ERβ Levels Research Articles

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.

Abstract PR-04: Association of HER2/GRB7 co-expression with Indigenous American ancestry and breast cancer clinical-pathological features

Abstract Introduction: Breast cancer is the most prevalent cancer in women worldwide. It is a heterogeneous disease that has been classified into intrinsic subtypes based on gene expression analyses. Previous studies have reported a higher proportion of HER2-positive (HER2+) tumors in Hispanics/Latinas, compared to Non-Hispanic White women, characterized by their aggressive phenotype. A gene expression analysis of breast tumor tissues from Colombian women reported that patients with a higher Indigenous American (IA) ancestry expressed higher mRNA levels of ERBB2, and of other genes in the HER2-amplicon region (17q12) such as GRB7. Although the expression of HER2 and GRB7 is highly correlated, it has been reported that these proteins might also be expressed independently. It is still unclear whether IA ancestry is associated with the co-expression of HER2/GRB7, or only with one of these two proteins. Aim of the study: To explore differences in HER2 and GRB7 protein expression according to genetic ancestry and the presentation of clinical-pathological features in Colombian women with breast cancer. Methods: A total of 517 Colombian breast cancer patients from different health institutions were included. For genetic ancestry estimation, 106 ancestry informative markers were genotyped from non-tumoral DNA. The protein expression of HER2 and GRB7 was evaluated in paraffin blocks with tumoral tissue by immunohistochemistry by a single pathologist. We tested the association between genetic ancestry and protein expression of HER2, GRB7, and the co-expression of both using logistic and multinomial logistic regression models. Results: Higher IA ancestry fractions were observed in patients with HER2+ compared to HER2- tumors (46% vs 41%, respectively, p = 0.013). We found higher odds of having HER2+ (OR=1.65, 95% CI, 1.05-2.63, p=0.0293) and GRB7+ tumors (OR=1.92, 95% CI, 1.03-3.56, p=0.0376) for every 25% increase in the IA ancestry proportion. The association between IA ancestry and HER2/GRB7 co-expression was influenced by estrogen receptor (ER) status. Patients with a higher IA ancestry (>42%) and with ER+ subtypes presented higher odds of having HER2+/GRB7- breast tumors (OR=3.59, 95% CI, 1.11-11.65, p=0.0327), whereas ER- patients with high IA ancestry tended to have HER2+/GRB7+ tumors (OR=2.27, 95% CI, 0.69 - 7.45, p=0.1730). The co-expression of these proteins was found associated with advanced clinical stages (p=0.018), less differentiated tumors (p=0.005), and a higher proliferation index (p<0.001), compared to HER2+/GRB7- and HER2-/GRB7- tumors. Conclusions: Our results confirm the previously reported association between IA ancestry and HER2 expression in breast tumors and suggest that HER2/GRB7 co-expression in patients with high AI ancestry may change according to the ER status. This event also correlates with unfavorable clinical-pathological features. Citation Format: Laura Rey Vargas, Lina Maria Bejarano, Juan Carlos Mejia Henao, Luz Fernanda Sua Villegas, Jhon Faustino Bastidas Andrade, Carlos Andrés Ossa Gómez, Laura Fejerman, Maria Carolina Sanabria Salas, Silvia Juliana Serrano Gomez. Association of HER2/GRB7 co-expression with Indigenous American ancestry and breast cancer clinical-pathological features [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR-04.

Effects of Fuke Qianjin Formula on hormones and their receptors and metabonomics study in uterine fibroids model rats

ObjectiveTo investigate the effects of different fractions from Fuke Qianjin Formula (妇科千金方, FKQJF) on uterine leiomyoma (UL) to determine the best fraction. MethodsFKQJF was extracted and isolated to obtain polysaccharides (FKP), flavonoids (FKF), and grease (FKG). 140 female SPF SD rats were divided into 14 groups [model (MOD), normal control (NC), Gouliuqing (GLQ), Mifepristone (MFST), FKQJF, low, medium, and high dose of polysaccharides (l-FKP, m-FKP, and h-FKP), low, medium, and high dose of flavonoids (l-FKF, m-FKF, and h-FKF), low, medium, and high dose of grease (l-FKG, m-FKG, and h-FKG)], and uterine fibroids model rats were treated with drugs for four weeks. Serum levels of estrogen and progesterone were measured using enzyme-linked immunoassay assay (ELISA) kits. The expression of estrogen receptor (ER-α, ER-β) and progesterone receptor (PR) in the uterus was observed using immunohistochemistry (IHC). Serum metabolite profiles and FKG were analyzed using gas chromatography-mass spectrometry (GC-MS). ResultsFKQJF, h-FKF, m-FKG, and h-FKG significantly downregulated the estrogen level in the uterine fibroid model rats (P < 0.01). FKQJF, h-FKF, and h-FKG significantly reduced the level of progesterone in the uterine fibroid model rats (P < 0.01). The levels of ER-α, ER-β, and PR in uterine fibroid model rats were significantly decreased by FKQJF and h-FKG (P < 0.01). The levels of ER-α, ER-β, and PR in the fibroid model rats were decreased by m-FKG (P < 0.05). Additionally, serum metabolism results revealed that h-FKG and FKQJF could regulate related endogenous metabolites and make the pathological indices of uterine fibroids in rats close to the normal group. Forty-six components were identified in the oil, accounting for 91.97% of the total oil components. ConclusionFKQJF and h-FKG showed a significant anti-myoma activity and significantly improved the pathological state of the uterus in rats with hysteromyoma. The mechanism of action may be related to the regulation of estrogen progesterone and its receptor in uterine fibroid model animals. These findings proved the effect of FKQJF on uterine leiomyoma and provided an experimental basis for its clinical research and application.

