Significance of dysregulated M2 macrophage and ESR2 in the ovarian metastasis of gastric cancer.

Background: Prognosis of gastric cancer (GC) patients with ovarian metastasis (OM) remains poor. We hereby characterized the role of tumor immune microenvironment (TIME) and identified potential key regulators in the OM with the aim of understanding its molecular basis to develop novel therapeutic targets. Methods: Transcriptomic analyses of paired primary and ovarian metastatic lesions of seven GC patients from Fudan University Shanghai Cancer Center uncovered and functionally annotated their differentially expressed genes (DEGs). CIBERSORT analysis revealed differential TIME between primary GCs and OMs, which was further validated by multiplex immunofluorescence (mIF). Unique overexpression of candidate regulator in OMs was validated by an immunohistochemical (IHC) staining-based cohort study and in vitro cell growth, migration and invasion assays were conducted to characterize its function in GC progression. Results: Functional enrichment analyses of DEGs between GCs and matched OMs revealed multiple significantly dysregulated immune-related and cancer-related pathways. Distinctive subsets of immune cells, especially M2 macrophage, were selectively enriched in metastatic lesions. mIF-based quantification further validated the overexpression of CD68+CD206+ M2 macrophage in the OMs. Estrogen receptor 2 (ESR2), which encodes estrogen receptor ß (ERß), was not only potentially correlated with M2 macrophage but also overexpressed in the OM of GC. ESR2 was up-regulated in cancerous tissue and its high expression correlated with younger age, more advanced lymph node metastasis and pathological stage, as well as a worse patient survival. IHC staining of ERß in the cohort of paired primary and metastatic GCs validated its selective overexpression in OMs. Small-interfering RNAs (siRNAs)-induced knockdown of ESR2 significantly inhibited the invasion and migration of both AGS and HGC-27 GC cell lines. Conclusions: Comparative RNA-sequencing analysis revealed the dysregulated TIME, M2 macrophage in particular, between primary GC and OM. ESR2 potentially correlated with M2 macrophage and played pro-oncogenic roles in GC progression and metastasis.

Estrogen receptor ß activation alleviates inflammatory bowel disease by suppressing NLRP3-dependent IL-1ß production in macrophages via downregulation of intracellular calcium level.

INTRODUCTION: Although several estrogen receptor ß (ERß) agonists have been reported to alleviate IBD, the pivotal mechanism remains obscure. OBJECTIVES: To examine the effects and mechanisms of ERß activation on cytokine/chemokine networks in colitis mice. METHODS: Dextran sulfate sodium salt (DSS) and trinitro-benzene-sulfonic acid (TNBS) were used to induce mouse colitis model. Multiple molecular biological methods were employed to evaluate the severity of mouse colitis and the level of cytokine and/or chemokine. RESULTS: Bioinformatics analysis, ELISA and immunofluorescence results showed that the targeted cytokines and/or chemokines associated with ERß expression and activation is IL-1ß, and the anti-colitis effect of ERß activation was significantly attenuated by the overexpression of AAV9-IL-1ß. Immunofluorescence analysis indicated that ERß activation led to most evident downregulation of IL-1ß expression in colonic macrophages as compared to monocytes and neutrophils. Given the pivotal roles of NLRP3, NLRC4, and AIM2 inflammasome activation in the production of IL-1ß, we examined the influence of ERß activation on inflammasome activity. ELISA and WB results showed that ERß activation selectively blocked the NLRP3 inflammasome assembly-mediated IL-1ß secretion. The calcium-sensing receptor (CaSR) and calcium signaling play crucial roles in the assembly of the NLRP3 inflammasome. WB and immunofluorescence results showed that ERß activation reduced intracellular CaSR expression and calcium signaling in colonic macrophages. Combination with CaSR overexpression plasmid reversed the suppressive effect of ERß activation on NLRP3 inflammasome assembly, and counteracting the downregulation of IL-1ß secretion. CONCLUSION: Our research uncovers that the anti-colitis effect of ERß activation is accomplished through the reduction of IL-1ß levels in colonic tissue, achieved by specifically decreasing CaSR expression in macrophages to lower intracellular calcium levels and inhibit NLRP3 inflammasome assembly-mediated IL-1ß production.

