P38 And JNK-regulated H2AX Phosphorylation Is Essential For Apoptosis In Chronic Myelogenous Leukemia Cells Induced By Resveratrol

Objective H2AX is one of the histone family, as a new tumor suppressor, its phosphorylation at Ser139 is essential mechanism for tumor cells apoptosis. Resveratrol, which is a natural polyphenolic phytoalexin and its potential chemopreventive and chemotherapeutic activities have been demonstrated in various carcinogenesis. The aim of the current study was to investigate whether resveratrol can induce H2AX(Serl39) phosphorylation and further study whether H2AX phosphorylation is required for the apoptosis of chronic myelogenous leukemia (CML) cells 。At the same time, the molecular mechanism that is involved in was searched.Methods K562 cells were dispose of different concentrations of resveratrol (0-100 umol/L), in order to choose the best concentration of resveratrol. after 48 h,western blot detected rH2AX expression level. In order to choose the best point in time, dealing K562 cells with resveratrol (60 umol/1), western blot detected different period (0-72 h) rH2AX、H2AX expression level; at the same time, flow cytometry detected apoptosis of K562 cells. Later, in order to prove that P38 and JNK pathway is involved in H2AX phosphorylation, we through inhibitors inhibit P38 and JNK kinase activity, as well as using siRNA transfection on P38 and Jnk genes, on the basis we use western blot test rH2AX protein. Finally, we use flow cytometry to test the changes of K562 cells’apoptosis after overexpression or knockdown H2ax genes. All data are presented as mean ± SEM.All analyses were performed using SPSS v19.0 software,P values of <0.05 was considered significant for all comparisons.Results Resveratrol induced H2AX phosphorylation at Serl39 (yH2AX) in a time-and dose-dependent manner, witch coincided with apoptosis of K562.The mitogen activated protein kinase (MAPK) family members P38 and c-Jun N-terminal kinase (JNK) were activated by resveratrol, and which accorded with H2AX phosphorylation. High levels of activated extracellular signal-regulated kinase (ERK), which is another MAPK family member, were found in K562 cells, and resveratrol blocked ERK activation. Furthermore, P38 and JNK inhibition using chemical inhibitors or P38 and Jnk knockdown using targeted siRNAs reduced resveratrol-induced H2AX phosphorylation. Overexpression of H2AX increased the resveratrol-induced apoptosis of K562 cells. Overexpression of H2AX-139m (Serl39 was mutated to block phosphorylation) inhibited resveratrol-induced apoptosis. Similarly. H2AX knockdown resulted in K562 cells that were resistant to resveratrol-induced apoptosis.Conclusion Resveratrol induced apoptosis of chronic myelogenous leukemia cells by P38 and JNK-regulated H2AX phosphorylation.