Role of reactive oxygen species generated by NADPH oxidase in the mechanism of activation of K+-Cl--cotransport by N-ethylmaleimide in HepG2 human hepatoma cells

K⁺-Cl^--cotransport (KCC) is ubiquitously present in all cells, and plays an essential role in ion and volume regulation. In this study we investigated the role of reactive oxygen species (ROS) in regulation of KCC in HepG2 human hepatoblastoma cells. N-ethylmaleimide (NEM), a KCC activator, induced Cl^--dependent K⁺ efflux, which was markedly prevented by KCC inhibitors (calyculin-A, genistein and BaCl₂), indicating that KCC is activated by NEM in the HepG2 cells. Treatment with NEM also induced a sustained increase in the level of intracellular ROS assessed by 2′,7′-dichlorofluorescein flourescence. Antioxidants, N-acetyl cysteine or N,N′-diphenyl-p-phenylenediamine significantly inhibited both ROS generation and KCC activation induced by NEM. The NEM-induced ROS production was significantly suppressed by inhibitors of NADPH oxidase (diphenylene iodonium, apocynin and neopterine). These inhibitors also significantly inhibited the NEM-induced KCC activation. Taken together, these results suggest that ROS generated by NADPH oxidase may mediate the NEM-induced activation of KCC in human hepatoma cells.     

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.     

Polyamines inhibit NADPH oxidase-mediated superoxide generation and putrescine prevents programmed cell death induced by polyamine oxidase-generated hydrogen peroxide

Our previous results indicate that during protoplast isolation an oxidative burst occurs [A.K. Papadakis and KA Roubelakis-Angelakis (1999) Plant Physiol 127:197–205] and that suppression of totipotency is correlated with reduced antioxidant activity and low redox state [A.K. Papadakis et al. (2001b) Plant Physiol 126:434–444]. Polyamines are known to affect cell development and to act as antioxidants. Polyamines applied during isolation of tobacco (Nicotiana tabacum L.) protoplasts reduced the accumulation of O₂^·– but not that of H₂O₂. This antioxidant effect is probably due to the inhibition of microsomal membrane NADPH oxidase, which occurred in a concentration-dependent manner, with spermine exerting the highest inhibitory effect. However, during protoplast culture, polyamine oxidase activity increased severalfold in spermidine- and spermine-treated protoplasts, concomitant with H₂O₂ titers. A cell death program was executed in untreated protoplasts, as documented by membrane malfunction, induced DNase activity, DNA fragmentation and a positive TUNEL reaction. Protoplast cell death was prevented in protoplasts treated with putrescine, but not by treatment with spermidine or spermine, which rather had the opposite effect. The data presented suggest that PAs may be implicated in the expression of plant protoplast totipotency.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H₂O₂-dependent pathway via reduction of the antioxidant defenses.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

Biochemical entities involved in reactive oxygen species generation by human spermatozoa

Summary.  Spermatozoa were the first cell type suggested to generate reactive oxygen species. However, a lack of standardization in sperm preparation techniques and the obfuscating impact of contaminating cell types in human ejaculates have made it difficult to confirm that mammalian germ cells do, in fact, make such reactive metabolites. By identifying, on a molecular level, those entities involved in reactive oxygen species generation and demonstrating their presence in spermatozoa, the role of redox chemistry in the control of sperm function can be elucidated. Two major proteins have apparently been identified in this context, namely, NOX5, a calcium-activated NADPH oxidase, and nitric oxide synthase. Understanding the involvement of these enzymes in sperm physiology is essential if we are to understand the causes of oxidative stress in the male germ line. Received May 2, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.     

The influence of phenobarbital on cytochromes and reactive oxygen species in erythropoietin producing HepG2 cells

Light absorption photometry of HepG2 cells treated with phenobarbital for enhancing the content of cytochrome P-450 and the synthesis of erythropoietin revealed an influence on all cytochromes detectable in the wavelength range between 400 and 620 nm. No correlation was found between specific changes of cytochrome P-450 absorption and increased EPO synthesis as proposed earlier by Fandrey et al. (Life Sci. (1990) 47, 127–134). In the present study, however, the increased erythropoietin synthesis could be related to a decreased intracellular hydroxyl radical level described as crucial for the oxygen regulated gene expression (Kietzmann et al., Biochem. J. (1998) 335, 425–432; Porwol et al., Eur. J. Biochem. (1998) 256, 16–23).     

