Imeglimin Exhibits Novel Anti-Inflammatory Effects on High-Glucose-Stimulated Mouse Microglia through ULK1-Mediated Suppression of the TXNIP-NLRP3 Axis.

Type 2 diabetes mellitus (T2DM) is an epidemiological risk factor for dementia and has been implicated in multifactorial pathologies, including neuroinflammation. In the present study, we aimed to elucidate the potential anti-inflammatory effects of imeglimin, a novel antidiabetic agent, on high-glucose (HG)-stimulated microglia. Mouse microglial BV2 cells were stimulated with HG in the presence or absence of imeglimin. We examined the effects of imeglimin on the levels of proinflammatory cytokines, intracellular reactive oxygen species (ROS), mitochondrial integrity, and components related to the inflammasome or autophagy pathways in these cells. Our results showed that imeglimin suppressed the HG-induced production of interleukin-1beta (IL-1ß) by reducing the intracellular ROS levels, ameliorating mitochondrial dysfunction, and inhibiting the activation of the thioredoxin-interacting protein (TXNIP)-NOD-like receptor family pyrin domain containing 3 (NLRP3) axis. Moreover, the inhibitory effects of imeglimin on the TXNIP-NLRP3 axis depended on the imeglimin-induced activation of ULK1, which also exhibited novel anti-inflammatory effects without autophagy induction. These findings suggest that imeglimin exerted novel suppressive effects on HG-stimulated microglia through the ULK1-TXNIP-NLRP3 axis, and may, thereby, contribute to the development of innovative strategies to prevent T2DM-associated cognitive impairment.

Potential of Forsythoside I as a therapeutic approach for acute lung injury: Involvement of TXNIP/NLRP3 inflammasome.

OBJECTIVE: To explore the role of Forsythoside I (FI) in acute lung injury (ALI) mouse and its underling mechanism. METHODS: The cell models of ALI are constructed by LPS induction. After pretreatment with different concentrations of FI, the lung injury is assessed by pathological changes of lung tissues and cell apoptosis. The cell viability, levels of pro-inflammatory cytokines, and the activation of TXNIP/NLRP3 pathway are inspected to investigate whether the effect of FI on inflammatory response is exerted by regulating the TXNIP/NLRP3 pathway. RESULTS: LPS induces inflammatory cell infiltration, tissue necrosis and pulmonary interstitial edema of mouse tissues, and LPS increases the protein concentration and levels of pro-inflammatory factors in mouse BALF. Additionally, enhanced cell apoptotic level, increased W/D ratio and MPO activity, as well as suppressed SOD activity are observed in LPS-induced mouse models. Those inflammation response, oxidative stress and lung injury can be attenuated by FI (12.5 mg/kg, 25 mg/kg, 50 mg/kg) in a dose-dependent manner. Meanwhile, both in vitro and in vivo studies reveal that FI can lead to suppressed TXNIP expression and inactivated NLRP3 inflammasomes. TXNIP is an upstream target of NLRP3, and FI mitigates ALI by decreasing TXNIP to block NLRP3 inflammasomes. CONCLUSION: FI protects against ALI through the mediation of TXNIP/NLRP3 inflammasome axis and therefore has a certain potential for ALI treatment.

Cytotoxic and radiosensitising effects of a novel thioredoxin reductase inhibitor in breast cancer.

