| Summary: | 博士 === 中山醫學大學 === 醫學研究所 === 101 === Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer mortality in the worldwide with approximately one million fatalities every year. 17β-estradiol (E2) is the most effective endogenous estrogen that controls the growth, differentiation and function of various tissues through two distinct intracellular receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ). Previous studies reported that, estrogen receptors (ERs) are expressed in normal human liver, chronic hepatitis and in benign hepatic tumor tissues. However, decreased ER was found in hepatocellular carcinoma (HCC) and their role in HCC is not fully understood. Thus the present study was aimed to investigate the molecular mechanism induced by overexpressing ERα in Hep3B cells. The human hepatoma cell line--Hep3B was maintained in MEM media (supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and penicillin/streptomycin at 37°C, in a 5% CO2 atmosphere). We first determined the apoptosis induction in Hep3B cells using DNA fragmentation assay and flow cytometry. Additionally, Western blotting assay was used to measure active caspase 3 and TNF-α expression in ERα transfected cells. To further understand the importance of Sp1 binding sites in TNFα promoter, Hep3B cells were co-transfected with ERα and wild type TNFα plasmid or TNFα with deleted Sp1 regions. Mithramycin (MA) was used to block Sp1 protein expression. Co-immuno-precipitation assay confirms the binding interaction between ERα and Sp1. In conclusion, we found that ERα and ERα plus E2 treatment increased apoptosis in Hep3B cells. Additionally, Western blotting assay showed increased active caspase 3 and TNF-α expression in ERα transfected cells. Deletion of both distant and proximal Sp1 site abolished the activity of ERα, similar results were observed by blocking Sp1 protein expression using MA. We demonstrate that ER alpha overexpression mediated apoptosis in Hep3B cells by binding with Sp1 proteins. Additionally, this ERα-Sp1 complex binds to the proximal and distal site of TNFα gene promoters and further induces active caspase 3 expression in a ligand dependent manner.
|
|---|
