Abstract
BackgroundThe selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted.MethodologyThe role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured.Conclusions/SignificanceIn cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner.
Highlights
The mammalian thioredoxin system consists of the selenoprotein thioredoxin reductase (TR), thioredoxin (Trx), and electron donor NADPH
We constructed an inducible cell line where we could express a C-terminal mutant of TR1 that was resistant to siRNA knockdown
Since the induced expression of the Sec-deficient C-terminal mutant TR1 construct did not elicit an alteration in redox status of Trx, we evaluated the cytotoxic response of RKO cells with endogenous TR1 as well as cells where the TR1 was attenuated with siRNA to reactive oxygen and nitrogen in the form of H2O2, the oxidized lipid 4-HNE, or the nitric oxide (NO) donor JS-K (Figure 4)
Summary
The mammalian thioredoxin system consists of the selenoprotein thioredoxin reductase (TR), thioredoxin (Trx), and electron donor NADPH. Sec-deficient TR1 has demonstrated pro-apoptotic activity in studies evaluating the role of TR1 in interferon and retinoic acid-induced apoptosis [17], as well as more recent supporting data that has demonstrated Sec-deficient TR1 species (designated SecTRAPs) are by themselves potent initiators of apoptosis in human cancer cell lines [16]. Apoptosis in these cases were hypothesized to be mediated by increased oxidative stress in the cells. A greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted
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