Induced peroxidase and cytoprotective enzyme expressions support adaptation of HUVECs to sustain subsequent H2O2 exposure.

H2O2 mediates autocrine and paracrine signaling in the vasculature and can propagate endothelial dysfunction. However, it is not clear how endothelial cells withstand H2O2 exposure and promote H2O2-induced vascular remodeling. To understand the innate ability of endothelial cells for sustaining excess H2O2 exposure, we investigated the genotypic and functional regulation of redox systems in primary HUVECs following an H2O2 treatment. Primary HUVECs were exposed to transient H2O2 exposure and consistent H2O2 exposure. Following H2O2 treatments for 24, 48 and 72 h, we measured O2(-) production, mitochondrial membrane polarization (MMP), and gene expressions of pro-oxidative enzymes, peroxidase enzymes, and cytoprotective intermediates. Our results showed that the 24 h H2O2 exposure significantly increased O2(-) levels, hyperpolarized MMP, and downregulated CAT, GPX1, TXNRD1, NFE2L2, ASK1, and ATF2 gene expression in HUVECs. At 72 h, HUVECs in both treatment conditions were shown to adapt to reduce O2(-) levels and normalize MMP. An upregulation of GPX1, TXNRD1, and HMOX1 gene expression and a recovery of NFE2L2 and PRDX1 gene expression to control levels were observed in both consistent and transient treatments at 48 and 72 h. The response of endothelial cells to excess levels of H2O2 involves a complex interaction amongst O2(-) levels, mitochondrial membrane polarization and anti- and pro-oxidant gene regulation. As a part of this response, HUVECs induce cytoprotective mechanisms including the expression of peroxidase and antioxidant enzymes along with the downregulation of pro-apoptotic genes. This adaptation assists HUVECs to withstand subsequent exposures to H2O2.

Adenanthin, a new inhibitor of thiol-dependent antioxidant enzymes, impairs the effector functions of human natural killer cells.

Natural killer (NK) cells are considered critical components of the innate and adaptive immune responses. Deficiencies in NK cell activity are common, such as those that occur in cancer patients, and they can be responsible for dysfunctional immune surveillance. Persistent oxidative stress is intrinsic to many malignant tumours, and numerous studies have focused on the effects of reactive oxygen species on the anti-tumour activity of NK cells. Indeed, investigations in animal models have suggested that one of the most important thiol-dependent antioxidant enzymes, peroxiredoxin 1 (PRDX1), is essential for NK cell function. In this work, our analysis of the transcriptomic expression pattern of antioxidant enzymes in human NK cells has identified PRDX1 as the most prominently induced transcript out of the 18 transcripts evaluated in activated NK cells. The change in PRDX1 expression was followed by increased expression of two other enzymes from the PRDX-related antioxidant chain: thioredoxin and thioredoxin reductase. To study the role of thiol-dependent antioxidants in more detail, we applied a novel compound, adenanthin, to induce an abrupt dysfunction of the PRDX-related antioxidant chain in NK cells. In human primary NK cells, we observed profound alterations in spontaneous and antibody-dependent NK cell cytotoxicity against cancer cells, impaired degranulation, and a decreased expression of activation markers under these conditions. Collectively, our study pinpoints the unique role for the antioxidant activity of the PRDX-related enzymatic chain in human NK cell functions. Further understanding this phenomenon will prospectively lead to fine-tuning of the novel NK-targeted therapeutic approaches to human disease.

Tissue thioredoxin reductase-1 expression in astrocytomas of different grades.

