Activation Of ERβ Research Articles

The protective effects of estrogen on biliary and liver damage are independent of ER‐β signaling in female Mdr2‐/‐ mice

BACKGROUND & AIMCholestatic liver diseases present with ductular reaction, biliary senescence, peribiliary inflammation and bridging fibrosis, and sex‐dependent differences in liver injury exist in different pathologies. The multidrug resistance 2 knockout (Mdr2‐/‐) mouse is a model of toxic bile‐induced cholangitis which show a higher degree of injury in female mice. Estrogen signals through its receptors, ER‐α and ER‐β, that form homodimers and act as transcription factors. Interestingly, ER‐α and ER‐β can have differing downstream responses. In a model of autoimmune cholestasis, inhibition of ER‐β reduces biliary damage, inflammation, immune cell infiltration and liver fibrosis in both male and female mice. We aimed to determine the role of estrogen/ER‐β signaling on biliary and liver damage in female Mdr2‐/‐ mice.METHODSStudies were performed in female wild‐type (WT) and Mdr2‐/‐ mice at 12 wks of age, with a subset of Mdr2‐/‐ mice subjected to ovariectomy (OVX, to reduce estrogen levels) or treated with PHTPP (ER‐β antagonist, 15 mg/kg BW/day by I.P. implanted minipump) for 1 wk. All studies utilized n=4 mice per group. We evaluated liver damage by H&E staining. Ductular reaction was measured by CK‐19 staining and semi‐quantification, and biliary senescence was imaged using p16 co‐staining with CK‐19 (to mark bile ducts). Peribiliary inflammation was evaluated using F4/80 (macrophage marker) staining and semi‐quantification, and qPCR for IL‐6 and TNF‐α in total liver samples. Immune cell infiltration was visualized by staining for CD3 (pan‐T cell marker) and CD20 (pan‐B cell marker). We determined liver fibrosis by (i) Sirius red staining and semi‐quantification, (ii) qPCR for TIMP1, Col1a1 and αSMA in total liver samples and (iii) co‐staining for desmin (hepatic stellate cell marker) with CK‐19. Human female control (avg. age 24.5 yr) and late‐stage, untreated PSC patient (avg. age 51.8 yr) serum samples were evaluated for estrogen levels by EIA.RESULTSOVX exacerbated liver damage, ductular reaction, biliary senescence, inflammation, immune response and liver fibrosis in female Mdr2‐/‐ mice, demonstrating a protective effect of estrogen signaling during cholangitis. Interestingly, female Mdr2‐/‐ mice treated with PHTPP (blocks ER‐β activity) had no change in any parameters of biliary damage or liver injury when compared to controls suggesting that estrogen’s protective effects are not mediated through ER‐β. Serum estrogen levels increased in female PSC samples versus control, potentially as a compensatory response or abnormal dysregulation as a consequence of injury.CONCLUSIONEstrogen may have a protective effect against biliary damage, inflammation, immune response and liver fibrosis during cholestasis. However, the protective effects are not dependent upon ER‐β signaling in female Mdr2‐/‐ mice and future work is ongoing to understand the role or ER‐α in this setting. Alteration of specific estrogen signaling mechanisms may be important for targeting cholestatic liver diseases in female patients.

Activity of Estrogen Receptor β Agonists in Therapy-Resistant Estrogen Receptor-Positive Breast Cancer.

BackgroundAmong women, breast cancer is the leading cause of cancer-related death worldwide. Estrogen receptor α-positive (ERα+) breast cancer accounts for 70% of all breast cancer subtypes. Although ERα+ breast cancer initially responds to estrogen deprivation or blockade, the emergence of resistance compels the use of more aggressive therapies. While ERα is a driver in ERα+ breast cancer, ERβ plays an inhibitory role in several different cancer types. To date, the lack of highly selective ERβ agonists without ERα activity has limited the exploration of ERβ activation as a strategy for ERα+ breast cancer.MethodsWe measured the expression levels of ESR1 and ESR2 genes in immortalized mammary epithelial cells and different breast cancer cell lines. The viability of ERα+ breast cancer cell lines upon treatments with specific ERβ agonists, including OSU-ERb-12 and LY500307, was assessed. The specificity of the ERβ agonists, OSU-ERb-12 and LY500307, was confirmed by reporter assays. The effects of ERβ agonists on cell proliferation, cell cycle, apoptosis, colony formation, cell migration, and expression of tumor suppressor proteins were analyzed. The expression of ESR2 and genes containing ERE-AP1 composite response elements was examined in ERα+ human breast cancer samples to determine the correlation between ESR2 expression and overall survival and that of putative ESR2-regulated genes.ResultsIn this study, we demonstrate the efficacy of highly selective ERβ agonists in ERα+ breast cancer cell lines and drug-resistant derivatives. ERβ agonists blocked cell proliferation, migration, and colony formation and induced apoptosis and S and/or G2/M cell-cycle arrest of ERα+ breast cancer cell lines. Also, increases in the expression of the key tumor suppressors FOXO1 and FOXO3a were noted. Importantly, the strong synergy between ERβ agonists and ERα antagonists suggested that the efficacy of ERβ agonists is maximized by combination with ERα blockade. Lastly, ESR2 (ERβ gene) expression was negatively correlated with ESR1 (ERα gene) and CCND1 RNA expression in human metastatic ERα+/HER2- breast cancer samples.ConclusionOur results demonstrate that highly selective ERβ agonists attenuate the viability of ERα+ breast cancer cell lines in vitro and suggest that this therapeutic strategy merits further evaluation for ERα+ breast cancer.

