2-氯脱氧腺苷(2-CDA)诱导人黑色素瘤A375细胞凋亡的机制探讨

Mechanism of A375 cell apoptosis induced by 2-chlorodeoxyadenosine(2-CDA)

目的探索2-氯脱氧腺苷(2-CDA)诱导人黑色素瘤A375细胞凋亡的机制。方法取对数生长期A375细胞,MTT比色法检测2-CDA对其体外增殖的抑制作用,Annexin V/PI双标法流式细胞术检测细胞凋亡情况,Western blotting检测细胞内蛋白水平的变化。结果 2-CDA对A375细胞增殖具有明显抑制作用,且随浓度增加及作用时间延长而逐渐增强,48h时的半数抑制浓度(IC50)为3.04μmol/L;Annexin V/PI双染并经流式细胞术检测显示2-CDA能够明显诱导细胞凋亡;Western blotting检测显示2-CDA能够显著下调Stat3的磷酸化水平,2-CDA作用后细胞凋亡信号蛋白caspase-3发生剪切而活化,导致其下游蛋白PARP发生剪切而诱导细胞凋亡。结论 2-CDA可通过抑制Stat3蛋白活性并激活Caspase-3而诱导A375细胞发生凋亡。

Objective The effect of nucleoside analogue,2-chlorodeoxyadenosine（2-CDA） on human malignant melanoma has not been fully studied heretofore.The aim of present study was to investigate the mechanisms of apoptosis of human melanoma A375 cells induced by 2-chlorodeoxyadenosine（2-CDA）.Methods A375 cells were treated with different doses（0,0.5,1,2,5μmol/L） of 2-CDA for different duration.The inhibitory effect on cell proliferation was measured by MTT assay.The cell apoptosis was detected by flow cytometry after annexin V-FITC/PI double staining.The expressions of p-Stat3,t-Stat3,Caspase-3,PARP and β-actin proteins were determined by Western blot.Results 2-CDA inhibited the proliferation of A375 cells in a time-and dose-dependent manner.After treatment for 48h,the IC50 of 2-CDA was 3.04μmol/L.Results of annexin V-FITC/PI double staining and flow cytometry indicated that 2-CDA effectively induced A375 apoptosis in a dose-dependent manner.The apoptotic rate of A375 cells was increased from 1.01% to 32.0% after treatment with 5μmol/L 2-CDA for 48h.The results of Western blotting showed that 2-CDA decreased the phosphorylation level of Stat3 kinase in time-and dose-dependent manner.When the cells treated with 5μmol/L 2-CDA for 36h,there was a prominent inhibitory effect on Stat3 kinase,indicated by a sharp decline of its phosphorylation level.At the same time,both the Caspase-3 and its client PARP,a hallmark of apoptosis,were all cleaved,implying that the cell apoptosis was induced in a caspase-dependent pathway.Conclusion 2-CDA may inhibit proliferation and induce apoptosis in human melanoma A375 cells by blocking the activity of Stat3 kinase and activating the Caspase-3/PARP apoptosis pathway.