Lysosomal and mitochondrial pathways in H2O2-induced apoptosis of alveolar type II cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (AT II) cell apoptosis remain poorly defined. Here we investigated the role of lysosomes as modulators of oxidant-mediated AT II cell apoptosis using an in vitro model of H(2)O(2)-stress. H(2)O(2) stress led to time-dependent increases in intracellular oxidants, mitochondrial membrane polarization, cytochrome c release, lysosomal rupture, and AT II cells apoptosis. Increased apoptosis was prevented by specific inhibition of the caspase cascade using the broad-spectrum caspase inhibitor z-VAD-fmk or a caspase 3 inhibitor, or by using functional inhibitors for cathepsin D (pepstatin A) or cathepsin B. Inhibition of cathepsin D also prevented mitochondrial permeabilization and cythocrome c release suggesting that lysosomal rupture precedes and is necessary for the activation of the mitochondrial pathway of cell death.     

Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities

H₂O₂ can freely crosses membranes and in the presence of Fe²⁺ (or Cu⁺) it is prone to participate in Fenton reaction. This study evaluated the concentration and time-dependent effects of H₂O₂-induced oxidative stress on MnSOD, Se:GPx and catalase and on aconitase. Acute and chronic H₂O₂ treatments were able to induce oxidative stress in HeLa cells as they significantly decreased aconitase activity and also caused a very significant decrease on antioxidant enzyme activities. The inhibition of enzyme activities was time- and concentration-dependent. Chronic treatment with 5 µM H₂O₂/h after 24 h was able to decrease all enzyme activities almost at the same level as the acute treatment. Acute and chronic treatments on antioxidant enzyme activities were prevented by cell treatment with ascorbic acid or N-acetylcysteine. These results indicate that antioxidant enzymes can also be affected by the same ROS they produce or neutralize if the time of exposure is long enough.     

EphA2 overexpression reduces H2O2-induced damage of lens epithelial cells

Age-related cataract (ARC) is a progressive lens opacification that occurs from middle to old age. Eph-receptor tyrosinekinase-type A2 (EphA2) has been reported to be associated with ARC. This work aims to investigate the molecular mechanism of EphA2 in ARC. We treated human lens epithelial cells (SRA01/04) with different concentration of H₂O₂ to induce lens epithelial cell damage. Then, we found that H₂O₂ treatment significantly suppressed cell viability and enhanced the expression of EphA2 in the SRA01/04 cells. H₂O₂ treatment repressed cell viability and enhanced the levels of reactive oxygen species (ROS) in SRA01/04 cells, which was partly abolished by EphA2 up-regulation. Moreover, EphA2 overexpression reduced H₂O₂-induced apoptosis of SRA01/04 cells. EphA2 up-regulation caused an up-regulation of Bcl-2, and repressed the expression of Bax and Cleaved-caspase-3 in the SRA01/04 cells following H₂O₂ treatment. In conclusion, our data confirm that EphA2 overexpression enhances cell viability and inhibits apoptosis in the H₂O₂-treated SRA01/04 cells, thereby reducing H₂O₂-induced damage of lens epithelial cells. Thus, this work provides new insights into the mechanism of EphA2 in ARC.     

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.     

Peroxiredoxin II is an essential antioxidant enzyme that prevents the oxidative inactivation of VEGF receptor-2 in vascular endothelial cells

Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.     

Peroxiredoxin 2, glutathione peroxidase,and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract

Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H₂O₂-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H₂O₂ concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H₂O₂, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H₂O₂-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.     

Pterostilbene protects against H2O2-induced oxidative stress by regulating GAS6/Axl signaling in HL-1 cells

Pterostilbene (PTE, trans-3,5-dimethoxy-4′-hydroxystilbene), a natural plant polyphenol, possesses numerous pharmacological effects, including antioxidant, antidiabetic, antiatherosclerotic, and neuroprotective aspects. This study aims to investigate whether PTE plays a protective role against oxidative stress injury by GAS6/Axl signaling pathway in cardiomyocytes. Hydrogen peroxide (H₂O₂)-induced oxidative stress HL-1 cells were used as models. The mechanism by which PTE protected oxidative stress is investigated by combining cell viability, cell ROS levels, apoptosis assay, molecular docking, quantitative real-time PCR, and western blot analysis. GAS6 shRNA was performed to investigate the involvement of GAS6/Axl pathways in PTE's protective role. The results showed that PTE treatment improved the cell morphology and viability, and inhibited the apoptosis rate and ROS levels in H₂O₂-injured HL-1 cells. Particularly, PTE treatment upregulated the levels of GAS6, Axl, and markers related to oxidative stress, apoptosis, and mitochondrial function related. Molecular docking showed that PTE and GAS6 have good binding ability. Taken together, PTE plays a protective role against oxidative stress injury through inhibiting oxidative stress and apoptosis and improving mitochondrial function. Particularly, GAS6/Axl axis is the surprisingly prominent in the PTE-mediated pleiotropic effects.     

