過度表現甲型雌性激素受體透過結合Sp1蛋白導致肝癌細胞凋亡

Estrogen receptor-α overexpression mediated apoptosis in hepatoma cells by binding with Sp1 protein

涂川洲(Chuan-Chou Tu)

指導教授 ： 吳文俊

中山醫學大學/醫學院/醫學研究所/博士(2013年)

https://doi.org/10.6834/CSMU.2013.00111

摘要

肝細胞癌（HCC）是在全世界第五位最常致死的癌症，每年約有一百萬人死於此疾病。17β-雌二醇（E2）是最強效的內生性雌激素，其通過兩個不同的細胞內受體，雌激素受體α（ERα）和雌激素受體β（ERβ），來控制各種組織的生長，分化之功能。之前的研究報告顯示，正常人體肝臟、慢性肝炎、良性肝腫瘤組織中存在有雌激素受體（ER）。然而，在肝細胞癌組織中發現其雌激素受體的表現量卻是減少的。雌激素受體在肝癌的發生所扮演的角色目前還不清楚。因此，本研究旨在探討在肝癌細胞株Hep3B過量表現雌激素受體α所產生的分子機轉。人類肝癌細胞株Hep3B保持在MEM培養基（10％胎牛血清，2mM L-穀胺醯胺，1.5 g/L的碳酸氫鈉，0.1 mM的非必需氨基酸，1.0 mM的丙酮酸鈉和青黴素/鏈黴素在37℃，5％ CO2的環境）。首先使用DNA片段化分析法和流式細胞儀來分析在Hep3B細胞誘導細胞凋亡的結果。此外，在轉染ERα的細胞株中使用西方墨點法是來測量活化的caspase 3和TNF-α的表達量。為了進一步了解TNFα啟動子Sp1結合點的重要性，於Hep3B細胞中分別轉染ERα和野生型TNFα質體，或刪除SP1區域的TNFα質體。使用mithromycin來阻止SP1蛋白的表達。也使用免疫共同沉澱實驗來證實ERα和SP1之間的互動關係。我們發現，在Hep3B細胞株加上ERα或ERα及E2會增加凋亡。此外，西方墨點法檢測結果顯示在ERα轉染的細胞，其活化的caspase 3和TNF-α的表現量增加。刪除了遠端及近端的SP1結合位置會使ERα失去活性，而透過使用mithramycin阻斷SP1蛋白表現也觀察到類似的結果。我們證明在Hep3B細胞中過度表現ERα會透過結合Sp1蛋白導致肝癌細胞凋亡。此外，此ERα與Sp1的複合物以配體依賴性的方式結合至TNFα基因啟動子的近端和遠端位置，並進一步誘導活性caspase 3的表現。

關鍵字

甲型雌性激素受體 ； Sp1蛋白 ； 肝癌細胞 ； 凋亡

並列摘要

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.

並列關鍵字

estrogen receptor-α ； Sp1 protein ； hepatoma cells ； apoptosis

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