Serum deprivation-induced reactive oxygen species production is mediated by Romo1

Serum deprivation-triggered increases in reactive oxygen species (ROS) are known to induce apoptotic cell death. However, the mechanism by which serum deprivation causes ROS production is not known. Since mitochondria are the main source of ROS and since mitochondrial ROS modulator 1 (Romo1) is involved in ROS production, we sought to determine if serum deprivation triggered ROS production through Romo1. To examine the relationship between Romo1 and the serum deprivation-triggered increase in ROS, we transfected Romo1 siRNA into various cell lines and looked for inhibition of mitochondrial ROS generation. Romo1 knockdown by Romo1 siRNA blocked the mitochondrial ROS production caused by serum deprivation, which originates in the mitochondrial electron transport chain. We also found that Romo1 knockdown inhibited serum deprivation-induced apoptosis. These findings suggest that Romo1-derived ROS play an important role in apoptotic cell death triggered by withdrawal of cell survival factors.     

Activation of mitochondrial-driven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species production

While the acquisition of apoptosis resistance is part of the differentiation program of skeletal muscle cells, differentiated muscle cells can undergo apoptosis in response to physiological or pathological stimuli. The generation of reactive oxygen species by mitochondria plays a major role in the control of apoptosis in many cell types. Indeed their involvement in controlling apoptosis in differentiated muscle cells, or in generating resistance to apoptosis remains unknown. Moreover, differentiated muscle cells specifically express the uncoupling protein-3, a mitochondrial protein potentially involved in controlling reactive oxygen species production. To study the role of mitochondrial reactive oxygen species in the control of apoptosis in skeletal muscle cells, L6E9 myoblasts and myotubes were exposed to staurosporine, an inducer of apoptosis via mitochondrial pathways. Staurosporine activated apoptotic pathways (i.e. caspase-3 and caspase-9) increasing reactive oxygen species in myoblasts and, to a minor extent, in myotubes. However, the increase in reactive oxygen species was not needed to induce apoptosis nor was it involved in the differential sensitization of myoblasts and myotubes to apoptosis. Moreover, expression of uncoupling protein-3 in myotubes did not affect reactive oxygen species production, although it produced a slight sensitization for staurosporine-induced apoptosis. Results indicate that apoptotic activation in skeletal muscle cells mainly involves reactive oxygen species-independent mechanisms and that mitochondrial uncoupling protein-3 is not protective either for reactive oxygen species production or for apoptotic activation in muscle cells.     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.     

Aggrecan degradation in chondrocytes is mediated by reactive oxygen species and protected by antioxidants

Reactive oxygen species (ROS) are implicated in aging of cartilage and in the pathogenesis of osteoarthritis. However, the biological role of chondrocytes-derived ROS has not been elucidated. An in-vitro model was developed to study the role of chondrocyte-derived ROS in cartilage matrix degradation. The primary articular chondrocytes were cultured and the aggrecan matrix was radiolabeled with 35-sulfate. The labeled aggrecan matrix was washed to remove unincorporated label and chondrocytes were returned to serum free balanced salt solution. The cell-monolayer-matrix sensitivity to oxidative damage due to either hydrogen peroxide or glucose oxidase was established by monitoring the release of labeled aggrecan into the medium. Lipopolysaccharide (LPS) treatment of chondrocytemonolayer enhanced the release of labeled aggrecan. Catalase significantly prevented the release of labeled aggrecan in LPS-chondrocyte cultures, suggesting a role for chondrocyte-derived hydrogen peroxide in aggrecan degradation. Superoxide dismutase or boiled catalase had no such inhibitory effect. The effect of several antioxidants on LPS-chondrocyte-dependent aggrecan degradation was examined. Hydroxyl radical scavengers (mannitol and thiourea) significantly decreased aggrecan degradation. A spin trapping agent N-tert-butyl-phenylnitrone (but not its inactive analog tert-butyl-phenylcarbonate) significantly decreased aggrecan degradation. Butylated hydroxytoluene also inhibited aggrecan degradation, whereas the other lipophilic antioxidant tested, propyl gallate, had a marked dose-dependent inhibitory effect. These data indicate that general antioxidants, hydroxyl radical scavengers, antioxidant vitamins, iron chelating agents, lipophilic antioxidants, and spin trapping agents can influence chondrocyte-dependent aggrecan degradation. These studies support the role of a chondrocyte-dependent oxidative mechanism in aggrecan degradation and indicate that antioxidants can prevent matrix degradation and therefore may have a preventive or therapeutic value in arthritis. The enhancement of oxidative activity in chondrocytes and its damaging effect on matrix may be an important mechanism of matrix degradation in osteoarthritis.     