Diarylpropionitrile inhibits melanogenesis via protein kinase A/cAMP-response element-binding protein/microphthalmia-associated transcription factor signaling pathway in α-MSH-stimulated B16F10 melanoma cells

Diarylpropionitrile (DPN), a selective agonist for estrogen receptor β (ERβ), has been reported to regulate various hormonal responses through activation of ERβ in tissues including the mammary gland and brain. However, the effect of DPN on melanogenesis independent of ERβ has not been studied. The aim of this study is to examine the possibility of anti-melanogenic effect of DPN and its underlying mechanism. Melanin contents and cellular tyrosinase activity assay indicated that DPN inhibited melanin biosynthesis in alpha-melanocyte stimulating hormone-stimulated B16F10 melanoma cell line. However, DPN had no direct influence on in vitro tyrosinase catalytic activity. On the other hand, 17β-estradiol had no effect on inhibition of melanogenesis, suggesting that the DPN-mediated suppression of melanin production was not related with estrogen signaling pathway. Immunoblotting analysis showed that DPN down-regulated the expression of microphthalmia-associated transcription factor (MITF), a central transcription factor of melanogenesis and its down-stream genes including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Also, DPN attenuated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). Additionally, DPN suppressed the melanin synthesis in UVB-irradiated HaCaT conditioned media culture system suggesting that DPN has potential as an anti-melanogenic activity in physiological conditions. Collectively, our data show that DPN inhibits melanogenesis via down-regulation of PKA/CREB/MITF signaling pathway.

Pharmacological Properties to Pharmacological Insight of Sesamin in Breast Cancer Treatment: A Literature-Based Review Study.

The use of dietary phytochemical rather than conventional therapies to treat numerous cancers is now a well-known approach in medical science. Easily available and less toxic dietary phytochemicals present in plants should be introduced in the list of phytochemical-based treatment areas. Sesamin, a natural phytochemical, may be a promising chemopreventive agent aiming to manage breast cancer. In this study, we discussed the pharmacological properties of sesamin that determine its therapeutics opportunity to be used in breast cancer treatment and other diseases. Sesamin is available in medicinal plants, especially in Sesamum indicum, and is easily metabolized by the liver. To better understand the antibreast cancer consequence of sesamin, we postulate some putative pathways related to the antibreast cancer mechanism: (1) regulation of estrogen receptor (ER-α and ER-β) activities, (2) suppressing programmed death-ligand 1 (PD-L1) overexpression, (3) growth factor receptor inhibition, and (4) some tyrosine kinase pathways. Targeting these pathways, sesamin can modulate cell proliferation, cell cycle arrest, cell growth and viability, metastasis, angiogenesis, apoptosis, and oncogene inactivation in various in vitro and animal models. Although the actual tumor intrinsic signaling mechanism targeted by sesamin in cancer treatment is still unknown, this review summarized that this phytoestrogen suppressed NF-κB, STAT, MAPK, and PIK/AKT signaling pathways and activated some tumor suppressor protein in numerous breast cancer models. Cotreatment with γ-tocotrienol, conventional drugs, and several drug carriers systems increased the anticancer potentiality of sesamin. Furthermore, sesamin exhibited promising pharmacokinetics properties with less toxicity in the bodies. Overall, the shreds of evidence highlight that sesamin can be a potent candidate to design drugs against breast cancer. So, like other phytochemicals, sesamin can be consumed for better therapeutic advantages due to having the ability to target a plethora of molecular pathways until clinically trialed standard drugs are not available in pharma markets.

Abstract P4-02-09: The lncRNA XIST mediates sensitivity to ERβ targeted therapies in triple negative breast cancer

