研究clusterin在人類肺癌H1299細胞的表現及功能

Study of the clusterin expression and functions in human lung cancer H1299 cells

曾昭榮(Chao-Jung Tseng)

指導教授 ： 許國堂

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00062

摘要

Clusterin為一種分泌型之異二聚體醣蛋白，它廣泛的表現在組織中以及在人類所有體液中都可發現它的存在。我們的研究結果得知可以促進clusterin表現的路徑有兩條：其一是將細胞處理蛋白質體抑制劑MG132，MG132會調控熱休克蛋白因子轉錄活化區而促進clusterin啟動子的活性；另一是將細胞處理組蛋白去乙醯酶抑制劑TSA，TSA會使得染色質裡的組蛋白高度乙醯化而讓許多基因及其蛋白增加表現量，其中包括了clusterin。而在H1299人類肺癌細胞中，以上兩條路徑都有反應；但在Huh-7人類肝癌細胞，只經由組蛋白乙醯化路徑而促進clusterin的表現，同樣地，在Huh-7人類肝癌細胞中，Lg-HDAg也是經由組蛋白高度乙醯化的路徑而增加clusterin的表現。
我們將clusterin穏定地表現在H1299細胞株，處理化療藥物後，利用MTS assay去偵測細胞的存活率，結果顯示穏定表現clusterin的細胞株會對Doxorubicin產生抗藥性；而對Gemcitabine有非常明顯的敏藥性，但處理Taxotere或Vincristine，clusterin的表現量不影響細胞對藥的感受性。另外，比較野生型及穏定表現clusterin的H1299細胞株，利用Gelatin zymography去觀察MMP-2及MMP-9表現的情形，結果發現穏定表現clusterin細胞株的MMP-2表現量明顯降低，另外我們再觀察內生性clusterin表現量就較高的Huh-7及H1355，除了Huh-7的MMP-9有微量表現，其餘都偵測不到MMP-2及MMP-9的表現。由以上結果得知：clusterin對於MMP-2的表現具有其關聯性，但兩者確切的相關性究竟為何，則有待後續實驗證明之。

關鍵字

群集素 ； 組蛋白乙醯化 ； 化學治療藥物 ； 基質金屬蛋白酵素-2

並列摘要

Clusterin is a secretory heterodimeric glycoprotein, which expressing in all tissues and can be found in all human fluids. To analyze the mechanism of clusterin induction, we generated the luciferase reporter gene constructs with 5’-regulatory sequence of clusterin gene. Our data showed that clusterin expression could be mediated by two pathways. The first pathway is through activation of heat-shock factor (HSF). Another pathway is epigenetic regulated by histone hyperacetylation of chromatin. In H1299 human lung cancer cell, both pathways are associated with clusterin induction; but in human liver cancer cell (Huh-7), only the histone acetylation pathway is exist. We also showed that expression of Lg-HDAg upregulating clusterin expression via histone acetylation in Huh-7 cells.
To study the function of clusterin in lung cancer cells, we generated the clusterin stably overexpression clone from H1299 cells and exposed to chemotherapeutic drugs for cytotoxicity assay. The results suggested that clusterin stably expressing cells increased resistance to doxorubicin but sensitive to gemcitabine treatments. There are no effect on treatment of the clusterin stably expressing cells with taxotere and vincristine when compare to wild type H1299 cells. We also investigated the expression of matrix metalloproteinase (MMP)-2 and -9 activities with clusterin stably expressing cells by gelatin zymography method, the results showed apparent reduction of MMP-2 in clusterin stably expressing cells. The data indicates that clusterin may act as a regulator for MMP-2 expression but the role of clusterin with MMPs gene needed to be further investigated in the future.

並列關鍵字

clusterin ； histone acetylation ； chemotherapeutic drugs ； MMP-2

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