Estrogen Pathways Research Articles

Subcellular localization and characterization of estrogenic pathway regulators and mediators in Atlantic salmon spermatozoal cells.

Much progress has been made regarding our understanding of aromatase regulation, estrogen synthesis partitioning and communication between the germinal and somatic compartments of the differentiating gonad. We now know that most of the enzymatic and signaling apparatus required for steroidogenesis is endogenously expressed within germ cells. However, less is known about the expression and localization of steroidogenic components within mature spermatozoa. We have assembled a sperm library presenting 197,015 putative transcripts. Co-expression clustering analysis revealed that 6687 genes were present at higher levels in sperm in comparison to fifteen other salmon tissue libraries. The sperm transcriptome is highly complex containing the highest proportion of unannotated genes (45%) of the tissues analyzed. Our analysis of highly expressed genes in late-stage sperm revealed dedication to tasks involving chromatin remodeling, flagellogenesis and proteolysis. In addition, using various different embedding and microscopic techniques, we examined the morphology of salmon spermatozoa and characterized expression and localization of several estrogenic regulatory and signaling proteins by immunohistochemistry. We provide evidence for the endogenous synthesis and localization of aromatase (CYP19A and CYP19B1) and potential mediators of estrogen [i.e., ER-alpha and soluble adenylyl cyclase (sAC)] or phosphate (i.e., CREB and FOXL2A) signaling. Partitioning of select transcripts that encode AR-beta, FSH and the LH receptor, but not AR-alpha, LH or the FSH receptor, further points to localized specificity of function in the steroidogenic circuitry of the sperm cell. These results open new avenues of investigation to further our understanding of the intra- and intercellular regulatory processes that guide sperm development and biology.

Endocrine Therapy of Estrogen Receptor-Positive Breast Cancer Cells: Early Differential Effects on Stem Cell Markers.

Endocrine therapy of breast cancer, which either deprives cancer tissue of estrogen or prevents estrogen pathway signaling, is the most common treatment after surgery and radiotherapy. We have previously shown for the estrogen-responsive MCF-7 cell line that exposure to tamoxifen, or deprivation of estrogen, leads initially to inhibition of cell proliferation, followed after several months by the emergence of resistant sub-lines that are phenotypically different from the parental line. We examined the early responses of MCF-7 cells following either exposure to 4-hydroxytamoxifen or deprivation of estrogen for periods of 2 days-4 weeks. Endocrine-sensitive or -resistant breast cancer cell lines were used to examine the expression of the stem cell gene SOX2, and the Wnt effector genes AXIN2 and DKK1 using quantitative PCR analysis. Breast cancer cell lines were used to assess the anti-proliferative effects (as determined by IC50 values) of Wnt pathway inhibitors LGK974 and IWP-2. Hormone therapy led to time-dependent increases of up to 10-fold in SOX2 expression, up to threefold in expression of the Wnt target genes AXIN2 and DKK1, and variable changes in NANOG and OCT4 expression. The cells also showed increased mammosphere formation and increased CD24 surface protein expression. Some but not all hormone-resistant MCF-7 sub-lines, emerging after long-term hormonal stress, showed up to 50-fold increases in SOX2 expression and smaller increases in AXIN2 and DKK1 expression. However, the increase in Wnt target gene expression was not accompanied by an increase in sensitivity to Wnt pathway inhibitors LGK974 and IWP-2. A general trend of lower IC50 values was observed in 3-dimensional spheroid culture conditions (which allowed enrichment of cells with cancer stem cell phenotype) relative to monolayer cultures. The endocrine-resistant cell lines showed no significant increase in sensitivity to Wnt inhibitors. Hormone treatment of cultured MCF-7 cells leads within 2 days to increased expression of components of the SOX2 and Wnt pathways and to increased potential for mammosphere formation. We suggest that these responses are indicative of early adaptation to endocrine stress with features of stem cell character and that this facilitates the survival of emerging hormone-resistant cell populations.

