E2/ERβ抑制PPARα基因以及功能於Hep3B肝癌細胞之分子機制探討; Investigation the mechanisms of E2/ERβ inhibited the PPARα tumor promotion functions in Hep3B cells

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Title:	E2/ERβ抑制PPARα基因以及功能於Hep3B肝癌細胞之分子機制探討;Investigation the mechanisms of E2/ERβ inhibited the PPARα tumor promotion functions in Hep3B cells
Authors:	蔡志豪;Chih-Hao Tsai
Contributors:	中國醫藥大學：基礎醫學研究所
Keywords:	雌性激素受體β;過氧化體增生活化受體α;肝癌;ERβ;PPARα;liver cancer
Date:	2009-07-16
Issue Date:	2009-08-11 14:35:52 (UTC+8)
Abstract:	PPARα屬於細胞核內賀爾蒙受體的一員，在許多研究中已經指出若因肥胖或是長期服用降血脂藥會因PPARα的ligands（如類降血脂藥物Fenofibrate或脂肪酸類）過度的刺激而產生氧化傷害以及開啟肝臟不正常增生基因的表現而導致肝癌的發生(Hepatocarcinogenesis)。在本實驗室先前研究已經指出在肝癌病人的組織中發現PPARα mRNA的表現量在癌區是高於非癌區，因此PPARα在肝癌發展中扮演著相當重要的角色。此外根據許多研究報告及統計資料指出，男性罹患肝癌的比率為女性的3-8倍；同時，我們之前發表的文獻亦說明雌激素(Estrogen,E2)，及雌激素接受體α(Estrogen Receptorα, ERα)具有抑制PPARα表現量甚至對於抑制肝癌Hep3B細胞增生及促凋亡有明顯的功效。然而，ERα 與ERβ 於不同癌症中可能扮演著相似或是相反的功能。因此本篇論文將探討ERβ是否也具有調控PPARα的作用? 本研究中發現過量表現ERβ之後，則發現PPARα的mRNA與蛋白質表現量受到壓制，PPARα的進核情形亦受到抑制，此外PPARα的活性指標Acyl-CoA oxidase (ACO)表現量則是下降。ERβ抑制PPARα所活化的增生現象，而降低了cyclin A,E,PCNA 與c-Fos的表現量。ERβ抑制survival proteins的表現，使得Bcl-xL, Bcl-2,p-Bad與Akt 表現下降；促進mitochondrial apoptotic pathway的調控蛋白質，使得Bad ,Bax,cyt.c,caspase 9與caspase 3表現量上升。除此之外在co-immunoprecipitation assay 中發現ERβ扮演著PPARα的corepressor角色，甚至在EMSA的實驗中發現ERβ會直接結合於PPRE序列阻止 PPARα的下游被開啟。綜合以上結果可以得知，E2/ERβ可被當作co-repressor除了與PPARα本身結合之外又與PPARα下游基因反應序列(PPRE)結合，抑制了PPARα的表現以及活性，甚至具有抑制Hep3B增生同時促進凋亡的功能。 Peroxisome proliferator-activated receptor-α (PPARα) is a member of the nuclear receptor superfamily. Administration of its ligands, fenofibrate and fatty acid, can cause hepatocarcinogenesis in rats and mice. Our previous studies demonstrated that PPARα mRNA expressed significantly higher in liver tumor part, and overexpressed ERα induced apoptosis but also inhibited PPARα expression and cell proliferation in Hep3B cell. However, ERα and ERβ may play similar or opposite functions in different cancers. Therefore, we aim to further determine the role of PPARα in hepatocarcinogenesis, and define how ERβ regulates the PPARα in Hep3B cells. Our data show the overexpressed ERβ not only overcome fenofibrate effect to induce the protein levels of Cyt.c, Caspase 9 and Caspase 3 but also inhibit the protein levels of Bcl-xL, Bcl-2, p-Bad, cyclin A, E and PCNA. All these effects cause the enhancement of mitochondrial dependent apoptotic pathway and the attenuation of cell proliferation. Moreover,the overexpressed ERβ not only reduced the level of mRNA, protein expression of PPARα, but also even its downstream Acyl-CoA oxidase (ACO). The EMSA was applied to identify the ERβ, actually mediates through the binding of PPARα promoter to repress PPARα promoter activity and gene expression. In addition, the direct interaction between ERβ and PPARα proteins was observed by co-immunoprecipitation assay. The E2/ERβ might even inhibit fenofibrate-induced the nuclear translocation effect of PPARα. Taken together, ERβ might directly downregulate PPARα gene expression and inhibit the nuclear translocation to suppress the proliferation and induce the apoptosis of Hep3B cells.
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