Thioredoxin (Trx) is a redox active protein that regulates several physiological and biochemical functions, such as growth, apoptosis and cellular defense. The function of Trx itself is regulated by thioredoxin reductase (TrxR). Studies performed in a variety of human primary tumors have shown that thioredoxin reductase 1 (TrxR1) is overexpressed in tumoral tissues compared with corresponding normal tissues. This study was designed to determine the expression of TrxR1 in astrocytoma tissues of different World Health Organization (WHO) grades (grade I-IV). The proliferative (Ki-67) and apoptotic indices of the specimens were also investigated for correlation analysis. Astrocytoma tissues were extracted from the histopathological specimens of 40 patients. These samples included seven histologically normal brain tissues that served as a control group and ten tumoral samples for each grade of astrocytoma (grade I-IV). The histologically normal brain tissues were obtained from the non-tumoral portions of the pathological specimens of grade I (2 cases), grade II (2 cases), grade III (2 cases) and grade IV (1 case) astrocytomas. TrxR1 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunostaining. The proliferative and apoptotic indices of the specimens were investigated by Ki-67 immunostaining and TUNEL assay, respectively. TrxR1 expression, as assessed by qRT-PCR, increased significantly with astrocytoma grade (p = 0.01). The immunostaining intensity of TrxR1 in grade IV astrocytomas was significantly greater than that in the control tissue and all other astrocytoma grades (p < 0.001). Similarly, immunostaining intensity of TrxR1 in the grade III astrocytomas was significantly greater than that in the control group and grade I astrocytomas (p < 0.001). All astrocytoma tissues showed more intense staining in ascending grades, but the differences between grade I and the control, grade II and the control, grades II and I, grades III and II were not statistically significant (p > 0.05). Ki-67 index values increased significant in accordance with grade progression (p = 0.01). The apoptotic index values were not significantly different in any group (p > 0.05); however, the differences between grade IV and the control and between grades IV and I were statistically significant (p < 0.05). Expression of TrxR1, as assessed by both qRT-PCR and immunostaining, correlated highly with both the astrocytoma grade and Ki-67 index.

Induction of complementary function reductase enzymes in colon cancer cells by dithiole-3-thione versus sodium selenite.

Cellular induction of reductase enzymes can alter the susceptibility of cells toward drugs and chemicals. In this study, we compared the capacity of a single dose of sodium selenite and 3H-1,2-dithiole-3-thione (D3T) to influence the drug-relevant reducing capacity of HT29 cells over time, and defined the protein-specific contribution to this activity on the basis of selected reaction monitoring mass spectrometry. Thioredoxin reductase 1 (TrxR1) protein levels and activity were inducible up to 2.2-fold by selenium. In contrast, selenium had only a minor influence on prostaglandin reductase 1 (PTGR1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) activity and protein levels. D3T, a strong Nrf2 inducer, induced all the reductases and additionally increased the cytotoxicity of hydroxymethylacylfulvene, a bioreductive DNA-alkylating drug. The data and experimental approaches allow one to define induction potency for reductase enzymes PTGR1, TrxR1, and NQO1 in HT29 cells and link these to changes in drug cytotoxicity.

Wobble decoding by the Escherichia coli selenocysteine insertion machinery.

Selenoprotein expression in Escherichia coli redefines specific single UGA codons from translational termination to selenocysteine (Sec) insertion. This process requires the presence of a Sec Insertion Sequence (SECIS) in the mRNA, which forms a secondary structure that binds a unique Sec-specific elongation factor that catalyzes Sec insertion at the predefined UGA instead of release factor 2-mediated termination. During overproduction of recombinant selenoproteins, this process nonetheless typically results in expression of UGA-truncated products together with the production of recombinant selenoproteins. Here, we found that premature termination can be fully avoided through a SECIS-dependent Sec-mediated suppression of UGG, thereby yielding either tryptophan or Sec insertion without detectable premature truncation. The yield of recombinant selenoprotein produced with this method approached that obtained with a classical UGA codon for Sec insertion. Sec-mediated suppression of UGG thus provides a novel method for selenoprotein production, as here demonstrated with rat thioredoxin reductase. The results also reveal that the E. coli selenoprotein synthesis machinery has the inherent capability to promote wobble decoding.

Zebularine inhibits the growth of A549 lung cancer cells via cell cycle arrest and apoptosis.

