靶向抑制组蛋白甲基转移酶G9a通过线粒体凋亡途径抑制肺腺癌细胞增殖  

Inhibition of G9a attenuates cell proliferation via the mitochondrial apoptosis pathway in lung adenocarcinoma

作　　者：万海军[1] 吕望[1] 虞莉[1] 周振宇[1] 胡耶基 胡坚[1] 

机构地区：[1]浙江大学医学院附属第一医院胸外科,杭州310003

出　　处：《中华肿瘤杂志》2017年第1期13-17,共5页Chinese Journal of Oncology

基　　金：吴阶平医学基金会临床科研专项资助基金（320.6750.14300）;浙江省中医药科学研究基金计划（2015ZA216）;浙江省中医药科技计划重点研究项目（2015ZZ007）

摘　　要：目的探讨小分子抑制剂BIX-01294靶向组蛋白甲基转移酶G9a抑制肺腺癌细胞增殖的作用及分子机制。方法应用四甲基偶氮唑蓝法和集落克隆实验检测BIX-01294对人肺腺癌细胞A549生长、增殖的影响，应用流式细胞术检测BIX-01294干预对细胞凋亡的影响，应用Western blot检测13IX-01294干预后，G9a催化产物H3K9me和H3K9me2以及细胞凋亡相关蛋白的表达。研究easpases抑制剂Z-VAD-FMK预处理细胞后，BIX-01294抑制A549细胞增殖是否呈凋亡依赖性。结果与空白对照组比较，组蛋白甲基转移酶G9a抑制剂BIX-01294抑制A549细胞的生长，呈剂量时间依赖关系。BIX-01294作用7d，空白对照组和10μmol／LBIX-01294组的集落形成数分别为（172．7±23．0）个和（42．5±8．7）个，差异有统计学意义（P〈0．05）。10μmol／LBIX-01294作用24h，A549细胞凋亡率为（47．6±8．4）％，与空白对照组[（7．2±3．6）％]比较，差异有统计学意义（P〈0．05）。10μmol／LBIX-01294干预24h，A549细胞中C9a催化产物H3K9me和H3K9me2的表达下降，线粒体凋亡途径蛋白Bax、Bak和cleaved caspase-9的表达上调，抗凋亡蛋白Bel-2的表达下降，死亡受体凋亡途径蛋白cleaved caspase-8的表达无变化，凋亡执行蛋白cleaved caspase-3和cleavedPARP的表达上调。Z-VAD-FMK预处理后，A549细胞中凋亡率和凋亡相关蛋白的表达下降。结论BIX-01294具有抑制肺腺癌细胞增殖的作用，激活线粒体途径诱导细胞凋亡是其抗肿瘤作用机制之一。Objective The aim of this study is to investigate the effect of G9a inhibitor BIX-01294 on attenuating cell proliferation in human lung adenoearcinoma A549 cell line and the underlying molecular mechanism. Methods Treated with BIX-01294, the growth and proliferation of A549 cells were detected by MTT assay and colony formation assay, and its impact on cell apoptosis was analyzed using flow cytometry. By Western blot, we explored the alterations in the expression of apoptosis-related proteins and the G9a eatalysate, H3K9me and H3K9me2. In addition, in the pretreatment with caspase inhibitor Z-VAD-FMK, we detected the apoptotic dependence of BIX-O1294 attenuating impact on A549 cell proliferation. Results Compared with the control group, the histone methyhransferase G9a inhibitor BIX-01294 attenuated cell proliferation in A549 ceils in a dose- and time-dependent manner. There were 42.5±8.7 colonies after BIX- 01294 （ 10 μmol/L） treatment for 7 days, while 172.7±23.0 colonies in the control group, with a statistical significance （P〈0.05）. After treatment with BIX-01294 （10 μmol/L） for 24 hours, the cell apoptotic rate was（47.6±8.4） %, with a significant difference in comparison with the control group [ （7.2± 3.6）%, P〈 0.05 ]. The expression of G9a catalysate, H3K9me and H3K9me2 was downregulated, the same with anti- apoptotic protein Bcl-2, while the proteins in mitochondrial apoptosis pathway, Bax, Bak and cleaved caspase-9, were upregulated, so was the expression of cleaved caspase-3 and cleaved PARP, and there was no alteration in the expression of cleaved caspase-8, which is a protein related with death receptor apoptosis pathway. Furthermore, after Z-VAD-FMK pretreatment, the cell apoptotic rate was decreased significantly,and the expression of apoptosis-related proteins were downregulated. Conclusions Our results indicate that BIX-01294 can attenuate cell proliferation in lung adenocarcinoma, and it can be considered as one of the underlying mechanisms, the apoptosis may be induce

关 键 词：肺肿瘤 腺癌 细胞凋亡 组蛋白甲基转移酶G9a BIX-01294 

分 类 号：R734.2[医药卫生—肿瘤]