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PubMed2005

Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii.

The role of reactive oxygen species (ROS) in cell communication, control of gene expression, and oxygen sensing is well established. Inappropriate regulation of ROS levels can damage cells, resulting in a diseased state. In Colletotrichum trifolii, a fungal pathogen of alfalfa, the mutationally activated oncogenic fungal Ras (DARas) elevates levels of ROS, causing abnormal fungal growth and development and eventual apoptotic-like cell death but only when grown under nutrient-limiting conditions. Remarkably, restoration to the wild-type phenotype requires only proline. Here, we describe a generally unrecognized function of proline: its ability to function as a potent antioxidant and inhibitor of programmed cell death. Addition of proline to DARas mutant cells effectively quenched ROS levels and prevented cell death. Treating cells with inhibitors of ROS production yielded similar results. In addition, proline protected wild-type C. trifolii cells against various lethal stresses, including UV light, salt, heat, and hydrogen peroxide. These observations appear to be general because proline also protected yeast cells from lethal levels of the ROS-generating herbicide methyl viologen (paraquat), suggesting a common protective role for proline in response to oxidative stress. The ability of proline to scavenge intracellular ROS and inhibit ROS-mediated apoptosis may be an important and broad-based function of this amino acid in responding to cellular stress, in addition to its well established role as an osmolyte.

Apoptosis↗

PubMed1996

Reactive oxygen species are downstream mediators of p53-dependent apoptosis.

Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis. Conversely, experiments under hypoxic conditions have suggested that apoptosis could occur in the absence of ROS. We sought to determine whether a central modulator of apoptosis, p53, regulates the levels of intracellular ROS and whether a rise in ROS levels is required for the induction of p53-dependent apoptosis. We transiently overexpressed wild-type p53, using adenoviral gene transfer, and identified cell types that were sensitive or resistant to p53-mediated apoptosis. Cells sensitive to p53-mediated apoptosis produced ROS concomitantly with p53 overexpression, whereas cells resistant to p53 failed to produce ROS. In sensitive cells, both ROS production and apoptosis were inhibited by antioxidant treatment. These results suggest that p53 acts to regulate the intracellular redox state and induces apoptosis by a pathway that is dependent on ROS production.

Adenoviruses, Human↗

PubMed1997

Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization.

Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires alpha(v)beta5 integrin, rather than alpha(v)beta3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both alpha(v)beta3 and alpha(v)beta5 integrins, only alpha(v)beta3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of alpha(v)beta5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, alpha(v)beta5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas alpha(v)beta3 was expressed basolaterally. Using quantitative fluorimaging to assess in vitro uptake of fluorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, alpha(v)beta5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for alpha(v)beta5 in phagocytosis, immunofluorescence experiments demonstrated codistribution of alpha(v)beta5 integrin with internalized ROS. Control experiments showed that blocking alpha(v)beta3 function with antibodies did not inhibit ROS phagocytosis and that alpha(v)beta3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor alpha(v)beta5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-alpha(v)beta5 complex. Alpha(v)beta5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.

Animals↗

PubMed1996

Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments). Evidence for ubiquitinylation of Gt and rhodopsin.

