Mitochondrial reactive oxygen species in cell death signaling

During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.     

TNFalpha and reactive oxygen species in necrotic cell death

Death receptors, including the TNF receptor-1 （TNF-RI）, have been shown to be able to initiate caspase-independent cell death. This form of ＂necrotic cell death＂ appears to be dependent on the generation of reactive oxygen species. Recent data have indicated that superoxide generation is dependent on the activation of NADPH oxidases, which form a complex with the adaptor molecules RIP1 and TRADD. The mechanism of superoxide generation further establishes RIP1 as the central molecule in ROS production and cell death initiated by TNFa and other death receptors. A role for the sustained JNK activation in necrotic cell death is also suggested. The sensitization of virus-infected cells to TNFα indicates that necrotic cell death may represent an alternative cell death pathway for clearance of infected cells.     

Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.     

Autophagy,programmed cell death and reactive oxygen species in sexual reproduction in plants

Autophagy is one of the major cellular processes of recycling of proteins, metabolites and intracellular organelles, and plays crucial roles in the regulation of innate immunity, stress responses and programmed cell death (PCD) in many eukaryotes. It is also essential in development and sexual reproduction in many animals. In plants, although autophagy-deficient mutants of Arabidopsis thaliana show phenotypes in abiotic and biotic stress responses, their life cycle seems normal and thus little had been known until recently about the roles of autophagy in development and reproduction. Rice mutants defective in autophagy show sporophytic male sterility and immature pollens, indicating crucial roles of autophagy during pollen maturation. Enzymatic production of reactive oxygen species (ROS) by respiratory burst oxidase homologues (Rbohs) play multiple roles in regulating anther development, pollen tube elongation and fertilization. Significance of autophagy and ROS in the regulation of PCD of transient cells during plant sexual reproduction is discussed in comparison with animals.     

MuRF1 activity is present in cardiac mitochondria and regulates reactive oxygen species production in vivo

MuRF1 is a previously reported ubiquitin-ligase found in striated muscle that targets troponin I and myosin heavy chain for degradation. While MuRF1 has been reported to interact with mitochondrial substrates in yeast two-hybrid studies, no studies have identified MuRF1’s role in regulating mitochondrial function to date. In the present study, we measured cardiac mitochondrial function from isolated permeabilized muscle fibers in previously phenotyped MuRF1 transgenic and MuRF1?/? mouse models to determine the role of MuRF1 in intermediate energy metabolism and ROS production. We identified a significant decrease in reactive oxygen species production in cardiac muscle fibers from MuRF1 transgenic mice with increased α-MHC driven MuRF1 expression. Increased MuRF1 expression in ex vivo and in vitro experiments revealed no alterations in the respiratory chain complex I and II function. Working perfusion experiments on MuRF1 transgenic hearts demonstrated significant changes in glucose oxidation. This is an factual error as written; however, total oxygen consumption was decreased. This data provides evidence for MuRF1 as a novel regulator of cardiac ROS, offering another mechanism by which increased MuRF1 expression may be cardioprotective in ischemia reperfusion injury, in addition to its inhibition of apoptosis via proteasome-mediate degradation of c-Jun. The lack of mitochondrial function phenotype identified in MuRF1?/? hearts may be due to the overlapping interactions of MuRF1 and MuRF2 with energy regulating proteins found by yeast two-hybrid studies reported here, implying a duplicity in MuRF1 and MuRF2’s regulation of mitochondrial function.     

Quinclorac-induced cell death is accompanied by generation of reactive oxygen species in maize root tissue

The importance of reactive oxygen species for herbicide quinclorac (3,7-dichloro-8-quinolinecarboxylic acid)-induced cell death in roots was investigated. This was in order to understand its mode of action in grass species grown in the dark. Under these dark conditions, quinclorac suppressed the shoot and root growth of maize (Zea mays L. cv. Honey Bantam) in a concentration-dependent manner (50muM), although the inhibition level was less than that observed under growth conditions in the light. Analysis of cell viability using Evans blue or fluorescein diacetate-propidium iodide (FDA-PI) staining showed that the maize root cells significantly lost their viability after 14h root treatment with 10muM quinclorac, but not 10muM 2,4-dichlorophenoxyacetic acid (2,4-D). Determination of reactive oxygen species (ROS) in maize roots using a superoxide anion (O(2)(-))-specific indicator, dihydroethidium (DHE), indicated that 50muM quinclorac induced a high level of O(2)(-) production in maize roots after 14h root treatment than that of either the control (non-treated) or with 50muM 2,4-D. Moreover, either cell death or ethane evolution, an indicator of lipid peroxide formation, in maize root segments was significantly enhanced by 50muM quinclorac, but not by 50muM 2,4-D. On the other hand, the 50muM 2,4-D treatment induced much higher ethylene and cyanide production in the root segments than with the 50muM quinclorac. These results suggest that quinclorac-induced cell death in maize roots may be caused by ROS and lipid peroxidation, but not by ethylene and its biosynthetic pathway-related substances including cyanide, which have been thought to be the causative factor of quinclorac-induced phytotoxicity in susceptible grass weeds such as Echinochloa, Digitaria, and Setaria.     