Radiotherapy is an effective treatment modality for breast cancer but, unfortunately, not all patients respond fully with a significant number experiencing local recurrences. Overexpression of thioredoxin and thioredoxin reductase has been reported to cause multidrug and radiation resistance - their inhibition may therefore improve therapeutic efficacy. Novel indolequinone compounds have been shown, in pancreatic cancer models, to inhibit thioredoxin reductase activity and exhibit potent anticancer activity. The present study evaluates, using in vitro breast cancer models, the efficacy of a novel indolequinone compound (IQ9) as a single agent and in combination with ionising radiation using a variety of endpoint assays including cell proliferation, clonogenic survival, enzyme activity, and western blotting. Three triple-negative breast cancer (MDA-MB-231, MDA-MB-468, and MDA-MB-436) and two luminal (MCF-7 and T47D) breast cancer cell lines were used. Results show that treatment with IQ9 significantly inhibited thioredoxin reductase activity, and inhibited cell growth and colony formation of breast cancer cells with IC50 values in the low micromolar ranges. Enhanced radiosensitivity of triple-negative breast cancer cells was observed, with sensitiser enhancement ratios of 1.20-1.43, but with no evident radiosensitisation of luminal breast cancer cell lines. IQ9 upregulated protein expression of thioredoxin reductase in luminal but not in triple-negative breast cancer cells which may explain the observed differential radiosensitisation. This study provides important evidence of the roles of the thioredoxin system as an exploitable radiobiological target in breast cancer cells and highlights the potential therapeutic value of indolequinones as radiosensitisers.***This study was not part of a clinical trial. Clinical trial registration number: N/A.

Changes in the Expression of TBP-2 in Response to Histone Deacetylase Inhibitor Treatment in Human Endometrial Cells.

Histone deacetylase inhibitors (HDACi) induce apoptosis preferentially in cancer cells by caspase pathway activation and reactive oxygen species (ROS) accumulation. Suberoylanilide hydroxamic acid (SAHA), a HDACi, increases apoptosis via altering intracellular oxidative stress through thioredoxin (TRX) and TRX binding protein-2 (TBP-2). Because ROS accumulation, as well as the redox status determined by TBP-2 and TRX, are suggested as possible mechanisms for endometriosis, we queried whether SAHA induces apoptosis of human endometrial cells via the TRX-TBP-2 system in endometriosis. Eutopic endometrium from participants without endometriosis, and ectopic endometrium from patients with endometriosis, was obtained surgically. Human endometrial stromal cells (HESCs) and Ishikawa cells were treated with SAHA and cell proliferation was assessed using the CCK-8 assay. Real-time PCR and Western blotting were used to quantify TRX and TBP-2 mRNA and protein expression. After inducing oxidative stress, SAHA was applied. Short-interfering TRX (SiTRX) transfection was performed to see the changes after TRX inhibition. The mRNA and protein expression of TBP-2 was increased with SAHA concentrations in HESCs significantly. The mRNA TBP-2 expression was decreased after oxidative stress, upregulated by adding 2.5 µM of SAHA. The TRX/TBP-2 ratio decreased, apoptosis increased significantly, and SiTRX transfection decreased with SAHA. In conclusion, SAHA induces apoptosis by modulating the TRX/TBP-2 system, suggesting its potential as a therapeutic agent for endometriosis.

Anti-oxidative and anti-adipogenic effects of caffeine in an in vitro model of Graves' orbitopathy.

Oxidative stress and adipogenesis play key roles in the pathogenesis of Graves' orbitopathy (GO). In this study, the therapeutic effects of caffeine on the reduction of oxidative stress and adipogenesis were evaluated in primary cultured GO orbital fibroblasts in vitro. Orbital fibroblasts were cultured from orbital connective tissues obtained from individuals with GO. Intracellular reactive oxygen species (ROS) levels induced by hydrogen peroxide or cigarette smoke extract and the expression of anti-oxidative enzymes were measured after caffeine treatment. After adipogenic differentiation and caffeine treatment, cells were stained with Oil Red O and the levels of peroxisome proliferator activator γ (PPARγ), C/EBPα, and C/EBPß were determined by western blot analysis. Hydrogen peroxide and cigarette smoke extract increased the levels of intracellular ROS and anti-oxidative enzymes, which decreased in a dose-dependent manner upon pretreatment with caffeine in GO orbital fibroblasts. Oil Red-O staining results revealed a decrease in lipid droplets; furthermore, PPARγ, C/EBPα, and C/EBPß protein expression levels were inhibited upon treatment with caffeine during adipocyte differentiation. In conclusion, caffeine decreased oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. These findings may contribute to the development of new types of caffeine-containing pharmacological agents for use in the management of GO.

