5氮杂胞苷联合干扰素-γ上调神经母细胞瘤细胞Caspase 8表达的研究

5-Azacytidine and IFN-gamma cooperate to upregulate Caspase-8 expression in neuroblastoma cells

目的:应用去甲基药5氮杂胞苷(5AZA)和/或干扰素(IFNγ)诱导神经母细胞瘤(neuroblastoma,NB)细胞Caspase 8的表达,并观察是否可以恢复NB细胞对肿瘤坏死因子相关凋亡诱导配体(TRAIL)的敏感性。方法:应用Western blot方法检测5AZA和/或IFNγ作用前后SY5Y细胞Caspase 8蛋白的表达;应用流式细胞术检测5AZA、IFNγ、TRAIL、5AZA和/或IFNγ+TRAIL、5AZA和/或IFNγ+Caspase 8抑制剂zIETD-FMK+TRAIL对NB细胞生长及凋亡的影响;应用比色法测定Caspase 8相对活性。结果:对TRAIL耐药的SY5Y细胞不表达Caspase 8,经5AZA和/或IFNγ处理后Caspase 8蛋白表达水平逐步增加;IFNγ与5AZA联合作用后其Caspase 8表达较单一用药明显增加。IFNγ和/或5AZA与TRAIL联用对SY5Y细胞有明显诱导凋亡作用。5AZA和/或IFNγ+TRAIL组的SY5Y细胞Caspase 8相对活性明显高于对照组、IFNγ组、5AZA组、TRAIL组及抑制剂组。结论:5AZA联合IFNγ可以明显上调NB细胞Caspase 8表达;表达Caspase 8的NB细胞对TRAIL的诱导凋亡作用敏感;TRAIL诱导NB细胞凋亡过程中伴随Caspase 8活性的增加。

Objective：To investigate if induction of Caspase 8 by demethylation agent 5-Azacytidine（5AZA）and/or γ-interferon（IFNγ） renders neuroblastoma（NB） cells sensitive to tumor necrosis factor related apoptosis inducing ligand（TRAIL）.Methods：Caspase 8 expression was monitored by Western blot analysis.The effects of 5AZA,IFNγ,TRAIL,5AZA and/or IFNγ ＋TRAIL,5AZA and/or IFNγ ＋ Caspase 8 inhibitor＋ TRAIL on the growth and apoptosis of NB cells were detected with flow cytometry.The relative Caspase 8 activity was measured with colorimetric assay.Results：Caspase 8 was undetectable in SH-SY5Y（SY5Y） cells which were resistant to TRAIL,but an increased expression of Caspase 8 was found after treatment with 5AZA and/or IFNγ.The level of Caspase 8 protein in IFNγ＋5AZA group was higher than that in single treatment group.Moreover,TRAIL combined with 5AZA and/or IFNγ induced apoptosis in SY5Y cells.The relative Caspase 8 activity of SY5Y cells in 5AZA and/or IFNγ＋TRAIL group was significantly higher than those of control group,5AZA group,IFNγ group,TRAIL group and Caspase 8 inhibitor group.Conclusion：5-AZA and IFNγ could cooperate to upregulate Caspase-8 expression in NB cells.TRAIL induced apoptosis in NB cells expressing Caspase 8 with an increase of relative Caspase 8 activity.