Inhibition Of Thioredoxin Reductase Activity Research Articles

Inhibition of thioredoxin reductase activity reduces the antioxidant defense capacity of human pluripotent stem cells under conditions of mild but not severe oxidative stress

Pro-oxidative shift in redox balance, usually termed as “oxidative stress”, can lead to different cell responses depending on its intensity. Excessive accumulation of reactive oxygen species (“oxidative distress”) can cause DNA, lipid and protein damage. Physiological oxidative stimulus (“oxidative eustress”), in turn, can favor cell proliferation and differentiation – the processes of paramount importance primarily for stem cells. Functions of antioxidant enzymes in cells is currently a focus of intense research, however the role of different antioxidant pathways in pluripotent cell responses to oxidative distress/eustress is still under investigation. In this study, we assessed the contribution of the thioredoxin reductase (TrxR)-dependent pathways to maintaining the redox homeostasis in human induced pluripotent stem cells and their differentiated progeny cells under basal conditions and under conditions of oxidative stress of varying intensity. Employing the genetically encoded H2O2 biosensor cyto-HyPer and two inhibitors of thioredoxin reductase (auranofin and Tri-1), we show that the reduced activity of TrxR-dependent enzymatic systems leads to the non-cytotoxic disruption of thiol-disulfide metabolism in the cytoplasm of both pluripotent and differentiated cells under basal conditions. Quantifying the cytoplasmic concentrations of peroxide establishing in H2O2-stressed cells, we demonstrate that TrxR-dependent pathways contribute to the antioxidant activity in the cell cytoplasm under conditions of mild but not severe oxidative stress in both cell lines tested. The observed effects may testify about a conservative role of the TrxR-controlled enzymatic systems manifested as a response to physiological redox stimuli rather than a protection against the severe oxidative stress.

Depletion of multidrug-resistant uropathogenic Escherichia coli BC1 by ebselen and silver ion.

Ebselen, an organo‐selenium compound with well‐characterized toxicology and pharmacology, recently exhibited potent antibacterial activity against glutathione (GSH)‐negative bacteria by disrupting redox homeostasis. In this paper, we show that ebselen and silver ion in combination exert strong bactericidal activity against multidrug‐resistant (MDR) uropathogenic Escherichia coli (UPEC) BC1, a model MDR GSH‐positive bacterium. The mechanisms were found to involve consumption of total intracellular GSH and inhibition of thioredoxin reductase activity, which was highly related to reactive oxygen species up‐regulation. Furthermore, the therapeutic efficacy of ebselen and silver ion against UPEC‐induced cystitis was assessed in a mouse model. Treatment with ebselen and silver ion significantly reduced bacterial loads, down‐regulated the expression levels of tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) on‐site and decreased white/red blood cell counts in mild cystitis model mice, which demonstrated the anti‐inflammatory property of these agents. In addition, ebselen and silver ion also exhibited significantly high protective ability (100%) against acute cystitis infections. These results together may lay the foundation for further analysis and development of ebselen and silver ion as antibacterial agents for treatment of MDR UPEC infections.

Novel approach for evaluating pharmaceuticals toxicity using Daphnia model: analysis of the mode of cytochrome P450-generated metabolite action after acetaminophen exposure

Because of its widespread use, the pharmaceutical acetaminophen (APAP) is frequently detected in aquatic environments. APAP can have serious physiological effects, such as reduced reproduction, low growth rates, and abnormal behavior, in aquatic organisms. However, the methods available for evaluation of the aquatic toxicity of APAP are of limited usefulness. The present study aimed to develop reliable and sensitive markers for evaluation of APAP toxicity using Daphnia as a model organism. We focused on N-acetyl-p-benzoquinoneimine (NAPQI) production from APAP via cytochrome P450 metabolism because NAPQI causes APAP toxicity. Daphnia magna were exposed to APAP (0, 50, or 100 mg/L for 12 h or 24 h), and the total metabolites were extracted and analyzed for NAPQI. Direct detection of NAPQI was difficult because of its high reactivity, and its peak was close to that for APAP. Therefore, we tried to identify molecular and biochemical indicators associated with NAPQI generation, elimination, and its interactions with macromolecules. We identified changes in CYP370A13 gene expression, glutathione depletion, inhibition of thioredoxin reductase activity, and production of reactive oxygen species as indicators of D. magna exposure to APAP. These indicators could be used to develop sensitive and accurate techniques to evaluate the environmental toxicity of APAP.