Zebularine (Zeb) is a DNA methyltransferase (DNMT) inhibitor to that has an anti-tumor effect. Here, we evaluated the anti-growth effect of Zeb on A549 lung cancer cells in relation to reactive oxygen species (ROS) levels. Zeb inhibited the growth of A549 cells with an IC50 of approximately 70 µM at 72 h. Cell cycle analysis indicated that Zeb induced an S phase arrest in A549 cells. Zeb also induced A549 cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm ), Bcl-2 decrease, Bax increase, p53 increase and activation of caspase-3 and -8. In contrast, Zeb mildly inhibited the growth of human pulmonary fibroblast (HPF) normal cells and lead to a G1 phase arrest. Zeb did not induce apoptosis in HPF cells. In relation to ROS level, Zeb increased ROS level in A549 cells and induced glutathione (GSH) depletion. The well-known antioxidant, N-acetyl cysteine (NAC) prevented the death of Zeb-treated A549 cells. Moreover, Zeb increased the level of thioredoxin reductase 1 (TrxR1) in A549 cells. While the overexpression of TrxR1 attenuated death and ROS level in Zeb-treated A549 cells, the downregulation of TrxR1 intensified death and ROS level in these cells. In conclusion, Zeb inhibited the growth of A549 lung cancer cells via cell cycle arrest and apoptosis. The inhibition was influenced by ROS and TrxR1 levels.

Expressed prostatic secretion biomarkers improve stratification of NCCN active surveillance candidates: performance of secretion capacity and TMPRSS2:ERG models.

PURPOSE: Active surveillance is a viable patient option for prostate cancer provided that a clinical determination of low risk and presumably organ confined disease can be made. To standardize risk stratification schemes the NCCN (National Comprehensive Cancer Network®) provides guidelines for the active surveillance option. We determined the effectiveness of expressed prostatic secretion biomarkers for detecting occult risk factors in NCCN active surveillance candidates. MATERIALS AND METHODS: Expressed prostatic secretion specimens were obtained before robot-assisted radical prostatectomy. Secretion capacity biomarkers, including total RNA and expressed prostatic secretion specimen volume, were measured by standard techniques. RNA expression biomarkers, including TXNRD1 mRNA, prostate specific antigen mRNA, TMPRSS2:ERG fusion mRNA and PCA3 mRNA, were measured by quantitative reverse-transcription polymerase chain reaction. RESULTS: Of the 528 patients from whom expressed prostatic secretions were collected 216 were eligible for active surveillance under NCCN guidelines. Variable selection on logistic regression identified 2 models, including one featuring types III and VI TMPRSS2:ERG variants, and one featuring 2 secretion capacity biomarkers. Of the 2 high performing models the secretion capacity model was most effective for detecting cases in this group that were up-staged or up-staged plus upgraded. It decreased the risk of up-staging in patients with a negative test almost eightfold and decreased the risk of up-staging plus upgrading about fivefold while doubling the prevalence of up-staging in the positive test group. CONCLUSIONS: Noninvasive expressed prostatic secretion testing may improve patient acceptance of active surveillance by dramatically reducing the presence of occult risk factors among those eligible for active surveillance under NCCN guidelines.

TrxR1 and GPx2 are potently induced by isothiocyanates and selenium, and mutually cooperate to protect Caco-2 cells against free radical-mediated cell death.

Currently, there is significant interest in the field of diet-gene interactions and the mechanisms by which food compounds regulate gene expression to modify cancer susceptibility. From a nutrition perspective, two key components potentially exert cancer chemopreventive effects: isothiocyanates (ITCs), present in cruciferous vegetables, and selenium (Se) which, as selenocysteine, is an integral part of selenoproteins. However, the role of these compounds in the expression of key selenoenzymes once the cancer process has been initiated still needs elucidation. Therefore, this investigation examined the effect of two forms of selenium, selenium-methylselenocysteine and sodium selenite, both individually and in combination with two ITCs, sulforaphane or iberin, on the expression of the two selenoenzymes, thioredoxin reductase 1 (TrxR1) and gastrointestinal glutathione peroxidase (GPx2), which are targets of ITCs, in Caco-2 cells. Co-treatment with both ITCs and Se induced expression of TrxR1 and GPx2 more than either compound alone. Moreover, pre-treatment of cells with ITC+Se enhanced cytoprotection against H(2)O(2)-induced cell death through a ROS-dependent mechanism. Furthermore, a single and double knockdown of TrxR1 and/or GPx2 suggested that both selenoproteins were responsible for protecting against H(2)O(2)-induced cell death. Together, these data shed new light on the mechanism of interactions between ITC and Se in which translational expression of the enhanced transcripts by the former is dependent on an adequate Se supply, resulting in a cooperative antioxidant protective effect against cell death.

