青蒿酯联合阿糖胞苷±柔红霉素对MLL基因重排白血病细胞株MV4-11凋亡的影响及其机制研究

The Mechanism of Artesunate Combined with Cytarabine and/or Daunorubicin on the Apoptosis of MV4-11 MLL-rearranged Acute Myeloid Leukemia Cell Line

目的:探讨青蒿酯(ARTS)联合柔红霉素(DNR)±阿糖胞苷(Ara-C)对MLL基因重排(MLL-r)急性髓系白血病(AML)细胞株MV4-11细胞增殖、凋亡的影响及作用机制。方法:采用CCK-8法检测ARTS、DNR、Ara-C单药及联用对MV4-11细胞增殖率及计算单药的IC50;流式细胞术检测各组细胞的凋亡情况及凋亡受体DR4、DR5的表达;Western blot检测各组细胞中Caspase-3、Caspase-9的表达。结果:ARTS、Ara-C、DNR均呈浓度依赖性抑制MV4-1l细胞增殖(r=0.99,r=0.90,r=0.97),48 h的IC50分别为0.31μg/ml、1.43μmol/L、22.47 nmol/L。ARTS 0.3μg/ml、Ara-C 1.0μmol/L、DNR 15 nmol/L作用于MV4-11细胞48 h的增殖率比较:3药联合组<2药联合组<单药组(均P<0.05);2药联合组细胞增殖率比较:ARTS+Ara-C组<ARTS+DNR组<Ara-C+DNR组,3药及2药相互作用的协同指数(CI)均<l。2药及3药联合处理细胞后,ARTS+DNR+Ara-C组细胞凋亡率高于Ara-C+DNR组、ARTS+DNR组(P<0.05),ARTS+DNR+Ara-C组细胞凋亡率与ARTS+Ara-C组相当(P>0.05),各组细胞中DR4和DR5的表达无差异(P>0.05)。与DNR+Ara-C组相比,ARTS+DNR+Ara-C组、ARTS+Ara-C组24 h Caspase-3表达下降,差异均有统计学意义(P<0.05),其中3药联合组细胞中Caspase-3表达下调最显著,但各组细胞中Caspase-9表达无明显改变。结论:体外研究显示ARTS+DNR+Ara-C、ARTS+Ara-C方案协同抑制MV4-11细胞增殖和诱导MV4-11细胞凋亡作用均强于传统方案Ara-C+DNR,其作用机制可能是通过下调Caspase-3表达发挥作用,对Caspase-9、DR4、DR5的表达无影响。

Objective:To investigate the effect and mechanism of artesunate(ARTS)combined with cytarabine(Ara-C)and/or daunorubicin(DNR)on the proliferation and apoptosis of MV4-11 human mixed-lineage leukemia rearranged(MLL-r)acute myeloid leukemia(AML)cell line.Methods:CCK-8 assay was used to detect the proliferation effect of individual or in combination of ARTS,DNR,Ara-C on MV4-11 cells.The IC50 of ARTS,DNR and Ara-C was calculated separately.The cell apoptosis and expression of receptors DR4 and DR5 were detected by flowcytometry.Western blot was used to detect the expression of Caspase-3 and Caspase-9 in each groups.Results:The inhibition effect of ARTS,Ara-C and DNR on the proliferation of MV4-11 were all dose-dependently(r=0.99,0.90 and 0.97,respectively).The IC50 of ARTS,Ara-C and DNR on MV4-11 for 48 hours were 0.31μg/ml,1.43μmol/L and 22.47 nmol/L,respectively.At the dose of ARTS 0.3μg/ml,Ara-C 1.0μmol/L and DNR 15 nmol/L,the proliferation rate for 48 hours of the tri-combination treatment was significantly lower than that of the bi-combination treatment,while both were significantly lower than that of the individual treatment(all P<0.05).In terms of bi-combination treatment,the cells proliferation rate for 48 hours of the ARTS+Ara-C group was significantly lower than that of the ARTS+DNR group,while both were significantly lower than that of the Ara-C+DNR group(all P<0.05).The cooperativity index(CI)of bi-and tri-combination treatment were all less than 1.After 48 hours of drug action,the cell apoptosis rate of the ARTS+DNR+Ara-C group was significantly higher than that of the Ara-C+DNR group,while both were significantly higher than that of the ARTS+DNR group(all P<0.05).M eanwhile,the was no statistical difference between the cells apoptotic rate of the ARTS+DNR+Ara-C group and the ARTS+Ara-C group(P>0.05).The expression of DR4 and DR5 also showed no difference between control group and drug group.Compared with the DNR+Ara-C group,the expressions of Caspase-3 were significantly down-regulated in both the ARTS+DNR+Ara-C group and the ARTS+Ara-C group(all P<0.05).The down-regulation of Caspase-3 expression was the most significantly in the combination group of three drugs,while the Caspase-9 expressions in different groups showed no apparent change.Conclusion:The in vitro study showed that tri-combination of ARTS+Ara-C+DNR and bicombination of ARTS+Ara-C could inhibit the proliferation and promote apoptosis of M V4-11 cell line.The inhibition effect of these two combinations were significantly superior to that of the traditional Ara-C+DNR treatment.The mechanism underlying this finding may be identified by the down regulation of Caspase-3,while no altered expression was observed of Caspase-9,DR4 and DR5.