High efficacy of tamoxifen-loaded L-lysine coated magnetic iron oxide nanoparticles in cell cycle arrest and anti-cancer activity for breast cancer therapy.

Introduction: Due to the side effects of drugs, the development of nanoscale drug delivery systems has led to a significant improvement in medicinal therapies due to drug pharmacokinetics changes, decreased toxicity, and increased half-life of the drug. This study aimed to synthesize tamoxifen (TMX)-loaded L-lysine coated magnetic iron oxide nanoparticles as a nano-carrier to investigate its cytotoxic effects and anti-cancer properties against MCF-7 cancer cells. Methods: Magnetic Fe3O4 nanoparticles were synthesized and coated with L-lysine (F-Lys NPs). Then, TMX was loaded onto these NPs. The characteristics of synthesized nanoparticles (F-Lys-TMX NPs) were evaluated by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), differential scanning calorimetry (DSC), vibrating sample magnetometer (VSM), and thermogravimetric analysis (TGA). The drug release was analyzed at pH 5.8 and pH 7.4. The MCF-7 cells were exposed to F-Lys-TMX NPs, F-Lys NPs, and TMX for 24, 48, and 72 hours. To evaluate the cytotoxic potential of designed nanoparticles, MTT and apoptosis assays, real-time PCR, and cell cycle analysis was carried out. Results: The F-Lys-TMX NPs had spherical morphology with a size ranging from 9 to 30 nm. By increasing the nanoparticles concentration and treatment time, more cell proliferation inhibition and apoptosis induction were observed in F-Lys-TMX NPs-treated cells compared to the TMX. The expression levels of ERBB2, cyclin D1, and cyclin E genes were down-regulated and expression levels of the caspase-3 and caspase-9 genes were up-regulated. Studies on the drug release revealed a slow and controlled pH-dependent release of the nanoparticles. Cell cycle analysis indicated that F-Lys-TMX NPs could arrest the cells at the G0/G1 phase. Conclusion: The findings suggest that F-Lys-TMX NPs are more effective and have the potential for cell proliferation inhibition and apoptosis induction compared to the TMX. Hence, F-Lys-TMX NPs can be considered as an anti-cancer agent against MCF-7 breast cancer cells.

Chronic exposure to nonylphenol induces oxidative stress and liver damage in male zebrafish (Danio rerio): Mechanistic insight into cellular energy sensors, lipid accumulation and immune modulation

Nonylphenol (NP), an environmentally persistent and toxic endocrine-disrupting chemical with estrogenic properties, has severe implications on humans and wildlife. Accumulating evidence demonstrates the toxic response of NP on the developmental process, nervous system, and reproductive parameters. Although NP exposure has been implicated in chronic liver injury, the underlying events associated with hepatic pathophysiology remain less investigated. Using male zebrafish (Danio rerio) as the model, the present study investigates the impact of environmentally relevant concentrations of NP (50 and 100 μg/L, 21 days) on hepatic redox homeostasis vis-à-vis cellular energy sensors, inflammatory response, and cell death involving a mechanistic insight into estrogen receptor (ER) modulation. Our results demonstrate that congruent with significant alteration in transcript abundance of antioxidant enzymes (SOD1, SOD2, Catalase, GPx1a, GSTα1), chronic exposure to NP promotes ROS synthesis, more specifically superoxide anions and H2O2 levels, and lipid peroxidation potentially through elevated NOX4 expression. Importantly, NP perturbation of markers associated with fatty acid biosynthesis (srebf1/fasn) and cellular energy-sensing network (sirt1/ampkα/pgc1α) indicates dysregulated energy homeostasis, metabolic disruption, and macrovesicular steatosis, albeit with differential sensitivity at the dose level tested. Besides, elevated p38-MAPK phosphorylation (activation) together with loss of ER homeostasis at both mRNA (esr1, esr2a, esr2b) and protein (ERα, ERβ) levels suggest that NP modulation of ER abundance may have a significant influence on hepatic events. Elevated expression of inflammatory markers (TLR4, p-NF-κB, TNF-α, IL-6, IL-1β, and NOS2) and pro-apoptotic and necrotic regulators, e.g., Bax, caspase- 8, -9 and cleaved PARP1 (50 kDa), indicate chronic inflammation and hepatotoxicity in NP-exposed males. Collectively, elevated oxidative stress, metabolic dysregulation and immune modulation may lead to chronic liver injury in organisms exposed to metabolic disrupting chemicals.

