三氧化二砷联合佛波酯对Kasumi-1细胞增殖的抑制作用及其机制

The inhibitory effect of As2O3 combined with phorbol ester on the proliferation of Kasumi-1 cells and its mechanism

目的 探讨三氧化二砷（As2O3）联合佛波酯（TPA）对急性早幼粒细胞白血病细胞系Kasumi-1细胞的增殖抑制作用及机制.方法 用200 nmol/L TPA、不同浓度As2O3单独及联合作用于Kasumi-1细胞,用CCK-8法观察细胞增殖抑制率,Annexin Ⅴ法测定细胞凋亡率,集落形成实验检测集落形成率,流式细胞术检测细胞分化及细胞周期改变,Western blot法检测P38及磷酸化P38蛋白的表达.结果 TPA联合不同浓度As2O3（0.2、2.0、20.0 μmol/L）作用于Kasumi-1细胞48 h的增殖抑制率分别为（25.56±7.29）％、（60.63±6.64）％、（73.37±2.15）％,细胞凋亡率为（61.65±2.62）％、（75.39± 1.04）％、（89.95±1.46）％,集落形成率为（76.17±2.06）％、（38.50±1.87）％、（18.53±2.20）％,与不同浓度As2O3单药组相比差异均有统计学意义（P＜0.05）;TPA可促使Kasumi-1细胞向单核巨噬细胞分化;TPA可使Kasumi-1细胞阻滞于G1期,As2O3可使Kasumi-1细胞阻滞于G2期,TPA可增强As2O3对细胞周期的影响;TPA与As2O3联用可增强As2O3促磷酸化P38蛋白表达.结论 TPA与As2O3联合作用能增强As2O3的促凋亡作用及对细胞周期的影响,增强其抗白血病作用.

Objective To investigate the inhibitory effect of arsenic trioxide （As2O3） combined with tetradecanoylphorbol acetate （TPA） on the proliferation of Kasumi-1 cell line and its mechanism.Methods Kasumi-1 cells were treated with 200 nmol/L TPA,different concentrations of As2O3 alone and combined with 200 nmol/L TPA.The proliferative inhibition rates were determined with CCK-8.Annexin V was adopted to detect apoptosis.Colony formation assay was used to determine the cloning efficiency.Flow cytometry was used to detect the cell differentiation and cell cycle changes.Western blot was employed to detect the expression of P38 and p-P38.Results The proliferation inhibition rates of Kasumi-1 cells by TPA combined with different concentrations of As2O3（0.2,2.0 and 20.0 mmol/L） for 48 h were （25.56±7.29）%,（60.63 ± 6.64）%,and （73.37±2.15）%,the apoptosis rates were （61.65 ±2.62）%,（75.39± 1.04）%,and （89.95± 1.46）%,and the colony formation rates were （76.17±2.06）%,（38.50± 1.87）%,and （18.53±2.20）%,respectively,compared with the different concentrations of As2O3 alone groups,the difference was statistically significant （P〈0.05）.Cells treated with both TPA and AS2O3 expressed more CD1 lb antigens compared with the cells exposed to As2O3 alone.TPA treated Kasumi-l cells were arrested at G1 phase compared with the control group,while As2O3 increased the percentage of Kasumi-1 cells in the G2 phase.Combination treatment increased the expression of p-P38 of Kasumi-1 cells compared with the cells exposed to As2O3 alone.Conclusions TPA can enhance the effect of As2O3 on inducing apoptosis and regulating cell cycle,thereby enhancing its anti-leukemia effect.