Attenuation Of Reactive Oxygen Species Research Articles

Mitochondria-mediated cardioprotection by trimetazidine in rabbit heart failure

Trimetazidine (TMZ) is used successfully for treatment of ischemic cardiomyopathy, however its therapeutic potential in heart failure (HF) remains to be established. While the cardioprotective action of TMZ has been linked to inhibition of free fatty acid oxidation (FAO) via 3-ketoacyl CoA thiolase (3-KAT), additional mechanisms have been suggested. The aim of this study was to evaluate systematically the effects of TMZ on calcium signaling and mitochondrial function in a rabbit model of non-ischemic HF and to determine the cellular mechanisms of the cardioprotective action of TMZ. TMZ protected HF ventricular myocytes from cytosolic Ca2+ overload and subsequent hypercontracture, induced by electrical and ß-adrenergic (isoproterenol) stimulation. This effect was mediated by the ability of TMZ to protect HF myocytes against mitochondrial permeability transition pore (mPTP) opening via attenuation of reactive oxygen species (ROS) generation by the mitochondrial electron transport chain (ETC) and uncoupled mitochondrial nitric oxide synthase (mtNOS). The majority of ROS generated by the ETC in HF arose from enhanced complex II-mediated electron leak. TMZ inhibited the elevated electron leak at the level of mitochondrial ETC complex II and improved impaired activity of mitochondrial complex I, thereby restoring redox balance and mitochondrial membrane potential in HF. While TMZ decreased FAO by ~15%, the 3-KAT inhibitor 4-bromotiglic acid did not provide protection against palmitic acid-induced mPTP opening, indicating that TMZ effects were 3-KAT independent. Thus, the beneficial effect of TMZ in rabbit HF was not linked to FAO inhibition, but rather associated with reduced complex II- and uncoupled mtNOS-mediated oxidative stress and decreased propensity for mPTP opening.

Attenuation of reactive oxygen species by antioxidants suppresses hypoxia-induced epithelial-mesenchymal transition and metastasis of pancreatic cancer cells

Hypoxia has been shown to promote metastasis of cancer cells through induction of epithelial-mesenchymal transition (EMT). It is also known to cause generation of reactive oxygen species (ROS). We investigated here the role of ROS in hypoxia-induced EMT and whether attenuation of ROS by antioxidants suppresses hypoxia-induced EMT and metastasis of human pancreatic cancer cells in a xenograft nude mouse model. PANC-1 and MiaPaCa-2 cells exposed to hypoxia (1% O(2)) showed increased ROS generation and characteristic changes of EMT such as morphological changes, enhanced invasiveness, and upregulation of EMT regulators, SLUG, SNAI1 and TWIST. The antioxidants N-acetylcysteine (NAC) and ebselen significantly suppressed EMT and the expression of EMT regulators during hypoxia. NAC abrogated activation of HIF-1α and NF-κB, both of which were found to play an active role in hypoxia-induced EMT. Administration of NAC to nude mice with orthotopic tumors suppressed the expression of EMT regulators in hypoxic areas and significantly inhibited hepatic metastasis. Together, the present findings demonstrate that attenuation of ROS by antioxidants suppresses hypoxia-induced EMT and metastatic phenotype, suggesting that antioxidants may be of therapeutic value in treating pancreatic cancers.

CREG mediated adventitial fibroblast phenotype modulation: A possible therapeutic target for proliferative vascular disease

Proliferative vascular diseases, of which neointimal formation is a key pathological feature, cause significant morbidity and mortality worldwide. Adventitia is the outermost connective tissue that surrounds an artery. In recent years, accumulating data indicate that adventitial fibroblasts participate in the formation of neointimal lesions. Our previous studies have demonstrated that cellular repressor of E1A-stimulated genes (CREG) plays critical roles in reducing neointimal hyperplasia by promoting vascular smooth muscle cell (VSMC) differentiation and grow arrest and inhibiting migration. Hence, it is plausible that genetical modification with CREG gene in adventitial fibroblasts might inhibit angiotensin II-induced transdifferentiation to myofibroblasts, proliferation and migration, as well as adventitial thickening, finally decreasing neointimal formation. Possible mechanisms may include CREG direct attenuation of reactive oxygen species derived from reduced cathepsin-dependent activation of NADPH oxidase and indirect suppression of the downstream of NADPH oxidase including ERK1/2, JNK and p38 MAPK pathways in adventitial fibroblasts. Therefore, CREG may be a potential therapeutic target for proliferative vascular diseases.