Mechanisms of Action of Phytoestrogens and Their Role in Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is a rare disease characterized by the development of adenomatous polyps in the colon and rectum already in adolescence. If left untreated, patients develop colorectal cancer (CRC) with a 100% probability. To date, the gold standard of FAP management is surgery, which is associated with morbidity and mortality. A chemopreventive agent capable of delaying, preventing and reversing the development of CRC has been sought. Several classes of drugs have been used but to date no chemopreventive drug has been found for the management of this disease. In recent years, the importance of estrogen receptors in FAP and CRC, particularly the ß subtype, has emerged. Indeed, the expression of the latter is strongly reduced in adenomatous polyps and CRC and is inversely correlated with the aggressiveness of the disease. Since phytoestrogens have a high affinity for this receptor, they have been suggested for use as chemopreventive agents in FAP and CRC. A combination of phytoestrogens and insoluble fibres has proved particularly effective. In this review, the various mechanisms of action of phytoestrogens were analyzed and the effectiveness of using phytoestrogens as an effective chemopreventive strategy was discussed.

Small molecule conjugates with selective estrogen receptor ß agonism promote anti-aging benefits in metabolism and skin recovery.

Estrogen is imperative to mammalian reproductivity, metabolism, and aging. However, the hormone activating estrogen receptor (ERs) α can cause major safety concerns due to the enrichment of ERα in female tissues and certain malignancies. In contrast, ERß is more broadly expressed in metabolic tissues and the skin. Thus, it is desirable to generate selective ERß agonist conjugates for maximizing the therapeutic effects of ERs while minimizing the risks of ERα activation. Here, we report the design and production of small molecule conjugates containing selective non-steroid ERß agonists Gtx878 or genistein. Treatment of aged mice with our synthesized conjugates improved aging-associated declines in insulin sensitivity, visceral adipose integrity, skeletal muscle function, and skin health, with validation in vitro. We further uncovered the benefits of ERß conjugates in the skin using two inducible skin injury mouse models, showing increased skin basal cell proliferation, epidermal thickness, and wound healing. Therefore, our ERß-selective agonist conjugates offer novel therapeutic potential to improve aging-associated conditions and aid in rejuvenating skin health.

Detection of isoflavones and phytoestrogen-rich plant extracts binding to estrogen receptor ß using a yeast-based fluorescent assay.

Alchemilla vulgaris L., Trifolium pratense L. and Glycyrrhiza glabra L. are important remedies in traditional medicine, known for many usages, including treating gynecological diseases. Despite folkloric use of the plant materials, there is a lack of scientific data to support their therapeutic application. The aims of the present study were to evaluate the relative binding affinities (RBAs) of plant-derived phytoestrogens for estrogen receptor ß (ERß) using fluorescent biosensor in yeast and to apply this assay for the assessment of the potential of plant materials towards ERs and treatment of estrogen-related disorders. Ligand-binding domain of ERß fused with yellow fluorescent protein (ERß LBD-YFP) was expressed in S. cerevisiae and fluorescence was detected by fluorimetry and fluorescence microscopy. Structural basis for experimental results was explored by molecular docking. Yeast-based fluorescent assay was successfully optimized and applied for identification of natural phenolic compounds and phytoestrogen-rich plant extracts that interact with ERß-LBD, making this biosensor a valuable tool for screening estrogenic potential of a variety of plant extracts. This assay can be used for preliminary testing of plant-derived or fungal extracts, but also other sources of environmental substances with ER-modulating activity in order to assess their possible effects on the female reproductive system.

ERß Regulation of Indian Hedgehog Expression in the First Wave of Ovarian Follicles.

