Trx System Research Articles

Thioredoxin System Protein Expression Is Associated with Poor Clinical Outcome in Adult and Paediatric Gliomas and Medulloblastomas

The thioredoxin (Trx) system is an important enzyme family that regulates cellular redox homeostasis. Protein expression of Trx system family members has been assessed in various cancers and linked to various clinicopathological variables, disease progression, treatment response and survival outcomes but information is lacking in brain tumours. Expression of the system was therefore examined, by immunohistochemistry in different brain tumour types, adult and paediatric cases, to determine if expression was of importance to clinical outcome. Trx system proteins were expressed, to variable levels, across all brain tumour types with significant variations in expression between different tumour types/grades/regions. High Trx reductase (TrxR) expression was linked to worse prognosis across all cohorts. High cytoplasmic TrxR expression was significantly associated with adverse overall survival (OS) in adult glioblastoma (P = 0.027) and paediatric low-grade glioma (LGG) patients (P = 0.012). High expression of nuclear TrxR, cytoplasmic and nuclear Trx and Trx-interacting protein (TxNIP) was associated with improved OS in paediatric LGGs (P = 0.031, P < 0.001, P = 0.044 and P = 0.018, respectively). For patients with high-grade gliomas, both high cytoplasmic TrxR and Trx expression were associated with poor OS (P = 0.002 and P = 0.007, respectively). In medulloblastoma, high expression of cytoplasmic TrxR and Trx and nuclear Trx was associated with worse prognosis (P = 0.013, P = 0.033 and P = 0.007, respectively); with cytoplasmic TrxR and nuclear Trx remaining so in multivariate analysis (P = 0.009 and P = 0.013, respectively). The consistent finding that high levels of cytoplasmic TrxR are associated with a worse prognosis across all cohorts suggests that TrxR is an important therapeutic target in brain cancers.

Paracrine regulation and improvement of β-cell function by thioredoxin

The failure of insulin-producing β-cells is the underlying cause of hyperglycemia in diabetes mellitus. β-cell decay has been linked to hypoxia, chronic inflammation, and oxidative stress. Thioredoxin (Trx) proteins are major actors in redox signaling and essential for signal transduction and the cellular stress response. We have analyzed the cytosolic, mitochondrial, and extracellular Trx system proteins in hypoxic and cytokine-induced stress using β-cell culture, isolated pancreatic islets, and pancreatic islet transplantation modelling low oxygen supply.Protein levels of cytosolic Trx1 and Trx reductase (TrxR) 1 significantly decreased, while mitochondrial Trx2 and TrxR2 increased upon hypoxia and reoxygenation. Interestingly, Trx1 was secreted by β-cells during hypoxia. Moreover, murine and human pancreatic islet grafts released Trx1 upon glucose stimulation. Survival of transplanted islets was substantially impaired by the TrxR inhibitor auranofin.Since a release was prominent upon hypoxia, putative paracrine effects of Trx1 on β-cells were examined. In fact, exogenously added recombinant hTrx1 mitigated apoptosis and preserved glucose sensitivity in pancreatic islets subjected to hypoxia and inflammatory stimuli, dependent on its redox activity. Human subjects were studied, demonstrating a transient increase in extracellular Trx1 in serum after glucose challenge. This increase correlated with better pancreatic islet function. Moreover, hTrx1 inhibited the migration of primary murine macrophages.In conclusion, our study offers evidence for paracrine functions of extracellular Trx1 that improve the survival and function of pancreatic β-cells.

Enhanced thioredoxin, glutathione and Nrf2 antioxidant systems by safflower extract and aceglutamide attenuate cerebral ischaemia/reperfusion injury.

