Abstract 2112: The role of reactive oxygen species (ROS) in cell death induced by thymidylate synthase inhibitors.

Abstract

Abstract Thymidylate synthase (TS) catalyzes the reductive transfer of a methyl group from N5,N10-methylenetetrahydrofolate (CH2H4PteGlu) to dUMP, forming dTMP and dihydrofolate (H2PteGlu). It is generally accepted that inhibition of the enzyme leads to a deficiency of dTMP, followed by genome damage and programmed cell death. As such, TS has long been viewed as an important target of anti-neoplastic agents, such as 5-fluorouracil (FUra) and ratitrexed (RTX), which function as inhibitors of the enzyme. Oxidative stress is well-recognized as having a central role in cellular response to a number of DNA damaging agents, and may be a proximate cause of therapy-induced cell death. Despite such recognition, little detail exists on the origin and regulation of reactive oxygen species (ROS) during TS-directed chemotherapy. We have undertaken a detailed examination of the origin, nature, and role of ROS in cell death mediated by TS inhibitors. In colon tumor cell line HCT116, fluorescence detection of H2O2 and O2•− (using H2DCFDA and DHE, respectively) showed that profound increases in ROS levels occur during drug exposure. These increases are attenuated by treatment with the antioxidant N-acetylcysteine (NAC), by addition of thymidine to the medium and by TS overproduction, indicating that it is indeed TS inhibition and the resulting dTMP deficiency that is responsible. Oxidative damage to DNA, as indicated by 8-oxo-guanine formation, is elevated in drug-treated cells. Apoptotic indices, as measured by TUNEL assays, parallel changes in ROS levels, i.e., they are induced in response to drug, and are inhibited by NAC, thymidine, and TS overproduction. We have identified NADPH oxidase (NOX) as the primary source of increased ROS following exposure to TS-directed agents, as indicated by observing decreased ROS production apoptotic indices in cells treated with NOX inhibitors. We have found that in response to TS inhibitors, NOX enzyme activity increases in association with induction of the transcript for p67phox, which specifically regulates the NOX2 isoform. These effects were reduced by thymidine and by TS overproduction. Based on these results, we conclude that augmentation of NOX2 activity via induction of p67phox mRNA expression is the proximate cause of programmed cell death elicited by dTMP deficiency. Citation Format: Ufuk Ozer, Karen W. Barbour, Franklin G. Berger. The role of reactive oxygen species (ROS) in cell death induced by thymidylate synthase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2013-2112