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 ERa (ERa (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 ERa and ERa plus 17b-estradiol treatment increased apoptosis in Hep3B cells. Additionally, western blotting showed increased expression of active caspase 3 and tumor necrosis factor a (TNFa (TNF)) in ERa-transfected cells. To further understand the importance of SP1-binding sites in the TNFa promoter, ERa-negative Hep3B cells were co-transfected with ERa and a wild-type TNFa plasmid or TNFa with deleted SP1 regions. Deletion of both distant and primal SP1 sites abolished the activity of ERa, and similar results were observed by blocking the expression of SP1 protein using mithramycin (MA). This result indicates that SP1 protein is essential for ERa-activated TNFa promoter activity. Co-immunoprecipitation assay further confirmed the binding interaction between ERa and SP1 in a ligand-dependent manner.In general, we demonstrate that the overexpression of ERa mediates apoptosis in ERa-negative Hep3B cells by the binding of ERa to SP1 protein. Additionally, this ERa-SP1 complex binds to the proximal and distal sites of the TNFa gene promoter and further induces the expression of active caspase 3 in a ligand-dependent manner.

miR‑939‑3p promotes epithelial‑mesenchymal transition and may be used as a prognostic marker in hepatocellular carcinoma

“…An increasing number of studies have demonstrated that ESR1 may act as a tumor suppressor in various cancer types (16,19). The present study demonstrated that the expression levels of ESR1 are downregulated in HCC cell lines compared with normal liver cells.…”

“…Hishida et al (18) predicted that ESR1 is a tumor suppressor gene in HCC by triple-combination array analysis. Additionally, Tu et al (19) demonstrated that ESR1 overexpression mediated apoptosis in Hep3B cells by binding with SP1 proteins. However, to the best of our knowledge, the effect of ESR1 on the metastasis of HCC cells has not been studied.…”

Bazedoxifene exhibits growth suppressive activity by targeting interleukin‐6/glycoprotein 130/signal transducer and activator of transcription 3 signaling in hepatocellular carcinoma

“…In a previous study, we reported bazedoxifene as a novel inhibitor of IL‐6/GP130 protein‐protein interactions using multiple ligand simultaneously docking and drug repositioning approaches. The Hep3B liver cancer cell line, which is considered estrogen receptor negative, was used to examine whether bazedoxifene is capable of inhibiting IL‐6‐induced STAT3 phosphorylation. Our results showed that IL‐6 induced STAT3 phosphorylation on tyrosine 705 in Hep3B cancer cells.…”

Fenofibrate induced PPAR alpha expression was attenuated by oestrogen receptor alpha overexpression in Hep3B cells

“…Immuno‐precipitation and immunoblotting were performed as previously described . Cell lysates were centrifuged at 12,000 g for 20 minutes and 100 μg of total protein were mixed with 2 μg of anti‐ERα, 20 µL of a protein protein G beads and the samples were rolled at 4°C for overnight.…”