Browsing by Subject "Estrogen"

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Involvement of Estrogen Receptor Beta 5 in the Progression of Glioma
(2013-05-01) Li, Wenjun; Shaohua Yang
Emerging evidence suggests a decline of ERβ expression in various peripheral cancers and ERβ has been proposed as a cancer brake that inhibits tumor cell growth and proliferation. In the current study, we have identified ERβ5 as the predominant isoform of ERβ in human glioma and its expression was significantly increased in human glioma as compared with non-neoplastic brain tissue. Hypoxia and activation of hypoxia inducible factor (HIF) increased ERβ transcription in U87 cells, suggesting elevated ERβ expression in glioma might be induced by the hypoxic stress in the tumor. Overexpression of either ERβ1 or ERβ5 increased PTEN expression and inhibited activation of the PI3K/AKT/mTOR pathway; ERβ5 also inhibited the MAPK/ERK pathway. In U87 cells, ERβ1 and ERβ5 decreased cell proliferation and decreased cells in the S+G2/M phase. Our findings suggest hypoxia induced ERβ5 expression in glioma as a self-protective mechanism against tumor proliferation and that ERβ5 might serve as a therapeutic target for the treatment of glioma. We also reported potential association between ERβ expression and outcomes of TMZ or tamoxifen treatment for GBM, which might be of practical clinical values.
Neuroprotective and neurotoxic outcomes of androgens and estrogens in an oxidative stress environment
(BioMed Central Ltd., 2020-03-29) Duong, Phong; Tenkorang, Mavis A. A.; Trieu, Jenny; McCuiston, Clayton; Rybalchenko, Nataliya; Cunningham, Rebecca L.
BACKGROUND: The role of sex hormones on cellular function is unclear. Studies show androgens and estrogens are protective in the CNS, whereas other studies found no effects or damaging effects. Furthermore, sex differences have been observed in multiple oxidative stress-associated CNS disorders, such as Alzheimer's disease, depression, and Parkinson's disease. The goal of this study is to examine the relationship between sex hormones (i.e., androgens and estrogens) and oxidative stress on cell viability. METHODS: N27 and PC12 neuronal and C6 glial phenotypic cell lines were used. N27 cells are female rat derived, whereas PC12 cells and C6 cells are male rat derived. These cells express estrogen receptors and the membrane-associated androgen receptor variant, AR45, but not the full-length androgen receptor. N27, PC12, and C6 cells were exposed to sex hormones either before or after an oxidative stressor to examine neuroprotective and neurotoxic properties, respectively. Estrogen receptor and androgen receptor inhibitors were used to determine the mechanisms mediating hormone-oxidative stress interactions on cell viability. Since the presence of AR45 in the human brain tissue was unknown, we examined the postmortem brain tissue from men and women for AR45 protein expression. RESULTS: Neither androgens nor estrogens were protective against subsequent oxidative stress insults in glial cells. However, these hormones exhibited neuroprotective properties in neuronal N27 and PC12 cells via the estrogen receptor. Interestingly, a window of opportunity exists for sex hormone neuroprotection, wherein temporary hormone deprivation blocked neuroprotection by sex hormones. However, if sex hormones are applied following an oxidative stressor, they exacerbated oxidative stress-induced cell loss in neuronal and glial cells. CONCLUSIONS: Sex hormone action on cell viability is dependent on the cellular environment. In healthy neuronal cells, sex hormones are protective against oxidative stress insults via the estrogen receptor, regardless of sex chromosome complement (XX, XY). However, in unhealthy (e.g., high oxidative stress) cells, sex hormones exacerbated oxidative stress-induced cell loss, regardless of cell type or sex chromosome complement. The non-genomic AR45 receptor, which is present in humans, mediated androgen's damaging effects, but it is unknown which receptor mediated estrogen's damaging effects. These differential effects of sex hormones that are dependent on the cellular environment, receptor profile, and cell type may mediate the observed sex differences in oxidative stress-associated CNS disorders.