A large number of reactive oxygen species (ROS) aggravate cerebral damage after ischaemia/reperfusion (I/R). Glutathione (GSH), thioredoxin (Trx) and nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) represent three major antioxidant systems and play vital roles in affecting each other in eliminating ROS. Identification of drugs targeting triple antioxidant systems simultaneously is vital for inhibiting oxidative damage after cerebral I/R. This study investigated the protective effect of safflower extract and aceglutamide (SAAG) against cerebral I/R injury through modulating multiple antioxidant systems of GSH, Trx and Nrf2 and identified each role of its component acegluatminde (AG) and safflower extract (SA) on these systems. Safflower extract and aceglutamide and its two components decreased neurological deficit scores, infarction rate, apoptosis and oxidative damage after cerebral I/R while enhanced cell viability, decreased reactive oxygen species and nitric oxide level in H2O2‐induced PC12 cell model. Importantly, compared to its two components, SAAG demonstrated more effective enhancement of GSH, Nrf2 and Trx systems and a better protection against cerebral I/R injury. The enhanced antioxidant systems prevented ASK1 activation and suppressed subsequent p38 and JNK cascade‐mediated apoptosis. Moreover, inhibition of Trx and Nrf2 systems by auranofin and ML385 abolished SAAG‐mediated protection, respectively. Thus, enhanced triple systems by SAAG played a better protective role than those by SA or AG via inhibition of ASK1 cascades. This research provided evidence for the necessity of combination drugs from the perspective of multiple antioxidant systems. Furthermore, it also offers references for the study of combination drugs and inspires novel treatments for ischaemic stroke.

Cross-talk between Bcr-abl and the Thioredoxin System in Chronic Myeloid Leukaemia: Implications for CML Treatment.

Chronic myeloid leukaemia (CML) is currently treated with inhibitors of the CML specific oncoprotein, bcr-abl. While this strategy is initially successful, drug resistance can become a problem. Therefore, new targets need to be identified to ensure the disease can be appropriately managed. The thioredoxin (Trx) system, comprised of Trx, thioredoxin reductase (TrxR), and NADPH, is an antioxidant system previously identified as a target for therapies aimed at overcoming drug resistance in other cancers. We assessed the effectiveness of TrxR inhibitors on drug resistant CML cells and examined links between TrxR and the bcr-abl cell-signalling pathway. Two TrxR inhibitors, auranofin and [Au(d2pype)2]Cl, increased intracellular ROS levels and elicited apoptosis in both sensitive and imatinib resistant CML cells. Inhibition of TrxR activity by these pharmacological inhibitors, or by specific siRNA, also resulted in decreased bcr-abl mRNA and protein levels, and lower bcr-abl downstream signalling activity, potentially enhancing the effectiveness of TrxR inhibitors as CML therapies. In addition, imatinib resistant CML cell lines showed upregulated expression of the Trx system. Furthermore, analysis of datasets showed that CML patients who did not respond to imatinib had higher Trx mRNA levels than patients who responded to treatment. Our study demonstrates a link between the Trx system and the bcr-abl protein and highlights the therapeutic potential of targeting the Trx system to improve CML patients’ outcomes.

Thioredoxin-1 distinctly promotes NF-κB target DNA binding and NLRP3 inflammasome activation independently of Txnip.

Antioxidant systems, such as the thioredoxin-1 (Trx1) pathway, ensure cellular redox homeostasis. However, how such systems regulate development and function of myeloid cells is barely understood. Here we show that in contrast to its critical role in T cells, the murine Trx1 system is dispensable for steady-state myeloid-cell hematopoiesis due to their capacity to tap the glutathione/glutaredoxin pathway for DNA biosynthesis. However, the Trx1 pathway instrumentally enables nuclear NF-κB DNA-binding and thereby pro-inflammatory responses in monocytes and dendritic cells. Moreover, independent of this activity, Trx1 is critical for NLRP3 inflammasome activation and IL-1β production in macrophages by detoxifying excessive ROS levels. Notably, we exclude the involvement of the Trx1 inhibitor Txnip as a redox-sensitive ligand of NLRP3 as previously proposed. Together, this study suggests that targeting Trx1 may be exploited to treat inflammatory diseases.

What is the role of the thioredoxin antioxidant complex in relation to HAM/TSP?

