Translocation of Ethanolamine Phosphoglyceride is Required for Initiation of Apoptotic Death in OLN-93 Oligodendroglial Cells

The possible interplay between extracellular signal-regulated protein kinase (ERK) activation and ethanolamine phosphoglycerides (PG) membrane bilayer translocation following oxidative stress (OS) (0.5 mM H₂O₂/0.05 mM Fe²⁺), was examined in oligodendroglia, OLN93, cells with altered plasma membrane PG composition. Cells supplemented with 50 μM docosahexaenoic acid (DHA, 22:6n3) to increase the number of potential double bond targets for OS in ethanolamine-PG (EPG) were compared to cells with diminished content of EPG, attained by the addition of 0.5 mM N,N-dimethylethanolamine (dEa). After 30 min OS, EPG translocation accompanied by sustained ERK activation and nuclear translocation culminating in apoptosis was found in DHA-supplemented cells in contrast to no EPG translocation, a brief ERK activation, but no nuclear translocation, and no cell death in DHA/dEa-supplemented cells. DHA/dEa-supplemented cells pretreated with the protein-tyrosine phosphatases inhibitor Na₃VO₄ followed by OS, although expressing a sustained ERK activation and nuclear translocation, failed to show apoptosis and lacked EPG translocation. In DHA-supplemented cells U0126, a MEK inhibitor, prevented ERK activation and EPG translocation and protected from cell death. These findings most likely indicate that ERK activation is an indispensable component for the signaling cascades leading to EPG translocation but only activation of the latter is leading to OS-induced apoptotic cell death.     

N-methyl bases of ethanolamine prevent apoptotic cell death induced by oxidative stress in cells of oligodendroglia origin

A major reason for brain tissue vulnerability to oxidative damage is the high content of polyunsaturated fatty acids (PUFAs). Oligodendroglia-like OLN 93 cells lack PUFAs and are relatively insensitive to oxidative stress. When grown in serum-free defined medium in the presence of 0.1 mM docosahexaenoic acid (DHA; 22:6 n-3) for 3 days, OLN 93 cells release in the medium 2.6-fold more thiobarbituric acid-reactive substances (TBARS) after a 30-min exposure to 0.1 mM H2O2 and 50 microM Fe2+. Release of TBARS was substantially decreased by approximately 20 and 30% on coincubation with either 1 mM N-monomethylethanolamine or N,N'-dimethylethanolamine (dEa), respectively. The protective effect of dEa was concentration- and time-dependent and was still visible after dEa removal, suggesting a long-lasting mechanism of protection. After 24 h following H2O2-induced stress, cell death monitored by cell sorting showed 16% of the cells in the sub-G1 area, indicative of apoptotic cell death. DHA-supplemented cultures showed 35% cell death, whereas cosupplements with dEa reduced cell death to 12%, indicating cell rescue. Although the exact mechanism for this protection is not known, the nature of the polar head group and the degree of unsaturation may determine the ultimate resistance of nerve cells to oxidative stress.     

Early ethanolamine phospholipid translocation marks stress-induced apoptotic cell death in oligodendroglial cells

The consequences of H(2)O(2)/Fe(2+)-induced oxidative stress on translocation of ethanolamine phosphoglyceride (EPG) and serine phosphoglyceride (SPG) were studied in an oligodendroglia-like cell line (OLN 93) following 3 days of supplementation with 0.1 mM docosahexaenoic acid (DHA) and a series of polar head group precursors, including N-monomethyl- and N,N-dimethylethanolamine at millimolar concentrations. Added DHA was predominantly esterified in EPG species and those cells enriched in DHA showed enhanced sensitivity to oxidative stress and eventually died by apoptosis. Co-supplements with ethanolamine and DHA resulted in a rapid, but transient, EPG translocation with a maximum at 30 min following stress, as characterized by a trinitrobenzenesulfonic acid reagent. There was no significant translocation of SPG as evidenced by annexin V binding. Unlike SPG, which is usually irreversibly translocated to subserve as a tag for phagocytosis, EPG acted as a signaling molecule with biphasic kinetic characteristics. N-Monomethyl- and N,N-dimethylethanolamine supplements reduced EPG synthesis, prevented its externalization and rescued cells from apoptotic death. Following stress, the fatty acid profile of the externalized EPG showed marked losses in polyunsaturated fatty acids and aldehydes compared with the remaining intracellular EPG. Prevention of EPG species selective translocation to the outer membrane leaflet by altering phospholipid asymmetry may be important in the mechanism of rescue from cell death.     