Abstract Background:. Poor patient outcomes in triple negative breast cancer (TNBC) largely stem from a lack of understanding of therapeutic vulnerabilities and an insufficient armamentarium of effective drugs. The standard of care for most early stage TNBC patients continues to be (neo)adjuvant chemotherapy and radiation. Identifying additional therapeutic options for this subset of patients represents a major unmet need. We and others demonstrated that estrogen receptor beta (ERβ) is expressed in about 20% of TN tumors. Prior research has shown that ligand-mediated activation of ERβ decreases proliferation, invasion, and migration in TNBC cell lines. In vivo, ERβ suppresses the growth of cell line xenograft models and prevents the development of metastatic lesions in a ligand dependent manner. Mechanistically, we found that ERβ repurposes EZH2 to suppress oncogenic NFκB signaling and that pharmacologic inhibition of EZH2 diminishes ERβ function and its anti-cancer effects. This research has led to an ongoing clinical trial (NCT03941730) assessing the efficacy of estradiol in ERβ+ TNBC patients with chemorefractory disease. As with all therapies, patients will exhibit de novo and acquired resistance and therefore we sought to understand the mechanisms of resistance to ERβ targeted therapies. Methods:. Using multiple models of ERβ positive TNBC, we developed ERβ resistant cell lines through chronic exposure to estradiol and the ERβ specific agonist LY500307 over a period of 8 months. We employed RNA sequencing to characterize the transcriptomic changes which occurred following ERβ resistance. We profiled XIST expression across multiple publicly available datasets, including TCGA, Metabric, BEAUTY, and GTEx. XIST expression was also modulated using CRISPR/Cas9 to assess subsequent effects on TNBC cell biology, ERβ function, and response to ERβ targeted therapies. Results:. Our resistant cell line models of ERβ positive TNBC maintained ERβ expression, but were no longer growth inhibited by ERβ agonists. RNAseq revealed substantial differences comparing the transcriptome of resistant versus sensitive cell lines, of which the most increased transcript in the resistant setting was the lncRNA XIST. XIST is best known for its role in X-chromosome inactivation through recruitment and association with the PRC2 complex, and thus EZH2. However, little is known about its functions in breast cancer. We therefore assessed the expression levels of XIST in breast tumors and cell lines. XIST expression was highly variable in breast cancer, was found in a proportion of all breast cancer sub-types and did not correlate with ERβ status. However, XIST expression was up-regulated in ERβ expressing cell lines following long term estrogen treatment. No effects on XIST expression were identified in multiple ERα positive models following estrogen, SERM/SERD treatment, or estrogen deprivation. Furthermore, upregulation of XIST was not associated with resistance to Paclitaxel or Doxorubicin, suggesting XIST is not a part of a broad resistance phenotype. Strikingly, CRISPR mediated knockout of XIST in ERβ resistant cells completely re-sensitized cells to ERβ targeted therapies suggesting that XIST expression is a critical component of ERβ resistance. Conclusions:. ERβ represents a relevant therapeutic target that is being tested in clinical trials. Using multiple in vitro models, we provide evidence that XIST expression is sufficient to induce resistance to. ERβ targeted therapies in TNBC and may therefore represent a relevant biomarker for patient stratification. Further strategies to suppress XIST expression may elicit anti-cancer effects on their own and may resensitize a sub-set of ERβ resistant tumors to ERβ agonists. Citation Format: Michael J Emch, Kirsten GM Aspros, Elizabeth S Bruinsma, Krishna R Kalari, Calley J Jones, Brandon W Simone, Matthew P Goetz, John R Hawse. The lncRNA XIST mediates sensitivity to ERβ targeted therapies in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-09.

The interplay between estrogen receptor beta and protein kinase C, a crucial collaboration for medulloblastoma cell proliferation and invasion

Medulloblastoma (MB) is the most common and aggressive pediatric intracranial tumor. Estrogen receptor β (ERβ) expression correlates with MB development and its phosphorylation modifies its transcriptional activity in a ligand-dependent or independent manner. Using in silico tools, we have identified several residues in ERβ protein as potential targets of protein kinases C (PKCs) α and δ. Using Daoy cells, we observed that PKCα and PKCδ associate with ERβ and induce its phosphorylation. The activation of ERβ promotes MB cells proliferation and invasion, and PKCs downregulation dysregulates these steroid receptor mediated processes. Our data suggest that these kinases may play a crucial role in the regulation of the ERβ transcriptional activity. Overexpression of both PKCα and PKCδ in MB biopsies samples supports their relevance in MB progression.

ERβ and Inflammation.

Estrogen, through the regulation of cytokine production, can act both as pro-inflammatory and anti-inflammatory signals dependent on the tissue context. In breast cancer cells, ERα is known to modulate inflammatory signaling through interaction with NFκB. Whether ERβ has a role in inflammation is less explored. Low levels of ERβ have been corroborated in several immune-related organs and, for example, in colonic epithelial cells. Specifically, an impact of ERβ on colitis and colitis-associated colorectal cancer (CRC) is experimentally supported, using ERβ-selective agonists, full-body ERβ knockout mice and, most recently, intestinal epithelial-specific knockout mice. An intricate crosstalk between ERβ and TNFα/NFκB signaling in the colon is supported, and ERβ activation appears to reduce macrophage infiltration also during high fat diet (HFD)-induced colon inflammation. Finally, the gut microbiota plays a fundamental role in the pathogenesis of colitis and ERβ has been indicated to modulate the microbiota diversity during colitis and colitis-induced CRC. ERβ is thus proposed to protect against colitis, by modulating NFκB signaling, immune cell infiltration, and/or microbiota composition. Selective activation of ERβ may therefore constitute a suitable preventative approach for the treatment of for example colitis-associated CRC.

Galangin 3-benzyl-5-methylether derivatives function as an adiponectin synthesis-promoting peroxisome proliferator-activated receptor γ partial agonist

The upregulation of adiponectin production has been suggested as a novel strategy for the treatment of metabolic diseases. Galangin, a natural flavonoid, exhibited adiponectin synthesis-promoting activity during adipogenesis in human bone marrow mesenchymal stem cells. In target identification, galangin bound both peroxisome proliferator-activated receptor (PPAR) γ and estrogen receptor (ER) β. Novel galangin derivatives were synthesized to improve adiponectin synthesis-promoting compounds by increasing the PPARγ activity of galangin and reducing its ERβ activity, because PPARγ functions can be inhibited by ERβ. Three galangin 3-benzyl-5-methylether derivatives significantly promoted adiponectin production by 2.88-, 4.47-, and 2.76-fold, respectively, compared to the effect of galangin. The most potent compound, galangin 3-benzyl-5,7-dimethylether, selectively bound to PPARγ (Ki, 1.7 μM), whereas it did not bind to ERβ. Galangin 3-benzyl-5,7-dimethylether was identified as a PPARγ partial agonist in docking and pharmacological competition studies, suggesting that it may have diverse therapeutic potential in a variety of metabolic diseases.

