Induction of apoptotic death by curcumin in human tongue squamous cell carcinoma SCC-4 cells is mediated through endoplasmic reticulum stress and mitochondria-dependent pathways

Curcumin from the rhizome of the Curcuma longa plant has been noted for its chemo-preventative and chemo-therapy activities, and it inhibits the growth of many types of human cancer cell lines. In this study, the mechanisms of cell death involved in curcumin-induced growth inhibition, including cell cycle arrest and induction of apoptosis in human tongue cancer SCC-4 cells, were investigated. Herein, we observed that curcumin inhibited cell growth of SCC-4 cells and induced cell death in a dose-dependent manner. Treatment of SCC-4 cells with curcumin caused a moderate and promoted the G(2) /M phase arrest, which was accompanied with decreases in cyclin B/CDK1 and CDC25C protein levels. Moreover, curcumin significantly induced apoptosis of SCC-4 cells with a decrease of the Bcl-2 level, reduction of mitochondrial membrane potential (ΔΨ(m) ), and promoted the active forms of caspase-3. Curcumin also promoted the releases of AIF and Endo G from the mitochondria in SCC-4 cells by using confocal laser microscope. Therefore, we suggest that curcumin induced apoptosis through a mitochondria-dependent pathway in SCC-4 cells. In addition, we also found that curcumin-induced apoptosis of SCC-4 cells was partly through endoplasmic reticulum stress. In conclusion, curcumin increased G(2) /M phase arrest and induced apoptosis through ER stress and mitochondria-dependent pathways in SCC-4 cells.     

Potentiation of tumor necrosis factor-alpha-induced cell death by rottlerin through a cytochrome-C-independent pathway

The protein kinase C (PKC) signal transduction pathway negatively regulates receptor-initiated cell death. In HeLa cells, tumor necrosis factor-alpha (TNF)-mediated cell death involved mitochondria and was blocked by the overexpression of Bcl-2. The PKC-specific inhibitor bisindolylmaleimide and the PKCdelta inhibitor rottlerin enhanced TNF-induced cell death. We have investigated if potentiation of TNF-induced cell death by rottlerin involved amplification of the mitochondrial pathway. TNF induced cleavage of the proapoptotic protein Bid and release of mitochondrial cytochrome c. Rottlerin enhanced activation of caspase-8 and cleavage of Bid. It also enhanced activation of caspase-9 but it did not increase cytochrome c in the cytosol. It, however, increased release of mitochondrial apoptosis-inducing factor (AIF) to the cytosol. Overexpression of Bcl-2 prevented release of both cytochrome c and AIF to the cytosol. Prolonged exposure (> or =6 h) of HeLa cells to rottlerin and TNF decreased the level of cytochrome c but not of AIF in the cytosol. These results suggest that rottlerin activates a cytochrome-c-independent cell death pathway to potentiate cell death by TNF.     

Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid-mediated apoptosis through Bak activation in ovarian cancer cells

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release

Defective Cu,Zn-superoxide dismutase (SOD1) is responsible for some types of amyotrophic lateral sclerosis, and ventral horn motor neurons (VMN) have been shown to die through a mitochondria-dependent apoptotic pathway after chronic exposure to high levels of reactive oxygen species (ROS). VMN are also selectively vulnerable to mild spinal cord injury (SCI); however, the involvement of SOD1, ROS, and apoptosis in their death has not been clarified. Mild compression SCI was induced in SOD1-overexpressing transgenic rats and wild-type littermates. Superoxide production, mitochondrial release of cytochrome c, and activation of caspase-9 were examined, and apoptotic DNA injury was also characterized. In the wild-type animals, increased superoxide production, mitochondrial release of cytochrome c, and cleaved caspase-9 were observed exclusively in VMN after SCI. Subsequently, a majority of VMN (75%) selectively underwent delayed apoptotic cell death. Transgenic animals showed less superoxide production, mitochondrial cytochrome c release, and caspase-9 activation, resulting in death of only 45% of the VMN. These results suggest that the ROS-initiated mitochondrial signaling pathway possibly plays a pivotal role in apoptotic VMN death after SCI and that increased levels of SOD1 in VMN reduce oxidative stress, thereby attenuating the activation of the pathway and delayed cell death.     

