Structure, Mechanism, and Inhibition of Aspergillus fumigatus Thioredoxin Reductase.

Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen [2-phenyl-1,2-benzoselenazol-3(2H)-one] is an inhibitor of A. fumigatus TrxR (Ki = 0.22 µM) and inhibits growth of Aspergillus spp., with in vitro MIC values of 16 to 64 µg/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of A. fumigatus growth.

Comparative Analyses of Cu-Zn Superoxide Dismutase (SOD1) and Thioredoxin Reductase (TrxR) at the mRNA Level between Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) Under Stress Conditions.

This study compared stress-induced expression of Cu-Zn superoxide dismutase (SOD1) and thioredoxin reductase (TrxR) genes in the European honeybee Apis mellifera L. and Asian honeybee Apis cerana F. Expression of both SOD1 and TrxR rapidly increased up to 5 h after exposure to cold (4 °C) or heat (37 °C) treatment and then gradually decreased, with a stronger effect induced by cold stress in A. mellifera compared with A. cerana. Injection of stress-inducing substances (methyl viologen, [MV] and H2O2) also increased SOD1 and TrxR expression in both A. mellifera and A. cerana, and this effect was more pronounced with MV than H2O2. Additionally, we heterologously expressed the A. mellifera and A. cerana SOD1 and TrxR proteins in an Escherichia coli expression system, and detection by SDS-PAGE, confirmed by Western blotting using anti-His tag antibodies, revealed bands at 16 and 60 kDa, respectively. Our results show that the expression patterns of SOD1 and TrxR differ between A. mellifera and A. cerana under conditions of low or high temperature as well as oxidative stress.

Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells.

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo.

Using chemical approaches to study selenoproteins-focus on thioredoxin reductases.

The study of selenocysteine-containing proteins is difficult due to the problems associated with the heterologous production of these proteins. These problems are due to the intricate recoding mechanism used by cells to translate the UGA codon as a sense codon for selenocysteine. The process is further complicated by the fact that eukaryotes and prokaryotes have different UGA recoding machineries. This review focuses on chemical approaches to produce selenoproteins and study the mechanism of selenoenzymes. The use of intein-mediated peptide ligation is discussed with respect to the production of the mammalian selenoenzymes thioredoxin reductase and selenoprotein R, also known as methionine sulfoxide reductase B1. New methods for removing protecting groups from selenocysteine post-synthesis and methods for selenosulfide/diselenide formation are also reviewed. Chemical approaches have also been used to study the enzymatic mechanism of thioredoxin reductase. The approach divides the enzyme into two modules, a large protein module lacking selenocysteine and a small, synthetic selenocysteine-containing peptide. Study of this semisynthetic enzyme has revealed three distinct enzymatic pathways that depend on the properties of the substrate. The enzyme utilizes a macromolecular mechanism for protein substrates, a second mechanism for small molecule substrates and a third pathway for selenium-containing substrates such as selenocystine.

Metabolism of selenium compounds catalyzed by the mammalian selenoprotein thioredoxin reductase.

The mammalian thioredoxin reductases (TrxR) are selenoproteins with a catalytic selenocysteine residue which in the oxidized enzyme forms a selenenylsulfide and in the reduced enzyme is present as a selenolthiol. Selenium compounds such as selenite, selenodiglutathione and selenocystine are substrates for the enzyme with low K(m)-values and the enzyme is implicated in reductive assimilation of selenium by generating selenide for selenoprotein synthesis. Redox cycling of reduced metabolites of these selenium compounds including selenide with oxygen via TrxR and reduced thioredoxin (Trx) will oxidize NADPH and produce reactive oxygen species inducing cell death at high concentrations explaining selenite toxicity. There is no free pool of selenocysteine since this would be toxic in an oxygen environment by redox cycling via thioredoxin systems. The importance of selenium compounds and TrxR in cancer and cardiovascular diseases both for prevention and treatment is discussed. A selenazol drug like ebselen is a direct substrate for mammalian TrxR and dithiol Trx and ebselen selenol is readily reoxidized by hydrogen peroxide and lipid hydroperoxides, acting as an anti-oxidant and anti-inflammatory drug.

