Oxidant-induced Cell Death Research Articles

Abstract 161: ABCB10 Plays a Role in Heme Synthesis and Mitochondrial δ-Aminolevulinic Acid Export

Introduction: Heme is essential for cardiovascular health, as it is a component of myoglobin, cytochrome P450, electron transport chain complexes, and antioxidant enzymes. The eight steps of heme synthesis are well characterized; however, it is not known how the synthetic intermediates are transported between mitochondria and cytosol. We hypothesized that the mitochondrial ATP-binding cassette protein-B10 (ABCB10) plays a role in heme synthesis by mediating the export of δ-aminolevulinic acid (ALA) out of the mitochondria. Results: Lentiviral shRNA-based knockdown of ABCB10 in H9c2 cardiac myoblasts significantly reduced cytosolic and mitochondrial heme levels and the activities of heme-containing proteins (catalase, peroxidase and complex IV), while their mRNA levels remained unchanged. The observed heme deficiency was not due to a defect in heme synthesis, iron incorporation, or enhanced heme degradation, as ABCB10 shRNA had no effect on the expression of enzymes mediating these steps. We then tested whether ABCB10 regulates mitochondrial export of ALA. Addition of exogenous ALA completely reversed the reduction in heme levels and normalized the activities of heme containing enzymes in cells with ABCB10 knockdown. However, overexpression of ALA synthase 1 (ALAS1), an enzyme catalyzing the production of ALA in the mitochondria, resulted in increased heme levels at baseline, but failed to restore heme content in ABCB10 shRNA-treated cells. To examine the role of ABCB10 in cardiac disease, we measured ABCB10 expression in the hearts of mice subjected to myocardial infarction and in explanted human hearts with ischemic cardiomyopathy. ABCB10 protein was significantly elevated in mouse and human hearts, and adenoviral overexpression of ABCB10 in H9c2 cardiac myoblasts significantly attenuated the production of reactive oxygen species (ROS), suggesting the protective role for ABCB10 in heart disease. Conclusions: Our studies place ABCB10 between the mitochondrial synthesis of ALA by ALAS1 and the cytosolic conversion of ALA into heme, suggesting that ABCB10 plays a role ALA export out of the mitochondria. Furthermore, its overexpression exerts protective effects against oxidant induced cell death in cardiac cells.

Estradiol protects adipose tissue–derived stem cells against H2O2‐induced toxicity

Oxidative stress is associated with various pathophysiological processes, including cell survival, adhesion, apoptosis, and cancer. In the present study, we aimed to evaluate the effects of H(2)O(2)-induced toxicity on adipose tissue-derived stem cells (ADSCs) and whether 17β-estradiol (E(2)) has protective effects on these cells. ADSCs derived from adult Sprague-Dawley rats were pretreated with different doses of E(2) for 24 h and then exposed to 200 µM H(2)O(2) for 4 h. Incubation of ADSCs with H(2)O(2)-decreased cell viability in a concentration-dependent fashion (p < 0.0001), whereas pretreatment of these cells with E(2) significantly reversed toxicity (p < 0.05), inhibited apoptotic changes, and decreased lipid peroxidation (p < 0.0005). Our findings suggest that E(2) protects ADSCs from oxidative-induced cell death, and therefore, it may be used to improve the survival rate and regenerative capacity of stem cells.

The Role of Endoplasmic Reticulum Ca2+-Independent Phospholipase A2γ in Oxidant-Induced Lipid Peroxidation, Ca2+ Release, and Renal Cell Death