A novel targeted approach to delineate a role for estrogen receptor-β in ameliorating murine mammary tumor-associated neuroinflammation.

Circulating estrogens in breast cancer patients and survivors are often extremely low due to menopause and estrogen-reducing cancer treatments. Simultaneously, circulating inflammatory markers, and inflammatory proteins in brains of rodent tumor models, can be elevated and correlate with debilitating neurological and psychological comorbidities. Because estrogen has anti-inflammatory properties in the brain, we hypothesized that mammary tumor-induced neuroinflammation is driven, in part, by reduced brain estrogen signaling. An ovariectomized mouse model of postmenopausal breast cancer utilizing the ERα-positive 67NR mammary tumor cell line was used for these experiments. A novel, orally bioavailable, and brain penetrant ERβ agonist was administered daily via oral gavage. Following treatment, estrogen-responsive genes were measured in brain regions. Central and circulating inflammatory markers were measured via RT-qPCR and a multiplex cytokine array, respectively. We present novel findings that peripheral mammary tumors alter estrogen signaling genes including receptors and aromatase in the hypothalamus, hippocampus, and frontal cortex. Mammary tumors induced peripheral and central inflammation, however, pharmacological ERβ activation was not sufficient to reduce this inflammation. Data presented here suggest that compensating for low circulating estrogen with ERβ brain activation is not sufficient to attenuate mammary tumor-induced neuroinflammation, and is therefore not a likely candidate for the treatment of behavioral symptoms in patients. The novel finding that mammary tumors alter estrogen signaling-related genes is a clinically relevant advancement to the understanding of how peripheral tumor biology modulates neurobiology. This is necessary to predict and prevent behavioral comorbidities (e.g., cognitive impairment) prevalent in cancer patients and survivors.

Total flavonoids of Drynariae Rhizoma regulates ER-p38 MAPK signaling pathway to improve scopolamine-induced learning and memory impairments in model mice

This study intended to explore the effect and mechanism of total flavonoids of Drynariae Rhizoma in improving scopola-mine-induced learning and memory impairments in model mice. Ninety four-month-old Kunming(KM) mice were randomly divided into six groups. The ones in the model group and blank group were treated with intragastric administration of normal saline, while those in the medication groups separately received the total flavonoids of Drynariae Rhizoma, Kangnaoshuai Capsules, donepezil, as well as total flavonoids of Rhizoma Drynariae plus estrogen receptor(ER) blocker by gavage. The mouse model of learning and memory impairments was established via intraperitoneal injection of scopolamine. Following the measurement of mouse learning and memory abilities in Morris water maze test, the hippocampal ERβ expression was detected by immunohistochemistry, and the expression levels of ERβ and phosphorylated p38(p-p38) in the hippocampus and B-cell lymphoma 2(Bcl-2), Bcl-2-associated death promoter(Bad), and cysteinyl aspartate-specific protease-3(caspase-3) in the apoptotic system were assayed by Western blot. The contents of malondia-ldehyde(MDA), superoxide dismutase(SOD), and nitric oxide(NO) in the hippocampus were then determined using corresponding kits. Compared with the control group, the model group exhibited significantly prolonged incubation period, reduced frequency of cros-sing the platform, shortened residence time in the target quadrant, lowered ERβ, Bcl-2 and SOD activity in the hippocampus, and increased p-p38/p38, Bad, caspase-3, MDA, and NO. Compared with the model group, the total flavonoids of Rhizoma Drynariae increased the expression of ERβ and SOD in the hippocampus, down-regulated the expression of neuronal pro-apoptotic proteins, up-re-gulated the expression of anti-apoptotic proteins, and reduced p-p38/p38, MDA, and NO. The effects of total flavonoids of Drynariae Rhizoma on the above indexes were reversed by ER blocker. It has been proved that the total flavonoids of Drynariae Rhizoma obviously alleviate scopolamine-induced learning and memory impairments in mice, which may be achieved by regulating the neuronal apoptotic system and oxidative stress via the ER-p38 mitogen-activated protein kinase(ER-p38 MAPK) signaling pathway.

Estrogen Receptor β Participates in Alternariol-Induced Oxidative Stress in Normal Prostate Epithelial Cells.

