Gambogic acid-induced G2/M phase cell-cycle arrest via disturbing CDK7-mediated phosphorylation of CDC2/p34 in human gastric carcinoma BGC-823 cells.
Molecular mechanisms of cell-cycle arrest caused by gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hanburryi tree, have been investigated using BGC-823 human gastric carcinoma cells as a model. Based on our 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of GA caused an irreversible arrest in the G(2)/M phase of the cell cycle. Western blot analysis demonstrated that GA-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in CDC2/p34 synthesis, which led to the accumulation of phosphorylated-Tyr(15) (inactive) form of CDC2/p34. Real-time PCR, western blot and kinase activity assays revealed that GA-induced reduction of CDC2/p34 expression was mediated through the inhibition of cyclin-dependent kinase (CDK)-activating kinase (CDK7/cyclin H) activity. In addition, GA-treated cells were shown to have a low level of CDK7 kinase-phosphorylated-Thr(161) CDC2/p34 (active). Taken together, our results suggested that the inhibited proliferation of GA-treated BGC-823 cells was associated with the decreased production of CDK7 mRNA and protein, which in turn, resulted in the reduction of CDK7 kinase activity. The reduced CDK7 kinase activity is responsible for the inactivation of CDC2/p34 kinase and the irreversible G(2)/M phase cell-cycle arrest of human gastric carcinoma BGC-823 cells.
Yu
Jun
J
Department of Physiology, China Pharmaceutical University, Nanjing 210009, People's Republic of China.
Guo
Qing-Long
QL
You
Qi-Dong
QD
Zhao
Li
L
Gu
Hong-Yan
HY
Yang
Yong
Y
Zhang
Hai-wei
HW
Tan
Zi
Z
Wang
Xiaotang
X
D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D013274:Stomach Neoplasms / Q000473:pathology*; D044004:Xanthones / Q000494:pharmacology*; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D044004:Xanthones; C052659:gambogic acid; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human