Application of Adverse Outcome Pathways to U.S. EPA’s Endocrine Disruptor Screening Program

Background:The U.S. EPA’s Endocrine Disruptor Screening Program (EDSP) screens and tests environmental chemicals for potential effects in estrogen, androgen, and thyroid hormone pathways, and it is one of the only regulatory programs designed around chemical mode of action.Objectives:This review describes the EDSP’s use of adverse outcome pathway (AOP) and toxicity pathway frameworks to organize and integrate diverse biological data for evaluating the endocrine activity of chemicals. Using these frameworks helps to establish biologically plausible links between endocrine mechanisms and apical responses when those end points are not measured in the same assay.Results:Pathway frameworks can facilitate a weight of evidence determination of a chemical’s potential endocrine activity, identify data gaps, aid study design, direct assay development, and guide testing strategies. Pathway frameworks also can be used to evaluate the performance of computational approaches as alternatives for low-throughput and animal-based assays and predict downstream key events. In cases where computational methods can be validated based on performance, they may be considered as alternatives to specific assays or end points.Conclusions:A variety of biological systems affect apical end points used in regulatory risk assessments, and without mechanistic data, an endocrine mode of action cannot be determined. Because the EDSP was designed to consider mode of action, toxicity pathway and AOP concepts are a natural fit. Pathway frameworks have diverse applications to endocrine screening and testing. An estrogen pathway example is presented, and similar approaches are being used to evaluate alternative methods and develop predictive models for androgen and thyroid pathways. https://doi.org/10.1289/EHP1304

Summary of the development the US Environmental Protection Agency's Medaka Extended One Generation Reproduction Test (MEOGRT) using data from 9 multigenerational medaka tests.

In response to various legislative mandates, the US Environmental Protection Agency (USEPA) formed its Endocrine Disruptor Screening Program (EDSP), which in turn, formed the basis of a tiered testing strategy to determine the potential of pesticides, commercial chemicals, and environmental contaminants to disrupt the endocrine system. The first tier of tests is intended to detect the potential for endocrine disruption mediated through estrogen, androgen, or thyroid pathways, whereas the second tier is intended to further characterize the effects on these pathways and to establish a dose-response relationship for adverse effects. One of these tier 2 tests, the Medaka Extended One Generation Reproduction Test (MEOGRT), was developed by the USEPA for the EDSP and, in collaboration with the Japanese Ministry of the Environment, for the Guidelines for the Testing of Chemicals of the Organisation for Economic Co-operation and Development (OECD). The MEOGRT protocol was iteratively modified based on knowledge gained after the successful completion of 9 tests with variations in test protocols. The present study describes both the final MEOGRT protocol that has been published by the USEPA and the OECD, and the iterations that provided valuable insights into nuances of the protocol. The various tests include exposure to 17β-estradiol, 4-t-octylphenol, o,p'- dichlorodiphenyltrichloroethane, 4-chloro-3-methylphenol, tamoxifen, 17β-trenbolone, vinclozolin, and prochloraz. Environ Toxicol Chem 2017;36:3387-3403. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Age, estrogen, and immune response in breast adenocarcinoma and adjacent normal tissue

ABSTRACTChronic inflammation promotes breast tumor growth and invasion by accelerating angiogenesis and tissue remodeling in the tumor microenvironment. There is a complex relationship between inflammation and estrogen, which drives the growth of 70 percent of breast tumors. While low levels of estrogen exposure stimulate macrophages and other inflammatory cell populations, very high levels are immune suppressive. Breast tumor incidence is increased by obesity and age, which interact to influence inflammatory cell populations in normal breast tissue. To characterize the impact of these factors on tumors and the tumor microenvironment, we measured gene expression in 195 breast adenocarcinomas and matched adjacent normal breast tissue samples collected at Akershus University Hospital (AHUS). Age and Body Mass Index (BMI) were independently associated with inflammation in adjacent normal tissue but not tumors. Estrogen Receptor (ER)-negative tumors had elevated macrophage expression compared with matched normal tissue, but ER-positive tumors showed an unexpected decrease in macrophage expression. We found an inverse relationship between the increase in tumor estrogen pathway expression compared with adjacent normal tissue and tumor macrophage score. We validated this finding in 126 breast tumor-normal pairs from the previously published METABRIC cohort. We developed a novel statistic, the Rewiring Coefficient, to quantify the rewiring of gene co-expression networks at the level of individual genes. Differential correlation analysis demonstrated distinct pathways were rewired during tumorigenesis. Our data support an immune suppressive effect of high doses of estrogen signaling in breast tumor microenvironment, suggesting that this effect contributes to the greater presence of prognostic and therapeutically relevant immune cells in ER-negative tumors.