We have little information about the definite role of the thioredoxin antioxidant complex system during viral infection, particularly during human T-cell lymphotropic virus type 1 (HTLV-1) infection and the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) state. Therefore, we conducted comprehensive next-generation sequencing (NGS) analysis to determine Trx system expression changes in three categories of subjects: sero-negative HTLV-1 individuals, asymptomatic HTLV-1 people and HAM/TSP patients. We found that Trx capacity is reduced in the HAM/TSP state compared to healthy individuals, which indicates increasing inflammation and reduction of apoptosis, which might contribute to the progression of inflammation in the spinal cord, which in turn may develop into the HAM/TSP state.

Thioredoxin and protein-disulfide isomerase selectivity for redox regulation of proteins in Corynebacterium glutamicum.

Thioredoxins (Trxs) and protein-disulfide isomerases (PDIs) are believed to play a pivotal role in ensuring the proper folding of proteins, facilitating appropriate functioning of proteins, and maintaining intracellular redox homeostasis in bacteria. Two thioredoxins (Trxs) and three thiol-disulfide isomerases (PDIs) have been annotated in Corynebacterium glutamicum. However, nothing is known about their functional diversity in the redox regulation of proteins. Thus, we here analyzed the Trx- and PDI-dependent redox shifts of ribonucleotide reductase (RNR), insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and several thiol-dependent peroxidases by measuring enzyme activity and thiol status in vitro. We found that the two Trxs and the three PDIs had activities in the cleavage of the disulfidebond, whereas the PDIs had a lower efficiency than the two Trxs. Trx2 could activate thiol-dependent peroxidases with an efficiency comparable with that of Trx1, but the PDIs were inefficient. The redox-active Cys-X-X-Cys motif harbored in both Trxs and PDIs was essential to supply efficiently the donor of reducing equivalents for protein disulfides. In addition, stress-responsive extracytoplasmic function (ECF)-sigma factor H (SigH)-dependent Trxs and PDIs expressions were observed. These results contributed importantly to our overall understanding of reducing functionality of the Trx and PDI systems, and also highlighted the complexity and plasticity of the intracellular redox network.

Upregulation of thioredoxin and its reductase attenuates arsenic trioxide‑induced growth suppression in human pulmonary artery smooth muscle cells by reducing oxidative stress.

The thioredoxin (Trx) system is an important enzymatic complex involved in cellular redox homeostasis. Arsenic trioxide (ATO; As2O3) is known to trigger cell death in vascular smooth muscle cells (VSMCs) via oxidative stress. In the present study, the effects of changes in thioredoxin1 (Trx1) and Trx reductase1 (TrxR1) on cell growth, death, reactive oxygen species (ROS), and glutathione (GSH) levels were evaluated in ATO‑treated human pulmonary artery smooth muscle cells (HPASMCs). ATO inhibited growth and induced cell death in the HPASMCs at 24h. Overexpression of Trx1 and TrxR1 using adenoviruses attenuated cell growth inhibition caused by ATO and partially prevented cell death. ATO increased ROS levels including the mitochondrial superoxide anion (O2•-) at 5min. Administration of adTrx1 or adTrxR1 reduced the increased mitochondrial O2•- level in these cells. HPASMCs treated with Trx1 or TrxR1 siRNA showed increases in ROS levels with or without treatment of ATO at 5min. Although ATO transiently increased GSH levels at 5min, Trx1 and TrxR1 siRNAs reduced the increased GSH levels in these cells. In addition, PX‑12 (a Trx1 inhibitor) and auranofin (a TrxR1 inhibitor) diminished the cellular metabolism in HPASMCs at 4h, accompanied by an increase in ROS level and a decrease in GSH level. In conclusion, upregulation of Trx1 and TrxR1 somewhat decreased cell growth inhibition and death in ATO‑treated HPASMCs, which was accompanied by reduced oxidative stress.

