新型青蒿素衍生物SM1044诱导Kasumi-1细胞凋亡及机制的研究

本研究探讨新型水溶性青蒿素衍生物SM1044对人急性髓系白血病M2b细胞株Kasumi-1的诱导凋亡作用及其机制。应用四甲基偶氮唑蓝(MTT)还原法观察SM1044对细胞生长的抑制作用;Annexin-Ⅴ/PI双标记流式细胞术、PI单标记流式细胞术分别检测细胞凋亡和细胞周期;Western blot检测加药处理后Kasumi-1细胞凋亡相关蛋白Caspase 3、PARP以及融合蛋白AML1-ETO的变化。结果显示:青蒿素衍生物SM1044可抑制Kasumi-1细胞的增殖,其抑制作用呈时间和剂量依赖性;1μmol/L SM1044作用24小时,生长抑制率达到50%,48小时的IC50值为0.17±0.067μmol/L;SM1044通过Caspase依赖的途径诱导Kasumi-1细胞凋亡,细胞凋亡率随药物浓度的增加而增加。细胞周期测定显示,SM1044阻滞细胞周期于G0/G1期,5μmol/LSM1044作用24小时使G0/G1期细胞由(58.33±4.46)%上升为(71.75±2.24)%;Western blot测定显示SM1044促使凋亡相关蛋白cCaspase 3(cleaved Caspase 3)、cPARP(cleaved PARP)表达水平增加,同时融合蛋白AML1-ETO表达降低。结论 :SM1044可有效抑制Kasumi-1细胞增殖,诱导细胞凋亡,该过程与cCaspase 3、cPARP蛋白的表达上调有关。SM1044还能阻滞细胞周期,将Kasumi-1细胞阻滞在G0/G1期;同时SM1044能明显引起融合蛋白AML1-ETO的表达降低,可作为SM1044作用的胞内靶点。

A new artemisinin derivative SM1044 induces apoptosis of Kasumi-1 cells and its mechanism

The aim of this study was to investigate the apoptosis-inducing effect of artemisinin derivative SM1044 on Kasumi-1 cells and its possible mechanism. Kasumi-1 cells were treated with different concentrations of SM1044, the cell viability was evaluated by MTT assay. Cell apoptosis and cell cycle progression were assessed by using flow cytometry with Annexin-V/PI double staining and flow cytometry with PI staining respectively. The expression of apoptosis-related proteins caspase 3, PARP and the fusion protein AML1-ETO were detected by Western blot. The results indicated that SM1044 inhibited cell growth of Kasumi-1 cells in time- and dose-dependent manners. After exposure of Kasumi-1 cells to 1 ixmol/L SM1044 for 24 hours, the cell viability was decreased to 50%. IC50 of SM1044 to Kasumi-1 cells at 48 hours was 0.17 ±0. 067 μmol/L. SM1044 induced cell apoptosis in a caspase-dependent manner, and the apoptotic rate of Kasumi-1 cells increased as SM1044 concentration increased. Flow cytometry with PI staining revealed that SM1044 induced cell cycle arrest, and the proportion of ceils in G0/G1 phase increased from 58.33 ± 4.46% to 71.75 ± 2.24% after exposure to 5 μmol/L SM1044 for 24 hours. Western blot showed that SM1044 increased the expression of apoptosis-related proteins cPARP and cleaved caspase 3 and also degraded the AML1-ETO fusion protein. It is concluded that SM1044 can inhibit the proliferation of Kasumi-1 cells, induce cell apoptosis which may be releted to the increased level of cleaved PARP and cleaved caspase 3. SM1044 can also induce cell arrest in G0/G1 phase. As the fusion protein AML1-ETO degrades obviously, it can be the potential target of SM1044 in Kasumi-1 cells.