Transcriptional regulation of mammalian selenoprotein expression.

BACKGROUND: Selenoproteins contain the twenty-first amino acid, selenocysteine, and are involved in cellular defenses against oxidative damage, important metabolic and developmental pathways, and responses to environmental challenges. Elucidating the mechanisms regulating selenoprotein expression at the transcriptional level is a key to understanding how these mechanisms are called into play to respond to the changing environment. METHODS: This review summarizes published studies on transcriptional regulation of selenoprotein genes, focused primarily on genes whose encoded protein functions are at least partially understood. This is followed by in silico analysis of predicted regulatory elements in selenoprotein genes, including those in the aforementioned category as well as the genes whose functions are not known. RESULTS: Our findings reveal regulatory pathways common to many selenoprotein genes, including several involved in stress-responses. In addition, tissue-specific regulatory factors are implicated in regulating many selenoprotein genes. CONCLUSIONS: These studies provide new insights into how selenoprotein genes respond to environmental and other challenges, and the roles these proteins play in allowing cells to adapt to these changes. GENERAL SIGNIFICANCE: Elucidating the regulatory mechanisms affecting selenoprotein expression is essential for understanding their roles in human diseases, and for developing diagnostic and potential therapeutic approaches to address dysregulation of members of this gene family.

Role of histidine-86 in the catalytic mechanism of ferredoxin:thioredoxin reductase.

Ferredoxin:thioredoxin reductase catalyzes the reduction of thioredoxins in plant chloroplasts using the [Fe2S2] ferredoxin as a one-electron donor and as such plays a central role in light regulation of oxygenic photosynthesis. The active-site comprises a [Fe4S4] cluster next to a redox-active disulfide that is cleaved in sequential one-electron steps and the combination of spectroscopic and crystallographic studies have revealed a catalytic mechanism involving novel site specific cluster chemistry in the oxidized, one-electron- and two-electron-reduced redox states. Histidine-86 has emerged as a potential proton donor/acceptor in the catalytic mechanism based on redox-related changes in the positioning of the imidazole ring during redox cycling and greatly decreased activity for the H86Y variant. Here we report on spectroscopic and redox characterization of the [Fe4S4] center in Synechocystis sp. PCC 6803 H86Y ferredoxin:thoredoxin reductase in the accessible redox states of both the as purified and N-ethylmaleimide-modified forms, using the combination of UV-visible absorption and variable-temperature magnetic circular dichroism, EPR, resonance Raman and Mössbauer spectroscopies. The results demonstrate that His86 is required for formation of the partially valence-localized [Fe4S4]2+ cluster that is the hallmark of two-electron-reduced intermediate. Taken together with the available structural data, the spectroscopic results indicate a functional role for His86 in protonation/deprotonation of the cluster-interacting thiol and anchoring the cluster interacting thiol in close proximity to the cluster in the two-electron-reduced intermediate.

Negation of the cancer-preventive actions of selenium by over-expression of protein kinase Cepsilon and selenoprotein thioredoxin reductase.