Oxidant-induced lipid peroxidation and cell death are major components of ischemia/reperfusion and toxicant injury. Our previous studies showed that renal proximal tubular cells (RPTCs) express Ca(2+)-independent phospholipase A(2)γ (iPLA(2)γ) in endoplasmic reticulum (ER) and mitochondria and that iPLA(2)γ is cytoprotective. Our present studies reveal the role of ER-iPLA(2)γ in oxidant-induced ER lipid peroxidation, Ca(2+) release, and cell death. Oxidant tert-butyl hydroperoxide (TBHP) caused ER lipid peroxidation and Ca(2+) release in isolated rabbit kidney cortex microsomes. ER-iPLA(2)γ inhibition, using bromoenol lactone (BEL), potentiated both oxidant-induced ER lipid peroxidation and Ca(2+) release. Assessment of fatty acids using electrospray ionization-mass spectrometry revealed that ER-iPLA(2)γ mediates the TBHP-induced release of arachidonic acid (20:4), linoleic acid (18:2), and their oxidized forms (18:2-OH, 18:2-OOH, 20:4-OH, 20:4-OOH, 20:4-(OH)(3). iPLA(2)γ inhibition also accelerated oxidant-induced ER Ca(2+) release in RPTC. Depletion of ER Ca(2+) stores in RPTC with thapsigargin, an ER Ca(2+) pump inhibitor, prior to TBHP exposure reduced necrotic cell death and blocked the potentiation of TBHP-induced necrotic cell death by BEL. Together, these data provide strong evidence that ER-iPLA(2)γ protects renal cells from oxidant-induced necrotic cell death by releasing unsaturated and/or oxidized fatty acids from ER membranes, thereby preserving ER membrane integrity and preventing ER Ca(2+) release.

Oxidant-Induced Cell Death and Nrf2-Dependent Antioxidative Response Are Controlled by Fra-1/AP-1

AP-1 (Jun/Fos) transcription factors play key roles in various biological processes, including cell death. Here we report a novel role for Fra-1 in oxidant-induced cell death controlled by modulating antioxidant gene expression. Fra-1-deficient (Fra-1(Δ/Δ)) mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts (PLFs) were remarkably resistant to H(2)O(2)- and diquat-induced cell death, compared to their wild-type (Fra-1(+/+)) counterparts. Fra-1 deficiency ablated oxidant-induced mitochondrion-dependent apoptosis. Fra-1(Δ/Δ) cells had elevated basal levels of antioxidant enzymes and intracellular glutathione (GSH), which were further stimulated by oxidants. Loss of Fra-1 led to an increased half-life of transcription factor Nrf2 and increased recruitment of this protein to the promoters of antioxidant genes and increased their expression. Depletion of intracellular GSH or RNA interference (RNAi)-mediated knockdown of Nqo1, Hmox1, and Nrf2 restored oxidant-induced cell death in Fra-1(Δ/Δ) cells. Thus, Fra-1 appears to increase susceptibility to oxidants and promotes cell death by attenuating Nrf2-driven antioxidant responses.

Mechanism of RPE Cell Death in α-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.

Nitric oxide induces cell death in canine cruciate ligament cells by activation of tyrosine kinase and reactive oxygen species

BackgroundThere is increasing evidence suggesting that development of progressive canine cranial cruciate ligament (CCL) rupture involves a gradual degeneration of the CCL itself, initiated by a combination of factors, ranging from mechanical to biochemical. To date, knowledge is lacking to what extent cruciate disease results from abnormal biomechanics on a normal ligament or contrary how far preliminary alterations of the ligament due to biochemical factors provoke abnormal biomechanics. This study is focused on nitric oxide (NO), one of the potential biochemical factors. The NO-donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in canine cranial and caudal cruciate ligament cells and to characterize signaling mechanisms during NO-stimulation.ResultsSodium nitroprusside increased apoptotic cell death dose- and time-dependently in cruciate ligamentocytes. Cells from the CCL were more susceptible to apoptosis than CaCL cells. Caspase-3 processing in response to SNP was not detected. Testing major upstream and signal transducing pathways, NO-induced cruciate ligament cell death seemed to be mediated on different levels. Specific inhibition of tyrosine kinase significantly decreased SNP-induced cell death. Mitogen activated protein kinase ERK1 and 2 are activated upon NO and provide anti-apoptotic signals whereas p38 kinase and protein kinase C are not involved. Moreover, data showed that the inhibition reactive oxygen species (ROS) significantly reduced the level of cruciate ligament cell death.ConclusionsOur data support the hypothesis that canine cruciate ligamentocytes, independently from their origin (CCL or CaCL) follow crucial signaling pathways involved in NO-induced cell death. However, the difference on susceptibility upon NO-mediated apoptosis seems to be dependent on other pathways than on these tested in the present study. In both, CCL and CaCL, the activation of the tyrosine kinase and the generation of ROS reveal important signaling pathways. In perspective, new efforts to prevent the development and progression of cruciate disease may include strategies aimed at reducing ROS.

