Fas-stimulated generation of reactive oxygen species or exogenous oxidative stress sensitize cells to Fas-mediated apoptosis

Inhibition of Fas-mediated apoptosis in B cell lymphomas by thiol antioxidants (glutathione and N-acetylcysteine) supported previous studies, suggesting that Fas-stimulated ROS generation may play a role in Fas-mediated apoptosis. Thus, the goal of the current study was to determine if Fas stimulation could induce ROS generation and what role, if any, it played in apoptosis. Fas crosslinking induced rapid generation of ROS (within 15 min) well before the appearance of characteristic apoptotic changes. Overexpression of catalase or superoxide dismutase suggested that Fas induced production of both superoxide anion and hydrogen peroxide. ROS generation was only observed, however, in cells that were sensitive to apoptosis and not in B cells inherently resistant to anti-Fas or in those in which resistance was induced by B cell receptor crosslinking. The exogenous addition of 250 microM hydrogen peroxide could reverse the resistant phenotype and sensitize cells to Fas-induced apoptosis. In Fas-sensitive cells, depletion of endogenous antioxidant defenses with buthionine sulfoximine increased the sensitivity to Fas-induced apoptosis, while overexpression of antioxidant enzymes and antiapoptotic proteins suggested a role for Fas-induced production of hydrogen peroxide in apoptosis. Further analysis suggested a redox-sensitive step early in Fas signaling at the level of initiator caspase (caspase-8) activation. Thus, the data suggest that the level of oxidative stress, either from exogenous sources or generated endogenously upon receptor stimulation, regulates the sensitivity to Fas-mediated apoptosis.     

Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis

During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.     

Analysis of mitochondrial generation and release of reactive oxygen species.

BACKGROUND: Reactive oxygen species (ROS) are mainly produced in mitochondria and are important contributors to many forms of cell death. ROS also function as second messengers within the cell and may constitute a signaling pathway from mitochondria to the cytoplasm and nucleus. The aim of the present study was to develop a protocol to detect changes in intra- and extramitochondrial releases of ROS, which could be used to analyze the role of mitochondria in cell signaling and cell death. METHODS: Fluorescence-based assays were used to measure (a) total production of ROS, (b) intramitochondrial ROS, (c) extramitochondrial hydrogen peroxide, and (d) superoxide outside inverted (inside-out) submitochondrial particles. ROS generation in the samples was increased or decreased by the addition of different substrates, enzymes, and inhibitors of the electron transport chain. RESULTS: The individual assays used were sensitive to increased (e.g., after addition of antimycin A; increased signal) and decreased (ROS scavenging; decreased signal) levels of ROS. In combination, the assays provided information about mitochondrial ROS generation and release dynamics from small samples of isolated mitochondria. CONCLUSIONS: The combination of fluorescent techniques described is a useful tool to study the role of ROS in cell death and in cellular redox signaling.     

Caudatin induces caspase-dependent apoptosis in human glioma cells with involvement of mitochondrial dysfunction and reactive oxygen species generation

Caudatin as one species of C-21 steroidal from Cynanchum bungei decne displays potential anticancer activity. However, the underlying mechanisms remain elusive. In the present study, the growth suppressive effect and mechanism of caudatin on human glioma U251 and U87 cells were evaluated in vitro. The results indicated that caudatin significantly inhibited U251 and U87 cell growth in both a time- and dose-dependent manner. Flow cytometry analysis revealed that caudatin-induced cell growth inhibition was achieved by induction of cell apoptosis, as convinced by the increase of Sub-G1 peak, PARP cleavage and activation of caspase-3, caspase-7 and caspase-9. Caudatin treatment also resulted in mitochondrial dysfunction which correlated with an imbalance of Bcl-2 family members. Further investigation revealed that caudatin triggered U251 cell apoptosis by inducing reactive oxygen species (ROS) generation through disturbing the redox homeostasis. Moreover, pretreatment of caspase inhibitors apparently weakens caudatin-induced cell killing, PARP cleavage and caspase activation and eventually reverses caudatin-mediated apoptosis. Importantly, caudatin significantly inhibited U251 tumour xenografts in vivo through induction of cell apoptosis involving the inhibition of cell proliferation and angiogenesis, which further validate its value in combating human glioma in vivo. Taken together, the results described above all suggest that caudatin inhibited human glioma cell growth by induction of caspase-dependent apoptosis with involvement of mitochondrial dysfunction and ROS generation.     

Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion.

