Department of Geriatrics, Renmin Hospital of Wuhan University, Wuchang District, Wuhan, PR China.
Biomed Pharmacother. 2013 Feb;67(1):78-87. doi: 10.1016/j.biopha.2012.10.015. Epub 2012 Nov 19.
The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has increasingly attracted worldwide attention for its antineoplastic potential. The cytotoxitic mechanisms, however, especially, the relative contribution of silenced genes reactivation by demethylation and enzyme-DNA adduct formation to the efficacy of 5-Aza-CdR is still a crucial unresolved question. In this investigation, we demonstrated that 5-Aza-CdR treatment resulted in growth suppression in a concentration and time-dependent manner and G2 phrase arrest - hallmarks of a DNA damage response in gastric cancer AGS cells. Formation of DNA double-strand breaks, as monitored by comet assay was examined in an ATM (ataxia-telangiectasia mutated)-dependent manner based on the fact that PI3K inhibitor Wortmannin abolished the action of cytotoxicity of 5-Aza-CdR. Upon treatment with 5-Aza-CdR, ATM activation was clearly associated with P53 phosphorylation at Ser(15), which was directly responsible for 5-Aza-CdR modified P21(Waf1/Cip1) expression. Further exploration revealed that demethylation of P16(INK4A) correlated with the strikingly down-regulated expressions of DNA methyltransferase 3A as well as 3B was, at least in part, attributed to the cytotoxicity of 5-Aza-CdR in AGS cells. Conclusively, these results greatly enhance our understanding of the mechanisms of cytotoxicity of 5-Aza-CdR and strongly provide the preclinical rationale for an assessment of 5-Aza-CdR to ameliorate patient outcome with gastric cancer.
DNA 甲基化抑制剂 5-氮杂-2'-脱氧胞苷（5-Aza-CdR）因其抗肿瘤潜力而受到全球越来越多的关注。然而，细胞毒性机制，特别是通过去甲基化沉默基因的重新激活和酶-DNA 加合物形成对 5-Aza-CdR 疗效的相对贡献，仍然是一个关键的未解决问题。在这项研究中，我们证明了 5-Aza-CdR 处理以浓度和时间依赖的方式导致生长抑制，并导致胃癌 AGS 细胞的 G2 期停滞——这是 DNA 损伤反应的标志。通过彗星试验检测 DNA 双链断裂的形成，这是基于 PI3K 抑制剂 Wortmannin 消除了 5-Aza-CdR 细胞毒性作用的事实，以 ATM（共济失调毛细血管扩张突变）依赖性方式进行检查。用 5-Aza-CdR 处理后，ATM 激活与 P53 在丝氨酸 15 位的磷酸化明显相关，这直接导致了 5-Aza-CdR 修饰的 P21(Waf1/Cip1)表达。进一步的探索表明，P16(INK4A)的去甲基化与 DNA 甲基转移酶 3A 和 3B 的表达显著下调相关，至少部分归因于 5-Aza-CdR 在 AGS 细胞中的细胞毒性。总之，这些结果大大提高了我们对 5-Aza-CdR 细胞毒性机制的理解，并为评估 5-Aza-CdR 改善胃癌患者预后提供了临床前依据。
