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eloid leukemia cells. In pancreatic cancer cell lines, Cuc B-Piceatannol induced G2/M phase arrest might be mediated by inhibiting activated JAK2, STAT3, and STAT5, increasing level of p21, and decreasing expression of cyclin A, cyclin B1. While in BEL-7402 human hepatocellular carcinoma cells, Cuc B induced S-phase arrest was considered to be due to its inhibition of cyclin D1 and Cdk1 expression but without affecting STAT3 phosphorylation. However, the detailed underlying mechanisms remain to be clear. Intracellular reactive oxygen species has been implicated in a wide range of biological activities and disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Cuc B induced intracellular ROS formation in SW480 cells, which played an important role in G2 cycle arrest and apoptosis. Cuc B induced mitochondrial ROS production also contributed to autophagy in HeLa cells. Excess ROS production could cause different kinds cell damage, including Cucurbitacin B Induced DNA Damage Causes G2/M Arrest the oxidative injury of DNA, which can via checkpoint activation induce cell cycle arrest. In the DNA damage response, activation of DNA damage checkpoints is firstly recognized by sensors proteins, followed by activation of a series effector kinases which target the major cell cycle control machinery. ATM and ATR, two important transducer proteins, play critical roles in DNA damage response by controlling the damage response through phosphorylation of effector proteins. Following their activation and phosphorylation, the downstream effectors such as Chk1, Chk2, p53 were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19643932 phosphorylated and activated, leading to further transmission of the checkpoint signals. By the interaction between the cyclin-dependent kinases and the cyclins, cells transit between different phases of cycle. CDKs was activated by dephosphorylation on Thr-14 and Tyr-15 through Cdc25, which complete phases transition. Cdc25 can be phosphorylated by Chk1/Chk2 on Ser-323 and Ser-216 and functionally inactivated by binding to 14-3-3 proteins. In the present study, we examined the effect and potential mechanisms of Cuc B on cell phases in A549 cells. We demonstrated that Cuc B causes G2/M phase cell cycle arrest in A549 cells, which is associated with DNA damage mediated by ATM-activated Chk1-Cdc25C-Cdk1 and p53-14-3-3-s parallel branches. The DNA damage was mediated by the accumulation of intracellular ROS formation. These findings dissect the role of ROS and DNA damage in Cuc B induced G2/M arrest, and might offer some potential therapeutic targets for this natural product. Materials and Methods Chemicals and antibodies Cucurbitacin B, purchased from ShunBo Biological Engineering Technology Co., Ltd, was dissolved in dimethyl sulfoxide to make 200 mM stock solutions and was kept at 220uC. The stock solutions were freshly diluted to the desired concentration just before use. N-acetyl-L-cysteine was purchased from Beyotime. Specific antibodies 2 Cucurbitacin B Induced DNA Damage Causes G2/M Arrest against GAPDH, phospho-Cdc25C-Ser-216, Cdc25C, phosphop53-Ser-15, phospho-p53-Ser-20, p53, phospho-STAT3-Tyr-705, STAT3, phospho-ATM-Ser-1981, phosphor-ATR-Ser-428, ATR, phospho-Chk1-Ser-345, Chk1, phosphor-Chk2-Thr-68, Chk2 were purchased from Cell Signaling Technology. Phospho-Cdk1Tyr-15, Cyclin B1, 14-3-3-s were from Santa Cruz Biotechnology. Cdk1 antibody was obtained from BD Transduction LaboratoriesTM. Antibodies for ATM and cH2AX were obtained from GeneTex and Millip