Post-transcriptional defects of antioxidant selenoenzymes cause oxidative stress under methylmercury exposure.

Methylmercury (MeHg) toxicity is a continuous environmental problem to human health. The critical role of oxidative stress in the pathogenesis of MeHg cytotoxicity has been clarified, but the molecular mechanisms underlying MeHg-mediated oxidative stress remain to be elucidated. Here we demonstrate a post-transcriptional effect of MeHg on antioxidant selenoenzymes by using a MeHg-susceptible cell line. MeHg-induced selenium deficiency leads to failure of the recoding of a UGA codon for selenocysteine and results in degradation of the major antioxidant selenoenzyme glutathione peroxidase 1 (GPx1) mRNA by nonsense-mediated mRNA decay (NMD), a cellular mechanism that detects the premature termination codon (PTC) located 5'-upstream of the last exon-exon junction and degrades PTC-containing mRNAs. In contrast, thioredoxin reductase 1 (TrxR1), another antioxidant selenoenzyme of the thioredoxin system, was likely skipped by NMD because of a UGA codon in the last exon. However, TrxR1 activity was decreased despite mRNA up-regulation, which was probably due to the synthesis of aberrant TrxR1 protein without selenocysteine. Changes in selenoenzyme GPx1 and TrxR1 mRNAs were observed earlier than was the incidence of oxidative stress and up-regulation of other antioxidant enzyme mRNAs. Results indicated that the MeHg-induced relative selenium-deficient condition affects the major antioxidant selenoenzymes GPx1 and TrxR1 through a post-transcriptional effect, resulting in the disturbance of cellular redox systems and the incidence of oxidative stress. Treatment with ebselen, a seleno-organic compound, effectively suppressed oxidative stress and protected cells against MeHg-induced relative selenium deficiency and cytotoxicity.

Modulation of thioredoxin reductase-2 expression in EAhy926 cells: implications for endothelial selenoprotein hierarchy.

BACKGROUND: We examined the expression of the mitochondrial selenoenzyme TrxR2 in the endothelial cell line EAhy926 under conditions known to modify its cytoplasmic counterpart TrxR1. METHODS: Cells were cultured with varying concentrations of selenite, sulforaphane or the Ca2+ ionophore A23187 for 72-h, prior to assay of TrxR concentration and activity. Further cultures underwent prolonged (7-day) Se-depletion before selenoprotein measurement. RESULTS: In Se-deficient cultures, neither Se, A23187 or sulforaphane affected TrxR2 concentration, while these treatments induced TrxR1 concentration (p<0.05). When co-incubated, optimal concentrations of Se (40 nM) and sulforaphane (4 microM) only modestly increased TrxR2 protein (approximately 1.3-fold), compared with TrxR1 (approximately 4-fold). In Se-deficient cells, TrxR activity was unaffected by sulforaphane or A23187. Prolonged Se-depletion caused a comparatively small reduction in TrxR2 (66% TrxR2 retained) against TrxR1 and glutathione peroxidase-1 activity (38% and 17% retained, respectively). CONCLUSIONS: The relative resistance of TrxR2 to Se-deprivation and induction by sulforaphane and A23187 suggests TrxR2 lies near the top of the selenoprotein hierarchy in EAhy926 cells and exhibits near maximum expression under a range of culture conditions. In Se deficiency an inactive (possibly truncated) TrxR1 is produced in response to stimulus by sulforaphane and A23187. GENERAL SIGNIFICANCE: These observations underpin a likely critical antioxidant role for TrxR2 and TrxR1 in the endothelium.

Exposure to monomethylarsonous acid (MMA(III)) leads to altered selenoprotein synthesis in a primary human lung cell model.