Abstract 11303: Reduced Level of ErbB Receptors in Non-Myocyte Left Ventricular Cells in Patients with Diabetes Mellitus Undergoing Coronary Artery Bypass Grafting Surgery Contributes to Vascular Dysfunction

Introduction: ErbB signaling contributes to the regulation of vascular function through endothelial cell differentiation, proliferation, survival, and regulation of barrier integrity. Patients with diabetes (DM) are at an increased risk for endothelial dysfunction and worse outcomes after CABG. We investigated the level of ErbB expression on CD31 endothelial cells and CD105+ cells in DM and non-DM patients undergoing CABG. Methods: With IRB approval, left ventricular (LV) epicardial biopsies and blood samples from 25 patients (non-DM=9, DM=16), were obtained during surgery. Flow cytometry was used to determine the cell surface expression of ErbB receptors on cardiac endothelial and CD105+ cells isolated from the LV. Subpopulations of white blood cells were determined in blood samples. Plasma levels of IL-6, IL-8, and TNFα were measured. Results: Lower levels of ErbB1 and ErbB2 receptors expression on endothelial cells isolated from LV biopsies were found in patients with DM compared to non-DM control, and the expression of ErbB receptors negatively correlated with HgbA1c level. There were fewer CD105+ cells in DM patients (122 vs 153 cell/mg tissue, P=.021). Trends toward a negative association were found between the expression of ErbB2 in CD105+ cells and the number of neutrophils, as well as, HgbA1c level. No differences were found between DM and non-DM patients in the number of white blood cell subsets or plasma levels of IL-6, IL-8 and TNFα. Conclusions: We demonstrated that DM and high glucose blood levels are associated with reduced expression of ErbB1 and ErbB2 on endothelial cells and decreased number of CD105+ cells in the LV. Cardiac CD105+ are endothelial progenitor cells that differentiate in response to ErbB ligands. A decreased pool of CD105 endothelial progenitors in combination with down-regulation of ErbB1 and ErbB2 receptors in endothelial cells represent a novel mechanism for vascular dysfunction and adverse remodeling seen in diabetic patients.

Abstract PO-057: Targeting ErbB2 degradation via the ubiquitin–proteasome pathway to inhibit the metastasis of pancreatic cancer

Abstract Objective: Pancreatic cancer is currently one of the most lethal human cancers and continues to be a major unsolved health problem in the 21st century. Since pancreatic cancer has a high metastatic potential, chemotherapy is inevitable for most pancreatic cancer patients, whereas the clinical responses to anticancer agents are not satisfied. In this work, we designed and synthesized a novel compound, termed as 6-MPA, and found that 6-MPA could improve the proteasomal degradation of ErbB2 pancreatic cancer cells. Methods: The inhibitory effects of 6-MPA on migration and invasion was measured by scratch experiment, wound healing assay and transwell experiment. Clone formation and CCK-8 assay was used to determine the cytotoxicity of 6-MPA. Real-time PCR and western blotting was used to determine the expression of indicated genes or proteins respectively. Immunoprecipitation was used to characterize protein–protein interactions. Results: 4, 8, and 16 μM of 6-MPA exhibited an inhibitory effects on the migration of SW1990 cells and the inhibition rates reached 40.2±10.1%, 58.2±11.2%, and 65.2±9.9% in vitro, respectively. RNA-seq showed that 6-MPA treatment manipulated ErbB2 level and its downstream PI3K/AKT/mTOR signaling pathways, which was verified by western blotting assay. Treatment with 6-MPA could enhance the interaction between ErbB2 and ubiquitin, and thus promote the proteasomal degradation of ErbB2. Conclusion: In this study, we obtained a novel small molecule 6-MPA, which could specifically degrade ErbB2 through the activation of protein ubiquitinination. And we found that 6-MPA possessed potent inhibitory effects on migration and invasion of pancreatic cancer cells. Therefore, our work illustrated a novel structure skeleton that could be used to target ErbB2, and potentially suppressed the metastasis of pancreatic cancer. Citation Format: Bo Zhang, Fei Teng, Nengming Lin. Targeting ErbB2 degradation via the ubiquitin–proteasome pathway to inhibit the metastasis of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-057.

