AZT对肝癌细胞端粒酶活性及相关蛋白表达的影响

背景与目的：端粒酶抑制剂抑制端粒酶活性的机制十分复杂,可能涉及多个蛋白的共同作用。本研究通过3’-叠氮脱氧胸苷（3’-azido-deoxythymidine,AZT）作用于人肝癌细胞SMMC-7721。比较AZT作用后癌细胞端粒酶活性及相关蛋白的变化,探讨AZT抑制端粒酶活性的可能机制。方法：利用噻唑蓝[3（4,5-demethyhhiazole-2-y1）-2,5-diphenyl tetrazolium-bromide,MTT]实验确定AZT作用的最佳作用时间和浓度;实时荧光定量端粒重复序列扩增法（real-time fluorescent quantitative TRAP assay,FQ-TRAP法）检测AZT作用后,SMMC-7721细胞端粒酶活性变化;用缺口末端标记技术（terminal deoxyribonucleotidyl transferse（TdT）-mediated biotin-16-dUTP nick-end labelling,TUNEL）法和流式细胞术检测AZT作用后．SMMC-7721细胞凋亡情况;单细胞激光拉曼光谱技术和蛋白质芯片-飞行时间质谱仪,检测AZT作用后特异蛋白质的变化。结果：AZT抑制癌细胞生长的最佳作用时间为48h,最佳作用浓度为20mmol／L;FQ-TRAP法检测发现,AZT作用后肝癌细胞端粒酶活性与对照组相比受到明显抑制（P=0．0001）;TUNEL法观察到AZT诱导肝癌细胞凋亡形态学改变．流式细胞术检测AZT诱导肝癌细胞凋亡率为13．5％：单细胞激光拉曼光谱技术观察到,AZT作用后有7个与蛋白代谢相关的特征峰变化;蛋白质芯片．飞行时间质谱仪检测发现,AZT作用后有24个蛋白分子在癌细胞中高表达,8个蛋白分子在癌细胞中低表达,32个差异蛋白均属于小分子蛋白。结论：AZT可抑制SMMC-7721细胞端粒酶活性,诱导凋亡与相关特异小分子蛋白有关。

Effects of 3'-azido-deoxythymidine on telomerase activity and protein expression of hepatocarcinoma cell line SMMC-7721

BACKGROUND & OBJECTIVE: The mechanism of inhibiting telomerase activity by telomerase inhibitors is very complex, and involves common actions of many proteins. This study was to investigate the effects of 3'-azido-deoxythymidine (AZT) on telomerase activity and protein expression of hepatocarcinoma cell line SMMC-7721, and explore possible mechanism of inhibiting telomerase activity in SMMC-7721 cells by AZT. METHODS: Optimized concentration and treatment time of AZT were detected by MTT assay. After treatment with AZT, the telomerase activity in SMMC-7721 cells was detected by real-time fluorescent quantitative TRAP (FQ-TRAP) assay; the apoptosis of SMMC-7721 cells was detected by TUNEL assay and flow cytometry (FCM); the changes of specific proteins were monitored by raman spectra assay and surface-enhanced laser desorption time of flight-mass spectrum (SELDI-TOF-MS) with protein chip assay. RESULTS: The optimized treatment time of AZT was 48 h, and the optimized concentration of AZT was 20 mmol/L. When treated with 20 mmol/L AZT for 48 h, the telomerase activity in SMMC-7721 cells was inhibited by 53.85% of control (P<0.001); apoptotic morphologic changes of SMMC-7721 cells were observed; the apoptosis rate of SMMC-7721 cells was 13.5%; 7 spectral peak of proteins were changed; 24 proteins were up-regulated and 8 were down-regulated in SMMC-7721 cells. All of the 32 distinct proteins were small molecular proteins. CONCLUSION: AZT can inhibit the telomerase activity and induce apoptosis of SMMC-7721 cells, which is related to specific small molecular proteins.