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- Novel protective properties of auranofin: Inhibition of human astrocyte cytotoxic secretions and direct neuroprotection. [JOURNAL ARTICLE]
- Life Sci 2013 Apr 26.
AIMS:Steroidal and non-steroidal anti-inflammatory drugs are used for treatment of peripheral inflammation, but they are not effective in neurodegenerative disorders. Gold compounds are also used to treat peripheral inflammation, but their effects on neuroimmune reactions are unknown. This study investigated the effects of gold compounds on astrocytic cell functions and assessed in vivo distribution of auranofin after its oral administration in mice. MAIN
METHODS:Auranofin and three other gold compounds were investigated for their ability to reduce the secretion of pro-inflammatory cytokines and cytotoxins produced by activated human astrocytic cells. Ability of the gold compounds to protect neuronal cells from glial cytotoxins and from oxidative damage induced by hydrogen peroxide was also studied. The in vivo distribution of auranofin was investigated using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). KEY
FINDINGS:Auranofin (0.1-5μM) inhibited the toxicity of stimulated primary human astrocytes and U-373 MG astrocytic cells towards human neuronal cells, but did not inhibit secretion of cytokines. Treatment of neuronal cells with high nanomolar to low micromolar concentrations of auranofin protected them from toxicity induced by hydrogen peroxide and supernatants of stimulated astrocytic cells through the upregulation of heme-oxygenase (HOX)-1. Aurothiomalate, aurothioglucose, and aurothiosulphate were ineffective in the assays used. Auranofin reached low micromolar concentrations in mouse brains following daily oral administration for one week.
SIGNIFICANCE:Since auranofin may protect neurons by inhibiting astrocyte toxicity and is also directly neuroprotective, it could be useful in neurodegenerative diseases where activation of astrocytes contributes to the neuronal loss.
- Reduction of dimethylarsinic acid to the highly toxic dimethylarsinous acid by rats and rat liver cytosol. [Journal Article, Research Support, Non-U.S. Gov't]
- Chem Res Toxicol 2013 Mar 18; 26(3):432-43.
Dimethylarsinic acid (DMAs(V)), the major urinary metabolite of inorganic arsenic, is weakly cytotoxic, whereas its reduced form, dimethylarsinous acid (DMAs(III)), is highly toxic. Although glutathione S-transferase omega 1 (GSTO1) and arsenic methyltransferase have been shown or thought to catalyze DMAs(V) reduction, their role in DMAs(V) reduction in vivo, or in cell extracts is uncertain. Therefore, the reduction of DMAs(V) to DMAs(III) in rats and in rat liver cytosol was studied to better understand its mechanism. To assess DMAs(V) reduction in rats, a novel procedure was devised based on following the accumulation of red blood cell (RBC)-bound dimethylarsenic (DMAs), which represents DMAs(III), in the blood of DMAs(V)-injected anesthetized rats. These studies indicated that rats reduced DMAs(V) to DMAs(III) to a significant extent, as in 90 min 31% of the injected 50 μmol/kg DMAs(V) dose was converted to DMAs(III) that was sequestered by the circulating erythrocytes. Pretreatment of rats with glutathione (GSH) depletors (phorone or BSO) delayed the elimination of DMAs(V) and the accumulation of RBC-bound DMAs, whereas the indirect methyltransferase inhibitor periodate-oxidized adenosine was without effect. Assessment of DMAs(V)-reducing activity of rat liver cytosol revealed that reduction of DMAs(V) required cytosolic protein and GSH and was inhibited by thiol reagents, GSSG and dehydroascorbate. Although thioredoxin reductase (TRR) inhibitors (aurothioglucose and Sb(III)) inhibited cytosolic DMAs(V) reduction, recombinant rat TRR plus NADPH, alone or when added to the cytosol, failed to support DMAs(V) reduction. On ultrafiltration of the cytosol through a 3 kDa filter, the reducing activity in the retentate was lost but was largely restored by NADPH. Such experiments also suggested that the reducing enzyme was larger than 100 kDa and was not GSTO1. In summary, reduction of DMAs(V) to the highly toxic DMAs(III) in rats and rat liver cytosol is a GSH-dependent enzymatic process, yet its mechanism remains uncertain.
- Thioredoxin-mimetic peptides (TXM) reverse auranofin induced apoptosis and restore insulin secretion in insulinoma cells. [Journal Article, Research Support, Non-U.S. Gov't]
- Biochem Pharmacol 2013 Apr 1; 85(7):977-90.