Exacerbation of Oxidative Stress-Induced Cell Death and Differentiation in Induced Pluripotent Stem Cells Lacking Heme Oxygenase-1

Embryonic stem cells (ESCs) are promising donor sources in cell therapies for various diseases. Although low levels of reactive oxygen species (ROS) are necessary for the maintenance of stem cells, increased ROS levels initiate differentiation and cell damage. We and others have previously demonstrated that heme oxygenase (HO)-1, a stress response protein with antioxidative and anti-inflammatory properties, plays critical protective functions in cardiovascular and other diseases. However, the functions of HO-1 in ESCs remain to be elucidated. Our goal was to investigate the roles of HO-1 in ESC survival and differentiation. Due to the lack of HO-1-deficient ESCs, we used Oct3/4, Sox2, c-Myc, and Klf4 retroviruses to reprogram mouse embryonic fibroblasts into induced pluripotent stem (iPS) cells of different HO-1 genotypes. These iPS-HO-1 cells exhibited characteristics of mouse ESCs (mESCs) and formed teratomas that were composed of cell types of all 3 germ layers after injected into severe combined immunodeficiency mice. In response to oxidant stress, iPS-HO-1(-/-) cells accumulated higher levels of intracellular ROS compared with D3 mESCs or iPS-HO-1(+/+) cells and were more prone to oxidant-induced cell death. Spontaneous differentiation experiments revealed that Oct4 levels were significantly lower in iPS-HO-1(-/-) cells after leukemia inhibitory factor withdrawal and removal of feeders. Further, during the course of spontaneous differentiation, iPS-HO-1(-/-) cells had enhanced Erk1/2 phosphorylation, which has been linked to ESC differentiation. By the loss-of-function approach using iPS-HO-1(-/-) cells, our results demonstrate that a lack of HO-1 renders iPS cells more prone to oxidative stress-induced cell death and differentiation.

Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs

In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury.

Lane-Hamilton Syndrome: Ferritin Protects Lung Macrophages Against Iron and Oxidation

Lysosomal disruption and consequent apoptosis have been implicated in lung diseases characterized by iron overload. Free reactive iron in lysosomes sensitizes cells to oxidative stress. Apoptosis is prevented by heavy-chain (H)-ferritin, which can incorporate lysosomal iron into ferritin molecules. Tumor necrosis factor (TNF)-α stimulates the synthesis of H-ferritin. Idiopathic pulmonary hemosiderosis presents with the accumulation of iron and the upregulation of ferritin synthesis. We therefore analyzed the lysosomal response to oxidants and the role of H-ferritin synthesis in lung macrophages (LMs) harvested from the first Swedish case, to our knowledge, of Lane-Hamilton syndrome. Iron-exposed murine macrophages were used as a reference. Both cell types were stimulated with TNF-α (or not), then iron was assessed cytochemically and by atomic absorption spectrophotometry. H-ferritin expression was analyzed by Western blot and reduced glutathione (GSH) by spectrofluorometry. Following exposure to hydrogen peroxide, lysosomal membrane integrity and DNA degradation were analyzed by flow cytometry, whereas morphologic signs of apoptosis and necrosis were assessed by light microscopy. GSH levels were approximately equal in LMs and murine macrophages. Although LMs contained much more iron than murine macrophages, lysosomal iron was bound in a harmless unreactive state by ample amounts of ferritin and hemosiderin, its lysosomal degradation product. Therefore, lysosomes of LMs were more oxidant resistant, and these cells were more adept at surviving oxidative stress. In both cell types, TNF-α prevented oxidant-induced lysosomal damage and cell death by upregulating synthesis of H-ferritin and GSH. Iron-overloaded LMs are equipped with an efficient armor of antioxidative mechanisms of which H-ferritin and hemosiderin seem to be particularly important.

