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Botanical Modulation of AhR-ERα by Crosstalk Inhibitors Promotes Estrogen (E2) Detoxification

串扰抑制剂对 AhR-ERα 的植物调节促进雌激素 (E2) 解毒

基本信息

批准号：
9610944
负责人：
Ryan Thomas Hitzman
金额：
$ 4.03万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-16 至 2021-08-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9610944
关键词：
Agonist Alkaline Phosphatase Animals Antibodies Aryl Hydrocarbon Receptor Benign Binding Biological Assay Botanical dietary supplements Botanicals Breast Cancer Risk Factor CYP1A1 gene CYP1B1 gene Cancer Etiology Cell Culture Techniques Cell Line Cells Cessation of life Chemopreventive Agent Collaborations Complex Cytochrome P450 DNA Binding Down-Regulation Drug Metabolic Detoxication Endometrial Carcinoma Enzymes Epigenetic Process Epimedium Estradiol Estrogen Antagonists Estrogen Metabolism Estrogen Receptor alpha Estrogen Receptors Estrogen receptor positive Estrogens Exhibits Failure Fluorescence Polarization Fractionation Fright Gene Expression Genetic Transcription Goals Health HepG2 Hormone replacement therapy Human Humulus Immunohistochemistry In Vitro Knowledge Lead Luciferases MCF7 cell MG132 Measures Mediating Menopausal Symptom Methods Methylation Microsomes Modeling Mutation Outcome Pathway interactions Pharmacologic Substance Postmenopause Progesterone Property Proteasome Inhibitor Quantitative Reverse Transcriptase PCR Quinones Rattus Receptor Cell Reporter Reporting Research Response Elements Risk Safety Signal Transduction Standardization Testing Tissue Stains Translating Up-Regulation Weight Woman Women&apos s Health Xenobiotics adduct aryl hydrocarbon receptor ligand carcinogenesis chemical carcinogenesis chromatin immunoprecipitation estrogenic genotoxicity hormone therapy horny goat weed in vivo inhibitor/antagonist insight malignant breast neoplasm novel chemoprevention

项目摘要

Botanical Modulation of AhR-ERα by Crosstalk Inhibitors Promotes Estrogen (E2) Detoxification Estrogen receptor (ER) positive breast cancer poses significant health risks for postmenopausal women, and is in part mediated by the estrogen (E2) metabolite, estradiol-3,4-quinone, which causes depurinating adducts and leads to mutations. P450 1B1 is the primary enzyme for the conversion of estradiol to the 4-hydroxylated product, which can result in a genotoxic 4-quinone, while P450 1A1, which is normally epigenetically inhibited by E2-activated estrogen receptor alpha (ERα), converts estradiol to a nongenotoxic 2-hydroxylated product. Activated aryl hydrocarbon receptor (AhR) induces degradation of ERα as well as the transcription of both CYP1A1 and CYP1B1, which are translated into P450 1A1 and 1B1 respectively. Activators and ligands of AhR (Crosstalk Inhibitors) have been shown to preferentially upregulate CYP1A1 and ultimately the nongenotoxic 2-hydroxylated estradiol metabolite (estrogen detoxification pathway), through downregulation of the epigenetic inhibition of CYP1A1. Women have increasingly turned to botanical dietary supplements (BDS), instead of traditional hormone replacement therapy (HRT), since the release of the findings that estrogen + progesterone increases breast cancer risk. Interestingly, icaritin, a bioactive compound from Epimedium sp., a botanical used for women's health purposes, has been shown to activate AhR. The hypothesis of this proposal is that some women's health botanicals contain antiestrogenic, AhR activating compounds which degrade ERα (Aim 1) and reverse epigenetic CYP1A1 inhibition to preferentially activate the estrogen detoxification pathway in an in vitro cell culture model (Aim 2) and an in vivo ovariectomized rat model (Aim 3). Inhibition of estrogen-dependent alkaline phosphatase activity in Ishikawa cells, and a failure to inhibit a fluorescent estradiol-ERα complex by botanical compounds in a fluorescence polarization enzyme assay will provide antiestrogenic compounds which do not act through ERα. These compounds will be subjected to in- cell western of MCF-7 (ER+) cells against an ERα antibody to find compounds which degrade ERα. Additional XRE-luciferase activity in HepG2 cells will indicate AhR activating compounds, while a decrease in methylation through a ChIP assay of DNMT and XRE, and qRT-PCR of CYP1A1/1B1 will show that the reversal of estradiol mediated epigenetic CYP1A1 inhibition leads to upregulation of the estrogen detoxification pathway by Crosstalk Inhibitors in vitro. This premise will be taken to an ovariectomized rat model involving E2 and botanical compounds before sacrificing and analyzing uterine weight, performing LC-MS analysis of the E2 metabolites, and quantifying ERα expression levels using immunohistochemistry. This research may reveal mechanisms by which some bioactive women's health BDS compounds exhibit antiestrogenic outcomes, and the importance of the E2 detoxification pathway in promoting wellness in an in vivo postmenopausal model.