Mutations of mitochondrial (mt) DNA accumulate during normal aging. The most frequent mutation is a 4,977-base pair deletion also called the common deletion, which is increased in photoaged skin. Oxidative stress may play a major role in the generation of large scale mtDNA deletions, but direct proof for this has been elusive. We therefore assessed whether the common deletion can be generated in vitro through UV irradiation and whether reactive oxygen species are involved in this process. Normal human fibroblasts were repetitively exposed to sublethal doses of UVA radiation and assayed for the common deletion employing a semiquantitative polymerase chain reaction technique. There was a time/dose-dependent generation of the common deletion, attributable to the generation of singlet oxygen, since the common deletion was diminished when irradiating in the presence of singlet oxygen quenchers, but increased when enhancing singlet oxygen half-life by deuterium oxide. The induction of the common deletion by UVA irradiation was mimicked by treatment of unirradiated cells with singlet oxygen produced by the thermodecomposition of an endoperoxide. These studies provide evidence for the involvement of reactive oxygen species in the generation of aging-associated mtDNA lesions in human cells and indicate a previously unrecognized role of singlet oxygen in photoaging of human skin.     

The role of external and matrix pH in mitochondrial reactive oxygen species generation

Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.     

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H₂O₂ release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (α-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1–0.2% of O₂ consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release.     

Reactive oxygen species and oxidative stress

Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity of the cell. For long, ROS have been considered as harmful by-products of the normal aerobic metabolism process of the mitochondria, implicated in a large variety of diseases. But there are now growing evidences that controlled ROS production also play physiological roles especially in regulating cell redox homeostasis and cell signaling. Biological ROS effects are now well documented. Data show that living organisms have not only adapted themselves to coexist with free radicals but have also developed mechanisms to use them advantageously. However their main sources and mechanisms of action remain poorly described. This review focuses on the main properties of ROS and their paradoxical effects.     

Induction of apoptosis by chemotherapeutic drugs without generation of reactive oxygen species.

Studies in a variety of cell types have suggested that cancer chemotherapy drugs induce tumor cell apoptosis in part by inducing formation of reactive oxygen species (ROS). Using human B lymphoma cells as the targets, we have found that apoptosis can be induced in the absence of any detectable oxidative stress. Apoptosis was induced with the chemotherapy drugs VP-16 and cisplatin. To determine whether oxidants are formed as part of the drug-induced apoptotic process, intracellular markers of oxidative stress were examined. These included measurement of (1) protein carbonyl groups by Western blot immunoassay, (2) protein methionine sulfoxide residues by amino acid analysis, (3) protein sulfhydryl oxidation by Western blot immunoassay, (4) F2-isoprostanes by GC/MS, and (5) intracellular ROS production using the oxidant-sensitive dyes DCFDA and dihydrorhodamine 123. Apoptosis was quantified using fluorescence microscopy to assess nuclear morphology. The results show that VP-16 and cisplatin induce extensive apoptosis in the absence of any detectable protein or lipid oxidation, measured in both the cytosolic and mitochondrial compartments of the cell. In contrast, H2O2, which kills the cells by nonapoptotic pathways, caused increases in both protein and lipid oxidation. Three different antioxidant compounds (N-acetyl cysteine, Tempol, and MnTBAP) failed to inhibit VP-16-induced apoptosis, while inhibiting H2O2-induced cell death. Only N-acetyl cysteine inhibited cisplatin-induced cell death and this is attributed to its known ability to react directly with and inactivate cisplatin before it enters the cell. The results demonstrate that, at least in B lymphoma cells, chemotherapy-induced apoptosis occurs using a mechanism that does not involve oxidants.     

Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.     

Production of reactive oxygen species in mitochondria of HeLa cells under oxidative stress

Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed ("dark") accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.     

Role of mitochondrial thiols of different localization in the generation of reactive oxygen species

The role of thiols of the outer and the inner membranes of mitochondria in the regulation of generation of reactive oxygen species (ROS) has been studied. It was found that N-ethylmaleimide (NEM), which penetrates through the mitochondrial membrane and binds thiols to form thioesters, at concentrations from 20 to 250 μM activates the production of superoxide anion and hydrogen peroxide during the oxidation of the substrates of complexes I and II of the respiratory chain. 5′,5′-Dithiobis-(2-nitrobenzoate) (DTNB), which does not penetrate into mitochondria and binds thiols to form disulfides, weakly activates hydrogen peroxide production during the oxidation of NAD-dependent substrates and inhibits the ROS production upon succinate oxidation. DTNB is particularly effective in inhibiting the menadione-induced formation of ROS. The differences in the ROS formation by these reagents are explained by the fact that they influence different thiol-containing proteins and enzymes. As distinct from NEM, which inhibits complex I of the respiratory chain, DTNB has no effect on the respiratory chain of mitochondria but can bind the SH-groups of NADH-quinone oxidoreductase, which is localized in the outer mitochondrial membrane and participates in the redox cycle of menadione. It was also shown that the ability to inhibit the ADP-stimulated respiration, a feature inherent in both reagents, does not significantly contribute to ROS production.     

