Mechanism of cell death induction by nitroxide and hyperthermia

Heat stress and nitroxides induce reactive oxygen species (ROS) and proapoptotic effects. The underlying mechanisms remain largely elusive. Here we report that Tempo (2,2,6,6-tetramethylpiperidine-N-oxyl) is a potent thermosensitizer for promoting cell death in human leukemia U937 cells. Treatment with Tempo (10 mM, 37 degrees C/30 min) and hyperthermia (44 degrees C/30 min) induced 30 and 70-80% apoptosis, respectively, through Bax-mediated cytochrome c release and DEVDase activation. The Tempo/heat combination also caused Bax-mediated cytochrome c release, but switched heat-induced apoptosis to the particular pyknotic cell death, resulting in the irreparable inhibition of proliferation. Tempo and heat stress, but not the combination, caused an early transient elevation of H2O2/O2*- and a late induction of only O2*-, respectively. Mitochondrial Ca2+ overloads were indistinguishable after any treatment. Heat stress induced the pan-caspase inhibitor zVAD-fmk-suppressible low-Deltapsi (mitochondrial membrane potential) in 75% of cells as a result of DEVDase activation. In contrast, Tempo yielded low-Deltapsi by deprivation of the mitochondrial H+ gradient. The combined treatment induced 97% zVAD-resistant low-Deltapsi cells through irreversible mitochondrial dysfunction. Together, thus, Tempo or heat stress induced Bax-mediated mitochondrial apoptosis with the possible help of ROS or mitochondrial Ca2+, and Tempo when combined with hyperthermia acts a sensitizer by inducing irreparable pyknotic cell death through irreversible mitochondrial dysfunction.     

Short-term and long-term effects of fatty acids in rat hepatoma AS-30D cells: the way to apoptosis

Arachidonic acid and, to a smaller extent, oleic acid at micromolar concentrations decreased the mitochondrial membrane potential within AS-30D rat hepatoma cells cultivated in vitro and increased cell respiration. The uncoupling effect of both fatty acids on cell respiration was partly prevented by cyclosporin A, blocker of the mitochondrial permeability transition pore. Arachidonic acid increased the rate of reactive oxygen species (ROS) production, while oleic acid decreased it. Both fatty acids induced apoptotic cell death of AS-30D cells, accompanied by the release of cytochrome c from mitochondria to the cytosol, activation of caspase-3 and association of proapoptotic Bax protein with mitochondria; arachidonic acid being a more potent inducer than oleic acid. Trolox, a potent antioxidant, prevented ROS increase induced by arachidonic acid and protected the cells against apoptosis produced by this fatty acid. It is concluded that arachidonic and oleic acids induce apoptosis of AS-30D hepatoma cells by the mitochondrial pathway but differ in the mechanism of their action: Arachidonic acid induces apoptosis mainly by stimulating ROS production, whereas oleic acid may contribute to programmed cell death by activation of the mitochondrial permeability transition pore.     

The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release,caspase activation,and mitochondrial dysfunction

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.     

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.     

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2*- and could be prevented by [Ca2+]i buffering. This study provides evidence that O2*- facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.     

Role of the mitochondrial permeability transition and cytochrome C release in hydrogen peroxide-induced apoptosis

We investigated the role of the mitochondrial inner membrane permeability transition and subsequent release of cytochrome c into the cytosol during oxidative stress-evoked apoptosis. Sublethal oxidative stress was applied by treating L929 cells with 0.5 mM H2O2 for 90 min. Then the cellular localization of cytochrome c was examined by immunofluorescent staining and Western blotting. H2O2 treatment caused the permeability transition and pore formation, resulting in membrane depolarization and translocation of cytochrome c from the mitochondria into the cytosol. Pretreatment with cyclosporin A and aristolochic acid (to inhibit pore formation) significantly attenuated a reduction of the mitochondrial membrane potential, as well as signs of apoptosis such as DNA fragmentation, increased plasma membrane permeability, and chromatin condensation. Therefore, exposure to H2O2 caused the opening of permeability transition pores in the inner mitochondrial membrane. An essential role of cytosolic cytochrome c in the execution of apoptosis was demonstrated by its direct microinjection into the cytosol, thus bypassing the need for cytochrome c release from the mitochondrial intermembrane space. Microinjection of cytochrome c caused caspase-dependent apoptosis.     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.     

