Gold(I) complexes determine apoptosis with limited oxidative stress in Jurkat T cells

In Jurkat T cells, S-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-beta-d-glucopyranoside (auranofin) and triethylphosphine gold(I) chloride (TepAu) induced apoptosis, as estimated by DNA fragmentation and visualised by fluorescence microscopy. Apoptosis was characterised by mitochondrial cytochrome c release which was not prevented by cyclosporin A. Apoptosis appeared to be triggered by inhibition exerted by gold(I) compounds on the cytosolic and mitochondrial isoforms of thioredoxin reductase, which determined a definite increase in hydrogen peroxide, whereas glutathione and its redox state were not modified. Total thiols showed a slight decrease, particularly in the presence of auranofin. However, no significant lipid peroxidation or nitric oxide formation were observed after incubation with gold(I) complexes, indicating that the cells had not been subjected to extensive oxidative stress. Interestingly, the gold(I) compound aurothiomalate was poorly effective, both in inhibiting thioredoxin reductase and in inducing apoptosis. These results demonstrate that the increased production of hydrogen peroxide determines an oxidative shift responsible for the occurrence of apoptosis and not involving lipid peroxidation.     

A gold(I) phosphine complex selectively induces apoptosis in breast cancer cells: implications for anticancer therapeutics targeted to mitochondria

Bis-chelated gold(I) phosphine complexes have shown great potential as anticancer agents, however, their efficacy has been limited by their high toxicity and lack of selectivity for cancer cells. Here, we have investigated the anticancer activity of a new bis-chelated Au(I) bidentate phosphine complex of the novel water soluble ligand 1,3-bis(di-2-pyridylphosphino)propane (d2pypp). We show that this gold complex [Au(d2pypp)(2)]Cl, at submicromolar concentrations, selectively induces apoptosis in breast cancer cells but not in normal breast cells. Apoptosis was induced via the mitochondrial pathway, which involved mitochondrial membrane potential depolarisation, depletion of the glutathione pool and caspase-3 and caspase-9 activation. The gold lipophilic complex was accumulated in mitochondria of cells, driven by the high mitochondrial membrane potential. To address the molecular basis of the observed selectivity between the two cell lines we investigated the effect of the gold complex on the thioredoxin/thioredoxin reductase system in normal and cancer breast cells. We show that [Au(d2pypp)(2)]Cl inhibits the activities of both thioredoxin and thioredoxin reductase and that this effect is more pronounced in the breast cancer cells. This difference may account for the selective cell death seen in the breast cancer cells but not in the normal cells. Our investigation has led to new insights into the mechanism of action of bis-chelated gold(I) diphosphine complexes and their future development as mitochondria targeted chemotherapeutics.     

Induction of mitochondrial permeability transition by auranofin,a gold(I)-phosphine derivative

1 Gold(I)-thiolate drugs are compounds that specifically interact with thiol and/or selenol groups and are essentially utilized in the treatment of rheumatoid arthritis. 2 Considering the importance of thiol groups in regulating mitochondrial membrane permeability, the effects of auranofin (S-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranoside), a second-generation gold drug, were studied on mitochondria isolated from rat liver. 3 Auranofin, at submicromolar concentrations, was able to induce the mitochondrial membrane permeability transition observed as swelling and loss of membrane potential. Both events are completely inhibited by cyclosporin A, the specific inhibitor of mitochondrial permeability transition. Calcium ions and energization by succinate are required for the occurrence of permeability transition. 4 By interacting with the active site selenol group, auranofin results as an extremely potent inhibitor of mitochondrial thioredoxin reductase, both isolated and in its mitochondrial environment. 5 It is concluded that auranofin, in the presence of calcium ions, is a highly efficient inducer of mitochondrial membrane permeability transition, potentially referable to its inhibition of mitochondrial thioredoxin reductase.     

The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes. III. Chlorotriethylphosphine gold(I)-induced alterations in isolated rat liver mitochondrial function