Increased activation of ovarian primordial follicles in Erß knockout (ErßKO) rats becomes evident as early as postnatal day 8.5. To identify the ERß-regulated genes that may control ovarian primordial follicle activation, we analyzed the transcriptome profiles of ErßKO rat ovaries collected on postnatal days 4.5, 6.5, and 8.5. Compared to wildtype ovaries, ErßKO ovaries displayed dramatic downregulation of Indian hedgehog (Ihh) expression. IHH-regulated genes, including Hhip, Gli1, and Ptch1, were also downregulated in ErßKO ovaries. This was associated with a downregulation of steroidogenic enzymes Cyp11a1, Cyp19a1, and Hsd17b1. The expression of Ihh remained very low in ErßKO ovaries despite the high levels of Gdf9 and Bmp15, which are known upregulators of Ihh expression in the granulosa cells of activated ovarian follicles. Strikingly, the downregulation of the Ihh gene in ErßKO ovaries began to disappear on postnatal day 16.5 and recovered on postnatal day 21.5. In rat ovaries, the first wave of primordial follicles is rapidly activated after their formation, whereas the second wave of primordial follicles remains dormant in the ovarian cortex and slowly starts activating after postnatal day 12.5. We localized the expression of Ihh mRNA in postnatal day 8.5 wildtype rat ovaries but not in the age-matched ErßKO ovaries. In postnatal day 21.5 ErßKO rat ovaries, we detected Ihh mRNA mainly in the activated follicles in the ovaries' peripheral regions. Our findings indicate that the expression of Ihh in the granulosa cells of the activated first wave of ovarian follicles depends on ERß.

Altered Glycolysis, Mitochondrial Biogenesis, Autophagy and Apoptosis in Peritoneal Endometriosis in Adolescents.

Energy metabolism plays a pivotal role in the pathogenesis of endometriosis. For the initial stages of the disease in adolescents, this aspect remains unexplored. The objective of this paper was to analyze the association of cellular and endosomal profiles of markers of glycolysis, mitochondrial biogenesis, apoptosis, autophagy and estrogen signaling in peritoneal endometriosis (PE) in adolescents. We included 60 girls aged 13-17 years in a case-control study: 45 with laparoscopically confirmed PE (main group) and 15 with paramesonephric cysts (comparison group). Samples of plasma and peritoneal fluid exosomes, endometrioid foci and non-affected peritoneum were tested for estrogen receptor (Erα/ß), hexokinase (Hex2), pyruvate dehydrogenase kinase (PDK1), glucose transporter (Glut1), monocarboxylate transporters (MCT1 and MCT2), optic atrophy 1 (OPA1, mitochondrial fusion protein), dynamin-related protein 1 (DRP1, mitochondrial fission protein), Bax, Bcl2, Beclin1, Bnip3, P38 mitogen-activated protein kinase (MAPK), hypoxia-inducible factor 1 (Hif-1α), mitochondrial voltage-dependent anion channel (VDAC) and transforming growth factor (TGFß) proteins as markers of estrogen signaling, glycolysis rates, mitochondrial biogenesis and damage, apoptosis and autophagy (Western-Blot and PCR). The analysis identified higher levels of molecules associated with proliferation (ERß), glycolysis (MCT2, PDK1, Glut1, Hex2, TGFß and Hif-1α), mitochondrial biogenesis (OPA1, DRP1) and autophagy (P38, Beclin1 and Bnip3) and decreased levels of apoptosis markers (Bcl2/Bax) in endometrioid foci compared to non-affected peritoneum and that in the comparison group (p < 0.05). Patients with PE had altered profiles of ERß in plasma and peritoneal fluid exosomes and higher levels of Glut1, MCT2 and Bnip3 in plasma exosomes (p < 0.05). The results of the differential expression profiles indicate microenvironment modification, mitochondrial biogenesis, estrogen reception activation and glycolytic switch along with apoptosis suppression in peritoneal endometrioid foci already in adolescents.

Estrogen α and ß Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model.