Abstract 4480: Evaluation of anti-tumor enone-based bioactive compounds as specific thioredoxin reductase inhibitors

Abstract The thioredoxin system is a promising anti-cancer therapeutic target. It is one of the main cellular thiol antioxidant systems and comprises of thioredoxin, thioredoxin reductase and NADPH. In particular, thioredoxin reductase is an attractive target with susceptibility to inhibition by electrophilic compounds due to its nucleophilic selenocysteine residue in the C-terminal active center. In our laboratory, a series of enone-based compounds were synthesized and evaluated for their possible selectivity to inhibit thioredoxin reductase. To this end, the compounds were first screened for their in vitro thioredoxin reductase and glutathione reductase inhibitory activities, and their anti-proliferative activities were assessed by the MTT cell viability assay. The effects of selected analogs on the activities of thioredoxin reductase, thioredoxin and glutathione reductase were next studied. Finally, the molecular events linking thioredoxin reductase inhibition to apoptosis induction were elucidated. A number of the enone-based structural analogs were identified to posses anti-proliferative activities against HCT116 colorectal and MCF7 breast carcinoma cells at GI50 values in the low micromolar range, as well as inhibit activity of rat thioredoxin reductase with half-maximal inhibitory concentrations in the nanomolar range. Furthermore, these compounds displayed relatively weaker in vitro glutathione reductase inhibitory activities with half-maximal inhibitory concentrations in the micromolar range. In corroboration with results obtained from the in vitro enzyme activity assays, the identified lead analogs exhibited significant dose-dependent cellular thioredoxin reductase inhibition accompanied by a near negligible lack of effect on cellular thioredoxin and glutathione reductase activity. Conversely, the parent compound was found to not only inhibit cellular thioredoxin reductase activity, but also caused an inhibition of cellular thioredoxin and glutathione reductase activities. Finally, at a molecular level, the identified lead analogs that exhibited selective inhibition against thioredoxin reductase were found to induce apoptosis through activation of JNK and p38 MAPK pathways. In conclusion, this study had evaluated and identified enone-based bioactive compounds that display selective and dose-dependent inhibition of thioredoxin reductase activity in susceptible cell lines, which led to activation of p38 and JNK MAPK signaling pathways that at least in part triggered the apoptotic cascade. Taken together, the findings have indicated the potential of designing and developing these compounds that exert their anti-tumor effects through selective inhibition of thioredoxin reductase. Citation Format: Kamila K. Kaminska, Joanne Jia-An Low, Wan-Ying Chua, Esther Woon, Eng-Hui Chew. Evaluation of anti-tumor enone-based bioactive compounds as specific thioredoxin reductase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4480. doi:10.1158/1538-7445.AM2015-4480

Enhanced bioaccessibility and transport of reduced coenzyme Q10 is dependent on GSH status in Caco‐2 human intestinal epithelial cells (1044.8)