Role of Thioredoxin 1 and Thioredoxin 2 on Proliferation of Human Adipose Tissue-Derived Mesenchymal Stem Cells

Thioredoxin (TRX) is a ubiquitous redox protein that is involved in numerous biological functions, including the first unique step in DNA synthesis. TRX provides control over a number of transcription factors affecting cell proliferation and death through a mechanism referred to as redox regulation. In mammals, there are at least 3 members of the TRX family: TRX1, TRX2, and sperm TRX. To investigate the role of TRX1 and TRX2 in human adipose tissue-derived mesenchymal stem cells (hADSC), we modulated TRX1 and TRX2 expressions in hADSC using a lentiviral gene transfer system and small interfering RNA technique. Reverse transcription-polymerase chain reaction analysis confirmed the changes in expression of TRX1 and TRX2 in lentivirus-transduced or small interfering RNA-transfected cells. Although overexpression of TRX1 and TRX2 did not affect the differentiation of hADSC into adipogenic and osteogenic lineages, it increased the proliferation of hADSC compared with control lentivirus-transduced cells, decreased reactive oxygen species production, and inhibited oxidant-induced cell death. Downregulation of TRX1 and TRX2 inhibited cell proliferation. The treatment of U0126 blocked TRX-induced increase in cell proliferation. Overexpression of TRX1 and TRX2 increased ERK1/2 phosphorylation, nuclear factor-kappaB activation, and β-catenin/Tcf promoter activities and inhibited lucine zipper tumor suppressor 2 expression. On the contrary, downregulation of TRX1 and TRX2 expression induced inhibition of ERK1/2 phosphorylation, nuclear factor-kappaB activation, and β-catenin/Tcf promoter activities and increased lucine zipper tumor suppressor 2 expression. Activation of Wnt signal increased ERK1/2 activities in hADSC. These results indicated that TRX1 and TRX2 regulate the proliferation and survival of hADSC; these processes are mediated by the activation of ERK1/2.

Regulation and Cytoprotective Role of Hexokinase III

BackgroundHexokinases (HKs) catalyze the first step in glucose metabolism. Of the three mammalian 100-kDa HK isoforms, HKI and II can bind to mitochondria and protect against cell death. HKIII does not bind mitochondria, and little is known about its regulation or cytoprotective effects. We studied the regulation of HKIII at the transcriptional and protein levels and investigated its role in cellular protection.Methodology/Principal FindingsWe show that like HKII, HKIII expression is regulated by hypoxia, but other factors that regulate HKII expression have no effect on HKIII levels. This transcriptional regulation is partially dependent on hypoxia-inducible factor (HIF) signaling. We also demonstrate regulation at the protein level, as mutations in putative N-terminal substrate binding residues altered C-terminal catalytic activity, suggesting that HKIII activity is governed, in part, by interactions between these two domains. Overexpression of HKIII reduced oxidant-induced cell death, increased ATP levels, decreased the production of reactive oxygen species (ROS), and preserved mitochondrial membrane potential. HKIII overexpression was also associated with higher levels of transcription factors that regulate mitochondrial biogenesis, and greater total mitochondrial DNA content. Attempts to target HKIII to the mitochondria by replacing its N-terminal 32-amino-acid sequence with the mitochondrial-targeting sequence of HKII led to protein aggregation, suggesting that this region is necessary to maintain proper protein folding and solubility.Conclusions/SignificanceThese results suggest that HKIII is regulated by hypoxia and there are functional interactions between its two halves. Furthermore, HKIII exerts protective effects against oxidative stress, perhaps by increasing ATP levels, reducing oxidant-induced ROS production, preserving mitochondrial membrane potential, and increasing mitochondrial biogenesis.