Inhibition of the thioredoxin system by PX-12 (1-methylpropyl 2-imidazolyl disulfide) impedes HIV-1 infection in TZM-bl cells.

Human immunodeficiency virus (HIV-1) entry is initiated by the binding between the viral envelope glycoprotein gp120 and the host receptor CD4, and followed by reduction of structural disulfides of gp120 and CD4. The host thioredoxin-1 (Trx1) efficiently reduces disulfides of gp120 and CD4 in vitro, and recently CD4-dependent HIV-1 entry was shown to be inhibited by anti-Trx1-antibodies, indicating a central role for Trx1. 1-methylpropyl-2-imidazolyl disulfide (PX-12) is a reversible inhibitor of the Trx1 system that may also cause a slow irreversible thioalkylation of Trx1. It was developed as an antitumor agent, however, the current study aimed to determine if it also has an anti-HIV-1 effect. We show that PX-12 has anti-HIV-1(IIIB) activity in TZM-bl cells, in fact, no virus was detected inside the cells in the presence of 10 µM PX-12. Moreover, PX-12 inhibited the enzymatic activity of Trx1 and the Trx1-dependent disulfide reduction of gp120. Microtubule polymerization and formation of acetylated microtubules were also inhibited, activities shown to be required for HIV-1 life cycle propagation. In conclusion, our data strengthens the notion that the early steps of the HIV-1 life cycle depends on the Trx1 system and indicate that the Trx1 system may be a rational drug target for HIV-1 treatment.

Binge ethanol and MDMA combination exacerbates HSP27 and Trx-1 (biomarkers of toxic cardiac effects) expression in right ventricle.

AIMS: Oxidative stress caused by exposure to drugs of abuse such as ethanol or 3, 4 methylenedioxymethamphetamine (MDMA) may derive from direct or indirect effects in many organs including the heart. The aim of the present work was to evaluate cardiac sympathetic activity and the expression and activation of two antioxidant proteins: heat shock protein27 (HSP27) and thioredoxin-1 (Trx-1) after voluntary binge ethanol consumption, alone and in combination with MDMA. MATERIAL AND METHODS: Adolescent mice received MDMA, ethanol or both. Drinking in the dark (DID) procedure was used as a model of binge. HSP27 expression and phosphorylation at serine 82 (pHSP27), Trx-1 expression, tyrosine hydroxylase (TH) and TH phosphorylated at serine 31 (pTH) were evaluated in adolescent mice 48 h and 7 days after treatments in the right ventricle. TH, HSP27 expression and phosphorylation and Trx-1 expression were measured by quantitative blot immunolabeling using specific antibodies. KEY FINDINGS: The expression of HSP27, pHSP27, Trx-1, total TH and pTH in the right ventricle was increased after binge ethanol or MDMA alone. In addition, the combination of binge ethanol + MDMA enhanced TH expression and phosphorylation versus their individual administration. SIGNIFICANCE: These results indicate that this combination could produce higher activation of sympathetic pathways, which could trigger an increased cell stress. On the other hand, increased HSP27, pHSP27 and Trx-1 expression in the right ventricle by ethanol + MDMA could be a protective mechanism to reduce the adverse effects of oxidative stress caused by both drugs of abuse.

Triptolide impairs thioredoxin system by suppressing Notch1-mediated PTEN/Akt/Txnip signaling in hepatocytes.

Triptolide (TP) is the main ingredient of Chinese herb Tripterygium wilfordii Hook f. (TWHF). Despite of its multifunction in pharmaceutics, accumulating evidences showed that TP caused obvious hepatotoxicity in clinic. The current study investigated the role of Notch1 signaling in TP-induced hepatotoxicity. Our data indicated that TP inhibited the protein expression of Notch1 and its active form Notch intracellular domain (NICD) leading to increased PTEN (phosphatase and tensin homolog deleted on chromosome ten) expression. Moreover, PTEN triggered Txnip (thioredoxin-interacting protein) activation by inhibiting Akt phosphorylation, which resulted in reduction of Trx (thioredoxin). In conclusion, TP caused liver injury through initiating oxidative stress in hepatocyte. This study indicated the potency of Notch1 to protect against TP-induced hepatotoxicity.