Estrogen, Estrogen Receptor and Lung Cancer.

Estrogen has been postulated as a contributor for lung cancer development and progression. We reviewed the current knowledge about the expression and prognostic implications of the estrogen receptors (ER) in lung cancer, the effect and signaling pathway of estrogen on lung cancer, the hormone replacement therapy and lung cancer risk and survival, the mechanistic relationship between the ER and the epidermal growth factor receptor (EGFR), and the relevant clinical trials combining the ER antagonist and the EGFR antagonist, to investigate the role of estrogen in lung cancer. Estrogen and its receptor have the potential to become a prognosticator and a therapeutic target in lung cancer. On the other hand, tobacco smoking aggravates the effect of estrogen and endocrine disruptive chemicals from the environment targeting ER may well contribute to the lung carcinogenesis. They have gradually become important issues in the course of preventive medicine.

Effects of environmental pollutants on signaling pathways in rat pituitary GH3 adenoma cells

An increased rate of acromegaly was reported in industrialized areas, suggesting an involvement of environmental pollutants in the pathogenesis and behavior of GH-secreting pituitary adenomas. Based on these premises, the aim of the study was to evaluate the effects of some widely diffused pollutants (i.e. benzene, BZ; bis(2-ethylhexyl) phthalate, DEHP and polychlorinated biphenyls, PCB) on growth hormone secretion, the somatostatin and estrogenic pathways, viability and proliferation of rat GH-producing pituitary adenoma (GH3) cells. All the pollutants induced a statistically significant increase in GH secretion and interfered with cell signaling. They all modulated the expression of SSTR2 and ZAC1, involved in the somatostatin signaling, and the expression of the transcription factor FOXA1, involved in the estrogen receptor signaling. Moreover, all the pollutants increased the expression of the CYP1A1, suggesting AHR pathway activation. None of the pollutants impacted on cell proliferation or viability. Present data demonstrate that exposure to different pollutants, used at in vivo relevant concentrations, plays an important role in the behavior of GH3 pituitary adenoma cells, by increasing GH secretion and modulating several cellular signaling pathways. These observations support a possible influence of different pollutants in vivo on the GH-adenoma aggressiveness and biological behavior.

Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation.

Cross-talk between estrogen receptor alpha (ERα) and signal transduction pathways plays an important role in the progression of endometrial cancer (EC). Here, we show that 17β-estradiol (E2) stimulation increases p21-activated kinase 4 (Pak4) expression and activation in ER-positive EC cells. The estrogen-induced Pak4 activation is mediated via the PI3K/AKT pathway. Estrogen increases Pak4 and phosphorylated-Pak4 (p-Pak4) nuclear accumulation, and Pak4 in turn enhances ERα trans-activation. Depletion or functional inhibition of Pak4 abrogates EC cell proliferation induced by E2, whereas overexpression of Pak4 rescues cell proliferation decreased by inhibiting the estrogen pathway. Pak4 knockdown decreases cyclin D1 expression and induces G1-S arrest. Importantly, Pak4 suppression inhibits E2 induced EC tumor growth in vivo, in a mouse xenograft model. These data demonstrate that estrogen stimulation increases Pak4 expression and activation, which in turn enhances ERα transcriptional activity and ERα-dependent gene expression, resulting in increased proliferation of EC cells. Thus inhibition of Pak4-ERα signaling may represent a novel therapeutic strategy against endometrial carcinoma.

A Mitochondrial Biomarker-Based Study of S-Equol in Alzheimer's Disease Subjects: Results of a Single-Arm, Pilot Trial.