Alternaria toxins are considered as emerging mycotoxins, however their toxicity has not been fully evaluated in humans. Alternariol (AOH), the most prevalent Alternaria mycotoxin, was previously reported to be genotoxic and to affect hormonal balance in cells; however, its direct molecular mechanism is not known. The imbalance in androgen/estrogen ratio as well as chronic inflammation are postulated as factors in prostate diseases. The environmental agents affecting the hormonal balance might participate in prostate carcinogenesis. Thus, this study evaluated the effect of two doses of AOH on prostate epithelial cells. We observed that AOH in a dose of 10 µM induces oxidative stress, DNA damage and cell cycle arrest and that this effect is partially mediated by estrogen receptor β (ERβ) whereas the lower tested dose of AOH (0.1 µM) induces only oxidative stress in cells. The modulation of nuclear erythroid-related factor 2 (Nrf2) was observed in response to the higher dose of AOH. The use of selective estrogen receptor β (ERβ) inhibitor PHTPP revealed that AOH-induced oxidative stress in both tested doses is partially dependent on activation of ERβ, but lack of its activation did not protect cells against AOH-induced ROS production or DNA-damaging effect in case of higher dose of AOH (10 µM). Taken together, this is the first study reporting that AOH might affect basic processes in normal prostate epithelial cells associated with benign and malignant changes in prostate tissue.

Elucidating the estrogen-like effects and biocompatibility of the herbal components in the Qing' E formula

Ethnopharmacological relevanceThe Qing' E Formula (QEF) is a compound preparation that was originally recorded in the ‘Prescriptions of the Bureau of Taiping People's Welfare Pharmacy’ during the Song Dynasty (10th century CE). It consists of four Chinese medicinal herbs, Eucommiae Cortex (Eucommia ulmoides), Psoraleae Fructus (Psoralea corylifolium), Juglandis Semen (Juglans regia), and Garlic Rhizoma. According to traditional Chinese medicine (TCM), QEF has the ability to tonify the kidney and strengthen muscle and bone. According to the ‘kidney governing bone’ theory in TCM, QEF is also used to treat the symptoms of climacteric syndrome, especially osteoporosis caused by reduced production of estrogen during the perimenopausal period; however, the therapeutic roles of the individual components of the QEF and their compatibility within the formula has not been investigated. Aim of the studyIn this study, the compatibility mechanism and estrogen-like action properties of the four herbal components in the QEF was elucidated according to the organizing principle of Chinese medicine formulas using both in vitro and in vivo models. Materials and methodsThe estrogen-like effects of QEF and its herbal components were investigated in MCF7 and HEK293 cells as well as ovariectomized (OVX) rats. The estrogen-like effects of the QEF and its components were analyzed in vitro using Cell Counting Kit-8 and Luciferase reporter gene assays. In the in vivo studies, the blood plasma levels of hormones, lipids, neurotransmitters, aromatase, superoxide dismutase (SOD), and malondialdehyde (MDA) were measured through enzyme-linked immunosorbent assays (ELISAs). The histological morphologies of the target organs after exposure to QEF were investigated by HE staining and immunohistochemical methods. The expression levels of estrogen pathway-related proteins and genes in the OVX rats were measured by Western blotting and real time quantitative PCR (RT-qPCR), respectively. ResultsThe in vitro results showed that the QEF, Eucommia (EC) and Psoralea (PF) promoted the proliferation of MCF-7 cells and upregulated the expression of ERα, ERβ and pS2 genes in the MCF-7 cells. Notably, the QEF demonstrated the most active estrogen-like effects compared to the individual ingredients. The in vivo results showed that the QEF, EC, and PF increased the uterine coefficient, upregulated the expression of both ERs (ERα and ERβ) in the uterus, and increased blood serum hormone levels. QEF and its individual components ameliorated menopausal-derived lipid metabolism dysfunction, increased neurotransmitter production by stimulating the adrenal glands, enhanced the antioxidant activity in the serum by increasing the concentration of SOD, reversed ovariectomy-derived atrophy in the uterus, and reduced the weight gain associated with estrogen reduction in the OVX rats. The QEF also antagonize the loss of appetite of OVX animals caused by feeding Psoralea alone, which could explain the compatibility mechanism of Qing' E Formula with reducing toxicity and increasing efficiency. ConclusionsThe estrogen-like effects of Eucommia and Psoralea were mainly mediated through activation of ERα and ERβ. The phytoestrogen components regulated hormone production and the expression of related proteins and genes, which indicated that these components exhibited estrogen-like therapeutic effects. However, the QEF showed the greatest estrogen-like effects compared to the individual components. Overall, this corroborated the therapeutic prowess of the QEF and clarified the pharmacodynamic interactions between the different components extracts in the QEF.

Estrogen receptor β alleviates inflammatory lesions in a rat model of inflammatory bowel disease via down-regulating P2X7R expression in macrophages.