The expression and clinical significance of ERβ/ERα in ovarian cancer: can we predict the effectiveness of platinum plus taxane therapy?

ERα and ERβ tumor expression was evaluated quantitatively by flow cytometry in a narrowly defined group (31 patients): stage III high-grade serous ovarian carcinoma (HGSOC), suboptimal surgical cytoreduction, front-line platinum plus taxane chemotherapy (front-line, six cycles). The median of progression-free survival (PFS) was 2 times greater (18 vs 8 months, p=0.04) and the recurrence risk (HR) was 2.2 times (95 % CI: 1.1-6.2, p=0.04) lower in the group with high (in more than 40% of the cells) vs low level of ERβ tumor expression. The statistically significant difference between PFS in the groups with high vs low tumor ERα expression was not revealed. A high level of ERβ and not ERα expression can predict the efficacy of front-line platinum plus taxane chemotherapy in stage III HGSOC patients. The status of estrogen receptor beta can be considered as one of the possible predictors for evaluating the effectiveness of ovarian cancer therapy.

Clinicopathological and Prognostic Significance of Klotho and Estrogen Receptors Expression in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is male-predominant cancer, but the underlying mechanism remains unclear. This study aimed to investigate the expression of the age-suppressing gene klotho and estrogen receptors (ERs) in HCC patients and analyze their association with clinicopathological variables and their effects on prognosis. The expression patterns of klotho, ERα, and ERβ were determined by tissue microarray and immunohistochemical technique, and their correlations with clinicopathological characteristics were investigated using univariate and multivariate analysis. Klotho expression was significantly lower in HCC than in the adjacent noncancerous tissues (52.7% (49/93) vs. 90.8% (79/87), P = .000), and its protein level in HCC tissue was negatively correlated with clinical staging, histological grade, and stage of the primary tumor (T) (P < .05). Whereas the expression of nuclear ERα and ERβ was higher in HCC than their corresponding non-neoplastic tissues (55.9% (52/93) vs. 35.6% (31/87), P = .006; 59.1% (55/93) vs. 43.7% (38/87), P = .038), and the level of nuclear ERα and ERβ in HCC tissue was inversely correlated with T stage, tumor size, and clinical staging (P < .05). Correlation analysis showed the expression level of klotho, which is positively correlated with that of nuclear ERα (r = 0.243, P = .019). Patients with klotho-positive tumors had longer survival than those with klotho-negative tumors (P = .002). Cox proportional hazards model analysis demonstrated that positive expression of klotho was an important factor indicating good prognosis (P = .003). Klotho, partially regulated by ERα-mediated estrogen pathway, acts as a tumor suppressor and might be a novel biomarker candidate for predicting progression and prognosis in HCC patients.

Cdc25A inhibits autophagy-mediated ferroptosis by upregulating ErbB2 through PKM2 dephosphorylation in cervical cancer cells

Cervical cancer is the leading cause of cancer-related deaths in women, and treatment for cervical cancer is very limited. Emerging evidence suggests that targeting ferroptosis is a promising way to treat cancer. Here, we investigated the role of ferroptosis in cervical cancer, with a focus on the Cdc25A/PKM2/ErbB2 axis. Cervical cancer cells were treated with sorafenib to induce ferroptosis. Cellular MDA/ROS/GSH/iron detection assays were used to measure ferroptosis. MTT assays were performed to assess cell viability. qRT-PCR, western blot, and immunostaining assays were performed to measure the levels of proteins. Autophagy was monitored by fluorescence microscopy. Nuclear and cytosolic fractions were isolated to examine the location of PKM2 modifications. Co-IP experiments were conducted to determine the Cdc25A/PKM2 interaction. ChIP assays were performed to measure the binding affinity between H3K9Ac and the ErbB3 promoter, and a dual luciferase assay was performed to examine the transcriptional activity of ErbB2. A nude mouse xenograft model was used to examine the effects of the Cdc25A/ErbB2 axis on tumour growth in vivo. Cdc25A was elevated in human cervical cancer tissues but was reduced during sorafenib-induced ferroptosis of cervical cancer cells. Overexpression of Cdc25A inhibited sorafenib-induced ferroptosis by dephosphorylating nuclear PKM2 and suppressing autophagy. Cdc25A regulated autophagy-induced ferroptosis by increasing ErbB2 levels via the PKM2–pH3T11–H3K9Ac pathway. Cdc25A increased the resistance of cervical cancer to sorafenib, while knockdown of ErbB2 blocked these effects. Cdc25A suppressed autophagy-dependent ferroptosis in cervical cancer cells by upregulating ErbB2 levels through the dephosphorylation of PKM2. These studies revealed that Cdc25A/PKM2/ErbB2 pathway-regulated ferroptosis could serve as a therapeutic target in cervical cancer.

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.