Selenium prevents cancer in some cases but fails to do so in others. Selenium's failure in this respect may be due to the development of resistance to its chemopreventive actions. Selenocompounds induce a variety of cancer-preventive actions in tumor cells, but these actions may be limited by the low concentrations of free selenocompounds able to reach cells from the plasma. Therefore, we have sought to identify the chemopreventive action requiring the lowest concentration of the redox-active form of selenium, methylseleninic acid (MSA). At submicromolar concentrations, MSA inhibited the malignant transformation of RWPE-1 prostate epithelial cells. In contrast, in already transformed prostate cancer cells, selenium in the micromolar range was required to inhibit cell growth and invasion and to induce apoptosis. The role of protein kinase C (PKC) in these cellular processes, especially the moderately selenium-sensitive PKCepsilon, was demonstrated using PKC-specific inhibitors and small interfering RNA. PKCepsilon levels inversely correlated with cellular sensitivity to MSA. An over-expression of PKCepsilon minimized MSA-induced inhibition of RWPE-1 cell transformation and induction of apoptosis. Thioredoxin reductase (TR), a selenoprotein, reversed the MSA-induced inactivation of PKC isoenzymes. High TR expression in advanced prostate cancer cells correlated with resistance to MSA. Furthermore, inhibition of TR by its specific inhibitor, auranofin, resulted in increased sensitivity of prostate cancer cells to MSA. Collectively, these results suggest that the cancer-preventive actions of selenium may be negated both by an over-expression of PKCepsilon, which is a redox-sensitive target for MSA, and by the selenoprotein TR, which reverses PKC sulfhydryl redox modification.

[Effects of parabolic flight on redox status in SH-SY5Y cells].

Space flight is known to produce a number of neurological disturbances. The etiology is unknown, but it may involve increased oxidative stress. A line of experimental evidence indicates that space flight may disrupt antioxidant defense system and result in increased oxidative stress. In vitro studies found that abundant of NO was produced in rat pheochromocytoma (PC12) cells, SHSY5Y neuroblastoma cells, and protein nitration was increased in PC12 cells within a simulated microgravity rotating wall bioreactor high aspect ratio vessel system or clinostat system. In the present study, we observed the change of redox status in SH-SY5Y cells after parabolic flight, and studied the effects of key redox molecule, thioredoxin (TRX), during the altered gravity. SH-SY5Y cells were divided into four groups: control cells, control cells transfected with TRX, flight cells and flight cells transfected with TRX. The expression levels of 3-nitrotyrosine (3-NT), inducible nitric oxide synthase (iNOS), TRX and thioredoxin reductase (TRXR) were observed by immunocytochemical method. It was shown that after parabolic flight, the staining of 3-NT and TRX were enhanced, while the expression level of TRXR was down-regulated compared with control. As for flight cells transfected with TRX, the staining of 3-NT and iNOS were weakened compared with flight cells. These results obtained suggest that altered gravity may increase protein nitration, down-regulate TRXR and elicit oxidative stress in SH-SY5Y cells, while TRX transfection could partly protect cells against oxidative stress induced by parabolic flight.

Thioredoxins and thioredoxin reductase in chloroplasts: A review.

The chloroplastic thioredoxins (Trxs), a family of thiol-disulphide oxidoreductases, are reduced by either ferredoxin (Fd)-dependent Trx reductase (FTR) or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Trx reductase (NTR). Two Trx systems are present in chloroplasts including Trxs, Trx-like proteins, and reductase FTR and NTRC. FTR is the main reductant for Trxs in chloroplasts, while the flavoprotein NTRC integrates NTR and Trx activity, and plays multiple roles in the Calvin cycle, the oxidative pentose phosphate pathway (OPPP), anti-peroxidation, tetrapyrrole metabolism, ATP and starch synthesis, and photoperiodic regulation. In addition, not only there exists a reduction potential transfer pathway across the thylakoid membrane, but also FTR and NTRC coordinate with each other to regulate chloroplast redox homeostasis. Herein, we summarise the physiological functions of these two Trx reduction systems, discuss how both regulate redox homeostasis in plant plastids, and emphasize the significance of chloroplast thioredoxin systems in maintaining photosynthetic efficiency in plants.

Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development.

Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heart-specific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart.

Oxidative stress and thioredoxin system.

