High fluence low‐power laser irradiation induces apoptosis via inactivation of Akt/GSK3β signaling pathway

High fluence low‐power laser irradiation (HF‐LPLI) is a newly discovered stimulus through generating reactive oxygen species (ROS) to trigger cell apoptosis. Activation of glycogen synthase kinase 3β (GSK3β) is proved to be involved in intrinsic apoptotic pathways under various stimuli. However, whether the proapoptotic factor GSK3β participates in HF‐LPLI‐induced apoptosis has not been elucidated. Therefore, in the present study, we investigated the involvement of GSK3β in apoptosis under HF‐LPLI treatment (120 J/cm², 633 nm). We found that GSK3β activation could promote HF‐LPLI‐induced apoptosis, which could be prevented by lithium chloride (a selective inhibitor of GSK3β) exposure or by GSK3β‐KD (a dominant‐negative GSK3β) overexpression. We also found that the activation of GSK3β by HF‐LPLI was due to the inactivation of protein kinase B (Akt), a widely reported and important upstream negative regulator of GSK3β, indicating the existence and inactivation of Akt/GSK3β signaling pathway. Moreover, the inactivation of Akt/GSK3β pathway depended on the fluence of HF‐LPLI treatment. Furthermore, vitamin c, a ROS scavenger, completely prevented the inactivation of Akt/GSK3β pathway, indicating ROS generation was crucial for the inactivation. In addition, GSK3β promoted Bax activation by down‐regulating Mcl‐1 upon HF‐LPLI treatment. Taken together, we have identified a new and important proapoptotic signaling pathway that is consisted of Akt/GSK3β inactivation for HF‐LPLI stimulation. Our research will extend the knowledge into the biological mechanisms induced by LPLI. J. Cell. Physiol. 226: 588–601, 2011. © 2010 Wiley‐Liss, Inc.     

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.     

Salt stress-induced programmed cell death in tobacco protoplasts is mediated by reactive oxygen species and mitochondrial permeability transition pore status

The status of mitochondrial permeability transition pore (PTP) and levels of reactive oxygen species (ROS) play key roles in regulating apoptosis in animal cells. To investigate if the PTP and cellular oxidation-reduction state are also involved in salt stress-induced programmed cell death (PCD) in tobacco (Nicotiana tabacum, cultivar BY-2) protoplasts, flow cytometry was used to simultaneously monitor ROS levels, PTP status and PCD. Increased ROS and decreased mitochondrial membrane potential (delta psi(m)) were observed before the appearance of PCD. Pre-treatment with an inhibitor of the PTP opening, cyclosporin A (CsA), effectively retarded the onset of PCD, the delta psi(m) decrease and the ROS content increase. Addition of ascorbic acid (AsA) during the salt stress significantly decreased the percentage of protoplasts undergoing PCD and ROS levels but increased delta psi(m). Hydrogen peroxide effectively induced the appearance of PCD and caused an increase in ROS and a decrease in delta psi(m). Pre-treatment of protoplasts with CsA weakened the effects of H2O2. All these results suggest that the open state of PTP and ROS are necessary elements for salt stress-induced PCD in tobacco protoplasts. The open states of PTP and ROS could promote each other suggesting that ROS could lead to a self-amplifying process. This positive feedback loop may act as an all-or-nothing switch, which is in good accordance with the hypothesis that PTP is an important coordinator and executioner of PCD in both animals and plants.     

Opening of the mitochondrial permeability transition pore induces reactive oxygen species production at the level of the respiratory chain complex I

We have investigated the consequences of permeability transition pore (PTP) opening on the rate of production of reactive oxygen species in isolated rat liver mitochondria. We found that PTP opening fully inhibited H(2)O(2) production when mitochondria were energized both with complex I or II substrates. Because PTP opening led to mitochondrial pyridine nucleotide depletion, H(2)O(2) production was measured again in the presence of various amounts of NADH. PTP opening-induced H(2)O(2) production began when NADH concentration was higher than 50 microm and reached a maximum at over 300 microm. At such concentrations of NADH, the maximal H(2)O(2) production was 4-fold higher than that observed when mitochondria were permeabilized with the channel-forming antibiotic alamethicin, indicating that the PTP opening-induced H(2)O(2) production was not due to antioxidant depletion. Moreover, PTP opening decreased rotenone-sensitive NADH ubiquinone reductase activity, whereas it did not affect the NADH FeCN reductase activity. We conclude that PTP opening induces a specific conformational change of complex I that (i) dramatically increases H(2)O(2) production so long as electrons are provided to complex I, and (ii) inhibits the physiological pathway of electrons inside complex I. These data allowed the identification of a novel consequence of permeability transition that may partly account for the mechanism by which PTP opening induces cell death.     