In corroboration of the hypothesized regulation of phototransduction proteins by the ubiquitin-dependent pathway, we identified free ubiquitin (8 kDa) and ubiquitin-protein conjugates (50 to >200 kDa; pI 5.3-6.8 by two-dimensional electrophoresis) in bovine rod outer segments (ROS). A 38-kDa ubiquitinylated protein and transducin (Gt) were eluted together from light-adapted ROS membranes with GTP. When ROS were dark-adapted, this 38-kDa ubiquitinylated species and Gt were readily solubilized in buffer lacking GTP. These data are consistent with ubiquitinylation of Gt and corroborate previous cell-free experiments identifying Gt as a substrate for ubiquitin-dependent proteolysis (Obin, M. S., Nowell, T., and Taylor, A. (1994) Biochem. Biophys. Res. Commun. 200, 1169-1176). Evidence for ubiquitinylation of rhodopsin (36 kDa), the (photo)receptor coupled to Gt, included (i) the presence in ROS membranes "stripped" of peripheral membrane proteins of numerous ubiquitin-protein conjugates, including two whose masses (44 and 50 kDa) are consistent with mono- and diubiquitinylated rhodopsin; (ii) catalysis by permeabilized ROS of 125I-labeled ubiquitin-protein conjugates whose masses (42, 50, and 58 kDa) suggest a "ladder" of mono-, di-, and triubiquitinylated rhodopsin; (iii) parallel mobility shifts on SDS-polyacrylamide gels of rhodopsin and these 125I-labeled ubiquitin-protein conjugates; and (iv) generation of enhanced levels of 125I-labeled ubiquitin-protein conjugates when stripped, detergent-solubilized ROS membranes (95% rhodopsin) were incubated with reticulocyte lysate. A functional ubiquitin-dependent pathway in ROS is demonstrated by the presence of (i) the ubiquitin-activating enzyme (E1); (ii) four ubiquitin carrier proteins (E214K, E220K, E225K, and E235K) and pronounced activity of E214K, an enzyme required for "N-end rule" proteolysis; (iii) ATP-dependent 26 S proteasome activity that rapidly degrades high mass 125I-labeled ubiquitin-ROS protein conjugates; and (iv) distinct ubiquitin C-terminal isopeptidase/hydrolase activities, including potent ubiquitin-aldehyde-insensitive activity directed at high mass ubiquitinylated moieties. Considered together, the data support a novel role for the ubiquitin-dependent pathway in the regulation of mammalian phototransduction protein levels and/or activities and provide the first identification of a non-calpain proteolytic system in photoreceptors.

Animals↗

PubMed1996

Reactive oxygen species mediate cytokine activation of c-Jun NH2-terminal kinases.

Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha) are known to induce production of reactive oxygen species (ROS), which have been suggested to act as second messengers. Here we demonstrate that ROS production by bovine chondrocytes upon cytokine stimulation induces c-jun expression. Since c-jun expression is regulated by its own gene product via phosphorylation by c-Jun NH2-terminal kinases (JNKs), we investigated if cytokines and ROS could modulate JNK activity in chondrocyte monolayer cultures. Treatment of bovine chondrocytes with both IL-1 and TNFalpha leads to rapid induction of JNK activity, stimulating JNK activity 7- and 20-fold, respectively. Importantly, the observation that antioxidant treatment antagonizes IL-1 and TNFalpha activation of JNK provides strong evidence that ROS can act as mediators of JNK activity. Moreover, potent activation of JNK is also observed by direct addition of the ROS hydrogen peroxide (H2O2) to the chondrocyte cultures. Nitric oxide (NO), a multifunctional ROS, also appears to simulate JNK, albeit to a lesser extent. These findings identify JNK as another molecular target for the actions of NO and H2O2. In addition, the inhibitory effect of diphenyleneiodonium on JNK activation implicates the involvement of flavonoid-containing enzymes in the ROS-mediated signaling process. Overstimulation of JNK activity by excessive production of ROS may, therefore, underlie pathological conditions such as arthritis and cancer.

Animals↗

PubMed1998

Nerve growth factor treatment prevents the increase in superoxide produced by epidermal growth factor in PC12 cells.

Stimulation of pheochromocytoma (PC12) cells with the mitogen epidermal growth factor (EGF) produced a rapid and robust accumulation of intracellular reactive oxygen species (ROS), an accumulation which, in other systems, has been shown to be essential for mitogenesis. Brief pretreatment of the cells with nerve growth factor (NGF) suppressed the EGF-mediated ROS increase. EGF failed to produce elevations in ROS in a PC12 variant stably expressing a dominant-negative p21(ras) construct (PC12-N17) or in cells pretreated with the MEK inhibitor PD098059. NGF failed to suppress the increase in ROS in the PC12 variant nnr5, which lacks p140(trk) receptors. The suppression of the increase in ROS by NGF was restored in nnr5 cells stably expressing p140(trk) (nnr5-trk), but NGF failed to prevent the increase in ROS in nnr cells expressing mutant p140(trk) receptors that lack binding sites for Shc and phospholipase Cgamma. Among several inhibitors of superoxide-generating enzymes, only the lipoxygenase inhibitor, nordihydroguaiaretic acid reduced EGF-mediated ROS accumulation. The inhibitory action of NGF on ROS production was mimicked by the nitric oxide donor, sodium nitroprusside, and was blocked by an inhibitor of nitric-oxide synthetase, L-nitroarginine methyl ester. These results suggest a novel mechanism for the rapid interruption of mitogenic signaling by the neurotrophin NGF.