Reductions in bioenergetic fluxes, mitochondrial enzyme activities, and mitochondrial number are observed in Alzheimer's disease (AD). Preclinical work indicates estrogen pathway signaling by either estrogen or selective β estrogen receptor (ERβ) agonists benefits these parameters. To assess whether an ERβ agonist could improve mitochondrial function in actual AD subjects, we administered S-equol (10 mg twice daily) to 15 women with AD and determined the platelet mitochondria cytochrome oxidase (COX) activity before initiating S-equol (lead-in), after two weeks of S-equol (active treatment), and two weeks after stopping S-equol (wash-out). Because the intra-individual variation of this enzyme across samples taken at different times was unknown we used a nonparametric, single-arm, dichotomous endpoint that classified subjects whose active treatment COX activity exceeded the average of their lead-in and wash-out measures as positive responders. Eleven positive responses were observed (p < 0.06). The implications of this finding on our null hypothesis (that S-equol does not influence platelet mitochondria COX activity) are discussed. To our knowledge, this is the first time a direct mitochondrial target engagement biomarker has been utilized in an AD clinical study.

Abstract 1005: Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer

Abstract The estrogen receptor (ER) promotes cell proliferation in non-small cell lung cancer (NSCLC). Since fibroblast growth factors (FGFs) are known regulators of stem cell markers in ER positive breast cancer, we investigated whether a link between the estrogen pathway, FGFs, and stem cell markers could be demonstrated in NSCLC animal models, human cells lines and tumor tissue. In the lungs of female FVB/N mice exposed to the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, NNK, treatment with the anti-estrogen fulvestrant and/or the aromatase inhibitor anastrozole blocked secretion of both FGF2 and FGF9, and reduced expression of the stem cell markers SOX2 and nanog. These effects were observed in the normal airway epilthelium as well as in both lung preneoplasias and lung adenomas. To investigate the effects of β-estradiol (E2), male mice were administered E2 in the drinking water during exposure to NNK for 4 weeks. In this mouse model, the incidence of carcinogen-induced lung preneoplasias increased 1.8-fold (p&amp;lt;0.05) with E2 exposure accompanied by increased expression of FGF2, FGF9, SOX2, and nanog in airway preneoplasias demonstrating a relationship between activation of the FGF pathway in the lungs, expansion of cells with a stem cell phenotype, and promotion of pre-cancerous changes. In FGFR1 normal copy number NSCLC cell lines that express multiple FGFR family members and secrete several FGFs, E2 treatment caused a significant increase (up to 4-fold; p&amp;lt;0.05) in release of FGF2, an effect that was completely blocked by fulvestrant. Treatment with fulvestrant also resulted in a 30% reduction in phosphorylation of fibroblast growth factor receptor substrate 2, the FGFR docking protein. Upon co-inhibition of ER and FGFRs using fulvestrant and the pan-FGFR inhibitor AZD4547, significantly enhanced (p&amp;lt;0.01) anti-proliferative effects were observed in FGFR1 normal copy number NSCLC cells that show low sensitivity to FGFR inhibitors as single agents. In NSCLC xenografts, the combination of AZD4547 (daily 12.5 mg/kg) and fulvestrant (30 mg/kg twice weekly) resulted in a significantly greater inhibition (67-85% decrease; p&amp;lt;0.05) of tumor growth and decreased expression of Ki67 and stem cell markers compared to each single agent treatment. Furthermore, tumor histology appeared more differentiated and up to a 25% decrease in malignant cellularity was observed with combination treatment compared to each single agent treatment. In NSCLC patient tumors, high ERB expression correlated with high FGFR1 expression (p&amp;lt;0.001). Taken together, these results suggest that interaction between the ER and FGFR pathways in NSCLC promotes a stem-like state. Combining an FGFR inhibitor with an ER pathway inhibitor could be exploited to increase the efficacy of FGFR inhibitors for NSCLC patients who lack FGFR genetic alterations. Supported by P50 CA090440 and the V-Foundation. Citation Format: Laura P. Stabile, Natalie J. Rothenberger, Mariya Farooqui, Sanja Dacic, Jill M. Siegfried. Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1005. doi:10.1158/1538-7445.AM2017-1005

Developmental Programming: Impact of Gestational Steroid and Metabolic Milieus on Mediators of Insulin Sensitivity in Prenatal Testosterone-Treated Female Sheep.