Anti-apoptotic effect of cGMP in cultured astrocytes: inhibition by cGMP-dependent protein kinase of mitochondrial permeable transition pore.

Reperfusion of cultured astrocytes with normal medium after exposure to H(2)O(2)-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H(2)O(2)-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H(2)O(2)-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca(2+)-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H(2)O(2)-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect.     

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.     

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.     

Retroactive pathway involving mitochondria in electroloaded cytochrome c-induced apoptosis. Protective properties of Bcl-2 and Bcl-XL

Cytochrome c release is thought to play an important role in the initiation of apoptosis. The nature of the control exerted by Bcl-2 and Bcl-XL on such a pathway is not precisely known. We addressed this issue by square-wave pulse electroloading of exogenous cytochrome c into Jurkat cells. Three hours after cytochrome c loading into the cells, characteristic phenotypes of apoptosis were observed. However, a significant drop in the mitochondrial membrane potential (Deltapsim) was also observed, while cytochrome c was generally considered to act downstream from the mitochondria. Related to the Deltapsim drop, there was a release of proapoptotic proteins such as AIF and Smac from the mitochondria. This release, as well as NAD(P)H and cardiolipids oxidation, are linked to previous caspase activation. Cytochrome c-linked caspase activation also led to potassium efflux out of the cell. Overexpression of Bcl-2 and Bcl-XL or N-acetyl-DEVD-aldehyde treatment not only prevented the mitochondrial membrane potential decrease, but also protected cells from the apoptosis directly induced by cytochrome c electroloading. Bcl-2 and Bcl-XL protection is based on the inhibition of the caspase-dependent retroactive pathway affecting the mitochondrial compartment.     

Peroxiredoxin III, a mitochondrion-specific peroxidase, regulates apoptotic signaling by mitochondria

Various proapoptotic stimuli increase the production of superoxide and H(2)O(2) by mitochondria. Whereas superoxide impairs mitochondrial function and is removed by Mn(2+)-dependent superoxide dismutase, the role and metabolism of mitochondrial H(2)O(2) during apoptosis have remained unclear. The effects on apoptotic signaling of depletion of peroxiredoxin (Prx) III, a mitochondrion-specific H(2)O(2)-scavenging enzyme, have now been investigated by RNA interference in HeLa cells. Depletion of Prx III resulted in increased intracellular levels of H(2)O(2) and sensitized cells to induction of apoptosis by staurosporine or TNF-alpha. The rates of mitochondrial membrane potential collapse, cytochrome c release, and caspase activation were increased in Prx III-depleted cells, and these effects were reversed by ectopic expression of Prx III or mitochondrion-targeted catalase. Depletion of Prx III also exacerbated damage to mitochondrial macromolecules induced by the proapoptotic stimuli. Our results suggest that Prx III is a critical regulator of the abundance of mitochondrial H(2)O(2), which itself promotes apoptosis in cooperation with other mediators of apoptotic signaling.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Hispidulin Induces Apoptosis Through Mitochondrial Dysfunction and Inhibition of P13k/Akt Signalling Pathway in HepG2 Cancer Cells

