Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells

Exposure of animals to hyperoxia results in respiratory failure and death within 72 h. Histologic evaluation of the lungs of these animals demonstrates epithelial apoptosis and necrosis. Although the generation of reactive oxygen species (ROS) is widely thought to be responsible for the cell death observed following exposure to hyperoxia, it is not clear whether they act upstream of activation of the cell death pathway or whether they are generated as a result of mitochondrial membrane permeabilization and caspase activation. We hypothesized that the generation of ROS was required for hyperoxia-induced cell death upstream of Bax activation. In primary rat alveolar epithelial cells, we found that exposure to hyperoxia resulted in the generation of ROS that was completely prevented by the administration of the combined superoxide dismutase/catalase mimetic EUK-134 (Eukarion, Inc., Bedford, MA). Exposure to hyperoxia resulted in the activation of Bax at the mitochondrial membrane, cytochrome c release, and cell death. The administration of EUK-134 prevented Bax activation, cytochrome c release, and cell death. In a mouse lung epithelial cell line (MLE-12), the overexpression of Bcl-XL protected cells against hyperoxia by preventing the activation of Bax at the mitochondrial membrane. We conclude that exposure to hyperoxia results in Bax activation at the mitochondrial membrane and subsequent cytochrome c release. Bax activation at the mitochondrial membrane requires the generation of ROS and can be prevented by the overexpression of Bcl-XL.     

Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation

Hyperoxia causes cell injury and death associated with reactive oxygen species formation and inflammatory responses. Recent studies show that hyperoxia-induced cell death involves apoptosis, necrosis, or mixed phenotypes depending on cell type, although the underlying mechanisms remain unclear. Using murine lung endothelial cells, we found that hyperoxia caused cell death by apoptosis involving both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) pathways. Hyperoxia-dependent activation of the extrinsic apoptosis pathway and formation of the death-inducing signaling complex required NADPH oxidase-dependent reactive oxygen species production, because this process was attenuated by chemical inhibition, as well as by genetic deletion of the p47(phox) subunit, of the oxidase. Overexpression of heme oxygenase-1 prevented hyperoxia-induced cell death and cytochrome c release. Likewise, carbon monoxide, at low concentrations, markedly inhibited hyperoxia-induced endothelial cell death by inhibiting cytochrome c release and caspase-9/3 activation. Carbon monoxide, by attenuating hyperoxia-induced reactive oxygen species production, inhibited extrinsic apoptosis signaling initiated by death-inducing signal complex trafficking from the Golgi apparatus to the plasma membrane and downstream activation of caspase-8. We also found that carbon monoxide inhibited the hyperoxia-induced activation of Bcl-2-related proteins involved in both intrinsic and extrinsic apoptotic signaling. Carbon monoxide inhibited the activation of Bid and the expression and mitochondrial translocation of Bax, whereas promoted Bcl-X(L)/Bax interaction and increased Bad phosphorylation. We also show that carbon monoxide promoted an interaction of heme oxygenase-1 with Bax. These results define novel mechanisms underlying the antiapoptotic effects of carbon monoxide during hyperoxic stress.     

Bcl-2 protects against hyperoxia-induced apoptosis through inhibition of the mitochondria-dependent pathway

Bcl-2 is an antiapoptotic molecule that prevents oxidative stress damage and cell death. We investigated the possible protective mechanisms mediated by Bcl-2 during hyperoxia-induced cell death in L929 cells. In these cells, hyperoxia promoted apoptosis without DNA fragmentation. Overexpression of Bcl-2 significantly protected cells from oxygen-induced apoptosis, as shown by measurement of lactate dehydrogenase release, quantification of apoptotic nuclei, and detection of Annexin-V-positive cells. Bcl-2 partially prevented mitochondrial damage and interfered with the mitochondrial proapoptotic signaling pathway: it reduced Bax translocation to mitochondria, decreased the release of cytochrome c, and inhibited caspase 3 activation. However, treatment with the caspase inhibitor Z-VAD.fmk failed to rescue the cells from death, indicating that protection provided by Bcl-2 was due not only to caspase inhibition. Bcl-2 also prevented the release of mitochondrial apoptotic inducing factor, a mediator of caspase-independent apoptosis, correlating with the absence of oligonucleosomal DNA fragmentation. In addition, Bcl-2-overexpressing cells showed significantly higher intracellular amounts of glutathione after 72 h of oxygen exposure. In conclusion, our results demonstrate that the overexpression of Bcl-2 is able to prevent hyperoxia-induced cell death, by affecting mitochondria-dependent apoptotic pathways and increasing intracellular antioxidant compounds.     