Aggrecan degradation in chondrocytes is mediated by reactive oxygen species and protected by antioxidants

Reactive oxygen species (ROS) are implicated in aging of cartilage and in the pathogenesis of osteoarthritis. However, the biological role of chondrocytes-derived ROS has not been elucidated. An in-vitro model was developed to study the role of chondrocyte-derived ROS in cartilage matrix degradation. The primary articular chondrocytes were cultured and the aggrecan matrix was radiolabeled with 35-sulfate. The labeled aggrecan matrix was washed to remove unincorporated label and chondrocytes were returned to serum free balanced salt solution. The cell-monolayer-matrix sensitivity to oxidative damage due to either hydrogen peroxide or glucose oxidase was established by monitoring the release of labeled aggrecan into the medium. Lipopolysaccharide (LPS) treatment of chondrocytemonolayer enhanced the release of labeled aggrecan. Catalase significantly prevented the release of labeled aggrecan in LPS-chondrocyte cultures, suggesting a role for chondrocyte-derived hydrogen peroxide in aggrecan degradation. Superoxide dismutase or boiled catalase had no such inhibitory effect. The effect of several antioxidants on LPS-chondrocyte-dependent aggrecan degradation was examined. Hydroxyl radical scavengers (mannitol and thiourea) significantly decreased aggrecan degradation. A spin trapping agent N-tert-butyl-phenylnitrone (but not its inactive analog tert-butyl-phenylcarbonate) significantly decreased aggrecan degradation. Butylated hydroxytoluene also inhibited aggrecan degradation, whereas the other lipophilic antioxidant tested, propyl gallate, had a marked dose-dependent inhibitory effect. These data indicate that general antioxidants, hydroxyl radical scavengers, antioxidant vitamins, iron chelating agents, lipophilic antioxidants, and spin trapping agents can influence chondrocyte-dependent aggrecan degradation. These studies support the role of a chondrocyte-dependent oxidative mechanism in aggrecan degradation and indicate that antioxidants can prevent matrix degradation and therefore may have a preventive or therapeutic value in arthritis. The enhancement of oxidative activity in chondrocytes and its damaging effect on matrix may be an important mechanism of matrix degradation in osteoarthritis.     

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.     

Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.     

The positive inotropic effect of endothelin-1 is mediated by mitochondrial reactive oxygen species

We have previously demonstrated the participation of reactive oxygen species (ROS) in the positive inotropic effect of a physiological concentration of Angiotensin II (Ang II, 1 nM). The objective of the present work was to evaluate the role and source of ROS generation in the positive inotropic effect produced by an equipotent concentration of endothelin-1 (ET-1, 0.4 nM). Isolated cat ventricular myocytes were used to measure sarcomere shortening with a video-camera, superoxide anion (()O(2)(-)) with chemiluminescence, and ROS production and intracellular pH (pH(i)) with epifluorescence. The ET-1-induced positive inotropic effect (40.4+/-3.1%, n=10, p<0.05) was associated to an increase in ROS production (105+/-29 fluorescence units above control, n=6, p<0.05). ET-1 also induced an increase in ()O(2)(-) production that was inhibited by the NADPH oxidase blocker, apocynin, and by the blockers of mitochondrial ATP-sensitive K(+) channels (mK(ATP)), glibenclamide and 5 hydroxydecanoic acid. The ET-1-induced positive inotropic effect was inhibited by apocynin (0.3 mM; 6.3+/-6.6%, n=13), glibenclamide (50 muM; 8.8+/-3.5%, n=6), 5 hydroxydecanoic acid (500 muM; 14.1+/-8.1, n=9), and by scavenging ROS with MPG (2 mM; 0.92+/-5.6%, n=8). ET-1 enhanced proton efflux (J(H)) carried by the Na(+)/H(+) exchanger (NHE) after an acid load, effect that was blocked by MPG. Consistently, the ET-induced positive inotropic effect was also inhibited by the NHE selective blocker HOE642 (5 muM; 9.37+/-6.07%, n=7). The data show that the effect of a concentration of ET-1 that induces an increase in contractility of about 40% is totally mediated by an intracellular pathway triggered by mitochondrial ROS formation and stimulation of the NHE.     

Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species

Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell (EC) membrane mediated by K_ATP channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating EC proliferation associated with the loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (mPMVECs) from wild-type, Kir6.2^–/–, and gp91^phox–/– mice. mPMVECs were labeled with PKH26 and cultured in artificial capillaries for 72 h at 5 dyn/cm² (flow adaptation), followed by 24 h of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared with wild-type mice in both types of gene-targeted mPMVECs. Cell proliferation was defined as the proliferation index (PI). After 72 h of flow, >98% of PKH26-labeled wild-type mPMVECs were at a single peak (PI 1.0) and the proportion of cells in the S+G₂/M phases were at 5.8% on the basis of cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and the ratio of cells in S+G₂/M phases were at 35%. Catalase, diphenyleneiodonium, and cromakalim markedly inhibited ROS production and cell proliferation in flow-adapted wild-type mPMVECs. Significant effects of ischemia were not observed in Kir6.2^–/– and gp91^phox–/– cells. ANG II activation of NADPH oxidase was unaffected by K_ATP gene deletion. Thus loss of shear stress in flow-adapted mPMVECs results in cell division associated with ROS generated by NADPH oxidase. This effect requires a functioning cell membrane K_ATP channel. cell signaling; ischemia; mechanotransduction; K_ATP channels; NADPH oxidase     

JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species

c-Jun N-terminal kinase (JNK), or stress-activated protein kinase, is an important member of the mitogen-activated protein kinase superfamily, the members of which are readily activated by many environmental stimuli. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important groups of free radicals that are capable of eliciting direct damaging effects or acting as critical intermediate signaling molecules, leading to oxidative and nitrosative stress and a series of biological consequences. Recently there has been an increasing amount of research interest focusing on the regulatory role of JNK activation in ROS-and RNS-induced cellular responses. In this review we will first summarize and discuss some recent findings regarding the signaling mechanisms of ROS-or RNS-mediated JNK activation. Second, we will talk about the role of JNK in ROS-or RNS-mediated cell death (both apoptosis and necrosis). Finally, we will analyze the emerging evidence for the involvement of ROS and RNS as mediators in tumor necrosis factor alpha-induced apoptosis. Taken together, the accumulating knowledge about the ROS/RNS-induced JNK signaling pathway has greatly advanced our understanding of the complex processes deciding the cellular responses to environmental stress.     

Mitochondrial production of reactive oxygen species

Numerous biochemical studies are aimed at elucidating the sources and mechanisms of formation of reactive oxygen species (ROS) because they are involved in cellular, organ-, and tissue-specific physiology. Mitochondria along with other cellular organelles of eukaryotes contribute significantly to ROS formation and utilization. This review is a critical account of the mitochondrial ROS production and methods for their registration. The physiological and pathophysiological significance of the mitochondrially produced ROS are discussed.     

Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species

Tumour–stroma interaction is a prerequisite for tumour progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumour-associated fibroblasts to MFs (myofibroblasts) by growth factors, for example TGFβ (transforming growth factor beta(). In this study, the question was addressed of whether fibroblast-associated NAD(P)H oxidase (NADH/NADPH oxidase), known to be activated by TGFβ1, is involved in the fibroblast-to-MF switch. The up-regulation of αSMA (alpha smooth muscle actin), a biomarker for MFs, is mediated by a TGFβ1-dependent increase in the intracellular level of ROS (reactive oxygen species). This report demonstrates two novel aspects of the TGFβ1 signalling cascade, namely the generation of ROS due to a biphasic NAD(P)H oxidase activity and a ROS-dependent downstream activation of p38 leading to a transition of dermal fibroblasts to MFs that can be inhibited by the selective NAD(P)H oxidase inhibitor apocynin. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-MF switch and may be important for chemoprevention in context of a ‘stromal therapy’ which was described earlier.     