Hispidulin is a flavonoid compound which is an active ingredient in a number of traditional Chinese medicinal herbs. However, it’s therapeutic activity remains poorly understood. The present study investigated the pro-apoptotic effects and mechanism by which Hispidulin induces apoptosis in human hepatoblastoma cancer (HepG2) cells. The results showed that Hispidulin induced cell death in a dose- and time-dependent manner in HepG2 cells whereas no toxic reaction was observed in normal human liver cells at indicated concentration. This study also demonstrated that Hispidulin induces apoptosis through mitochondrial dysfunction, which is characterized by decreased Bcl-2/Bax ratio, disrupted mitochondrial membrane potential and increased release of cytochrome C and activated capase-3. Our results also showed that mitochondrial dysfunction was triggered by Hispidulin-induced excessive ROS generation. Hispidulin also significantly inhibited Akt activation. ROS inhibitor NAC abrogated the inhibitory effect of Hispidulin on P13k/Akt signalling pathway and the proapoptotic effect in HepG2 cells. Our results demonstrate for the first time that Hispidulin induces apoptosis in HepG2 cells and suggested that the pro-apoptotic effect of Hispidulin was mediated through mitochondrial dysfunction and inhibition of P13k/Akt signalling pathway. Since no toxic effect was observed when normal liver cells were treated with Hispidulin, Hispidulin may have the potential to be used as therapeutic for liver cancer.     

Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib,a novel proteasome inhibitor,in human H460 non-small cell lung cancer cells

Bortezomib, a proteasome inhibitor, shows substantial anti-tumor activity in a variety of tumor cell lines, is in phase I, II, and III clinical trials and has recently been approved for the treatment of patients with multiple myeloma. The sequence of events leading to apoptosis following proteasome inhibition by bortezomib is unclear. Bortezomib effects on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration in the mitochondrial membrane potential (Delta psi m), and release of cytochrome c from mitochondria. With human H460 lung cancer cells, bortezomib exposure at 0.1 microM showed induction of apoptotic cell death starting at 24 h, with increasing effects after 48-72 h of treatment. After 3-6 h, an elevation in ROS generation, an increase in Delta psi m, and the release of cytochrome c into the cytosol, were observed in a time-dependent manner. Co-incubation with rotenone and antimycin A, inhibitors of mitochondrial electron transport chain complexes I and III, or with cyclosporine A, an inhibitor of mitochondrial permeability transition pore, resulted in inhibition of bortezomib-induced ROS generation, increase in Delta psi m, and cytochrome c release. Tiron, an antioxidant agent, blocked the bortezomib-induced ROS production, Delta psi m increase, and cytochrome c release. Tiron treatment also protected against the bortezomib-induced PARP protein cleavage and cell death. Benzyloxycarbonyl-VAD-fluoromethyl ketone, an inhibitor of pan-caspase, did not alter the bortezomib-induced ROS generation and increase in Delta psi m, although it prevented bortezomib-induced poly(ADP-ribose) polymerase cleavage and apoptotic death. In PC-3 prostate carcinoma cells (with overexpression of Bcl-2), a reduction of bortezomib-induced ROS generation, Delta psi m increase was correlated with cellular resistance to bortezomib and the attenuation of drug-induced apoptosis. The transient transfection of wild type p53 in p53 null H358 cells caused stimulation of the bortezomib-induced apoptosis but failed to enhance ROS generation and Delta psi m increase. Thus ROS generation plays a critical role in the initiation of the bortezomib-induced apoptotic cascade by mediation of the disruption of Delta psi m and the release of cytochrome c from mitochondria.     

Oxidative stress is involved in the permeabilization of the inner membrane of brain mitochondria exposed to hypoxia/reoxygenation and low micromolar Ca2+

