白藜芦醇通过下调SIX1抑制人白血病HL-60细胞增殖和诱导细胞凋亡

Proliferation inhibition and apoptosis induction in human leukemia HL-60 cells by resveratrol via down-regulating SIX1

目的:探讨白藜芦醇(resveratrol,RSV)对人白血病HL-60细胞增殖的影响及其机制。方法:CCK-8法检测RSV对HL-60细胞增殖的影响。碘化丙啶(propidium iodide,PI)单染流式细胞术检测RSV对HL-60细胞周期分布的影响;Annexin V-FITC/PI双染法观察RSV对HL-60细胞凋亡的影响。Western blot检测HL-60细胞中的细胞周期素D1(CyclinD1)、凋亡相关蛋白p53和p21、SIX1、酪氨酸蛋白激酶Ⅱα(CK2α)蛋白的表达水平,以及过表达SIX1后给予RSV处理对HL-60细胞中CyclinD1、p53、p21表达水平的影响。结果:(1)CCK-8实验结果显示,不同浓度的RSV(12.5μmol/L、25.0μmol/L、50.0μmol/L、100.0μmol/L)处理不同时间(24 h、48 h、72 h),HL-60细胞的存活率随着RSV作用浓度的增加和作用时间的延长逐渐降低(P<0.01)。(2)流式细胞术检测结果显示,与对照组相比,RSV处理后HL-60细胞凋亡增加(P<0.05),亚G;期细胞增多(P<0.05),G;/M期细胞减少(P<0.05)。(3)Western blot结果表明,与对照组相比,RSV作用HL-60细胞后,细胞周期蛋白CyclinD1水平降低(P<0.05),凋亡相关的p53、p21蛋白水平升高(P<0.05)。(4)与对照组相比,RSV处理后的HL-60细胞中SIX1和CK2α蛋白的表达水平降低(P<0.01),且过表达SIX1后抵消了RSV对HL-60细胞中CyclinD1、p53、p21的作用。结论:RSV可能通过下调SIX1抑制HL-60细胞增殖、诱导其凋亡。

Objective:To explore the effect of Resveratrol (RSV) on the proliferation of human leukemia HL-60 cell and its mechanism.Methods:The effect of RSV on the proliferation of HL-60 cells was detected by CCK-8 method.Flow cytometry with propidium iodide(PI)staining was used to detect the effect of RSV on HL-60 cell cycle distribution.Annexin V-FITC/PI staining was used to observe the effect of RSV on apoptosis of HL-60 cells.Western blot was used to detect the expression levels of CyclinD1,apoptosis-related protein p53 and p21,SIX1 and tyrosine protein kinaseⅡα(CK2α)in HL-60 cells,and the effect of RSV treatment on the expression levels of CyclinD1,p53 and P21 after overexpression of SIX1 in HL-60 cells.Results:(1)In the CCK-8 tests,HL-60 cells were treated with RSV of different concentrations(12.5μmol/L,25.0μmol/L,50.0μmol/L,100.0μmol/L)and different times(24 h,48 h,72 h),the results showed that the survival rate of HL-60 cells decreased with the increasing of RSV concentration and the prolongation of RSV treatment time(P<0.01).(2)Flow cytometry results showed that the apoptosis of HL-60 cells increased after RSV treatment compared with those of the control group(P<0.05).Moreover,the cells in sub-G;phase increased(P<0.05)and the cells in G;/M phase decreased(P<0.05).(3)Western blot results showed the level of cell cycle protein CyclinD1 decreased(P<0.05),and the levels of apoptosis-related protein p53and p21 increased after RSV treatment(P<0.05).(4) Compare with the control group,the expression levels of SIX1 and CK2αprotein decreased in HL-60 cells after RSV treatment,and overexpression of SIX1 inhibited the effect of RSV on CyclinD1,p53,and p21 in HL-60 cells.Conclusion:RSV may inhibit cell proliferation and induce apoptosis by down-regulating SIX1 in HL-60 cells.