Sulfocoumarins as dual inhibitors of human carbonic anhydrase isoforms IX/XII and of human thioredoxin reductase

Abstract

The hypothesis that sulfocoumarin acting as inhibitors of human carbonic anhydrase (CA, EC 4.2.1.1) cancer-associated isoforms hCA IX and – hCA XII is being able to also inhibit thioredoxin reductase was verified and confirmed. The dual targeting of two cancer cell defence mechanisms, i.e. hypoxia and oxidative stress, may both contribute to the observed antiproliferative profile of these compounds against many cancer cell lines. This unprecedented dual anticancer mechanism may lead to a new approach for designing innovative therapeutic agents.     

The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Effect of Auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.     

Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.     

Impairment of thioredoxin reductase activity by oxidative stress in human rheumatoid synoviocytes

The thioredoxin/thioredoxin reductase system is strongly induced in patients with rheumatoid arthritis (RA). We have investigated the impact on TR activity of doses of superoxide anion generated by the hypoxanthine (HX)/xanthine oxidase (XO) system and by hydrogen peroxide, H₂O₂, for various times and compared the findings with synoviocytes obtained from osteoarthritis (OA) patients. At baseline, TR activity in RA cells was significantly higher than in OA cells (2.31 ± 0.65 versus 0.74 ± 0.43 mUnit/mg protein, p < 0.01). HX/XO and H₂O₂ in RA cells decreased TR activity, which was found to be unchanged in OA cells. H₂O₂ and superoxide anion caused a time-dependent accumulation of oxidized TR and induced the formation of carbonyl groups in TR protein in RA cells rather than OA cells, and oxidized the selenocysteine of the active site. The oxidation in TR protein was irreversible in RA cells but not in OA cells. In conclusion, we report that the oxidative aggression generates modifications in the redox status of the active site of the TR and induces an alteration of the Trx/TR system, concomitant with those of the other antioxidant systems that could explain the causes of oxidative stress related to RA disease.     

Synthesis of peptide substrates for mammalian thioredoxin reductase.

Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.     

Overexpression of thioredoxin reductase 1 inhibits migration of HEK-293 cells

Background information. TrxR (thioredoxin reductase), in addition to protecting against oxidative stress, plays a role in the redox regulation of intracellular signalling pathways controlling, among others, cell proliferation and apoptosis. The aim of the present study was to determine whether TrxR1 is involved in the regulation of cell migration. Results. Stably transfected HEK‐293 (human embryonic kidney) cells which overexpress cytosolic TrxR1 (HEK‐TrxR15 and HEK‐TrxR11 cells) were used in the present study. We found that the stimulation of cell motility induced by PKC (protein kinase C) activators, PMA and DPhT (diphenyltin), was inhibited significantly in the HEK‐TrxR15 and HEK‐TrxR11 cells compared with control cells. The overexpression of TrxR1 also inhibited characteristic morphological changes and reorganization of the F‐actin cytoskeleton induced by PMA and DPhT. In addition, the selective activation of PKCδ by DPhT was inhibited in cells that overexpressed cytosolic TrxR1. Furthermore, rottlerin, a selective inhibitor of PKCδ, and PKCδ siRNA (small interfering RNA), suppressed the morphological changes induced by DPhT in the control cells. Conclusions. The overexpression of TrxR1 inhibits migration of HEK‐293 cells stimulated with PMA and DPhT. Moreover, our observations suggest that this effect is mediated by the inhibition of PKCδ activation.     

Evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine as an irreversible inhibitor of mammalian thioredoxin reductase1

Context: Thioredoxin reductase (TrxR) is up-regulated in a number of human malignant cells and becomes a promising target for anticancer drug development.

Objective: To evaluate N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC), a potent anticancer agent against melanoma, as an inhibitor of mammalian TrxR1.

Material and methods: The mechanism of inhibition against TrxR1 was investigated using substrate protection, dialysis and liquid chromatography–tandem mass spectrometry.

Results: NACC inhibits TrxR1 in a time and concentration dependent manner. The K_i and k_inact of NACC against TrxR1 were determined to be 80?μM and 0.178?min^?1, respectively. The inhibition occurred only in the presence of NADPH and persisted after extensive dialysis. The tandem mass spectrometric analysis demonstrated that the selenocysteine rather than cysteine residue at the active site was p-chlorophenyl carbamoylated by NACC. Inhibition of intracellular TrxR by NACC in cultured melanoma cells was observed.

Discussion and conclusion: NACC which irreversibly inhibits TrxR1 by forming a covalent bond with selenocysteine can be an effective tool in the study of TrxR1.     

Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress‐induced premature senescence

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. By using a proteomic‐based approach, here we identify TrxR1 as a caveolar membrane‐resident protein. We show that caveolin 1, the structural protein component of caveolae, is a TrxR1‐binding protein by demonstrating that the scaffolding domain of caveolin 1 (amino acids 82–101) binds directly to the caveolin‐binding motif (CBM) of TrxR1 (amino acids 454–463). We also show that overexpression of caveolin 1 inhibits TrxR activity, whereas a lack of caveolin 1 activates TrxR, both in vitro and in vivo. Expression of a peptide corresponding to the caveolin 1 scaffolding domain is sufficient to inhibit TrxR activity. A TrxR1 mutant lacking the CBM, which fails to localize to caveolae and bind to caveolin 1, is constitutively active and inhibits oxidative‐stress‐mediated activation of the p53/p21^Waf1/Cip1 pathway and induction of premature senescence. Finally, we show that caveolin 1 expression inhibits TrxR1‐mediated cell transformation. Thus, caveolin 1 links free radicals to activation of the p53/p21^Waf1/Cip1 pathway and induction of cellular senescence by acting as an endogenous inhibitor of TrxR1.     

