Zebularine引發細胞週期停滯及凋亡之研究

Abstract

DNA甲基化是許多癌症的指標，DNA methyltransferases（DNMT）抑制劑可抑制DNMT重新表現抑癌基因。Zebularine除了抑制DNA甲基化外，有研究顯示其可增加癌細胞對輻射之敏感度。本實驗中發現Zebularine可透過ATR之訊息傳遞磷酸化H2AX，推測其可引發DNA damage，同時也可見p53、p21蛋白表現與細胞週期停滯於G1。Zebularine可活化調控細胞能量平衡之酵素AMPK。AMPK抑制劑compound C或AMPKα si-RNA 抑制AMPK之活化除了觀察到H2AX之磷酸化、p53與p21之表現增加外，還可促進細胞凋亡與抑制癌細胞生長；反之，活化AMPK可抑制p53蛋白之表現。本實驗結果顯示Zebularine可分別引起DNA damage和活化AMPK。AMPK之活化在Zebularine引起細胞毒殺作用扮演保護性之角色，因此合併Zebularine與AMPK抑制劑，能在癌症治療上提供更好的治療方式。

Aberrant DNA hypermethylation is a frequent finding in tumor cells. DNA methyltransferases（DNMTs）inhibitors may reactivate tumor suppressor genes and makes it an effective anticancer strategy. In addition to its demethylating function, DNMT inhibitor, zebularine was also reported to enhance tumor cell radiosensibility. In this study, we found that zebularine induces H2AX phosphorylation through ATR signaling, suggesting the induction of DNA damage by zebularine. Phoshphorylation of p53 at Ser15、overexpression of p53 and p21 proteins and cell cycle G1 arrest were also seen. We assessed the role of an intracellular energy balancing enzyme, AMPK in DNA damage as well. Zebularine activated AMPK, and the inhibition of AMPK by the inhibitor compound C or AMPKα si-RNA resulted in an increase of H2AX phosphorylation and p53 expression. Expression of the constitutive active form of AMPK down-regulated zebularine-induced p53 expression. Our data demostrated that zebularine separately caused DNA damage and activated AMPK. Activation of AMPK may protect the cytotoxic effect of zebularine. Therefore, the combination of zebularine and AMPK inhibitors could be a novel chemotherapeutic strategy.