Nuclear Permeable Ruthenium(II) β-Carboline Complexes Induce Autophagy To Antagonize Mitochondrial-Mediated Apoptosis

The role of autophagy in cancer development and response to cancer therapy has been a subject of debate. Here we demonstrate that a series of ruthenium(II) complexes containing a β-carboline alkaloid as ligand can simultaneously induce autophagy and apoptosis in tumor cells. These Ru(II) complexes are nuclear permeable and highly active against a panel of human cancer cell lines, with complex 3 displaying activities greater than those of cisplatin. The antiproliferative potentialities of 1-3 are in accordance with their relative lipophilicities, cell membrane penetration abilities, and in vitro DNA binding affinities. Complexes 1-3 trigger release of reactive oxygen species (ROS) and attenuation of ROS by scavengers reduced the sub-G1 population, suggesting ROS-dependent apoptosis. Inhibition of ROS generation also reduces autophagy, indicating that ROS triggers autophagy. Further studies show that suppression of autophagy using pharmacological inhibitors (3-methyladenine and chloroquine) enhances apoptotic cell death.

Robinetinidol-(4β→8)-epigallocatechin 3-O-gallate, A Galloyl Dimer Prorobinetinidin From Acacia Mearnsii De Wild, Effectively Protects Human Neuroblastoma SH-SY5Y Cells Against Acrolein-Induced Oxidative Damage

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde to which humans are exposed in many situations and has been implicated in neurodegenerative diseases such as Alzheimer's disease. A galloyl dimer prorobinetinidin from Acacia mearnsii De Wild, robinetinidol-(4beta-->8)-epigallocatechin 3-O-gallate (REO), has antioxidant properties and could protect brain against acrolein-induced oxidative damage. In this study, the molecular basis of acrolein-induced cytotoxicity in human neuroblastoma SH-SY5Y cells and the modulating effects of REO were examined. Our results indicate that REO protects SH-SY5Y cells from acrolein-induced damage by the attenuation of reactive oxygen species, the remediation of NADPH oxidase activity, the enhancement of the glutathione system, and the prevention of protein oxidation/nitration and lipid peroxidation. In order to determine the effects of REO on mitochondrial events, mitochondrial membrane potentials (Delta Psim) and caspase cascades downstream of mitochondria were assessed. REO inhibited the collapse of Delta Psi m, suggesting that REO reduces the mitochondrial dysfunction associated with acrolein treatment. REO also inhibited caspase-3 activation, which can be triggered by mitochondrial malfunctions. Furthermore, REO induced a significant reduction in the level of phospho-JNK, which is known as an apoptotic mediator in acrolein-induced neuronal cell death. Our results indicate that REO protects neurons from the deleterious effects of acrolein via the attenuation of oxidative stress, NADPH oxidase activity, GSH depletion, protein oxidation/nitration, lipid peroxidation, mitochondrial dysfunction, JNK activation, and caspase activity. These findings suggest that REO could be potentially useful as a protective agent for people exposed to acrolein.

Induction of Reactive Oxygen Species-mediated Autophagy by a Novel Microtubule-modulating Agent

Autophagy is being increasingly implicated in both cell survival and death. However, the intricate relationships between drug-induced autophagy and apoptosis remain elusive. Here we demonstrate that a tubulin-binding noscapine analog, (R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]-di-oxolo[4,5-g]isoquinoline (Red-Br-nos), exerts a novel autophagic response followed by apoptotic cell death in human prostate cancer PC-3 cells. Red-Br-nos-induced autophagy was an early event detectable within 12 h that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. Red-Br-nos-triggered release of reactive oxygen species (ROS) and attenuation of ROS by tiron, a ROS scavenger, reduced the sub-G(1) population suggesting ROS-dependent apoptosis. Abrogation of ROS also reduced autophagy indicating that ROS triggers autophagy. Pharmacological and genetic approaches to inhibit autophagy uncovered the protective role of Red-Br-nos-induced autophagy in PC-3 cells. Direct effects of the drug on mitochondria viz. disruption of normal cristae architecture and dissipation of mitochondrial transmembrane potential revealed a functional link between ROS generation, autophagy, and apoptosis induction. This is the first report to demonstrate the protective role of ROS-mediated autophagy and induction of caspase-independent ROS-dependent apoptosis in PC-3 cells by Red-Br-nos, a member of the noscapinoid family of microtubule-modulating anticancer agents.