Se-methylselenocysteine induces apoptosis mediated by reactive oxygen species in HL-60 cells

Recent studies have implicated apoptosis as one of the most plausible mechanisms of the chemopreventive effects of selenium compounds, and reactive oxygen species (ROS) as important mediators in apoptosis induced by various stimuli. In the present study, we demonstrate that Se-methylselenocysteine (MSC), one of the most effective selenium compounds at chemoprevention, induced apoptosis in HL-60 cells and that ROS plays a crucial role in MSC-induced apoptosis. The uptake of MSC by HL-60 cells occurred quite early, reaching the maximum within 1 h. The dose-dependent decrease in cell viability was observed by MSC treatment and was coincident with increased DNA fragmentation and sub-G(1) population. 50 microM of MSC was able to induce apoptosis in 48% of cell population at a 24 h time point. Moreover, the release of cytochrome c from mitochondria and the activation of caspase-3 and caspase-9 were also observed. The measurement of ROS by dichlorofluorescein fluorescence revealed that dose- and time-dependent increase in ROS was induced by MSC. N-acetylcysteine, glutathione, and deferoxamine blocked cell death, DNA fragmentation, and ROS generation induced by MSC. Moreover, N-acetylcysteine effectively blocked caspase-3 activation and the increase of the sub-G(1) population induced by MSC. These results imply that ROS is a critical mediator of the MSC-induced apoptosis in HL-60 cells.     

Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells

Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-L-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.     

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.     

Zinc induces mitogen-activated protein kinase activation mediated by reactive oxygen species in rice roots.

It is well known that zinc (Zn) is one of the micronutrients essential for normal growth and development of plants. However, the molecular mechanisms responsible for the regulation of plant growth by Zn are still not completely understood. The aim of this study was to investigate the signalling transduction pathways activated by Zn. We show that Zn elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot analysis, we suggest that Zn-activated 40- and 42-kDa MBP kinases are mitogen-activated protein kinases (MAPK). Pre-treatment of rice roots with reactive oxygen species (ROS) scavenger, sodium benzoate, was able to effectively prevent Zn-induced MAPK activation. However, phosphoinositide 3-kinase (PI-3K) inhibitor, LY294002, was unable to inhibit Zn-induced MAPK activation. These results suggest that the ROS may function in the Zn-triggered MAPK signalling pathway in rice roots.     

LINE‐1 hypomethylation induced by reactive oxygen species is mediated via depletion of S‐adenosylmethionine

Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H₂O₂ for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H₂O₂‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H₂O₂‐treated cells were explored. Viabilities of cells treated with H₂O₂ were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H₂O₂‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H₂O₂‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H₂O₂‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H₂O₂‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H₂O₂, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.     

线粒体电子传递链电子漏的化学发光测定

本实验用差速离心法分离正常大鼠肝脏和心肌线粒体 ,以lucigenin (探测超氧阴离子 )与luminol (探测过氧化氢 )为探剂 ,用化学发光法测定METC电子漏的生成。在反应体系中加入外源底物 ,其发光强度明显高于空白对照 (体系中无线粒体 )。在肝线粒体体系中 ,无论是lucigenin还是luminol诱发的发光 ,琥珀酸底物引起的发光强均要高于丙酮酸 /苹果酸引起的发光强度。在心肌线粒体 luminol体系中也有与肝线粒体相似的结果 ,在心肌线粒体 lucigenin体系中 ,加入外源底物丙酮酸 /苹果酸诱发的发光强度高于琥珀酸诱发的发光强度     

Isoalantolactone induces reactive oxygen species mediated apoptosis in pancreatic carcinoma PANC-1 cells

Isoalantolactone, a sesquiterpene lactone compound possesses antifungal, antibacteria, antihelminthic and antiproliferative activities. In the present study, we found that isoalantolactone inhibits growth and induces apoptosis in pancreatic cancer cells. Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of reactive oxygen species, cardiolipin oxidation, reduced mitochondrial membrane potential, release of cytochrome c and cell cycle arrest at S phase. N-Acetyl Cysteine (NAC), a specific ROS inhibitor restored cell viability and completely blocked isoalantolactone-mediated apoptosis in PANC-1 cells indicating that ROS are involved in isoalantolactone-mediated apoptosis. Western blot study showed that isoalantolactone increased the expression of phosphorylated p38 MAPK, Bax, and cleaved caspase-3 and decreased the expression of Bcl-2 in a dose-dependent manner. No change in expression of phosphorylated p38 MAPK and Bax was found when cells were treated with isoalantolactone in the presence of NAC, indicating that activation of these proteins is directly dependent on ROS generation. The present study provides evidence for the first time that isoalantolactone induces ROS-dependent apoptosis through intrinsic pathway. Furthermore, our in vivo toxicity study demonstrated that isoalantolactone did not induce any acute or chronic toxicity in liver and kidneys of CD1 mice at dose of 100 mg/kg body weight. Therefore, isoalantolactone may be a safe chemotherapeutic candidate for the treatment of human pancreatic carcinoma.     