Prenatal testosterone (T) excess in sheep leads to peripheral insulin resistance (IR), reduced adipocyte size, and tissue-specific changes, with liver and muscle but not adipose tissue being insulin resistant. To determine the basis for the tissue-specific differences in insulin sensitivity, we assessed changes in negative (inflammation, oxidative stress, and lipotoxicity) and positive mediators (adiponectin and antioxidants) of insulin sensitivity in the liver, muscle, and adipose tissues of control and prenatal T-treated sheep. Because T excess leads to maternal hyperinsulinemia, fetal hyperandrogenism, and functional hyperandrogenism and IR in their female offspring, prenatal and postnatal interventions with antiandrogen, flutamide, and the insulin sensitizer rosiglitazone were used to parse out the contribution of androgenic and metabolic pathways in programming and maintaining these defects. Results showed that (1) peripheral IR in prenatal T-treated female sheep is related to increases in triglycerides and 3-nitrotyrosine, which appear to override the increase in high-molecular-weight adiponectin; (2) liver IR is a function of the increase in oxidative stress (3-nitrotyrosine) and lipotoxicity; (3) muscle IR is related to lipotoxicity; and (4) the insulin-sensitive status of visceral adipose tissue appears to be a function of the increase in antioxidants that likely overrides the increase in proinflammatory cytokines, macrophages, and oxidative stress. Prenatal and postnatal intervention with either antiandrogen or insulin sensitizer had partial effects in preventing or ameliorating the prenatal T-induced changes in mediators of insulin sensitivity, suggesting that both pathways are critical for the programming and maintenance of the prenatal T-induced changes and point to potential involvement of estrogenic pathways.

Rapid conceptus elongation in the pig: An interleukin 1 beta 2 and estrogen-regulated phenomenon.

Establishment and maintenance of pregnancy in the pig involves activating many physiological, cellular, and molecular signaling pathways between the developing conceptus and hormonally regulated maternal endometrium. Rapid elongation of the pig trophoblast allows for the establishment of sufficient placental surface area for the transport of nutrients to the fetus throughout pregnancy. Estrogens secreted by the conceptus during elongation act on uterine epithelia to induce secretion of uterine factors required for conceptus development and for preventing endocrine secretion of prostaglandin F2α, which would cause luteolysis. Thus, trophoblast expansion within the uterine lumen during early gestation is an essential process for implantation and maintenance of pregnancy in species with an epitheliochorial form of placentation. In the pig, rapid conceptus elongation involves the unique expression of interleukin-1 beta 2 (IL1B2), which establishes pro-inflammatory effects that may be tempered by the spatiotemporal secretion of estrogen from the conceptuses. The present review provides current information on pig conceptus remodeling and signaling via estrogen and IL1B2 pathways, as well as endometrial responses to those conceptus factors leading to establishment of pregnancy.

Uterine ERα epigenetic modifications are induced by the endocrine disruptor endosulfan in female rats with impaired fertility

High ERα activity may disrupt the window of uterine receptivity, causing defective implantation. We investigated whether implantation failures prompted by endosulfan are associated with aberrant ERα uterine expression and DNA methylation status during the pre-implantation period. ERα-dependent target genes that play a crucial role in the uterine receptivity for embryo attachment and implantation were also investigated. Newborn female rats received corn oil (vehicle, Control), 6 μg/kg/d of endosulfan (Endo6) or 600 μg/kg/d of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5, and 7. On PND90, females were made pregnant and on gestational day 5 (GD5, pre-implantation period) uterine samples were collected. ERα expression was assessed at protein and mRNA levels by immunohistochemistry and real time RT-PCR, respectively. ERα transcript variants mRNA containing alternative 5’-untranslated regions (5′UTRs) were also evaluated. We searched for predicted transcription factors binding sites in ERα regulatory regions and assessed their methylation status by Methylation-Sensitive Restriction Enzymes-PCR technique (MSRE-PCR). The expression of the ERα-dependent uterine target genes, i.e. mucin-1 (MUC-1), insulin-like growth factor-1 (IGF-1), and leukemia inhibitory factor (LIF), was assessed by real time RT-PCR. Both doses of endosulfan increased the expression of ERα and its transcript variants ERα-OS, ERα-O, ERα-OT and ERα-E1. Moreover, a decreased DNA methylation levels were detected in some ERα regulatory regions, suggesting an epigenetic up-regulation of it transcription. ERα overexpression was associated with an induction of its downstream genes, MUC-1 and IGF-1, suggesting that endosulfan might alter the uterine estrogenic pathway compromising uterine receptivity. These alterations could account, at least in part, for the endosulfan-induced implantation failures.