Monomethylarsonous acid (MMA(III)), a trivalent metabolite of arsenic, is highly cytotoxic and recent cell culture studies suggest that it might act as a carcinogen. The general consensus of studies indicates that the cytotoxicity of MMA(III) is a result of increased levels of reactive oxygen species (ROS). A longstanding relationship between arsenic and selenium metabolism has led to the use of selenium as a supplement in arsenic exposed populations, however the impact of organic arsenicals (methylated metabolites) on selenium metabolism is still poorly understood. In this study we determined the impact of exposure to MMA(III) on the regulation of expression of TrxR1 and its activity using a primary lung fibroblast line, WI-38. The promoter region of the gene encoding the selenoprotein thioredoxin reductase 1 (TrxR1) contains an antioxidant responsive element (ARE) that has been shown to be activated in the presence of electrophilic compounds. Results from radiolabeled selenoproteins indicate that exposure to low concentrations of MMA(III) resulted in increased synthesis of TrxR1 in WI-38 cells, and lower incorporation of selenium into other selenoproteins. MMA(III) treatment led to increased mRNA encoding TrxR1 in WI-38 cells, while lower levels of mRNA coding for cellular glutathione peroxidase (cGpx) were detected in exposed cells. Luciferase activity of TrxR1 promoter fusions increased with addition of MMA(III), as did expression of a rat quinone reductase (QR) promoter fusion construct. However, MMA(III) induction of the TRX1 promoter fusion was abrogated when the ARE was mutated, suggesting that this regulation is mediated via the ARE. These results indicate that MMA(III) alters the expression of selenoproteins based on a selective induction of TrxR1, and this response to exposure to organic arsenicals that requires the ARE element.

Oxidative Stress Gene Expression Profile Correlates with Cancer Patient Poor Prognosis: Identification of Crucial Pathways Might Select Novel Therapeutic Approaches.

The role of altered redox status and high reactive oxygen species (ROS) is still controversial in cancer development and progression. Intracellular levels of ROS are elevated in cancer cells suggesting a role in cancer initiation and progression; on the contrary, ROS elevated levels may induce programmed cell death and have been associated with cancer suppression. Thus, it is crucial to consider the double-face of ROS, for novel therapeutic strategies targeting redox regulatory mechanisms. In this review, in order to derive cancer-type specific oxidative stress genes' profile and their potential prognostic role, we integrated a publicly available oxidative stress gene signature with patient survival data from the Cancer Genome Atlas database. Overall, we found several genes statistically significant associated with poor prognosis in the examined six tumor types. Among them, FoxM1 and thioredoxin reductase1 expression showed the same pattern in four out of six cancers, suggesting their specific critical role in cancer-related oxidative stress adaptation. Our analysis also unveiled an enriched cellular network, highlighting specific pathways, in which many genes are strictly correlated. Finally, we discussed novel findings on the correlation between oxidative stress and cancer stem cells in order to define those pathways to be prioritized in drug development.

Interplay between the chalcone cardamonin and selenium in the biosynthesis of Nrf2-regulated antioxidant enzymes in intestinal Caco-2 cells.

Selenoenzymes and nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated phase II enzymes comprise key components of the cellular redox and antioxidant systems, which show multiple interrelations. Deficiency of the micronutrient selenium (Se) and impaired biosynthesis of selenoproteins have been reported to result in induction of Nrf2 target genes. Conversely, transcription of the selenoenzymes glutathione peroxidase 2 (GPx2) and thioredoxin reductase 1 (TrxR1) is up-regulated upon Nrf2 activation. Here, we have studied the interplay between Se and the secondary plant metabolite cardamonin, an Nrf2-activating chalcone, in the regulation of Nrf2-controlled antioxidant enzymes. Se-deficient and Se-repleted (sodium selenite-supplemented) human intestinal Caco-2 cells were exposed to cardamonin. Uptake of cardamonin by the Caco-2 cells was independent of their Se status. Cardamonin strongly induced gene expression of GPx2 and TrxR1. However, cardamonin treatment did not result in elevated GPx or TrxR activity and protein levels, possibly relating to a concomitant down-regulation of O-phosphoseryl-tRNA(Sec) kinase (PSTK), an enzyme involved in translation of selenoprotein mRNAs. On the other hand, induction of the Nrf2-regulated enzyme heme oxygenase 1 (HO-1) by cardamonin was diminished in Se-replete compared to Se-deficient cells. Our findings suggest that cardamonin interferes with the biosynthesis of Nrf2-regulated selenoenzymes, in contrast to the Nrf2-activating isothiocyanate compound sulforaphane, which has been shown earlier to synergize with Se-mediated cytoprotection. Conversely, the cellular Se status apparently affects the cardamonin-mediated induction of non-selenoprotein antioxidant enzymes such as HO-1.

TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-Ðºß inhibition.

Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Introduction of the proteasome-inhibitor bortezomib has improved MM prognosis and survival; however hypoxia-induced or acquired bortezomib resistance remains a clinical problem. This study highlighted the role of thioredoxin reductase 1 (TrxR1) in the hypoxia-induced and acquired bortezomib resistance in MM. Higher TrxR1 gene expression correlated with high-risk disease, adverse overall survival, and poor prognosis in myeloma patients. We demonstrated that hypoxia induced bortezomib resistance in myeloma cells and increased TrxR1 protein levels. Inhibition of TrxR1 using auranofin overcame hypoxia-induced bortezomib resistance and restored the sensitivity of hypoxic-myeloma cells to bortezomib. Hypoxia increased NF-Ðºß subunit p65 nuclear protein levels and TrxR1 inhibition decreased hypoxia-induced NF-Ðºß p65 protein levels in the nucleus and reduced the expression of NF-кß-regulated genes. In addition, higher TrxR1 protein levels were observed in bortezomib-resistant myeloma cells compared to the naïve cells, and its inhibition using either auranofin or TrxR1-specific siRNAs reversed bortezomib resistance. TrxR1 inhibition reduced p65 mRNA and protein expression in bortezomib-resistant myeloma cells, and also decreased the expression of NF-кß-regulated anti-apoptotic and proliferative genes. Thus, TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance by inhibiting the NF-Ðºß signaling pathway. Our findings demonstrate that elevated TrxR1 levels correlate with the acquisition of bortezomib resistance in MM. We propose considering TrxR1-inhibiting drugs, such as auranofin, either for single agent or combination therapy to circumvent bortezomib-resistance and improve survival outcomes of MM patients.

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.

Postprandial oxidative stress is modulated by dietary fat in adipose tissue from elderly people.

We have investigated whether dietary fat modifies the postprandial oxidative stress in adipose tissue of elderly people. Twenty participants received three diets for 4 weeks each: SFA-rich diet, Mediterranean (Med) diet enriched in MUFA with virgin olive oil, and a low-fat, high-carbohydrate diet enriched in n-3 PUFA (α-linolenic acid from plant origin) (CHO-PUFA diet). After 12 h of fasting, volunteers received a breakfast reflecting the fatty acid composition of the diet ingested in the preceding dietary period. Med diet induced higher postprandial SOD2 and TrxR mRNA levels, and CHO-PUFA diet induced higher GPx1 and TrxR mRNA levels compared with SFA-rich diet. Med and CHO-PUFA breakfasts induced a postprandial increase in plasma reduced glutathione (GSH), and a greater postprandial GSH/oxidized glutathione ratio compared to the SFA-rich diet. Our study suggests that the consumption of Med and CHO-PUFA diets may reduce postprandial oxidative stress compared to an SFA-rich diet, which may be due to higher antioxidant enzymes gene expression in adipose tissue.

Deep-sea water containing selenium provides intestinal protection against duodenal ulcers through the upregulation of Bcl-2 and thioredoxin reductase 1.