A Dual PI3K/HDAC Inhibitor Downregulates Oncogenic Pathways in Hematologic Tumors In Vitro and In Vivo.

Purpose: To investigate the efficacy and mechanism of compound 23, a PI3K/HDAC dual-target inhibitor, on hematologic tumor cells in vitro and in vivo. Methods: The MTS Kit was used to study the antiproliferative effects in vitro. Western blot was used to analyze the involved signaling pathways. Flow cytometry was used to analyze apoptosis and the cell cycle. The antiproliferative effects were evaluated in vivo using EL4 and A20 xenograft models. The CCLE database was used to analyze gene expression. Results: Compound 23 significantly inhibited the proliferation of hematologic tumors; it simultaneously regulated PI3K/HDAC pathways and induced apoptosis and G1-phase arrest in EL4, NB4, and A20 cells in vitro. When tested in vivo, compound 23 significantly inhibited the proliferation of EL4 and A20. The expression levels of ErbB2 and ErbB3 decreased in hematologic tumors compared with it in solid tumors. Conclusion: Compound 23 modulates the PI3K/HDAC pathway, which results in significant inhibition of hematologic tumor proliferation in vivo and in vitro. The differential levels of ERBB2 and ERBB3 might be related to the difference in the effect of compound 23 on hematologic tumors and solid tumors.

FASN inhibition sensitizes metastatic OSCC cells to cisplatin and paclitaxel by downregulating cyclin B1.

To investigate the potential effect of fatty acid synthase (FASN) inhibitor orlistat to enhance the effectiveness of chemotherapy drugs widely used to treat oral squamous cell carcinomas (OSCC), such as 5-fluorouracil, cisplatin, and paclitaxel. The OSCC SCC-9 LN-1metastatic cell line, which expresses high levels of FASN, was used for drug combination experiments. Cell viability was analyzed by crystal violet staining and automatic cell counting. Apoptosis and cell cycle were analyzed by flow cytometry with Annexin-V/7-AAD and propidium iodide staining, respectively. Cyclin B1, Cdc25C, Cdk1, FASN, and ERBB2levels were assessed by Western blotting. Finally, cell scratch and transwell assays were performed to assess cell migration and invasion. Inhibition of FASN with orlistat sensitized SCC-9 LN-1 cells to the cytotoxic effects of paclitaxel and cisplatin, but not 5-fluorouracil, which was accompanied by a significant reduction in cyclin B1. The suppression of proliferation, migration, and invasion of SCC-9 LN-1 cells induced by orlistat plus cisplatin or paclitaxel was not superior to the effects of chemotherapy drugs alone. Our results suggest that orlistat enhances the chemosensitivity of SCC-9 LN-1 cells to cisplatin and paclitaxel by downregulating cyclin B1.

Sexually dimorphic expression of sexual differentiation genes in the internal genital organs of Japanese quail embryos

Japanese quail (Coturnix japonica) is an avian model used to evaluate the reproductive and developmental toxicity of chemicals. The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail, NIES-L, which may be a better model because of its highly inbred characteristics. To understand sexual differentiation of the reproductive organs and the value of using NIES-L quails for avian toxicity assessment, we profiled estradiol and androgen plasma levels by enzyme-linked immunosorbent assay; the mRNA levels of estrogen receptor-α (ERα), ERβ, and androgen receptor (AR) in the gonads, Müllerian ducts, Wolffian ducts; and the mRNA levels of steroidogenic enzymes, cholesterol side chain cleavage enzyme (P450scc), 17α-hydroxylase/C17-20 lyase (P45017α, lyase), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and aromatase (P450arom), anti-Müllerian hormone (AMH), and AMH receptor type 2 (AMHR2) in the gonads of NIES-L Japanese quails on embryonic days 9, 12, and 15 using a real-time quantitative PCR method. The plasma estradiol concentration was higher in females than males on these embryonic days, but no sex difference was found in the plasma androgens. The mRNA levels of all examined steroidogenic enzymes were significantly higher in female than male embryos. In particular, the P450arom mRNA levels showed a striking sex difference: P450arom was expressed in female but not male gonads. In contrast, the AMH and AMHR2 mRNA levels in the gonads were higher in males than females. The ERα, ERβ, and AR mRNA levels increased in the left female gonad and peaked on embryonic day 15, but not in the left and right male gonads; therefore, there was a female-biased sex difference. The ERα, ERβ, and AR mRNA levels in the left Müllerian duct, but not in the right Müllerian duct, of females increased and peaked on embryonic day 15, which resulted in asymmetric mRNA levels. The Wolffian ducts expressed ERα, ERβ, and AR in both sexes, and no sex difference or asymmetry of mRNA levels was found. The information obtained from this study helps elucidate the molecular endocrinological basis of sexual dimorphism formation of reproductive organs and clarify the value of NIES-L quails for toxicity assessment.