1,4‐butanediyl‐bismethanethiosulfonate (BMTS) induces apoptosis through reactive oxygen species‐mediated mechanism

Although methane sulfonate compounds are widely used for the protein modification for their selectivity of thiol groups in proteins, their intracellular signaling events have not yet been clearly documented. This study demonstrated the methane sulfonate chemical 1,4‐butanediyl‐bismethanethiosulfonate (BMTS)‐induced cascades of signals that ultimately led to apoptosis of Jurkat cells. BMTS induced apoptosis through fragmentation of DNA, activation of caspase‐9 and caspase‐3, and downregulation of Bcl‐2 protein with reduction of mitochondrial membrane potential. Moreover, BMTS intensely and transiently induced intracellular reactive oxygen species (ROS) production and ROS produced by BMTS was mediated through mitochondria. We also found that a reducing agent dithiothreitol (DTT) and an anti‐oxidant N‐acetyl cysteine (NAC) inhibited BMTS‐mediated caspase‐9 and ‐3 activation, ROS production and induction of Annexin V/propidium iodide double positive cells, suggesting the involvement of ROS in the apoptosis process. Therefore, this study further extends our understanding on the basic mechanism of redox‐linked apoptosis induced by sulfhydryl‐reactive chemicals. J. Cell. Biochem. 108: 1059–1065, 2009. © 2009 Wiley‐Liss, Inc.     

Protein lysine‐Nζ alkylation and O‐phosphorylation mediated by DTT‐generated reactive oxygen species

Reactive oxygen species (ROS) play crucial roles in physiology and pathology. In this report, we use NMR spectroscopy and mass spectrometry (MS) to demonstrate that proteins (galectin-1, ubiquitin, RNase, cytochrome c, myoglobin, and lysozyme) under reducing conditions with dithiothreitol (DTT) become alkylated at lysine-N_ζ groups and O-phosphorylated at serine and threonine residues. These adduction reactions only occur in the presence of monophosphate, potassium, trace metals Fe/Cu, and oxygen, and are promoted by reactive oxygen species (ROS) generated via DTT oxidation. Superoxide mediates the chemistry, because superoxide dismutase inhibits the reaction, and hydroxyl and phosphoryl radicals are also likely involved. While lysine alkylation accounts for most of the adduction, low levels of phosphorylation are also observed at some serine and threonine residues, as determined by western blotting and MS fingerprinting. The adducted alkyl group is found to be a fragment of DTT that forms a Schiff base at lysine N_ζ groups. Although its exact chemical structure remains unknown, the DTT fragment includes a SH group and a –CHOH–CH₂– group. Chemical adduction appears to be promoted in the context of a well-folded protein, because some adducted sites in the proteins studied are considerably more reactive than others and the reaction occurs to a lesser extent with shorter, unfolded peptides and not at all with small organic molecules. A structural signature involving clusters of positively charged and other polar groups appears to facilitate the reaction. Overall, our findings demonstrate a novel reaction for DTT-mediated ROS chemistry with proteins.     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

Ultraviolet irradiation induces autofluorescence enhancement via production of reactive oxygen species and photodecomposition in erythrocytes

Ultraviolet (UV) light has a significant influence on human health. In this study, human erythrocytes were exposed to UV light to investigate the effects of UV irradiation (UVI) on autofluorescence. Our results showed that high-dose continuous UVI enhanced erythrocyte autofluorescence, whereas low-dose pulsed UVI alone did not have this effect. Further, we found that H₂O₂, one type of reactive oxygen species (ROS), accelerated autofluorescence enhancement under both continuous and pulsed UVI. In contrast, continuous and pulsed visible light did not result in erythrocyte autofluorescence enhancement in the presence or absence of H₂O₂. Moreover, NAD(P)H had little effect on UVI-induced autofluorescence enhancement. From these studies, we conclude that UVI-induced erythrocyte autofluorescence enhancement via both UVI-dependent ROS production and photodecomposition. Finally, we present a theoretical study of this autofluorescence enhancement using a rate equation model. Notably, the results of this theoretical simulation agree well with the experimental data further supporting our conclusion that UVI plays two roles in the autofluorescence enhancement process.