Hu Songliu, Fu Songbin, Xu Xiangying, Chen Lin, Xu Jianyu, Li Bin, Qu Yuanyuan, Yu Hongyang, Lu Shan, Li Wenxin
Cell Physiol Biochem. 2015;37(3):1219-30. doi: 10.1159/000430245. Epub 2015 Sep 30.
BACKGROUND/AIMS: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We investigated the effects of YM155, a small molecule inhibitor of survivin expression, on the radiosensitivity of human non-small cell lung cancer (NSCLC) cell lines and elucidated a relationship between the cellular localization of survivin and DNA double-strand break repair.
The cellular distribution of survivin was determined by Western blotting of subcellular fractions and by immunofluorescent staining in A549 NSCLC cells. Radiation-induced DNA damage was evaluated based on histone H2AX phosphorylation and foci formation. The relationship between the cellular localization of survivin and DNA double-strand break repair was analyzed by Western blotting and co-immunoprecipitations.
YM155 down-regulated survivin expression in NSCLC cells in a concentration- and time-dependent manner. An in vitro clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to radiation. After irradiation, we observed a rapid accumulation of survivin in the nucleus. An immunofluorescent analysis of histone x03B3;-H2AX demonstrated that the inhibition of survivin expression by YM155 resulted in impaired DNA double-strand break repair. Co-immunoprecipitation assays using nuclear extracts revealed an interaction between survivin, Ku70, x03B3;-H2AX, and DNA-PKcs. Furthermore, S2056 autophosphorylation of DNA-PKcs was reduced in survivin-depleted cells.
These results suggested that YM155 sensitized NSCLC cells to radiation, at least in part by inhibiting DNA repair and enhancing apoptosis via the down-regulation of survivin expression. YM155 pretreatment inhibited DNA-PKcs autophosphorylation at S2056. Nuclear survivin was involved in DNA double-strand break repair via interactions with members of the DNA double-strand break repair machinery.
背景/目的：生存素是凋亡抑制蛋白家族的成员，是癌症治疗中一个有吸引力的靶点。我们研究了生存素表达的小分子抑制剂YM155对人非小细胞肺癌（NSCLC）细胞系放射敏感性的影响，并阐明了生存素的细胞定位与DNA双链断裂修复之间的关系。
通过亚细胞组分的蛋白质印迹法和A549 NSCLC细胞中的免疫荧光染色来确定生存素的细胞分布。基于组蛋白H2AX磷酸化和焦点形成评估辐射诱导的DNA损伤。通过蛋白质印迹法和共免疫沉淀分析生存素的细胞定位与DNA双链断裂修复之间的关系。
YM155以浓度和时间依赖性方式下调NSCLC细胞中生存素的表达。体外克隆形成存活试验表明，YM155增加了NSCLC细胞对辐射的敏感性。照射后，我们观察到生存素在细胞核中快速积累。组蛋白γ-H2AX的免疫荧光分析表明，YM155对生存素表达的抑制导致DNA双链断裂修复受损。使用核提取物的共免疫沉淀试验揭示了生存素、Ku70、γ-H2AX和DNA-PKcs之间的相互作用。此外，在生存素缺失的细胞中，DNA-PKcs的S2056自磷酸化降低。
这些结果表明，YM155使NSCLC细胞对辐射敏感，至少部分是通过抑制DNA修复并通过下调生存素表达增强细胞凋亡。YM155预处理抑制了DNA-PKcs在S2056处的自磷酸化。核生存素通过与DNA双链断裂修复机制的成员相互作用参与DNA双链断裂修复。