H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis.

H2O2 treatment on U937 cells leads to the block of glycolytic flux and the inactivation of glyceraldehyde-3-phosphate-dehydrogenase by a posttranslational modification (possibly ADP-ribosylation). Glycolysis spontaneously reactivates after 2 h of recovery from oxidative stress; thereafter cells begin to undergo apoptosis. The specific ADP-ribosylation inhibitor 3-aminobenzamide inhibits the stress-induced inactivation of glyceraldehyde-3-phosphate-dehydrogenase and the block of glycolysis; concomitantly, it anticipates and increases apoptosis. Exogenous block of glycolysis (i.e., by culture in glucose-free medium or with glucose analogs or after NAD depletion), turns the transient block into a stable one: this results in protection from apoptosis, even when downstream cell metabolism is kept active by the addition of pyruvate. All this evidence indicates that the stress-induced block of glycolysis is not the result of a passive oxidative damage, but rather an active cell reaction programmed via ADP-ribosylation for cell self-defense.     

Ferulic acid (FA) protects human retinal pigment epithelial cells from H2O2‐induced oxidative injuries

The aim of present study is to investigate whether Ferulic acid (FA), a natural polyphenol antioxidant, was able to protect ARPE‑19 cells from hydrogen peroxide (H₂O₂)‑induced damage, and elucidate the underlying mechanisms. Our results revealed that FA pre‐treatment for 24 hours can reverse cell loss of H₂O₂‐induced ARPE‐19 cells via the promotion of cell proliferation and prevention of apoptosis, as evidenced by 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) assay, respectively. Moreover, the addition of FA (5 mM) can decrease Bax and cleaved caspase‐3 protein expression, but increase Bcl‐2 protein expression in ARPE‐19 cells. Furthermore, H₂O₂‐induced oxidative stress in ARPE‐19 cells was significantly alleviated by FA, illustrated by reduced levels of ROS and MDA. In addition, the attenuated antioxidant enzymes activities of (SOD, CAT and GPX) and GSH level were reversed almost to the normal base level by the pre‐addition of FA for 24 hours. In all assays, FA itself did not exert any effect on the change of the above parameters. These novel findings indicated that FA effectively protected human ARPE‐19 cells from H₂O₂‐induced oxidative damage through its pro‐proliferation, anti‐apoptosis and antioxidant activity, suggesting that FA has a therapeutic potential in the prevention and treatment of AMD.     

Neuroprotective effects of triterpenoid saponins from Medicago sativa L. against H2O2-induced oxidative stress in SH-SY5Y cells

Medicago sativa L. is a forage legume plant widely distributed in all continents. Six new triterpenoid saponins, Medicagosides A-F (1–6) and five known ones (7–11) were isolated from M. sativa. Their structures were determined via HRESIMS, 1D and 2D NMR analysis. Biologically, all the isolates displayed neuroprotective activities against H₂O₂-induced damage in SH-SY5Y cells. Among them, compounds 1, 3–5 and 10 exhibited striking neuroprotective activities at 100 μM, restoring cell viability range from 79.66% to 89.03%, relative to 79.46% (100 μM) of Trolox used as the positive control.     

Tanshinone IIA protects lens epithelial cells from H2O2-induced injury by upregulation of lncRNA ANRIL

Tanshinone IIA is a lipophilic diterpene extracted from the Salvia miltiorrhiza bunge, possessing antiapoptotic and antioxidant activities. The purpose of this study was to explore the effects of Tanshinone IIA on age-related nuclear cataract. Human lens epithelial cell line SRA01/04 was subjected to H ₂O ₂ to mimic a cell model of cataract. Cell Counting Kit-8 assay, flow cytometer, and reactive oxygen species (ROS) detection were performed to evaluate the effect of Tanshinone IIA pretreatment on SRA01/04 cells injured by H ₂O ₂. Besides, the real-time quantitative polymerase chain reaction was used to assess the expression of long noncoding RNA (lncRNA) antisense noncoding RNA in the INK4 locus (ANRIL). Western blot analysis was performed to detect the expression of core proteins involved in cell survival and nuclear factor-κB (NF-κB) pathway. H ₂O ₂ significantly decreased SRA01/04 cells viability, whereas increased apoptosis and ROS generation. This phenomenon was coupled with the upregulated p53, p21, Bax, cleaved caspase-3, and the downregulated cyclinD1, CDK4, and Bcl-2. Tanshinone IIA pretreatment protected SRA01/04 cells against H ₂O ₂-induced injury. In the meantime, the expression of lncRNA ANRIL was upregulated by Tanshinone IIA. And, the protective effects of Tanshinone IIA on H ₂O ₂-stimulated SRA01/04 cells were abolished when lncRNA ANRIL was silenced. Moreover, the elevated expression of lncRNA ANRIL induced by Tanshinone IIA was abolished by BAY 11-7082 (an inhibitor of NF-κB). To conclude, Tanshinone IIA protects SRA01/04 cells from apoptosis triggered by H ₂O ₂. Tanshinone IIA confers its protective effects possibly via modulation of NF-κB signaling and thereby elevating the expression of lncRNA ANRIL.     