QUANTITATIVE PROTEOMIC INVESTIGATION OF ESTROGENIC ENDOCRINE-DISRUPTING EFFECTS IN THE RAT UTERUS USING SYSTEMS BIOLOGY
(2014-03) Sahyouni, Fatima; Szarka, Szabolcs; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo
Endocrine disrupting chemicals (EDCs) are a class of chemicals that interfere with the biological actions of hormones, and there has been significant public concern about EDCs in the environment adversely affecting both wildlife and human health. They can alter processes regardless of whether they are related to reproduction. The mammalian uterus is one of the most sensitive organs for estrogenicity, but the widely used uterotrophic rat assay to assess known and potential EDCs merely considers the uterine weight gain endpoint. In this presentation, we focus on the quantitative proteomic investigation of estrogenic endocrine disruption in the rat uterus utilizing a comprehensive systems biology approach. Using 17β-estradiol (E2), an endogenous estrogen, will serve as a reference for subsequent studies of agents with estrogenic potential. Purpose (a): To validate potential markers of estrogenicity of discovery identified differentially expressed estrogen-induced proteins in rat uterine tissue using quantitative proteomics. Methods (b): Ovariectomized rats were treated short-term with subcutaneous E2 injections using corn oil as a vehicle. Approximately 10 mg of tissue were dissected from the uterus of vehicle-treated control and E2-treated animals for proteomic analyses. Uterine proteins were extracted with 8M urea for 30 minutes and subsequently processed by reduction, alkylation and digestion for mass spectrometry analysis. The samples were analyzed using a hybrid linear ion trap–Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source and connected to a nanoflow liquid chromatography system. MS/MS data was searched against a composite UniProt rat protein database using the Mascot software. Quantitation was performed using an MS-based total precursor intensity approach using the Scaffold software. Additionally, the differentially expressed proteins were mapped to signaling networks and biological processes using Ingenuity Pathway Analysis (IPA). Results (c): The mammalian uterus increases its weight due to fluid imbibition and cell proliferation by exogenously administered estrogenic compounds. With the observation of weight gain in the treated uterus compared to non-treated control rats, we confirmed E2’s uterotrophic effects for our subsequent proteomics study. Estrogen-regulated proteins were identified using an MS-based label-free quantitative approach. With p<0.05 considered statistically significant and >2-fold change as threshold, 135 proteins were differentially regulated by the hormone. Of these significantly differentially regulated proteins, 97 were up-regulated in E2-treated uteri and 38 were down-regulated in E2-treated uteri. When these 135 proteins were submitted for bioinformatic pathway analysis, 131 proteins were mapped into 14 networks that merged into E2-regulated pathways. Major molecular processes involve metabolic pathways, steroid signaling, and inflammatory signaling. Top networks include post-translational modification, protein folding, carbohydrate metabolism, cell death and survival, cancer, and endocrine system disorders. Implicated diseases include endocrine system and metabolic disorders. Proteotypic peptides from proteins that were strongly influenced by E2 administration have been selected for targeted validation studies. Conclusions (d): In addition to confirming the expected increase in wet uterine weights, we have derived interaction networks that mechanistically dissect E2’s uterotrophic effect at the proteome level. We have selected proteotypic peptides of strongly regulated proteins for future targeted validation as a potential biomarker panel for estrogenicity. (Supported by the Robert A. Welch Foundation, BK-0031, and the NIH grant AG031535).