Estrogen receptor beta (ERβ) agonists could inhibit inflammation in animal models of inflammatory bowel disease (IBD). However, the mechanism underlying such effect and the potential role of P2 × 7 receptor (P2X7R) remains unclear. In the present study, we examined whether the effect of ERβ activation in IBD rats was related to P2X7R. Overexpression of ERβ using a recombinant lentivirus in IBD rats improved the IBD-like symptoms, including weight loss, disease activity index (DAI) scores, and inflammatory responses. ERβ agonists DPN and ERB-041 attenuated P2X7R expression in macrophages from colitis rats and in a murine macrophage cell line (RAW264.7) in response to either lipopolysaccharide (LPS) or adenosine triphosphate (ATP). DPN and ERB-041 also blocked increased production of TNF-α, IL-6, and IL-1β in the rectocolon of colitis rats. The two ERβ agonists reversed LPS- and ATP-induced up-regulation of P2X7R and its downstream proteins, including NLRP3, ASC, caspase-1, and pro-IL-1β, in RAW264.7 cells. Also, in both the rectocolon of colitis rats and RAW264.7 cells, ERβ agonists reversed the up-regulation of extracellular regulated protein kinases (ERK), the Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3 (STAT3), but not up-regulation of serine threonine kinase or cAMP-response element binding protein (CREB). Blockade of JAK2 or STAT3 phosphorylation significantly reduced the ability of DPN to down-regulate P2X7R expression and the ability of ERB-041 and DPN to inhibit IL-1β release from RAW264.7 cells. We found that ERβ and P2X7R co-localized in the macrophages of rat rectocolon and in RAW264.7 cells. Deletion of macrophages from colitis rats with clodronate abolished the inhibitory effect of DPN. These results suggest that ERβ plays an important anti-inflammatory role in IBD rats by down-regulating P2X7R expression and inhibiting IL-1β release from macrophages through the JAK2/STAT3 signaling pathway.

Effects and mechanism of estrogen receptor β agonist on the migration and oxidative stress of human umbilical vein endothelial cell under high glucose condition

Objective: To investigate the effects and related mechanism of estrogen receptor β (ERβ) agonist on the migration and oxidative stress of human umbilial vein endothelial cells (HUVECs) under high glucose condition. Methods: The experimental research method was adopted. HUVECs were routinely cultured and passaged, and then cells of the logarithmic growth phase were collected for the subsequent experiments. The cells were divided into three groups according to the random number table, including normal control group (cultured with Roswell Park Memorial Institute 1640 cell culture medium (the same cell culture medium below) containing 5.5 mmol/L D-glucose), high glucose alone group (cultured with cell culture medium containing 25.0 mmol/L D-glucose alone), and high glucose+ERβ agonist diarylpropionitrile (DPN) group (cultured with cell culture medium containing 25.0 mmol/L D-glucose and 10 μmol/L DPN). Scratch test was conducted to detect the cell migration rate in the 3 groups at 24 h post scratching. Fluorescent probe method was used to detect the reactive oxygen species (ROS, denoted by red fluorescence intensity) of cells in the 3 groups on 5 d post culture. Western blotting was used to detect the protein expression levels of vascular endothelial growth factor (VEGF) and superoxide dismutase 2 (SOD2) of cells in the 3 groups on 5 d post culture. In the above-mentioned experiments, cells were grouped and cultured correspondingly as before, the number of samples in each group was 5. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: At 24 h post scratching, the cell migration rate in high glucose alone group was (36±5)%, which was significantly lower than (76±4)% of normal control group and (65±5)% of high glucose+DPN group (t=14.511, 9.603, P<0.01), and the cell migration rate in high glucose+DPN group was significantly lower than that in normal control group (t=3.943, P<0.01). On 5 d post culture, the level of ROS of cells in high glucose alone group (1.81±0.12) was significantly increased compared with normal control group and high glucose+DPN group (1.00±0.14, 0.91±0.15, t=9.679, 10.549, P<0.01), while the level of ROS of cells in normal control group and high glucose+DPN group were close (t=1.031, P>0.05). On 5 d post culture, the protein expression levels of VEGF and SOD2 of cells in high glucose alone group were significantly lower than the levels of normal control group (t=14.175, 13.787, P<0.01) and high glucose+DPN group (t=6.321, 17.750, P<0.01). The protein expression level of VEGF of cells in high glucose+DPN group was significantly lower than the level of normal control group (t=7.206, P<0.05), while the protein expression level of SOD2 of cells in high glucose+DPN group was significantly higher than the level of normal control group (t=2.890, P<0.05). Conclusions: The activation of ERβ can improve the inhibition of HUVECs migration induced by high glucose and alleviate oxidative stress injury induced by high glucose, which may be achieved by promoting the expression of VEGF and SOD2.

Apigenin Inhibits the Histamine-Induced Proliferation of Ovarian Cancer Cells by Downregulating ERα/ERβExpression.

BackgroundApigenin (APG), a natural flavonoid, can affect the development of a variety of tumors, but its role in ovarian cancer remains unclear. There has been an increasing amount of evidence supporting the vital role played by mast cells and the bioactive mediators they release, as components of the tumor microenvironment, in the progression of ovarian cancer (OC); however, the mechanism warrants further exploration.Methods and ResultsIn this study, a combination of transcriptomics analysis and application of TCGA database was performed, and we found that the expression of genes related to mast cell degranulation in ovarian cancer tissues changed remarkably. We then explored whether histamine, a major constituent of mast cell degranulation, could affect the development of ovarian cancer through immunohistochemistry analysis and cell proliferation assays. The results showed that a certain concentration of histamine promoted the proliferation of ovarian cancer cells by upregulating the expression of estrogen receptor α (ERα)/estrogen receptor β (ERβ). Additionally, we found that the inhibition of ERα or the activation of ERβ could inhibit the proliferation of ovarian cancer cells induced by histamine through real-time PCR and western blot assays. Finally, we demonstrated the attenuation effect imparted by apigenin in histamine-mediated ovarian cancer via the PI3K/AKT/mTOR signaling pathway.ConclusionOur research revealed that apigenin decelerated ovarian cancer development by downregulating ER-mediated PI3K/AKT/mTOR expression, thus providing evidence of its applicability as a potentially effective therapeutic agent for ovarian cancer treatment.