Studies on the phenolic profiling, anti-oxidant and cytotoxic activity of Indian honey: in vitro evaluation

Commercial honey types were screened for phenolic profile and anti-oxidant capacity. Phenolic profiling was done using high performance liquid chromatography, which was further corroborated with electro spray ionisation-mass spectroscopy. Dihydroxy benzoic acid, caffeic acid, ferulic acid and cinnamic acid were the major phenolic constituents found in the honey samples. The anti-oxidant content and free-radical scavenging effect of honey were established using various assays. Total anti-oxidant potential and free-radical scavenging ability varied among the honey varieties and exhibited significant correlation with their phenolic content. Further, honey samples with richly abundant phenolic content were found to limit oxidant-induced cell death more effectively. Cytotoxic studies of a selected sample on a breast cancer cell displayed growth inhibition, depending on the concentration used. Cell cycle analysis indicated increasing accumulation of cells at the sub-G1 phase. These results summarise the phenolic profile and anti-oxidant and cytotoxic potential of Indian honey samples for the first time.

Oxidative stress enhances granulocytic differentiation in HL 60 cells, an acute promyelocytic leukemia cell line

This paper studied the effects of physiologically available oxidants on HL 60 differentiation induced by all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). Hydrogen peroxide (15 μM) and taurine chloramine (200 μM) induced HL 60 differentiation, which was detected by CD11b expression and superoxide production. Cd11b and p67phox mRNA expression was also augmented by these oxidants. In contrast, reducing chemicals, such as dithiothreitol, 2,3-dimercapto-1-propanol and N-acetylcysteine inhibited CD11b expression. Notably, DMSO inhibited methionine sulfoxide reductase activity, induced heme oxygenase-1 (ho-1) mRNA and enhanced oxidant-induced cell death, which indicated that DMSO intensified oxidative stress. After the addition of oxidants, ho-1 expression preceded the cd11b expression. Vicinal dithiol-reactive phenylarsine oxide (50 nM) also increased CD11b expression induced by DMSO or ATRA. These observations suggested that oxidative stress enhanced granulocytic differentiation of HL 60 cells and that leukaemic cell differentiation was affected by cellular redox status.

Mitochondria-Targeted Antioxidant Peptides

Overproduction of reactive oxygen species (ROS) under pathophysiologic conditions is part of the disease process. These ROS are released from different sources, and in particular from mitochondria. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are unclear, oxidative stress seems to play an important role. ROS are essential to cell function, but adequate levels of antioxidant defenses are required in order to avoid the harmful effects that excessive ROS production can produce. Mitochondrial oxidative stress damage and dysfunction contribute to a number of cell pathologies that manifest themselves through a range of conditions. The antioxidants available until now have not proved to be particularly effective against many of these disorders. It is possible that these antioxidants do not reach the sites of free radical generation, especially when mitochondria are the primary source of ROS. Recent developments in mitochondria-targeted antioxidants have moved closer to providing protection against mitochondrial oxidative damage. The SS (Szeto-Schiller) peptide antioxidants represent a novel approach that employs the targeted delivery of antioxidants to the inner mitochondrial membrane. These SS peptides scavenge hydrogen peroxide and peroxynitrite and inhibit lipid peroxidation. By reducing mitochondrial ROS, they inhibit mitochondrial permeability transition and cytochrome c release, thus preventing oxidant-induced cell death. Preclinical studies support the use of these peptides for ischemia-reperfusion injury and neurodegenerative disorders. Although peptides have often been considered to be poor drug candidates, the few that have been studied are promising agents for the treatment of diseases.