The role of reactive oxygen species and autophagy in safingol-induced cell death

Safingol is a sphingolipid with promising anticancer potential, which is currently in phase I clinical trial. Yet, the underlying mechanisms of its action remain largely unknown. We reported here that safingol-induced primarily accidental necrotic cell death in MDA-MB-231 and HT-29 cells, as shown by the increase in the percentage of cells stained positive for 7-aminoactinomycin , collapse of mitochondria membrane potential and depletion of intracellular ATP. Importantly, safingol treatment produced time- and concentration-dependent reactive oxygen species (ROS) generation. Autophagy was triggered following safingol treatment, as reflected by the formation of autophagosomes, acidic vacuoles, increased light chain 3-II and Atg biomarkers expression. Interestingly, scavenging ROS with N-acetyl--cysteine could prevent the autophagic features and reverse safingol-induced necrosis. Our data also suggested that autophagy was a cell repair mechanism, as suppression of autophagy by 3-methyladenine or bafilomycin A1 significantly augmented cell death on 2-5 μ safingol treatment. In addition, Bcl-xL and Bax might be involved in the regulation of safingol-induced autophagy. Finally, glucose uptake was shown to be inhibited by safingol treatment, which was associated with an increase in p-AMPK expression. Taken together, our data suggested that ROS was the mediator of safingol-induced cancer cell death, and autophagy is likely to be a mechanism triggered to repair damages from ROS generation on safingol treatment.     

The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species

Oxidative stress mediated by reactive oxygen species (ROS) is linked to degenerative conditions in humans and damage to an array of cellular components. However, it is unclear which molecular target(s) may be the primary "Achilles' heel" of organisms, accounting for the inhibitory action of ROS. Rli1p (ABCE1) is an essential and highly conserved protein of eukaryotes and archaea that requires notoriously ROS-labile cofactors (Fe-S clusters) for its functions in protein synthesis. In this study, we tested the hypothesis that ROS toxicity is caused by Rli1p dysfunction. In addition to being essential, Rli1p activity (in nuclear ribosomal-subunit export) was shown to be impaired by mild oxidative stress in yeast. Furthermore, prooxidant resistance was decreased by RLI1 repression and increased by RLI1 overexpression. This Rlip1 dependency was abolished during anaerobicity and accentuated in cells expressing a FeS cluster-defective Rli1p construct. The protein's FeS clusters appeared ROS labile during in vitro incubations, but less so in vivo. Instead, it was primarily (55)FeS-cluster supply to Rli1p that was defective in prooxidant-exposed cells. The data indicate that, owing to its essential nature but dependency on ROS-labile FeS clusters, Rli1p function is a primary target of ROS action. Such insight could help inform new approaches for combating oxidative stress-related disease.     

The mitochondrial membrane protein FgLetm1 regulates mitochondrial integrity,production of endogenous reactive oxygen species and mycotoxin biosynthesis in Fusarium graminearum

Deoxynivalenol (DON) is a mycotoxin produced in cereal crops infected with Fusarium graminearum. DON poses a serious threat to human and animal health, and is a critical virulence factor. Various environmental factors, including reactive oxygen species (ROS), have been shown to interfere with DON biosynthesis in this pathogen. The regulatory mechanisms of how ROS trigger DON production have been investigated extensively in F. graminearum. However, the role of the endogenous ROS‐generating system in DON biosynthesis is largely unknown. In this study, we genetically analysed the function of leucine zipper‐EF‐hand‐containing transmembrane 1 (LETM1) superfamily proteins and evaluated the role of the mitochondrial‐produced ROS in DON biosynthesis. Our results show that there are two Letm1 orthologues, FgLetm1 and FgLetm2, in F. graminearum. FgLetm1 is localized to the mitochondria and is essential for mitochondrial integrity, whereas FgLetm2 plays a minor role in the maintenance of mitochondrial integrity. The ΔFgLetm1 mutant demonstrated a vegetative growth defect, abnormal conidia and increased sensitivity to various stress agents. More importantly, the ΔFgLetm1 mutant showed significantly reduced levels of endogenous ROS, decreased DON biosynthesis and attenuated virulence in planta. To our knowledge, this is the first report showing that mitochondrial integrity and endogenous ROS production by mitochondria are important for DON production and virulence in Fusarium species.     

