69P - Reactive oxygen species induced by OSU-A9 inhibit the growth of duodenal cancer and gastric cancer cells through dephosphorylating intranuclear pyruvate kinase muscle isozyme M2.

Pyruvate kinase M2 (PKM2) is a key enzyme responsible for the final step of glycolysis. Whether and how the pyruvate kinase M2 is involved in reactive oxygen species (ROS)-mediated cytotoxicity of gastrointestinal cancer is unknown. One duodenal cancer cell line AZ521, and two gastric cancer cell lines NUGC and SCM-1 and were treated with OSU-A9 which is known to induce cytotoxicity of acute myeloid leukemia through ROS generation. The in vitro cytotoxic and proapoptotic activities of OSU-A9 was evaluated by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin V – propidium iodide staining, respectively. Overexpression experiment was performed by transfection with indicated plasmid using Lipofectamine 2000 according to the manufacturer's protocol. OSU-A9 induced a dose- and time-dependent cytotoxicity and apoptosis in duodenal cancer and gastric cancer cells through ROS generation. The IC50 of OSU-A9 at 24 and 48 h for AZ-521, NUGC and SCM-1 were 2.68 and 1.83, 3.34 and 2.81, and 2.71 and 2.36 mM, respectively. Pretreatment with ROS scavenger rescued the cancer cells from apoptosis and concomitant PARP cleavage, implicating a key role of ROS in OSU-A9-induced cell death. Furthermore, OSU-A9-mediated ROS down-regulated pTyr105-PKM2 which occurred in cell nucleus rather than in cytoplasm. Ectopic overexpression of PKM-2 partially overcame the cytotoxicity of OSU-A9, which implied a role of phosphorylated PKM2 beyond glycolysis in survival of duodenal cancer and gastric cancer cells. This study shows that ROS-mediated intranuclear PKM2 dephosphorylation, in part, contributes to the anticancer activity of OSU-A9 in duodenal cancer and gastric cancer. Differential down-regulation of phosphorylated PKM2 between nucleus and cytoplasm suggests a non-glycolytic role of PKM2 in cell survival response to ROS stress. The authors. The Ministry of Health and Welfare, Taiwan. All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]