Honokiol Alleviates Oxidative Stress-Induced Neurotoxicity via Activation of Nrf2.

Honokiol (Hon), a polyphenol and main active ingredient from the bark of Magnolia officinalis, has been documented as having multiple pharmacological functions, including neuroprotection. However, the mechanisms underlying its neuroprotective effects are not well-defined. In this study, we reported that Hon attenuates the H2O2- or 6-hydroxydopamine (6-OHDA)-induced apoptosis of PC12 cells by increasing the glutathione level and upregulating a multitude of cytoprotective proteins, including heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1, thioredoxin 1, and thioredoxin reductase 1. Further studies reveal that Hon promotes transcription factor Nrf2 nuclear translocation and activation. Moreover, the cytoprotection of Hon was antagonized by silence of Nrf2 expression, highlighting the fact that Nrf2 is critically engaged in the cellular functions of Hon. Taken together, our study identified that Hon is an effective agonist of Nrf2 in the neuronal system and displays potent neuroprotection against oxidative stress-mediated PC12 cell damage. These findings indicate that Hon is promising for further development as a therapeutic drug against oxidative stress-related neurodegenerative disorders.

Babesia microti thioredoxin 3 is an effective antioxidant and involved in the response to antiprotozoal drugs.

The intra-erythrocytic apicomplexan Babesia microti is the predominant pathogen that causes human babesiosis, an infectious disease that occurs worldwide. B. microti relies on the antioxidant including thioredoxin system to maintain the redox balance during the erythrocytic stage. In the present study, the full-length B. microti thioredoxin 3 (BmTrx3) gene was cloned, expressed in vitro, and its response to antiprotozoal drugs were tested. The full-length BmTrx3 was 663 bp and contained an intact open reading frame of 567 bp. The encoded polypeptide was 188 amino acids and the predicted molecular weight of the protein was 21.7 kDa. A conserved thioredoxin-like family domain was found in BmTrx3. The expression of BmTrx3 was upregulated on both the third and eighth day post-infection in mice, whereas expression was downregulated during the beginning and later stages. Western blot analysis showed that mouse anti-BmTrx3 serum could recognize the native BmTrx3 in parasite lysates and that the mouse anti-B. microti serum could recognize the recombinant BmTrx3 protein. Immunofluorescence microscopy showed that BmTrx3 localized in the cell cytoplasm of B. microti merozoites in B. microti-infected red blood cells. The results of bovine insulin reduction assay indicated the enzyme activity of the purified recombinant BmTrx3 protein. The anti-malaria drug chloroquine significantly inhibited the expression of BmTrx3, however, another anti-malaria drug qunine, and a known anti-babesiosis drug clindamycin, induced significantly higher upregulation of BmTrx3 mRNA. The results of the present study demonstrate that BmTrx3 is a functional enzyme with antioxidant activity and may be involved in the response of B. microti to anti-parasite drugs.

Asbestos modulates thioredoxin-thioredoxin interacting protein interaction to regulate inflammasome activation.