Bcl-2 Family Members and Functional Electron Transport Chain Regulate Oxygen Deprivation-Induced Cell Death

The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-X(L) prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-X(L) to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F(1)F(0)-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-X(L), cells devoid of mitochondrial DNA (rho degrees cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax(-/-) bak(-/-) mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation.     

Apaf-1 deficiency confers resistance to ultraviolet-induced apoptosis in mouse embryonic fibroblasts by disrupting reactive oxygen species amplification production and mitochondrial pathway

Apoptosis requires tightly regulated cell death pathways. The signaling pathways that trigger a cell to undergo apoptosis after UV radiation are cell type specific and are currently being defined. Here, we have used pharmacological and genetic tools to demonstrate the decisive part of the mitochondrial pathway in UVC-induced apoptosis in mouse embryo fibroblasts (MEFs). UVC-induced apoptosis proceeded independent of the activation of death receptor components. In contrast, soon after UV radiation, MAPK activation and generation of reactive oxygen species (ROS) increased, followed by a decline in mitochondrial membrane potential (MMP) and cytochrome c release, as well as activation of caspase-9 and -3 and the upregulation of p47-phox. Deficiency of apaf-1, a critical member of the apoptosome, dramatically abolished all the UV-induced signal deterioration and cell death. In parallel, UVC-induced apoptosis was largely attenuated by either DN-caspase-9 or Bcl-X(L) overexpression. Pretreatment of cells with N-acetylcysteine or catalase but not Tempol decreased UVC-induced MAPK activation and apoptosis. Inhibition of JNK and caspase attenuated p47-phox upregulation. Altogether, we have for the first time demonstrated the critical role of Apaf-1 in the regulation of MAPK, ROS, and MMP in UVC-radiated MEFs and propose that the amplification feedback loop among mitochondrial signal molecules culminates in the demise of the cell.     

FLIP inhibits endothelial cell apoptosis during hyperoxia by suppressing Bax

High oxygen tension (hyperoxia) causes pulmonary cell death, involving apoptosis, necrosis, or mixed death phenotypes, though the underlying mechanisms remain unclear. In mouse lung endothelial cells (MLEC) hyperoxia activates both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) apoptotic pathways. We examined the hypothesis that FLIP, an inhibitor of caspase-8, can protect endothelial cells against the lethal effects of hyperoxia. Hyperoxia caused the time-dependent downregulation of FLIP in MLEC. Overexpression of FLIP attenuated intracellular reactive oxygen species generation during hyperoxia exposure, by downregulating extracellular-regulated kinase-1/2 activation and p47(phox) expression. FLIP prevented hyperoxia-induced trafficking of the death-inducing signal complex from the Golgi apparatus to the plasma membrane. Furthermore, FLIP blocked the activations of caspase-8/Bid, caspases -3/-9, and inhibited the mitochondrial translocation and activation of Bax, resulting in protection against hyperoxia-induced cell death. Under normoxic conditions, FLIP expression increased the phosphorylation of p38 mitogen-activated protein kinase leading to increased phosphorylation of Bax during hyperoxic stress. Furthermore, FLIP expression markedly inhibited protein kinase C activation and expression of distinct protein kinase C isoforms (alpha, eta, and zeta), and stabilized an interaction of PKC with Bax. In conclusion, FLIP exerted novel inhibitory effects on extrinsic and intrinsic apoptotic pathways, which significantly protected endothelial cells from the lethal effects of hyperoxia.     

Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt pathways in lung epithelial cells

Oxygen toxicity is one of the major risk factors in the development of the chronic lung disease or bronchopulmonary dysplasia in premature infants. Using proteomic analysis, we discovered that mitochondrial aldehyde dehydrogenase (mtALDH or ALDH2) was downregulated in neonatal rat lung after hyperoxic exposure. To study the role of mtALDH in hyperoxic lung injury, we overexpressed mtALDH in human lung epithelial cells (A549) and found that mtALDH significantly reduced hyperoxia-induced cell death. Compared with control cells (Neo-A549), the necrotic cell death in mtALDH-overexpressing cells (mtALDH-A549) decreased from 25.3 to 6.5%, 50.5 to 9.1%, and 52.4 to 15.1% after 24-, 48-, and 72-h hyperoxic exposure, respectively. The levels of intracellular and mitochondria-derived reactive oxygen species (ROS) in mtALDH-A549 cells after hyperoxic exposure were significantly lowered compared with Neo-A549 cells. mtALDH overexpression significantly stimulated extracellular signal-regulated kinase (ERK) phosphorylation under normoxic and hyperoxic conditions. Inhibition of ERK phosphorylation partially eliminated the protective effect of mtALDH in hyperoxia-induced cell death, suggesting ERK activation by mtALDH conferred cellular resistance to hyperoxia. mtALDH overexpression augmented Akt phosphorylation and maintained the total Akt level in mtALDH-A549 cells under normoxic and hyperoxic conditions. Inhibition of phosphatidylinositol 3-kinase (PI3K) activation by LY294002 in mtALDH-A549 cells significantly increased necrotic cell death after hyperoxic exposure, indicating that PI3K-Akt activation by mtALDH played an important role in cell survival after hyperoxia. Taken together, these data demonstrate that mtALDH overexpression attenuates hyperoxia-induced cell death in lung epithelial cells through reduction of ROS, activation of ERK/MAPK, and PI3K-Akt cell survival signaling pathways.     