The novel thioredoxin reductase inhibitor A-Z2 triggers intrinsic apoptosis and shows efficacy in the treatment of acute myeloid leukemia

Chemoresistance and high incidence of relapse in acute myeloid leukemia (AML) patients are associated with thioredoxin (Trx) overexpression. Thus, targeting the Trx system has emerged as a promising approach to treating AML. Both arsenicals and azelaic acid (AZA) are thioredoxin reductase (TrxR) inhibitors and possess antileukemic effects. In this study, to exploit agents with higher potency and lower toxicity, we got some organic arsenicals and further synthesized a series of targeted compounds by binding AZA to organic arsenicals, and then screened the most effective one, N-(4-(1, 3, 2-dithiarsinan-2-yl) phenyl)-azelamide (A-Z2). A-Z2 showed a stronger inhibitory effect against TrxR activity and in AML cell lines than did AZA or arsenicals. Additionally, A-Z2 was less toxic to healthy cells compared with traditional chemotherapeutic drugs. A-Z2 induces apoptosis by collapsing of mitochondrial membrane potential, reducing ATP level, releasing of cytochrome c and TNF-α, activating of caspase 9, 8 and 3. Analysis of the mechanism revealed that A-Z2 activates the intrinsic apoptotic pathway by directly selectively targeting TrxR/Trx and indirectly inhibiting NF-κB. A-Z2's better efficacy and safety profile against arsenicals and azelaic acid were also evident in vivo. A-Z2 had better plasma stability and biological activity in rats. A-Z2-treated mice displayed significant symptom relief and prolonged survival in a patient-derived xenograft (PDX) AML model. Herein, our study provides a novel antitumor candidate and approach for treating AML.

Age-Associated Changes in Antioxidants and Redox Proteins of Rat Heart.

Oxidative stress and decline in cellular redox regulation have been hypothesized to play a key role in cardiovascular aging; however, data on antioxidant and redox regulating systems in the aging heart are controversial. The aim of the present study was to examine the effect of aging on critical antioxidant enzymes and two major redox-regulatory systems glutathione (GSH) and thioredoxin (Trx) system in hearts from adult (6-month-old), old (15-month-old), and senescent (26-month-old) rats. Aging was associated with a non-uniform array of changes, including decline in contents of reduced GSH and total mercaptans in the senescent heart. The activities of Mn-superoxide dismutase (SOD2), glutathione peroxidase (GPx), glutathione reductase (GR), and thioredoxin reductase (TrxR) exhibited an age-related decline, whereas catalase was unchanged and Cu,Zn-superoxide dismutase (SOD1) displayed only slight decrease in old heart and was unchanged in the senescent heart. GR, Trx, and peroxiredoxin levels were significantly reduced in old and/or senescent hearts, indicating a diminished expression of these proteins. In contrast, SOD2 level was unchanged in the old heart and was slightly elevated in the senescent heart. Decline in GPx activity was accompanied by a loss of GPx level only in old rats, the level in senescent heart was unchanged. These results indicate age-related posttranslational protein modification of SOD2 and GPx. In summary, our data suggest that changes are more pronounced in senescent than in old rat hearts and support the view that aging is associated with disturbed redox balance that could alter cellular signaling and regulation.

C57BL/6J mice upregulate catalase to maintain the hydrogen peroxide buffering capacity of liver mitochondria