BACKGROUND: Asbestos exposure is related to various diseases including asbestosis and malignant mesothelioma (MM). Among the pathogenic mechanisms proposed by which asbestos can cause diseases involving epithelial and mesothelial cells, the most widely accepted one is the generation of reactive oxygen species and/or depletion of antioxidants like glutathione. It has also been demonstrated that asbestos can induce inflammation, perhaps due to activation of inflammasomes. METHODS: The oxidation state of thioredoxin was analyzed by redox Western blot analysis and ROS generation was assessed spectrophotometrically as a read-out of solubilized formazan produced by the reduction of nitrotetrazolium blue (NTB) by superoxide. Quantitative real time PCR was used to assess changes in gene transcription. RESULTS: Here we demonstrate that crocidolite asbestos fibers oxidize the pool of the antioxidant, Thioredoxin-1 (Trx1), which results in release of Thioredoxin Interacting Protein (TXNIP) and subsequent activation of inflammasomes in human mesothelial cells. Exposure to crocidolite asbestos resulted in the depletion of reduced Trx1 in human peritoneal mesothelial (LP9/hTERT) cells. Pretreatment with the antioxidant dehydroascorbic acid (a reactive oxygen species (ROS) scavenger) reduced the level of crocidolite asbestos-induced Trx1 oxidation as well as the depletion of reduced Trx1. Increasing Trx1 expression levels using a Trx1 over-expression vector, reduced the extent of Trx1 oxidation and generation of ROS by crocidolite asbestos, and increased cell survival. In addition, knockdown of TXNIP expression by siRNA attenuated crocidolite asbestos-induced activation of the inflammasome. CONCLUSION: Our novel findings suggest that extensive Trx1 oxidation and TXNIP dissociation may be one of the mechanisms by which crocidolite asbestos activates the inflammasome and helps in development of MM.

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

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

The effects of chromium(VI) on the thioredoxin system: implications for redox regulation.

Hexavalent chromium [Cr(VI)] compounds are highly redox active and have long been recognized as potent cytotoxins and carcinogens. The intracellular reduction of Cr(VI) generates reactive Cr intermediates, which are themselves strong oxidants, as well as superoxide, hydrogen peroxide, and hydroxyl radical. These probably contribute to the oxidative damage and effects on redox-sensitive transcription factors that have been reported. However, the identification of events that initiate these signaling changes has been elusive. More recent studies show that Cr(VI) causes irreversible inhibition of thioredoxin reductase (TrxR) and oxidation of thioredoxin (Trx) and peroxiredoxin (Prx). Mitochondrial Trx2/Prx3 are more sensitive to Cr(VI) treatment than cytosolic Trx1/Prx1, although both compartments show thiol oxidation with higher doses or longer treatments. Thiol redox proteomics demonstrate that Trx2, Prx3, and Trx1 are among the most sensitive proteins in cells to Cr(VI) treatment. Their oxidation could therefore represent initiating events that have widespread implications for protein thiol redox control and for multiple aspects of redox signaling. This review summarizes the effects of Cr(VI) on the TrxR/Trx system and how these events could influence a number of downstream redox signaling systems that are influenced by Cr(VI) exposure. Some of the signaling events discussed include the activation of apoptosis signal regulating kinase and MAP kinases (p38 and JNK) and the modulation of a number of redox-sensitive transcription factors including AP-1, NF-κB, p53, and Nrf2.

Induction of heme oxygenase-1 by Macleaya cordata extract and its constituent sanguinarine in RAW264.7 cells.

Macleaya cordata (plume poppy) is used in traditional Chinese medicine for its anti-inflammatory and antibacterial activities. In this study, we examined whether M. cordata extract and/or its major alkaloid constituents, protopine, allocryptopine, sanguinarine and chelerythrine activate the Nrf2 signalling pathway which regulates the expression of cytoprotective enzymes including heme oxygenase-1 (HO-1) and thioredoxin 1. In murine macrophage RAW264.7 cells, M. cordata extract increased both mRNA and protein levels of HO-1. Of the alkaloids examined, only sanguinarine appeared to be responsible for these effects. At the concentration of 2 µM, sanguinarine induced nuclear accumulation of Nrf2, increased the expression of Hmox1 gene encoding HO-1 and elevated HO-1 protein levels. Sanguinarine-induced Hmox1 mRNA expression was suppressed by SB203580, a pharmacologic inhibitor of p38 mitogen-activated protein kinases (p38 MAPKs). The upregulation of HO-1 in RAW264.7 cells by sanguinarine was, however, accompanied by decrease in cell viability. Nonetheless, sanguinarine at micromolar, non-cytotoxic concentrations elevated protein levels of HO-1 and thioredoxin 1 in primary cultures of human hepatocytes. We conclude that sanguinarine may, under appropriate conditions, increase the capacity of the enzymatic antioxidant defence system via activation of the p38 MAPK/Nrf2 pathway.