Mitochondrial dysfunction in CD47-mediated caspase-independent cell death: ROS production in the absence of cytochrome c and AIF release

Ligation of CD47 by its natural ligand thrombospondin (TSP), or cross-linking by CD47 antibodies, triggers caspase-independent cell death in normal and leukemic cells. This kind of cell death is characterised by the cytoplasmic events of apoptosis including externalisation of phosphatidylserines and mitochondria swelling. We report herein selective mitochondrial changes in CD47-dependent cell death of T cells. After T cell stimulation via CD47, a rapid mitochondrial transmembrane potential (deltapsi(m)) disruption is accompanied by the production of reactive oxygen species (ROS) and phosphatidylserine exposure. Surprisingly, mitochondrial dysfunction does not induce cytochrome c or AIF release. Moreover, the dying cells do not exhibit caspase-3 activation and display intact nuclei without any large-scale, or oligonucleosomal DNA fragmentation. We conclude that DeltaPsi(m) loss and ROS production are an early step in CD47-dependent killing and neither cytochrome c, nor AIF are implicated in this new cell death pathway.     

Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria

Caspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cyt c is sufficient to activate the Apaf-caspase-9 apoptosome in vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway.     

Bid-induced release of AIF from mitochondria causes immediate neuronal cell death

Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.     

Caspase-dependent and -independent cell death induced by 3-nitropropionic acid in rat cortical neurons

Mitochondria play a critical role in cell death by releasing apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), from the intermembrane space into the cytoplasm. Because mitochondrial dysfunction has been shown to be involved in several neurodegenerative diseases, mitochondrial toxins are largely used to model these disorders. These include 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, which has been used to model Huntington's disease and was previously reported by us to induce apoptotic cell death through caspase activation. In the present study, we evaluated the involvement of caspase-independent neuronal cell death induced by 3-NP (1 mM) and the effect of z-VDVAD-fmk, an inhibitor of caspase-2, using cortical neurons in culture. Our results highly suggest that 3-NP induces both caspase-dependent and -independent cell death. We showed that z-VDVAD-fmk prevented both caspase-2 and -3-like activities evoked by 3-NP, but only partly prevented chromatin fragmentation/condensation. However, z-VDVAD-fmk did not avoid 3-NP-induced release of cytochrome c or AIF from mitochondria nor did it affect the levels of mitochondrial Bax. Furthermore, 3-NP-mediated decrease in plasma membrane integrity was not affected by z-VDVAD-fmk. Under these conditions, the inhibitor prevented the caspase-dependent cell death.     

Celastrol inhibits growth and induces apoptotic cell death in melanoma cells via the activation ROS-dependent mitochondrial pathway and the suppression of PI3K/AKT signaling

Celastrol has been reported to possess anticancer effects in various cancers; however, the precise mechanism underlying ROS-mediated mitochondria-dependent apoptotic cell death triggered by celastrol treatment in melanoma cells remains unknown. We showed that celastrol effectively induced apoptotic cell death and inhibited tumor growth using tissue culture and in vivo models of B16 melanoma. In addition to apoptotic cell death in B16 cells, several apoptotic events such as PARP cleavage and activation of caspase were confirmed. Pretreatment with caspase inhibitor modestly attenuated the celastrol-induced increase in PARP cleavage and sub-G1 cell population, implying that caspases play a partial role in celastrol-induced apoptosis. Moreover, ROS generation was detected following celastrol treatment. Blocking of ROS accumulation with ROS scavengers resulted in inhibition of celastrol-induced Bcl-2 family-mediated apoptosis, indicating that celastrol-induced apoptosis involves ROS generation as well as an increase in the Bax/Bcl-2 ratio leading to release of cytochrome c and AIF. Importantly, silencing of AIF by transfection of siAIF into cells remarkably attenuated celastrol-induced apoptotic cell death. Moreover, celastrol inhibited the activation of PI3K/AKT/mTOR signaling cascade in B16 cells. Our data reveal that celastrol inhibits growth and induces apoptosis in melanoma cells via the activation of ROS-mediated caspase-dependent and -independent pathways and the suppression of PI3K/AKT signaling.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

Flavopiridol induces mitochondrial-mediated apoptosis in murine glioma GL261 cells via release of cytochrome c and apoptosis inducing factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration; 3. decrease expression of cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and 5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs from the mechanism of flavopiridolinduced cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug kills different types of glioma cells by different mitochondrial-dependent pathways.     

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.     

Mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury: protection by cyclosporin A

Hyperoxia is known to induce extensive alveolar cell death by still poorly defined mechanisms. In this study, the mitochondria-dependent cell death pathway was explored during hyperoxia-induced lung injury in mice. We observed a progressive release of cytochrome c from the mitochondria into the cytosol of alveolar cells. This release was accompanied by the translocation of the proapoptotic protein Bax from cytosol to mitochondria without detectable activation of caspase-3. As cytochrome c release can be induced by mitochondrial membrane alteration and permeability transition (MPT), mice were treated with cyclosporin A, which specifically inhibits MPT. Cyclosporin A treatment prevented mitochondrial release of cytochrome c during hyperoxia and concomitantly preserved mitochondria from extensive swelling and crista disorganization, as assessed by electron microscopy analysis of alveolar epithelial cells. These morphological and biochemical observations correlated with decreased lung tissue damage, as evaluated by morphological score and lung weight. In conclusion, mitochondrial damage and cytochrome c release are important linked events in hyperoxia-induced lung injury and can be efficiently blocked by cyclosporin A.     

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.     

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

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

CD437, a synthetic retinoid, induces apoptosis in human respiratory epithelial cells via caspase-independent mitochondrial and caspase-8-dependent pathways both up-regulated by JNK signaling pathway

The synthetic retinoid-related molecule CD437-induced apoptosis in human epithelial airway respiratory cells: the 16HBE bronchial cell line and normal nasal epithelial cells. CD437 caused apoptosis in S-phase cells and cell cycle arrest in S phase. Apoptosis was abolished by caspase-8 inhibitor z-IETD-fmk which preserved S-phase cells but was weakly inhibited by others selective caspase-inhibitors, indicating that caspase-8 activation was involved. z-VAD and z-IETD prevented the nuclear envelope fragmentation but did not block the chromatin condensation. The disruption of mitochondrial transmembrane potential was also induced by CD437 treatment. The translocation of Bax to mitochondria was demonstrated, as well as the release of cytochrome c into the cytosol and of apoptosis-inducing factor (AIF) translocated into the nucleus. z-VAD and z-IETD did not inhibit mitochondrial depolarization, Bax translocation or release of cytochrome c and AIF from mitochondria. These results suggest that CD437-induced apoptosis is executed by two converging pathways. AIF release is responsible for chromatin condensation, the first stage of apoptotic cell, via a mitochondrial pathway independent of caspase. But final stage of apoptosis requires the caspase-8-dependent nuclear envelope fragmentation. In addition, using SP600125, JNK inhibitor, we demonstrated that CD437 activates the JNK-MAP kinase signaling pathway upstream to mitochondrial and caspase-8 pathways. Conversely, JNK pathway inhibition, which suppresses S-phase apoptosis, did not prevent cell cycle arrest within S phase, confirming that these processes are triggered by distinct mechanisms.     

A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis

Granulysin is a cytolytic molecule released by CTL via granule-mediated exocytosis. In a previous study we showed that granulysin induced apoptosis using both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granulysin-induced apoptosis of tumor cells. Granulysin-induced death is significantly inhibited by Bcl-2 overexpression and is associated with a rapid (1-5 h) loss of mitochondrial membrane potential, which is not mediated by ceramide generation and is not inhibited by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C(18)) ceramide is truly associated with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexpression. The nuclear morphology of cells dying after granulysin treatment in the presence of caspase inhibitors suggested the involvement of mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We demonstrate using confocal microscopy that AIF is translocated from mitochondria to the nucleus during granulysin-induced apoptosis. The majority of Bcl-2 transfectants are protected from granulysin-induced cell death, mitochondrial membrane potential loss, and AIF translocation, while a small percentage are not protected. In this small percentage the typical nuclear apoptotic morphology is delayed, being of the AIF type at 5 h time, while at longer times (12 h) the normal apoptotic morphology is predominant. These and previous results support a key role for the mitochondrial pathway of apoptosis, and especially for AIF, during granulysin-induced tumoral cell death.     

Mitochondria-dependent apoptosis and cellular pH regulation

Mitochondria play a critical role in apoptosis induction in response to myriad stimuli. These organelles release proteins into the cytosol which trigger caspase activation or perform other functions relevant to apoptosis, including cytochrome c (cyt-c), caspases, AIF, and SMAC (Diablo). The mechanisms by which these proteins escape from mitochondria remain enigmatic. Moreover, it is unclear whether release of these proteins versus disturbances in core mitochondrial functions represents the cell death commitment mechanism. In this regard, suppression of apoptosis using broad-spectrum caspase inhibitory compounds has been reported in many circumstances to prevent the morphological and biochemical manifestations of apoptosis, and yet not protect cells from death and not preserve clonigenic survival. Thus, while mitochondrial damage can be coupled to caspase activation pathways, cell death commitment often occurs upstream of caspase activation when mitochondria-dependent cell death pathways are invoked. Here, we review evidence implicating dysregulation of cellular pH as a component of the cell death mechanism involving mitochondria. Cell Death and Differentiation (2000) 7, 1155 - 1165