Nuclear factor erythroid-2 related factor-2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small-cell lung cancer (NSCLC). In this study, we demonstrate that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth and results in increased sensitivity to chemotherapeutic drug induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional nonactive site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E 0 ) of the Trx1 active site (؊230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an ␣ helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the nonactive site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.Thioredoxin (Trx1) 1 is a ubiquitous 12-kDa protein that functions as a reductant for ribonucleotide reductase, peroxiredoxins, and transcription factors (e.g. Fos, Jun, NF-B, p53), controlling key aspects of cell proliferation and survival (1-4). The active site of Trx1, WCGPC, is conserved among species from cyanobacteria to humans (5). The active site cysteines are readily accessible on the surface of the protein and become oxidized to a disulfide upon reduction of a target protein. This disulfide is cycled back to the dithiol by Trx reductase (6).Unlike Trxs from lower species, mammalian Trx1 contains additional conserved cysteine residues (at positions 62, 69, and 73 of human Trx1; See Fig. 1). Whether these non-active site Cys residues have biologic function is unknown. Cys-73 was present as an intermolecular disulfide bond (Trx1 homodimer) in x-ray crystal studies (7), suggesting a possible function for Cys-73. However, a mutant Trx1 bearing a serine at this position still appeared as a homodimer in the crystal structure, suggesting that Cys-73 was not essential for dimerization (7). More recently, S-glutathionylation of Trx1 at Cys-73 has been found during oxidative stress (8). In addition, S-nitrosylation of Cys-69 has recently been described (9).The midpoint potential (E 0 ) for the active site dithiol of Trx is available for several lower species (10 -14) but not for mammals. Equilibrium with NADPH in the presence of a catalytic amount of Trx reductase, where it is assumed that each mole of NADPH consumed translates into ...

In type 2 diabetes, hyperglycemia and increased sympathetic drive may alter mitochondria energetic/redox properties, decreasing the organelle’s functionality. These perturbations may prompt or sustain basal low-cardiac performance and limited exercise capacity. Yet the precise steps involved in this mitochondrial failure remain elusive. Here, we have identified dysfunctional mitochondrial respiration with substrates of complex I, II, and IV and lowered thioredoxin-2/glutathione (GSH) pools as the main processes accounting for impaired state 4→3 energetic transition shown by mitochondria from hearts of type 2 diabetic db/db mice upon challenge with high glucose (HG) and the β-agonist isoproterenol (ISO). By mimicking clinically relevant conditions in type 2 diabetic patients, this regimen triggers a major overflow of reactive oxygen species (ROS) from mitochondria that directly perturbs cardiac electro-contraction coupling, ultimately leading to heart dysfunction. Exogenous GSH or, even more so, the fatty acid palmitate rescues basal and β-stimulated function in db/db myocyte/heart preparations exposed to HG/ISO. This occurs because both interventions provide the reducing equivalents necessary to counter mitochondrial ROS outburst and energetic failure. Thus, in the presence of poor glycemic control, the diabetic patient’s inability to cope with increased cardiac work demand largely stems from mitochondrial redox/energetic disarrangements that mutually influence each other, leading to myocyte or whole-heart mechanical dysfunction.

Exogenously added ROS (reactive oxygen species) cause generalized oxidation of cellular components, whereas endogenously generated ROS induced by physiological stimuli activate discrete signal transduction pathways. Compartmentation is an important aspect of such pathways, but little is known about its role in redox signalling. We measured the redox states of cytosolic and nuclear Trx1 (thioredoxin-1) and mitochondrial Trx2 (thioredoxin-2) using redox Western blot methodologies during endogenous ROS production induced by EGF (epidermal growth factor) signalling. The glutathione redox state was measured by HPLC. Results showed that only cytosolic Trx1 undergoes significant oxidation. Thus EGF signalling involves subcellular compartmental oxidation of Trx1 in the absence of a generalized cellular oxidation.