title: Estrogen receptor α (ESR1) over-expression mediated apoptosis in Hep3B cells by binding with SP1 proteins.
abstract: Previous studies have reported that estrogen receptors (ERs) are expressed in normal human liver, chronic hepatitis, and benign hepatic tumor tissues. However, decreased expression of ERs can be observed in hepatocellular carcinoma (HCC) and the role of ERs in HCC is not fully understood. Thus, the present study aimed to investigate the molecular mechanism induced by the overexpression of ERα (ERα (ESR1)) in Hep3B cells. We first detected the induction of apoptosis in ER-negative Hep3B cells using DNA fragmentation assay and flow cytometry. We found that ERα and ERα plus 17β-estradiol treatment increased apoptosis in Hep3B cells. Additionally, western blotting showed increased expression of active caspase 3 and tumor necrosis factor α (TNFα (TNF)) in ERα-transfected cells. To further understand the importance of SP1-binding sites in the TNFα promoter, ERα-negative Hep3B cells were co-transfected with ERα and a wild-type TNFα plasmid or TNFα with deleted SP1 regions. Deletion of both distant and primal SP1 sites abolished the activity of ERα, and similar results were observed by blocking the expression of SP1 protein using mithramycin (MA). This result indicates that SP1 protein is essential for ERα-activated TNFα promoter activity. Co-immunoprecipitation assay further confirmed the binding interaction between ERα and SP1 in a ligand-dependent manner. In general, we demonstrate that the overexpression of ERα mediates apoptosis in ERα-negative Hep3B cells by the binding of ERα to SP1 protein. Additionally, this ERα-SP1 complex binds to the proximal and distal sites of the TNFα gene promoter and further induces the expression of active caspase 3 in a ligand-dependent manner.
authors.affiliation: Institute of Medical and Molecular Toxicology and Institute of Medicine, Chung Shan University, Taichung, Taiwan Division of Chest Medicine, Department of Internal Medicine, Armed Force Taichung General Hospital, Taichung, Taiwan.
authors.forename: Chuan-Chou
authors.initials: CC
authors.lastname: Tu
authors.forename: V Bharath
authors.initials: VB
authors.lastname: Kumar
authors.forename: Cecilia Hsuan
authors.initials: CH
authors.lastname: Day
authors.forename: Wei-Wen
authors.initials: WW
authors.lastname: Kuo
authors.forename: Su-Peng
authors.initials: SP
authors.lastname: Yeh
authors.forename: Ray-Jade
authors.initials: RJ
authors.lastname: Chen
authors.forename: Chen-Rong
authors.initials: CR
authors.lastname: Liao
authors.forename: Hsiao-Yu
authors.initials: HY
authors.lastname: Chen
authors.forename: Fuu-Jen
authors.initials: FJ
authors.lastname: Tsai
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: Wu
authors.forename: Chih-Yang
authors.initials: CY
authors.lastname: Huang
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D047628:Estrogen Receptor alpha / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D047628:Estrogen Receptor alpha; D016329:Sp1 Transcription Factor; D014409:Tumor Necrosis Factor-alpha
keywords: ERα; Hep3B cells; SP1; TNFα; apoptosis; caspase 3