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

Abstract

BackgroundPyruvate 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. MethodsOne 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. ResultsOSU-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 48h for AZ-521, NUGC and SCM-1 were 2.68 and 1.83, 3.34 and 2.81, and 2.71 and 2.36mM, 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. ConclusionsThis 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. Legal entity responsible for the studyThe authors. FundingThe Ministry of Health and Welfare, Taiwan. DisclosureAll authors have declared no conflicts of interest.