RAPID LABEL-FREE QUANTITATIVE PROTEOMICS OF ESTROGENIC ENDOCRINE-DISRUPTING EFFECTS IN THE RAT UTERUS USING A SYSTEMS BIOLOGY APPROACH
(2013-04-12) Sahyouni, Fatima
Purpose: To identify estrogen-induced differential protein expression in rat uterine tissue using a rapid label-free proteomics and systems biology approach. Methods: Ovariectomized rats were treated short-term with subcutaneous E2 injections using corn oil as a vehicle. Approximately 10 mg of tissue were dissected from the uterus of vehicle-treated control and E2-treated animals for proteomic analyses. Uterine proteins were extracted with 8M urea for 30 minutes and subsequently processed by reduction, alkylation and digestion for mass spectrometry analysis. The samples were analyzed using a hybrid linear ion trap-Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source and connected to a nanoflow liquid chromatography system. MS/MS data was searched against a composite IPI rat protein database containing both forward and randomized sequences using the Mascot software. Quantitation was performed using an MS/MS-based total ion currents (TICs) approach using the Scaffold software. Additionally, Ingenuity Pathway Analysis (IPA) was utilized to derive interaction networks among the identified proteins. Results: The mammalian uterus increases its weight due to fluid imbibition and cell proliferation by exogenously administered estrogenic compounds. With the observation of weight gain in the treated uterus compared to non-treated control rats, we confirmed E2's uterotrophic effects for our subsequent proteomics study. Out of a total of 262 identified proteins, 163 proteins were differentially regulated (with p<0.05 considered statistically significant) by the hormone. Of the 163 proteins that were significantly regulated, 153 were up-regulated in E2-treated uteri and 10 were down-regulated in E2-treated uteri. These 163 proteins were submitted and mapped into 19 networks that merged into E2-regulated pathways. Top networks included molecular transport, carbohydrate metabolism, cancer, developmental disorders and cellular function and maintenance. Implicated diseases were endocrine system and metabolic disorders. Top signaling pathways involved metabolic pathways, steroid signaling and actin cytoskeleton signaling. Conclusions: In addition to the expected increase in wet uterine weights, we have elucidated and organized a large number of E2-induced protein expression changes into interaction networks. Metabolism and developmental disorders were implicated as the top networks. (Supported by the Robert A. Welch Foundation, BK-0031, and the NIH grant AG031535)
Synergy 2010: Annual Research Report
(2010-01-01)
The Phenotype of Cells Expressing Estrogen Receptor Alpha in the Nucleus of the Solitary Tract
(2018-05) Horn, Christopher L.; Mifflin, Steve W.; Schreihofer, Ann M.; Cunningham, J. Thomas; Phillips, Nicole R.
Estrogen protects females from hypertension. The nucleus tractus solitarius (NTS) is a hindbrain site involved in the regulation of blood pressure, however little is known about estrogen receptors within the NTS. The purpose of these studies was to determine the phenotype of the cells expressing estrogen alpha receptors in the nucleus tractus solitarius. Four female Sprague-Dawley rats were transcardially perfused with 4% paraformaldehyde and hindbrains harvested. In coronal sections containing the NTS (40μm thick), immunohistochemistry was performed to determine which type of cells were expressed with estrogen receptor alpha (ERα) expressing cells. We used the anti-ERα antibody with an antibody for each protein of interest: anti-tyrosine hydroxylase (TH), anti-glial fibrillary acidic protein (GFAP), anti-NeuN, and anti-Iba-1. Sections were captured using an Olympus BX41 Fluorescence Microscope and analyzed using ImageJ. The NTS was divided into 2 regions: sections caudal to the area postrema (caudal) and sections lying below the area postrema (sub-postrema, SP) and the number of immunoreactive neurons in each region counted and expressed as an average number of labeled neurons per section±SEM. The number of sections analyzed ranged from 5-10 per individual in caudal and 2-4 per individual in SP. At sacrifice, females were in estrus (1), diestrus (2) or proestrus (3). NeuN in SP NTS (n=4) was observed in 151±53 and ERα in 50±21 neurons per section. Colocalization of ERα and NeuN in SP NTS was observed in 11±6 neurons per section (about 7%). NeuN in caudal NTS was observed in 59±7 and ERα 27±3 neurons per section. Colocalization of ERα and NeuN in caudal NTS was observed in 4±1 neurons per section (about 7%). TH in SP NTS (n=6) was observed in 49±8 and ERα in 51±12 neurons per section. Colocalization of ERα and TH in SP NTS was observed in 26±4 neurons per section (about 53%). TH in caudal NTS was observed in 26±6 and ERα 29±7 neurons per section. Colocalization of ERα and TH in caudal NTS was observed in 17±4 neurons per section (about 51%). Due to the quantity and shape of GFAP immunoreactive cells in the NTS (n=4), we were not able to count the number cells. Colocalization of ERα and GFAP expressing cells were not observed in our study. Cells expressing Iba1 were not observed in the later trials of our study (n=4). ERα is expressed on a subset of catecholaminergic NTS neurons, as well as non-catecholaminergic neurons. Since the NTS catecholaminergic neurons contribute to responses to stress (e.g., hypoxia), this finding could provide a substrate for estrogen-mediated cardiovascular protection in females.
