5氮杂胞苷诱导NB细胞Caspase8表达及对TRAIL敏感性的研究

Induction of Caspase 8 by 5-Azacytidine renders NB cells sensitive to TRAIL

目的:探讨5氮杂胞苷(5Azacytidine)对肿瘤坏死因子相关凋亡诱导配体(tumornecrosisfactorrelatedapoptosisinducingligand,TRAIL)诱导神经母细胞瘤(neuroblastoma,NB)细胞株SHSY5Y凋亡的影响及其发生机制。方法:应用RTPCR方法检测5氮杂胞苷作用前后SY5Y细胞Caspase8的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测TRAIL、5氮杂胞苷+TRAIL、5氮杂胞苷+Caspase8抑制剂+TRAIL对SY5Y细胞生长及凋亡的影响。结果:SY5Y细胞不表达Caspase8,5氮杂胞苷作用1、3和5d的SY5Y细胞Caspase8表达逐渐增加;SY5Y细胞对TRAIL不敏感,经5氮杂胞苷诱导表达Caspase8的SY5Y细胞对TRAIL敏感,并呈现一定的时间和剂量依赖性。联合用药组细胞凋亡率分别为(28.6±4.0)%、(38.5±4.2)%和(42.3±6.2)%,与单独用TRAIL组[(9.5±2.0)%、(10.7±1.8)%和(11.2±1.5)%]比较,差异有统计学意义,F值分别为41.131、45.014和49.405,P值均为0.000;Caspase8抑制剂组细胞凋亡率分别为(11.8±2.0)%、(13.5±2.9)%和(15.1±3.4)%,与相应浓度的5氮杂胞苷+TRAIL组相比,细胞凋亡率明显降低,F值分别为41.131、45.014和49.405,P值均为0.000,说明Caspase8抑制剂可明显降低TRAIL对表达Caspase8的SY5Y细胞的杀伤作用。结论:5氮杂胞苷能增强SY5Y细胞对TRAIL的敏感性,其发生机制可能与Caspase8表达上调有关。

OBJECTIVE：To study the effect of demethylation agent 5-Azacytidine on TRAIL-induced apoptosis in neuroblastoma cells and its possible molecular mechanisms. METHODS： The expression of Caspase 8 mRNA was detected by RT-PCR after treatment of 5-Azacytidine for 1,3 and 5 days. The effects of TRAIl., 5-Azacytidine＋TRAiL, 5-Azacytidine＋Caspase 8 inhibitor＋TRAIL on the growth and apoptosis of SH-SY5Y cells were detected by the methods of MTT and FCM. RESULTS： Expression of Caspase 8 was not detected in SY5Y cells but an increased expression of Caspase 8 was found after treatment with 5-Azacytidine. SY5Y cells were not sensitive to TRAIL, but SY5Y cells treated by 5-Azacytidine were sensitive to TRAIL and showed time and dose dependency of TRAIL-induced apoptosis. The apoptosis rates of the TRAIL ＋ 5Azacytidine group [ （ 28.6 ± 4. 0 ） %, （ 38. 5 ± 4.2） %, （42.3 ± 6. 2） % ] were significantly higher than those of the TRAIl. alone group [ （ 9.5 ± 2.0 ） %, （ 10. 7 ± 1.8 ）%, （11.2± 1.5）%], F= 41. 131,45. 014, 49. 405, P = 0. 000. The apoptosis rates of the Caspase 8 inhibitor group [（11.8± 2.0） %, （ 13. 5 ± 2. 9 ） %, （ 15. 1± 3.4 ） % ] were significantly lower than those of the 5-Azacytidine ＋ TRAIL group, F= 41. 131,45.014, 49.405, P= 0.000. The killing effect of TRAIL on Caspase 8-expressing SY5Y cells was diminished by Caspase 8 inhibitor. CONCLUSION： 5-Azacytidine can increase TRAIL antitumor activity on SY5Y cells and its mechanisms may be involved in the up-regulation of Caspase 8 gene.