Immunoproteasome deficiency alters retinal proteasome’s response to stress

Our previous work demonstrated that immunoproteasome is up-regulated in the retina and brain in response to injury that does not involve an inflammatory response (J. Neurochem. 2008; 106:158). These results suggest additional non-immune functions for the immunoproteasome in the cellular stress response pathway. The present study further investigates the potential involvement of the immunoproteasome in responding to the chronic stress of aging or oxidant exposure in the retina and cultured retinal pigment epithelial (RPE) cells from knock-out mice missing either one (lmp7(-/-)) or two (lmp7(-/-)/mecl-1(-/-)) immunoproteasome subunits. We show that aging and chronic oxidative stress up-regulates immunoproteasome in the retina and RPE from wild-type mice. No up-regulation of LMP2 was observed in retinas or RPE lacking MECL-1 and/or LMP7, suggesting that the full complement of immunoproteasome subunits is required to achieve maximal up-regulation in response to stress. We also show that RPE deficient in immunoproteasome are more susceptible to oxidation-induced cell death, supporting a role for immunoproteasome in protecting from oxidative stress. These results provide key mechanistic insight into novel aspects of proteasome biology and are an important first step in identifying alternative roles for retinal immunoproteasome that are unrelated to its role in the immune response.

DJ-1 protects against oxidative damage by regulating the thioredoxin/ASK1 complex

DJ-1 is a multifunctional protein linked to recessively inherited Parkinson's disease (PD) due to loss of function mutations. Among its activities is anti-oxidant property leading to cytoprotection under oxidative stress conditions. A key effector of oxidant-induced cell death is the MAP3 kinase apoptosis signal-regulating kinase 1 (ASK1) which is bound to and inhibited by thioredoxin 1 (Trx1) under basal conditions. Upon oxidative stimuli, however, ASK1 dissociates from this physiological inhibitor and is activated. In the present study, we investigated the role of DJ-1 in regulating Trx1/ASK1 interaction. Over-expression of DJ-1 suppressed ASK1 activation in response to H 2O 2 in a time-dependent manner. Wild-type DJ-1, but not the PD-associated L166P mutant, prevented the dissociation of ASK1 from Trx1 in response to H 2O 2. Among cysteine mutants of DJ-1, C46S, C53S, and C106S, only C106S failed to inhibit this dissociation implying that cysteine 106 is essential for Trx1/ASK1 regulation. Furthermore, compared to wild-type mice, DJ-1 null mouse brain homogenates and embryonic fibroblasts were more susceptible to oxidant-induced dissociation of ASK1 from Trx1, activation of the downstream kinase c-Jun N-terminal kinase, and to cell death. These findings point to yet another mechanism through which DJ-1 has anti-oxidant and cytoprotective properties by regulating the Trx1/ASK1 complex and controlling the availability of ASK1 to effect apoptosis.

Zinc inhibits oxidative stress-induced iron signaling and apoptosis in Caco-2 cells

Studies in humans and animals have suggested negative interactions of iron and zinc during their intestinal absorption. Further, zinc seems to prevent iron-induced oxidative damage in rats, which was hypothesized to be through the modulation of the intracellular iron signaling pathway. The aim of this study was, therefore, to understand the effects of zinc on oxidant-induced iron signaling and cell death in human enterocyte-like Caco-2 cells. We demonstrate that zinc decreases glucose/glucose oxidase (H 2O 2-generating system)-induced iron uptake and inhibits iron-regulatory protein 1 activation and divalent metal ion transporter 1 expression. There was also a concomitant decrease in oxidant-induced intracellular labile iron and restoration of ferritin and metallothionein expression. Further, zinc enhanced the Bcl-2/Bax ratio and reduced caspase-3 activity, leading to inhibition of apoptosis. Interestingly, bathophenanthroline disulfonic acid, an extracellular iron chelator, emulated the effects of zinc except for the reduced ferritin levels. These results suggest that zinc inhibits apoptosis by reducing oxidant-induced iron signaling in Caco-2 cells.

Synergistic Chondroprotective Effect of α-Tocopherol, Ascorbic Acid, and Selenium as well as Glucosamine and Chondroitin on Oxidant Induced Cell Death and Inhibition of Matrix Metalloproteinase-3—Studies in Cultured Chondrocytes