Oxidative stress plays an important role in the modulation of several important physiological functions. On the other side, oxidative stress is accountable for development of many unphysiological changes, which can be deleterious for cells. Consequently, at the present time there is increased interest about study mechanisms and changes evocated by oxidative stress. Despite the highly oxidizing environment (21% oxygen, at sea level), at normal conditions, the cell cytoplasm of all aerobic organisms is reduced and proteins contain free sulfhydryl groups. In the cytoplasm, two major systems were identificated responsible for maintaining a reduced state: thioredoxin and glutathione/glutaredoxin system. Thioredoxin in bacteria, thanks to the low redox potential is the major dithiol reductant in the cytosol, or an advanced equivalent to dithiothreitol of cells (Holmgren 1985). Thioredoxin system acts the dominant role in many physiological processes (see below) and it is also a cell antioxidant.

Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome.

Thioredoxin reductases (Txnrd) maintain intracellular redox homeostasis in most organisms. Metazoan Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1-/- cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated that primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd-/- and txnrd+/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however, mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells.

Contribution of thioredoxin reductase to T-cell mitogenesis and NF-kappaB DNA-binding promoted by selenite.

Although the essential role of selenium for cellular immune responses is obvious, delineation of the functions is lacking because selenium can either promote or inhibit cell growth, cytokine production, and activation of transcription factor nuclear factor-kappaB (NF-kappaB). Studies with human thioredoxin-1 (Trx-1)-transgenic (Tg) mice were conducted to evaluate the relationship between stimulation of T-cell mitogenic response by sodium selenite and the intracellular Trx-1 levels, and the activities of selenoenzymes and NF-kappaB-DNA binding. Concanavalin A-induced mitogenesis of wild-type mouse splenic cells was stimulated by exposure to low levels of selenite (0.02-0.1 microM), with augmentation of NF-kappaB-DNA binding activity. Treatment with NF-kappaB nuclear translocation inhibitor SN50 or thioredoxin reductase (TR) inhibitor aurothioglucose depressed this stimulatory action. The mitogenic response of Trx-1-Tg mouse splenic cells was enhanced by exposure to relatively high levels of selenite (> or = 0.05 microM), compared with the wild-type mouse. Selenite also augmented TR activity but not cellular glutathione peroxidase activity in the Trx-1-overexpressed cells. These results suggest that the stimulation of T-cell mitogenic response by the physiological levels of selenite is predominantly caused by increased TR activity, which may lead to reduction of Trx-1 dependent on the intracellular expression level and promotion of DNA binding of NF-kappaB.

Thioredoxin reductase mediates cell death effects of the combination of beta interferon and retinoic acid.

Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN-beta-all-trans-retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.

Thioredoxin in the cardiovascular system.

The thioredoxin (TRX) system (TRX, TRX reductase, and NADPH) is a ubiquitous thiol oxidoreductase system that regulates cellular reduction/oxidation (redox) status. The impairment of cell redox state alters multiple cell pathways, which may contribute to the pathogenesis of cardiovascular disorders including hypertension, atherosclerosis, and heart failure. In this manuscript, we review the essential roles that TRX plays by limiting oxidative stress directly via antioxidant effects and indirectly by protein-protein interactions with key signaling molecules such as thioredoxin interacting protein (TXNIP). TRX and its endogenous regulators may represent important future targets to develop clinical therapies for diseases associated with oxidative stress.

An event of alternative splicing affects the expression of the NTRC gene, encoding NADPH-thioredoxin reductase C, in seed plants.

The NTRC gene encodes a NADPH-dependent thioredoxin reductase with a joint thioredoxin domain, exclusive of photosynthetic organisms. An updated search shows that although most species harbor a single copy of the NTRC gene, two copies were identified in different species of the genus Solanum, Glycine max and the moss Physcomitrella patens. The phylogenetic analysis of NTRCs from different sources produced a tree with the major groups of photosynthetic organisms: cyanobacteria, algae and land plants, indicating the evolutionary success of the NTRC gene among photosynthetic eukaryotes. An event of alternative splicing affecting the expression of the NTRC gene was identified, which is conserved in seed plants but not in algae, bryophytes and lycophytes. The alternative splicing event results in a transcript with premature stop codon, which would produce a truncated form of the enzyme. The standard splicing/alternative splicing (SS/AS) transcripts ratio was higher in photosynthetic tissues from Arabidopsis, Brachypodium and tomato, in line with the higher content of the NTRC polypeptide in these tissues. Moreover, environmental stresses such as cold or high salt affected the SS/AS ratio of the NTRC gene transcripts in Brachypodium seedlings. These results suggest that the alternative splicing of the NTRC gene might be an additional mechanism for modulating the content of NTRC in photosynthetic and non-photosynthetic tissues of seed plants.