Glioblastoma multiforme (GBM) is a malignant tumor with a higher prevalence in men and a higher survival rate in transmenopausal women. It exhibits distinct areas influenced by changing environmental conditions. This study examines how these areas differ in the levels of estrogen receptors (ERs) which play an important role in the development and progression of many cancers, and whose expression levels are often correlated with patient survival. This study utilized two research models: an in vitro model employing the U87 cell line and a second model involving tumors resected from patients (including tumor core, enhancing tumor region, and peritumoral area). ER expression was assessed at both gene and protein levels, with the results validated using confocal microscopy and immunohistochemistry. Under hypoxic conditions, the U87 line displayed a decrease in ERß mRNA expression and an increase in ERα mRNA expression. In patient samples, ERß mRNA expression was lower in the tumor core compared to the enhancing tumor region (only in males when the study group was divided by sex). In addition, ERß protein expression was lower in the tumor core than in the peritumoral area (only in women when the study group was divided by sex). Immunohistochemical analysis indicated the highest ERß protein expression in the enhancing tumor area, followed by the peritumoral area, and the lowest in the tumor core. The findings suggest that ER expression may significantly influence the development of GBM, exhibiting variability under the influence of conditions present in different tumor areas.

Loss of ERß Disrupts Gene Regulation in Primordial and Primary Follicles.

Loss of ERß increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERß in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erß knockout (Erßnull) mouse ovaries, and their transcriptomes were compared with those of control Erßfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erßnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERß has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erßnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erßfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erß-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erßnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erßnull PrFs were downregulated. Such deregulation of genes in Erßnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erßnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERß either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.

Novel estrogen receptor ß/histone deacetylase dual-targeted near-infrared fluorescent probes as theranostic agents for imaging and treatment of prostate cancer.

Estrogen receptor (ER) ß and histone deacetylases (HDACs), when overexpressed, are associated closely with the occurrence and development of prostate cancer and are, therefore, considered important targets and biomarkers used in the clinical treatment of prostate cancer. The present study involved the design and synthesis of the first ERß and HDAC dual-target near-infrared fluorescent probe with both imaging capacity and antitumor activity for prostate cancer. Both P1 and P2 probes exhibited excellent ERß selectivity, with P1 being almost exclusively selective for ERß compared to ERα. In addition, P1 exhibited good optical properties, such as strong near-infrared emission, large Stokes shift, and better anti-interference ability, along with excellent imaging ability for living cells. P1 also exhibited potent inhibitory activity against HDAC6 and DU-145 cells, with IC50 values of 52 nM and 0.96 µM, respectively. Further, P1 was applied successfully for the in vivo imaging of prostate cancer in a mouse model, and significant in vivo antitumor efficacy was achieved. The developed dual-target NIR fluorescent probe is expected to serve as an effective tool in the research on prostate cancer, leading to novel insights for the theranostic study of diseases related to ERß and HDACs.

ERß-activated LINC01018 promotes endometriosis development by regulating the CDC25C/CDK1/CyclinB1 pathway.

Endometriosis refers to as an estrogen-dependent disease. Estrogen receptor ß (ERß), the main estrogen receptor subtype which is encoded by the estrogen receptor 2 (ESR2) gene, can mediate the action of estrogen in endometriosis. Although selective estrogen receptor modulators can target the ERß, they are not specific due to the wide distribution of ERß. Recently, long noncoding RNAs have been implicated in endometriosis. Therefore, we aim to explore and validate the downstream regulatory mechanism of ERß, and to investigate the potential role of long intergenic noncoding RNA 1018 (LINC01018) as a nonhormonal treatment for endometriosis. Our study demonstrates that the expression levels of ESR2 and LINC01018 are increased in ectopic endometrial tissues and reveals a significant positive correlation between the ESR2 and LINC01018 expression. Mechanistically, ERß directly binds to an estrogen response element located in the LINC01018 promoter region and activates LINC01018 transcription. Functionally, ERß can regulate the CDC25C/CDK1/CyclinB1 pathway and promote ectopic endometrial stromal cell proliferation via LINC01018 in vitro. Consistent with these findings, the knockdown of LINC01018 inhibits endometriotic lesion proliferation in vivo. In summary, our study demonstrates that the ERß/LINC01018/CDC25C/CDK1/CyclinB1 signaling axis regulates endometriosis progression.