Genistein modulation of seizure: involvement of estrogen and serotonin receptors.

Genistein, a major source of phytoestrogen exposure for humans and animals, has been shown to mediate neuroprotection in Alzheimer's disease and status epilepticus. In the present study, we investigated the effect of genistein on pentylenetetrazole-induced seizures in ovariectomized mice and the possible involvement of estrogenic and serotonergic pathways in the probable effects of genistein. Intraperitoneal (i.p.) administration of genistein (10mg/kg) significantly increased the seizure threshold 30min prior to induction of seizures 14 days after ovariectomy surgery. Administration of fulvestrant (1mg/kg, i.p.), an estrogen receptor antagonist, completely reversed the anticonvulsant effect of genistein (10mg/kg) in ovariectomized mice. Administration of the antagonist of serotonin receptor (5-HT3), tropisetron (10mg/kg, i.p.), eliminated the anticonvulsant effect of genistein, whereas co-administration of m-chlorophenylbiguanide (5-HT3 receptor agonist; 1mg/kg) and a non-effective dose of genistein (5mg/kg) increased the seizure threshold. To conclude, it seems that estrogenic/serotonergic systems might be involved in the anticonvulsant properties of genistein.

Seasonal variations of aromatase and estrogen receptors expression in the testis of free-ranging sand rats

An increasing number of studies revealed the importance of estrogen in male reproduction. However, most research was conducted in laboratory rodents subjected to standardized environmental conditions. Therefore, seasonal regulations of estrogen pathways remain poorly understood under natural conditions. Using immunohistochemistry, the expression of several molecules involved in the functioning of testis (i.e. 17-β estradiol [E2], P450 aromatase, estrogen receptors ESR1, ESR2, and GPER1 [also known as GPR30]) were investigated in free-ranging fat sand rats, Psammomys obesus, during the breeding and resting seasons. Leydig cells showed a strong immunoreactivity for aromatase in the testis sampled during the breeding season only; however, E2, ESR1, ESR2 and GPER1 were present during both seasons. Sertoli cells showed a positive signal for E2 and ESR2 during the breeding season; though, all molecules, except GPER1, were present during the resting season. Spermatogonia were reactive for E2, ESR2 and GPER1 during the breeding season and for ESR1 and GPER1 during the resting season. During both seasons, spermatocytes-I presented a moderate reactivity for E2, ESR1, ESR2 and a strong reactivity for GPER1; aromatase was detected during the resting season only. Spermatids and spermatozoa were present exclusively during breeding season and were reactive for all molecules; except round spermatids that were negative for aromatase. The functioning of the testis depends on finely tuned stimulation and inhibition systems. Our results suggest that differential expression of aromatase, ESR1, ESR2, and GPER1 across cells types is involved in the seasonal activation/inactivation cycle of spermatogenesis in a free-ranging species.