The Role of Estrogen and Estrogen Analogues in Friedreich’s Ataxia Cytoprotection
(2014-05-01) Richardson, Timothy E.; Simpkins, James W.
Friedreich’s ataxia (FRDA) is the most common form of inherited ataxia in the world, affecting roughly 1:50,000 people in the United States. It is inherited in an autosomal recessive manner due to a GAA trinucleotide repeat expansion in the first intron of the FXN gene on chromosome 9q13-21, causing gene silencing and a functional absence of the mitochondrial-localizing protein frataxin. The frataxin protein is responsible for the assembly of iron-sulfur centers in mitochondrial proteins, including the electron transport chain complex I-III, heme synthesis, as well as removing iron from around the mitochondria, preventing the formation of reactive oxygen species (ROS). The loss of FXN function causes an accumulation of mitochondrial iron and ROS, as well as impaired function of Fe-S centers in mitochondrial proteins, leading to mitochondrial damage and a decrease in activity of mitochondrial complexes I-III. The damaged mitochondria are unable to match ATP production to the cell’s energy requirements, resulting in cell death. High energy use cell types, such as neurons and cardiac myocytes, depend almost entirely on oxidative phosphorylation, leaving them especially vulnerable to the mitochondrial damage caused, and it is for this reason that these tissues are the most severely affected by the pathogenesis of FRDA. Cellular models of Friedreich’s Ataxia have employed L-buthionine (S,R)-sulfoximine (BSO), a chemotherapeutic agent which blocks the rate limiting step of de novo glutathione (GSH) synthesis, catalyzed by gamma-glutamylcysteine synthetase. Studies have shown that donor fibroblasts from Friedreich’s Ataxia patients are extremely susceptible to this BSO induced oxidative stress, while fibroblasts from healthy patients are not, due the presence of functional frataxin to absorb the increased load of cellular ROS when GSH is inhibited. Currently, there are few effective treatment modalities for FRDA. Historically, treatment has been focused on palliative care: patient counseling, genetic counseling for prospective parents, speech therapy, physical therapy, wheelchair and other ambulatory device use, propranolol for tremors, dantrolene sodium for muscle spasms and symptomatic treatment for heart disease and diabetes. Recently, antioxidant and mitochondria specific iron chelation therapy have both been proposed as possible therapies to treat the root cause of FRDA. Iron chelation therapy works by a similar principal, removing the iron from around the mitochondria, preventing the formation of free radicals and preventing the associated mitochondrial damage. The neuroprotective effects of 17β-estradiol (E2) have been clearly documented for more than a decade in a variety of disease states involving mitochondrial disruption, but the exact mechanism of action is currently poorly understood. Although the neuroprotective effects of estrogens have never been tested in an FRDA model and FRDA shows no gender-bias in incidence, some epidemiologic studies of FRDA have shown a better prognosis in female patients. Since there is a simple genetic test to determine the presence of FRDA in the children of silent FRDA carriers, it is possible to determine the presence of Friedreich’s ataxia in newborns, years before the cardio- and neurodegeneration and clinical symptoms begin, a time window during which nonfeminizing estrogens and other antioxidants could potentially be clinically useful. Estrogens are putative candidate drugs to provide a neuroprotective effect in Friedreich’s ataxia. The ability of phenolic estrogens to protect against the oxidative damage of ROS, coupled with the possibility that they maintain the integrity of the oxidative phosphorylation process makes them ideal for the treatment of the underlying cellular dysfunction, not just the symptoms of FRDA. This study will determine if E2 and estrogen-like compounds can protect human FRDA fibroblasts from oxidative insults in vitro. In addition, we will attempt to determine the exact mechanism by which E2 acts and investigate the possibility of any synergistic effects with other compounds proposed as putative treatments for FRDA.