Yeast cell death during DNA damage arrest is independent of caspase or reactive oxygen species

CDC13 encodes a telomere-binding protein that prevents degradation of telomeres. cdc13-1 yeast grown at the nonpermissive temperature undergo G2/M arrest, progressive chromosome instability, and subsequent cell death. Recently, it has been suggested that cell death in the cdc13-1 mutant is an active process characterized by phenotypic hallmarks of apoptosis and caspase activation. In this work, we show that cell death triggered by cdc13-1 is independent of the yeast metacaspase Yca1p and reactive oxygen species but related to cell cycle arrest per se. Inactivating YCA1 or depleting reactive oxygen species does not increase viability of cdc13-1 cells. In turn, caspase activation does not precede cell death in the cdc13-1 mutant. Yca1p activity assayed by cell binding of mammalian caspase inhibitors is confounded by artifactual labeling of dead yeast cells, which nonspecifically bind fluorochromes. We speculate that during a prolonged cell cycle arrest, cdc13-1 cells reach a critical size and die by cell lysis.     

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.     

RIP1-dependent reactive oxygen species production executes artesunate-induced cell death in renal carcinoma Caki cells

Artesunate is a well-known anti-malarial drug originated from artemisinin as a Chinese herb and has been reported to have anti-cancer potential in many cancer cells. In the present study, we examined the efficacy of artesunate against the renal carcinoma Caki cells and explored its mechanism of cytotoxicity. A steep decline in cell viability within 18 h was recorded upon artesunate exposure, but pretreatment of z-VAD-FMK had no effect on the loss of the cell viability by artesunate. On the other hand, necrostatin-1 pretreatment and knockdown of RIP-1 significantly reduced the cytotoxicity of artesunate against Caki cell. Moreover, the generation of mitochondrial ROS prompted by artesunate was found to be the principle mechanism of cell death. Pretreatment with necrostatin-1 or knockdown of RIP-1 inhibited the generation of ROS by artesunate, resulting in the protection of the cells from artesunate toxicity. Moreover, the similar results were observed in the case of other renal carcinoma cell lines (ACHN and A498). The results suggest that artesunate induces the generation of ROS and cell death in RIP1-dependent manner. Therefore, our data suggest that artesunate could induce RIP1-dependent cell death in human renal carcinoma.     

Stress response, cell death and signalling: the many faces of reactive oxygen species

Plants respond to pathogens and abiotic stresses by transient increases in the production of reactive oxygen species (ROS) and ion fluxes, which activate both local programmed cell death and systemic increases in stress- and pathogen-resistance. The present essay explores the emerging complexity of the multiple roles that ROS play in intra- and intercellular communication in both stressed and unstressed organisms.     

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.     

A novel protein Jpk induces bacterial cell death through reactive oxygen species

Jpk, a trans-acting regulatory factor associating with the position-specific regulatory element of Hoxa-7, has been reported to induce cell death in both prokaryotic and eukaryotic cells upon overexpression. The N- and C-terminal deleted variants of Jpk were constructed and then the toxicity of each construct was analyzed by checking the viability of the cells and the concomitant morphological changes through electron microscopy following the expression. The N-terminus of Jpk harboring transmembrane domain seemed to be more toxic to bacterial cell than C-terminus and the morphology of bacterial cells expressing N-terminal Jpk was similar to that induced by full length Jpk. The toxicity caused by Jpk protein in bacterial cell was through the production of ROS, which was decreased by an antioxidant (DTT) in a concentration dependent manner. The finding described in this study provides valuable clues on the relationship between Jpk toxicity and ROS generation.     

Down-regulated reactive oxygen species by HSP90 in 3HK-induced SKN-SH cell death

In this present study, we show that 3HK induced reactive oxygen species (ROS) accumulation and after caspase activation lead to apoptotic cell death. Pretreatment with N-acetylcysteine (NAC), an effective antioxidant, significantly attenuated 3HK-induced apoptosis by way of a reduction of ROS accumulation and caspase activity. SKN-SN cells were protected from 3HK-induced cytotoxicity by heat shock protein (HSP). HSP effectively attenuated 3HK-mediated ROS accumulation and apoptosis. In addition, the protective effect of HSP90 was abolished by pretreatment with HSP90 anti-sense oligonucleotides, but not when pretreated with anti-senses for other HSPs. These results suggest that HSP90 protects SKN-SH cells from 3HK-induced cytotoxicity by reducing ROS levels and caspase activity.