Recently, the bioavailability of orally administered reduced CoQ10 (CoQH) was reported to be markedly greater than that of oxidized CoQ10 (CoQ10) in healthy adults. We have investigated the basis for the influence of redox state on absorption using the coupled in vitro digestion/Caco‐2 cell model. Aliquots of CoQH and CoQ10 in hydrogenated castor oil were added to yogurt before simulated gastric and small intestinal digestion. Partitioning of CoQH in aqueous (i.e., bioaccessible) fraction of chyme was significantly greater than that of CoQ10. Similarly, when diluted bioaccessible fraction was added to the apical compartment of monolayers of Caco‐2 cells, uptake and trans‐epithelial transport of CoQH were 2.3 and 3.4 times greater than that of CoQ10, respectively. Reduction of cellular GSH significantly decreased both uptake and transport of CoQH and CoQ10 by Caco‐2 cells, although the extent of inhibition was much greater for CoQH. This suggests that maintaining a reduced intracellular environment is essential for efficient absorption of the CoQ. Auranofin‐mediated inhibition of thioredoxin reductase activity also decreased apical uptake and transport of CoQH, but not CoQ10. However, this differential effect was associated with auranofin‐associated oxidation and degradation of micellar CoQH in apical medium rather than changes in intracellular GSH status. These data suggest that the increased bioavailability of CoQH compared to CoQ10 results from its greater micellarization, more efficient uptake, and enhanced trans‐epithelial transport across the epithelial barrier, and that the cellular processes are dependent on a high ratio of GSH to GSSG for maintaining CoQ in the reduced state.

Strong inhibition of thioredoxin reductase by highly cytotoxic gold(I) complexes. DNA binding studies

Biological properties of a series of aminophosphine-thiolate gold(I) complexes [Au(SR)(PPh2NHpy)] [Ph2PNHpy=2-(diphenylphosphinoamino)pyridine; HSR=2-mercaptopyridine (2-HSpy) (3), 2-mercaptonicotinic acid (2-H2-mna) (4), 2-thiouracil (2-HTU) (5) or 2-thiocytosine (2-HTC) (6)] and [Au(SR){PPh2NH(Htrz)}] [Ph2PNH(Htrz)=3-(diphenylphosphinoamino)-1,2,4-triazole]; HSR=2-mercaptopyridine (2-HSpy) (7), 2-thiocytosine (2-HTC) (8) or 6-thioguanine (6-HTG) (9) have been studied. Their antitumor properties have been tested in vitro against two tumor human cell lines, HeLa (derived from cervical cancer) and MCF-7 (derived from breast cancer), using a metabolic activity test (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT). Some of them showed excellent cytotoxic activity. With the aim to obtain more information about the mechanisms of action of these derivatives, the interactions of complexes 3, 5, 7 and 9 with thioredoxin reductase in HeLa cells were studied. They showed a potent inhibition of thioredoxin reductase activity. In order to complete this study, interactions of the complexes with calf thymus (CT-) DNA and with different bacterial DNAs, namely the plasmid pEMBL9 and the promoter region of the furA (ferric uptake regulator A) gene from Anabaena sp. PCC 7120 were investigated. Although interactions of complexes with CT-DNA have been verified, none of them cause significant changes in its structure.

Abstract 3677: Prostate cancer progression decreases the cancer prevention by selenium: relation to overexpression of protein kinase Cε and selenoprotein thioredoxin reductase.