Animals↗

PubMed1999

Hydrogen peroxide activates p70(S6k) signaling pathway.

We investigated a possible role of reactive oxygen species (ROS) in p70(S6k) activation, which plays an important role in the progression of cells from G(0)/G(1) to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts that encode for components of the protein synthetic machinery. Treatment of mouse epidermal cell JB6 with H(2)O(2) generated extracellularly by glucose/glucose oxidase led to the activation of p70(S6k) and p90(Rsk) and to phosphorylation of p42(MAPK)/p44(MAPK). The activation of p70(S6k) and p90(Rsk) was dose-dependent and transient, maximal activities being in extracts treated for 15 and 30 min, respectively. Further characterization of ROS-induced activation of p70(S6k) using specific inhibitors for p70(S6k) signaling pathway, rapamycin, and wortmannin revealed that ROS acted upstream of the rapamycin-sensitive component FRAP/RAFT and wortmannin-sensitive component phosphatidylinositol 3-kinase, because both inhibitors caused the inhibition of ROS-induced p70(S6k) activity. In addition, Ca(2+) chelation also inhibited ROS-induced activation of p70(S6k), indicating that Ca(2+) is a mediator of p70(S6k) activation by ROS. However, down-regulation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive protein kinase C (PKC) by chronic pretreatment with TPA or a specific PKC inhibitor Ro-31-8220 did not block the activation of p70(S6k) by ROS, indicating that the activation of TPA-responsive PKC was not required for stimulation of p70(S6k) activity by H(2)O(2) in JB6 cells. Exposure of JB6 cells to platelet-derived growth factor or epidermal growth factor led to a rapid increase in H(2)O(2), phosphorylation, and activation of p70(S6k), which were antagonized by the pretreatment of catalase. Taken together, the results suggest that ROS act as a messenger in growth factor-induced p70(S6k) signaling pathway.

Androstadienes↗

PubMed2000

Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade.

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha, although the events through which TNF-alpha induces ROS generation are not yet well characterized. We therefore investigated selected candidates likely to mediate TNF-alpha-induced ROS generation. Consistent with the role of Rac in that process, stable expression of Rac(Asn-17), a dominant negative Rac1 mutant, completely blocked TNF-alpha-induced ROS generation. To understand better the mediators downstream of Rac, we investigated the involvement of cytosolic phospholipase A(2) (cPLA(2)) activation and metabolism of the resultant arachidonic acid (AA) by 5-lipoxygenase (5-LO). TNF-alpha-induced ROS generation was blocked by inhibition of cPLA(2) or 5-LO, but not cyclooxygenase, suggesting that TNF-alpha-induced ROS generation is dependent on synthesis of AA and its subsequent metabolism to leukotrienes. Consistent with that hypothesis, TNF-alpha Rac-dependently stimulated endogenous production of leukotriene B(4) (LTB(4)), while exogenous application of LTB(4) increased levels of ROS. In contrast, application of leukotrienes C(4), D(4), and E(4) or prostaglandin E(2) had little effect. Our findings suggest that LTB(4) production by 5-LO is situated downstream of the Rac-cPLA(2) cascade, and we conclude that Rac, cPLA(2), and LTB(4) play pivotal roles in the ROS-generating cascade triggered by TNF-alpha.

Animals↗

PubMed2001

Reactive oxygen species are downstream products of TRAF-mediated signal transduction.

Members of the TNFR (tumor necrosis factor receptor) superfamily are involved in regulating activation and differentiation of cells as well as cell survival and programmed cell death/apoptosis. Multimerization of TNFRs can lead to recruitment of TRAFs (TNFR-associated factors) by the receptors resulting in activation of kinases and transcription factors, such as c-Jun N-terminal kinase and nuclear factor kappaB (NF-kappaB). Signal transduction triggered by TNF-alpha also induces an increase in intracellular reactive oxygen species (ROS). ROS have been suggested to play a role in NF-kappaB activation, which is thought to promote cell survival. However, oxidation of proteins and lipids by ROS can also result in apoptosis. The processes generating intracellular ROS and the mechanism(s) regulating the cellular redox status have not been fully elucidated. We investigated whether TRAFs play a role in controlling intracellular ROS levels. Our results indicate that recruitment of TRAFs to the plasma membrane of human embryonic kidney (HEK) 293 cells is crucial for activation of signaling pathways, which regulate ROS production in mitochondria. TRAF-mediated changes in ROS levels enhanced NF-kappaB activation but were not dependent on NF-kappaB-inducing kinase. Consistent with its anti-apoptotic function, Bcl-x(L) interfered with TRAF-mediated ROS generation but not NF-kappaB activation. Taken together, our results suggest a novel role of TRAFs in signal transduction pathways triggered by TNFR-related proteins, which balance cell survival and apoptosis by regulating the electron transport in mitochondria.

