探討Vorinostat透過表觀機制調控抑制非小細胞肺癌端粒酶之活性

Vorinostat, SAHA, represses telomerase activity via epigenetic regulation of telomerase reverse transcriptase in non-small cell lung cancer cells.

李建德(CHIEN-TE LI)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學研究所/博士(2011年)

https://doi.org/10.6834/CSMU.2011.00130

摘要

Vorinostat (又稱為suberoylanilide hydroxamic acid, SAHA)是屬於一種組蛋白去乙醯基化的抑制劑，並且是臨床試驗中一種新興種類的抗癌藥物。Vorinostat是可以導致細胞生長停滯及分化，並且無論在細胞內或細胞外的實驗皆證實會造成多種類的腫瘤的細胞凋亡。近期的報導指出，在許多癌症上，人類端粒反轉錄酶的表現量[human telomerase reverse transcriptase (hTERT)]可以當作一個癌症的治療指標。在此我們使用染色體端粒酶重覆增幅步驟技術[Telomerase repeat amplification protocol (TRAP) assay]分析在處理vorinostat 後的A549肺癌細胞的端粒酶活性差異。結果顯示vorinostat透過降低A549肺癌細胞的hTERT的表現量，而抑制端粒酶的活性。透過甲基化專一性PCR (methylation-specific PCR )以及亞硫酸鹽定序(bisulfate sequencing) hTERT啟動子上甲基化程度得知，vorinostat主要是透過表觀基因(epigenetic)機制而造成降低hTERT的表現。結果得知，vorinostat會經由去降低DNA的甲基轉移酶的表現量而使hTERT啟動子上特定區位的CpG island產生去甲基化反應。而DNA甲基酶包含DNMT1 及DNMT3b 皆會受到vorinostat 處理後而減少表現。再者, 利用染色體免疫沉澱實驗 (Chromatin immunoprecipitation assay, ChIP)分析hTERT啟動子活化或非活化染色質標誌的差異。由結果顯示，vorinostat會減少非活化態的染色質標誌dimethyl-H3K9，同步會去降低DNMT1 和DNMT3b的關聯性結合。由以上結果中得知，可以使用vorinostat當作特異性抗癌治療，並會經由表觀機制的影響而導致端粒酶的活性下降。

關鍵字

vorinostat ； 肺癌細胞 ； 端粒酶活性 ； 人類端粒酶逆轉錄酶表觀基因調控

並列摘要

Vorinostat (suberoylanilide hydroxamic acid, SAHA), a class of histone deacetylase (HDAC) inhibitors, represents an emerging class of anticancer agents currently progressing in clinical trials. It causes cell growth inhibition, differentiation, and apoptosis of many tumor types in vitro and in vivo. Recently, it was reported that hTERT is one of the targets for cancer therapy in cancer cells. Telomerase repeat amplification protocol (TRAP) assay was used to analyze the expression of hTERT after vorinostat treatment in the A549 lung cancer cells. Vorinostat inhibited telomerase activity by reducing the expression of human telomerase reverse transcriptase (hTERT) in A549 human lung cancer cells. The epigenetic regulation mechanism is responsible for the repression of hTERT by vorinostat, analyzed through the methylation-specific PCR and bisulfite sequencing of the hTERT promoter. Vorinostat induced the demethylation of site-specific CpGs on the promoter region of hTERT, which was caused by the down-regulation of DNA methyltransferases. DNA methyltransferases (DNMT1 and DNMT3b) were also decreased in vorinostat-treated A549 cancer cells. Furthermore, chromatin immunoprecipitation (ChIP) analysis of the hTERT promoter revealed that vorinostat decreased the level of inactive chromatin markers dimethyl-H3K9, and the declined binding of DNMT1 and DNMT3b were associated. The novel insights showed that vorinostat down-regulated telomerase via epigenetic alteration in lung cancer lead to vorinostat-mediated cancer-specific therapies.

並列關鍵字

vorinostat ； lung cancer ； telomerase activity ； hTERT ； epigenetic regulation

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