The effect of reactive oxygen species on cardiomyocyte differentiation of pluripotent stem cells

The coordination of metabolic shift with genetic circuits is critical to cell specification, but the metabolic mechanisms that drive cardiac development are largely unknown. Reactive oxygen species (ROS) are not only the by-product of mitochondrial metabolism, but play a critical role in signalling cascade of cardiac development as a second messenger. Various levels of ROS appear differential and even oppose effect on selfrenewal and cardiac differentiation of pluripotent stem cells (PSCs) at each stage of differentiation. The intracellular ROS and redox balance are meticulous regulated by several systems of ROS generation and scavenging, among which mitochondria and the NADPH oxidase (NOX) are major sources of intracellular ROS involved in cardiomyocyte differentiation. Some critical signalling modulators are activated or inactivated by oxidation, suggesting ROS can be involved in regulation of cell fate through these downstream targets. In this review, the literatures about major sources of ROS, the effect of ROS level on cardiac differentiation of PSCs, as well as the underlying mechanism of ROS in the control of cardiac fate of PSC are summarised and discussed.     

N-Nitrosopiperidine and N-Nitrosodibutylamine induce apoptosis in HepG2 cells via the caspase dependent pathway

The human hepatoma cell line (HepG2) exhibited a dose and time-dependent apoptotic response following treatment with N-Nitrosopiperidine (NPIP) and N-Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24 h treatment with NDBA (3.5 mM), whereas it was necessary to use high doses of NPIP (45 mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase-8) and intrinsic pathway (caspase-9) could be implicated in the N-Nitrosamines-induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N-acetylcysteine (NAC) did not block NPIP-induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.     

NADPH oxidase produces reactive oxygen species and maintains survival of rat astrocytes

Reactive oxygen species (ROS) produced by activated astrocytes have been considered to be involved in the pathogenesis of neurodegenerative diseases, while NADPH oxidase is an essential enzyme involved in ROS-mediated signal transduction. The goal of the present study was to determine whether NADPH oxidase plays a role in ROS generation and cell survival in rat astrocytes. We found that the release of ROS in rat astrocytes was significantly increased by stimulation with calcium ionophore or opsonized zymosan, which are known to trigger a respiration burst in phagocytes by the NADPH oxidase pathway. Further study indicated that diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, significantly suppressed the increase of ROS release caused by the calcium ionophore or opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI dose- and time-dependently decreased the viability of normal astrocytes, whereas exogenous supplementation of H2O2 can reverse the survival of DPI-treated astrocytes. For the first time, our results suggest that NADPH oxidase is an important enzyme for the generation of ROS in astrocytes, and the ROS generated by NADPH oxidase play an essential role in astrocyte survival.     

Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production

Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition.     

Evidence of cell damage induced by major components of a diet-compatible mixture of oxysterols in human colon cancer CaCo-2 cell line

Cholesterol oxidation products, termed oxysterols, have been shown to be more reactive than unoxidized cholesterol, possessing marked pro-inflammatory and cytotoxic effects in a number of cells and tissues. Oxysterols, absorbed with the diet as products of cholesterol auto-oxidation, have recently been suggested to potentially interfere with homeostasis of the mucosal intestinal epithelium, by promoting and sustaining irreversible damage.     

Proton irradiation induced reactive oxygen species promote morphological and functional changes in HepG2 cells

The increased use of proton therapy has led to the need of better understanding the cellular mechanisms involved. The aim of this study was to investigate the effects induced by the accelerated proton beam in hepatocarcinoma cells. An existing facility in IFIN-HH, a 3 MV Tandetron? accelerator, was used to irradiate HepG2 human hepatocarcinoma cells with doses between 0 and 3 Gy. Colony formation was used to assess the influence of radiation on cell long-term replication. Also, the changes induced at the mitochondrial level were shown by increased ROS and ATP levels as well as a decrease in the mitochondrial membrane potential. An increased dose has induced DNA damages and G₂/M cell cycle arrest which leads to caspase 3/7 mediated apoptosis and senescence induction. Finally, the morphological and ultrastructural changes were observed at the membrane level and the nucleus of the irradiated cells. Thus, proton irradiation induces both morphological and functional changes in HepG2 cells.     

