities, rendering the cells with an increased sensitivity to tamoxifen. The observations suggest that the TMEM97/sigma 2 receptor is a novel regulator of ERα activities in breast tumor cell growth.

Acetylcholine, Another Factor in Breast Cancer.

Acetylcholine (ACh) is a neurotransmitter that regulates multiple functions in the nervous system, and emerging evidence indicates that it could play a role in cancer progression. However, this function is controversial. Previously, we showed that organophosphorus pesticides decreased the levels of the enzyme acetylcholinesterase in vivo, increasing ACh serum levels and the formation of tumors in the mammary glands of rats. Furthermore, we showed that ACh exposure in breast cancer cell lines induced overexpression of estrogen receptor alpha (ERα), a key protein described as the master regulator in breast cancer. Therefore, here, we hypothesize that ACh alters the ERα activity through a ligand-independent mechanism. The results here reveal that the physiological concentration of ACh leads to the release of Ca+2 and the activity of MAPK/ERK and PI3K/Akt pathways. These changes are associated with an induction of p-ERα and its recruitment to the nucleus. However, ACh fails to induce overexpression of estrogen-responsive genes, suggesting a different activation mechanism than that of 17ß-estradiol. Finally, ACh promotes the viability of breast cancer cell lines in an ERα-dependent manner and induces the overexpression of some EMT markers. In summary, our results show that ACh promotes breast cancer cell proliferation and ERα activity, possibly in a ligand-independent manner, suggesting its putative role in breast cancer progression.

Application of machine learning algorithms in drug screening

At present, in the medical field, drug screening is usually performed using in vivo drug experiments. However, it is very time-consuming and laborious to conduct in vivo experiments on a large number of drugs to be screened one by one. This paper attempts to propose using machine learning algorithms to perform preliminary screening of a large number of compounds to be screened and their molecular structures to reduce the workload of in vivo experiments. Among them, it is internationally recognized that there is an important association between breast cancer progression and the alpha subtype of the estrogen receptor. Anti-breast cancer drug candidates with excellent efficacy need to contain compounds that can better antagonize ERα activity. In this paper, the research object is narrowed down from compounds to the molecular structure of the compounds, and then the random forest regression algorithm is used to develop the molecular structure-ERα activity prediction model. Molecular structures with significant effects on biological activity were screened from molecular structure descriptors in numerous compounds. Four different kernel functions were used to conduct comparative experiments, and finally a support vector regression algorithm based on radial basis kernel function was established, which realized the quantitative prediction of compounds on biological activity of ERα, and could find potential compounds beneficial to breast cancer treatment. This is a novel, computer-based method for preliminary drug screening, which can help medical researchers effectively narrow the scope of experiments and achieve more accurate optimization of drugs.

Naringenin protects pancreatic β cells in diabetic rat through activation of estrogen receptor β

Naringenin is a citrus flavonoid that potently improves metabolic parameters in animal models of metabolic disorders, such as type 2 diabetes. Estrogen receptor (ER) activation promotes β cell function and survival, thereby improving systemic glucose metabolism. In this study, we used a luciferase reporter assay, isolated rat islets and a diabetic rat model to investigate the effects of naringenin on ER signaling and the underlying mechanism of naringenin-mediated improvement of islet function in diabetes. Naringenin specifically activated ERβ without affecting the activity of ERα, G protein-coupled estrogen receptor (GPER) or estrogen-related receptor (ERR) α/β/γ. Additionally, treatment with naringenin enhanced glucose-stimulated insulin secretion in isolated rat islets. This effect was abrogated by PHTPP, an ERβ antagonist. Transcriptomic analysis revealed that naringenin upregulated the expression of genes, such as Pdx1 and Mafa, which are closely linked to improved β-cell function. In consistence, single administration of naringenin to normal rats elevated plasma insulin levels and improved glucose responses. These beneficial effects were blocked by PHTPP. In streptozocin-nicotinamide induced diabetic rats, treatment for 2 weeks with naringenin alone, but not in combination with PHTPP, significantly restored pancreatic β cell mass and improved glucose metabolism. Collectively, these data support that naringenin specifically activate ERβ to improve insulin secretion in the primary rat islets. Furthermore, naringenin administration also protected β cell function and reversed glucose dysregulation in diabetic rats. These beneficial effects are at least partially dependent on the ERβ pathway.

Estrogen receptor α interaction of zearalenone and its phase I metabolite α-zearalenol in combination with soy isoflavones in hERα-HeLa-9903 cells

Risk assessment primarily relies on toxicological data of individual substances, with limited information on combined effects. Recent in vitro experiments using Ishikawa cells, an endometrial carcinoma cell line expressing both estrogen receptor isoforms, demonstrated interactive effects of phyto- and mycoestrogens. The mycoestrogens, zearalenone (ZEN), and α-zearalenol (α-ZEL) exhibited significantly enhanced estrogenic responses in the presence of isoflavones (ISF), depending on substance ratios and concentrations. This study investigated the impact of phyto- and mycoestrogen combinations on estrogenic response following OECD guideline 455, utilizing hERα-HeLa-9903 cells. Test substances included mycoestrogens (ZEN and α-ZEL) and isoflavones (genistein (GEN), daidzein (DAI), and S-equol (EQ), a gut microbial metabolite of DAI). Mycoestrogens were tested in the range of 0.001 to 100 nM, while isoflavones were used at concentrations 1000 times higher based on relevant occurrence ratios. Results showed that ZEN and α-ZEL induced ERα-dependent luciferase expression in concentrations above 1 nM and 0.01 nM, respectively. However, ISF caused a superinduction of the luciferase signal above 1 µM. A superinduction is characterized by an unusually strong or heightened increase in the activity of the luciferase enzyme. This signal is not affected by the estrogen receptor antagonist 4-hydroxytamoxifen (4-OH-TAM), which was additionally used to verify whether the increase of signal is a true reflection of receptor activation. This superinduction was observed in all combinations of ZEN and α-ZEL with ISFs. Contrary to the luciferase activity findings, RT-qPCR experiments and a stability approach revealed lower real ERα activation by ISFs than measured in the ONE-Glo™ luciferase test system. In conclusion, the OECD protocol 455 appears unsuitable for testing ISFs due to their superinduction of luciferase and interactions with the test system, resulting in experimental artifacts. Further studies are necessary to explore structure–activity relationships within polyphenols and clarify the test system’s applicability.