Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2015/10/06/mol.115.099614.DC1 1521-0111/88/6/970–981$25.00 http://dx.doi.org/10.1124/mol.115.099614 MOLECULAR PHARMACOLOGY Mol Pharmacol 88:970–981, December 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Oxidative Stress and Response to Thymidylate Synthase-Targeted Antimetabolites s Ufuk Ozer, Karen W. Barbour, Sarah A. Clinton, and Franklin G. Berger Department of Biological Sciences, and Center for Colon Cancer Research, University of South Carolina, Columbia, South Carolina Received April 23, 2015; accepted October 5, 2015 Downloaded from ABSTRACT Thymidylate synthase (TYMS; EC 2.1.1.15) catalyzes the (NOX), which catalyzes one-electron reduction of O2 to generate d2 reductive methylation of 2ʹ-deoxyuridine-5ʹ-monophosphate superoxide (O2 ), is a significant source of increased ROS (dUMP) by N5,N10-methyhlenetetrahydrofolate, forming dTMP levels in drug-treated cells. However, gene expression profiling for the maintenance of DNA replication and repair. Inhibitors of revealed a number of other redox-related genes that may TYMS have been widely used in the treatment of neoplastic contribute to ROS generation. TYMS inhibitors also induce a disease. A number of fluoropyrimidine and folate analogs have protective response, including activation of the transcription molpharm.aspetjournals.org been developed that lead to inhibition of the enzyme, resulting in factor nuclear factor E2-related factor 2 (NRF2), a critical dTMP deficiency and cell death. In the current study, we have mediator of defense against oxidative and electrophilic stress. examined the role of oxidative stress in response to TYMS Our results show that exposure to TYMS inhibitors induces inhibitors. We observed that intracellular reactive oxygen spe- oxidative stress that leads to cell death, while simultaneously cies (ROS) concentrations are induced by these inhibitors and generating a protective response that may underlie resistance promote apoptosis. Activation of the enzyme NADPH oxidase against such death. 1995; Berger and Berger, 2006; Barbour and Berger, 2008). Introduction at ASPET Journals on September 26, 2021 Fluoropyrimidines such as 5-fluorouracil (FUra) and 5-fluoro- Thymidylate synthase (TYMS, EC 2.1.1.15) catalyzes the 2ʹ-deoxyuridine (FdUrd) are metabolized within cells to the reductive methylation of 2ʹ-deoxyuridine-5ʹ-monophosphate ʹ 5 10 potent TYMS inhibitor 5-fluoro-2 -deoxyuridylic acid and have (dUMP) by N N -methylenetetrahydrofolate (CH2H4PteGlu), been important in the therapy of a variety of cancers, in- to form dTMP and dihydrofolate (Carreras and Santi, 1995; cluding those of the ovary, breast, and gastrointestinal tract Berger and Berger, 2006). The enzyme is indispensable to (Chu et al., 2003; Longley et al., 2003). Folate-based inhibitors DNA synthesis and is therefore critical for replication and of TYMS have been designed on the basis of information from repair of the cellular genome in growing cells. As a conse- the structures of the natural substrates and the enzyme’s quence, it has been an attractive target at which chemother- active site cleft (Carreras and Santi, 1995; Bertino, 1997; apeutic drugs are directed (Chu et al., 2003; Longley et al., Takemura and Jackman, 1997). Indeed, these inhibitors were 2003; Wilson et al., 2014). Inhibitors of TYMS, including both among the first to be developed using such rationally based fluoropyrimidine and folate analogs, reduce de novo synthesis criteria. Several, including raltitrexed (RTX), LY231514 of dTMP, which leads to impaired DNA replication, genome [(2R)-2-[[4-[2-(2-amino-4-oxo-1,7-dihydropyrrolo[2,3-d]pyrimidin- damage, and programmed cell death (Carreras and Santi, 5-yl)ethyl]benzoyl]amino]pentanedioic acid], ZD9331 [(2S)-2-[[4-[(2,7-dimethyl-4-oxo-1H-quinazolin-6-yl)methyl-prop- This work was supported by the National Cancer Institute [Grant 2-ynylamino]-2-fluorobenzoyl]amino]-4-(2H-tetrazol-5-yl) CA44013]; and the National Institutes of Health Centers of Biomedical butanoic acid], and GW1843U89 [(S)-2-(5-(((3-methyl-1- Research Excellence program of the National Institute of General Medical oxo-1,2-dihydrobenzo[f]quinazolin-9-yl)methyl)amino)-1- Sciences [Grant GM103336]. The Microarray Core Facility receives partial support from South Carolina IDeA Networks of Biomedical Research oxoisoindolin-2-yl)pentanedioic acid], have advanced to clinical Excellence [Grant P20GM103499]. trials, and have demonstrated significant activity against a dx.doi.org/10.1124/mol.115.099614. s This article has supplemental material available at molpharm. variety of neoplasms (Bertino, 1997; Takemura and Jackman, aspetjournals.org. 1997). ABBREVIATIONS: APO, apocyanin; DCFDA, 