Erratum.

Gastric cancer is the fourth most common type of cancer and the second highest leading cause of cancer-related deaths worldwide. It has already been established that miR-133a is involved in gastric cancer. In this study, we investigated the molecular mechanisms by which miR-133a inhibits the proliferation of gastric cancer cells. We analyzed the proliferative capacity of human gastric cancer cells SNU-1 using an MTT assay. Cell apoptosis was determined using flow cytometry. The expression levels of ERBB2, p-ERK1/2, and p-AKT in SNU-1 cells were determined using Western blot analysis. To confirm that ERBB2 is a direct target of miR-133a, a luciferase reporter assay was performed. Results showed that miR-133a overexpression inhibited SNU-1 cell proliferation and increased apoptosis. ERBB2 was a direct target of miR-133a, and it was negatively regulated by miR-133a. Interestingly, ERBB2 silencing has a similar impact to miR-133a overexpression, in that it significantly induced apoptosis and inhibited ERK and AKT activation. Our study showed that miR-133a inhibits the proliferation of gastric cancer cells by downregulating the expression of ERBB2 and its downstream signaling molecules p-ERK1/2 and p-AKT. Therefore, miR-133a might be used as a therapeutic target for treating gastric cancer.

MP39-11 ESTROGEN RECEPTOR Β PROMOTES RENAL CELL CARCINOMA MALIGNANCY AND TKI RESISTANCE VIA REGULATING LNCRNA AND CIRCRNA ASSOCIATED CERNA NETWORK

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-11 ESTROGEN RECEPTOR Β PROMOTES RENAL CELL CARCINOMA MALIGNANCY AND TKI RESISTANCE VIA REGULATING LNCRNA AND CIRCRNA ASSOCIATED CERNA NETWORK Yixi Hu, Jie Ding, Junfei Gu, Zhenwei Han, Wenbiao Ren, Edward M. Messing, Chawnshang Chang, Jean V. Joseph, and Shuyuan Yeh Yixi HuYixi Hu More articles by this author , Jie DingJie Ding More articles by this author , Junfei GuJunfei Gu More articles by this author , Zhenwei HanZhenwei Han More articles by this author , Wenbiao RenWenbiao Ren More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Jean V. JosephJean V. Joseph More articles by this author , and Shuyuan YehShuyuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Kidney cancer accounts for at least 3% of malignant diseases, and the incidence and mortality appear to be rising. Recent studies indicated that the estrogen receptor beta (ERβ), but not the ER alpha, could affect the progression of renal cell carcinoma (RCC), however, its downstream signals remain to be further elucidated. METHODS: Human 786-O, A498, ACHN, and Caki-1 RCC cell lines were used. The RNAs of 10 nM E2 treated and shERβ transfected 786-O cells were isolated for RNA-Seq and non-coding RNA analysis. Invasion assays were performed using the matrigel transwell and 3D invasion assays. The q-PCR analysis and western blot were performed to evaluate the RNA and the protein expressions. HOTAIR promoter was constructed into the pGL3 for luciferase assays. Protein-DNA complexes were detected by Chromatin immunoprecipitation (ChIP) assay. RNA pull-down assays were performed to detect the binding of putative miRNAs. A preclinical xenograft mouse RCC model was used to validate ERβ signal-promoted ceRNA network. RESULTS: RNAseq data of RCC patients were retrieved from the TCGA database and analysis results showed high expressions of ERβ and its down-stream signals were associated with advanced stages and poor prognosis in RCC patients. Mechanism dissection revealed that targeting ERβ with ERβshRNA and stimulating the transactivation of ERβ with 17β-estradiol or environmental endocrine disrupting chemicals, resulted in altering the lncRNA HOTAIR expression. The ERβ-modulated HOTAIR can function via antagonizing several miRNAs (miR-138, -200c, -204, or -217) to impact various oncogenes, including ADAM9, CCND2, EZH2, VEGFA, VIM, ZEB1, and ZEB2, to promote RCC invasion. In addition, we found ERβ can suppress the expression of the circular RNA, circATP2B1, which then led to a reduced miR-204-3, increased fibronectin 1 (FN1) expression, and enhanced RCC cell invasion. Our results further showed that the expression levels of those ERβ regulated long non-coding RNAs (lncRNAs), circRNAs, and miRNAs could predict the malignancy or TKI resistance of RCC. CONCLUSIONS: We provide evidence that expression levels of ERβ and ERβ-regulated CeRNA network could be used to predict metastasis and overall survival in RCC patients. The identification of the ERβ-lncRNA or circRNA regulated signal pathways may provide physicians with new biomarkers and/or therapeutic targets to better control RCC progression in the future. Source of Funding: This work was partially supported by URMC Urology Department Research Fund and George Whipple H. Professorship Endowment © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e708-e709 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yixi Hu More articles by this author Jie Ding More articles by this author Junfei Gu More articles by this author Zhenwei Han More articles by this author Wenbiao Ren More articles by this author Edward M. Messing More articles by this author Chawnshang Chang More articles by this author Jean V. Joseph More articles by this author Shuyuan Yeh More articles by this author Expand All Advertisement Loading ...

