Liu Zhaoyun, Luo Qianfu, Guo Chunbao
Laboratory of Surgery, Children's Hospital of Chongqing Medical University, 136 Zhongshan 2nd Rd, Chongqing, 400014 P. R. China.
Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, P. R. China.
Cancer Cell Int. 2015 Mar 30;15:34. doi: 10.1186/s12935-015-0188-y. eCollection 2015.
ATF2 mediated cytochrome c release is the formation of a channel with some unknown factors larger than that of the individual proteins. BHS-only proteins (BH3s), such as Bim, could induce BAX and VDAC, forming a new channel. According to this facts, we can speculated that there is possible signal relationship with BH3s and ATF2, which is associated with mitochondrial-based death programs.
The growth inhibitory effects of mitochondrial ATF2 were tested in cancer cell lines B16F10, A549, EG7, and LL2. Apoptosis was measured by flow cytometry. The effects of ATF2 and levels of apoptosis regulatory proteins were measured by Western blotting. The interaction of proteins were evaluated by immunoprecipitation analysis. The in vivo antitumor activity of mitochondrial ATF2 were tested in xenograft B16F10 models.
Genotoxic stress enabled mitochondrial ATF2 accumulation, perturbing the HK1-VDAC1 complex, increasing mitochondrial permeability, and promoting apoptosis. ATF2 inhibition strongly reduced the conformational activation of Bim, suggesting that Bim acts downstream of ATF2. Although Bim downregulation had no effect on ATF2 activation, Bim knockdown abolished VDAC1 activation; the failure of VDAC1 activation in Bim-depleted cells could be reversed by the BH3-only protein mimic ABT-737. We also demonstrate that silencing of ATF2 in B16F10 cells increases both the incidence and prevalence of tumor xenografts in vivo, whereas stably mitochondrial ATF2 transfection inhibited B16F10 tumor xenografts growth.
Altogether, these results show that ATF2 is a component of the apoptosis machinery that involves a hierarchical contribution of ATF2, Bim, and VDAC1. Our data offer new insight into the mechanism of mitochondrial ATF2 in mitochondrial apoptosis.
ATF2介导的细胞色素c释放是由一些未知因子形成的通道，其大小比单个蛋白质大。仅含BH3结构域的蛋白质（BH3s），如Bim，可诱导BAX和VDAC，形成新的通道。基于这些事实，我们推测BH3s与ATF2之间可能存在信号关系，这与基于线粒体的死亡程序相关。
在癌细胞系B16F10、A549、EG7和LL2中测试线粒体ATF2的生长抑制作用。通过流式细胞术检测细胞凋亡。通过蛋白质印迹法检测ATF2的作用和凋亡调节蛋白的水平。通过免疫沉淀分析评估蛋白质之间的相互作用。在异种移植B16F10模型中测试线粒体ATF2的体内抗肿瘤活性。
基因毒性应激使线粒体ATF2积累，扰乱HK1-VDAC1复合物，增加线粒体通透性，并促进细胞凋亡。ATF2抑制强烈降低Bim的构象活化，表明Bim在ATF2下游起作用。虽然Bim下调对ATF2活化没有影响，但Bim敲低消除了VDAC1活化；在Bim缺失细胞中VDAC1活化的失败可被仅含BH3结构域的蛋白质模拟物ABT-737逆转。我们还证明，在B16F10细胞中沉默ATF2会增加体内肿瘤异种移植的发生率和患病率，而稳定的线粒体ATF2转染抑制B16F10肿瘤异种移植的生长。
总之，这些结果表明ATF2是凋亡机制的一个组成部分，涉及ATF2、Bim和VDAC1的分级作用。我们的数据为线粒体ATF2在线粒体凋亡中的机制提供了新的见解。