Cross-interference of two model peroxisome proliferators in peroxisomal and estrogenic pathways in brown trout hepatocytes

Peroxisome proliferators cause species-specific effects, which seem to be primarily transduced by peroxisome proliferator-activated receptor alpha (PPARα). Interestingly, PPARα has a close interrelationship with estrogenic signaling, and this latter has already been promptly activated in brown trout primary hepatocytes. Thus, and further exploring this model, we assess here the reactivity of two PPARα agonists in direct peroxisomal routes and, in parallel the cross-interferences in estrogen receptor (ER) mediated paths. To achieve these goals, three independent in vitro studies were performed using single exposures to clofibrate – CLF (50, 500 and 1000μM), Wy-14,643 – Wy (50 and 150μM), GW6471 – GW (1 and 10μM), and mixtures, including PPARα agonist or antagonist plus an ER agonist or antagonist. Endpoints included gene expression analysis of peroxisome/lipidic related genes (encoding apolipoprotein AI – ApoAI, fatty acid binding protein 1 – Fabp1, catalase – Cat, 17 beta-hydroxysteroid dehydrogenase 4 – 17β-HSD4, peroxin 11 alpha – Pex11α, PPARαBb, PPARαBa and urate oxidase – Uox) and those encoding estrogenic targets (ERα, ERβ-1 and vitellogenin A – VtgA). A quantitative morphological approach by using a pre-validated catalase immunofluorescence technique allowed checking possible changes in peroxisomes. Our results show a low responsiveness of trout hepatocytes to model PPARα agonists in direct target receptor pathways. Additionally, we unveiled interferences in estrogenic signaling caused by Wy, leading to an up-regulation VtgA and ERα at 150μM; these effects seem counteracted with a co-exposure to an ER antagonist. The present data stress the potential of this in vitro model for further exploring the physiological/toxicological implications related with this nuclear receptor cross-regulation.

Effects of Dietary Isoflavones, Age, and Stroke on Estrogen Pathway Activation in Male Rats

The effects of stroke can cause long lasting impairment on not only one's physical health but their quality of life and everyday function. Dietary modifications provide a potential solution to aid in neuroprotection and allow enhanced functional recovery post‐stroke. Previous investigation tested this, and found the addition of dietary soy compounds does show significantly improved functional recovery through behavioral tests in our rat models. Further studies aimed to identify specific effects on gene expression on the dietary inclusion of isoflavones alone, using Daidzein and Genistein, known neuroprotective estrogenic receptor activators. We not only examined the effect of isoflavone content on altered gene expression, but also age as another potential mediating factor either promoting or inhibiting anti‐inflammatory and neuroprotective pathways. In doing so, both adult and aged male Hooded Long Evan rats were separated into distinct dietary protein groups containing either Sodium Caseinate (CAS), or Sodium Caseinate with the isoflavones daidzein and genistein (CAS+ISO) two weeks before a permanent unilateral middle cerebral artery occlusion (MCAO). Tissue samples were collected from the injured hemisphere on Day +3 (n=6) and Day +0 (n=4; received no MCAO). Data were collected via a qPCR array after RNA extraction and cDNA synthesis, following the manufacturer's protocol guidelines (Qiagen). Expression of mRNA was compared between treatment groups based on age, isoflavone content, and stroke. Changes in mRNA expression greater than 2‐fold were considered significant. Detailed results will be presented on the poster.Support or Funding InformationNIH NCCAM (1R15AT006593; JLC) and DuPont Nutrition and Health

Leptin is overexpressed in the tumor microenvironment of obese patients with estrogen receptor positive breast cancer.

The present study aimed to investigate the potential role of leptin in the progression of breast cancer and the associated cell proliferation signalling pathway(s). A total of 44 female patients diagnosed with breast cancer and 24 healthy donors from Ain Shams University Hospitals (Cairo, Egypt) were enrolled in the present study. The present study assessed leptin expression in breast cancer tissues at the gene and protein level using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. The results demonstrate that the expression of leptin was significantly higher in tissue of breast cancer samples from obese patients than overweight and control samples (P<0.001). ELISA results indicated a significant increase (P<0.001) of leptin expression in obese patients. To investigate whether there is any difference in leptin expression between the peripheral and tumor microenvironment blood of patients with breast cancer, the concentration of leptin was assessed in plasma from both using ELISA assays. The results demonstrated a statistically significant increase in the level of leptin in plasma samples from the tumor microenvironment of obese patients with estrogen receptor positive (ER+) breast cancer, compared with peripheral plasma samples. Furthermore, the leptin gene was overexpressed in obese ER+ breast cancer tissue. RT-qPCR was also performed to assess the expression of genes involved in proliferation pathways including leptin receptor (LEPR), aromatase, mitogen activated protein kinase (MAPK) and signal transducer and activator of transcription-3 (STAT3). A positive association between leptin expression, LEPR, aromatase, MAPK and STAT3 was detected in tissue samples of patients with breast cancer. The current study concluded that leptin may enhance breast cancer progression by inducing the expression of JAK/STAT3, ERK1/2 and estrogen pathways in obese patients breast cancer.