The steroid hormone ADIOL promotes learning by reducing neural kynurenic acid levels.

Reductions in brain kynurenic acid levels, a neuroinhibitory metabolite, improve cognitive function in diverse organisms. Thus, modulation of kynurenic acid levels is thought to have therapeutic potential in a range of brain disorders. Here we report that the steroid 5-androstene 3ß, 17ß-diol (ADIOL) reduces kynurenic acid levels and promotes associative learning in Caenorhabditis elegans We identify the molecular mechanisms through which ADIOL links peripheral metabolic pathways to neural mechanisms of learning capacity. Moreover, we show that in aged animals, which normally experience rapid cognitive decline, ADIOL improves learning capacity. The molecular mechanisms that underlie the biosynthesis of ADIOL as well as those through which it promotes kynurenic acid reduction are conserved in mammals. Thus, rather than a minor intermediate in the production of sex steroids, ADIOL is an endogenous hormone that potently regulates learning capacity by causing reductions in neural kynurenic acid levels.

ER-ß accelerates the process of primary osteoporosis by promoting VEGFA-mediated apoptosis of osteoblasts.

Primary osteoporosis (POP) is a widespread and severe disorder of bone metabolism characterized by reduced bone mass and destruction of bone structure, frequently inducing fracture risk and imposing a heavy economic burden on public life. The development of POP partially revolves around the estrogen receptor ß (ER-ß), one of the major mediator receptors of estrogen that influences apoptosis in a range of cells. We performed KEGG and GO analysis by mining the transcriptomic dataset of POP samples showing significant enrichment of differentially expressed genes (DEGs) in multiple apoptosis-related pathways. The results of the Spearman correlation analysis and Protein-Protein Interaction (PPI) Networks screening of hub genes indicated that vascular endothelial growth factor A (VEGFA) may be a key target of ER-ß in controlling osteoblast apoptosis. Further, we carried out high-throughput sequencing of ESR2-silenced MC3T3-E1 cells and noticed a substantial suppression in VEGFA expression and all apoptosis-related pathways. In addition, we determined the cell cycle and apoptosis by constructing a VEGFA-silenced cell model utilizing flow cytometry (FCM), and the results showed that ER-ß could regulate the osteoblast cycle and thus promote osteoblast apoptosis by promoting VEGFA expression. And Western blot results showed that apoptosis was most likely realized through the regulation of downstream apoptosis markers c-JUN (c-Jun N-terminal kinase, JNK) and GADD45G (Growth Arrest and DNA Damage-Inducible Protein 45 gamma). The effects of ESR2 and VEGFA on the proliferation of osteoblasts were lastly assessed using the cell counting kit- 8 (CCK-8) assay. In conclusion, this study identifies that the roles of ER-ß in the regulation of osteoblast apoptosis are closely related to VEGFA and provides a new target for POP treatment.

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

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 ß 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 ERß without affecting the activity of ERα, G protein-coupled estrogen receptor (GPER) or estrogen-related receptor (ERR) α/ß/γ. Additionally, treatment with naringenin enhanced glucose-stimulated insulin secretion in isolated rat islets. This effect was abrogated by PHTPP, an ERß antagonist. Transcriptomic analysis revealed that naringenin upregulated the expression of genes, such as Pdx1 and Mafa, which are closely linked to improved ß-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 ß cell mass and improved glucose metabolism. Collectively, these data support that naringenin specifically activate ERß to improve insulin secretion in the primary rat islets. Furthermore, naringenin administration also protected ß cell function and reversed glucose dysregulation in diabetic rats. These beneficial effects are at least partially dependent on the ERß pathway.

Loss of ERß in Aging LXRαß Knockout Mice Leads to Colitis.