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate; DHE, dihydroethidium; DPI, diphenyleneiodonium; dThd, deoxythymidine; dUMP, 2ʹ-deoxyuridine-5ʹ-monophosphate; FdUrd, 5-fluoro-2ʹ-deoxyuridine; FDXR, ferredoxin reductase; FUra, 5-fluorouracil; GW1843U89, (S)-2-(5-(((3-methyl-1-oxo-1,2-dihydrobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxoisoindolin-2-yl)pentanedioic acid; LY231514, (2R)- 2-[[4-[2-(2-amino-4-oxo-1,7-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioic acid; NAC, N-acetylcysteine; NOX, NADPH d2 oxidase; NRF2, nuclear factor E2-related factor 2; O2 , superoxide; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; ROS, reactive oxygen species; RTX, raltitrexed; siRNAs, small interfering RNAs; TYMS, thymidylate synthase; VAS, VAS-2870, VAS-2870, 3-benzyl- 7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine; ZD9331, (2S)-2-[[4-[(2,7-dimethyl-4-oxo-1H-quinazolin-6-yl)methyl-prop-2-ynylamino]-2-flu- orobenzoyl]amino]-4-(2H-tetrazol-5-yl)butanoic acid. 970 Oxidative Stress and Thymidylate Synthase Inhibitors 971 The mechanism by which dTMP deficiencies lead to the For measurement of ROS by flow cytometry, cells were grown to death of cells exposed to TYMS inhibitors is of central interest. 80% confluence in 60-mm plates and incubated in fluorescence- Over a decade ago, Hwang et al. (Hwang et al., 2001) demon- activated cell-sorting staining buffer (phosphate-buffered saline with strated that induction of reactive oxygen species (ROS) in 1% bovine serum albumin) at 37°C for 15 minutes in 50 nM DCFDA or FUra-treated cells results from increased expression of the DHE. After incubation, cells were washed twice with fluorescence- activated cell-sorting buffer and analyzed by flow cytometry (FC 500; mitochondrial enzyme ferredoxin reductase (FDXR), and is Beckman Coulter, Fort Collins, CO). A shift to the right indicates required for p53-dependent apoptotic response to drug. Stud- increased ROS levels. ies by Liu and Chen (2002) similarly suggested that FDXR Measurement of Apoptotic Cell Death. Cells were treated with promotes ROS-mediated apoptosis in response to FUra in TYMS inhibitors and inhibitory agents at the indicated concentrations a p53-dependent fashion. Despite these interesting studies for 48 hours. Apoptotic indices were determined by TUNEL assays as well as others showing increased ROS levels after expo- using the In Situ Cell Death Detection Kit, POD (Roche Applied sure to FUra (Akhdar et al., 2009; Matsunaga et al., 2010; Science, Indianapolis, IN). Cells were stained according to the Lamberti et al., 2012), we still lack a detailed mechanistic manufacturer’s instructions, counterstained with hematoxylin, and understanding of how exposure to TYMS inhibitors alters viewed under a light microscope at 400Â magnification. Apoptotic the intracellular redox environment, and how cells respond nuclei were counted manually, based on staining and morphology, and to such alterations. Currently, we have examined ROS- the apoptotic index was calculated as the ratio of apoptotic/total cells. At least 1000 cells from several microscopic fields were counted in each Downloaded from mediated oxidative stress and its function in apoptotic cell determination. death induced by TYMS inhibitors. We have identified the NADPH Oxidase Activity Assay. For each reaction, 105 cells superoxide-generating enzyme NADPH oxidase (NOX) as an were suspended in 500 ml reaction buffer [50 mM phosphate buffer important source of increased ROS levels during drug expo- (pH 7.0), 1 mM EGTA, 150 mM sucrose]. NADPH oxidase activity was sure. In addition, we have found that cells mount a protective detected by lucigenin-derived chemiluminescence with 100 mM response that includes activation of the transcription factor NADPH as substrate and 5 mM lucigenin. Cells were incubated at nuclear factor E2-related factor 2 (NRF2) and its downstream 37°C for 10 minutes. Chemiluminescence was measured using a molpharm.aspetjournals.org target genes, which may diminish the cytotoxic impact of luminometer (Promega, Madison WI) and expressed as arbitrary light 5 TYMS inhibitors. units per 10 cells. RNA Extraction and Quantitative Reverse-Transcription Materials and Methods Polymerase Chain Reaction. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Germantown, MD) with the addition of Reagents. FUra, FdUrd, deoxythymidine (dThd), folinic acid, RNase Free DNase (Qiagen) to eliminate contaminating genomic and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich DNA. The RNA concentration was determined by measuring the (St. Louis, MO). RTX was obtained from AstraZeneca (Macclesfield, absorbance at 260 nm and 280 nm (NanoDrop ND-1000 Spectropho- Cheshire, UK). Diphenyleneiodonium