Here, we demonstrate that the upregulation of catalase is required to compensate for the loss of nicotinamide nucleotide transhydrogenase (NNT) to maintain hydrogen peroxide (H2O2) steady-state levels in C57BL/6J liver mitochondria. Our investigations using the closely related mouse strains C57BL/6NJ (6NJ; +NNT) and C57BL/6J (6J; -NNT) revealed that NNT is required for the provision of NADPH and that the upregulation of isocitrate dehydrogenase-2 (IDH2) activity is not enough to compensate for the absence of NNT, which is consistent with previous observations. Intriguingly, despite the absence of NNT, 6J mitochondria had rates of H2O2 production (58.56 ± 3.79 pmol mg−1 min−1) that were similar to samples collected from 6NJ mice (72.75 ± 14.26 pmol mg−1 min−1) when pyruvate served as the substrate. However, 6NJ mitochondria energized with succinate produced significantly less H2O2 (59.95 ± 2.13 pmol mg−1 min−1) when compared to samples from 6J mice (116.39 ± 20.74 pmol mg−1 min−1), an effect that was attributed to the presence of NNT. Further investigations into the H2O2 eliminating capacities of these mitochondria led to the novel observation that 6J mitochondria compensate for the loss of NNT by upregulating catalase. Indeed, 6NJ and 6J mitochondria energized with pyruvate or succinate displayed similar rates for H2O2 elimination, quenching ~84% and ~86% of the H2O2, respectively, in the surrounding medium within 30 s. However, inclusion of palmitoyl-CoA, an NNT inhibitor, significantly limited H2O2 degradation by 6NJ mitochondria only (~55% of H2O2 eliminated in 30 s). Liver mitochondria from 6J mice treated with palmitoyl-CoA still cleared ~80% of the H2O2 from the surrounding environment. Inhibition of catalase with triazole compromised the capacity of 6J mitochondria to maintain H2O2 steady-state levels. By contrast, disabling NADPH-dependent antioxidant systems had a limited effect on the H2O2 clearing capacity of 6J mitochondria. Liver mitochondria collected from 6NJ mice, on the other hand, were more reliant on the GSH and TRX systems to clear exogenously added H2O2. However, catalase still played an integral in eliminating H2O2 in 6NJ liver mitochondria. Immunoblot analyses demonstrated that catalase protein levels were ~7.7-fold higher in 6J mitochondria. Collectively, our findings demonstrate for the first time that 6J liver mitochondria compensate for the loss of NNT by increasing catalase levels for the maintenance of H2O2 steady-state levels. In general, our observations reveal that catalase is an integral arm of the antioxidant response in liver mitochondria.

Distinct Roles of Shewanella oneidensis Thioredoxin in Regulation of Cellular Responses to Hydrogen and Organic Peroxides.

The thioredoxin (Trx) and glutaredoxin (Grx) antioxidant systems are deeply involved in bacterial response to oxidative stress, but to date, we know surprisingly little about the roles of these systems in response to reactive oxygen species (ROS) other than hydrogen peroxide (H2O2). In this study, we used Shewanella oneidensis, an environmental bacterium, as a research model to investigate the roles of Trx and Grx in oxidative stress response because it has functionally intertwined ROS responsive regulators OxyR and OhrR. We found that Trx1 is the major thiol/disulfide redox system and that in its absence a Grx system becomes essential under normal conditions. Although overshadowed by Trx1 in the wild type, Trx2 can fully replace Trx1 in physiology when overproduced. Trx1 is required for OxyR to function as a repressor but, more importantly, plays a critical role in the cellular response to organic peroxide (OP) by mediating the redox status of OhrR but not OP scavenger OhrA. While none of the trx and grx genes are OxyR dependent, trxA and trxC are affected by OhrR indirectly. Additional data suggest that depletion of glutathione is likely the cue to trigger induced expression of trxA and trxC These findings underscore the particular importance of Trx in the bacterial OP stress response.IMPORTANCE The Trx and Grx systems are deeply involved in bacterial responses to H2O2-induced oxidative stress. However, little is known about their roles in response to other ROS, such as organic peroxides (OPs). In this study, we used S. oneidensis as a research model to investigate the interplay between Trx/Grx and OxyR/OhrR. We show that Trxs mediate the redox status of transcriptional OP-responding regulator OhrR. Although none of the trx or grx genes are directly controlled by OxyR or OhrR, expression of trxA and trxC is induced by tert-butyl hydroperoxide (t-BHP). We further show that the trxA and trxC genes respond to effects of glutathione (GSH) depletion rather than oxidation. These findings underscore the particular importance of Trx in the bacterial OP stress response.

Olean-28,13b-olide 2 plays a role in cisplatin-mediated apoptosis and reverses cisplatin resistance in human lung cancer through multiple signaling pathways