Abstract Selenium effectively prevents cancer in some cases, but fails in other cases. It could be possible that cancer-preventive actions of selenium are diminished by tumor progression, thus making this an important issue to study. In previous studies, tumor cell sensitivity to selenium was determined with commonly used, highly malignant prostate cancer cell lines such as DU145, PC-3, and LNCaP, which were originally cloned from different patients. These cell lines help us understand the mechanistic details of selenium-induced cellular effects, but due to their different genetic backgrounds, it is difficult to establish mechanisms related to cellular sensitivity to selenium. Previously, others have cloned a family of human prostate cancer cell lines with a common lineage showing progression characteristics from a low to high degree of malignancy and mimicking different stages of tumor progression seen in human prostate cancer. For this study, we used these prostate cancer cell lines with increasing tumorigenicity and invasive ability: WPE1-NA22 showing the lowest, WPE1-NB14 and WPE1-NB11 showing intermediate, and WPE1-NB26 showing the greatest. The low tumorigenic cell line, WPE1-NA22 resembles noninvasive prostatic intra-epithelial neoplasia and represents a good target for chemoprevention in humans. In this study, we found that WPE1-NA22 cells required a lower concentration of methylseleninic acid (MSA) for inhibition of cell growth (anchorage-dependent and anchorage-independent), induction of apoptosis, and inhibition of matrigel invasion. On the contrary, highly malignant cell line WPE1-NB26 required higher concentration of MSA to induce these effects, whereas moderately malignant cell lines WPE1-NB14 and WPE1-NB11 required intermediate levels of MSA. We have previously shown that protein kinase Cε (PKCε) is a molecular target for MSA and that the redox modification of this kinase is reversed by thioredoxin reductase (TR) system. Therefore, we determined whether there is an increase in expression of these two enzymes in tumor progression relating to a decrease in sensitivity to MSA. Western immunoblot analysis revealed low levels of PKCε, TR1, and TR2 in low tumorigenic WPE1-NA22 cells, while their levels were higher in advanced malignant cell lines correlating with their sensitivity to MSA. A conditional overexpression of PKCε in WPE1-NA22 cells resulted in an increase of both growth in soft agar and matrigel invasion and a decrease in sensitivity to MSA. An inhibition of TR activity in WPE1-NB26 resulted in an increase in sensitivity to MSA. Conceivably, there is an inverse correlation between the sensitivity to selenium-induced cancer prevention and prostate tumor progression involving an overexpression of PKCε and TR. Citation Format: Rayudu Gopalakrishna, Barsegh A. Barseghian, Carleen Sarksian, Jason E. Schiffman, David V. Rayudu, Usha Gundimeda. Prostate cancer progression decreases the cancer prevention by selenium: relation to overexpression of protein kinase Cε and selenoprotein thioredoxin reductase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2013-3677

Inhibition of thioredoxin reductase by lanthanum chloride

Lanthanum chloride (LaCl3) is a good inhibitor of both the cytosolic and mitochondrial thioredoxin reductase with IC50 values of 1.75 and 7.46μM, respectively. On the contrary, the related enzyme glutathione reductase is not inhibited by lanthanum ions even at relatively high concentrations. In the presence of LaCl3, steady-state kinetics shows a non-competitive type of inhibition with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) as a substrate suggesting no interaction of this ion with the thiol/selenol active site of thioredoxin reductase. Comparison of the electrostatic surface potential of thioredoxin reductase shows that the presence of a trivalent cation such as La3+ decreases the negatively charged area of the enzyme surface particularly in the region closed to the NADPH binding site. Human ovarian carcinoma cells (A2780 cells) incubated with lanthanum ions show a noticeable inhibition of thioredoxin reductase activity indicating the ability of this ion to reach the active site of the enzyme even in a cellular setting. In addition, A2780 cells treated with LaCl3 show an increase in reactive oxygen species production in part dependent on thioredoxin reductase inhibition.

Spectroscopic and electrochemical characterization of gold(I) and gold(III) complexes with glyoxaldehyde bis(thiosemicarbazones): cytotoxicity against human tumor cell lines and inhibition of thioredoxin reductase activity

Complexes [Au(2)(H(2)Gy3DH)(2)]Cl(2) (1), [Au(H(2)Gy3Me)]Cl(3) (2) and [Au(H(2)Gy3Et)]Cl(3) (3) were obtained with glyoxaldehyde bis(thiosemicarbazone) (H(2)Gy3DH) and its N(3)-methyl (H(2)Gy3Me) and N(3)-ethyl (H(2)Gy3Et) derivatives. The bis(thiosemicarbazones) and their gold(I) and gold(III) complexes exhibited anti-proliferative activity against HL-60, Jurkat (leukemia) and MCF-7 (breast cancer) cells at 10 μmol L(-1). Complex (2) was able to in vitro inhibit thioredoxin reductase (TrxR) activity, which suggests that inhibition of TrxR could be part of its mechanism of action.

