Dong Xiaorong, Tong Fan, Qian Cai, Zhang Ruiguang, Dong Jihua, Wu Gang, Hu Yu
a Cancer Center, Union Hospital, Tongji Medical College, HuaZhong University of Science and Technology, Wuhan, 430022, China.
Radiat Res. 2015 Jan;183(1):82-93. doi: 10.1667/RR13682.1. Epub 2014 Dec 23.
Recently several laboratories have reported that radiation induces senescence in endothelial cells. Senescent cells can secrete multiple growth-regulatory proteins, some of which affect tumor growth, survival, invasion or angiogenesis. The purpose of this study was to explore the mechanisms of radiation-induced senescence and its effects on angiogenesis in human umbilical vein endothelial cells (HUVECs). HUVECs were either pretreated with or without PS1145 prior to irradiation with 0-8 Gy. PS1145 is a novel, highly specific small-molecule inhibitor of nuclear factor kappa B essential modulator (NEMO). MTT assays showed that in HUVECs untreated with PS1145, there was an increase in the number of radiation-induced senescence-like endothelial cells 5 days after 8 Gy irradiation, while pretreatment with PS1145 significantly ameliorated the induction in senescence of HUVECs compared to the control group. Electrophoretic mobility shift assay (EMSA) showed that pretreatment with PS1145 inhibited the radiation-induced NF-κB activation, which regulates cell fate in response to genotoxic stress. In addition, Western blotting demonstrated less translocation of p65 from cytoplasm to nucleus. Furthermore, real-time polymerase chain reaction (PCR) showed that pretreatment with PS1145 inhibited the increase of mRNA expressions of interleukin-6 (IL-6) and p53-induced death domain (PIDD) protein, which have been show to play crucial roles in both senescence and apoptosis (P < 0.05). TUNEL staining revealed an increase in apoptotic HUVECs in the group pretreated with PS1145 after irradiation. The series of functional assays further showed that radiation-induced senescence-like HUVECs had malfunctions in migration, invasion and formation of capillary-like structures, compared with the sham-irradiated and untreated, irradiated groups. Taken together, these findings indicate that the angiogenic capacity of radiation-induced senescence-like HUVECs decreased, and that irradiation caused vascular endothelial cells to gain a senescence-like phenotype through the DSB/NEMO/NF-κB signal pathway. The data suggests that NEMO may be a critical switch that regulates cellular senescence and apoptosis caused by exposure to radiation, and provides new clues for the clinical potential of the combination of radiotherapy and angiogenesis inhibitors.
最近，几个实验室报告称辐射可诱导内皮细胞衰老。衰老细胞能分泌多种生长调节蛋白，其中一些会影响肿瘤生长、存活、侵袭或血管生成。本研究的目的是探讨辐射诱导人脐静脉内皮细胞（HUVECs）衰老的机制及其对血管生成的影响。在用0 - 8 Gy辐射之前，HUVECs分别用或不用PS1145进行预处理。PS1145是一种新型、高度特异性的核因子κB必需调节因子（NEMO）小分子抑制剂。MTT分析表明，在未用PS1145处理的HUVECs中，8 Gy辐射5天后辐射诱导的衰老样内皮细胞数量增加，而与对照组相比，用PS1145预处理可显著改善HUVECs的衰老诱导情况。电泳迁移率变动分析（EMSA）表明，用PS1145预处理可抑制辐射诱导的NF-κB激活，NF-κB可调节细胞对基因毒性应激的命运。此外，蛋白质免疫印迹法显示p65从细胞质向细胞核的转位减少。此外，实时聚合酶链反应（PCR）表明，用PS1145预处理可抑制白细胞介素-6（IL-6）和p53诱导的死亡结构域（PIDD）蛋白mRNA表达的增加，这两种蛋白在衰老和凋亡中均起关键作用（P < 0.05）。TUNEL染色显示，辐射后用PS1145预处理的组中凋亡的HUVECs增加。一系列功能分析进一步表明，与假照射和未照射组相比，辐射诱导的衰老样HUVECs在迁移、侵袭和形成毛细血管样结构方面存在功能障碍。综上所述，这些发现表明，辐射诱导的衰老样HUVECs的血管生成能力下降，并且辐射通过DSB/NEMO/NF-κB信号通路使血管内皮细胞获得衰老样表型。数据表明，NEMO可能是调节辐射暴露引起的细胞衰老和凋亡的关键开关，并为放疗与血管生成抑制剂联合应用的临床潜力提供了新线索。