Negative inotropic response to cerium in ventricular papillary muscle is mediated by reactive oxygen species

This study was performed with the objective of assessing the mechanical response of the myocardium to different levels of cerium and delineation of the mechanism underlying the mediation of the functional changes. Rat ventricular papillary muscle was used as the experimental model. Isolated papillary muscles were exposed to different concentrations of CeCl₃ and the force of contraction was measured using a force transducer. Experiments have revealed that the negative inotropic response to CeCl₃ was proportional to its concentration. The inotropic changes were found to be completely reversible at concentrations ≤5μM, and partially reversible at higher concentrations. Neutralization of cerium-induced inotropic changes by the superoxide anion scavenger superoxide dismutase (SOD) at concentrations ≤5 μM indicates that the mechanical changes are mediated by reactive oxygen species. At higher concentrations of Ce³⁺, SOD partially reversed the contractile changes. The beneficial effect of SOD was seen only if the muscles were pretreated with the scavenger prior to the addition of cerium chloride.     

Laser mediated production of reactive oxygen and nitrogen species; implications for therapy

Laser therapy has gained wide acceptance applications to many medical disciplines. The side effect-effects from laser therapy involve the potential for interaction with cellular and extracellular matrix molecules to generate reactive oxygen species and reactive nitrogen species which in turn can initiate lipid peroxidation, protein damage or DNA modification. These issues are addressed in this short overview in the context of experimental models of laser-induced thrombosis.     

The production of reactive oxygen species by dietary flavonols

Flavonols are a group of naturally occurring compounds which are widely distributed in nature where they are found glycosylated primarily in vegetables and fruits. A number of studies have found both anti- and prooxidant effects for many of these compounds. The most widely studied because of their ubiquitous nature have been quercetin, a B-dihydroxylated and myricetin, a B-trihydroxylated flavonol. Some of their prooxidant properties have been attributed to the fact that they can undergo autooxidation when dissolved in aqueous buffer. Studying a number of factors affecting autooxidation, we found the rate of autooxidation for both quercetin and myricetin to be highly pH dependent with no autooxidation detected for quercetin at physiologic pH. Both the addition of iron for the two flavonols and the addition of iron followed by SOD for quercetin at physiologic pH. Both the addistantially. Neither kaempferol, a monohydroxylated flavonol nor rutin, a glycosylated quercetin showed any ability to autooxidize. The results with rutin differ from what we expected based on the B-ring structural similarity to quercetin. The autooxidation of quercetin and myricetin was further studied by electron spin resonance spectroscopy (ESR). Whereas quercetin produced a characteristic DMPO-OH radical, it was not detected below a pH of 9. However, the addition of iron allowed the signal to be detected at a pH as low as 8.0. On the other hand, myricetin autooxidation yielded a semiquinone signal which upon the addition of iron, converted to a DMPO-OH signal detected at a pH of 7.5. In a microsome-NADPH system, quercetin produced an increase in oxygen utilization and with ESR, an ethanol-derived radical signal which could be completely suppressed by catalase indicating the dependence of the signal on hydrogen peroxide. These studies demonstrate that the extracellular production of active oxygen species by dietary flavonols is not likely to occur in vivo but the potential for intracellular redox cycling may have toxicologic significance.     

Anti-colorectal cancer activity of macrostemonoside A mediated by reactive oxygen species

Macrostemonoside A (MSS.A), an active steroidal saponin from Allium macrostemon Bung has been shown to possess anti-coagulation and anti-obesity effects. However, the functional role of MSS.A on tumor growth has not been elucidated. We found that MSS.A significantly inhibited human colorectal cancer cell growth in Caco2 and SW480 cells. Incubation of SW480 cells with MSS.A for 48 h resulted in cell cycle arrest. Moreover, MSS.A dose-dependently induced apoptosis in SW480 cells as shown by increased AnnexinV positively stained cell population, caspase activation, increased pro-apoptotic and reduced anti-apoptotic Bcl-2 family protein levels. Treatment of SW480 cells with MSS.A resulted in increased reactive oxygen species (ROS) generation. However, pre-incubation of SW480 cells with antioxidant N-acetylcysteine (NAC) attenuated the ROS generation and anti-colorectal cancer activities of MSS.A. Lastly, intra-peritoneal injections of MSS.A significantly inhibited tumor formation in BALB/c nude mice carcinogenesis xenograft model by reduced tumor volume and tumor weight when treated at dosages of 10, 50 or 100 mg/kg daily for 35 days compared with PBS control. Taken together, our results indicate that MSS.A suppressed colorectal cancer growth and induced cell apoptosis by inducing ROS production, and that MSS.A may have therapeutic relevance in the treatment of human colorectal cancer.