Dietary lipids modulate methylmercury toxicity in Atlantic salmon.

This experiment aimed to study the molecular toxicity of methylmercury (MeHg) in liver, brain and white muscle of Atlantic salmon fed a diet based on fish oil (FO, high dietary n-3/n-6 ratio) compared to an alternative diet mainly based on vegetable oil (VO, low dietary n-3/n-6 ratio). Juvenile salmon were fed decontaminated diets or the FO and VO diets enriched with 5 mg Hg/kg (added as MeHg) for three months. The dietary lipid composition affected the fatty acid composition in the tissues, especially in liver and white muscle. After 84 days of exposure, the liver accumulated three times as much MeHg as the brain and white muscle. Vitamin C content and heme oxygenase, tubulin alpha (TUBA) and Cpt1 transcriptional levels all showed significant effects of MeHg exposure in the liver. TBARS, α-tocopherol, γ-tocopherol, and the transcriptional levels of thioredoxin, heme oxygenase, TUBA, PPARB1, D5D and D6D showed an effect of dietary lipid composition in liver tissue. Effects of dietary lipids were observed in brain tissue for MT-A, HIF1, Bcl-X and TUBA. Interaction effects between MeHg exposure and dietary lipid composition were observed in all tissues. Our data suggest that dietary fats have modulating effects on MeHg toxicity in Atlantic salmon.

Possible role for the thioredoxin system in the protective effects of probucol in the pancreatic islets of diabetic rats.

1. Probucol, a lipid-lowering agent with a potent anti-oxidant action, protects diabetic pancreatic islets by an as yet unknown mechanism. Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the ubiquitous thiol oxidoreductase thioredoxin (TRX), has been associated with oxidative stress in diabetic rat islets. The aim of the present study was to examine the effects of probucol on diabetic islet function and expression of TRX and TXNIP. 2. Thirty rats were randomly assigned to one of three groups: a normal control group, a diabetic group and a probucol-treated diabetic group. After 8 weeks treatment with probucol (500 mg/kg per day), plasma malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activity were determined using chemical colourimetric methods. In addition, the expression of insulin, TRX and TXNIP in islets was analysed using immunohistochemical, western blot and reverse transcription-polymerase chain reaction methods. 3. The expression of TRX and insulin in islets and plasma SOD and CAT activity were lower, but the expression of TXNIP in islets and plasma MDA were higher, in diabetic compared with normal control rats. Upregulated expression of TRX and insulin and downregulated expression of TXNIP were observed in probucol-treated diabetic rats. Probucol treatment increased plasma SOD, decreased plasma MDA and improved hypoinsulinaemia in diabetic rats. 4. The results indicate that treatment with probucol decreases TXNIP expression and increases TRX expression, which may alleviate hypoinsulinaemia by reducing oxidative stress. Therefore, probucol shows promise as a supplemental therapy for islet protection in Type 2 diabetes mellitus.

The intracellular redox stress caused by hexavalent chromium is selective for proteins that have key roles in cell survival and thiol redox control.

Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr intermediates that can directly oxidize some cell components and can promote the generation of reactive oxygen and nitrogen species. Inhalation is a major route of exposure which directly exposes the bronchial epithelium. Previous studies with non-cancerous human bronchial epithelial cells (BEAS-2B) demonstrated that Cr(VI) treatment results in the irreversible inhibition of thioredoxin reductase (TrxR) and the oxidation of thioredoxins (Trx) and peroxiredoxins (Prx). The mitochondrial Trx/Prx system is somewhat more sensitive to Cr(VI) than the cytosolic Trx/Prx system, and other redox-sensitive mitochondrial functions are subsequently affected including electron transport complexes I and II. Studies reported here show that Cr(VI) does not cause indiscriminant thiol oxidation, and that the Trx/Prx system is among the most sensitive of cellular protein thiols. Trx/Prx oxidation is not unique to BEAS-2B cells, as it was also observed in primary human bronchial epithelial cells. Increasing the intracellular levels of ascorbate, an endogenous Cr(VI) reductant, did not alter the effects on TrxR, Trx, or Prx. The peroxynitrite scavenger MnTBAP did not protect TrxR, Trx, Prx, or the electron transport chain from the effects of Cr(VI), implying that peroxynitrite is not required for these effects. Nitration of tyrosine residues of TrxR was not observed following Cr(VI) treatment, further ruling out peroxynitrite as a significant contributor to the irreversible inhibition of TrxR. Cr(VI) treatments that disrupt the TrxR/Trx/Prx system did not cause detectable mitochondrial DNA damage. Overall, the redox stress that results from Cr(VI) exposure shows selectivity for key proteins which are known to be important for redox signaling, antioxidant defense, and cell survival.

Oxidation of thiols and modification of redox-sensitive signaling in human lung epithelial cells exposed to Pseudomonas pyocyanin.

The aim of this study was to examine the effects of pyocyanin exposure on mitochondrial GSH, other cellular thiols (thioredoxin-1, Trx-1), and oxidant-sensitive signaling pathways hypoxia inducible factor (HIF-1α) and heme oxygenase (HO-1) in A549 and HBE cell lines. A549 human type II alveolar epithelial cells and human bronchial epithelial (HBE) cells were treated with varying concentrations of pyocyanin extracted from Pseudomonas aeruginosa bacteria. Cytoplasmic and mitochondrial thiols and oxidant sensitive signal transduction proteins (HIF-1α and HO-1) were measured. Exposure to pyocyanin generated reactive oxygen species (ROS) in cellular mitochondria and altered total cellular glutathione (GSH). Pyocyanin, at concentrations present in conditions in vivo, increased oxidized Trx-1 in A549 human type II alveolar epithelial cells and HBE cells by 184 and 74%, respectively. Oxidized mitochondrial glutathione (GSSG) was elevated more than twofold in both cell types. Pyocyanin also increased the cellular oxidant-sensitive proteins HIF-1α and HO-1. Data indicate that pyocyanin-induced alterations in mitochondrial and cytosolic thiols, as well as oxidant-sensitive proteins, may contribute to P. aeruginosa-mediated lung injury.

In vitro and in vivo effects of D-allose: up-regulation of thioredoxin-interacting protein in head and neck cancer cells.

OBJECTIVES: This study was aimed to investigate the relationship between the antiproliferative effects of D-allose and the up-regulation of thioredoxin-interacting protein (TXNIP) in head and neck cancer cells. METHODS: For the in vitro study, 5 oral squamous cell carcinoma cell lines (Ca9-22, HSC-3, HSC-4, SAS, and KON) were treated with 25 mmol/L D-allose. For the in vivo study, HSC-3 cells were used in a xenograft model with female athymic nude mice (BALB/c nu/nu; 5 to 6 weeks old). RESULTS: Inhibition of cell growth by D-allose was noted in HSC-3 and Ca9-22 cells, along with significant induction of TXNIP. Although TXNIP up-regulation was also evident, albeit to a lesser extent, in the remaining cell lines, D-allose did not inhibit their growth. With the HSC-3 line, the cell survival fractions decreased and TXNIP expression increased in a D-allose dose-dependent manner. The antiproliferative effects were partially suppressed by concomitant D-glucose treatment, which also reduced TXNIP expression. In the in vivo experiment, the tumor volume at day 15 after D-allose treatment was reduced to 61% of that of the control group. CONCLUSIONS: This study showed that D-allose exerts growth inhibitory effects on head and neck cancer cells in vitro and in vivo. The sugar may act as an antiproliferative agent via TXNIP induction and thus may be useful as a novel anticancer drug.