Redox regulation of cellular stress response in neurodegenerative disorders.

There is increasing evidence that reactive oxygen species (ROS) are not only toxic but play an important role in cellular signaling and in the regulation of gene expression. A number of biochemical and physiologic stimuli, such as perturbation in redox status, expression of misfolded proteins, altered glyc(osyl)ation and glucose deprivation, overloading of products of polyunsaturated fatty acid peroxidation (Hydroxynonenals, HNE) or cholesterol oxidation and decomposition, can disrupt redox homeostasis, impose stress and subsequently lead to accumulation of unfolded or misfolded proteins in brain cells. Alzheimer's (AD), Parkinson's (PD), Huntington's disease (HD), Amyothrophic lateral sclerosis (ALS) and Friedreich ataxia (FRDA) are major neurological disorders associated with production of abnormal proteins and, as such, belong to the so called "protein conformational diseases". The Central Nervous System has evolved highly specific signaling pathways called the unfolded protein response to cope with the accumulation of unfolded or misfolded proteins. Recent discoveries of the mechanisms of cellular stress signaling have led to major new insights into the diverse processes that are regulated by cellular stress response. Thus, the pathogenic dysfunctional aggregation of proteins in non-native conformations is associated with metabolic derangements and excessive production of ROS. The brain response to detect and control metabolic or oxidative stress is accomplished by a complex network of "longevity assurance processes" integrated to the expression of genes termed vitagenes. Heat shock proteins are a highly conserved system responsible for the preservation and repair of correct protein conformation. Heme oxygenase-1, a inducible and redox-regulated enzyme, is currently considered as having an important role in cellular antioxidant defense. A neuroprotective effect, due to its heme degrading activity, and tissue-specific antioxidant effects due to its products CO and biliverdin, this latter being further reduced by biliverdin reductase in bilirubin is an emerging concept. There is a current interest in dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiology of Alzheimer disease, with a chronic inflammatory response, brain injury and beta-amyloid associated pathology. Curcumin and ferulic acid, two powerful antioxidants, the first from the curry spice turmeric and the second a major constituent of fruit and vegetables, have emerged as strong inducers of the heat shock response. Food supplementation with curcumin and ferulic acid is considered a nutritional approach to reduce oxidative damage and amyloid pathology in Alzheimer disease. This review summarizes the complex regulation of cellular stress signaling and its relevance to human physiology and disease.

The role of thioredoxin reductase activity in selenium-induced cytotoxicity.

The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.

Thioredoxin reductase plays a critical role in IFN retinoid-mediated tumor-growth control in vivo.

The IFN and retinoic acid (RA) combination suppresses cell growth by inducing apoptosis in the cultured tumor cells. Using a genetic technique, we have isolated several "genes associated with retinoid-IFN-induced mortality" (GRIM) that participate in this death pathway. One such gene, GRIM-12, encodes the redox enzyme thioredoxin reductase (TR). Antisense-mediated inhibition of TR abrogates cell death. To test the in vivo relevance of TR for growth suppression, we have conducted the following study. A wild-type TR or a catalytically defective mutant were expressed in MCF-7 breast carcinoma cells and transplanted into athymic nude mice. These mice were treated with IFN-beta and all-trans RA combination. Tumors expressing the vector or wild-type TR were readily suppressed by the IFN/RA combination. In contrast, the tumors bearing a mutant TR were resistant to regression. We further show that markers of apoptosis are stimulated in the regressing tumors. These studies show a prominent role for TR in tumor-growth suppression.