Therapeutic hypothermia cardioprotection via Akt- and nitric oxide-mediated attenuation of mitochondrial oxidants

Therapeutic hypothermia (TH) is a promising cardioprotective treatment for cardiac arrest and acute myocardial infarction, but its cytoprotective mechanisms remain unknown. In this study, we developed a murine cardiomyocyte model of ischemia-reperfusion injury to better determine the mechanisms of TH cardioprotection. We hypothesized that TH manipulates Akt, a survival kinase that mediates mitochondrial protection by modulating reactive oxygen species (ROS) and nitric oxide (NO) generation. Cardiomyocytes, isolated from 1- to 2-day-old C57BL6/J mice, were exposed to 90 min simulated ischemia and 3 h reperfusion. For TH, cells were cooled to 32 degrees C during the last 20 min of ischemia and the first hour of reperfusion. Cell viability was evaluated by propidium iodide and lactate dehydrogenase release. ROS production was measured by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate and mitochondrial membrane potential (DeltaPsim) by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazoly-carbocyanine iodide (JC-1). Phospho (p)-Akt (Thr308), p-Akt (Ser473), and phosphorylated heat shock protein 27 (p-HSP27) (Ser82) were analyzed by Western blot analysis. TH attenuated reperfusion ROS generation, increased NO, maintained DeltaPsim, and decreased cell death [19.3 + or - 3.3% (n = 11) vs. 44.7 + or - 2.7% (n = 10), P < 0.001]. TH also increased p-Akt during ischemia before reperfusion. TH protection and attenuation of ROS were blocked by the inhibition of Akt and NO synthase but not by a cGMP inhibitor. HSP27, a regulator of Akt, also exhibited increased phosphorylation (Ser82) during ischemia with TH. We conclude that TH cardioprotection is mediated by enhanced Akt/HSP27 phosphorylation and enhanced NO generation, resulting in the attenuation of ROS generation and the maintenance of DeltaPsim following ischemia-reperfusion.

Cardioprotective Effect of ‘Methylamine Irisolidone’, a New Compound, in Hypoxia/Reoxygenation Injury in Cultured Rat Cardiac Myocytes

'Methylamine irisolidone' (=5,7-dihydroxy-6-methoxy-3-(4-methoxyphenyl)-8-[(methylamino)methyl]-4H-[1]benzopyran-4-one), a new compound, is a structurally modified kakkalide with good water solubility. In this study, we investigated its effect on hypoxia/reoxygenation (H/R) injury in cultured rat cardiac myocytes. The results showed that methylamine irisolidone could significantly inhibit lactate dehydrogenase (LDH) release, enhance the mitochondrial membrane potential, decrease intracellular calcium (Ca(2+)) associated with the attenuation of reactive oxygen species (ROS) generation, reduce contents of malondialdehyde (MDA), and increase the activity of superoxide dismutase (SOD) after H/R in a dose-dependent manner. The present study demonstrated that methylamine irisolidone can directly protect cardiomyocytes against H/R injury, primarily as a result of reduction of the intracellular Ca(2+) overload coincident with an attenuation of ROS generation and ROS-mediated lipid peroxidation, which may contribute to the preservation of mitochondrion function and antioxidant against H/R injury.

Genome-Wide Fitness and Expression Profiling Implicate Mga2 in Adaptation to Hydrogen Peroxide

Caloric restriction extends lifespan, an effect once thought to involve attenuation of reactive oxygen species (ROS) generated by aerobic metabolism. However, recent evidence suggests that caloric restriction may in fact raise ROS levels, which in turn provides protection from acute doses of oxidant through a process called adaptation. To shed light on the molecular mechanisms of adaptation, we designed a series of genome-wide deletion fitness and mRNA expression screens to identify genes involved in adaptation to hydrogen peroxide. Combined with known transcriptional interactions, the integrated data implicate Yap1 and Skn7 as central transcription factors of both the adaptive and acute oxidative responses. They also identify the transcription factors Mga2 and Rox1 as active exclusively in the adaptive response and show that Mga2 is essential for adaptation. These findings are striking because Mga2 and Rox1 have been thought to control the response to hypoxic, not oxidative, conditions. Expression profiling of mga2Δ and rox1Δ knockouts shows that these factors most strongly regulate targets in ergosterol, fatty-acid, and zinc metabolic pathways. Direct quantitation of ergosterol reveals that its basal concentration indeed depends on Mga2, but that Mga2 is not required for the decrease in ergosterol observed during adaptation.