From in vivo models of stroke it is known that ischemia/reperfusion induces oxidative stress that is accompanied by deterioration of brain mitochondria. Previously, we reported that the increase in Ca2+ induces functional breakdown and morphological disintegration in brain mitochondria subjected to hypoxia/reoxygenation (H/R). Protection by ADP indicated the involvement of the mitochondrial permeability transition pore in the mechanism of membrane permeabilization. Until now it has been unclear how reactive oxygen species (ROS) contribute to this process. We now report that brain mitochondria which had been subjected to H/R in the presence of low micromolar Ca2+ display low state 3 respiration (20% of control), loss of cytochrome c, and reduced glutathione levels (75% of control). During reoxygenation, significant mitochondrial generation of hydrogen peroxide (H2O2) was detected. The addition of the membrane permeant superoxide anion scavenger TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) suppressed the production of H2O2 by brain mitochondria metabolizing glutamate plus malate by 80% under normoxic conditions. TEMPOL partially protected brain mitochondria exposed to H/R and low micromolar Ca2+ from decrease in state 3 respiration (from 25% of control to 60% of control with TEMPOL) and permeabilization of the inner membrane. Membrane permeabilization was obvious, because state 3 respiration could be stimulated by extramitochondrial NADH. Our data suggest that ROS and Ca2+ synergistically induce permeabilization of the inner membrane of brain mitochondria exposed to H/R. However, permeabilization can only partially be prevented by suppressing mitochondrial generation of ROS. We conclude that transient deprivation of oxygen and glucose during temporary ischemia coupled with elevation in cytosolic Ca2+ concentration triggers ROS generation and mitochondrial permeabilization, resulting in neural cell death.     

Vitamin D sensitizes breast cancer cells to the action of H2O2: mitochondria as a convergence point in the death pathway

Calcitriol, the hormonal form of vitamin D3, sensitizes breast cancer cells to reactive oxygen species (ROS)-dependent cytotoxicity induced by various anticancer modalities. This effect could be due to increased generation of ROS and/ or to increased sensitivity of the target cells to ROS. This work examined the effect of calcitriol on the damage inflicted on breast cancer cells by the direct action of ROS represented by H2O2. Treatment of MCF-7 cells with H2O2 resulted in activation of caspase 7 as well as induction of caspase-independent cell death. Both were enhanced by 48-72 h of pretreatment with calcitriol. This effect was not due to modulation of H2O2 degradation or to a specific effect on *OH-mediated cytotoxicity. The H2O2-induced drop in mitochondrial membrane potential and release of cytochrome c were enhanced by calcitriol. These findings indicate that calcitriol sensitizes breast cancer cells to ROS-induced death by affecting event(s) common to both caspase-dependent and -independent modes of cell death upstream to mitochondrial damage.     

Critical upstream signals of cytochrome C release induced by a novel Bcl-2 inhibitor

Cytochrome c release is a central step in the apoptosis induced by many death stimuli. Bcl-2 plays a critical role in controlling this step. In this study, we investigated the upstream mechanism of cytochrome c release induced by ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a recently discovered small molecule inhibitor of Bcl-2. HA14-1 was found to induce cytochrome c release from the mitochondria of intact cells but not from isolated mitochondria. Cytochrome c release from isolated mitochondria requires the presence of both HA14-1 and exogenous Ca(2+). This suggests that both mitochondrial and extramitochondrial signals are important. In intact cells, treatment with HA14-1 caused Ca(2+) spike, change in mitochondrial membrane potential (Delta psi(m)) transition, Bax translocation, and reactive oxygen species (ROS) generation prior to cytochrome c release. Pretreatment with either EGTA acetoxymethyl ester or vitamin E resulted in a significant decrease in cytochrome c release and cell death induced by HA14-1. Furthermore pretreatment with RU-360, an inhibitor of the mitochondrial Ca(2+) uniporter, or with EGTA acetoxymethyl ester, but not with vitamin E, prevented the HA14-1-induced Delta psi(m) transition and Bax translocation. This suggests that ROS generation is an event that occurs after the Delta psi(m) transition and Bax translocation. Together these data demonstrate that the Ca(2+) spike, mitochondrial Bcl-2 presensitization, and subsequent Delta psi(m) transition, Bax translocation, and ROS generation are important upstream signals for cytochrome c release upon HA14-1 stimulation. The involvement of endoplasmic reticulum and mitochondrial signals suggests both organelles are crucial for HA14-1-induced apoptosis.     

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.