The intrinsic apoptotic pathway is required for lipopolysaccharide-induced lung endothelial cell death

LPS has been implicated in the pathogenesis of endothelial cell death associated with Gram-negative bacterial sepsis. The binding of LPS to the TLR-4 on the surface of endothelial cells initiates the formation of a death-inducing signaling complex at the cell surface. The subsequent signaling pathways that result in apoptotic cell death remain unclear and may differ among endothelial cells in different organs. We sought to determine whether LPS and cycloheximide-induced cell death in human lung microvascular endothelial cells (HmVECs) was dependent upon activation of the intrinsic apoptotic pathway and the generation of reactive oxygen species. We found that cells overexpressing the anti-apoptotic protein Bcl-X(L) were resistant to LPS and cycloheximide-induced death and that the proapoptotic Bcl-2 protein Bid was cleaved following treatment with LPS. The importance of Bid was confirmed by protection of Bid-deficient (bid(-/-)) mice from LPS-induced lung injury. Neither HmVECs treated with the combined superoxide dismutase/catalase mimetic EUK-134 nor HmVECs depleted of mitochondrial DNA (rho(0) cells) were protected against LPS and cycloheximide-induced death. We conclude that LPS and cycloheximide-induced death in HmVECs requires the intrinsic cell death pathway, but not the generation of reactive oxygen species.     

Normal function of HERG K+ channels expressed in HEK293 cells requires basal protein kinase B activity

In this study, we show that ultraviolet B radiation (UVB)-induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl-2 inhibited UVB-induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)-induced cell death. It also prevented the subsequent activation of procaspase-3 and -8 as well as Bid cleavage in UVB-treated cells. Comparative analysis of UVB and FasL death pathways revealed a differential role and mechanism of caspase activation, with the UVB-induced activation of procaspase-8 only being a bystander cytosolic event rather than a major initiator mechanism, as is the case for the FasL-induced cell death. Our results suggest that Bcl-2 overexpression, by preventing reactive oxygen species production, helps indirectly to maintain the integrity of lysosomal membranes, and therefore inhibits the release of cathepsins, which contribute to the cytosolic activation of procaspase-8 in UVB-irradiated keratinocytes.     

Reactive oxygen and nitrogen species induce cell apoptosis via a mitochondria-dependent pathway in hyperoxia lung injury

Hyperoxia-induced lung injury limits the application of mechanical ventilation on rescuing the lives of premature infants and seriously ill and respiratory failure patients, and its mechanisms are not completely understood. In this article, we focused on the relationship between hyperoxia-induced lung injury and reactive oxygen species (ROS), reactive nitrogen species (RNS), mitochondria damage, as well as apoptosis in the pulmonary epithelial II cell line RLE-6TN. After exposure to hyperoxia, the cell viability was significantly decreased, accompanied by the increase in ROS, nitric oxide (NO), inflammatory cytokines, and cell death. Furthermore, hyperoxia triggered the loss of mitochondrial membrane potential (▵Ψm), thereby promoting cytochrome c to release from mitochondria to cytoplasm. Further studies conclusively showed that the Bax/Bcl-2 ratio was enlarged to activate the mitochondria-dependent apoptotic pathway after hyperoxia treatment. Intriguingly, the effects of hyperoxia on the level of ROS, NO and inflammation, mitochondrial damage, as well as cell death were reversed by free radical scavengers N-acetylcysteine and hemoglobin. In addition, a hyperoxia model of neonatal Sprague-Dawley (SD) rats presented the obvious characteristics of lung injury, such as a decrease in alveolar numbers, alveolar mass edema, and disorganized pulmonary structure. The effects of hyperoxia on ROS, RNS, inflammatory cytokines, and apoptosis-related proteins in lung injury tissues of neonatal SD rats were similar to that in RLE-6TN cells. In conclusion, mitochondria are a primary target of hyperoxia-induced free radical, whereas ROS and RNS are the key mediators of hyperoxia-induced cell apoptosis via the mitochondria-dependent pathway in RLE-6TN cells.     