Lung cancer, similar to other chronic diseases, occurs due to perturbations in multiple signaling pathways. Mono-targeted therapies are not ideal since they are not likely to be effective for the treatment and prevention of lung cancer, and are often associated with drug resistance. Therefore, the development of multi-targeted agents is required for novel lung cancer therapies. Thioredoxin reductase (TrxR or TXNRD1) is a pivotal component of the thioredoxin (Trx) system. Various types of tumor cells are able to overexpress TrxR/Trx proteins in order to maintain tumor survival, and this overexpression has been shown to be associated with clinical outcomes, including irradiation and drug resistance. Emerging evidence has indicated that oleanolic acid (OA) and its derivatives exhibit potent anticancer activity, and are able to overcome drug resistance in cancer cell lines. In the present study, it was demonstrated that a novel synthesized OA family compound, olean-28,13b-olide 2 (OLO-2), synergistically enhanced cisplatin (CDDP)-mediated apoptosis, led to the activation of caspase-3 and the generation of reactive oxygen species (ROS), induced DNA damage, and inhibited the activation of the extracellular-signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3), AKT and nuclear factor-κB (NF-κB) pathways in human multidrug-resistant A549/CDDP lung adenocarcinoma cells. Subsequent analyses revealed that OLO-2 inhibited P-glycoprotein (P-gp or ABCB1) and TrxR by reducing their expression at the protein and mRNA levels, and by suppressing P-gp ATPase and TrxR activities. Further biological evaluation indicated that OLO-2 significantly reduced Trx and excision repair cross-complementary1 (ERCC1) protein expression and significantly inhibited the proliferation of drug-sensitive (A549) and multidrug-resistant (A549/CDDP) non-small cell lung cancer (NSCLC) cells, but had no effect on non-tumor lung epithelial-like cells. In addition, the present study demonstrated, for the first time, to the best of our knowledge, that overexpressing or knocking down TrxR in NSCLC cells enhanced or attenuated, respectively, the resistance of NSCLC cells against CDDP, which indicated that TrxR plays an important role in CDDP resistance and functions as a protector of NSCLC against chemotherapeutic drugs. OLO-2 treatment also exhibited up to 4.6-fold selectivity against human lung adenocarcinoma cells. Taken together, the results of the present study shed light on the drug resistance-reversing effects of OLO-2 in lung cancer cells.

The Thioredoxin System is Regulated by the ASK-1/JNK/p38/Survivin Pathway During Germ Cell Apoptosis.

The aim is to explore the mechanism of the apoptosis signal-regulating kinase-1 (ASK-1) signaling pathway and the involvement of the thioredoxin (Trx) system during testicular ischemia reperfusion injury (tIRI) by using ASK-1 specific inhibitor, NQDI-1. Male Sprague-Dawley rats (n = 36, 250–300 g) were equally divided into 3 groups: sham, tIRI, and tIRI + NQDI-1 (10 mg/kg, i.p, pre-reperfusion). For tIRI induction, the testicular cord and artery were occluded for 1 h followed by 4 h of reperfusion. Histological analyses, protein immunoexpression, biochemical assays, and real-time PCR were used to evaluate spermatogenesis, ASK-1/Trx axis expression, enzyme activities, and relative mRNA expression, respectively. During tIRI, ipsilateral testes underwent oxidative stress indicated by low levels of superoxide dismutase (SOD) and Glutathione (GSH), increased oxidative damage to lipids and DNA, and spermatogenic damage. This was associated with induced mRNA expression of pro-apoptosis genes, downregulation of antiapoptosis genes, increased caspase 3 activity and activation of the ASK-1/JNK/p38/survivin apoptosis pathway. In parallel, the expression of Trx, Trx reductase were significantly reduced, while the expression of Trx interacting protein (TXNIP) and the NADP+/ nicotinamide Adenine Dinucleotide phosphate (NADPH) ratio were increased. These modulations were attenuated by NQDI-1 treatment. In conclusion, the Trx system is regulated by the ASK-1/Trx/TXNIP axis to maintain cellular redox homeostasis and is linked to tIRI-induced germ cell apoptosis via the ASK-1/JNK/p38/survivin apoptosis pathway.