ANESTHETIC PRECONDITIONING CONFERS ACUTE CARDIOPROTECTION VIA UP-REGULATION OF MANGANESE SUPEROXIDE DISMUTASE AND PRESERVATION OF MITOCHONDRIAL RESPIRATORY ENZYME ACTIVITY

The cellular and molecular mechanisms that underlie cardioprotection against I/R by anesthetic-induced preconditioning (APC) require further elucidation. Using isoflurane as a representative anesthetic, we evaluated the hypothesis that APC induces myocardial protection against I/R by attenuation of excessive reactive oxygen species and restoration of mitochondrial bioenergetics through postischemic up-regulation of manganese superoxide dismutase (MnSOD) expression and preservation of respiratory enzyme activity. Pentobarbital anesthetized open-chest Sprague-Dawley rats were subject to 30-min left coronary artery occlusion, followed by 120-min reperfusion. Before ischemia, rats were randomly assigned to receive 0.9% saline, two cycles of brief coronary artery occlusion and reperfusion, or a 30-min exposure to 1.0 minimum alveolar concentration isoflurane in the absence or presence of a specific mitochondrial adenosine triphosphate-sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate; a membrane-permeable superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl; or a NOS inhibitor, N(G)-nitro-L-arginine methyl ester. Isoflurane exposure induced an initial increase in myocardial superoxide (O2-), but not NO level. It also significantly decreased infarct size and restored mitochondrial respiratory enzyme activity or ATP production in I/R rat hearts, along with suppression of the O2- surge at reperfusion and increase in MnSOD expression or enzyme activity. These protective effects were abrogated by 5-hydroxydecanoate or 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl, but not by N(G)-nitro-L-arginine methyl ester pretreatment. These results suggest that opening of mitochondrial KATP channel, followed by O2- signaling, induces postischemic augmentation of MnSOD and preservation of mitochondrial respiratory enzyme activities, leading to attenuated cardiac O2- surge and restored ATP production during reperfusion, and underlie APC-induced cardioprotection.

PROTECTIVE EFFECT OF N-ACETYLCYSTEINE ON HYPEROXIA-INDUCED LUNG INJURY AND ITS INTERACTION WITH P38 MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING PATHWAY

INTRODUCTION: N-Acetylcysteine (NAC) is an effective oxidation inhibitor, but the protection of NAC in hyperoxia-induced lung injury is unknown. OBJECTIVE: The objective of this study was to explore the protective effect of NAC on hyperoxia-induced lung injury and change of p38 mitogen-activated protein kinase (MAPK) expression caused by NAC treatment. METHODS: Forty Wistar rats were randomly assigned to room air (A), hyperoxia injury (B), hyperoxia + NAC (C), hyperoxia + SB203580 (D), or hyperoxia + NAC + SB203580 (E). The lung wet/dry ratio, pathology, and location and quantity of p38 protein were detected. RESULTS: Although pathologic changes in group B included severe alveolar edema with inflammatory cell aggregation and red blood cell leakage, the lung micrographic pictures in groups C, D, and E were improved significantly compared with group B; p38-positive cells increased in group B compared with that in group A and labeled in many types cells in lung tissue, especially in infiltrative inflammatory cells. In groups C, D, and E, the positive cells remarkably decreased compared with those in group B; the quantity of p38 MAPK was higher in group B than in group A, and p38 expression in groups C, D, and E decreased significantly compared with group B but was higher than that in the control group. There was no significant difference of p38 quantity among the 3 groups. CONCLUSIONS: Reactive oxygen species activated phospho-p38 MAPK signaling pathway, and NAC and SB203580 treatments reduced the extent of hyperoxia-induced lung injury, as evidenced by reduction of the wet/dry ratio and lung pathology. NAC may exert a protective effect on hyperoxia-induced lung injury through attenuation of reactive oxygen species–induced p38 MAPK activation.