Impact of pubertal onset on region-specific Esr2 expression.

In female rats, pubertal onset is associated with maturation of the medial prefrontal cortex (mPFC) and mPFC-mediated behaviours. These behavioural and anatomical changes are likely a result of the effects of oestrogens at the nuclear oestrogen receptor (ER)β, which is expressed at higher levels than the ERα isoform in the adult mPFC. Researchers have previously quantified ERβ protein and Esr2 RNA in rodents during early postnatal development and adulthood, although an adolescent-specific trajectory of this receptor in the mPFC has not been documented. Given that Esr2 expression can fluctuate in the presence or absence of oestrogens, puberty and the subsequent rise in gonadal hormones could influence levels of ERβ in the adolescent brain. To further explore this, we used RNAscope® technology to quantify the amount of Esr2 mRNA in pre-pubertal adolescent, recently post-pubertal adolescent and adult female rats. We show that Esr2 expression decreases significantly in the mPFC, striatum and motor cortex between pre-pubertal adolescence and adulthood. In the mPFC, this decrease occurs rapidly at pubertal onset, with no significant decrease in Esr2 levels between the recently post-pubertal and adult cohort. By contrast, the striatum and motor cortex had no significant differences in the amount of Esr2 mRNA between pre- and post-pubertal females. Insofar as the amount of Esr2 expression is proportional to functional ERβ, these results suggest ERβ decreases in a region-specific pattern in response to pubertal onset and highlight a role for this receptor in the maturational events that occur in the female rat mPFC at puberty.

Biocompatibility evaluation of a 3D-bioprinted alginate-GelMA-bacteria nanocellulose (BNC) scaffold laden with oriented-growth RSC96 cells

Peripheral nerve injury can cause various degrees of damage to the morphological structure and physiological function of the peripheral nerve. At present, compared with “gold standard” autologous nerve transplantation, tissue engineering has certain potential for regeneration and growth; however, achieving oriented guidance is still a challenge. In this study, we used 3D bioprinting to construct a nerve scaffold of RSC96 cells wrapped in sodium alginate/gelatin methacrylate (GelMA)/bacterial nanocellulose (BNC) hydrogel. The 5% sodium alginate+5% GelMA+0.3% BNC group had the thinnest lines among all groups after printing, indicating that the inherent shape of the scaffold could be maintained after adding BNC. Physical and chemical property testing (Fourier transform infrared, rheometer, conductivity, and compression modulus) showed that the 5% alginate+5% GelMA+0.3% BNC group had better mechanical and rheological properties. Live/dead cell staining showed that no mass cell death was observed on days 1, 3, 5, and 7 after printing. In the 5% alginate+5% GelMA group, the cells grew and formed linear connections in the scaffold. This phenomenon was more obvious in the 5% alginate+5% GelMA+0.3% BNC group. In the 5% alginate+5% GelMA+0.3% BNC group, S-100β immunofluorescence staining and cytoskeleton staining showed oriented growth. Polymerase chain reaction (PCR) array results showed that mRNA levels of related neurofactors ASCL1, POU3F3, NEUROG1, DLL1, NOTCH1 and ERBB2 in the 5%GelMA+0.3%BNC group were higher than those of other groups. Four weeks after implantation in nude mice, RSC96 cells grew and proliferated well, blood vessels grew, and S-100β immunofluorescence was positive. These results indicate that a 3D-bioprinted sodium alginate/GelMA/BNC composite scaffold can improve cell-oriented growth, adhesion and the expression of related factors. This 3D-bioprinted composite scaffold has good biocompatibility and is expected to become a new type of scaffold material in the field of neural tissue engineering.

17β-Estradiol alleviates intervertebral disc degeneration by inhibiting NF-κB signal pathway