Estrogen induces CXCR4 overexpression and CXCR4/CXL12 pathway activation in lung adenocarcinoma cells in vitro

ABSTRACTPurpose: This study was designed to investigate whether estradiol is related to the expression of the chemokine receptor CXCR4 and its activation in lung adenocarcinoma in vitro, since lung adenocarcinomas from premenopausal women have shown high levels of CXCR4, and this expression has been associated with worse prognosis and poor survival. Methods: The effect of 17-β-estradiol (E2) (0.03 nM-10 nM) on CXCR4 expression was analyzed in lung adenocarcinoma cell lines (SK-LU-1, H1435, H23, A549) by immunofluorescence after 24 and 72-h poststimulation. Tamoxifen treatment was applied to corroborate the estrogenic effect. The wound-healing assay was performed to investigate whether E2 treatment increased CXCR4/CXL12 pathway activity. A549 and SK-LU-1 cells were stimulated with E2, CXCL12, and CXCL12 combined with E2. Tamoxifen and AMD3100 were applied to corroborate estrogen and chemokine pathway activation. Results: Estradiol stimulated significantly CXCR4 overexpression in all the cell lines analyzed in a dose- and a time-dependent manner. Tamoxifen treatment inhibited the CXCR4 overexpression observed in estrogen-treated groups, demonstrating that estrogen strongly influences CXCR4 expression in lung adenocarcinoma cells. Cells treated with E2, CXCL12 and E2 combined with CXCL12 exhibited significant cell migration, which was suppressed when tamoxifen and AMD3100 were present. Conclusion: Overexpression of CXCR4 induced by estrogen and the activity of CXCL12/CXCR4 pathway could be a new mechanism by which this hormone supports tumor progression and metastasis. These findings may partly explain the worse prognosis observed in premenopausal women and suggest considering the role of estrogen in lung cancer for the design of more specific treatment schemes.

SIRT1-dependent AMPK pathway in the protection of estrogen against ischemic brain injury.

Stroke is a major cause of mortality and disability, especially for postmenopausal women. In view of the protective action of estrogen, hormone therapy remains the only effective way to limit this risk. The objective of this study was to investigate the efficiency and underlying mechanisms of estrogen neuroprotection. Subcutaneous injection of 17β-estradiol in rats after ovariectomy (OVX) was used to manipulate estrogen level and explore the effects of estrogen in cerebral ischemic damage both in vivo and in vitro. Silent mating type information regulation 2 homolog 1 (SIRT1) knockout mice and adenosine monophosphate (AMP)-activated kinase (AMPK) inhibitor Compound C were also used to investigate the underlying pathway of estrogen. Estrogen deficiency induced by OVX aggravated brain infarction in experimentally induced cerebral ischemia rats, whereas estrogen pretreatment reduced ischemia-induced cerebral injuries. Neurons of estrogen deficiency models were susceptible to apoptosis under oxygen-glucose deprivation (OGD). In contrast, neurons with estrogen-supplemented serum exhibited restored resistance to cell apoptosis. In OGD neurons, estrogen promoted AMPK activation through estrogen receptor α, and neuroprotection of estrogen was prevented by AMPK inhibition. Estrogen increased SIRT1 expression and activation, and estrogen-induced AMPK activation disappeared in SIRT1 knockout neurons. Moreover, estrogen-induced neuroprotection was abolished in SIRT1 knockout mice and AMPK-inhibited rats. Our data support that estrogen protects against ischemic stroke through preventing neuron death via the SIRT1-dependent AMPK pathway.