Anticancer activity of a Gold(I) phosphine thioredoxin reductase inhibitor in multiple myeloma

Multiple myeloma (MM), the second most common haematological malignancy, is a clonal plasma B-cell neoplasm that forms within the bone marrow. Despite recent advancements in treatment, MM remains an incurable disease. Auranofin, a linear gold(I) phosphine compound, has previously been shown to exert a significant anti-myeloma activity by inhibiting thioredoxin reductase (TrxR) activity. A bis-chelated tetrahedral gold(I) phosphine complex [Au(d2pype)2]Cl (where d2pype is 1,2-bis(di-2-pyridylphosphino)ethane) was previously designed to improve the gold(I) compound selectivity towards selenol- and thiol-containing proteins, such as TrxR. In this study, we show that [Au(d2pype)2]Cl significantly inhibited TrxR activity in both bortezomib-sensitive and resistant myeloma cells, which led to a significant reduction in cell proliferation and induction of apoptosis, both of which were dependent on ROS. In clonogenic assays, treatment with [Au(d2pype)2]Cl completely abrogated the tumourigenic capacity of MM cells, whereas auranofin was less effective. We also show that [Au(d2pype)2]Cl exerted a significant anti-myeloma activity in vivo in human RPMI8226 xenograft model in immunocompromised NOD/SCID mice. The MYC oncogene, known to drive myeloma progression, was downregulated in both in vitro and in vivo models when treated with [Au(d2pype)2]Cl. This study highlights the “proof of concept” that improved gold(I)-based compounds could potentially be used to not only treat MM but as an alternative tool to understand the role of the Trx system in the pathogenesis of this blood disease.

Quercetin protects human granulosa cells against oxidative stress via thioredoxin system

Granulosa Cells (GCs) are sensitive to excessive production of reactive oxygen species (ROS). Quercetin (QUR) is a free radical scavenger which can alleviate oxidative stress through nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/antioxidant response element (ARE) pathway and thioredoxin (Trx) system. We aimed to explore the probable protective role of QUR on cultured human GCs treated with hydrogen peroxide (H2O2) as an inducer of oxidative stress. MTT assay was applied for evaluating the cell cytotoxicity of QUR and H2O2. The rate of apoptotic cells and intracellular ROS generation were determined by Annexin V-FITC/PI staining and 2′-7′-dichlorodihydrofluorescein diacetate fluorescent probes (DCFH-DA), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blot analysis were used to evaluate the gene and protein expression of Nrf2 and kelch-like ech-associated protein 1 (Keap1)1. The Nrf2 and Trx activities were measured by Enzyme-linked Immunosorbent Assay (ELISA). The results indicated that QUR pretreatment can decrease ROS production and apoptosis induced by H2O2. In addition, QUR increased Nrf2 gene and protein expression, as well as its nuclear translocation. Moreover, in QUR-treated group, a lower level of Keap1 protein was observed, which was not reported as significant. The results also indicated a significant correlation between the expression of Nrf2 and Keap1 in QUR-treated group. Further, QUR protected GCs from oxidative stress by increasing Trx gene expression and activity. This study suggests that QUR as a supplementary factor may protect GCs from oxidative stress in diseases related to this condition.

Glutathione-glutaredoxin is an efficient electron donor system for mammalian p53R2–R1-dependent ribonucleotide reductase

Deoxyribonucleotides are DNA building blocks and are produced de novo by reduction of ribose to deoxyribose. This reduction is catalyzed by ribonucleotide reductase (RNR), a heterodimeric tetramer enzyme in mammalian cells, having one of two free radical-containing subunits called R2 and p53R2. R2 is S-phase specific and used for DNA replication, whereas p53R2 functions in DNA repair and mitochondrial DNA synthesis. The larger RNR subunit, R1, has catalytically active cysteine thiols in its buried active site and a C-terminal swinging arm, with a Cys-Leu-Met-Cys sequence suggested to act as a shuttle dithiol/disulfide for electron transport. After each catalytic cycle the active site contains a disulfide, which has to be reduced for turnover. Thioredoxin (Trx) and glutaredoxin (Grx) systems have been implicated as electron donors for the RNR disulfide reduction via the swinging arm. Using mouse R1-R2 and R1-p53R2 complexes, we found here that the catalytic efficiency of the GSH-Grx system is 4-6 times higher than that of the Trx1 system. For both complexes, the Vmax values for Grx are strongly depended on GSH concentrations. The GSH disulfide resulting from the Grx reaction was reduced by NADPH and GSH reductase and this enzyme was essential because reaction with GSH alone yielded only little activity. These results indicate that C-terminal shuttle dithiols of mammalian R1 have a crucial catalytic role and that the GSH-Grx system favors the R1-p53R2 enzyme for DNA replication in hypoxic conditions, mitochondrial DNA synthesis, and in DNA repair outside the S-phase.

