Prevented Reactive Oxygen Species Generation Research Articles

Simultaneously deplete reactive oxygen species and inhibit pyroptosis by dopamine/thioketal-containing polymers delivering disulfiram in combination with Cu(II) for acute glaucoma

Glaucoma is a worldwide prominent cause of irreversible vision damage, which is characterized by progressive loss of retinal ganglion cells (RGCs) and currently no effective treatment is available till far. Overexpression of reactive oxygen species (ROS) contribute to RGCs death. Moreover, pyroptosis mediate RGCs death also played a key role in neurodegeneration of glaucoma. However, there is no treatment available for glaucoma that can simultaneously prevent ROS generation and inhibit pyroptosis. Herein, bioinformatics analysis was firstly applied to identify the upregulation of a key protein N-terminal gasdermin D (N-GSDMD) can result in membrane pore formation in RGCs, leading to pyroptosis. Thereafter, disulfiram (DSF), a novel N-GSDMD inhibitor, was screened out which could be in combination with Cu(II) (DSF+Cu(II)) to significantly inhibit RGCs pyroptosis. To translate this effect in vivo, a ROS scavenging biodegradable polymer containing dopamine and thioketal bonds was designed to deliver DSF as nanoparticles (DSF-NPs). Once DSF-NPs were internalized by RGCs, the high intracellular ROS could break up the thioketal bonds to release dopamine for depleting ROS and simultaneously release DSF which could work in combination with Cu(II) for inhibiting RGCs pyroptosis. In vitro, DSF-NPs+Cu(II) was proved to protect R28 cells significantly better than DSF+Cu(II) under oxygen and glucose deprivation (OGD) conditions. In vivo, DSF-NPs could accumulate in the RGCs of an ischemia/reperfusion (I/R) mouse model after intravitreal injection, which further worked together with Cu(II) to significantly inhibit RGCs pyroptosis. Taken together, DSF-NPs+Cu(II) can selectively inhibit pyroptosis of RGCs, resulting in considerable protection from high intraocular pressure injury, providing a new therapeutic strategy for acute glaucoma.

Anti-Inflammatory Activity of Ferula assafoetida Oleo-Gum-Resin (Asafoetida) against TNF-α-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs).

Inflammation is the body's biological reaction to endogenous and exogenous stimuli. Recent studies have demonstrated several anti-inflammatory properties of Ferula species. In this paper, we decided to study the anti-inflammatory effect of ethanolic extract of Ferula assafoetida oleo-gum-resin (asafoetida) against TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in a flat-bottom plate and then treated with ethanolic extract of asafoetida (EEA, 0-500 μg/ml) and TNF-α (0-100 ng/ml) for 24 h. We used the MTT test to assess cell survival. In addition, the LC-MS analysis was performed to determine the active substances. HUVECs were pretreated with EEA and then induced by TNF-α. Intracellular reactive oxygen species (ROS) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs were evaluated with DCFH-DA and CFSE fluorescent probes, respectively. Gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin and surface expression of ICAM-1 protein were measured using real-time PCR and flow cytometry methods, respectively. While TNF-α significantly increased intracellular ROS formation and PBMC adhesion to TNF-α-induced HUVECs, the pretreatment of HUVECs with EEA (125 and 250 μg/ml) significantly reduced the parameters. In addition, EEA pretreatment decreased TNF-α-induced mRNA expression of VCAM-1 and surface protein expression of ICAM-1 in the target cells. Taken together, the results indicated that EEA prevented ROS generation, triggered by TNF-α, and inhibited the expression of VCAM-1 and ICAM-1, leading to reduced PBMC adhesion. These findings suggest that EEA can probably have anti-inflammatory properties.

Miktoarm Star Polymers with Environment-Selective ROS/GSH Responsive Locations: From Modular Synthesis to Tuned Drug Release through Micellar Partial Corona Shedding and/or Core Disassembly.

