紫草素对人白血病MV4-11细胞的增殖抑制和促凋亡作用

Proliferation inhibition and pro-apoptotic effects of shikonin on human leukemia MV4-11 cells

目的:探讨紫草素对FMS样酪氨酸激酶3受体基因内部串联重复序列(FLT3-ITD)突变急性髓系白血病(AML)MV4-11细胞的增殖抑制和促凋亡作用,并初步阐明其分子机制。方法:生长状态良好的MV4-11细胞分为二甲基亚砜(DMSO)组和不同浓度(0.5、1.0、2.0、4.0和8.0μmol·L-1)紫草素组,处理细胞24和48 h,采用CCK-8法检测各组细胞增殖抑制率,并计算半数抑制浓度(IC 50)。MV4-11细胞分为空白对照组、DMSO组和不同浓度(0.25、0.50和1.00μmol·L-1)紫草素组,处理细胞48和72 h,采用羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记法检测各组细胞增殖率。MV4-11细胞分为DMSO组和不同浓度(0.702、1.404和2.808μmol·L-1)紫草素组,处理细胞48 h,采用流式细胞术检测各组细胞凋亡率。MV4-11细胞分为DMSO组和不同浓度(0.351、0.702和1.404μmol·L-1)紫草素组,处理细胞48 h,Real-time PCR法检测各组细胞中微小RNA-155(miR-155)的表达水平。结果:CCK-8法和CFSE法检测,与DMSO组比较,不同浓度紫草素组细胞增殖抑制率明显升高(P<0.05或P<0.01),增殖率明显降低(P<0.05),且呈剂量依赖性;24和48 h时IC 50分别为1.743和1.404μmol·L-1。流式细胞术检测,与DMSO组比较,不同浓度紫草素组细胞凋亡率明显升高(P<0.01),且呈剂量依赖性。Real-time PCR法检测,与DMSO组比较,不同浓度紫草素组细胞中miR-155表达水平明显降低(P<0.01),1.404μmol·L-1紫草素组细胞中miR-155表达水平降低超过75%。结论:紫草素对人FLT3-ITD突变AML MV4-11细胞具有增殖抑制和促凋亡作用,并可下调miR-155表达,提示紫草素可能作为FLT3-ITD突变AML的潜在治疗药物之一。

Objective:To discuss the proliferation inhibition and apoptosis induction of shikonin on the FMS-like tyrosine kinase-3 receptor internal tandem duplication(FLT3-ITD)mutated acute myeloid leukemia(AML)MV4-11 cells,and to preliminarily clarify the molecular mechanisms.Methods:The MV4-11 cells were divided into DMSO group and different concentrations(0.5,1.0,2.0,4.0,and 8.0μmol·L-1)of shikonin groups,and treated for 24 and 48 h.The inhibitory rate of proliferation was analyzed by CCK-8 assay,and half inhibitory concentration(IC50)was calculated.The MV4-11 cells were divided into blank control group,DMSO group,and different concentrations(0.25,0.50,and 1.00μmol·L-1)of shikonin groups,and treated for 48 and 72 h;the proliferation rate of cells was analyzed by carbox fluorescenceindiacetate succinimidyl este(CFSE).The MV4-11 cells were divided into DMSO group and different concentrations(0.702,1.404,and 2.808μmol·L-1)of shikonin groups,and treated for 48 h;the apoptotic rate was determined by flow cytometry.The MV4-11 cells were divided into DMSO group and different concentrations(0.351,0.702,and 1.404μmol·L-1)of shikonin groups,and treated for 48 h;the microRNA-155(miR-155)expression level was detected by Real-time PCR.Results:The results of CCK-8 and CFSE methods indicated that the inhibitory rates of proliferation of MV4-11 cells in different concentrations of shikonin groups were increased compared with DMSO grpup(P<0.05 or P<0.01),and the proliferation rates were decreased(P<0.05 or P<0.01)in a concentration-dependent manner;the IC50 of 24 and48 hwere 1.743 and 1.404μmol·L-1,respectively.The flow cytometry results showed that the apoptotic rates of the cells in different concentrations of shikonin groups were increased compared with DMSO group(P<0.01)in a concentration-dependent manner.The Real-time PCR results showed that the expression levels of miR-155 in the cells in different concentrations of shikonin groups were decreased significantly(P<0.01),and the expression level in 1.404μmol·L-1 shikonin group was decreased by more than 75%.Conclusion:Shikonin could inhibit the proliferation and promote the apoptosis of FLT3-ITD mutated AML MV4-11 cells,and down-regulate the expression of miR-155,suggesting that shikonin may be one of the potential therapeutic drugs for FLT3-ITD mututed AML.