Liver X receptors (LXRα and LXRß) are oxysterol-activated nuclear receptors that play key roles in cholesterol homeostasis, the central nervous system, and the immune system. We have previously reported that LXRαß-deficient mice are more susceptible to dextran sodium sulfate (DSS)-induced colitis than their WT littermates, and that an LXR agonist protects against colitis in mice mainly via the regulation of the immune system in the gut. We now report that both LXRα and LXRß are expressed in the colonic epithelium and that in aging LXRαß-/- mice there is a reduction in the intensity of goblet cells, mucin (MUC2), TFF3, and estrogen receptor ß (ERß) levels. The cytoplasmic compartment of the surface epithelial cells was markedly reduced and there was a massive invasion of macrophages in the lamina propria. The expression and localization of ß-catenin, α-catenin, and E-cadherin were not changed, but the shrinkage of the cytoplasm led to an appearance of an increase in staining. In the colonic epithelium there was a reduction in the expression of plectin, a hemidesmosome protein whose loss in mice leads to spontaneous colitis, ELOVL1, a fatty acid elongase protein coding gene whose overexpression is found in colorectal cancer, and non-neuronal choline acetyltransferase (ChAT) involved in the regulation of epithelial cell adhesion. We conclude that in aging LXRαß-/- mice, the phenotype in the colon is due to loss of ERß expression.

Evaluating estrogenic activity of isoflavones in miso using yeast two-hybrid method.

Estrogenic activity in miso was evaluated without in vivo animal experimentation using in vitro method with yeast in a two-hybrid method because of its similarities with human cells. First, the recombinant yeast containing genes of human estrogen receptor (hER) was prepared for modeling human cells. Subsequently, standard solutions of 17ß-estradiol and isoflavone (1.0 × 10-12 - 1.0 × 10-6 ) were assayed using the yeast. Their yeast produces ß-glucosidase according to the concentrations of their solutions. Therefore, the estrogenic activity can be evaluated using recombinant yeast for the yeast two-hybrid method. Results show that 17ß-estradiol has affinity to bind with Y187-αα. Genistein has affinity to bind with Y187-ßß. Daidzein, genistein, and glycitein in miso were 2.0-2.2 times the average concentrations of miso. Particularly, Mame miso had the highest concentration of isoflavones among all miso samples. Isoflavone in miso samples showed estrogenic activity against Y187-ßß. Mame miso had particularly high activity (1.97 U/OD660 1.0) against Y187-ßß modeling hERßß. Finally, the interaction of the human estrogen receptors was analyzed with 17ß-estradiol and isoflavones using Y187 strains. Isoflavone inhibited the estrogenic activity of 17ß-estradiol using Y187-αα. However, the estrogenic activity of 17ß-estradiol against Y187-αß and Y187-ßß, which model hER-αß and hER-ßß, was activated by isoflavone. Results showed genistein as the antagonist of estrogenic activity within 17ß-estradiol against hERαα. However, it is an agonist of the activity within 17ß-estradiol against hERαß and hERßß. The yeast two-hybrid method has some potential for assessing the estrogenic activity of isoflavone in foods as a human model. PRACTICAL APPLICATION: Today, isoflavones in foods must be evaluated using in vivo methods such as animal experimentation because the estrogenic activities of isoflavones are agonist or antagonist with 17ß-estradiol against estrogen receptors. Because animal experimentation is time-consuming and expensive, isoflavones in foods can be evaluated using yeast, a eukaryote that has similarity to human cells, while obviating in vivo methods. The yeast two-hybrid method is useful to assay the estrogenic activity of isoflavones in foods.

11-Ethoxyviburtinal improves chronic restraint stress-induced anxiety-like behaviors in gender-specific mice via PI3K/Akt and E2 /ERß signaling pathways.