Chlorotriethylphosphine gold(I) (TEPAu) is an organo-gold compound that has therapeutic activity in animal models of rheumatoid arthritis. Initial studies have suggested that TEPAu is a potent cytotoxic compound in vitro against a variety of cultured cell types and isolated hepatocytes. Mitochondrial dysfunction induced by this compound has been suggested as a primary biochemical alteration which may result in lethal cell injury in isolated hepatocytes. The purpose of this study was, therefore, to determine the mechanism of TEPAu-induced dysfunction of isolated rat liver mitochondria. TEPAu induced a rapid, concentration-related collapse of the mitochondrial inner membrane potential (EC50 = 24.7 +/- 2.5 microM) which was potentiated in Ca2+ loaded mitochondria (EC50 = 11.3 +/- 3.8 microM). TEPAu-induced collapse of the membrane potential was partially inhibited in the presence of ruthenium red or EGTA. TEPAu caused the rapid release of mitochondrially sequestered Ca2+ which was not inhibited by ruthenium red and, thus, was not via a reversal of the Ca2+ uniporter. TEPAu caused mitochondrial swelling, increased permeability of the inner membrane, and the oxidation/hydrolysis of endogenous mitochondrial pyridine nucleotides. Addition of exogenous ATP slightly reversed the effects of TEPAu on pyridine nucleotides. TEPAu-induced mitochondrial alterations were reversed or inhibited by exposure to the sulfhydryl reducing agent, dithiothreitol. Also, the TEPAu-induced collapse of the mitochondrial membrane potential was partially inhibited by dibucaine, a non-specific inhibitor of phospholipases. These data suggest that TEPAu-induced mitochondrial dysfunction is sulfhydryl dependent. TEPAu-induced mitochondrial dysfunction results in dissipation of the potential difference across the inner mitochondrial membrane which inhibits mitochondrial oxidative phosphorylation. The mechanism by which TEPAu induces the collapse of the membrane potential may be mediated by a sulfhydryl-dependent increase in permeability of the inner membrane to protons.     

Benzimidazol-2-ylidene gold(I) complexes are thioredoxin reductase inhibitors with multiple antitumor properties

Gold(I) complexes such as auranofin have been used for decades to treat symptoms of rheumatoid arthritis and have also demonstrated a considerable potential as new anticancer drugs. The enzyme thioredoxin reductase (TrxR) is considered as the most relevant molecular target for these species. The here investigated gold(I) complexes with benzimidazole derived N-heterocyclic carbene (NHC) ligands represent a promising class of gold coordination compounds with a good stability against the thiol glutathione. TrxR was selectively inhibited by in comparison to the closely related enzyme glutathione reductase, and all complexes triggered significant antiproliferative effects in cultured tumor cells. More detailed studies on a selected complex revealed a distinct pharmacodynamic profile including the high increase of reactive oxygen species formation, apoptosis induction, strong effects on cellular metabolism (related to cell surface properties, respiration, and glycolysis), inhibition of mitochondrial respiration and activity against resistant cell lines.     

Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.     

Auranofin increases apoptosis and ischaemia-reperfusion injury in the rat isolated heart

Auranofin, an antirheumatic gold compound, is an inhibitor of selenocysteine enzymes, such as thioredoxin reductase and glutathione peroxidase. These enzymes play an important role in protecting cardiac tissue from oxidative stress generated during ischaemia-reperfusion. Auranofin (100 mg/kg) was administered to rats and their hearts were subjected to an in vitro model of ischaemia-reperfusion. The activity of thioredoxin reductase and glutathione peroxidase was determined in liver and heart tissues in an attempt to correlate enzymatic activity with heart recovery after ischaemia-reperfusion. There was significantly less thioredoxin reductase activity in rat liver extracts, whereas the level of glutathione activity remained unchanged, demonstrating that the dose of auranofin used was able to selectively inhibit one of these enzyme systems. Rats administered auranofin displayed significantly impaired recovery from ischaemic insult. The end diastolic pressure was increased, whereas the rate pressure product was significantly decreased. The level of postischaemic apoptosis was also assessed by examining caspase-3 activity in tissue homogenates. Auranofin significantly increased the degree of postischaemic apoptosis, leading to poor postischaemic recovery.     

Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, ‘auranofin-like’ gold(I) complexes all containing the [Au(PEt₃)]⁺ synthon and the ligands: Cl⁻, Br⁻, cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.     

4-hydroxynonenal induces mitochondrial oxidative stress, apoptosis and expression of glutathione S-transferase A4-4 and cytochrome P450 2E1 in PC12 cells