The thioredoxin reductase/thioredoxin system (TrxR/Trx1) plays a major role in protecting cells from oxidative stress. Disruption of the TrxR-Trx1 system keeps Trx1 in the oxidized state leading to cell death through activation of the ASK1-Trx1 apoptotic pathway. The potential mechanism and ability of tri- and tetra-oligopeptides derived from the canonical -CxxC- motif of the Trx1-active site to mimic and enhance Trx1 cellular activity was examined. The Trx mimetics peptides (TXM) protected insulinoma INS 832/13 cells from oxidative stress induced by selectively inhibiting TrxR with auranofin (AuF). TXM reversed the AuF-effects preventing apoptosis, and increasing cell-viability. The TXM peptides were effective in inhibiting AuF-induced MAPK, JNK and p38(MAPK) phosphorylation, in correlation with preventing caspase-3 cleavage and thereby PARP-1 dissociation. The ability to form a disulfide-bridge-like conformation was estimated from molecular dynamics simulations. The TXM peptides restored insulin secretion and displayed Trx1 denitrosylase activity. Their potency was 10-100-fold higher than redox reagents like NAC, AD4, or ascorbic acid. Unable to reverse ERK1/2 phosphorylation, TXM-CB3 (NAc-Cys-Pro-Cys amide) appeared to function in part, through inhibiting ASK1-Trx dissociation. These highly effective anti-apoptotic effects of Trx1 mimetic peptides exhibited in INS 832/13 cells could become valuable in treating adverse oxidative-stress related disorders such as diabetes.
- Stimulation of human TRPA1 channels by clinical concentrations of the antirheumatic drug auranofin. [Journal Article, Research Support, Non-U.S. Gov't]
- Am J Physiol Cell Physiol 2013 Feb 15; 304(4):C354-61.
Gold compounds, which were widely used to treat rheumatoid arthritis, have been recently used as experimental agents for tumor treatment. Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1) is a Ca(2+)-permeable ion channel that senses acute and inflammatory pain signals. Electrophilic compounds such as mustard oil and cinnamaldehyde activate TRPA1 by interacting with TRPA1 cysteine residues. Here we investigate the effects of the gold compound auranofin (AUR) on TRPA1 channels. Intracellular Ca(2+) and whole cell patch-clamp recordings were performed on human embryonic kidney cells transiently expressed with TRPA1, TRP melastatin 8 (TRPM8), and vanilloid type TRP (TRPV1-4) channels. AUR stimulated TRPA1 in a concentration-dependent manner with a half-maximum potency of around 1.0 μM. The AUR-induced response was effectively blocked by HC030031, a TRPA1 antagonist. On the other hand, AUR failed to activate TRPM8 and TRPV1-4 channels, which are highly expressed in sensory neurons as nociceptors. The stimulatory effect on TRPA1 channels depended on the C414, C421, C621, and C633 cysteine residues and not on the inhibition of thioredoxin reductase by AUR. Moreover, AUR effectively activated TRPA1 channels expressed in human differentiated neuroblastoma cell lines. The study shows that AUR is a potent stimulator of TRPA1 channels.
- The thioredoxin reductase-thioredoxin system is involved in the entry of tetanus and botulinum neurotoxins in the cytosol of nerve terminals. [Journal Article, Research Support, Non-U.S. Gov't]
- FEBS Lett 2013 Jan 16; 587(2):150-5.
Tetanus and botulinum neurotoxins cause paralysis by cleaving SNARE proteins within the cytosol of nerve terminals. They are endocytosed inside acidic vesicles and the pH gradient across the membrane drives the translocation of their metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-thioredoxin reductase-thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins.
- Susceptibility of human head and neck cancer cells to combined inhibition of glutathione and thioredoxin metabolism. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
- PLoS One 2012; 7(10):e48175.
Increased glutathione (GSH) and thioredoxin (Trx) metabolism are mechanisms that are widely implicated in resistance of cancer cells to chemotherapy. The current study determined if simultaneous inhibition of GSH and Trx metabolism enhanced cell killing of human head and neck squamous cell carcinoma (HNSCC) cells by a mechanism involving oxidative stress. Inhibition of GSH and Trx metabolism with buthionine sulfoximine (BSO) and auranofin (AUR), respectively, induced significant decreases in clonogenic survival compared to either drug alone in FaDu, Cal-27 and SCC-25 HNSCC cells in vitro and in vivo in Cal-27 xenografts. BSO+AUR significantly increased glutathione and thioredoxin oxidation and suppressed peroxiredoxin activity in vitro. Pre-treatment with N-acetylcysteine completely reversed BSO+AUR-induced cell killing in FaDu and Cal-27 cells, while catalase and selenium supplementation only inhibited BSO+AUR-induced cell killing in FaDu cells. BSO+AUR decreased caspase 3/7 activity in HNSCC cells and significantly reduced the viability of both Bax/Bak double knockout (DKO) and DKO-Bax reconstituted hematopoietic cells suggesting that necrosis was involved. BSO+AUR also significantly sensitized FaDu, Cal-27, SCC-25 and SQ20B cells to cell killing induced by the EGFR inhibitor Erlotinib in vitro. These results support the conclusion that simultaneous inhibition of GSH and Trx metabolism pathways induces oxidative stress and clonogenic killing in HNSCCs and this strategy may be useful in sensitizing HNSCCs to EGFR inhibitors.
