高三尖杉酯碱对白血病细胞凋亡、增殖的影响及对Ras/Raf/MEK通路的抑制作用

Effect of homoharringtonine on lapoptosis and proliferation of leukemia cells and its inhibitory effect on Ras/Raf/MEK pathway

目的探讨高三尖杉酯碱(HHT)对白血病细胞凋亡、增殖的影响及对Ras/Raf/MEK通路的抑制作用。方法取对数生长期K562、Kasum-1细胞进行研究,分别用0、10、20、50 ng/ml的HHT处理。采用MTT检测相对细胞活力,计算24、48、72 h时细胞IC50,采用克隆形成实验检测检测细胞克隆数量,采用流式细胞仪检测细胞凋亡水平,采用蛋白质印迹法检测Ras/Raf/MEK通路蛋白和凋亡相关蛋白相对水平。结果24、48、72 h时HHT对K562细胞IC50分别为80.69、65.28、50.76 ng/ml,HHT对Kasum-1细胞IC50分别为85.16、70.11、51.14 ng/ml。培养24、48、72 h时,10、20、50 ng/ml HHT细胞存活率均低于0 ng/ml,20、50 ng/ml HHT细胞存活率均低于10 ng/ml,50 ng/ml HHT细胞存活率均低于20 ng/ml HHT,差异有统计学意义(P<0.05)。10、20、50 ng/ml HHT细胞克隆数均低于0 ng/ml,20、50 ng/ml HHT细胞克隆数均低于10 ng/ml HHT,50 ng/ml HHT细胞克隆数均低于20 ng/ml HHT,差异有统计学意义(P<0.05)。10、20、50 ng/ml HHT细胞凋亡率均高于0 ng/ml HHT,20、50 ng/ml HHT细胞凋亡率均高于10 ng/ml HHT,50 ng/ml HHT细胞凋亡率均高于20 ng/ml HHT,差异有统计学意义(P<0.05)。10、20、50 ng/ml HHT RAS、p-cRAF、p-MEK蛋白表达量均低于0 ng/ml HHT,20、50 ng/ml HHT RAS、p-cRAF、p-MEK蛋白表达量均低于10 ng/ml HHT,50 ng/ml HHT RAS、p-cRAF、p-MEK蛋白表达量均低于20 ng/ml HHT,差异有统计学意义(P<0.05)。10、20、50 ng/ml HHT Survivan蛋白表达量均低于0 ng/ml HHT,Cleaved caspase-3、Cleaved PARP蛋白表达量均高于0 ng/ml HHT;20、50 ng/ml HHT Survivan蛋白表达量均低于10 ng/ml HHT,Cleaved caspase-3、Cleaved PARP蛋白表达量均高于10 ng/ml HHT、50 ng/ml HHT Survivan蛋白表达量均低于20 ng/ml HHT,Cleaved caspase-3、Cleaved PARP蛋白表达量均高于20 ng/ml HHT,差异有统计学意义(P<0.05)。结论HHT可抑制Ras/Raf/MEK通路活性,抑制白血病细胞增殖和促进白血病细胞凋亡。

Objective To investigate the effect of homoharringtonine(HHT)on apoptosis and proliferation of leukemia cells and its inhibitory effect on Ras/Raf/MEK pathway.Methods The logarithmic growth phase K562 and Kasum-1 cells were taken for study and treated with HHT of 0,10,20,and 50 ng/ml,respectively.MTT was used to detect relative cell viability,clone formation assay was used to detect the number of cell clones,cell IC50 was calculated at 24,48,and 72 hours,flow cytometry was used to detect the level of apoptosis,and Western blot was used to detect the relative levels of Ras/Raf/MEK pathway proteins and apoptosis-related proteins.Results At 24,48,and 72 hours,the IC50 of HHT on K562 cells was 80.69,65.28,and 50.76 ng/ml,respectively.The IC50 of HHT on Kasum-1 cells was 85.16,70.11,and 51.14 ng/ml,respectively.When cultured for 24,48,and 72 h,the survival rate of 10,20 and 50 ng/ml HHT cells was lower than 0 ng/ml,and the survival rate of 20 and 50 ng/ml HHT cells was lower than 10 ng/ml.The survival rate of 50 ng/ml HHT cells was lower than that of 20 ng/ml HHT cells,and the differences were statistically significant(P<0.05).The clones of 10,20 and 50 ng/ml HHT cells were all lower than 0 ng/ml;the clones of 20 and 50 ng/ml HHT cells were all lower than 10 ng/ml HHT cells;the clones of 50 ng/ml HHT cells were all lower than 20 ng/ml HHT cells,and the differences were statistically significant(P<0.05).The apoptosis rate of 10,20,and 50 ng/ml HHT cells was higher than that of 0 ng/ml HHT cells,the apoptosis rate of 20 and 50 ng/ml HHT cells was higher than that of 10 ng/ml HHT cells,and the apoptosis rate of 50 ng/ml HHT cells was higher than that of 20 ng/ml HHT cells,and the differences were statistically significant(P<0.05).The expression levels of RAS,p-cRAF,and p-MEK protein at 10,20,and 50 ng/ml HHT were all lower than 0 ng/ml HHT,and the expression levels of RAS,p-cRAF,and p-MEK protein at 20 and 50 ng/ml HHT were all lower than 10 ng/ml HHT.The expression levels of RAS,p-cRAF,and P-MEK at 50 ng/ml HHT were lower than those of 20 ng/ml HHT,and the differences were statistically significant(P<0.05).Survivan expression levels at 10,20,and 50 ng/ml HHT were lower than 0 ng/ml HHT,Cleaved caspase-3 and Cleaved PARP were higher than 0 ng/ml HHT.Survivan expression at 20 and 50 ng/ml HHT was lower than that at 10 ng/ml HHT.Survivan expression levels were lower than 10,and 50 ng/ml HHT,respectively,Cleaved caspase-3 and Cleaved PARP expression levels were higher than 20 ng/ml HHT.The protein expression levels of Cleaved caspase-3 and Cleaved PARP were higher than 20 ng/ml HHT,and the differences were statistically significant(P<0.05).Conclusion HHT can inhibit the activity of Ras/Raf/MEK pathway,inhibit the proliferation of leukemia cells,and promote apoptosis of leukemia cells.