Apoptosis↗

PubMed2001

Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR.

Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitric-oxide synthase inhibitors diphenyleneiodonium and N(G)methyl-l-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-gamma-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.

Androstadienes↗

PubMed2003

Nerve growth factor protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a phosphatidylinositol 3-kinase-dependent manner.

The survival signal elicited by the phosphatidylinositol 3-kinase (PI3K)/Akt1 pathway has been correlated with inactivation of pro-apoptotic proteins and attenuation of the general stress-induced increase in reactive oxygen species (ROS). However, the mechanisms by which this pathway regulates intracellular ROS levels remain largely unexplored. In this study, we demonstrate that nerve growth factor (NGF) prevents the accumulation of ROS in dopaminergic PC12 cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves PI3K/Akt-dependent induction of the stress response protein heme oxygenase-1 (HO-1). The effect of NGF was mimicked by induction of HO-1 expression with CoCl(2); by treatment with bilirubin, an end product of heme catabolism; and by infection with a retroviral expression vector for human HO-1. The relevance of HO-1 in NGF-induced ROS reduction was further demonstrated by the evidence that cells treated with the HO-1 inhibitor tin-protoporphyrin or infected with a retroviral expression vector for antisense HO-1 exhibited enhanced ROS release in response to 6-OHDA, despite the presence of the neurotrophin. Inhibition of PI3K prevented NGF induction of HO-1 mRNA and protein and partially reversed its protective effect against 6-OHDA-induced ROS release. By contrast, cells transfected with a membrane-targeted active version of Akt1 exhibited increased HO-1 expression, even in the absence of NGF, and displayed a greatly attenuated production of ROS and apoptosis in response to 6-OHDA. These observations indicate that the PI3K/Akt pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.

Adrenergic Agents↗

PubMed2003

Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells.

Bortezomib, a proteasome inhibitor, shows substantial anti-tumor activity in a variety of tumor cell lines, is in phase I, II, and III clinical trials and has recently been approved for the treatment of patients with multiple myeloma. The sequence of events leading to apoptosis following proteasome inhibition by bortezomib is unclear. Bortezomib effects on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration in the mitochondrial membrane potential (Delta psi m), and release of cytochrome c from mitochondria. With human H460 lung cancer cells, bortezomib exposure at 0.1 microM showed induction of apoptotic cell death starting at 24 h, with increasing effects after 48-72 h of treatment. After 3-6 h, an elevation in ROS generation, an increase in Delta psi m, and the release of cytochrome c into the cytosol, were observed in a time-dependent manner. Co-incubation with rotenone and antimycin A, inhibitors of mitochondrial electron transport chain complexes I and III, or with cyclosporine A, an inhibitor of mitochondrial permeability transition pore, resulted in inhibition of bortezomib-induced ROS generation, increase in Delta psi m, and cytochrome c release. Tiron, an antioxidant agent, blocked the bortezomib-induced ROS production, Delta psi m increase, and cytochrome c release. Tiron treatment also protected against the bortezomib-induced PARP protein cleavage and cell death. Benzyloxycarbonyl-VAD-fluoromethyl ketone, an inhibitor of pan-caspase, did not alter the bortezomib-induced ROS generation and increase in Delta psi m, although it prevented bortezomib-induced poly(ADP-ribose) polymerase cleavage and apoptotic death. In PC-3 prostate carcinoma cells (with overexpression of Bcl-2), a reduction of bortezomib-induced ROS generation, Delta psi m increase was correlated with cellular resistance to bortezomib and the attenuation of drug-induced apoptosis. The transient transfection of wild type p53 in p53 null H358 cells caused stimulation of the bortezomib-induced apoptosis but failed to enhance ROS generation and Delta psi m increase. Thus ROS generation plays a critical role in the initiation of the bortezomib-induced apoptotic cascade by mediation of the disruption of Delta psi m and the release of cytochrome c from mitochondria.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed2003

Production of reactive oxygen species by mitochondria: central role of complex III.