Destabilization of CARP mRNAs by aloe-emodin contributes to caspase-8-mediated p53-independent apoptosis of human carcinoma cells

Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.     

Loss of Mcl-1 protein and inhibition of electron transport chain together induce anoxic cell death

How cells die in the absence of oxygen (anoxia) is not understood. Here we report that cells deficient in Bax and Bak or caspase-9 do not undergo anoxia-induced cell death. However, the caspase-9 null cells do not survive reoxygenation due to the generation of mitochondrial reactive oxygen species. The individual loss of Bim, Bid, Puma, Noxa, Bad, caspase-2, or hypoxia-inducible factor 1beta, which are potential upstream regulators of Bax or Bak, did not prevent anoxia-induced cell death. Anoxia triggered the loss of the Mcl-1 protein upstream of Bax/Bak activation. Cells containing a mitochondrial DNA cytochrome b 4-base-pair deletion ([rho(-)] cells) and cells depleted of their entire mitochondrial DNA ([rho(0)] cells) are oxidative phosphorylation incompetent and displayed loss of the Mcl-1 protein under anoxia. [rho(0)] cells, in contrast to [rho(-)] cells, did not die under anoxia. However, [rho(0)] cells did undergo cell death in the presence of the Bad BH3 peptide, an inhibitor of Bcl-X(L)/Bcl-2 proteins. These results indicate that [rho(0)] cells survive under anoxia despite the loss of Mcl-1 protein due to residual prosurvival activity of the Bcl-X(L)/Bcl-2 proteins. Collectively, these results demonstrate that anoxia-induced cell death requires the loss of Mcl-1 protein and inhibition of the electron transport chain to negate Bcl-X(L)/Bcl-2 proteins.     

Oxidative stress-mediated apoptosis. The anticancer effect of the sesquiterpene lactone parthenolide

The sesquiterpene lactone parthenolide, the principal active component in medicinal plants, has been used conventionally to treat migraines, inflammation, and tumors. However, the antitumor effects of parthenolide and the mechanism(s) involved are poorly understood. We found that parthenolide effectively inhibits hepatoma cell growth in a tumor cell-specific manner and triggers apoptosis of hepatoma cells. Parthenolide triggered apoptosis in invasive sarcomatoid hepatocellular carcinoma cells (SH-J1) as well as in other ordinary hepatoma cells at 5-10 microm concentrations and arrested the cell growth (at G(2)/M) at sublethal concentrations (1-3 microm). During parthenolide-induced apoptosis, depletion of glutathione, generation of reactive oxygen species, reduction of mitochondrial transmembrane potential, activation of caspases (caspases-7, -8, and -9), overexpression of GADD153 (an oxidative stress or anticancer agent inducible gene), and subsequent apoptotic cell death was observed. This induced apoptosis could be effectively inhibited or abrogated by an antioxidant N-acetyl-l-cysteine, whereas l-buthionine-(S,R)-sulfoximine enhanced it. Furthermore, stable overexpression of GADD153 sensitized the cells to apoptosis induced by parthenolide, and this susceptibility could be reversed by transfection with an antisense to GADD153. Parthenolide did not alter the expression of Bcl-2 or Bcl-X(L) proteins during apoptosis in hepatoma cells. Oxidative stress may contribute to parthenolide-induced apoptosis and to GADD153 overexpression in a glutathione-sensitive manner. The sensitivity of tumor cells to parthenolide appears to result from the low expression level of the multifunctional detoxification enzyme glutathione S-transferase pi. Finally, parthenolide and its derivatives may be useful chemotherapeutic agents to treat these invasive cancers.     

Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis

Fluorescent protein based signaling probes are emerging as valuable tools to study cell signaling because of their ability to provide spatio- temporal information in non invasive live cell mode. Previously, multiple fluorescent protein probes were employed to characterize key events of apoptosis in diverse experimental systems. We have employed a live cell image based approach to visualize the key events of apoptosis signaling induced by zerumbone, the active principle from ginger Zingiber zerumbet, in cancer cells that enabled us to analyze prominent apoptotic changes in a hierarchical manner with temporal resolution. Our studies substantiate that mitochondrial permeabilisation and cytochrome c dependent caspase activation dominate in zerumbone induced cell death. Bax activation, the essential and early event of cell death, is independently activated by reactive oxygen species as well as calpains. Zerumbone failed to induce apoptosis or mitochondrial permeabilisation in Bax knockout cells and over-expression of Bax enhanced cell death induced by zerumbone confirming the essential role of Bax for mitochondrial permeabilsation. Simultaneous inhibition of reactive oxygen species and calpain is required for preventing Bax activation and cell death. However, apoptosis induced by zerumbone was prevented in Bcl 2 and Bcl-XL over-expressing cells, whereas more protection was afforded by Bcl 2 specifically targeted to endoplasmic reticulum. Even though zerumbone treatment down-regulated survival proteins such as XIAP, Survivin and Akt, it failed to affect the pro-apoptotic proteins such as PUMA and BIM. Multiple normal diploid cell lines were employed to address cytotoxic activity of zerumbone and, in general, mammary epithelial cells, endothelial progenitor cells and smooth muscle cells were relatively resistant to zerumbone induced cell death with lesser ROS accumulation than cancer cells.     

The role of Bcl-X(S) in radiation sensitivity

Bcl-X(S) is a pro-apoptosis member of the Bcl2 family that has been shown to induce cell death and enhance chemosensitivity. We have investigated the effect of Bcl-X(S) overexpression on radiation sensitivity. Using a tetracycline-repressible system, we found that removal of tetracycline for 16 h induced Bcl-X(S) and reduced the surviving fraction of NIH 3T3 cells to 25%. However, radiation sensitivity was not significantly affected by Bcl-X(S) expression; the mean inactivation doses for Bcl-X(S) repressed and Bcl-X(S) induced cells were 2.7 +/- 0.3 and 2.3 +/- 0.1 Gy, respectively. We conclude that Bcl-X(S) induces cell death without affecting radiation sensitivity. These results suggest that mitochondrial pathways to apoptosis may not have a significant role in survival after irradiation.     

Intersectin-1s regulates the mitochondrial apoptotic pathway in endothelial cells

Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.     

Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.     

Regulation of ionizing radiation-induced apoptosis by a manganese porphyrin complex

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Therefore, compounds that scavenge reactive oxygen species may confer regulatory effects on apoptosis. Superoxide dismutase (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. We investigated the effects of the manganese (III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP), a cell-permeable SOD mimetic, on ionizing radiation-induced apoptosis. Upon exposure to 2 Gy of gamma-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 5 microM MnTMPyP for 2 h with regard to apoptotic parameters, cellular redox status, mitochondria function, and oxidative damage to cells. MnTMPyP effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular reactive oxygen species were higher and the [NADPH]/[NADP(+)+NADPH] ratio was lower in control cells compared to MnTMPyP-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of reactive oxygen species, and the reduction of ATP production were significantly higher in control cells compared to MnTMPyP-treated cells. MnTMPyP pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that MnTMPyP may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of reactive oxygen species.     

Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression

Pancreatic β cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in β cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic β cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.     

Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins

Mitochondria are dynamic organelles that undergo continual fusion and fission to maintain their morphology and functions, but the mechanism involved is still not clear. Here, we investigated the effect of mitochondrial oxidative stress triggered by high-fluence low-power laser irradiation (HF-LPLI) on mitochondrial dynamics in human lung adenocarcinoma cells (ASTC-a-1) and African green monkey SV40-transformed kidney fibroblast cells (COS-7). Upon HF-LPLI-triggered oxidative stress, mitochondria displayed a fragmented structure, which was abolished by exposure to dehydroascorbic acid, a reactive oxygen species scavenger, indicating that oxidative stress can induce mitochondrial fragmentation. Further study revealed that HF-LPLI caused mitochondrial fragmentation by inhibiting fusion and enhancing fission. Mitochondrial translocation of the profission protein dynamin-related protein 1 (Drp1) was observed following HF-LPLI, demonstrating apoptosis-related activation of Drp1. Notably, overexpression of Drp1 increased mitochondrial fragmentation and promoted HF-LPLI-induced apoptosis through promoting cytochrome c release and caspase-9 activation, whereas overexpression of mitofusin 2 (Mfn2), a profusion protein, caused the opposite effects. Also, neither Drp1 overexpression nor Mfn2 overexpression affected mitochondrial reactive oxygen species generation, mitochondrial depolarization, or Bax activation. We conclude that mitochondrial oxidative stress mediated through Drp1 and Mfn2 causes an imbalance in mitochondrial fission-fusion, resulting in mitochondrial fragmentation, which contributes to mitochondrial and cell dysfunction.