The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves peroxiredoxin 3 oxidation
Introduction
Thioredoxin reductase (TrxR) is a selenoprotein that plays a critical role in maintaining redox homeostasis in cells through the NADPH-dependent reduction of thioredoxin (Trx) [1], [2]. One of the major antioxidant roles of Trx is to reduce a ubiquitous family of thiol peroxidases known as peroxiredoxins (Prxs). These enzymes decompose peroxides using a highly reactive cysteine thiolate in their active site [3]. In the presence of peroxides the Prx active site cysteine forms a disulfide bond with a neighbouring cysteine residue, which Trx reduces to complete the catalytic cycle. Mammals have six Prxs, with Prx3 localised to the mitochondrion. Prx3 is kept reduced through the action of mitochondrial Trx2 and TrxR2, and this coupled system is seen as important in protecting mitochondria from H2O2 generated by respiratory complexes during metabolism [4].
There is growing evidence that these coupled mitochondrial antioxidant enzyme systems may also play a role in the regulation of apoptosis. Overexpression of Prx3 provides protection against induction of apoptosis by serum deprivation, hypoxia and cytotoxic drugs [5], [6], [7], [8], [9]. The proposed mechanism is scavenging of H2O2 that may otherwise promote the release of pro-apoptotic factors from mitochondria. Consistent with this hypothesis we observed Prx3 oxidation during the initiation of receptor-mediated apoptosis [10]. We have also found that pro-apoptotic isothiocyanates are able to inhibit TrxR and cause selective Prx3 oxidation at concentrations that trigger apoptosis [11]. The mechanism of Prx3 oxidation in both models is currently unclear, but may be due to an increase in mitochondrial H2O2 generation or impairment of the TrxR/Trx system. Previous studies have shown that lowering of Prx3 levels with siRNA resulted in increased cellular levels of H2O2, and this sensitized cells to the induction of apoptosis by staurosporine or TNF [8]. Similar deficiencies in Trx2 [12], [13] or TrxR2 [14] also promote apoptosis in some cell types or organs. Prx3 has previously been identified as a target gene of c-Myc essential for the transformation associated with this oncogenic transcription factor [5], and overexpression of Prx3 has been reported in breast and lung carcinomas, mesothelioma and hepatocellular carcinomas [15], [16], [17], [18]. A recent study has also revealed a link between Fanconi anemia and deregulation of Prx3 function [19].
Several anti-cancer agents have been identified as TrxR inhibitors [20], [21], [22], [23], [24], [25]. Auranofin, an organic gold compound widely used for the treatment of rheumatoid arthritis [26], has also been tested as an anti-cancer agent along with other gold (I) compounds [27], [28], [29], [30]. The pharmacological effect of auranofin is thought to be due to its high reactivity with cellular nucleophiles such as selenocysteine and cysteine, making auranofin a potent inhibitor of TrxR [31], [32]. The predominantly cytosolic isoform TrxR1 has also been detected in the intermembrane space of mitochondria, where its inhibition by auranofin may inversely correlate to apoptosis through interactions with Cu,Zn-superoxide dismutase [33], illustrating the complexity of compartmentalization and interactions in subcellular redox system networks. Rigobello et al. have undertaken a series of studies on the ability of auranofin to trigger apoptosis in cultured cells, and propose a general model in which TrxR inhibition causes oxidative stress in the mitochondria that leads to apoptosis [34], [35], [36], [37]. Here we have examined the effect of auranofin treatment on cytoplasmic and mitochondrial Prxs, and show selective oxidation of mitochondrial Prx3 at doses that induce apoptosis. We also used mouse embryonic fibroblasts deficient in Bax and Bak to delineate a specific role for this mitochondrial pathway in auranofin-mediated apoptosis.
Section snippets
Reagents
Cell culture materials RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin, and geneticin were from Gibco BRL (Auckland, New Zealand). Auranofin was from ICN Biomedicals Inc (Costa Mesa, CA, USA). Human TNF was from R&D Systems (Minneapolis, MN, USA). Monoclonal antibody to cytochrome c (clone 7H8.2C12) was from BD Biosciences (San Jose, CA, USA). Rabbit polyclonal antibodies to Prx1, 2, 3 and Prx-SO2H were from Ab Frontier (Seoul, Korea). Hybond-PVDF membrane and enhanced
Inhibition of TrxR by auranofin correlates closely with induction of apoptosis at doses that cause oxidation of Prx3
Jurkat T-lymphoma cells were treated with auranofin at a range of concentrations, whereupon TrxR inhibition, Prx oxidation and viability were assessed. Auranofin had an IC50 of 0.2 μM for total cellular TrxR activity after 30 min, with virtually complete loss of activity at doses in excess of 1 μM auranofin (Fig. 1A). Separation of the cells into cytoplasmic and mitochondrial fractions indicated that auranofin had slightly greater efficacy against cytosolic (predominantly TrxR1) than mitochondrial
Discussion
In this study we have shown that auranofin caused selective oxidation of mitochondrial Prx3 at concentrations that were able to trigger apoptosis. Prx3 oxidation was detectable well before major apoptotic events could be measured, and it still occurred when apoptosis was blocked by overexpression of Bcl-2 or by the removal of the pro-apoptotic mediators Bax and Bak. This indicates that Prx3 oxidation was a direct effect of auranofin exposure rather than a consequence of downstream apoptotic
Acknowledgements
This project was supported by the Maurice and Phyllis Paykell Trust. A.G.C. and K.K.B. are recipients of Top Achiever Doctoral scholarships from the Tertiary Education Commission of New Zealand.
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