通过串扰抑制剂对AhR-ERα的植物性调节促进雌激素（E2）去甲基化雌激素受体（ER）阳性乳腺癌对绝经后妇女构成显著的健康风险，部分由雌激素（E2）代谢产物雌二醇-3，4-醌介导，导致脱嘌呤加合物并导致突变。P450 1B 1是将雌二醇转化为4-羟基化雌二醇的主要酶。产物，其可导致遗传毒性4-醌，而P450 1A 1，其通常在表观遗传学上被抑制通过E2激活的雌激素受体α（ERα），将雌二醇转化为无遗传毒性的2-羟基化产物。激活的芳香烃受体（AhR）诱导ERα降解以及两者的转录 CYP 1A 1和CYP 1B 1，分别翻译为P450 1A 1和1B 1。的活化剂和配体 AhR（串扰抑制剂）已被证明优先上调CYP 1A 1，并最终上调CYP 1A 1。非遗传毒性的2-羟基化雌二醇代谢物（雌激素解毒途径），通过下调 CYP 1A 1的表观遗传抑制。越来越多的妇女转向植物性膳食补充剂（BDS），而不是传统的激素替代疗法（HRT），因为研究结果的释放，雌激素+ 孕酮增加乳腺癌风险。有趣的是，淫羊藿苷，一种来自淫羊藿属的生物活性化合物，一用于女性健康目的的植物，已被证明可以激活AhR。这个假设建议是，一些妇女的健康植物含有抗雌激素，AhR激活化合物，降解ERα（Aim 1）并逆转表观遗传CYP 1A 1抑制，优先激活雌激素体外细胞培养模型（Aim 2）和体内卵巢切除大鼠模型（Aim 3）中的解毒途径。石川细胞雌激素依赖性碱性磷酸酶活性的抑制，在荧光偏振酶分析中，植物化合物的荧光雌二醇-ER α复合物将提供不通过ERα起作用的抗雌激素化合物。这些化合物将受到- 针对ERα抗体对MCF-7（ER+）细胞进行细胞Western分析，以寻找降解ERα的化合物。额外 HepG 2细胞中的XRE-荧光素酶活性将指示AhR活化化合物，而甲基化的降低将指示AhR活化化合物。通过DNMT和XRE的ChIP测定以及CYP 1A 1/1B 1的qRT-PCR，将显示逆转雌二醇介导的表观遗传CYP 1A 1抑制导致雌激素解毒途径上调通过体外串扰抑制剂。这一前提将被用于涉及E2和E2的卵巢切除大鼠模型。在处死和分析子宫重量之前，对E2进行LC-MS分析，代谢产物，并使用免疫组织化学定量ERα表达水平。这项研究可能揭示某些生物活性女性健康BDS化合物表现出抗雌激素结果的机制，以及在体内绝经后模型中，E2解毒途径在促进健康方面的重要性。