Restraining reactive oxygen species in Listeria monocytogenes promotes the apoptosis of glial cells

Objectives: Listeria monocytogenes is a facultative anaerobic foodborne pathogen that can traverse the blood–brain barrier and cause brain infection. L. monocytogenes infection induces host cell apoptosis in several cell types. In this study, we investigated the apoptosis of human glioma cell line U251 invaded by L. monocytogenes and evaluated the function of bacterial reactive oxygen species (ROS) during infection.

Methods: Bacterial ROS level was reduced by carrying out treatment with N-acetyl cysteine (NAC) and diphenyleneiodonium chloride (DPI). After infection, the apoptosis of U251 cells was examined by flow cytometry assay and propidium iodide staining.

Results: DPI and NAC efficiently decreased ROS level in L. monocytogenes without affecting bacterial growth. Moreover, the apoptosis of glial cells was enhanced upon invasion of DPI- and NAC-pretreated L. monocytogenes.

Discussion: Results indicate that the apoptosis of glial cells can be induced by L. monocytogenes, and that the inhibition of bacterial ROS increases the apoptosis of host cells.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Capsaicin induces apoptosis by generating reactive oxygen species and disrupting mitochondrial transmembrane potential in human colon cancer cell lines

Although genetic factors are a well-known cause of colorectal cancer, environmental factors contribute more to its development. Despite advances in the fields of surgery, radiotherapy and chemotherapy, the cure rates for colon cancer have not substantially improved over the past few decades. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the principal pungent ingredient of hot chili pepper, has exhibited an anti-tumor effect in many cell types. However, the mechanisms responsible for the anti-tumor effect of capsaicin are not yet completely understood. In this study, we investigated whether capsaicin induces apoptosis in colon cancer cell lines. Capsaicin decreased cell viability in a dose-dependent manner in Colo320DM and LoVo cells. In addition, capsaicin produced cell morphology changes and DNA fragmentation, decreased the DNA contents, and induced phosphatidylserine translocation, which is a hallmark of apoptotic cell death. We showed that capsaicin-induced apoptosis is associated with an increase in ROS generation and a disruption of the mitochondrial transmenbrane potential. A possible mechanism of capsaicin-induced apoptosis is the activation of caspase 3, a major apoptosis-executing enzyme. Treatment with capsaicin induced a dramatic increase in caspase 3 activity, as assessed by the cleavage of Ac-DEVD-AMC, a fluorogenic substrate. In conclusion, our results clearly showed that capsaicin induced apoptosis in colon cancer cells. Although the actual mechanisms of capsaicin-induced apoptosis remain uncertain, it may be a beneficial agent for colon cancer treatment and chemoprevention.     

Fatty acids as modulators of the cellular production of reactive oxygen species

Long-chain nonesterified ("free") fatty acids (FFA) and some of their derivatives and metabolites can modify intracellular production of reactive oxygen species (ROS), in particular O(2)(-) and H(2)O(2). In mitochondria, FFA exert a dual effect on ROS production. Because of slowing down the rate of electron flow through Complexes I and III of the respiratory chain due to interaction within the complex subunit structure, and between Complexes III and IV due to release of cytochrome c from the inner membrane, FFA increase the rate of ROS generation in the forward mode of electron transport. On the other hand, due to their protonophoric action on the inner mitochondrial membrane ("mild uncoupling effect"), FFA strongly decrease ROS generation in the reverse mode of electron transport. In the plasma membrane of phagocytic neutrophils and a number of other types of cells, polyunsaturated FFA stimulate O(2)(-) generation by NADPH oxidase. These effects of FFA can modulate signaling functions of ROS and be, at least partly, responsible for their proapoptotic effects in several types of cells.