AimTo investigate the effect of 17β-Estradiol (E2) on intervertebral disc degeneration (IVDD) and the related mechanism. Materials and methodsImmunohistochemistry was used to detect the expression of estrogen receptor β (ERβ) within intervertebral discs of humans and rats. After that, rat IVDD model was established by needle puncture and bilateral ovariectomy. Then, the serum E2 level was detected by enzyme linked immunosorbent assay, and the degree of IVDD was evaluated by X-ray, magnetic resonance imaging, hematoxylin and eosin staining, and Safranin O-Fast Green staining. Finally, we used immunohistochemistry and immunofluorescence staining to determine the effect of E2 on nuclear factor kappa-B (NF-κB) signal pathway both in vivo and in vitro. Key findingsWe identified that IVDD was associated with lower levels of ERβ and ERβ levels were inversely correlated with IVDD. The histological staining and radiological results showed that E2 supplement could alleviate IVDD progression. Additionally, immunohistochemistry staining demonstrated that E2 could inhibit nucleus pulposus cell (NPC) apoptosis, matrix metalloproteinases (MMPs) synthesis, and degradation of extracellular matrix (ECM) by inhibiting the activation of NF-κB signal pathway. Furthermore, immunofluorescence staining showed that the above effects of E2 on the NF-κB signal pathway could be blocked by the estrogen receptor antagonist ICI182780 in vitro. Finally, inhibition of NF-κB signal pathway by BAY11-7082 could reduce MMPs synthesis and ECM degradation of NPCs. SignificanceCollectively, these findings indicated that E2 could effectively ameliorate IVDD by inhibiting NPC apoptosis via inhibition of NF-κB signal pathway.

P-365 Pre-selected for an award: Altered endometrial oestrogen-responsiveness and aberrant expression of cell-fate markers may contribute to the aetiology of recurrent pregnancy loss

Abstract Study question Do women with recurrent pregnancy loss (RPL) have an aberrant expression of oestrogen receptor-β (ERβ) and cell-fate markers during the window of implantation (WOI) endometrium? Summary answer Women with RPL are found to have significantly altered levels of ERβ and Ki-67 in the WOI endometrium, possibly resulting in anti-proliferative and anti-angiogenic effects. What is known already RPL affects 1% of all women and has been associated with altered endometrial angiogenesis and proliferation when compared with the endometrium of healthy fertile women. RPL can be subcategorised into recurrent loss of anembryonic pregnancy, fetal loss (following evidence of a fetal heartbeat) and recurrent implantation failure (RIF). ERβ is the only oestrogen-receptor (ER) known to be expressed in the vascular endothelium of the endometrium and is the dominant ER during the WOI. It has an important role in endometrial regeneration and is proposed to regulate the angiogenic and vascular changes that occur in embryo implantation. Study design, size, duration This pilot case-control study took place at the Liverpool Women’s Hospital and included 38 women; 29 who suffered RPL and 9 controls with proven fertility (≥2 healthy pregnancies). Of the RPL group, 9 had recurrent loss of anembryonic pregnancy, 10 had recurrent fetal loss and 10 had RIF. Endometrial samples were collected during the WOI (cycle day 22+/-2). Participants/materials, setting, methods To determine whether markers of endometrial cell proliferation and oestrogen-responsiveness are associated with RPL, we assessed the immuno-staining for ERβ, progesterone receptor (PR) and cell-fate marker Ki-67 in endometrial biopsies during the WOI using immunohistochemistry. A semi-quantitative immuno-staining score was used to assess the endometrial glands, stroma, luminal epithelium, perivascular and vascular endothelium compartments separately. Statistical differences between groups were calculated by non-parametric tests and significance level set at p &amp;lt; 0.05. Main results and the role of chance During the WOI, the endometrial epithelium of women with RIF and recurrent anembryonic pregnancy loss showed significantly higher levels of ERβ when compared with fertile controls (p = 0.01 and p = 0.01, respectively). This may indicate an anti-proliferative process occurring at the site of implantation with very early pregnancy losses. In contrast, with women with recurrent fetal loss, a significantly lower level of ERβ was found within the vascular endothelium when compared with the fertile controls (p &amp;lt; 0.01). This supports the theory that increased oxygen levels may compromise trophoblastic invasion, thereby leading to fetal loss. The presence of Ki-67 (a marker of proliferation) was significantly lower within the vascular endothelium of all types of RPL recurrent anembryonic loss (p = 0.02), RIF (p = 0.02) and recurrent fetal loss (p &amp;lt; 0.01). These findings suggest ineffective endometrial angiogenesis in RPL, resulting in a suboptimal endometrial microenvironment. PR was found to be significantly reduced (p &amp;lt; 0.01) in the perivascular area of women with RIF versus fertile controls. Since decidualisation and preparation of the endometrium for a successful implantation is controlled by critical target genes downstream of PR, this alteration in PR may be an important feature of their defective endometrial phenotype. Limitations, reasons for caution Samples analysed were taken from the functional endometrium and therefore the results do not reflect the basalis. The WOI was identified using history and histological appearance, rather than timing with ovulation. Although we detected statistical significance, generalisation of the results requires further studies with larger sample size. Wider implications of the findings This data provides novel insight into the biological correlates of clinical types of RPL and suggests that specific alterations in the regulation of endometrial cell fate and oestrogen- responsiveness are associated with different types of RPL. This highlights possible new therapies for RPL, such as selective oestrogen receptor modulators (SERMs). Trial registration number Not applicable