Enhancement of catalase activity by repetitive low-grade H2O2 exposures protects fibroblasts from subsequent stress-induced apoptosis

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H2O2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m2) or H2O2 (7.5mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H2O2. Cell viability was also significantly better preserved in VST cells than VC cells after UV or H2O2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H2O2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.     

A role for PtdIns(4,5)P2 and PIP5Kalpha in regulating stress-induced apoptosis

The phosphoinositide phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) is essential for many cellular processes and is linked to the etiology of numerous human diseases . PtdIns(4,5)P(2) has been indirectly implicated as a negative regulator of apoptosis ; however, it is unclear if apoptotic stimuli negatively regulate PtdIns(4,5)P(2) levels in vivo. Here, we show that two apoptotic-stress stimuli, hydrogen peroxide (H(2)O(2)) and UV irradiation, cause PtdIns(4,5)P(2) depletion during programmed cell death independently of and prior to caspase activation. Depletion of PtdIns(4,5)P(2) is essential for apoptosis because maintenance of PtdIns(4,5)P(2) levels by overexpression of PIP5Kalpha rescues cells from H(2)O(2)-induced apoptosis. PIP5Kalpha expression promotes both basal and sustained ERK1/2 activation after H(2)O(2) treatment, and importantly, pharmacological inhibition of ERK1/2 signaling blocks PIP5Kalpha-mediated cell survival. H(2)O(2) induces tyrosine phosphorylation and translocation of PIP5Kalpha away from its substrate at the plasma membrane, and both are dependent upon the activity of c-src family kinases. Furthermore, constitutively active c-src enhances tyrosine phosphorylation of PIP5Kalpha in vivo and is sufficient for the translocation of PIP5Kalpha away from the plasma membrane. These observations demonstrate that certain apoptotic stimuli initiate an essential signaling pathway during cell death, and this pathway leads to caspase-independent downregulation of PIP5Kalpha and its product PtdIns(4,5)P(2).     

Mitochondrial extracellular signal-regulated kinases 1/2 (ERK1/2) are modulated during brain development

Intracellular activation and trafficking of extracellular signal-regulated protein kinases (ERK) play a significant role in cell cycle progression, contributing to developmental brain activities. Additionally, mitochondria participate in cell signalling through energy-linked functions, redox metabolism and activation of pro- or anti-apoptotic proteins. The purpose of the present study was to analyze the presence of ERK1/2 in mitochondria during rat brain development. Immunoblotting, immune electron microscopy and activity assays demonstrated that ERK1/2 are present in fully active brain mitochondria at the outer membrane/intermembrane space fraction. Besides, it was observed that ERK1/2 translocation to brain mitochondria follows a developmental pattern which is maximal between E19-P2 stages and afterwards declines at P3, just before maximal translocation to nucleus, and up to adulthood. Most of mitochondrial ERK1/2 were active; upstream phospho-MAPK/ERK kinases (MEK1/2) were also detected in the brain organelles. Mitochondrial phospho-ERK1/2 increased at 1 microm hydrogen peroxide (H(2)O(2)) concentration, but it decreased at higher 50-100 microm H(2)O(2), almost disappearing after the organelles were maximally stimulated to produce H(2)O(2) with antimycin. Our results suggest that developmental mitochondrial activation of ERK1/2 cascade contributes to its nuclear translocation effects, providing information about mitochondrial energetic and redox status to the proliferating/differentiating nuclear pathways.     

ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-alpha expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-alpha, which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-alpha blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.     

Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms

Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.     

HSP25 overexpression attenuates oxidative stress-induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase

HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2.     

Compensatory activation of ERK1/2 in Atg5-deficient mouse embryo fibroblasts suppresses oxidative stress-induced cell death