Gold(I) complexes with thiosemicarbazones: Cytotoxicity against human tumor cell lines and inhibition of thioredoxin reductase activity

Complexes [Au(H2Ac4DH)Cl]∙MeOH ( 1) [Au(H 22Ac4Me)Cl]Cl ( 2) [Au(H 22Ac4Ph)Cl]Cl∙2H 2O ( 3) and [Au(H 22Bz4Ph)Cl]Cl ( 4) were obtained with 2-acetylpyridine thiosemicarbazone (H2Ac4DH), its N(4)-methyl (H2Ac4Me) and N(4)-phenyl (H2Ac4Ph) derivatives, as well as with N(4)-phenyl 2-benzoylpyridine thiosemicarbazone (H2Bz4Ph). The compounds were cytotoxic to Jurkat (immortalized line of T lymphocyte), HL-60 (acute myeloid leukemia), MCF-7 (human breast adenocarcinoma) and HCT-116 (colorectal carcinoma) tumor cell lines. Jurkat and HL-60 cells were more sensitive than MCF-7 and HCT-116 cells. Upon coordinating to the gold(I) metal centers in complexes ( 2) and ( 4), the cytotoxic activity of the H2Ac4Me and H2Bz4Ph ligands increased against the HL-60 and Jurkat tumor cell lines. 2 was more active than auranofin against both leukemia cells. Most of the studied compounds were less toxic than auranofin to peripheral blood mononuclear cells (PBMC). All compounds induced DNA fragmentation in HL-60 and Jurkat cells indicating their pro-apoptotic potential. Complex ( 2) strongly inhibited the activity of thioredoxin reductase (TrxR), which suggests inhibition of TrxR to be part of its mechanism of action.

Inhibition of Glutathione and Thioredoxin Metabolism Enhances Sensitivity to Perifosine in Head and Neck Cancer Cells

The hypothesis that the Akt inhibitor, perifosine (PER), combined with inhibitors of glutathione (GSH) and thioredoxin (Trx) metabolism will induce cytotoxicity via metabolic oxidative stress in human head and neck cancer (HNSCC) cells was tested. PER induced increases in glutathione disulfide (%GSSG) in FaDu, Cal-27, and SCC-25 HNSCCs as well as causing significant clonogenic cell killing in FaDu and Cal-27, which was suppressed by simultaneous treatment with N-acetylcysteine (NAC). An inhibitor of GSH synthesis, buthionine sulfoximine (BSO), sensitized Cal-27 and SCC-25 cells to PER-induced clonogenic killing as well as decreased total GSH and increased %GSSG. Additionally, inhibition of thioredoxin reductase activity (TrxRed) with auranofin (AUR) was able to induce PER sensitization in SCC-25 cells that were initially refractory to PER. These results support the conclusion that PER induces oxidative stress and clonogenic killing in HNSCC cells that is enhanced with inhibitors of GSH and Trx metabolism.

Thioredoxin reductase inactivation as a pivotal mechanism of ifosfamide in cancer therapy

Thioredoxin reductase reduces thioredoxin, thereby contributing to multiple cellular events related to carcinogenesis including cell proliferation, apoptosis, and cell signaling. This selenium-containing oxidoreductase is over-expressed in many malignant cells and has been proposed as a target for cancer therapy. Ifosfamide is an oxazaphosphorine alkylating agent with a broad spectrum of antineoplastic activity. The purpose of this study is to test the hypothesis that anticancer efficacy of ifosfamide may rely on its ability to inhibit thioredoxin reductase in tumor. To inspect the consequence of thioredoxin reductase inhibition by ifosfamide on tumor cell proliferation, mice bearing hepatoma 22 (H22) cells in ascites were injected with 350 mg/kg ifosfamide. Thioredoxin reductase activity was maximally inhibited by half at 6 h, and a subsequent pronounced cellular proliferation inhibition due to cell cycle arrest in G 1 phase was found. Moreover, at 6 h, except thioredoxin reductase inhibition, ifosfamide did not affect cell cycle or other measured antioxidant enzymes activity in the tumor cells. Intriguingly, when these cells were injected into healthy mice, they totally lost the capacity of causing either ascitic or solid tumors. Thioredoxin reductase inhibition could also be found in solid H22 tumor by 62%, bladder by 74% and kidney by 37% at 6 h. Overall, these observations provide direct evidence that inhibition of thioredoxin reductase activity in malignant cells by ifosfamide is highly associated with its anticancer effect and the mechanism of ifosfamide systemic toxicity may be related to multi-organ inhibition of thioredoxin reductase activity.