Cadmium-induced apoptosis of primary epithelial lung cells: Involvement of Bax and p53, but not of oxidative stress

The lung is a target organ for cadmium (Cd) toxicity. Apoptosis induced by cadmium acetate (CdAc) was studied in alveolar type 2 cells and Clara cells isolated from rat lung. Relatively low concentrations of CdAc (1–10 μmol/L) induced apoptosis after exposure for 20 h. Type 2 cells were more sensitive than Clara cells to Cd-induced apoptosis and loss of cell viability. On exposure to 10 μmol/L CdAc, the levels of the apoptosis-modulating proteins p53 and Bax were increased at 2 h and 5–12 h, respectively. The expression of p53 preceded the expression of Bax and the apoptotic process. The exposure to 10 μmol/L CdAc did not significantly increase the formation of cellular reactive oxygen species (ROS). However, after exposure to a high concentration of CdAc (100 μmol/L), a 30% increase of the ROS level was observed. No significant nitric oxide production was measured following CdAc exposure. Catalase, superoxide dismutase, dimethyl sulfoxide, or tetramethylthiourea did not protect against Cd-induced apoptosis. In conclusion, the results show that Clara cells and type 2 cells are sensitive to Cd-induced apoptosis. Increased levels of p53 and Bax are suggested to be involved in the apoptosis. The apoptosis did not appear to be mediated by oxidative pathways. This revised version was published online in August 2006 with corrections to the Cover Date.     

Suppression of miR-181a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression

Background

Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H₂O₂. The expression of HKII in MSCs exposed to H₂O₂ was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H₂O₂ on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H₂O₂-induced apoptosis of MSC was evaluated.

Results

H₂O₂ (600 μM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H₂O₂ increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H₂O₂-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H₂O₂.

Conclusions

These findings suggest that H₂O₂-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.     

Neuroprotective effect of polysaccharide separated from Perilla frutescens Britton var. acuta Kudo against H2O2-induced oxidative stress in HT22 hippocampus cells

This study was carried out to evaluate the neuroprotective activity of polysaccharide extracts isolated from Perilla frutescens (PEPF) in H₂O₂-treated HT22 hippocampus cells. The PEPF treatment was found to increase the anti-oxidant activities of HT22 hippocampus cells. PEPF treatment resulted in a significant protection of HT22 hippocampus cells against H₂O₂-induced neurotoxicity, this protection ultimately occurred through an inhibition of ROS-mediated intracellular Ca²⁺ levels leading to MAPKs and NF-κB, as well as the accumulation of PI3K/AKT and Nrf2-mediated HO-1/NQO1 pathways. Furthermore, PEPF not only decreased the expression of Bax, cytochrome c, and cleaved caspases-3, -8, and -9, but also increased the expression of PARP and Bcl-2 in the H₂O₂-treated HT22 hippocampus cells, which overall contributed to the neuroprotective action. PEPF retains its mitochondrial membrane potential and reduces the elevated levels of sub-G1 phase and apoptotic morphological features induced by H₂O₂. It also reduces the malondialdehyde levels and enhances the intracellular SOD activity.     

Glutathione S-transferases as antioxidant enzymes: small cell lung cancer (H69) cells transfected with hGSTA1 resist doxorubicin-induced apoptosis

It has been suggested that the alpha-class glutathione S-transferases (GSTs) protect various cell types from oxidative stress and lipid peroxidation (LPO). In order to examine the protective role of alpha-class GST isozyme hGSTA1-1 against doxorubicin (DOX)-induced lipid peroxidation, cytotoxicity, and apoptosis, human small cell lung cancer (SCLC) H69 cells were stably transfected with hGSTA1. Immunological and biochemical characterization of hGSTA1-transfected cells revealed the expression of functionally active hGSTA1-1 localized near the cellular plasma membranes. hGSTA1-transfected cells acquired significantly increased resistance to the DOX-induced cytotoxicity by suppressing lipid peroxidation levels in these cells. Overexpression of hGSTA1-1 in cells inhibited DOX-mediated depletion of GSH and higher GSH levels were found in DOX-treated hGSTA1-transfected cells as compared with empty vector-transfected controls. hGSTA1-1 overexpression also provided protection to cells from DOX-induced apoptosis by inhibiting phosphorylation of c-Jun-N-terminal kinases (JNK), caspase-3 activation, and by preserving the levels of anti-apoptotic protein Bcl-2. These results are consistent with the idea that the alpha-class GSTs provide protection against oxidative stress by attenuating lipid peroxidation and these enzymes can modulate signaling for apoptosis.