荷载白细胞介素-24条件增殖腺病毒诱导黑色素瘤细胞凋亡的机制

The tumor-selective replicating adenovirus ZD55-interleukin-24 induces B cell lymphoma/leukemia-2 S-nitrosylation decline leading to melanoma cell apoptosis

目的 探讨荷载白细胞介素（IL）-24的条件增殖腺病毒ZD55-IL-24诱导黑色素瘤细胞凋亡的分子机制.方法 ZD55-IL-24感染人黑色素瘤A375细胞.Western blot和免疫沉淀技术检测细胞的IL-24、B淋巴细胞瘤/白血病-2（bcl-2）亚硝基化、bcl-2泛素化、bcl-2蛋白表达水平及半胱氨酰天冬氨酸特异性蛋白酶（Caspase）蛋白活性;加入不同浓度的一氧化氮（NO）调节剂改变bcl-2亚硝基化水平,检测其对bcl-2泛素化水平和Caspase蛋白活性及黑色素瘤细胞凋亡的影响.结果 ZD55-IL-24导致A375细胞bcl-2亚硝基化水平下调,泛素化水平上调,bcl-2蛋白水平下降,Caspase 信号通路激活,黑色素瘤细胞凋亡,在作用72 h后细胞凋亡达高峰,细胞凋亡率由对照组的11％上升到72％;NO供体SNP可促进bcl-2亚硝基化、抑制bcl-2泛素化水平从而上调bcl-2蛋白水平,细胞凋亡率由68％下降到19％;SNP拮抗剂DTT则加速ZD55-IL-24介导的肿瘤细胞凋亡,细胞凋亡率上升到93％;NO清除剂PTIO也同样促进黑色素瘤细胞的凋亡,细胞凋亡率上升到85％.结论 IL-24通过抑制bcl-2亚硝基化促进其泛素化降解,导致黑色素瘤细胞凋亡.

Objective To investigate tumor-selective replicating adenovirus ZD55-interleukin （IL）-24 induces B-cell lymphoma/leukemia-2 （bcl-2） S-nitrosylation decline and further facilitates bcl-2 ubiquitin degradation,leading to melanoma cell apoptosis.Methods ZD55-IL-24 was transfected into melanoma A375 cells,bcl-2 S-nitrosylation,bcl-2 ubiquitination,bcl-2 protein level and downstream Caspase protein were detected by Western blotting and Co-IP.Furthermore,nitric oxide （NO） regulator single nucleotide polymorphism （SNP）,PTIO and DTT were used to coffirm the relationship between bcl-2 S-nitrosylation and its ubiquitination.Results ZD55-IL-24 induced the bcl-2 protein level decline in time-dependent manner.Moreover,ZD55-IL-24 decreased bcl-2 S-nitrosylation and increased bcl-2 ubiquitination,which initiated the Caspase signal pathway activation and promoted melanoma cell apoptosis from 11% to 72% ; NO donor SNP promoted bcl-2 S-nitrosylation but restrained bcl-2 ubiquitination,which restored bcl-2 protein level.Moreover,melanoma cell apoptosis decreased from 68% to 19%.In contrast,SNP antagonist DTT reversed the above results by enhancing cell apoptosis to 93 %.NO inhibitor PTIO similarly increased melanoma cell apoptosis to 85%.Conclusion ZD55-IL-24 reduced bcl-2 S-nitrosylation and facilitated bcl-2 ubiquitin proteasome degradation,thereby resulting in melanoma cell apoptosis.