Phosphate increases mitochondrial reactive oxygen species release

The effects of inorganic phosphate (P_i), the main intracellular membrane permeable anion capable of altering mitochondrial pH gradients (ΔpH), were measured on mitochondrial H₂O₂ release. As expected, P_i decreased ΔpH and increased the electric membrane potential (ΔΨ). Mitochondrial H₂O₂ release was stimulated by P_i and also by its structural analogue arsenate. However, acetate, another membrane-permeable anion, did not stimulate mitochondrial H₂O₂ release. The stimulatory effect promoted by P_i was prevented by CCCP, which decreases transport of P_i across the inner mitochondrial membrane, indicating that P_i must be in the mitochondrial matrix to stimulate H₂O₂ release. In conclusion, we found that P_i and arsenate stimulate mitochondrial reactive oxygen release, an effect that may contribute towards oxidative stress under conditions such as ischemia/reperfusion, in which high-energy phosphate bonds are hydrolyzed.     

BCI induces apoptosis via generation of reactive oxygen species and activation of intrinsic mitochondrial pathway in H1299 lung cancer cells

The compound(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1 H-inden-1-one(BCI) is known as an inhibitor of dual specific phosphatase 1/6 and mitogen-activated protein kinase. However, its precise anti-lung cancer mechanism remains unknown. In this study, the effects of BCI on the viability of non-small cell lung cancer cell lines NCI-H1299, A549, and NCI-H460 were evaluated. We confirmed that BCI significantly inhibited the viability of p53(-) NCI-H1299 cells as compared to NCI-H460 and A549 cells, which express wild-type p53. Furthermore, BCI treatment increased the level of cellular reactive oxygen species and pre-treatment of cells with N-acetylcysteine markedly attenuated BCI-mediated apoptosis of NCI-H1299 cells. BCI induced cellular morphological changes, inhibited viability, and produced reactive oxygen species in NCI-H1299 cells in a dose-dependent manner. BCI induced processing of caspase-9, caspase-3, and poly ADP-ribose polymerase as well as the release of cytochrome c from the mitochondria into the cytosol. In addition, BCI downregulated Bcl-2 expression and enhanced Bax expression in a dose-dependent manner in NCI-H1299 cells. However, BCI failed to modulate the expression of the death receptor and extrinsic factor caspase-8 and Bid, a linker between the intrinsic and extrinsic apoptotic pathways in NCI-H1299 cells. Thus, BCI induces apoptosis via generation of reactive oxygen species and activation of the intrinsic pathway in NCI-H1299 cells.     

Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

Nogo-A is originally identified as an inhibitor of axon regeneration from the CNS myelin. Nogo-A is mainly expressed by oligodendrocytes, and also by some neuronal subpopulations, particularly in the developing nervous system. Although extensive studies have uncovered regulatory roles of Nogo-A in neurite outgrowth inhibition, precursor migration, neuronal homeostasis, plasticity and neurodegeneration, its cell-autonomous functions in neurons are largely uncharacterized. Here, we show that HIV-1 trans-activating-mediated amino-Nogo-A protein transduction into cultured primary cortical neurons achieves an almost complete neuroprotection against oxidative stress induced by exogenous hydrogen peroxide (H(2)O(2)). Endogenously expressed neuronal Nogo-A is significantly downregulated upon H(2)O(2) treatment. Furthermore, knockdown of Nogo-A results in more susceptibility to acute oxidative insults and markedly increases neuronal death. Interacting with peroxiredoxin 2 (Prdx2), amino-Nogo-A reduces reactive oxygen species (ROS) generation and extracellular signal-regulated kinase phosphorylation to exert neuroprotective effects. Structure-function mapping experiments reveal that, out of NiG-Δ20, a novel region comprising residues 290-562 of amino-Nogo-A is indispensable for preventing oxidative neuronal death. Moreover, mutagenesis analysis confirms that cysteine residues 424, 464 and 559 are involved in the inhibition of ROS generation and neuroprotective role of amino-Nogo-A. Our data suggest that neuronal Nogo-A might play a cell-autonomous role in improving neuronal survival against oxidative insult through interacting with Prdx2 and scavenging of ROS.     

Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling

Mitochondria are proposed to play an important role in hypoxic cell signaling. One currently accepted signaling paradigm is that the mitochondrial generation of reactive oxygen species (ROS) increases in hypoxia. This is paradoxical, because oxygen is a substrate for ROS generation. Although the response of isolated mitochondrial ROS generation to [O(2)] has been examined previously, such investigations did not apply rigorous control over [O(2)] within the hypoxic signaling range. With the use of open-flow respirometry and fluorimetry, the current study determined the response of isolated rat liver mitochondrial ROS generation to defined steady-state [O(2)] as low as 0.1 microM. In mitochondria respiring under state 4 (quiescent) or state 3 (ATP turnover) conditions, decreased ROS generation was always observed at low [O(2)]. It is concluded that the biochemical mechanism to facilitate increased ROS generation in response to hypoxia in cells is not intrinsic to the mitochondrial respiratory chain alone but may involve other factors. The implications for hypoxic cell signaling are discussed.