Date of Award

1-1-2013

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Berger, Franklin G.

Abstract

Thymidylate synthase (TS) catalyzes the reductive transfer of a methyl group from N5,N10-methylenetetrahydrofolate (CH2-THF) to dUMP, forming dTMP and dihydrofolate (DHF). 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 (5-FU) and raltitrexed (RTX), which lead to inhibition 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 for the increased ROS levels. 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. We have identified NADPH oxidase (NOX) as the primary source of increased ROS following exposure to TS-directed agents, as indicated by the observation of decreased ROS production and apoptotic indices in presence of 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. To substantiate and expand results obtained from HCT116, we used other colorectal cancer cells, including HCT15, SW480, DLD-1, LoVo, MOSER and LS180. We have determined that colon cancer cells exhibit diverse responses in both basal and drug-inducible levels of apoptosis, NOX activity and mRNA of NOX2 regulatory subunits, implicating the heterogeneity in colorectal cancer cells. To understand whether other factors are involved in TS inhibitors-mediated ROS generation, we investigated the effects of NFκB on drug-mediated ROS accumulation in HCT116 cells. Loss of NFκB has no effects on increase in NOX activity mediated by TS inhibitors. It has no effects on drug-induced ROS and p67phox mRNA expression, either. However, we have determined that NFκB is an important suppressor for basal ROS formation in correlation with the induction of p67phox mRNA. 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 in HCT116 cells. NFκB has no role in such effects. Each colorectal cancer cells might have different mechanism to undergo ROS-mediated cell death.

Rights

© 2013, Ufuk Ozer

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