Heat Shock Protein 27 Protects L929 Cells from Cisplatin-Induced Apoptosis by Enhancing Akt Activation and Abating Suppression of Thioredoxin Reductase Activity

Heat shock protein 27 (Hsp27) is up-regulated in multiple malignancies and implicated in cisplatin resistance. It is attempted to know how Hsp27 endues cell with cisplatin resistance by interfering with upstream of both apoptosis signal-regulating kinase 1 (ASK1)/p38 mitogen-activated protein kinase-activated apoptotic signaling and serine/threonine kinase Akt-dependent survival signaling. The mouse L929 cells stably transfected with human Hsp27 or its dominant-negative mutant and the human cervical cancer HeLa cells transfected with Hsp27 siRNA were used. The cisplatin-induced apoptosis and activation of ASK1, p38, and Akt were compared in control cells, cells overexpressing Hsp27, and cells with their endogenous Hsp27 knocked down. Hsp27 effectively protected the cells from cisplatin-induced DNA fragmentation. The p38 inhibitors obviously decreased whereas Akt inhibitors markedly increased the apoptotic fraction in cisplatin-treated cells. Overexpression of Hsp27 doubly enhanced the drug-induced Akt activation while substantially depressing the drug-induced activation of ASK1 and p38. Knockdown of the endogenous Hsp27 in HeLa cells resulted in the effects opposite to that observed in the Hsp27-overexpressing cells. Enhancement of Akt activation is associated with complex formation between Akt and Hsp27, whereas depression of ASK1/p38 activation is attributed to a reversion of the drug-induced inhibition of thioredoxin reductase activity and subsequent oxidation of thioredoxin. Hsp27 endues cells with cisplatin resistance via depression of the drug-induced ASK1/p38 activation and enhancement of the drug-induced Akt activation. This study revealed the intervention of Hsp27 in upstream of both ASK1/p38 apoptotic signaling and phosphatidylinositol 3-kinase/Akt survival signaling. Therefore, the inhibition of Hsp27 may be a novel strategy of cancer chemotherapy.

Characterization of Alternative Cytosolic Forms and Cellular Targets of Mouse Mitochondrial Thioredoxin Reductase

Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction.

Motexafin Gadolinium Disrupts Zinc Metabolism in Human Cancer Cell Lines

To gain a better understanding of the mechanism of action of the metal cation-containing chemotherapeutic drug motexafin gadolinium (MGd), gene expression profiling analyses were conducted on plateau phase human lung cancer (A549) cell cultures treated with MGd. Drug treatment elicited a highly specific response that manifested in elevated levels of metallothionein isoform and zinc transporter 1 (ZnT1) transcripts. A549 cultures incubated with MGd in the presence of exogenous zinc acetate displayed synergistic increases in the levels of intracellular free zinc, metallothionein transcripts, inhibition of thioredoxin reductase activity, and cell death. Similar effects were observed in PC3 prostate cancer and Ramos B-cell lymphoma cell lines. Intracellular free zinc levels increased in response to treatment with MGd in the absence of exogenous zinc, indicating that MGd can mobilize bound intracellular zinc. These findings lead us to suggest that an important component of the anticancer activity of MGd is related to its ability to disrupt zinc metabolism and alter cellular availability of zinc. This class of compounds may provide insight into the development of novel cancer drugs targeting control of intracellular free zinc and the roles that zinc and other metal cations play in biochemical pathways relevant to cancer.