硫氧还蛋白还原酶1(TrxR1)在胃癌中的表达及对胃癌细胞生长的影响

目的:探讨胃癌组织硫氧还蛋白还原酶1(TrxR1)表达与生存时间的关系及其对胃癌细胞生长的影响。方法:用Real-time PCR法检测76例胃癌组织及癌旁TrxR1 mRNA表达,并分析其与胃癌患者临床病理特征及预后的关系;随机选取3例胃癌组织及癌旁组织,采用免疫组化法、Western blot法检测TrxR1蛋白表达。采用Western blot法和Real-time PCR法检测胃癌细胞系及人胃粘膜上皮细胞中TrxR1的表达。采用小RNA干扰序列(siRNA)处理AGS细胞,根据处理方法不同将AGS细胞分为3组:阴性对照组:转染NC-siRNA、TRXR1 siRNA干扰1组:转染TRXR1-siRNA1、TRXR1 siRNA干扰2组:转染TRXR1-siRNA2。使用Real-time PCR法检测各组AGS细胞中TrxR1 mRNA的表达,克隆形成试验和MTT法检测AGS细胞生长情况。结果:胃癌组织中TrxR1 mRNA和蛋白表达量均显著性上调,TrxR1主要定位于细胞质中。TrxR1高表达与患者TNM分期及淋巴结转移有关,且TrxR1高表达组患者的中位生存时间短于低表达组...     

Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the host's organism. In this article we report the crystal structure (at 2.2 A resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N-terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C-terminal domain, forming a dimer with a twisted "W" shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed.     

Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system

2-Cys peroxiredoxin (Prx) is a novel cellular peroxidase that reduces peroxides in the presence of thioredoxin, thioredoxin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH) and that functions in H(2)O(2)-mediated signal transduction. Recent studies have shown that 2-cys Prx can be inactivated by cysteine overoxidation in conditions of oxidative stress. Therefore, peroxidase activity, rather than the protein level, of 2-cys Prx is the more important measure to predict its cellular function. Here, we introduce a modified activity assay method for mammalian 2-cys Prx based on yeast nonselenium thioredoxin reductase. Yeast thioredoxin reductase is expressed in Escherichia coli cells and purified at high yield (40 mg/L of culture broth) as an active flavoprotein by combined diethyl aminoethyl (DEAE) and phenyl hydrophobic chromatography. The optimal concentrations of yeast thioredoxin and thioredoxin reductase required to achieve maximum mammalian 2-cys Prx activity are 3.0 and 1.5 microM, respectively. This modified assay method is useful for measuring 2-cys Prx activity in cell lysates and can also be adapted for a 96-well plate reader for high-throughput screening of chemical compounds that target 2-cys Prx.     

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.     

Involvements of mitochondrial thioredoxin reductase (TrxR2) in cell proliferation

Mammalian cells contain two forms of thioredoxin reductase (TrxR), cytosolic TrxR1 and mitochondrial TrxR2. To investigate the biological roles of TrxR2, we generated stable HeLa cell lines expressing a dominant negative form of TrxR2 (TrxR2DN) under the control of the tetracycline-off system. We observed that TrxR2DN-induced cells, following stimulation with EGF, produced more hydrogen peroxide than uninduced cells. The extent of protein tyrosine phosphorylation of many proteins including ERK was higher in TrxR2DN-induced cells than in uninduced cells when stimulated with fetal bovine serum or EGF. Induction of TrxR2DN also resulted in the increased rate of progression of G1 to S phase in cell cycle and cell proliferation and affected the expression of many proteins involved in cell cycle. These results suggest that TrxR2 participates in the regulation of protein tyrosine phosphorylation and cell growth as a component of the mitochondria specific H2O2-eliminating system that includes peroxiredoxin III and thioredoxin 2.     

Effect of thioredoxin reductase 1 on glucocorticoid receptor activity in human outer root sheath cells

Alopecia areata (AA) is a common disease of patchy hair loss on the scalp that can progress to cover the entire scalp and eventually the entire body. Intralesional injection of corticosteroids is the first-line therapy for adult patients, however some patients do not respond to glucocorticoid treatment effectively. To delineate the molecular mechanism underlying glucocorticoid insensitivity, we examined the expression of glucocorticoid receptor (GR) and thioredoxin reductase 1 (TrxR1). In some case of glucocorticoid-resistant AA patients, the expression of TrxR1 was decreased in outer root sheath (ORS). We then investigated the effect of TrxR1 on GR activity using recombinant adenoviruses. Overexpression of TrxR1 markedly increased GR activity in ORS cells cultured in vitro. In addition, TrxR1 protected GR activity against H(2)O(2). Finally, TrxR1-enhanced GR activity was significantly inhibited by the overexpression of dominant negative form of Trx (Trx(C32S/C35S)). These results suggest that decreased TrxR1 may be one putative cause for glucocorticoid resistance in AA, through the impact on intracellular redox system.