The thioredoxin system and cancer therapy: a review.

Thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH are key members of the Trx system that is involved in redox regulation and antioxidant defense. In recent years, several researchers have provided information about the roles of the Trx system in cancer development and progression. These reports indicated that many tumor cells express high levels of Trx and TrxR, which can be responsible for drug resistance in tumorigenesis. Inhibition of the Trx system may thus contribute to cancer therapy and improving chemotherapeutic agents. There are now a number of effective natural and synthetic inhibitors with chemotherapy applications possessing antitumor activity ranging from oxidative stress induction to apoptosis. In this article, we first described the features and functions of the Trx system and then reviewed briefly its correlations with cancer. Finally, we summarized the present knowledge about the Trx/TrxR inhibitors as anticancer drugs.

Thioredoxin inhibitor PX-12 induces mitochondria-mediated apoptosis in acute lymphoblastic leukemia cells.

Imbalances in redox homeostasis have been described to be involved in the development, progression and relapse of leukemia. As the thioredoxin (Trx) system, one of the major cellular antioxidant networks, has been implicated in acute lymphoblastic leukemia (ALL), we investigated the therapeutic potential of Trx inhibition in ALL. Here, we show that the Trx inhibitor PX-12 reduced cell viability and induced cell death in a dose- and time-dependent manner in different ALL cell lines. This antileukemic activity was accompanied by an increase in reactive oxygen species (ROS) levels and enhanced PRDX3 dimerization. Pre-treatment with the thiol-containing ROS scavenger N-acetylcysteine (NAC), but not with non-thiol-containing scavengers α-tocopherol (α-Toc) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP), significantly rescued PX-12-induced cell death. Furthermore, PX-12 triggered activation of BAK. Importantly, knockdown of BAK reduced PX-12-stimulated ROS production and cell death. Similarly, silencing of NOXA provided significant protection from PX-12-mediated cell death. The relevance of mitochondria-mediated, caspase-dependent apoptosis was further supported by data showing that PX-12 triggered cleavage of caspase-3 and that addition of the broad-range caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD.fmk) potently blocked cell death upon PX-12 treatment. This study provides novel insights into the mechanisms of PX-12-induced cell death in ALL and further highlights the therapeutic potential of redox-active compounds in ALL.

Upregulated thioredoxin and its reductase prevent H2O2-induced growth inhibition and death in human pulmonary artery smooth muscle cells

The thioredoxin (Trx) system controls cellular redox in vascular smooth muscle cells. The present study investigated the roles of Trx1 and Trx reductase1 (TrxR1) proteins in regulation of cell growth, death, reactive oxygen species (ROS) and glutathione (GSH) levels in hydrogen peroxide (H2O2)-treated human pulmonary artery smooth muscle (HPASM) cells. H2O2 induced growth inhibition and cell death in HPASM cells over 24 h. Overexpression of Trx1 and TrxR1 using adenoviruses significantly weakened cell growth inhibition and cell death caused by H2O2. Increases in ROS levels including mitochondrial superoxide anion (O2•−) were observed as early as 5–30 min after H2O2 addition. Administration of adTrxR1 attenuated H2O2-induced increases in ROS levels at 30–180 min. adTrx1 and adTrxR1 significantly reduced the increases in O2•− level in H2O2-treated HPASM cells at 24 h. Furthermore, HPASM cells transfected with Trx1 or TrxR1 siRNA showed increases in ROS levels with or without H2O2 at 5 min. While H2O2 transiently decreased GSH level at 5 min, Trx1 and TrxR1 siRNA intensified the decrease in GSH level. In conclusion, upregulation of Trx1 and TrxR1 significantly attenuated cell growth inhibition and death in H2O2-treated HPASM cells. As a whole, Trx-related adenoviruses diminished H2O2-induced ROS level in HPASM cells whereas Trx-related siRNAs increased ROS levels and decreased GSH level in these cells.