Induction of G2/M arrest by pseudolaric acid B is mediated by activation of the ATM signaling pathway.

AbstractAim:The aim of this study was to investigate the mechanism of pseudolaric acid B (PLAB)-induced cell cycle arrest in human melanoma SK-28 cells.Methods:Cell growth inhibition was detected by MTT assay, the cell cycle was analyzed by flow cytometry, and protein expression was examined by Western blot analysis.Results:PLAB inhibited the growth of human melanoma cells and induced G₂/M arrest in SK-28 cells, accompanied by an up-regulation of Cdc2 phosphorylation and a subsequent down-regulation of Cdc2 expression. Furthermore, PLAB decreased the expression of Cdc25C phosphatase and increased the expression of Wee1 kinase. Meanwhile, a reduction in Cdc2 activity was partly due to induction of the expression of p21^waf1/cip1 in a p53-dependent manner. In addition, PLAB activated the checkpoint kinase, Chk2, and increased the expression of p53, two major targets of ATM kinase. These effects were inhibited by caffeine, an ATM kinase inhibitor. We also found that PLAB significantly enhanced ATM kinase activity.Conclusion:Taken together, these results suggest that PLAB induced G₂/M arrest in human melanoma cells via a mechanism involving the activation of ATM, and the effect of PLAB on Cdc2 activity was mediated via interactions with the Chk2-Cdc25C and p53 signalling pathways, two distinct downstream pathways of ATM. PLAB may be a promising chemopreventive agent for treating human melanoma.Acta Pharmacologica Sinica (2009) 30: 442–450; doi: 10.1038/aps.2009.20 [ABSTRACT FROM AUTHOR]