Branched architectures with asymmetric polymeric arms provide an advantageous platform for the construction of tailored nanocarriers for therapeutic interventions. Simple and adaptable synthetic methodologies to amphiphilic miktoarm star polymers have been developed in which spatial location of reactive oxygen species (ROS) and glutathione (GSH) responsive entities is articulated to be on the corona shell surface or inside the core. The design of such architectures is facilitated through versatile building blocks and selected combinations of ring-opening polymerization, Steglich esterification, and alkyne-azide click reactions. Soft nanoparticles from aqueous self-assembly of these stimuli responsive miktoarm stars have low critical micelle concentrations and high drug loading efficiencies. Partial corona shedding upon response to ROS is accompanied by an increase in drug release, without significant changes to overall micelle morphology. The location of the GSH responsive unit at the core leads to micelle disassembly and complete drug release. Curcumin loaded soft nanoparticles show higher efficiencies in preventing ROS generation in extracellular and cellular environments, and in ROS scavenging in human glioblastoma cells. The ease in synthetic elaboration and an understanding of structure-property relationships in stimuli responsive nanoparticles offer a facile venue for well-controlled drug delivery, based on the extra- and intracellular concentrations of ROS and GSH.

Ketamine-induced attenuation of reactive oxygen species in zebrafish is prevented by acetyl l-carnitine in vivo

Ketamine, an anesthetic, is a non-competitive antagonist of the calcium-permeable N-methyl-d-aspartate (NMDA) receptor. High concentrations of ketamine have been implicated in cardiotoxicity and neurotoxicity. Often, these toxicities are thought to be mediated by reactive oxygen species (ROS). However, findings to the contrary showing ketamine reducing ROS in mammalian cells and neurons in vitro, are emerging. Here, we determined the effects of ketamine on ROS levels in zebrafish larvae in vivo. Based on our earlier studies demonstrating reduction in ATP levels by ketamine, we hypothesized that as a calcium antagonist, ketamine would also prevent ROS generation, which is a by-product of ATP synthesis. To confirm that the detected ROS in a whole organism, such as the zebrafish larva, is specific, we used diphenyleneiodonium (DPI) that blocks ROS production by inhibiting the NADPH Oxidases (NOX). Upon 20 h exposure, DPI (5 and 10 μM) and ketamine at (1 and 2 mM) reduced ROS in the zebrafish larvae in vivo. Using acetyl l-carnitine (ALCAR), a dietary supplement, that induces mitochondrial ATP synthesis, we show elevated ROS generation with increasing ALCAR concentrations. Combined, ketamine and ALCAR counter-balanced ROS generation in the larvae suggesting that ketamine and ALCAR have opposing effects on mitochondrial metabolism, which may be key to maintaining ROS homeostasis in the larvae and affords ALCAR the ability to prevent ketamine toxicity. These results for the first time show ketamine’s antioxidative and ALCAR’s prooxidative effects in a live vertebrate.

Noradrenaline Acting on Alpha1 Adrenoceptor as well as by Chelating Iron Reduces Oxidative Burden on the Brain: Implications With Rapid Eye Movement Sleep.

The noradrenaline (NA) level in the brain is reduced during rapid eye movement sleep (REMS). However, upon REMS deprivation (REMSD) its level is elevated, which induces apoptosis and the degeneration of neurons in the brain. In contrast, isolated studies have reported that NA possesses an anti-oxidant property, while REMSD reduces lipid peroxidation (LP) and reactive oxygen species (ROS). We argued that an optimum level of NA is likely to play a physiologically beneficial role. To resolve the contradiction and for a better understanding of the role of NA in the brain, we estimated LP and ROS levels in synaptosomes prepared from the brains of control and REMS deprived rats with or without in vivo treatment with either α1-adrenoceptor (AR) antagonist, prazosin (PRZ) or α2-AR agonist, clonidine (CLN). REMSD significantly reduced LP and ROS in synaptosomes; while the effect on LP was ameliorated by both PRZ and CLN; ROS was prevented by CLN only. Thereafter, we evaluated in vitro the effects of NA, vitamin E (Vit E), vitamin C (Vit C), and desferrioxamine (DFX, iron chelator) in modulating hydrogen peroxide (H2O2)-induced LP and ROS in rat brain synaptosomes, Neuro2a, and C6 cells. We observed that NA prevented ROS generation by chelating iron (inhibiting a Fenton reaction). Also, interestingly, a lower dose of NA protected the neurons and glia, while a higher dose damaged the neurons and glia. These in vitro and in vivo results are complementary and support our contention. Based on the findings, we propose that REMS maintains an optimum level of NA in the brain (an antioxidant compromised organ) to protect the latter from continuous oxidative onslaught.