Differential Effects of Estrogen Receptor Alpha and Beta on Endogenous Ligands of Peroxisome Proliferator-Activated Receptor Gamma in Papillary Thyroid Cancer.

PurposeThe inhibition of estrogen receptor alpha (ERα) or the activation of ERβ can inhibit papillary thyroid cancer (PTC), but the precise mechanism is not known. We aimed to explore the role of ERα and ERβ on the production of endogenous peroxisome proliferator-activated receptor gamma (PPARγ) ligands in PTC.Methods2 PTC cell lines, 32 pairs of PTC tissues and matched normal thyroid tissues were used in this study. The levels of endogenous PPARγ ligands 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), 13-S-hydroxyoctadecadienoic acid (13(S)-HODE), and15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) were measured by ELISA.ResultsThe levels of PGJ2 and 15(S)-HETE were significantly reduced in PTC, but 13(S)-HODE was not changed. Activation of ERα or inhibition of ERβ significantly downregulated the production of PGJ2, 15(S)-HETE and 13(S)-HODE, whereas inhibition of ERα or activation of ERβ markedly upregulated the production of these three ligands. Application of endogenous PPARγ ligands inhibited growth, induced apoptosis of cancer cells, and promoted the efficacy of chemotherapy.ConclusionThe levels of endogenous PPARγ ligands PGJ2 and 15(S)-HETE are significantly decreased in PTC. The inhibition of ERα or activation of ERβ can inhibit PTC by stimulating the production of endogenous PPARγ ligands to induce apoptosis in cancer cells.

Retraction Note: Dioscin induces prostate cancer cell apoptosis through activation of estrogen receptor-\u03b2

Recent researches have shown that estrogen receptor-β (ERβ) activator may be a potent anticancer agent for prostate cancer (PCa), and our previous study also indicated that dioscin can upregulate the expression of ERβ in MC3T3-E1 cell. In the present work, the activity and mechanism of dioscin, a natural product, against PCa were investigated. The results showed that dioscin markedly inhibited cell viability, colony formation, motility and induced apoptosis in PC3 cells. Moreover, dioscin disrupted the formation of PC3 cell-derived mammospheres and reduced aldehyde dehydrogenase (ALDH) level and the CD133+/CD44+ cells, indicating that dioscin had a potent inhibitory activity on prostate cancer stem cells (PCSCs). In vivo results also showed that dioscin significantly suppressed the tumor growth of PC3 cell xenografts in nude mice. Furthermore, mechanism investigation showed that dioscin markedly upregulated ERβ expression level, subsequently increased prolyl hydroxylase 2 level, decreased the levels of hypoxia-inducible factor-1α, vascular endothelial growth factor A and BMI-1, and thus induced cell apoptosis by regulating the expression levels of caspase-3 and Bcl-2 family proteins. In addition, transfection experiment of ERβ-siRNA further indicated that diosicn showed excellent activity against PCa in vitro and in vivo by increasing ERβ expression level. The co-immunoprecipitation (Co-IP) results further suggested that dioscin promoted the interaction of c-ABL and ERβ, but did not change c-ABL expression. Moreover, the molecular docking assay showed that dioscin processed powerful affinity toward to ERβ mainly through the strong hydrogen bonding and hydrophobic effects, and the actions of dioscin on ERβ activation and tumor cells inhibition were significantly weakened in the mutational (Phe-336, Phe-468) PC3 cells. Collectively, these findings proved that dioscin exerted efficient anti-PCa activity via activation of ERβ, which should be developed as an efficient candidate in clinical for treating this cancer in the future.

Abstract 3094: Estrogen receptor β agonist depletes ovarian cancer stem cells via repressing epithelial to mesenchymal transition

Abstract Ovarian cancer is considered a hormone sensitive tumor. A healthy ovary expresses both Estrogen Receptors alpha (ERα) and beta (ERβ). However, although expression of ERα fairly remains constant, the expression of ERβ gradually decreases as the cells undergo malignant transformation and furthers with the progression of cancer. ERα is thought to promote expression of genes associated with cell survival and proliferation enhancing tumor growth. In contrast, the ability of ERβ to suppress orthotropic ovarian xenograft development supported the role of ERβ as a ‘tumor suppressor' in EOC tumorigenesis. Thus, use of ERβ agonists might prove to be therapeutically beneficial in ovarian cancer. Our studies reveal that activation of ERβ by using a newly developed ERβ agonist, WT-IV-012, is able to suppress the subpopulation of ovarian cancer stem cells. Mechanistically, WT-IV-012 inhibits epithelial to mesenchymal transition (EMT)-mediated CSC expansion, probably by downregulating ERα, which is a key regulator in the maintenance of stemness of CSCs. Taken together, our data indicate that suppressed expression of ERβ is critical to maintaining stemness of ovarian cancer stem cells, in turn, contributing to cancer relapse and chemoresistance. ERβ agonist would be able to limit the CSC subpopulation and prevent tumor relapse and metastasis. Citation Format: Ananya Banerjee, Shurui Cai, Na Li, Kousalya Lavudi, Tejinder Pal, Xuetao Bai, Qi-En Wang. Estrogen receptor β agonist depletes ovarian cancer stem cells via repressing epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3094.