Arsenicals inhibit thioredoxin reductase in cultured rat hepatocytes.

Thioredoxin reductase (TR), an NADPH-dependent flavoenzyme that catalyzes the reduction of many disulfide-containing substrates, plays an important role in the cellular response to oxidative stress. Trivalent arsenicals, especially methyl As that contains trivalent arsenic (MAs(III)), are potent noncompetitive inhibitors of TR purified from mouse liver. Because MAs(III) is produced in the biomethylation of As, it was postulated that the extent of inhibition of TR in cultured rat hepatocytes would correlate with the intracellular concentration of methyl As. Exposure of cultured hepatocytes to inorganic As(III) (iAs(III)), MAs(III), or aurothioglucose (ATG, a competitive inhibitor of TR activity) for 30 min caused a concentration-dependent reduction in TR activity. The estimated IC(50) was >>100 microM for iAs(III), approximately 10 microM for ATG, and approximately 3 microM for MAs(III). In hepatocytes exposed to 1 microM MAs(III) for up to 24 h, the inhibition of TR activity was maximal ( approximately 40%) after exposure for 15 min. After exposure for 3 h [when most MAs(III) has been converted to dimethyl As (DMAs)], TR activity in these cells had returned to control levels. Notably, exposure of the cell to 50 microM DMAs(III) did not affect TR activity. In hepatocytes exposed to 10 microM iAs(III) for up to 24 h, the inhibition of TR activity was progressive; at 24 h, activity was reduced approximately 35%. Following exposure to iAs(III) or MAs(III), the extent of inhibition of TR activity correlated strongly with the intracellular concentration of MAs. Taken together, these results suggest that arsenicals formed in the course of cellular metabolism of As are potent inhibitors of TR activity. In particular, MAs(III), an intermediate in the metabolic pathway, is an especially potent inhibitor of TR. Hence, the capacity of cells to produce or consume the intermediates in the pathway for As methylation may be an important determinant of susceptibility to the toxic effects of As.

Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.

The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2-14 microM) of reduced thioredoxin, with 0.125 microM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37 degrees C. Although the treatment of endothelial cells with 0.2-12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70-85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H2O2-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 degrees C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H2O2-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.

Inhibition Of Thioredoxin Reductase (E.C. 1.6.4.5.) By Antitumor Quinones

Thioredoxin reductase (TR) is a widely distributed flavoenzyme that provides reduced thioredoxin, a dithiol hydrogen donor for protein disulfide reduction and for the reduction of ribonucleotides to deoxyribonucleotides, the first unique step of DNA synthesis. Antitumor quinones were found to exhibit time- and concentration-dependent inhibition of purified rat liver TR that requires the presence of NADPH. Diaziquone initially shows competitive inhibition of the enzyme with 5,5'-dithiobis 2-nitrobenzoic acid as substrate with a Ki of 7.5 microM, which becomes non-competitive after 1 hour incubation with NADPH with a Ki of 0.5 microM. Doxorubicin shows non-competitive inhibition both initially and after 1 hr incubation with NADPH, with Ki values of 10 microM and 0.5 microM, respectively. Electron spin resonance spectroscopy showed the formation of semiquinone free radicals by TR incubated under anaerobic conditions with doxorubicin or diaziquone and NADPH. Redox cycling and formation of oxygen radicals does not play a major role in the inhibition of TR by antitumor quinones as shown by the minor effect on inhibition of removing O2, and the lack of effect of superoxide dismutase and catalase. Diaziquone causes time- and concentration-dependent inhibition of TR activity in intact A204 human rhabdomyosarcoma cells that is associated with growth inhibition. The results suggest that inhibition of TR by antitumor quinones could contribute to their growth inhibitory properties.