Overproduction of reactive oxygen species and impaired antioxidant defence accompanied by chronic inflammatory processes may impair joint health. Pro‑inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) stimulate the expression of metalloproteinases which degrade the extracellular matrix. Little is known regarding the potential synergistic effects of natural compounds such as α‑tocopherol (α-toc), ascorbic acid (AA) and selenium (Se) on oxidant induced cell death. Furthermore studies regarding the metalloproteinase-3 inhibitory activity of glucosamine sulfate (GS) and chondroitin sulfate (CS) are scarce. Therefore we have studied the effect of α-toc (0.1–2.5 µmol/L), AA (10–50 µmol/L) and Se (1–50 nmol/L) on t-butyl hydroperoxide (t‑BHP, 100–500 µmol/L)-induced cell death in SW1353 chondrocytes. Furthermore we have determined the effect of GS and CS alone (100–500 µmol/L each) and in combination on MMP3 mRNA levels and MMP3 secretion in IL-1β stimulated chondrocytes. A combination of α-toc, AA, and Se was more potent in counteracting t‑BHP‑induced cytotoxicity as compared to the single compounds. Similarly a combination of CS and GS was more effective in inhibiting MMP3 gene expression and secretion than the single components. The inhibition of MMP3 secretion due to GS plus CS was accompanied by a decrease in TNF-α production. Combining natural compounds such as α-toc, AA, and Se as well as GS and CS seems to be a promising strategy to combat oxidative stress and cytokine induced matrix degradation in chondrocytes.

Nitric Oxide Induces Cell Death by Regulating Anti-Apoptotic BCL-2 Family Members

Nitric oxide (NO) activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are completely protected from NO-induced cell death. The individual loss of the BH3-only proteins, Bim, Bid, Puma, Bad or Noxa, or Bid knockdown in Bim−/−/Puma−/− MEFs, does not prevent NO-induced cell death. Our data show that the anti-apoptotic protein MCL-1 undergoes ASK1-JNK1 mediated degradation upon exposure to NO, and that cells deficient in either Ask1 or Jnk1 are protected against NO-induced cell death. NO can inhibit the mitochondrial electron transport chain resulting in an increase in superoxide generation and peroxynitrite formation. However, scavengers of ROS or peroxynitrite do not prevent NO-induced cell death. Collectively, these data indicate that NO degrades MCL-1 through the ASK1-JNK1 axis to induce BAX/BAK-dependent cell death.

Synthetic Triterpenoids Attenuate Cytotoxic Retinal Injury: Cross-talk between Nrf2 and PI3K/AKT Signaling through Inhibition of the Lipid Phosphatase PTEN

Evidence implicating oxidative stress in the pathogenesis of age-related macular degeneration suggests that antioxidant therapy could play a role in preventing its progression. The aim of this study was to determine whether derivatives of the triterpenoid (TP) 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO; CDDO-imidazolide [-Im], CDDO-ethylamide [-EA], and CDDO-trifluoroethylamide [-TFEA]) confer cytoprotection from oxidative- and photooxidative-induced cellular damage and to explore the molecular mechanisms of this cytoprotection. Retinal pigment epithelial and retinal photoreceptor cell lines were treated with TP derivatives. Induction of Nrf2 signaling was measured by reporter assay. Cytoprotection was quantified by MTT assay. To determine whether TPs confer in vivo cytoprotection, BALB/c mice were pretreated with CDDO-TFEA, and retinal degeneration was induced by light exposure. To explore the association of TPs with PTEN, a biotinylated derivative of CDDO (CDDO-Bt) was used. Treatment with CDDO-Im-, -TFEA-, or -EA-induced Nrf2 signaling and TP pretreatment protected retinal cell lines from oxidant-induced cell death. The antioxidant and cytoprotective potential of these compounds was then examined in vivo. Treatment of BALB/c mice with CDDO-TFEA induced the Nrf2-regulated transcripts glcl and trx1 in retinal tissue and was protective from photooxidative retinal damage. Treatment with CDDO-Im leads to phosphorylation of AKT. CDDO-Bt directly binds cysteine 124 within PTEN's active site and inhibits PTEN's lipid phosphatase activity in vitro. Thus the stimulation of AKT activity is mediated by TP inhibition of PTEN activity. These studies highlight the potential of TPs in retinal cytoprotection and implicate PTEN inhibition as a target in cytoprotection.