IFN-γ联合阿霉素或依托泊苷增强TRAIL对神经母细胞瘤细胞的诱导凋亡作用

Combination of IFNγand Etoposide or Doxorubicin Enhances Tumor Necrotic Factor-related Aptoptosis-inducing Ligand-induced Apoptosis of Neuroblastoma Cells

目的:探讨半胱-天冬氨酸蛋白酶8(Caspase 8)和死亡受体(DR)在肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导神经母细胞瘤(NB)细胞凋亡中的作用。方法:应用RT-PCR方法检测γ干扰素(IFN-γ)作用前后NB细胞Caspase 8 mRNA的表达;应用Western blot方法检测Caspase 8、DR4和DR5蛋白表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞术检测IFN-γ,TRAIL,IFN-γ+TRAIL,Caspase 8抑制剂+TRAIL及IFN-γ+阿霉素/依托泊苷+TRAIL对NB细胞株生长及凋亡的影响。结果:IFN-γ在NB细胞株SKNDZ中诱导了Caspase 8mRNA及蛋白表达。SY5Y细胞对TRAIL不敏感,而IFN-γ与TRAIL或阿霉素,依托泊苷联用对SY5Y细胞有明显诱导凋亡作用。IFN-γ+TRAIL组SY5Y细胞早期凋亡率(23.09+2.35)%高于TRAIL组[(6.15±0.54)%(P<0.01)],但低于IFN-γ+阿霉素/依托泊苷+TRAIL组[(43.41±6.46)%/(38.86±7.29)%,P<0.01]。阿霉素或依托泊苷可以诱导NB细胞株DR5蛋白表达,但未诱导DR4蛋白表达。IFNγ诱导后表达Caspase 8的SKNDZ细胞对TRAIL的诱导凋亡作用仍不敏感,而阿霉素或依托泊苷处理后同时表达DR5的SKNDZ细胞对TRAIL的诱导凋亡作用相对敏感。IFN-γ+阿霉素/依托泊苷+TRAIL组SKNDZ细胞早期凋亡率(11.54±2.49)%/(13.38±1.65)%高于IFN-γ+TRAIL组(P<0.01)。结论:IFN-γ上调Caspase 8表达及化疗药阿霉素或依托泊苷诱导DR5表达可以恢复NB细胞对TRAIL的敏感性,Caspase 8和DR5在TRAIL诱导NB细胞凋亡中起着十分关键的作用。

Objective： This study aims to evaluate the role of Caspase 8 and death receptor （ DR ） in TNF-related apoptosis-inducing ligand （ TRAIL ）-induced apoptosis of neuroblastoma （ NB ） cell lines. Methods： Caspase 8 mRNA expression in NB cells before and after treatment with IFNγ was monitored through reverse transcriptase-polymerase chain reaction. Caspase 8, DR5, and DR4 protein expression was monitored through Western blot analysis. The effects of IFNγ, TRAIL, IFNγ ＋ TRAIL, Caspase 8 inhibitor ＋ TRAIL, and IFNγ ＋ etoposide/doxombicin ＋ TRAIL on the growth and apoptosis of NB cells were detected through MTT and flow cytometry. Results： The expression of Caspase 8 mRNA and protein was induced by IFN7 in the NB cell line SKNDZ. SY5Y cells were resistant to TRAIL. However, the combination treatment of IFN7 ＋ TRAIL and IFNy ＋ etoposide/doxorubicin ＋ TRAIL significantly induced cell apoptosis in SY5Y cells. The early stage apoptosis rate of SY5Y cells in the IFN7 ＋ TRAIL group [ （ 23.09 ± 2.35 ） % ] was significantly higher than that in the TRAIL group [ （ 6.15 ±0.54 ） % ] （ P 〈 0.01 ）, but was significantly lower than that in the IFN7 ＋ dox/eto ＋ TRAIL group [ （ 43.41 ± 6.46 ） ] %/ （ 38.86±7.29 ） % ] （ P 〈 0.01 ）. Moreover, etoposide or doxorubicin induced DR5 but not DR4 in NB cell lines. SKNDZ cells expressing Caspase 8 after treatment with IFNγ were still resistant to TRAIL but subsequently became sensitive to TRAIL after the induction of DR5 through the treatment of etoposide or doxombicin. The early stage apoptosis rate of the IFNγ ＋ dox/eto ＋ TRAIL group [ （ 11.54 ± 2.49 ） %/ （ 13.38± 1.65 ） % ] was significantly higher than that of the IFNy ＋ TRAIL group （ P 〈 0.01 ）. Conclusion： Sensitization of NB cells to TRAIL may be mediated by the upregulation of Caspase 8 with IFNγ and DR5 with etoposide or doxombicin. The results suggest that Caspase 8 and DR5 play key roles in TRAIL-induced apoptosis of NB cells.