钠钾ATP酶抑制剂通过调节DNA损伤感应复合体Mre11/Rad50/Nbs1的表达诱导肝癌HepG2细胞周期阻滞  

Na^+,K^+-ATPase inhibitor induces cell cycle arrest in liver cancer HepG2 cells by regulating expression of DNA damage Mre11/Rad50/Nbs1 complex

作　　者：徐忠伟[1] 王凤梅[2] 王聪聪[1] 单娜娜[1] 徐瑞成[1,3] 

机构地区：[1]中国人民武装警察部队后勤学院,天津300309 [2]天津市第三中心医院,天津300170 [3]天津市职业与环境危害生物标志物重点实验室,天津300309

出　　处：《中国药理学通报》2016年第3期323-327,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81273552);武警后勤学院博士启动金项目(No WHB201501)

摘　　要：目的研究钠钾ATP酶抑制剂华蟾毒配基(cinobufagin)对人肝癌HepG2细胞DNA损伤修复阻断及其发生机制。方法免疫组化分析人肝癌临床组织标本、正常肝组织中钠钾ATP酶α1亚单位的表达;以人肝癌细胞HepG2为靶细胞,实验分为对照组、5μmol·L^(-1)华蟾毒配基作用6、12和24 h组;单细胞电泳检测DNA双链断裂,Real time-PCR和Western blot检测损伤修复基因Mre11、Rad50、Nbs1和p53的表达变化,流式细胞术检测细胞周期。结果肝癌组织钠钾ATP酶α1亚单位表达与癌旁组织相比明显升高(P<0.05);华蟾毒配基诱导HepG2细胞DNA断裂的发生率与作用时间高度相关,作用时间延长断裂效应更加明显(P<0.05),Mre11,Nbs1,Rad50和p53表达随药物作用时间延长逐渐升高(P<0.05),流式细胞术分析细胞周期对照组S期细胞比例为(21.32±4.21)%,5μmol·L^(-1)华蟾毒配基处理6 h后为(33.25±5.72)%,处理12 h后为(56.72±6.29)%,作用24 h后为(67.32±9.42)%。结论华蟾毒配基激活Mre11/Rad50/Nbs1损伤感应复合体介导肝癌Hep G2细胞周期阻滞。Aim To explore the relationship between Mre11/Rad50/Nbs1(MRN) complex focus formation and DNA double-strand breaks( DSBs) caused by cinobufagin in human hepatocellular carcinoma HepG2 cells.Methods The Na^+,K^+-ATPase α1 subunit expression level in liver cancer tissues was detected by immunohistochemistry.After HepG2 cells were treated with 5μmol·L^(-1) cinobufagin for 6,12 and 24 h,the drug-induced DSBs were assessed by single cell gel electrophroesis(SCGE),the gene transcription and protein levels of Mrel1,Nbs1,Rad50 and p53 were evaluated by Real time-PCR and Western blot.The cell cycle in parallel was analyzed by flow cytometry.Results The Na^+,K^+-ATPase α1 subunit expression level in liver cancer tissues was significantly increased compared with the tissue adjacent to carcinoma(P<0.05).The 5μmol·L^(-1) cinobufagin could induce the DSBs in a time-dependent manner(P<0.05),and it could upregulate the gene expression levels of Mre11,Nbs1,Rad50 and p53 in HepG2 cells(P<0.05).The proportions of HepG2 cells in S phase were(21.32 ±4.21) % in the control group,and(33.25±5.72)%,(56.72 ±6.29)% and(67.32±9.42)% in HepG2 cells treated with 5 μmol·L^(-1) cinobufagin for 6,12 and 24 h,respectively.The proportions of cells in S phase in cinobufagin groups were significantly increased compared with the control group(P<0.05).Conclusion Cinobufagin could induce the cell cycle arrest in liver cancer HepG2 cells by activation of Mre11/Rad50 /Nbs1 Complex.

关 键 词：华蟾毒配基 HEPG2细胞 DNA双链断裂 钠钾ATP酶 细胞周期 细胞周期相关蛋白 

分 类 号：R284.1[医药卫生—中药学] R282.74[医药卫生—中医学]