An excessive and sustained increase in reactive oxygen species (ROS) production and oxidative stress have been implicated in the pathogenesis of many diseases. In the present study, we have demonstrated that 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, alters glutathione (GSH) pools and induces oxidative stress in PC12 cells in culture. This increase was accompanied by alterations in subcellular ROS and glutathione (GSH) metabolisms. The GSH homeostasis was affected as both mitochondrial and extramitochondrial GSH levels, GSH peroxidase and glutathione reductase activities were inhibited and glutathione S-transferase (GST) activity was increased after 4-HNE treatment. A concentration- and time-dependent increase in cytochrome P450 2E1 (CYP 2E1) activity in the mitochondria and postmitochondrial supernatant was also observed. 4-HNE-induced oxidative stress also caused an increase in the expression of GSTA4-4, CYP2E1 and Hsp70 proteins in the mitochondria. Increased oxidative stress in PC12 cells initiated apoptosis as indicated by the release of mitochondrial cytochrome c, activation of poly-(ADP-ribose) polymerase (PARP), DNA fragmentation and decreased expression of antiapoptotic Bcl-2 proteins. Mitochondrial respiratory and redox functions also appeared to be affected markedly by 4-HNE treatment. These results suggest that HNE-induced oxidative stress and apoptosis might be associated with altered mitochondrial functions and a compromised GSH metabolism and ROS clearance.     

The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes : II. Triethylphosphine gold chloride-induced alterations in mitochondrial function

Triethylphosphine gold complexes have therapeutic activity in the treatment of rheumatoid arthritis. Many of these compounds are also highly cytotoxic in vitro to a variety of tumor and non-tumor cell lines. Triethylphosphine gold chloride (TEPAu) is highly cytotoxic to isolated rat hepatocytes at concentrations greater than 25 microM. The earliest changes that could be detected in hepatocytes included bleb formation in the plasma membrane, alterations in the morphology of mitochondria, and rapid decreases in cellular ATP and oxygen consumption. The degradation of ATP could be followed sequentially through ADP and AMP and was ultimately accounted for entirely as xanthine. The sum of adenine and xanthine-derived nucleotides remained constant throughout the experiments. TEPAu (50 microM) caused a significant decrease in the hepatocyte ATP/ADP ratio and energy charge within 5 min. The antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), which blocked TEPAu-induced malondialdehyde formation but not cell death, also had no effect on the decreases in oxygen consumption, ATP, ATP/ADP ratio, or energy charge. In isolated rat liver mitochondria, TEPAu (1 microM) caused significant reductions in carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP) (uncoupled)-stimulated respiration. TEPAu (5 microM) inhibited state 3 respiration and the respiratory control ratio without affecting state 4 respiration and caused a rapid dissipation of the mitochondrial-membrane hydrogen-ion gradient (membrane potential). Concentrations greater than 5 microM also inhibited state 4 respiration. TEPAu caused a concentration-dependent inhibition of FCCP-stimulated respiration with pyruvate/malate and succinate as substrates but had not effect on ascorbate/tetramethyl-p-phenylenediamine-supported respiration. The inhibition of state 4 respiration and FCCP-stimulated respiration by TEPAu (10 microM) could be reversed by the addition of 2 mM dithiothreitol. Dithiothreitol also partially protected cells from TEPAu-induced injury and reversed the TEPAu-induced depletion in cellular ATP. These data indicate that TEPAu may be acting functionally as a respiratory site II inhibitor, similar to antimycin. The reversal of TEPAu-induced inhibition of mitochondrial respiration and cell lethality by dithiothreitol suggests that mitochondrial thiols may be involved.     

Role of mitochondrial permeability transition in human renal tubular epithelial cell death induced by aristolochic acid

Aristolochic acid (AA), a natural nephrotoxin and carcinogen, can induce a progressive tubulointerstitial nephropathy. However, the mechanism by which AA causes renal injury remains largely unknown. Here we reported that the mitochondrial permeability transition (MPT) plays an important role in the renal injury induced by aristolochic acid I (AAI). We found that in the presence of Ca(2+), AAI caused mitochondrial swelling, leakage of Ca(2+), membrane depolarization, and release of cytochrome c in isolated kidney mitochondria. These alterations were suppressed by cyclosporin A (CsA), an agent known to inhibit MPT. Culture of HK-2 cell, a human renal tubular epithelial cell line for 24 h with AAI caused a decrease in cellular ATP, mitochondrial membrane depolarization, cytochrome c release, and increase of caspase 3 activity. These toxic effects of AAI were attenuated by CsA and bongkrekic acid (BA), another specific MPT inhibitor. Furthermore, AAI greatly inhibited the activity of mitochondrial adenine nucleotide translocator (ANT) in isolated mitochondria. We suggested that ANT may mediate, at least in part, the AAI-induced MPT. Taken together, these results suggested that MPT plays a critical role in the pathogenesis of HK-2 cell injury induced by AAI and implied that MPT might contribute to human nephrotoxicity of aristolochic acid.     