Despite of the increasing evidence that oxidative stress may induce non-apoptotic cell death or autophagic cell death, the mechanism of this process is unclear. Here, we report a role and a down-stream molecular event of Atg5 during oxidative stress-induced cell death. Compared to wild type (WT) cells, Atg5-deficient mouse embryo fibroblasts (Atg5-/- MEFs) and Atg5 knockdown HT22 neuronal cells were more resistant to cell death induced by H2O2. On the contrary, Atg5-/- MEFs were as sensitive to tumor necrosis factor (TNF)-alpha and cycloheximide as WT cells, and were more sensitive to cell death triggered by amino acid-deprivation than WT MEFs. Treatment with H2O2 induced the recruitment of a GFP-LC3 fusion protein and conversion of LC3 I to LC3 II, correlated with the extent of autophagosome formation in WT cells, but much less in Atg5-deficient cells. Among stress kinases, ERK1/2 was markedly activated in Atg5-/- MEFs and Atg5 knockdown HT22 and SH-SY5Y neuronal cells. The inhibition of ERK1/2 by MEK1 inhibitor (PD98059) or dominant negative ERK2 enhanced the susceptibility of Atg5-/- MEFs to H2O2-induced cell death. Further, reconstitution of Atg5 sensitized Atg5-/- MEFs to H2O2 and suppressed the activation of ERK1/2. These results suggest that the inhibitory effect of Atg5 deficiency on cell death is attributable by the compensatory activation of ERK1/2 in Atg5-/- MEFs during oxidative stress-induced cell death.     

c-Jun N-terminal kinase mediates hydrogen peroxide-induced cell death via sustained poly(ADP-ribose) polymerase-1 activation

Reactive oxygen species (ROS) have been closely associated with both apoptotic and non-apoptotic/necrotic cell death. Our previous study has illustrated that c-Jun-N-terminal kinase 1 (JNK1) is the main executor in hydrogen peroxide (H(2)O(2))-induced nonapoptotic cell death. The main objective of this study is to further elucidate the molecular mechanisms downstream of JNK1 in H(2)O(2)-induced cell death. In this study, poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair protein, was readily activated by H(2)O(2) and inhibition of PARP-1 activation by either a pharmacological or genetic approach offered significant protection against H(2)O(2)-induced cell death. More importantly, H(2)O(2)-mediated PARP-1 activation is subject to regulation by JNK1. Suppression of JNK1 activation by a chemical inhibitor or genetic deletion markedly suppressed the late-phase PARP-1 activation induced by H(2)O(2), suggesting that JNK1 contributes to the sustained activation of PARP-1. Such findings were supported by the temporal pattern of nuclear translocation of activated JNK and a direct protein-protein interaction between JNK1 and PARP-1 in H(2)O(2)-treated cells. Finally, in vitro kinase assay suggests that PARP-1 may serve as the direct phosphorylation target for JNK1. Taken together, data from our study reveal a novel underlying mechanism in H(2)O(2)-induced nonapoptotic cell death: JNK1 promotes a sustained PARP-1 activation via nuclear translocation, protein-protein interaction and PARP-1 phosphorylation.     

Role of TRPM2 in H(2)O(2)-Induced Cell Apoptosis in Endothelial Cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.     

β-lapachone significantly increases the effect of ionizing radiation to cause mitochondrial apoptosis via JNK activation in cancer cells

Background

β-lapachone (β-lap), has been known to cause NQO1-dependnet death in cancer cells and sensitize cancer cells to ionizing radiation (IR). We investigated the mechanisms underlying the radiosensitization caused by β-lap.

Methodology/Principal Findings

β-lap enhanced the effect of IR to cause clonogenic cells in NQO1⁺-MDA-MB-231 cells but not in NQO1⁻-MDA-MB-231 cells. β-lap caused apoptosis only in NQO1⁺ cells and not in NQO1⁻ cells and it markedly increased IR-induced apoptosis only in NQO1⁺ cells. Combined treatment of NQO1⁺ cells induced ROS generation, triggered ER stress and stimulated activation of ERK and JNK. Inhibition of ROS generation by NAC effectively attenuated the activation of ERK and JNK, induction of ER stress, and subsequent apoptosis. Importantly, inhibition of ERK abolished ROS generation and ER stress, whereas inhibition of JNK did not, indicating that positive feedback regulation between ERK activation and ROS generation triggers ER stress in response to combined treatment. Furthermore, prevention of ER stress completely blocked combination treatment-induced JNK activation and subsequent apoptotic cell death. In addition, combined treatment efficiently induced the mitochondrial translocation of cleaved Bax, disrupted mitochondrial membrane potential, and the nuclear translocation of AIF, all of which were efficiently blocked by a JNK inhibitor. Caspases 3, 8 and 9 were activated by combined treatment but inhibition of these caspases did not abolish apoptosis indicating caspase activation played a minor role in the induction of apoptosis.

Conclusions/Significance

β-lap causes NQO1-dependent radiosensitization of cancer cells. When NQO1⁺ cells are treated with combination of IR and β-lap, positive feedback regulation between ERK and ROS leads to ER stress causing JNK activation and mitochondrial translocation of cleaved Bax. The resultant decrease in mitochondrial membrane leads to translocation of AIF and apoptosis.     

