维奈克拉增强MDS细胞系对地西他滨化疗敏感性机制的实验研究

Experimental Study on the Mechanism of Venetoclax Enhancing the Sensitivity of MDS Cell Lines to Decitabine Chemotherapy

目的研究维奈克拉(venetoclax,VCX)对骨髓增生异常综合征(myelodysplastic syndromes,MDS)细胞系地西他滨(decitabine,DAC)化疗敏感性的可能作用机制。方法CCK-8法检测不同浓度VCX对MDS细胞(SKM-1和MUTZ-1细胞系)增殖活力的影响;将MUTZ-1细胞根据不同处理分为4组:对照组、VCX组、DAC组和VCX+DAC组;Annexin V-FITC/PI法检测各组细胞凋亡率;Western blotting检测细胞中凋亡相关蛋白[细胞色素C(cytochrome C),裂解型半胱天冬酶3(cleaved Caspase-3)表达水平],B细胞白血病/淋巴瘤2(B cell leukemia/lymphoma-2,Bcl-2)与Bcl-2相关蛋白X(Bax)比值;JC-1线粒体膜电位检测试剂盒检测各组SKM-1和MUTZ-1细胞的线粒体膜电位;H2DCF-DA荧光探针法检测细胞中活性氧(reactive oxygen species,ROS)含量;Western blotting检测细胞中自噬相关蛋白Beclin1,P62和LC3-Ⅱ/LC3-Ⅰ比值。结果随VCX浓度升高,MUTZ-1细胞增殖活性明显降低,且呈浓度依赖性(F=0.003,P=0.001)。与对照组(4.28%±1.66%)相比,VCX组(13.75%±3.02%),DAC组(12.39%±4.16%)和VCX+DAC组(18.10%±3.50%)细胞凋亡率明显增高,差异有统计学意义(F=45.782,P<0.05)。与对照组(1.01±0.02,1.04±0.02,1.01±0.04)相比,VCX组(1.67±0.05,2.23±0.10,0.43±0.05),DAC组(1.62±0.08,1.85±0.06,0.49±0.07)和VCX+DAC组(3.24±0.10,3.81±0.19,0.13±0.01)细胞中凋亡相关蛋白cytochrome C,cleaved Caspase-3蛋白表达及Bcl-2/Bax比值明显升高,差异均有统计学意义(F=116.384,282.069,248.035,均P<0.05)。与对照组(2.05±0.34,8.78±1.37)相比,VCX组(8.72±1.26,14.02±1.45),DAC组(8.44±2.13,13.20±2.41)和VCX+DAC组(15.66±2.90,26.45±1.53)细胞线粒体膜电位及ROS含量明显升高,差异具有统计学意义(F=66.782,69.071,均P<0.05)。与对照组(1.05±0.04,1.02±0.08,1.01±0.07)相比,VCX组(1.62±0.15,2.60±0.19,0.56±0.15),DAC组(1.67±0.17,2.45±0.20,0.54±0.14)和VCX+DAC组(3.72±0.21,3.58±0.27,0.13±0.09)自噬相关蛋白Beclin1,LC3-Ⅱ/LC3-Ⅰ表达明显升高,而P62蛋白表达则明显降低,差异均有统计学意义(F=118.257,209.422,236.92,均P<0.05)。与DAC组比较,VCX+DAC组细胞凋亡率、cytochrome C,cleaved Caspase-3,Beclin1蛋白表达水平,LC3-Ⅱ/LC3-Ⅰ比值和ROS含量均明显降低(t=2.473,28.564,17.291,16.115,7.021,9.319);线粒体膜电位、Bcl-2/Bax比值和P62蛋白表达均明显升高(t=4.621,9.244,4.278),差异具有统计学意义(均P<0.05)。DAC组和VCX组细胞中上述检测指标差异均无统计学意义(均P>0.05)。结论VCX可能通过调节细胞凋亡、自噬和氧化应激来促进MDS细胞对DAC的化疗敏感性。

Objective To investigate the possible mechanism of action of venetoclax(VCX)susceptibility to chemotherapy in the myelodysplastic syndrome(MDS)cell line decitabine(DAC).Methods The CCK-8 method detected the effect of different concentrations of VCX on the proliferation and viability of MDS cells(SKM-1 and MUTZ-1 cell lines).MUTZ-1 cells were divided into 4 groups according to different treatments:control group,VCX group,DAC group and VCX+DAC group.The apoptosis rates of MUTZ-1 cells in each group were detected by Annexin V-FITC/PI method.Western blotting measured the ratio of apoptosis-associated protein(cytochrome C,cleaved Caspase-3)in cells,B-cell leukemia/lymphoma-2,Bcl-2,to Bcl-2-related protein X(Bax).Detection of mitochondrial membrane potential of SKM-1 and MUTZ-1 cells by JC-1 method.H2DCF-DA fluorescent probe method to detect the content of reactive oxygen species(ROS)in cells.Western blotting measured the ratio of autophagy-related proteins Beclin1,P62 and LC3-II./LC3-I.in cells.Results With the increase of VCX concentration,the proliferative activity of MUTZ-1 cells decreased significantly,and it was concentration-dependent.Compared with the control group(4.28%±1.66%),the apoptosis rate of VCX group(13.75%±3.02%),DAC group(12.39%±4.16%)and VCX+DAC group(18.10%±3.50%)was significantly higher,and the difference was statistically significant(F=45.782,P<0.05).Compared with the control group(1.01±0.02,1.04±0.02,1.01±0.04),the VCX group(1.67±0.05,2.23±0.10,0.43±0.05),the DAC group(1.62±0.08,1.85±0.06,0.49±0.07).In the VCX+DAC group(3.24±0.10,3.81±0.19,0.13±0.01),apoptosisrelated protein cytochrome C,cleaved Caspase-3 expression and Bcl-2/Bax ratio were increased significantly,the differences were statistically significant(F=116.384,282.069,248.035,all P<0.05).Compared with control group(2.05±0.34,8.78±1.37),VCX group(8.72±1.26,14.02±1.45),DAC group(8.44±2.13,13.20±2.41),VCX+DAC group(15.66±2.90,26.45±1.53)mitochondrial membrane potential and ROS contents were significantly increased,and the differences were statistically significant(F=66.782,69.071,all P<0.05).Compared with control group(1.05±0.04,1.02±0.08,1.01±0.07),VCX group(1.62±0.15,2.60±0.19,0.56±0.15),DAC group(1.67±0.17,2.45±0.20,0.54±0.14),in VCX+DAC group(3.72±0.21,3.58±0.27,0.13±0.09),the expression of autophagy related protein Beclin1,LC3-Ⅱ/LC3-Ⅰwas significantly increased,while the expression of P62 protein was significantly decreased,with statistical significance(F=118.257,209.422,236.92,all P<0.05).Compared with DAC group,apoptosis rate,cytochrome C,cleaved Caspase-3,Beclin1 protein expression level,LC3-Ⅱ/LC3-Ⅰratio and ROS content in VCX+DAC group were significantly decreased(t=2.473,28.564,17.291,16.115,7.021,9.319).Mitochondrial membrane potential,Bcl-2/Bax ratio and P62 protein expression were significantly increased(t=4.621,9.244,4.278),the differences were statistically significant,respectively(all P<0.05).There was no statistical significance in the above indexes between DAC group and VCX group(all P>0.05).Conclusion VCX may promote chemotherapy sensitivity of MDS cells to DAC by regulating apoptosis,autophagy,and oxidative stress.