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.

Animals↗

PubMed2003

Regulation of necrosis of H9c2 myogenic cells upon transient energy deprivation. Rapid deenergization of mitochondria precedes necrosis and is controlled by reactive oxygen species, stress kinase JNK, HSP72 and ARC.

Subjecting myogenic H9c2 cells to transient energy deprivation leads to a caspase-independent death with typical features of necrosis. Here we show that the rupture of cytoplasmic membrane, the terminal event in necrosis, is shortly preceded by rapid depolarization of mitochondrial membranes. The rapid deenergization of mitochondria critically depended upon prior generation of reactive oxygen species (ROS) during ATP depletion stage. Accordingly, expression of catalase prevented mitochondrial depolarization and averted subsequent necrosis. Interestingly, trifluoperazine, a compound that protects cells from ischemic insults, prevented necrosis of H9c2 cells through inhibition of ROS production. Other factors that regulated the mitochondrial membrane depolarization and subsequent loss of plasma membrane integrity include a stress kinase JNK activated at early steps of recovery from ATP depletion, as well as an apoptotic inhibitory protein ARC. Accordingly, inhibition of JNK or overexpression of ARC prevented mitochondrial depolarization and rescued H9c2 cells from necrosis. ROS and JNK affected mitochondrial deenergization and necrosis independently of each other since inhibition of ROS production did not prevent activation of JNK, whereas inhibition of JNK did not suppress ROS accumulation. Therefore, JNK activation and ROS production represent two independent pathways that control mitochondrial depolarization and subsequent necrosis of cells subjected to transient energy deprivation. Overexpression of ARC, although preventing mitochondrial depolarization, did not affect either JNK activation or production of ROS. The major heat shock protein Hsp72 inhibited JNK-related steps of necrotic pathway but did not affect ROS accumulation. Interestingly, mitochondrial depolarization and subsequent necrosis can be suppressed by an Hsp72 mutant Hsp72DeltaEEVD, which lacks chaperone function but can efficiently suppress JNK activation. Thus, Hsp72 is directly implicated in a signaling pathway, which leads to necrotic death.

Adenosine Triphosphate↗

PubMed2004

Doxorubicin paradoxically protects cardiomyocytes against iron-mediated toxicity: role of reactive oxygen species and ferritin.

The cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX) is attributed to reactions between iron and reactive oxygen species (ROS) that lead to oxidative damage. We found that DOX forms ROS in H9c2 cardiomyocytes, as shown by dichlorodihydrofluorescein oxidation and the expression of stress-responsive genes such as catalase or aldose reductase. DOX also increased ferritin levels in these cells, particularly the H subunit. A considerable increase in ferritin mRNA levels showed that DOX acted at transcriptional level, but an additional potential mechanism was identified as the down-regulation of iron regulatory protein-2, post-transcriptional inhibitor of ferritin synthesis. Pretreatment with DOX protected H9c2 cells against the damage induced by subsequent exposure to ferric ammonium citrate, and experiments with (55)Fe revealed that the protection was due to the deposition of iron in ferritin. Cytoprotection was also observed when DOX was replaced by glucose/glucose oxidase, a source of H(2)O(2), thus suggesting that DOX increases ferritin synthesis through the action of ROS. This concept was supported by three more lines of evidence. (i) DOX-induced ferritin synthesis was blocked by N-acetylcysteine, a scavenger of ROS. (ii) Mitoxantrone, a ROS-forming analogue, similarly induced ferritin expression and protected the cells against iron toxicity. (iii) 5-Iminodaunorubicin, an analogue lacking ROS-forming activity, did not induce ferritin synthesis or protect the cells against iron toxicity. These results characterize a paradoxically beneficial link between anthracycline-derived ROS, increased ferritin synthesis, and resistance to iron-mediated damage. The role of iron and ROS in anthracycline-induced cardiotoxicity may, therefore, be more complex than previously believed.

Animals↗

PubMed2004

Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.