Cysteine Dioxygenase 1 Mediates Erastin-Induced Ferroptosis in Human Gastric Cancer Cells

BACKGROUND: Ferroptosis is a recently discovered form of iron-dependent nonapoptotic cell death. It is characterized by loss of the activity of the lipid repair enzyme, glutathione peroxidase 4 (GPX4), and accumulation of lethal reactive lipid oxygen species. However, we still know relatively little about ferroptosis and its molecular mechanism in gastric cancer (GC) cells. Here, we demonstrate that erastin, a classic inducer of ferroptosis, induces this form of cell death in GC cells and that cysteine dioxygenase 1 (CDO1) plays an important role in this process. METHODS: We performed quantitative real-time polymerase chain reaction, Western blotting, cell viability assay, reactive oxygen species (ROS) assay, glutathione assay, lipid peroxidation assay, RNAi and gene transfection, immunofluorescent staining, dual-luciferase reporter assay, transmission electron microscopy, and chromatin immunoprecipitation assay to study the regulation of ferroptosis in GC cells. Mouse xenograft assay was used to figure out the mechanism in vivo. RESULTS: Silencing CDO1 inhibited erastin-induced ferroptosis in GC cells both in vitro and in vivo. Suppression of CDO1 restored cellular GSH levels, prevented ROS generation, and reduced malondialdehyde, one of the end products of lipid peroxidation. In addition, silencing COO1 maintained mitochondrial morphologic stability in erastin-treated cells. Mechanistically, c-Myb transcriptionally regulated CDO1, and inhibition of CDO1 expression upregulated GPX4 expression. CONCLUSIONS: Our findings give a better understanding of ferroptosis and its molecular mechanism in GC cells, gaining insight into ferroptosis-mediated cancer treatment.

Comparison of protective effect of ascorbic acid on redox and endocannabinoid systems interactions in in vitro cultured human skin fibroblasts exposed to UV radiation and hydrogen peroxide

The mechanisms of biological activity of commonly used natural compounds are constantly examined. Therefore, the aim of this study was to compare ascorbic acid efficacy in counteracting the consequences of UV and hydrogen peroxide treatment on lipid mediators and their regulative action on antioxidant abilities. Skin fibroblasts exposed to UVA and UVB irradiation, treated with hydrogen peroxide and ascorbic acid. The redox system was estimated through reactive oxygen species (ROS) generation (electron spin resonance spectrometer) and antioxidants level/activity (HPLC/spectrometry) which activity was evaluated by the level of phospholipid metabolites: 4-hydroxynonenal, malondialdehyde, 8-isoprostanes and endocannabinoids (GC/LC-MS) in the human skin fibroblasts. Protein and DNA oxidative modifications were also determined (LC). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), its activators and inhibitors as well as pro/anti-apoptotic proteins and endocannabinoid receptors was examined (Western blot) and collagen metabolism was evaluated by collagen biosynthesis and prolidase activity (spectrometry). UVA and UVB irradiation and hydrogen peroxide treatment enhanced activity of xanthine and NADPH oxidases resulting in ROS generation as well as diminution of antioxidant phospholipid protection (glutathione peroxidase-glutathione-vitamin E), what led to increased lipid peroxidation and decreased endocannabinoids level. Dysregulation of cannabinoid receptors expression and environment of transcription factor Nrf2 caused apoptosis induction. Ascorbic acid partially prevented ROS generation, antioxidant capacity diminution and endocannabinoid systems disturbances but only slightly protected macromolecules such as phospholipid, protein and DNA against oxidative modifications. However, ascorbic acid significantly prevented decrease in collagen type I biosynthesis. Ascorbic acid in similar degree prevents UV (UVA and UVB) and hydrogen peroxide-dependent redox imbalance. However, this antioxidant cannot efficiently protect cellular macromolecules and avert metabolic dysregulation leading to apoptosis.

Reversing Electron Transport in Ischemia and Beyond

Accumulation of the TCA cycle metabolite succinate in ischemic tissues has been reported for almost 50 years. However, the patho-physiologic importance of this event is still poorly understood. In 2014, it was reported that accumulated succinate is a key driver of Reactive Oxygen Species (ROS) generation at reperfusion [PMID: 25383517]. This has led to proposals that inhibiting ischemic succinate accumulation may afford therapeutic benefit in ischemia-reperfusion (IR) injury. However, the reversal of respiratory complex II (Cx-II, succinate dehydrogenase), which is the proposed route of ischemic succinate accumulation, may serve a beneficial role. Namely, by using fumarate as an alternate electron acceptor (in lieu of O2), reverse electron transfer (RET) at Cx-II allows complex I (Cx-I) to continue functioning. As such, H+ pumping by Cx-I provides a potential source of mitochondrial membrane potential during ischemia, preventing the catabolism of glycolytic ATP by reversal of the ATP synthase. Consistent with a beneficial role, succinate accumulation is widely documented in hypoxic tissues of diving mammals. A prediction from this modified viewpoint, is that the timing of Cx-II inhibition relative to IR injury may yield differential outcomes. Namely prophylactic Cx-II inhibition may lose any benefit from RET at Cx-II during ischemia. Conversely, Cx-II inhibition only at reperfusion will be beneficial by preventing ROS generation. Data supporting this hypothesis will be discussed, along with compelling new data regarding the precise mechanism of succinate accumulation in ischemia, which may not be as straightforward as initially thought.

The crown plays an important role in maintaining quality of harvested pineapple

Pineapple is a unique fruit partly because it has a beautiful crown and consumers around the world regard the crown as an integral part of pineapple fruit. However, farmers in some countries detach the crown at harvest and use it for propagation. It is not clear whether the detachment of crown affects quality of harvested pineapple. This study shows that decrowning aggravated internal browning by 55.2% and reduced SSC/TA ratio by 2.2, following 9-d storage, suggesting that decrowning deteriorated quality of the flesh and shortened shelf-life of fruit. Furthermore, decrowning increased reactive oxygen species (ROS), malondialdehyde (MDA), and phenolics levels, and upregulated polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) gene expression and activity, suggesting keeping crown intact prevented ROS generation and lipid peroxidation, and inhibiting phenolics biosynthesis and oxidation. Moreover, decrowning increased endogenous GAs(GA1 and GA4) and decreased endogenous ABA in pineapple tissues, suggesting crown is important for keeping balance between GAs and ABA. Exogenous application of ABA inhibited IB in pineapple with intact crown more effectively than in decrowned one, ABA application to crown only controlled IB as effectively as to both crown and fruit, and decrowning following ABA application to crown significantly compromised the efficacy. These suggest that the crown is the main source of endogenous ABA and that IB control depends on continuous supply of ABA from the crown. This study provides possibility for effectively controlling IB.

Rice protein suppresses ROS generation and stimulates antioxidant gene expression via Nrf2 activation in adult rats

To elucidate the effects of rice protein on the detoxification and antioxidant defense via the Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, adult rats were fed casein and rice protein under cholesterol-free and -enriched dietary conditions. Nrf2 proteins and gene expressions were stimulated by rice proteins with respect to caseins accompanied by up-regulating the expression of gene encoding antioxidant and phase II detoxification in the rice protein groups. In the liver, compared with caseins, rice proteins significantly increased hepatic contents of reduced glutathione (GSH) and mRNA levels of glutamate cysteine ligase catalytic subunit (GCLC), glutamate cysteine ligase modulatory subunit (GCLM), glutathione S-transferase (GST), heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), whereas hepatic accumulations of reactive oxygen species (ROS) and oxidized glutathione (GSSG) were markedly depressed in adult rats fed rice proteins. The present study demonstrates the endogenous antioxidant potential of rice proteins via mechanism through which Nrf2 activation plays an essential role in inducing Phase II antioxidants/detoxification enzymes and preventing ROS generation. This novel antioxidative function of rice protein is independent of dietary cholesterol during in adult rats.

An interaction of renin-angiotensin and kallikrein-kinin systems contributes to vascular hypertrophy in angiotensin II-induced hypertension: in vivo and in vitro studies.

The kallikrein-kinin and renin-angiotensin systems interact at multiple levels. In the present study, we tested the hypothesis that the B1 kinin receptor (B1R) contributes to vascular hypertrophy in angiotensin II (ANG II)–induced hypertension, through a mechanism involving reactive oxygen species (ROS) generation and extracellular signal-regulated kinase (ERK1/2) activation. Male Wistar rats were infused with vehicle (control rats), 400 ng/Kg/min ANG II (ANG II rats) or 400 ng/Kg/min ANG II plus B1 receptor antagonist, 350 ng/Kg/min des-Arg9-Leu8-bradykinin (ANGII+DAL rats), via osmotic mini-pumps (14 days) or received ANG II plus losartan (10 mg/Kg, 14 days, gavage - ANG II+LOS rats). After 14 days, ANG II rats exhibited increased systolic arterial pressure [(mmHg) 184±5.9 vs 115±2.3], aortic hypertrophy; increased ROS generation [2-hydroxyethidium/dihydroethidium (EOH/DHE): 21.8±2.7 vs 6.0±1.8] and ERK1/2 phosphorylation (% of control: 218.3±29.4 vs 100±0.25]. B1R expression was increased in aortas from ANG II and ANG II+DAL rats than in aortas from the ANG II+LOS and control groups. B1R antagonism reduced aorta hypertrophy, prevented ROS generation (EOH/DHE: 9.17±3.1) and ERK1/2 phosphorylation (137±20.7%) in ANG II rats. Cultured aortic vascular smooth muscle cells (VSMC) stimulated with low concentrations (0.1 nM) of ANG II plus B1R agonist exhibited increased ROS generation, ERK1/2 phosphorylation, proliferating-cell nuclear antigen expression and [H3]leucine incorporation. At this concentration, neither ANG II nor the B1R agonist produced any effects when tested individually. The ANG II/B1R agonist synergism was inhibited by losartan (AT1 blocker, 10 µM), B1R antagonist (10 µM) and Tiron (superoxide anion scavenger, 10 mM). These data suggest that B1R activation contributes to ANG II-induced aortic hypertrophy. This is associated with activation of redox-regulated ERK1/2 pathway that controls aortic smooth muscle cells growth. Our findings highlight an important cross-talk between the DABK and ANG II in the vascular system and contribute to a better understanding of the mechanisms involved in vascular remodeling in hypertension.

Hepatoprotective Effects of Terminalia chebula Fruit Extract against 2-AAF–Induced Hepatic Damage in Albino Mice: Role of MDR1 and COX-2

Terminalia chebula extracts were evaluated on 2-acetylaminofluorene (2-AAF)-induced hepatocellular carcinoma in mice. The 25 mg.kg−1 b.w. 2-AAF treatment showed liver aberration and up-regulation of multidrug resistance-1 (MDR1), generation of reactive oxygen species (ROS) and cyclooxygenase-2 (COX-2) expression via phosphorylation of Akt-MAPKs and nuclear translocation of NF-κB. Pre-administration of 50 mg.kg−1 TCE along with 25 mg.kg−1 2-AAF inhibited the expression of MDR1 by preventing ROS generation and COX-2 expression through Akt and MAPK signaling pathway. T. chebula may overcome the 2-AAF-induced oxidative stress and drug resistance in the hepatic tissue of mice and prevent the possible neoplastic transformation leading to hepatocarcinoma.

