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

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.