Hydrogen peroxide induces apoptotic-like cell death in coelomocytes of Themiste petricola (Sipuncula)

Apoptosis is an active form of cell death that plays a critical role in physiological and pathological conditions of multicellular organisms. These conditions include development, organogenesis, and elimination of infected, mutated, or damaged cells. Sipunculan cells may respond to changes in environmental exposure to oxidative stress by induction of apoptotic cell death. In coelomocytes of the sipunculan worm Themiste petricola, we evaluated morphological and biochemical changes that were induced by hydrogen peroxide (H2O2) and that could be compatible with an apoptotic-like phenotype. At an exposure of 100 mM H2O2, coelomocytes exhibited several morphological hallmarks of apoptosis such as chromatin condensation, nuclear segmentation, cell volume decrease, membrane blebbing, and formation of apoptotic bodies. Biochemical evidences of apoptotic-like cell death included exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane and oligonucleosomal DNA fragmentation. In addition, exposure of coelomocytes to H2O2 induced a rapid massive loss of mitochondrial membrane potential and of the acidic pH of lysosomes. Overall, our results showed that, in sipunculan coelomocytes, H2O2 can induce changes compatible with an apoptotic-like phenotype. The finding of an oxidative-stress-induced apoptotic-like phenotype in a sipunculan worm may indicate that this kind of cell death process participates in regulation of cell number during physiological and pathological situations, including immune responses.     

Apoptotic cell death induced by hydrogen peroxide in NT2 parental and mitochondrial DNA depleted cells

Oxidative stress has been implicated in several pathologies associated with degenerative processes. Mitochondria are involved in cell death by necrosis or apoptosis due to a large load of Ca2+, the formation of reactive oxygen species (ROS), mitochondrial depolarization and the release of cytochrome c that initiates the caspase cascade. Nevertheless, the role of mitochondria in cell death processes induced by hydrogen peroxide (H2O2) has not been fully established. In this study, we analyzed the cytotoxic effect of H2O2 on rho+ human teratocarcinoma (NT2) cells and on mitochondria-DNA depleted rho0 NT2 cells, lacking functional mitochondria. The cells were exposed to H2O2 for 24 h and cell viability was dose-dependently decreased in both cell lines upon H2O2 exposure, although cell susceptibility was higher in rho0 NT2 cells. Moreover a decrease in mitochondrial membrane potential (Deltapsi(m)), mitochondrial cytochrome c release, caspases activation and DNA fragmentation were largely induced by H2O2 and occurred in both cell lines. Nevertheless, increased cell toxicity in rho0 cells upon H2O2 exposure was accompanied by a higher activation of the effector caspases-3 and -6. The data support that, in general, no differences were observed in cells containing functional (rho+) or non-functional (rho0) mitochondria upon H2O2-induced apoptotic cell death.     

The role of p38 MAPK and JNK in Arsenic trioxide-induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.     

Association of increased bcl-2 expression with rescue from tumor necrosis factor-alpha-induced cell death in the oligodendrocyte cell line OLN-93

The present study investigated the effects of flupirtine (Katadolon) on tumor necrosis factor (TNF)-alpha-mediated cell death and Bcl-2 expression in the permanent rat oligodendrocyte cell line OLN-93 (OLN cells). TNF-alpha (500 U/ml) induced apoptosis of OLN cells, which was confirmed by DNA fragmentation using an in situ end-labeling technique and ultrastructural analysis. Flupirtine significantly reduced the rate of spontaneous cell death of OLN cells already at low concentrations; TNF-alpha-mediated apoptosis was suppressed only with higher concentrations of flupirtine (100 microM:). Expression of Bcl-2 protein and mRNA in OLN cells was detected by immunocytochemistry, western blot, and RT-PCR. Quantitative analysis of western blots revealed an approximately 2. 5-fold up-regulation of Bcl-2 protein during TNF-alpha treatment. Furthermore, addition of 10 or 100 microM: flupirtine before incubation with TNF-alpha led to an approximately threefold increase of Bcl-2 expression. Exposure of OLN cells to flupirtine alone moderately augmented the expression of Bcl-2 protein. Our data demonstrate that flupirtine up-regulates the expression of Bcl-2 protein in OLN cells; this Bcl-2 induction is associated with a reduced rate of TNF-alpha-induced cell death.