Redox-sensitive signaling factors regulate multiple cellular processes, including proliferation, cell cycle, and prosurvival signaling cascades, suggesting their potential as molecular targets for anticancer agents. It is logical-to set constraints that a molecular target should meet at least one of the following criteria: (1) inhibition of prosurvival signaling pathways; (2) inhibition of cell cycle progression; or (3) enhancement of the cytotoxic effects of anticancer agents. Therefore, we hypothesized that thioredoxin reductase 1 (TR), a component of several redox-regulated pathways, might represent a potential molecular target candidate in response to agents that induce oxidative stress. To address this issue, permanent cell lines overexpressing either the wild-type (pCXN2-myc-TR-wt) or a CysSer mutant (pCXN2-mye-mTR) TR gene were used, as were parental HeLa cells treated with 1-methyl-1-propyl-2-imidazolyl disullide (IV-2), a pharmacologic inhibitor of TR. Cells were exposed to the oxidative stressors, H2O2 and ionizing radiation (IR), and analyzed for changes in signal transduction, cell cycle, and cytotoxicity. Analysis of HeLa cells overexpressing the pCXN2-mye-TR-wt gene showed increased basal activity of nuclear factor kappaB (NFkappaB) and activator protein (AP-1), whereas HeLa cells expressing a pCXN2-myc-mTR gene and HeLa cells treated with IV-2 were unable to induce NFkappaB or AP-1 activity following H2O2 or IR exposure. Fluorescence-activated cell sorting analysis showed a marked accumulation of PCXN2-myc-mTR cells in the late G, phase, whereas pCXN2-myc-TR-wt cells showed a decreased G, subpopulation. Chemical inhibition of TR with IV-2 also completely inhibited cellular proliferation at concentrations between 10 and 25 mumol/L, resulting in a G, phase cell cycle arrest consistent with the results from cells expressing the pCXN2myc-mTR gene. Following exposure to H2O2 and IR, pCXN2-myc-MTRand IV-2-treated cells were significantly more sensitive to oxidative stress-induced cytotoxicity as measured by clonogenic survival assays. Finally, IV-2-treated cells showed increased tumor cell death when treated with 1120, and IR. These results identify TR as a potential target to enhance the cytotoxic effects of agents that induce oxidative stress, including IR.