Dual effect of ebselen on mitochondrial permeability transition

This study reports an investigation on the effect of the seleno-organic compound ebselen on rat liver mitochondria. We show that low concentrations of ebselen induced an increase in rat liver mitochondrial membrane permeability, resulting in swelling and loss of membrane potential. These effects were mediated by the opening of the permeability transition pore. They required Ca(2+), were independent of pyridine nucleotide oxidation, and involved the oxidation of thiol groups. Ebselen pore induction is apparently promoted by the glutathione peroxidase mimicking activity of the drug. Opposite effects, that is, inhibition of both pore opening and thiol oxidation, were observed when concentrations higher than 20 micro M were used. These data demonstrate that ebselen is able to modulate the opening of the permeability transition pore and that it might be a critical event for both the proapoptotic and cytoprotective activities of the drug.     

Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

Mechanism of cytotoxic action of perfluorinated acids: II. Disruption of mitochondrial bioenergetics

PFAs and derivatives due to perfect technological properties are broadly applied in industry and consumer goods, and in consequence widely disseminated, environmentally bioaccumulative and found at ppb level in human serum.Earlier we revealed that in vitro cytotoxicity increases with chain length (CF₆-CF₁₄). The compounds dissipate plasma membrane potential and acidify of cytosol. Here we determine whether there is an association between the protonophoric uncoupling of respiration and disruption of bioenergetics caused by CF₆-CF₁₂ on HCT116 cell apoptosis.Again the effects were stronger for longer molecules. Incubation of cells with CF₁₀ stimulated time-dependent generation of reactive oxygen species, opening of mitochondrial permeability transition (MPT) pore, release of cytochrome c, activation of caspases and depletion of intracellular level of ATP occurring in intrinsic pathway of apoptosis. Incubation with decanoic acid (DA) did not lead to mitochondrial dysfunctions neither to cell cycle disturbances. Synchronized removal of the phosphorylated state of Akt, ERK1/2 and PKCδ/θ kinases by CF₁₀ suggests presence of concerted action to uninhibit Bad protein activation and a cascade of intrinsic pathway of apoptosis. Blocking MPT pore by cyclosporin A (CsA) led to a reduction of mitochondrial potential dissipation (mtΔΨ). Such cells neither showed cytochrome c release nor the downstream activation of caspase-9 and caspase-3. Our results confirm that mitochondria play a crucial role in perfluorochemicals induced apoptosis by releasing apoptotic signals through MPT pore.     

The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves peroxiredoxin 3 oxidation

Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-α-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.     

Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca²⁺-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.     

Luteolin protects osteoblastic MC3T3-E1 cells from antimycin A-induced cytotoxicity through the improved mitochondrial function and activation of PI3K/Akt/CREB

Luteolin is a flavonoid found in many herbal extracts including celery, green pepper, parsley, perilla leaf and seeds, and chamomile. Antimycin A (AMA) is an inhibitor of the mitochondrial electron transport chain. In the present study, the protective effect of luteolin on AMA-induced cell damage was investigated in osteoblastic MC3T3-E1 cells. Luteolin significantly increased the viability of MC3T3-E1 cells in the presence of AMA and the effect of luteolin in increasing cell viability was completely prevented by the presence of LY294002, Akt inhibitor, or auranofin, suggesting that the effect of luteolin might be partly mediated from PI3K, Akt, and thioredoxin reductase. Pre-treatment with luteolin prior to AMA exposure significantly prevented mitochondrial membrane potential dissipation, ATP loss, inactivation of complex I and IV, ROS production, inactivation of thioredoxin reductase, intracellular calcium elevation, and cytochrome c release induced by AMA. Moreover, luteolin increased activities of PI3K (phosphoinositide 3-kinase) and Akt (protein kinase B), and CREB (cAMP-response element-binding protein) phosphorylation inhibited by AMA treatment. Collectively, these results suggest that luteolin protects MC3T3-E1 cells from AMA-induced cell damage through the improved mitochondrial function and activation of PI3K/Akt/CREB.     

Sublethal concentrations of diverse gold compounds inhibit mammalian cytosolic thioredoxin reductase (TrxR1)