Poor Endometrial Proliferation After Clomiphene is Associated With Altered Estrogen Action.

Suboptimal endometrial thickening is associated with lower pregnancy rates and occurs in some infertile women treated with clomiphene. To examine cellular and molecular differences in the endometrium of women with suboptimal vs optimal endometrial thickening following clomiphene. Translational prospective cohort study from 2018 to 2020 at a university-affiliated clinic. Reproductive age women with unexplained infertility treated with 100 mg of clomiphene on cycle days 3 to 7 who developed optimal (≥8mm; n = 6, controls) or suboptimal (<6mm; n = 7, subjects) endometrial thickness underwent preovulatory blood and endometrial sampling. The main outcome measures were endometrial tissue architecture, abundance and location of specific proteins, RNA expression, and estrogen receptor (ER) α binding. The endometrium of suboptimal subjects compared with optimal controls was characterized by a reduced volume of glandular epithelium (16% vs 24%, P = .01), decreased immunostaining of markers of proliferation (PCNA, ki67) and angiogenesis (PECAM-1), increased immunostaining of pan-leukocyte marker CD45 and ERβ, but decreased ERα immunostaining (all P < .05). RNA-seq identified 398 differentially expressed genes between groups. Pathway analysis of differentially expressed genes indicated reduced proliferation (Z-score = -2.2, P < .01), decreased angiogenesis (Z-score = -2.87, P < .001), increased inflammation (Z-score = +2.2, P < .01), and ERβ activation (Z-score = +1.6, P < .001) in suboptimal subjects. ChIP-seq identified 6 genes bound by ERα that were differentially expressed between groups (P < .01), some of which may play a role in implantation. Women with suboptimal endometrial thickness after clomiphene exhibit aberrant ER expression patterns, architectural changes, and altered gene and protein expression suggesting reduced proliferation and angiogenesis in the setting of increased inflammation.

Differential Expression of Estrogen Receptors in the Hypothalamus Underlies the Bimodal Effects of Estradiol on Luteinizing Hormone Release

Low 17β-estradiol (E2) levels suppress luteinizing hormone (LH) release, while high E2 stimulates an LH surge in the positive-feedback required for ovulation. Kisspeptin (Kp) neurons in the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei of hypothalamus have been implicated in E2 positive- and negative-feedback effects, respectively. However, how E2 differentially regulates these two neuronal populations remains unknown. We investigated whether neurons in the AVPV and ARC are differently responsive to changes in E2 levels and the regulatory role of estrogen receptors (ERs). Ovariectomized (OV) rats received oil or E2 at doses of 4 (OVE-4), 40 (OVE-40), or 80 (OVE-80) µg/kg. Rats on diestrus (DI) and proestrus (PRO) were used as physiological controls. OV rats were also treated with 0.02, 0.2 or 2.0 mg/kg of propyl pyrazole triol (PPT), or 6 mg/kg of diarylpropionitrile (DPN). Serial blood samples were collected for hormonal measurements. Brains were processed for immunohistochemistry and qPCR analyses in the AVPV and ARC. The E2 doses gradually increased plasma E2, with PRO levels being attained in OVE-80 rats. OVE-80 rats displayed a PRO-like LH surge, while LH levels were constantly suppressed in OVE-4 rats. Progesterone receptor (PR) was used as an index of E2 responsiveness. PR expression was increased in the AVPV of PRO and OVE-80 rats, associated with c-Fos expression and occurrence of LH surge. In the ARC, both low and high E2 induced PR expression and reduced the number of Kp-immunoreactive (ir) neurons, consistent with the negative feedback effects on LH. E2 at 4 or 80 µg/kg equally induced PR expression in 90% of ARC Kp-ir neurons. Despite the higher sensitivity to E2 in the ARC, the percentage of neurons expressing ERα was lower in the ARC compared with AVPV. However, Esr1 expression was 2-fold higher in the ARC than in AVPV for low E2 levels, whereas both Esr1 and Esr2 were more expressed in the AVPV under high E2 status. Notably, Esr1/Esr2 ratio was twice as high in the ARC as in the AVPV regardless of E2 levels, suggesting a stronger ERβ inhibition over ERα in the AVPV. Accordingly, ERα selective activation with PPT increased PR in the ARC at the doses of 0.2 and 2.0 mg/kg, reducing plasma LH, while only the highest dose was able to stimulate PR expression in the AVPV. ERβ activation with DPN, in turn, had no effect in OV rats but amplified the induction of AVPV PR and the size of the LH surge in OVE-80 rats. Thus, we provide evidence that ARC and AVPV neurons are responsive to low and high E2 levels, respectively. ARC is 10 times more sensitive to ERα activation than AVPV, whereas ERβ positively modulates AVPV responsiveness to high E2. These differential responses to E2 seem to be related to differences in the relative Esr1 and Esr2 expression in the ARC and AVPV. Our findings suggest that hypothalamic differences in the relative expression of ERs play a key role in the bimodal regulation of LH release by E2.