- Selenium supplementation protects trophoblast cells from oxidative stress. [Journal Article]
- Placenta 2012 Dec; 33(12):1012-9.
Oxidative stress is a key feature in the pathogenesis of pre-eclampsia and antioxidants have been proposed as a potential therapy in the treatment of this important complication of pregnancy. In this report selenium supplementation was used to up-regulate the antioxidant enzymes glutathione peroxidase and thioredoxin reductase and the protective effect that this had on cellular metabolism during oxidative stress was examined. Bewo and Jeg-3 trophoblast cells were supplemented with organic and inorganic forms of selenium and 3 forms of peroxide in a range of doses were utilised to generate oxidative stress. Thioredoxin reductase and glutathione peroxidase activity were maximally expressed after supplementation with 100 nM NaSe and 500 nM SeMethionine. Application of H₂O₂ in the range of 200-400 μM for 24h resulted in significant (p<0.001) inhibition of cellular activity, an effect negated by Se supplementation. Tert-butyl H₂O₂ and cumene H₂O₂ concentrations between 30 and 50 uM similarly inhibited cellular activity and this could be significantly (p<0.001) reversed by Se supplementation. Auranofin, a specific inhibitor of thioredoxin reductase and glutathione peroxidase was used to prove that the protective effect generated by Se supplementation was due to up regulation of these enzymes. These studies provide direct evidence that selenium supplementation can up-regulate endogenous antioxidant systems and protects trophoblast cells from oxidative stress. This may inform the development of future therapies for pre-eclampsia and emphasises the importance of selenium adequacy during pregnancy.
- Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose. [Journal Article, Research Support, Non-U.S. Gov't]
- J Biol Chem 2012 Nov 2; 287(45):38210-9.
Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.
- The biological activity of auranofin: implications for novel treatment of diseases. [Journal Article, Research Support, Non-U.S. Gov't, Review]
- Inflammopharmacology 2012 Dec; 20(6):297-306.
More than 30 years ago, auranofin was developed for the treatment of rheumatoid arthritis as a substitution for the injectable gold compounds aurothiomalate and aurothioglucose. Both the ease of oral administration over intramuscular injections and more potent anti-inflammatory effects in vitro made auranofin seem like an excellent substitute for the traditional injectable gold compounds. Despite efficacy in the treatment of both rheumatoid arthritis and psoriasis, currently, auranofin is seldom used as a treatment for patients with rheumatoid arthritis as more novel anti-rheumatic medications have become available. Despite the decline in its clinical applications, research on auranofin has continued as it shows promise in the treatment of several different diseases. In recent years, advances in technology have allowed researchers to use molecular techniques to identify novel mechanisms of action of auranofin. Additionally, researchers are discovering potential new applications of auranofin. Dual inhibition of inflammatory pathways and thiol redox enzymes by auranofin makes it a new candidate for cancer therapy and treating microbial infections. This review will summarize recently obtained data on the mechanisms of action of auranofin, and potential new applications of auranofin in the treatment of various diseases, including several types of leukaemia, carcinomas, and parasitic, bacterial, and viral infections.
- Total reflection X-ray fluorescence spectrometry as a tool for the quantification of gold and platinum metallodrugs: determination of recovery rates and precision in the ppb concentration range. [Journal Article, Validation Studies]
- J Pharm Biomed Anal 2012 Nov.:713-7.
Total reflection X-ray spectrometry is an instrumental technique based on X-ray fluorescence, which offers detection limits low enough to quantify trace element concentrations with negligible interference from matrix components. The technique is well established in material sciences and now reaches out to extended applications in life sciences and pharmaceutical quality control. In our study we focused on possible applications for the quantification of gold and platinum containing active ingredients in trace concentrations (ppb range) in a matrix of biological origin (cell suspensions). General aspects of sample preparation as well as selected important method performance parameters (precision, recovery rates) were investigated. Overall, TXRF represents an useful option to quantify metals in ppb concentrations with acceptable precision and recovery.