One reason why pancreatic cancer is so aggressive and unresponsive to treatments is its resistance to apoptosis. We report here that reactive oxygen species (ROS) are a prosurvival, antiapoptotic factor in pancreatic cancer cells. Human pancreatic adenocarcinoma MIA PaCa-2 and PANC-1 cells generated ROS, which was stimulated by growth factors (serum, insulin-like growth factor I, or fibroblast growth factor-2). Growth factors also stimulated membrane NAD(P)H oxidase activity in these cells. Both intracellular ROS and NAD(P)H oxidase activity were inhibited by antioxidants tiron and N-acetylcysteine and the inhibitor of flavoprotein-dependent oxidases, diphenylene iodonium, but not by inhibitors of various other ROS-generating enzymes. Using Rho(0) cells deficient in mitochondrial DNA, we showed that a nonmitochondrial NAD(P)H oxidase is a major source of growth factor-induced ROS in pancreatic cancer cells. Among proteins that have been implicated in NAD(P)H oxidase activity, MIA PaCa-2 and PANC-1 cells do not express the phagocytic gp91(phox) subunit but express several nonphagocytic oxidase (NOX) isoforms. Transfection with Nox4 antisense oligonucleotide inhibited NAD(P)H oxidase activity and ROS production in MIA PaCa-2 and PANC-1 cells. Inhibiting ROS with the antioxidants, Nox4 antisense, or MnSOD overexpression all stimulated apoptosis in pancreatic cancer cells as measured by internucleosomal DNA fragmentation, phosphatidylserine externalization, cytochrome c release, and effector caspase activation. The results show that growth factor-induced ROS produced by NAD(P)H oxidase (probably Nox4) protect pancreatic cancer cells from apoptosis. This mechanism may play an important role in pancreatic cancer resistance to treatment and thus represent a novel therapeutic target.

Antioxidants↗

PubMed2001

Ozone exposure and the production of reactive oxygen species by bronchoalveolar cells in humans.

Exposure to ozone injures respiratory epithelium, and the mechanisms may involve the generation of reactive oxygen species (ROS). This study tested the hypothesis that ozone exposure increases the airway burden of ROS to a greater degree in smokers than nonsmokers, and that this effect is independent of ozone-induced changes in spirometry. Healthy subjects were selected as either responders (decrement in FEV1 > 15%) or nonresponders (decrement in FEV1 < 5%) to ozone; each underwent 2 exposures to ozone and 1 to air, with bronchoalveolar lavage (BAL) performed 30 min (early) and 18 h (late) after exposure. Release of superoxide anion (O2(-)) was used as a measure of ROS release by all BAL cells, and flow cytometry was used to detect ROS production in alveolar macrophages (AM) only. Recovery of AM was approximately threefold greater in smokers than nonsmokers. Unstimulated, but not stimulated, cells obtained by BAL from smokers released approximately twofold greater amounts of O2(-) than cells from nonsmokers, both early and late after ozone exposure (p =.012 and p =.046, respectively). Stimulated, but not unstimulated, ROS generation by AM from smokers increased following ozone exposure, but the ozone effect was not significant. ROS production by AM decreased in nonsmokers (air vs. ozone late, p =.014). Total protein, albumin, and immunoglobulin M (IgM) increased in BAL fluid, consistent with an increase in epithelial permeability. In addition, the concentration of alpha2-macroglobulin increased approximately threefold 18 h after exposure in nonsmokers (p <.001). No relationship was found between measures of ROS production and lung function responsiveness to ozone. These studies suggest the airways of smokers experience a greater burden of ROS than those of nonsmokers following ozone exposure.

Administration, Inhalation↗

PubMed2006

Uncoupling the relationship between fatty acids and longevity.

Research into different species has verified the negative correlation between longevity and the level of reactive oxygen species (ROS) production. ROS creates oxidative damage and, consequently, fuels the aging process. As such, the astonishing longevity of avian species correlates well with their lower levels of ROS production, in comparison to mammals of similar size. Apart from this inter-species difference, caloric restriction (CR) is a widely-documented means of increasing intra-species longevity, and it works by decreasing ROS production. However, little is known about the mechanisms responsible, either for the retardation of aging in CR or for the longevity of long-living species. Recent findings have shown an increase in uncoupling protein (UCP) activity with lower ROS levels, after CR stress. These UCPs are stimulated by fatty acids. Moreover, in numerous studies, fatty acids have been demonstrated to generate a reduction in ROS generation. Thus, the decreased ROS production seen in both CR and longer lifespan may occur via up-regulation of free fatty acid stimulation of UCP activity. Consequently, free fatty acids may play an important regulatory role in longevity, by reducing ROS, via actions on UCPs.

Aging↗