Thioredoxin reductase (TrxR) reduces thioredoxin (Trx), thereby contributing to cellular redox balance, facilitating the synthesis of deoxy-ribose sugars for DNA synthesis, and regulating redox-sensitive gene expression. Auranofin is a gold compound that potently inhibits TrxR. This inhibition is one suspected mechanism of auranofin’s therapeutic benefit in the treatment of rheumatoid arthritis. The use of other gold compounds to treat cancer or inflammatory disease may rely on their ability to inhibit TrxR. In the current study, we tested the hypothesis that a variety of gold compounds may inhibit TrxR.Methods: We exposed rat-TrxR1 to auranofin, gold sodium thiomalate, sodium aurothiosulfate, triphenyl phosphine gold chloride, or gold acetate, and measured TrxR activity ex vivo. We then compared TrxR1 inhibitory levels of gold compounds to those that inhibited mitochondrial activity of THP1 monocytes and OSC2 epithelial cells, estimated by succinate dehydrogenase activity.Results: All gold compounds inhibited TrxR1 at concentrations ranging from 5 to 4000 nM (50% inhibitory concentration). The oxidation state of gold did not correlate with inhibitory potency, but ligand configuration was important. Au(I)-phosphine compounds (triphenyl phosphine gold chloride and auranofin) were the most potent inhibitors of TrxR. All TrxR1 inhibitory concentrations were sublethal to mitochondrial activity in both THP1 and OSC2 cells.Conclusions: Diverse types of gold compounds may be effective inhibitors of TrxR1 at concentrations that do not suppress cellular mitochondrial function. Inhibition may be optimized to some degree by altering the ligand configuration of the compounds. These results support future study of a variety of Au compounds for therapeutic development as inhibitors of TrxR1.     

Mitochondrial permeability transition as the critical target of N-acetyl perfluorooctane sulfonamide toxicity in vitro.

Perfluorooctanyl compounds with active functional groups have been shown to disrupt mitochondrial bioenergetics by three distinct mechanisms: protonophoric uncoupling of mitochondrial respiration, induction of the mitochondrial permeability transition (MPT), or a nonselective increase in membrane permeability. The purpose of this investigation was to identify the initial target and specific sequence of events associated with the N-acetyl substituted perfluorooctanesulfonamides induced MPT. N-acetyl-perfluorooctanesulfonamide (FOSAA), N-ethyl-N-acetyl-perfluorooctanesulfonamide (N-Et FOSAA), perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS), and N-ethyl-N-(2-ethoxy)-perfluorooctanesulfonamide (N-Et FOSE) were added individually to liver mitochondria freshly isolated from Sprague-Dawley rats. Mitochondrial swelling and cytochrome c release were recorded spectrophotometrically, oxygen uptake was monitored with a Clark-type oxygen electrode, and reactive oxygen species (ROS) were monitored by dichlorodihydrofluorescein diacetate (H(2)DCFDA) fluorescence. FOSAA (45 microM) and N-Et FOSAA (7.5 microM) induced calcium-dependent mitochondrial swelling, the release of cytochrome c, inhibition of uncoupled mitochondrial respiration, and ROS generation, all of which were inhibited by cyclosporin-A (CsA). PFOA (200 microM) displayed slight CsA sensitive activity, but neither PFOS (10 microM) nor N-Et FOSE (70 microM) induced the MPT. Results of this investigation demonstrate two important findings: (1) MPT induction is specific to the N-acetyl substituted perfluorooctanesulfonamides and, (2) the sequence of events is initiated by induction of the MPT, which causes the release of cytochrome c as well as other cofactors leading to inhibition of respiration and ROS generation. The toxicity of N-acetyl perfluorooctanyl compounds may therefore reflect the mitochondrial dysfunction, which is compounded by the ensuing oxidative injury.     

Induction of the mitochondrial permeability transition by selenium compounds mediated by oxidation of the protein thiol groups and generation of the superoxide

The cancer chemopreventive effect of selenium compounds cannot be fully explained by the role of selenium as a component of antioxidant enzymes, suggesting that other mechanisms, such as thiol oxidation or free radical generation, also underlie this effect. The toxicities of six different selenium compounds (selenite, selenate, selenocystine, selenocystamine, selenodioxide, and selenomethionine) have now been compared in HepG2 human hepatoma cells and isolated rat liver mitochondria. Selenite, selenocystine, and selenodioxide induced apoptosis in HepG2 cells and mediated oxidation of protein thiol groups in both HepG2 cells and isolated mitochondria. Selenocystamine oxidized protein thiol groups in isolated mitochondria and crude extracts of HepG2 cells but not in intact HepG2 cells, suggesting that this compound is not able to cross the cell membrane. The selenium compounds capable of oxidizing thiol groups also induced the mitochondrial permeability transition (MPT) in isolated mitochondria. Furthermore, they generated the superoxide (O(2) .-) on reaction with glutathione in the presence of mitochondria, and an O(2) .-) scavenger inhibited their induction of the MPT. These results suggest that the pro-apoptotic action of selenium compounds is mediated by both thiol oxidation and the generation of O(2) .-), both of which contribute to opening of the MPT pore.