活性氧在GDC-0152诱导NB4细胞凋亡和自噬中的作用

Role of Reactive Oxygen Species in GDC-0152-Induced Apoptosis and Autophagy of NB4 cells

目的:探讨活性氧(Reactive oxygen species,ROS)在GDC-0152诱导急性早幼粒细胞白血病细胞系NB4细胞凋亡和自噬过程中的作用。方法:不同浓度GDC-0152联合Z-VAD-FMK作用于NB4细胞,采用CCK8法检测细胞增殖活力;流式细胞术检测细胞凋亡率、细胞自噬及ROS水平;Cyto-ID染色荧光显微镜观察细胞自噬及流式细胞术检测荧光表达;Western blot检测自噬相关蛋白LC3B的表达。结果:GDC-0152处理NB4细胞后细胞增殖抑制率和细胞凋亡率增加(P<0.05);GDC-0152可诱导NB4细胞ROS水平升高;Cyto-ID染色后应用荧光显微镜观察和流式细胞术检测发现,GDC-0152可以增加NB4细胞自噬(P<0.05);Western blot结果显示,GDC-0152可增加NB4细胞LC3B表达,且促进LC3BⅠ向LC3BⅡ转化;与GDC-0152(100 ng/ml)相比,GDC-0152(100 ng/ml)联合ROS抑制剂YCG063(10μmol/L)后细胞凋亡率降低且自噬减少(P<0.05)。结论:GDC-0152通过诱导NB4细胞凋亡及自噬抑制细胞增殖。ROS能促进GDC-0152诱导的NB4细胞凋亡和自噬。

Objective:To investigate the effect of reactive oxygen species(ROS)on GDC-0152-induced apoptosis and autophagy of acute promyelocytic leukemia cell line NB4.Methods:Different concentrations of GDC-0152 combined with Z-VAD-FMK was applied to NB4 cells.Cell proliferation was detected by CCK8 method.Apoptosis rate,autophagy and ROS level were detected by flow cytometry.The autophagy was observed by Cyto-ID staining fluorescence microscopy,and flow cytometry were used to detect the fluorescence expression.The expression of autophagy-related protein LC3 B was detected by Western blot.Results:GDC-0152 increased proliferation inhibition rate and apoptosis rate in NB4 cells(P<0.05);GDC-0152 induced increase of ROS level of NB4 cells;GDC-0152 increased autophagy of NB4 cells that was found by Cyto-ID staining fluorescence microscopy and flow cytometry(P<0.05).Western blot showed that GDC-0152 increased LC3 B expression in NB4 cells and promoted the conversion of LC3 BI to LC3 BII;as compared with GDC-0152(100 ng/ml),GDC-0152(100 ng/ml)combined with ROS inhibitor YCG063(10μmol/L)decreased apoptosis and autophagy(P<0.05).Conclusion:GDC-0152 inhibits cell proliferation by inducing apoptosis and autophagy of NB4 cells.ROS can promote GDC-0152-induced apoptosis and autophagy of NB4 cells.