摘要
目的:探讨一氧化氮(NO)在肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导人甲状腺癌细胞凋亡中的作用。方法:S-亚硝基化生物素转化法、一氧化氮合酶(NOS)活性及NO产物测定法检测各组细胞3-磷酸甘油醛脱氢酶(GAPDH)S-亚硝基化与NO产物关系;蛋白质印迹法检测各组细胞及细胞核中GAPDH蛋白表达;台盼蓝染色、DNA裂解分析、Caspase-3活性测定法检测各组FRO细胞凋亡。结果:20ng/mLTRAIL处理FRO细胞0～24h,可见NOS活性及作为NO氧化产物的硝酸盐和亚硝酸盐水平呈时间依赖性增加,于4h开始显著增高,P=0.008;12h达高峰。iNOS抑制剂L-NAME抑制TRAIL诱导的GAPDHS-亚硝基化及其随后的细胞核转位,但不影响TRAIL诱导的GAPDH表达。L-NAME显著抑制TRAIL诱导的人甲状腺癌细胞凋亡和Caspase-3活性,P值均<0.001。结论:NO介导的GAPDHS-亚硝基化修饰和随后的细胞核转位可能参与了TRAIL诱导的人甲状腺癌细胞凋亡。
OBJECTIVE：To investigate the effect of NO on apoptosis of human thyroid cancer cells induced by tumor necrosis fac- tor-related apoptosis-inducing ligand（TRAIL）. METHODS： The S- nitrosylation biotin switch assay and measurement of NOS activity and NO production were used to investigate the relationship be- tween GAPDH S-nitrosylation and NO production in each group; Western blotting analysis was employed for GAPDH protein ex- pression in total cell lysates and nuclear fractions. Trypan blue stai- ning, nuclear DNA fragmentation, and Caspase-3 activity were measured to determine the apoptosis of human thyroid cancer cells. RESULTS： The analyses of FRO cells treated with 20 ng/mL TRAIL for 0--24 hours showed that their NOS activities and levels of nitrite/nitrate as NO-oxidation products were increased in a time- response dependent fashion, with a significant increase starting at 4 h treatment （P 0. 008） and a peak at 12 h. The iNOS inhibitor L-NAME suppressed TRAIL induced S-nitrosylation of GAPDH and its subsequent nuclear translocation, but did not influence TRAIL-induced GAPDH expression. On the other hand, L-NAME obviously inhibited cell apoptosis （P〈0. 001） and Caspase-3 activity （P〈0. 001） induced by TRAIL. CONCLUSION： NO-mediated .% nitrosylation of GAPDH and its subsequent nuclear translocation may be involved in TRAIL-induced human thyroid cancer ceil apoptosis. Chin J Cancer Prey Treat ,2008,15（22） ：1691-1694
出处
《中华肿瘤防治杂志》
CAS
2008年第22期1691-1694,共4页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(30740086)
沈阳市科委支持项目(1063316-1-00)