Deep-sea water (DSW), which is rich in micronutrients and minerals and with antioxidant and anti-inflammatory qualities, may be developed as marine drugs to provide intestinal protection against duodenal ulcers. We determined several characteristics in the modified DSW. We explored duodenal pressure, oxygenation, microvascular blood flow, and changes in pH and oxidative redox potential (ORP) values within the stomach and duodenum in response to tap water (TW, hardness: 2.48 ppm), DSW600 (hardness: 600 ppm), and DSW1200 (hardness: 1200 ppm) in Wistar rats and analyzed oxidative stress and apoptosis gene expressions by cDNA and RNA microarrays in the duodenal epithelium. We compared the effects of drinking DSW, MgCl2, and selenium water on duodenal ulcers using pathologic scoring, immunohistochemical analysis, and Western blotting. Our results showed DSW has a higher pH value, lower ORP value, higher scavenging H2O2 and HOCl activity, higher Mg2+ concentrations, and micronutrients selenium compared with TW samples. Water infusion significantly increased intestinal pressure, O2 levels, and microvascular blood flow in DSW and TW groups. Microarray showed DSW600, DSW1200, selenium water upregulated antioxidant and anti-apoptotic genes and downregulated pro-apoptotic gene expression compared with the TW group. Drinking DSW600, DSW1200, and selenium water but not Mg2+ water significantly enhanced Bcl-2 and thioredoxin reductase 1 expression. Bax/Bcl-2/caspase 3/poly-(ADP-ribose)-polymerase signaling was activated during the pathogenesis of duodenal ulceration. DSW drinking reduced ulcer area as well as apoptotic signaling in acetic acid-induced duodenal ulcers. DSW, which contains selenium, provides intestinal protection against duodenal ulcers through the upregulation of Bcl-2 and thioredoxin reductase 1.

Mitochondrial thioredoxin reductase inhibition, selenium status, and Nrf-2 activation are determinant factors modulating the toxicity of mercury compounds.

The thioredoxin system has essential functions in the maintenance of cellular redox homeostasis in the cytosol, nucleus, and mitochondria. Thioredoxin (Trx) and thioredoxin reductase (TrxR) are targets for mercury compounds in vitro and in vivo. This study aimed at understanding mechanistically how the mitochondrial and cytosolic thioredoxin systems were affected by mercurials, including the regulation of TrxR transcription. The effects of coexposure to selenite and mercurials on the thioredoxin system were also addressed. Results in HepG2 cells showed that TrxR1 expression was enhanced by Hg(2+), whereas exposure to MeHg decreased expression. Selenite exposure also increased the expression of TrxR1 and resulted in higher specific activity. Coexposure to 2 µM selenite and up to 5 µM Hg(2+) increased even further TrxR1 expression. This synergistic effect was not verified for MeHg, because TrxR1 expression and activity were reduced. Analysis of Nrf-2 translocation to the nucleus and TrxR mRNA suggests that induction of TrxR1 transcription was slower upon exposure to MeHg in comparison to Hg(2+). Subcellular fractions showed that MeHg affected the activity of the thioredoxin system equally in the mitochondria and cytosol, whereas Hg(2+) inhibited primarily the activity of TrxR2. The expression of TrxR2 was not upregulated by any treatment. These results show important differences between the mechanisms of toxicity of Hg(2+) and MeHg and stress the narrow range of selenite concentrations capable of antagonizing mercury toxicity. The results also highlight the relevance of the mitochondrial thioredoxin system (TrxR2 and Trx2) in the development of mercury toxicity.

Gambogic acid induces apoptosis in hepatocellular carcinoma SMMC-7721 cells by targeting cytosolic thioredoxin reductase.

The thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus and TrxR2 in mitochondria, are essential mammalian selenocysteine (Sec)-containing flavoenzymes with a unique C-terminal -Gly-Cys-Sec-Gly active site. TrxRs are often overexpressed in a number of human tumors, and the reduction of their expression in malignant cells reverses tumor growth, making the enzymes attractive targets for anticancer drug development. Gambogic acid (GA), a natural product that has been used in traditional Chinese medicine for centuries, demonstrates potent anticancer activity in numerous types of human cancer cells and has entered phase II clinical trials. We discovered that GA may interact with TrxR1 to elicit oxidative stress and eventually induce apoptosis in human hepatocellular carcinoma SMMC-7721 cells. GA primarily targets the Sec residue in the antioxidant enzyme TrxR1 to inhibit its Trx-reduction activity, leading to accumulation of reactive oxygen species and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 in cells attenuates the cytotoxicity of GA, whereas knockdown of TrxR1 sensitizes cells to GA. Targeting of TrxR1 by GA thus discloses a previously unrecognized mechanism underlying the biological action of GA and provides useful information for further development of GA as a potential agent in the treatment of cancer.

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death.

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death.