The physiological relevance of these changes will be explored in future work

f p53 was also significantly up-regulated in the meantime. 14-3-3-s is a p53-regulated downstream inhibitor of G2/M progression. Following p53 activation by Cuc B, the expression of 14-3-3-s was significantly increased. In G2 checkpoint control, Ser-216 phosphorylation and 14-3-3 binding negatively regulates Cdc25C. Immunoprecipitation assay showed that the binding of phosphorylation Cdc25C on Ser-216 and 14-3-3-s was increased. These results suggested that ATM-p53-14-3-3-s participated in the G2/M checkpoint in Cuc B induced DNA damage. conditions, the expression of STAT3 was investigated. We found that Cuc B did not affect the expression of STAT3, nor it affect the phosphorylation of STAT3 at Tyr-705. Thus, these data suggested that Cuc B induced ROS generation in A549 cells but showed no effect on STAT3 under our experimental conditions. Cuc B induced G2/M arrest and DNA DSBs was mediated by ROS Cuc B induced ROS formation has been shown to mediate autophagy, depletion of the G-actin pool and G2 phase arrest and apoptosis. Here, we test the role of ROS in G2/M arrest and DNA DSBs in A549 cells. NAC, a general antioxidant and a precursor of glutathione, could almost completely reverse Cuc B induced G2/M phase arrest. NAC pretreatment was able to block the phosphorylation of cH2AX, suggesting the involvement of ROS in Cuc B-mediated DNA damage. We further determined the effect of NAC on Cuc B induced phosphorylation level of G2/M regulatory proteins such as Chk1 on Ser-345, Cdc25C on Ser-216, Cdk1 on Tyr-15, p53 on Ser-15, and the expression of 14-3-3-s. Results showed that Cuc PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19643932 B induced up-regulation and phosphorylation of these proteins were all significantly reversed by NAC pretreatment. These data demonstrated that Cuc B causes DNA damage and G2/M checkpoint mediated by ROS generation in ATM-Chk1-Cdc25C-Cdk1 cascade. ATM knockdown reversed Cuc B induced G2/M Arrest To further establish the role of ATM in Cuc B-mediated G2/M phase arrest, transiently transfect A549 cells with ATM siRNA was performed. ATM siRNA transfection dramatically reversed Cuc B induced ATM activation and G2/M phase arrest. The ATM activated Chk1-Cdc25C-Cdk1 pathway was further investigated. Cuc B induced phosphorylation of Chk1 on Ser-345, phosphorylation of Cdc25C on Ser-216, and phosphorylation p53 on Ser-15 were all inhibited by ATM knockdown. Similarly, Cuc mediated ATM downstream effector of p53, 14-3-3-s expression is down-regulated by ATM siRNA. Furthermore, Cuc B up-regulated Cyclin B1 was also reversed by ATM siRNA. To test the effect of ATM siRNA on Cuc B induced Cdk1 and Cyclin B1 interactions, IP was performed. Compared with Cuc B treated group, a dramatic decrease of Cyclin B 1-bound Cdk1 was observed in ATM knockdown and Cuc B co-treatment. Discussion More attention has been paid to the anti-cancer effect of cucurbitacins in recent years. Inducing cell cycle arrest by cucurbitacins has been well established while the detailed mechanisms and pathways are largely to be clear. Cuc B, one of the widely MedChemExpress 480-44-4 investigated cucurbitacins, cause different phase cell cycle arrest in different cancer cells. Previous data suggested that Cuc B caused cell cycle arrest by blocking the STAT3 signaling pathway, which resulted in reduced expression of downstream targets, such as Cyclin B1, Cyclin A. In SW480 cells, Cuc B induced G2 arrest and apoptosis through a STAT3-independent but ROS-dependent mechanism. In this study, we showed that Cuc B induced G2/M arrest in a