Induction of G2/M arrest by pseudolaric acid B is mediated by activation of the ATM signaling pathway.
OBJECTIVE
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 G(2)/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.


CONCLUSIONS
Taken together, these results suggest that PLAB induced G(2)/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.
Meng
Ai-guo
AG
Department of Biochemistry, Hebei Medical University, Shijiazhuang, China.
Jiang
Ling-ling
LL
D064007:Ataxia Telangiectasia Mutated Proteins; D016203:CDC2 Protein Kinase; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000032:analysis / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D064447:Checkpoint Kinase 2; D019926:Cyclin B / Q000032:analysis; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000032:analysis; D018844:Cyclin-Dependent Kinases; D004268:DNA-Binding Proteins / Q000502:physiology*; D004224:Diterpenes / Q000494:pharmacology*; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D008545:Melanoma / Q000473:pathology; D009687:Nuclear Proteins / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis; D025521:Tumor Suppressor Proteins / Q000502:physiology*; D020687:cdc25 Phosphatases / Q000032:analysis
C534699:CCNB1 protein, human; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D019926:Cyclin B; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D004224:Diterpenes; D004365:Drugs, Chinese Herbal; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D002110:Caffeine; C058391:pseudolaric acid B; D064447:Checkpoint Kinase 2; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases