Human OXR1 maintains mitochondrial DNA integrity and counteracts hydrogen peroxide-induced oxidative stress by regulating antioxidant pathways involving p21

The oxidation resistance gene 1 (OXR1) prevents oxidative stress-induced cell death by an unknown pathway. Here, depletion of human OXR1 (hOXR1) sensitized several human cell lines to hydrogen peroxide-induced oxidative stress, reduced mtDNA integrity, and increased apoptosis. In contrast, depletion of hOXR1 in cells lacking mtDNA showed no significant change in ROS or viability, suggesting that OXR1 prevents intracellular hydrogen peroxide-induced increase in oxidative stress levels to avoid a vicious cycle of increased oxidative mtDNA damage and ROS formation. Furthermore, expression of p21 and the antioxidant genes GPX2 and HO-1 was reduced in hOXR1-depleted cells. In sum, these data reveal that human OXR1 upregulates the expression of antioxidant genes via the p21 signaling pathway to suppress hydrogen peroxide-induced oxidative stress and maintain mtDNA integrity.     

Oxidation resistance 1 is a novel senolytic target

The selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.     

Ferulic acid enhances the radiation sensitivity of lung and liver carcinoma cells by collapsing redox homeostasis: mechanistic involvement of Akt/p38 MAPK signalling pathway

Abstract

The major drawback of anticancer therapy is the development of resistance against drugs and radiation at the later phase of treatment which may lead to recurrences of the disease. Therefore, strategy was taken to enhance radiation sensitivity of lung (A549) and liver (HepG2) carcinoma cells by treatment with ferulic acid (FA) prior to irradiation. FA pre-treatment initially decreased reactive oxygen species (ROS) level in carcinoma cells which induced reductive stress and cytostasis. To overcome this stress, cellular mechanism increased the Keap1 level to down-regulate nuclear localisation of Nrf2 and its dependent antioxidant system. The antioxidant system reached the lowest level after 3 and 6?h of FA treatment in A549 and HepG2 cells respectively. As endogenous ROS were still being generated at same rate, ROS level was clearly higher than control which changed the reductive stress to oxidative stress. Exposure to γ-radiation in this condition further increased ROS level and caused radio-sensitisation of carcinoma cells. Combination of irradiation (IR) and FA activated mitochondrial apoptotic pathway and concomitantly inhibited the cell cycle progression and survival pathway over the IR group. Moreover, the combination treatment showed significant tumour regression, caspase 3 activation and nuclear fragmentation in tumour tissue compared to radiation alone. In contrast, FA pre-treatment protected peripheral blood mononuclear cells (PBMC) and normal lung fibroblast WI38 cells from radiation damage. Together, combination treatment offers effective strategy of killing cancer cells and demonstrates its potential for increasing the efficacy of radio-therapy.     

The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins

Background  

The NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1.     

A novel fine tuning scheme of miR-200c in modulating lung cell redox homeostasis

Oxidative stress induces miR-200c, the predominant microRNA (miRNA) in lung tissues; however, the antioxidant role and biochemistry of such induction have not been clearly defined. Therefore, a lung adenocarcinoma cell line (A549) and a normal lung fibroblast (MRC-5) were used as models to determine the effects of miR-200c expression on lung antioxidant response. Hydrogen peroxide (H₂O₂) upregulated miR-200c, whose overexpression exacerbated the decrease in cell proliferation, retarded the progression of cells in the G2/M-phase, and increased oxidative stress upon H₂O₂ stimulation. The expression of three antioxidant proteins, superoxide dismutase (SOD)-2, haem oxygenase (HO)-1, and sirtuin (SIRT) 1, was reduced upon H₂O₂ stimulation in miR-200c-overexpressed A549 cells. This phenomenon of increased oxidative stress and antioxidant protein downregulation also occurs simultaneously in miR-200c overexpressed MRC-5 cells. Molecular analysis revealed that miR-200c inhibited the gene expression of HO-1 by directly targeting its 3′-untranslated region. The downregulation of SOD2 and SIRT1 by miR-200c was mediated through zinc finger E-box-binding homeobox 2 (ZEB2) and extracellular signal-regulated kinase 5 (ERK5) pathways, respectively, where knockdown of ZEB2 or ERK5 decreased the expression of SOD2 or SIRT1 in A549 cells. LNA anti-miR-200c transfection in A549 cells inhibited the endogenous miR-200c expression, resulting in increased expressions of antioxidant proteins, reduced oxidative stress and recovered cell proliferation upon H₂O₂ stimulation. These findings indicate that miR-200c fine-tuned the antioxidant response of the lung cells to oxidative stress through several pathways, and thus this study provides novel information concerning the role of miR-200c in modulating redox homeostasis of lung.     

The Anopheles gambiae Oxidation Resistance 1 (OXR1) Gene Regulates Expression of Enzymes That Detoxify Reactive Oxygen Species

Background

OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage.

Methodology/Principal Findings

OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT) and glutathione peroxidase (Gpx) expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK) gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response.

Conclusion

The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS) in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection.     

N-acetylcysteine amide,a thiol antioxidant,prevents bleomycin-induced toxicity in human alveolar basal epithelial cells (A549)

Abstract

Bleomycin (BLM), a glycopeptide antibiotic from Streptomyces verticillus, is an effective antineoplastic drug. However, its clinical use is restricted due to the wide range of associated toxicities, especially pulmonary toxicity. Oxidative stress has been implicated as an important factor in the development of BLM-induced pulmonary toxicity. Previous studies have indicated disruption of thiol-redox status in lungs (lung epithelial cells) upon BLM treatment. Therefore, this study focused on (1) investigating the oxidative effects of BLM on lung epithelial cells (A549) and (2) elucidating whether a well-known thiol antioxidant, N-acetylcysteine amide (NACA), provides any protection against BLM-induced toxicity. Oxidative stress parameters, such as glutathione (GSH), malondialdehyde (MDA), and antioxidant enzyme activities were altered upon BLM treatment. Loss of mitochondrial membrane potential (ΔΨm), as assessed by fluorescence microscopy, indicated that cytotoxicity is possibly mediated through mitochondrial dysfunction. Pretreatment with NACA reversed the oxidative effects of BLM. NACA decreased the reactive oxygen species (ROS) and MDA levels and restored the intracellular GSH levels. Our data showed that BLM induced A549 cell death by a mechanism involving oxidative stress and mitochondrial dysfunction. NACA had a protective role against BLM-induced toxicity by inhibiting lipid peroxidation, scavenging ROS, and preserving intracellular GSH and ΔΨm. NACA can potentially be developed into a promising adjunctive therapeutic option for patients undergoing chemotherapy with BLM.     

The quality of the antioxidant defence system in term and preterm twin neonates

Abstract

Objective: Multiple pregnancy is associated with an enhanced metabolism and demand for O2, which may lead to the overproduction of reactive oxygen species and the development of oxidative stress. The degree of oxidative damage depends on the level of the antioxidant protection system of the foetus. The objective of the study was to identify the relationship between the state of the maturity and the antioxidant status of twin neonates. Investigations of the umbilical cord blood were carried out to detect differences in the antioxidant defence system between mature and premature twin neonates.

Methods: The activities of the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzymes, the levels of reduced glutathione (GSH), protein carbonyls and oxidized lipids and the total antioxidant capacity of the plasma were determined.

Results: The level of lipid peroxidation was significantly higher in the premature neonates. An increase in the total antioxidant capacity was accompanied by a decrease in the damaged protein concentration. Significantly elevated activities of GPx alone were observed in the premature twins, though the GSH content too tended to be increased. The activity of SOD was decreased in the premature neonates.

Discussion: The antioxidant status of twin neonates are mainly influenced by maturity. We suggest that the level of lipid peroxidation might be of clinical value as a marker of pre- and perinatal distress in twins.     

Oxidative stress balance is dysregulated and represents an additional target for treating cholangiocarcinoma

Background: Pancreatico-biliary malignancies exhibit similar characteristics, including obesity-related features and poor prognosis, and require new treatment strategies. Oxidative stress is known to induce DNA damage and carcinogenesis, and its reduction is viewed as being favorable. However, it also has anti-infection and anti-cancer functions that need to be maintained. To reveal the effect of oxidative stress on cancer progression, we evaluated oxidative stress and anti-oxidative balance in pancreatic cancer (PC) and cholangiocarcinoma (CC) patients, as well as the effect of add-on antioxidant treatment to chemotherapy in a mouse cholangiocarcinoma model.

Methods: We recruited 84?CC and 80?PC patients who were admitted to our hospital. Serum levels of reactive oxygen metabolites (ROM) and the anti-oxidative OXY-adsorbent test were determined and the balance of these tests was defined as an oxidative index. A diabetic mouse-based cholangiocarcinoma model was utilized to evaluate the effects of add-on antioxidant therapy on cholangiocarcinoma chemotherapy.

Results: Serum ROM was higher and anti-oxidant OXY was lower in CC patients with poor outcomes. These parameters were not significantly different in PC patients. In mice, vitamin E administration induced antioxidant hemeoxygenase (HO)-1 protein expression in cancer tissue, while the number of stem-like cells increased. l-carnitine administration improved intestinal microbiome and biliary acid balance, upregulated the hepatic mitochondrial membrane uptake related gene Cpt1 in non-cancerous tissue, and did not alter stem-like cell numbers.

Conclusion: Oxidative stress balance was dysregulated in cholangiocarcinoma with poor outcome. The mitochondrial function-supporting agent l-carnitine is a good candidate to control oxidative stress conditions.     

Methylglyoxal induces glycation and oxidative stress in Saccharomyces cerevisiae

Purpose

Hyperglycemia causes abnormal accumulation of methylglyoxal (MGO) and concomitant DNA, protein glycation. These pathophysiological changes further leads to diabetic complications. Yeast Saccharomyces cerevisiae is one of the best model to study MGO-induced glycation modifications. The aim of the present study was to investigate the effect of MGO on protein, DNA glycation, and oxidative stress markers using S. cerevisiae as a system.

Methods

Saccharomyces cerevisiae cells were incubated with 8 mM of MGO for 4 h and 24 h. After incubation, protein and DNA samples were isolated from the lysed cells. The samples were analyzed for various glycation (fructosamine, β-amyloid, free amino group, free thiol group, and hyperchromic shift analysis) and oxidative stress markers (total antioxidant potential, catalase, glutathione, and lipid peroxidation).

Results

MGO (8 mM) acted as a potent glycating agent, causing protein and DNA glycation in treated yeast cells. The glycation markers fructosamine and β-amyloid were significantly elevated when incubated for 4 h as compared to 24 h. Oxidative stress in the glycated yeast cells alleviated cellular antioxidant capacity and reduced the cell viability.

Conclusion

MGO caused significant glycation modifications of proteins and DNA in yeast cells. It also triggered increase in intracellular oxidative stress. MGO-induced protein, DNA glycation, and oxidative stress in S. cerevisiae indicate the suitability of the yeast model to study various biochemical pathways involved in diabetic complications and even conformational pathologies.

     

Bioregulators: unlocking their potential role in regulation of the plant oxidative defense system

Key message

Plant bioregulators play an important role in managing oxidative stress tolerance in plants. Utilizing their ability in stress sensitive crops through genetic engineering will be a meaningful approach to manage food production under the threat of climate change.

Abstract

Exploitation of the plant defense system against oxidative stress to engineer tolerant plants in the climate change scenario is a sustainable and meaningful strategy. Plant bioregulators (PBRs), which are important biotic factors, are known to play a vital role not only in the development of plants, but also in inducing tolerance in plants against various environmental extremes. These bioregulators include auxins, gibberellins, cytokinins, abscisic acid, brassinosteroids, polyamines, strigolactones, and ascorbic acid and provide protection against the oxidative stress-associated reactive oxygen species through modulation or activation of a plant’s antioxidant system. Therefore, exploitation of their functioning and accumulation is of considerable significance for the development of plants more tolerant of harsh environmental conditions in order to tackle the issue of food security under the threat of climate change. Therefore, this review summarizes a new line of evidence that how PBRs act as inducers of oxidative stress resistance in plants and how they could be modulated in transgenic crops via introgression of genes. Reactive oxygen species production during oxidative stress events and their neutralization through an efficient antioxidants system is comprehensively detailed. Further, the use of exogenously applied PBRs in the induction of oxidative stress resistance is discussed. Recent advances in engineering transgenic plants with modified PBR gene expression to exploit the plant defense system against oxidative stress are discussed from an agricultural perspective.

     

Stress induction and mitochondrial localization of Oxr1 proteins in yeast and humans

Reactive oxygen species (ROS) are critical molecules produced as a consequence of aerobic respiration. It is essential for cells to control the production and activity of such molecules in order to protect the genome and regulate cellular processes such as stress response and apoptosis. Mitochondria are the major source of ROS within the cell, and as a result, numerous proteins have evolved to prevent or repair oxidative damage in this organelle. The recently discovered OXR1 gene family represents a set of conserved eukaryotic genes. Previous studies of the yeast OXR1 gene indicate that it functions to protect cells from oxidative damage. In this report, we show that human and yeast OXR1 genes are induced by heat and oxidative stress and that their proteins localize to the mitochondria and function to protect against oxidative damage. We also demonstrate that mitochondrial localization is required for Oxr1 protein to prevent oxidative damage.     

Targeting oxidative stress response by green tea polyphenols: clinical implications

Abstract

Green tea polyphenols, the most interesting constituent of green tea leaves, have been shown to have both pro-oxidant and antioxidant properties. Both pro-oxidant and antioxidant properties are expected to contribute to modulation of oxidative stress response under ideal optimal dosage regimens. Exposure to a low concentration of a pro-oxidant prior to exposure to oxidative stress induces the expression of genes that code for proteins that induce adaptation in a subsequent oxidative stress. On the other hand, exposure to an antioxidant concurrently with exposure to the oxidative stress affords protection through free radical scavenging or through other indirect antioxidant mechanisms. In any case, the optimal conditions that afford protection from oxidative stress should be defined for any substance with redox properties. Green tea polyphenols, being naturally occurring substances, seem to be an ideal option for the modulation of oxidative stress response. This paper reviews available data on the pro-oxidant and antioxidant properties of green tea polyphenols focusing on their potential on the modulation of oxidative stress response.     

Dietary antioxidants provide differential subcellular protection in epithelial cells

Abstract

This study aimed to evaluate the organelle-specific antioxidant/pro-oxidant actions of clinically important dietary antioxidants against oxidative stress. An in vitro cellular model was employed to investigate the antioxidant/pro-oxidant effects of various concentrations (1, 10 and 100 μM) of ascorbic acid, α-tocopherol and β-carotene during H₂O₂-induced oxidative stress. Damage to nuclear and mitochondrial genomes was analyzed by quantitative polymerase chain reaction and oxidation of membrane lipids was measured via colorimetric assays. The key findings were: (i) dietary antioxidants conferred a dose-dependent protective effect (with a pro-oxidant shift at higher concentrations); (ii) the protection conferred to different sub-cellular organelles is highly specific to the dietary antioxidant; (iii) the mtDNA is highly sensitive to oxidative attack compared to nDNA (P < 0.05); and (iv) mtDNA protection conferred by dietary antioxidants was required to improve protection against oxidative-induced cell death. This study shows that antioxidant-induced protection of mtDNA is an important target for future oxidative stress therapies.     

Oxidative stress in myelin sheath: The other face of the extramitochondrial oxidative phosphorylation ability

Abstract

Oxidative phosphorylation (OXPHOS) is not only the main source of ATP for the cell, but also a major source of reactive oxygen species (ROS), which lead to oxidative stress. At present, mitochondria are considered the organelles responsible for the OXPHOS, but in the last years we have demonstrated that it can also occur outside the mitochondrion. Myelin sheath is able to conduct an aerobic metabolism, producing ATP that we have hypothesized is transferred to the axon, to support its energetic demand.

In this work, spectrophotometric, cytofluorimetric, and luminometric analyses were employed to investigate the oxidative stress production in isolated myelin, as far as its respiratory activity is concerned. We have evaluated the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), markers of lipid peroxidation, as well as of hydrogen peroxide (H₂O₂), marker of ROS production. To assess the presence of endogenous antioxidant systems, superoxide dismutase, catalase, and glutathione peroxidase activities were assayed. The effect of certain uncoupling or antioxidant molecules on oxidative stress in myelin was also investigated.

We report that isolated myelin produces high levels of MDA, 4-HNE, and H₂O₂, likely through the pathway composed by Complex I–III–IV, but it also contains active superoxide dismutase, catalase, and glutathione peroxidase, as antioxidant defense. Uncoupling compounds or Complex I inhibitors increase oxidative stress, while antioxidant compounds limit ROS generation.

Data may shed new light on the role of myelin sheath in physiology and pathology. In particular, it can be presumed that the axonal degeneration associated with myelin loss in demyelinating diseases is related to oxidative stress caused by impaired OXPHOS.     

KEAP1基因及其突变位点对非小细胞肺癌细胞株作用的实验初步研究

摘要 目的：研究KEAP1基因及KEAP1基因突变位点对肺癌细胞株的作用。方法：通过Western blot 方法,比较携带KEAP1 基因突变的肺癌细胞株（A549,NCI-H460,NCI-H838）与KEAP1 基因野生型的肺癌细胞株（NCI-H292, NCI-H1299, 95D, SPC-A1）之间,NRF2基因与NRF2下游基因HO-1的蛋白表达水平,检测并比较两组细胞的活性氧（ROS）含量;以新发现的非小细胞肺癌（NSCLC）病人的 KEAP1 体细胞突变作为模板构建 KEAP1 突变质粒,对自身存在 KEAP1 突变的肺癌细胞株A549,通过改造的 pMSCV 逆病毒转染体系,分别构建过表达空载,野生型及突变型 KEAP1 的 A549 稳定细胞株,比较过表达不同质粒的细胞间丙二醛（MDA）含量及 NRF2 下游抗氧化等相关基因的表达水平;通过克隆形成实验检测细胞增殖情况。结果：KEAP1基因突变组肺癌细胞株与KEAP1基因无突变的对照组细胞株相比,NRF2和HO-1蛋白表达显著增高,活性氧水平显著降低（P<0.01）;过表达野生型KEAP1 与过表达空载的 A549细胞相比,NRF2 及其下游基因转录水平表达显著下降（P<0.01）,蛋白水平表达下降,细胞内丙二醛水平显著增高（P<0.01）,克隆形成率显著降低（P<0.01）,而过表达突变型 KEAP1 与过表达空载的 A549细胞相比,NRF2 及其下游基因表达、细胞内丙二醛水平、克隆形成率均无显著差异（P>0.05）。结论：KEAP1基因具有抑癌作用,其突变为失活型突变,突变后KEAP1/NRF2通路激活,KEAP1基因突变可能通过改变细胞的氧化应激水平,促进肺癌的发生发展。