Inhibition Of DNA-PKcs Activity By Vanillin Derivative, And Its Anti-proliferation And Mechanisms Against Cancer Cells

AIM: Our study is to screen the effective chemical of anticancer from vanillin derivatives, and to evaluate its activity of antiproliferation and investigate its mechanisms. METHODS: MTT, colony-forming ability assays and single cell gel electrophoresis were used to analyze the cytotoxicity, cell proliferation, radiosensitivity and DNA damage. In vitro phosphorylation of p53 peptide substrate was performed to detect DNA-PKcs kinase activity. Flow cytometry, Hoechst33258 / FDA fluorescents staining and DNA-Ladder were used to measure apoptosis, and transmission electron microscopy was used to detect autophagic cell death. ROS level was detected by fluorescent probes. Western blot analysis was used to determine protein changes. Spindle structure was observed by immunostaining with anti-α-tubublin antibody detected by confocal laser scanning microscopy. RESLUTS: We had found an effective antiproliferative and DNA-PKcs inhibitor BVAN3208 from vanillin derivatives, and applied for a patent protection for this chemical. BVAN3208 could cleave DNA-PKcs and inhibited its kinase activity. BVAN3208 exhibited an effective antiproliferation against more than ten kinds of different detected cancer cell lines, and less cytotoxicity on normal cell lines as comprared to cancer cell lines. BVAN3208 sensitized cancer cells to ionizing radiation. BVAN3208 induced ROS, resulted in DNA strand breaks and inhibited DNA rejoining. Several types of cell death, including apoptosis, autophygic cell death and mitotic catastrophe, were triggered by BVAN3208. Abnormal spindle structure, multiplecentrosome and G2/M arrest were observed in BVAN3208 treated cancer cells. BVAN3208 inhibited the phosphorylation of Akt protein, which in turn led to the decreased phosphorylation of GSK3β and Bad proteins. CONCLUSION: We have obtained an effective anticancer and radiosensitization chemical BVAN3208 from vanillin derivatives, which acts as an inhibitor of DNA-PKcs. The anticancer activity of BVAN3208 might be achieved through at least two mechanistic pathways: 1) sensitizing cancer cells to radiotherapy or chemotherapy; 2) exhibiting direct anti-proliferation effect via targeting DNA-PKcs/Akt pathway.