Exogenous hydrogen sulfide protects H9c2 cardiac cells against high glucose-induced injury by inhibiting the activities of the p38 MAPK and ERK1/2 pathways

Hyperglycemia is a risk factor for the development of diabetic cardiovascular complications, which are associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we demonstrate the inhibitory effects of exogenous hydrogen sulfide (H₂S) on the activation of the MAPK pathway. The aim of the present study was to determine whether exogenous H₂S prevents high glucose (HG)-induced injury by inhibiting the activation of the p38 MAPK and extracellular signal-regulated kinase (ERK)1/2 (members of MAPK) pathways in cardiomyoblasts (H9c2 cells). The findings of the present study demonstrated that the treatment of H9c2 cells with HG (35 mM glucose) for 24 h not only significantly induced injury, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (MMP), but also upregulated the expression levels of phosphorylated (p)-p38 MAPK and p-ERK1/2. The increased expression levels of p-p38 MAPK and p-ERK1/2 were markedly reduced by pre-treatment of the H9c2 cells with 400 µM sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to 35 mM glucose. Importantly, pre-treatment of the cells with 400 µM NaHS or 3 µM SB203580 (a selective inhibitor of p38 MAPK) or 15 µM U0126 (a selective inhibitor of ERK1/2) attenuated the HG-induced cardiomyocyte injury, leading to an increase in cell viability and a decrease in the number of apoptotic cells, preventing ROS generation, as well as the loss of MMP. In addition, pre-treatment of the cells with 1,000 µM N‑acetyl‑L‑cysteine (a ROS scavenger) prior to exposure to HG ameliorated the HG-induced cytotoxicity. Taken together, the data from the present study demonstrate for the first time, to our knowledge, that exogenous H2S exerts a protective effect against HG‑induced injury by inhibiting the activation of the p38 MAPK and ERK1/2 pathways and preventing oxidative stress in H9c2 cells.

Ceramides increase mitochondrial ROS generation via altered mitochondrial dynamics in skeletal muscle

The role of reactive oxygen species (ROS) as mediators of chronic diseases is increasingly well established. Ceramides are known to induce mitochondrial ROS production, which likely explains some of the deleterious effects of ceramide accumulation. We propose that ceramide accrual in skeletal muscle induces mitochondrial fission, which is requisite for ceramide‐induced ROS generation. We found that exogenous ceramide treatment and endogenous ceramide biosynthesis both increased mitochondrial fission and increased expression of dynamin‐related protein 1 (Drp1), which is a prime mediator of mitochondrial fission. Moreover, ceramide accrual increased H2O2 production in muscle cells. Inhibition of serine palmitoyltransferase (SPT) via pharmacological (myriocin) and genetic (SPT2 siRNA) inhibition prevented increased ROS production in palmitate‐treated cells. Also, lack of Drp1 (siRNA) prevented mitochondrial fission with ceramide treatment and completely prevented ROS generation. Altogether, these findings suggest ceramide‐induced mitochondrial fission is an important mediator of the observed ROS generation in the presence of ceramide‐rich cells. Given the multiple chronic diseases that are affected by ceramide metabolism, such as insulin resistance, these findings may lead to improved and novel therapies. This work was supported by a BYU Undergraduate Research Award (BJT) and a BYU Mentoring Environment Grant (BTB).

ENOS Inhibition is Protective against Oxygen Glucose Deprivation in Brain Microvascular Endothelial Cells by Preempting eNOS Uncoupling

Reactive oxygen species (ROS) have been implicated in cellular injury due to oxygen glucose deprivation (OGD). Our objective was to determine whether uncoupling of endothelial nitric oxide synthase (eNOS) contributes to cell injury in endothelial cells exposed to OGD. Brain microvascular endothelial cells from rat (RBMEC) and human (HBMEC) were cultured and exposed either to 2 hours of OGD or normoxia ± N ω-nitro-1-arginine methyl ester (NAME). RBMEC and HBMEC viabilities were reduced by OGD and rescued by NAME dose-dependently. Low temperature native PAGE revealed reduced eNOS monomer/dimer ratio in RBMEC following OGD compared to control. Fluorescence measurements of dihydroethidium (DHE), a ROS-sensitive fluoroprobe, displayed increased ROS generation in RBMEC following OGD compared to control that was abolished by NAME. In conclusion, eNOS inhibition prevented ROS generation via eNOS uncoupling and afforded protection against OGD in endothelial cells.

Hyperglycemia Enhances IGF-I–Stimulated Src Activation via Increasing Nox4-Derived Reactive Oxygen Species in a PKCζ-Dependent Manner in Vascular Smooth Muscle Cells

IGF-I–stimulated sarcoma viral oncogene (Src) activation during hyperglycemia is required for propagating downstream signaling. The aim of the current study was to determine the mechanism by which hyperglycemia enhances IGF-I–stimulated Src activation and the role of NADPH oxidase 4 (Nox4) and protein kinase C ζ (PKCζ) in mediating this response in vascular smooth muscle cells (VSMCs). Nox4 expression was analyzed in VSMCs exposed to hyperglycemia. The role of Nox4-derived reactive oxygen species (ROS) in IGF-I–stimulated Src activation was investigated via knockdown of Nox4. Different isoforms of PKC were screened to investigate their role in hyperglycemia-induced Nox4. The oxidation of Src was shown to be a prerequisite for its activation in response to IGF-I during hyperglycemia. Hyperglycemia induced Nox4, but not Nox1, and p22 phagocyte oxidase (p22phox) expression and IGF-I stimulated Nox4/p22phox complex formation, leading to increased ROS generation. Knockdown of Nox4 prevented ROS generation and impaired the oxidation and activation of Src in response to IGF-I, whereas knockdown of Nox1 had no effect. PKCζ was shown to mediate the hyperglycemia-induced increase in Nox4 expression. The key observations in cultured VSMCs were confirmed in the diabetic mice. Nox4-derived ROS is responsible for the enhancing effect of hyperglycemia on IGF-I–stimulated Src activation, which in turn amplifies IGF-I–linked downstream signaling and biological actions.

Carfilzomib Interacts Synergistically with Histone Deacetylase Inhibitors in Mantle Cell Lymphoma Cells In Vitro and In Vivo

Interactions between the proteasome inhibitor carfilzomib and the histone deacetylase (HDAC) inhibitors vorinostat and SNDX-275 were examined in mantle cell lymphoma (MCL) cells in vitro and in vivo. Coadministration of very low, marginally toxic carfilzomib concentrations (e.g., 3-4 nmol/L) with minimally lethal vorinostat or SNDX-275 concentrations induced sharp increases in mitochondrial injury and apoptosis in multiple MCL cell lines and primary MCL cells. Enhanced lethality was associated with c-jun-NH,-kinase (JNK) 1/2 activation, increased DNA damage (induction of λH2A.X), and ERK1/2 and AKT1/2 inactivation. Coadministration of carfilzomib and histone deacetylase inhibitors (HDACI) induced a marked increase in reactive oxygen species (ROS) generation and G(2)-M arrest. Significantly, the free radical scavenger tetrakis(4-benzoic acid) porphyrin (TBAP) blocked carfilzomib/HDACI-mediated ROS generation, λH2A.X formation, JNK1/2 activation, and lethality. Genetic (short hairpin RNA) knockdown of JNK1/2 significantly attenuated carfilzomib/HDACI-induced apoptosis, but did not prevent ROS generation or DNA damage. Carfilzomib/HDACI regimens were also active against bortezomib-resistant MCL cells. Finally, carfilzomib/vorinostat coadministration resulted in a pronounced reduction in tumor growth compared with single agent treatment in an MCL xenograft model associated with enhanced apoptosis, λH2A.X formation, and JNK activation. Collectively, these findings suggest that carfilzomib/HDACI regimens warrant attention in MCL.