Anxiety disorder is a chronic and disabling psychiatric disorder that is more prevalent in females than in males. 11-Ethoxyviburtinal is an iridoid extracted from Valeriana jatamansi Jones, which has anxiolytic potential. The aim of the present work was to study the anxiolytic efficacy and mechanism of 11-ethoxyviburtinal in gender-specific mice. We first evaluated the anxiolytic-like efficacy of 11-ethoxyviburtinal in chronic restraint stress (CRS) mice of different sexes through behavioral experiments and biochemical indexes. In addition, network pharmacology and molecular docking were used to predict potential targets and important pathways for the treatment of anxiety disorder with 11-ethoxyviburtinal. Finally, the influence of 11-ethoxyviburtinal on phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, estrogen receptor ß (ERß) expression, and anxiety-like behavior in mice was verified by western blotting, immunohistochemistry staining, antagonist intervention methods, and behavioral experiments. 11-ethoxyviburtinal alleviated the anxiety-like behaviors induced by CRS and inhibited neurotransmitter dysregulation and HPA axis hyperactivity. It inhibited the abnormal activation of the PI3K/Akt signaling pathway, modulated estrogen production, and promoted ERß expression in mice. In addition, the female mice may be more sensitive to the pharmacological effects of 11-ethoxyviburtinal. 11-ethoxyviburtinal may exert its anxiolytic-like effects through PI3K/Akt and E2/ERß signaling pathways. Meanwhile, by comparing the male and female mice, gender differences may affect the therapy and development of anxiety disorder.

Breast cancer progression and kynurenine pathway enzymes are induced by hexachlorobenzene exposure in a Her2-positive model.

Breast cancer is one of the leading cancers among women worldwide. Given the evidence that pesticides play an important role in breast cancer, interest has grown in pesticide impact on disease progression. Hexachlorobenzene (HCB), an aryl hydrocarbon receptor (AhR) ligand, promotes triple-negative breast cancer cell migration and invasion. Estrogen receptor ß (ERß) inhibits cancer motility, while G protein-coupled ER (GPER) modulates the neoplastic transformation. Tryptophan is metabolized through the kynurenine pathway by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), with kynurenine signaling activation often predicting worse prognosis in cancer. In this context, we examined the HCB (0.005; 0.05; 0.5 and 5 µM) effect on LM3 cells, a human epidermal growth factor receptor 2 (HER2)-positive breast cancer model. Results show that HCB increases IDO and TDO mRNA levels and promotes cell viability, proliferation and migration through the AhR pathway. Moreover, HCB boosts mammosphere formation, vascular endothelial growth factor and cyclooxygenase-2 expression and reduces IL-10 levels. For some parameters, U-shaped or inverted U-shaped dose-response curves are shown. HCB alters ER levels, reducing ERß while increasing GPER. These results demonstrate that exposure to environmentally relevant concentrations of HCB up-regulates the kynurenine pathway and dysregulates ERß and GPER levels, collaborating in HER2-positive breast cancer progression.

ERß promoted invadopodia formation-mediated non-small cell lung cancer metastasis via the ICAM1/p-Src/p-Cortactin signaling pathway.

In a previous study, our research group observed that estrogen promotes the metastasis of non-small cell lung cancer (NSCLC) through the estrogen receptor ß (ERß). Invadopodia are key structures involved in tumor metastasis. However, it is unclear whether ERß is involved in the promotion of NSCLC metastasis through invadopodia. In our study, we used scanning electron microscopy to observe the formation of invadopodia following the overexpression of ERß and treatment with E2. In vitro experiments using multiple NSCLC cell lines demonstrated that ERß can increase the formation of invadopodia and cell invasion. Mechanistic studies revealed that ERß can upregulate the expression of ICAM1 by directly binding to estrogen-responsive elements (EREs) located on the ICAM1 promoter, which in turn can enhance the phosphorylation of Src/cortactin. We also confirmed these findings in vivo using an orthotopic lung transplantation mouse model, which validated the results obtained from the in vitro experiments. Finally, we examined the expressions of ERß and ICAM1 using immunohistochemistry in both NSCLC tissue and paired metastatic lymph nodes. The results confirmed that ERß promotes the formation of invadopodia in NSCLC cells through the ICAM1/p-Src/p-Cortactin signaling pathway.