ROS dependent manner without affecting STAT3 in A549 cells: Cuc B induced ROSmediated DNA damage, which activated G2/M phase checkpoint through ATM-activated Chk1-Cdc25C-Cdk1 and -p53-14-3-3-s cascades. The anti-proliferative effect of Cuc B on cancer cells has been reported everywhere. Similar to its effect on other reported cancer cells, Cuc B could significantly inhibit A549 cells proliferation and growth in a dose- and time- dependent manner. Though low concentrations of Cuc B showed no significant effect on A549 cell proliferation after 24 h treatment, prolonged treatment significantly inhibited cancer cells proliferation and colony formation clearly demonstrating that Cuc B is a potent cytotoxic compound. It could exert cytotoxicity at very low concentrations. STAT3, one of the seven members of the STAT transcription factor protein family, has been implicated as a potential target for cancer therapy. Activation of STAT3 signaling could up-regulate Cyclin B1, c-Myc, Bcl-x and regulating cell growth and survival. Chk1 knockdown reversed Cuc B induced G2/M phase arrest To dissect the 480-44-4 downstream effector in Cuc B mediated G2/M phase arrest, the role of Chk1 was examined with Chk1 siRNA. Similar to that of ATM siRNA, Cuc B- induced G2/M arrest in A549 cells was significantly decreased by Chk1 siRNA treatment. Furthermore, Cuc B caused phosphorylation of the Chk1 downstream effector Cdc25C on Ser-216 and Cdk1 on Tyr15 were also inhibited. Cuc B induced ROS generation and did PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645691 not affect STAT3 phosphorylation Recent studies have shown that Cuc B induced intracellular ROS formation in HeLa, SW480, and B16F10 cells. We investigated whether Cuc B induced ROS production in A549 cells. Cuc B significantly induced ROS formation in a dose dependent manner in A549 cell which could be inhibited by NAC pretreatment. Cucurbitacins has been identified as effective small molecular inhibitor of STAT3 and could dramatically suppress STAT3 phosphorylation. To test whether STAT3 play a role under our experimental Cucurbitacin B Induced DNA Damage Causes G2/M Arrest STAT3 inhibition has been found to induce cell cycle arrest and apoptosis in STAT3-positive tumor cells. Furthermore, STAT3 has been identified as one of the important molecular target for cucurbitacins. In this study, we found that low concentrations of Cuc B did not affect STAT3 phosphorylation in A549 cells. Thus, proposing that Cuc B induced G2/M phase arrest in a STAT3 independent manner. Many chemotherapeutic agents have been shown to induce DNA damage response in cancer cells. The effect of cucurbitacins on DNA damage remains to be clear. In present study, we firstly found that Cuc B induced DNA damage in A549 cells in a doseand time- dependent manner as evidenced by the long tails in comet assay, which was further confirmed 
by the upregulated expression of cH2AX, a biomarker/sensor for DNA damage. Similar results were also observed in Cuc B treated MCF-7 cells. 7 Cucurbitacin B Induced DNA Damage Causes G2/M Arrest These results suggested that DNA damage induction might be a common effect of Cuc B. PI3 Kinase-related kinase family members, ATM and ATR are the key protein kinases response DNA damage to activate checkpoints. Response to DNA damage, ATM and ATR are the firstly activated by autophosphorylation. In present study, ATR was not affected after Cuc B exposure, but enhanced phosphorylation of ATM on Ser-1981 and phosphorylation histone H2AX on Ser-139 was observed suggesting the