Loss of a Conserved Tyrosine Residue of Cytochrome b Induces Reactive Oxygen Species Production by Cytochrome bc1

Production of reactive oxygen species (ROS) induces oxidative damages, decreases cellular energy conversion efficiencies, and induces metabolic diseases in humans. During respiration, cytochrome bc(1) efficiently oxidizes hydroquinone to quinone, but how it performs this reaction without any leak of electrons to O(2) to yield ROS is not understood. Using the bacterial enzyme, here we show that a conserved Tyr residue of the cytochrome b subunit of cytochrome bc(1) is critical for this process. Substitution of this residue with other amino acids decreases cytochrome bc(1) activity and enhances ROS production. Moreover, the Tyr to Cys mutation cross-links together the cytochrome b and iron-sulfur subunits and renders the bacterial enzyme sensitive to O(2) by oxidative disruption of its catalytic [2Fe-2S] cluster. Hence, this Tyr residue is essential in controlling unproductive encounters between O(2) and catalytic intermediates at the quinol oxidation site of cytochrome bc(1) to prevent ROS generation. Remarkably, the same Tyr to Cys mutation is encountered in humans with mitochondrial disorders and in Plasmodium species that are resistant to the anti-malarial drug atovaquone. These findings illustrate the harmful consequences of this mutation in human diseases.

244 DIAGNOSTIC AND PROGNOSTIC ROLE OF SCCA-IGM IN HEPATOCELLULAR CARCINOMA (HCC)

generation of reactive oxygen species (ROS), phosphorylation/ inactivation of GSK-3b as well as phosphorylation of PI-3K, ERK1/2, JNK1/2, p38MAPK and STAT3. Invasiveness of cancer cells was prevented either by inhibiting ERK1/2, PI3K or JNK1/2 as well as by preventing ROS generation. Finally, OSM was expressed “in vivo” in tumor cells of HCC in areas also positive for hypoxia-related antigens. Conclusions: OSM, which is expressed in human HCC cells and peritumoral cirrhotic tissue, can induce EMT in human hepatic cancer cell lines and stimulate their increased invasiveness through the involvement of redox-dependent activation of EMT-related critical kinases and transcription factors.

Neurodegenerative influence of oxidative stress in the retina of a murine model of diabetes

Aims/hypothesisDiabetic retinopathy is a progressive neurodegenerative disease, but the underlying mechanism is still obscure. Here, we focused on oxidative stress in the retina, and analysed its influence on retinal neurodegeneration, using an antioxidant, lutein.MethodsC57BL/6 mice with streptozotocin-induced diabetes were constantly fed either a lutein-supplemented diet or a control diet from the onset of diabetes, and their metabolic data were recorded. In 1-month-diabetic mice, reactive oxygen species (ROS) in the retina were measured using dihydroethidium and visual function was evaluated by electroretinograms. Levels of activated extracellular signal-regulated kinase (ERK), synaptophysin and brain-derived neurotrophic factor (BDNF) were also measured by immunoblotting in the retina of 1-month-diabetic mice. In the retinal sections of 4-month-diabetic mice, histological changes, cleaved caspase-3 and TUNEL staining were analysed.ResultsLutein did not affect the metabolic status of the diabetic mice, but it prevented ROS generation in the retina and the visual impairment induced by diabetes. ERK activation, the subsequent synaptophysin reduction, and the BDNF depletion in the diabetic retina were all prevented by lutein. Later, in 4-month-diabetic mice, a decrease in the thickness of the inner plexiform and nuclear layers, and ganglion cell number, together with increase in cleaved caspase-3- and TUNEL-positive cells, were avoided in the retina of lutein-fed mice.Conclusions/interpretationThe results indicated that local oxidative stress that has a neurodegenerative influence in the diabetic retina is prevented by constant intake of a lutein-supplemented diet. The antioxidant, lutein may be a potential therapeutic approach to protect visual function in diabetes.