Zinc inhibits ethanol-induced HepG2 cell apoptosis

Alcohol consumption produces a variety of metabolic alterations in liver cells, associated with ethanol oxidation and with nonoxidative metabolism of ethanol, among others apoptosis of hepatocytes. As zinc is known as a potent antioxidant and an inhibitor of cell apoptosis, the aim of this paper was to investigate whether zinc supplementation could inhibit ethanol-induced HepG2 apoptosis, and whether this inhibition was connected with attenuation of oxidative stress and modulation of FasR/FasL system expression. The results indicated that zinc supplementation significantly inhibited ethanol-induced HepG2 cell apoptosis (measured by cytochrome c release from mitochondria and caspase-3 activation) by attenuation of reactive oxygen species (ROS) production, increase in the cellular level of GSH, inhibition of ethanol-induced sFasR and FasL overexpression and caspase-8 activation. These results indicate that zinc can inhibit ethanol-induced hepatocyte apoptosis by several independent mechanisms, among others by an indirect antioxidative effect and probably by inhibition of caspase-8 and caspase-9 activation.

Baicalein attenuates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells

It has been suggested that baicalein, a flavonoid obtained from the Scutellaria root, exerts a protective role on neurons against several neuronal insults. However, the protective mechanisms underlying this protective effect remain largely unknown. Our results indicate that baicalein protects SH-SY5Y cells, a dopaminergic neuronal cell line, from 6-hydroxydopamine (6-OHDA)-induced damage by the attenuation of reactive oxygen species (ROS). In order to determine the effects of baicalein on mitochondrial events, mitochondrial membrane potentials (Δ Ψ m) and caspase cascades downstream of mitochondria were assessed. Baicalein inhibited the collapse of Δ Ψ m, suggesting that baicalein reduces the mitochondrial dysfunction associated with 6-OHDA treatment. Baicalein also inhibited caspase-9 and caspase-3 activation, which can be triggered by mitochondrial malfunctions. Furthermore, baicalein induced a significant reduction in the level of phospho-JNK, which is known as an apoptotic mediator in 6-OHDA-induced neuronal cell death. Our results indicate that baicalein protects neurons from the deleterious effects of 6-OHDA via the attenuation of oxidative stress, mitochondrial dysfunction, caspase activity, and JNK activation.

Epigenetic silencing of manganese superoxide dismutase (SOD-2) in KAS 6/1 human multiple myeloma cells increases cell proliferation

The generation of reactive oxygen species (ROS) by mitochondrial electron transport chain (ETC) and oxidative phosphorylation activity, has been linked to modifications of multiple molecular processes, including lipid peroxidation, signaling pathway and transcription factor modulation, and oxidative damage to DNA. Oxidative damage by endogenous ROS has been associated with the etiology of various pathological states. There are numerous reports that levels of manganese superoxide dismutase enzyme (MnSOD), an antioxidant enzyme responsible for the attenuation of ROS, are lowered in cancer cells, but the reasons for this reduction are poorly defined. Epigenetic silencing of genes involved in tumor suppression and DNA repair is known to occur in a variety of malignant cell types. Here we report that in the human multiple myeloma cell line KAS 6/1, the SOD-2 gene, encoding manganese superoxide dismutase, is epigenetically silenced as a result of promoter hypermethylation. The DNA methyltransferase inhibitor Zebularine reverses SOD-2 promoter methylation, increasing gene expression and enzyme levels. Infection of KAS 6/1 cells with a recombinant adenovirus carrying the MnSOD cDNA reduced the cell proliferation rate by approximately one-half, confirming the detrimental effects of epigenetic silencing of SOD-2 expression.

Protective effect of erdosteine against hypochlorous acid-induced acute lung injury and lipopolysaccharide-induced neutrophilic lung inflammation in mice.

The effect of erdosteine, a mucoactive drug, on hypochlorous acid (HOCl)-induced lung injury, and the lipopolysaccharide (LPS)-induced increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the airway, was investigated. Male BALB/c mice were orally administered erdosteine (3-100 mgkg(-1)), ambroxol hydrochloride (ambroxol) (3-30 mgkg(-1)), S-carboxymethyl-L-cysteine (S-CMC) (100-600 mgkg(-1)) or prednisolone (10 mgkg(-1)), 1 h before intratracheal injection of HOCl or LPS. In the HOCl-injected mice, erdosteine markedly suppressed increases in the ratios of lung wet weight to bodyweight and lung dry weight to bodyweight, whereas the other mucoactive drugs ambroxol and S-CMC had little effect. Erdosteine also inhibited the LPS-induced neutrophil influx, although it did not affect the increased level of TNF-alpha in the bronchoalveolar lavage fluid. The results suggest that attenuation of reactive oxygen species and neutrophil recruitment is involved in the clinical efficacy of erdosteine in the treatment of chronic bronchitis.