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.     

The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.     

Interaction of HCV core protein with 14-3-3epsilon protein releases Bax to activate apoptosis

Through protein-protein binding assays, we found that HCV core protein interacted with 14-3-3epsilon protein. Interestingly, the expression of HCV core protein induced apoptosis in 293T cells. The apoptosis induced by core expression is accompanied by translocation of Bax from cytosol to mitochondria, disruption of mitochondrial membrane potential, cytochrome c release, and activation of caspase-9 and caspase-3. Furthermore, over-expression of 14-3-3epsilon inhibited the core-induced apoptosis and Bax translocation to mitochondria. These results indicate that HCV core protein induces the Bax-mediated apoptosis by interacting with 14-3-3epsilon protein in 293T cells. As a mechanism of apoptosis induction by HCV core, we propose that the interaction of HCV core with 14-3-3epsilon causes the dissociation of Bax from the Bax/14-3-3epsilon complex in cytosol, and the free Bax protein provokes activation of the mitochondrial apoptotic pathway.     

Bax and the mitochondrial permeability transition cooperate in the release of cytochrome c during endoplasmic reticulum-stress-induced apoptosis

Endoplasmic reticulum (ER) stress induces apoptosis by mechanisms that are not fully clear. Here we show that ER stress induced by the Ca(2+)-ATPase inhibitor thapsigargin (THG) activates cytochrome c-dependent apoptosis through cooperation between Bax and the mitochondrial permeability transition (MPT) in human leukemic CEM cells. Pharmacological inhibition of the MPT as well as small interfering RNA (siRNA) knockdown of the MPT core component cyclophilin D blocked cytochrome c release and caspase-dependent apoptosis but did not prevent Bax activation, translocation or N-terminal exposure in mitochondria. siRNA knockdown of Bax also blocked THG-mediated cytochrome c release and apoptosis, but did not prevent MPT activation and resulted in caspase-independent cell death. Our results show that ER-stress-induced cell death involves a caspase and Bax-dependent pathway as well as a caspase-independent MPT-directed pathway.     

Bax Contains Two Functional Mitochondrial Targeting Sequences and Translocates to Mitochondria in a Conformational Change- and Homo-oligomerization-driven Process

The apoptosis gateway protein Bax normally exists in the cytosol as a globular shaped monomer composed of nine α-helices. During apoptosis, Bax translocates to the mitochondria, forms homo-oligomers, and subsequently induces mitochondrial damage. The mechanism of Bax mitochondrial translocation remains unclear. Among the nine α-helices of Bax, helices 4, 5, 6, and 9 are capable of targeting a heterologous protein to mitochondria. However, only helices 6 and 9 can independently direct the oligomerized Bax to the mitochondria. Although Bax mitochondrial translocation can still proceed with mutations in either helix 6 or helix 9, combined mutations completely abolished mitochondrial targeting in response to activating signals. Using a proline mutagenesis scanning analysis, we demonstrated that conformational changes were sufficient to cause Bax to move from the cytosol to the mitochondria. Moreover, we found that homo-oligomerization of Bax contributed to its mitochondrial translocation. These results suggest that Bax is targeted to the mitochondria through the exposure of one or both of the two functional mitochondrial targeting sequences in a conformational change-driven and homo-oligomerization-aided process.     

Resveratrol induces p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated Bax protein oligomerization on mitochondria to initiate cytochrome c release and caspase activation

Resveratrol, a naturally occurring phytoalexin, is known to induce apoptosis in multiple cancer cell types, but the underlying molecular mechanisms remain unclear. Here, we show that resveratrol induced p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated translocation of Bax to mitochondria where it underwent oligomerization to initiate apoptosis. Resveratrol treatment promoted interaction between Bax and XIAP in the cytosol and on mitochondria, suggesting that XIAP plays a critical role in the activation and translocation of Bax to mitochondria. This process did not involve p53 but required accumulation of Bim and t-Bid on mitochondria. Bax primarily underwent homo-oligomerization on mitochondria and played a major role in release of cytochrome c to the cytosol. Bak, another key protein that regulates the mitochondrial membrane permeabilization, did not interact with p53 but continued to associate with Bcl-xL. Thus, the proapoptotic function of Bak remained suppressed during resveratrol-induced apoptosis. Caspase-9 silencing inhibited resveratrol-induced caspase activation, whereas caspase-8 knockdown did not affect caspase activity, suggesting that resveratrol induces caspase-9-dependent apoptosis. Together, our findings characterize the molecular mechanisms of resveratrol-induced caspase activation and subsequent apoptosis in cancer cells.     

Role of mitochondria in aspirin-induced apoptosis in human gastric epithelial cells

This study was undertaken to determine whether the Bcl-2 family proteins and Smac are regulators of aspirin-mediated apoptosis in a gastric mucosal cell line known as AGS cells. Cells were incubated with varying concentrations of acetylsalicylic acid (ASA; 2-40 mM), with or without preincubation of caspase inhibitors. Apoptosis was characterized by Hoechst staining and DNA-histone-associated complex formation. Antiapoptotic Bcl-2, proapoptotic Bax and Bid, Smac, and cytochrome-c oxidase (COX IV) were analyzed by Western blot analyses from cytosol and mitochondrial fractions. ASA downregulated Bcl-2 protein expression and induced Bax translocation into the mitochondria and cleavage of Bid. In contrast, expression of Smac was significantly decreased in mitochondrial fractions of ASA-treated cells. Bax and Bid involvement in apoptosis regulation was dependent on caspase activation, because caspase-8 inhibition suppressed Bax translocation and Bid processing. Caspase-9 inhibition prevented Smac release from mitochondria. Additionally, increased expression of the oxidative phosphorylation enzyme COX IV was observed in mitochondrial fractions exposed to ASA at concentrations >5 mM. Although caspase-8 inhibition had no effect on aspirin-induced apoptosis and DNA-histone complex formation, caspase-9 inhibition significantly decreased both of these events. We conclude that Bcl-2 protein family members and Smac regulate the apoptotic pathway in a caspase-dependent manner. Our results indicate also that mitochondrial integration and oxidative phosphorylation play a critical role in the pathogenesis of apoptosis in human gastric epithelial cells.     

Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria

Hepatitis B virus X protein (HBx) is essential for viral replication and plays an important role in viral pathogenesis. HBx transactivates many viral and cellular genes and participates in cellular signal transduction pathways, proliferation, and apoptosis. In the present study, we report that HBx induces apoptosis by enhancing the translocation of Bax to mitochondria, followed by inducing the loss of mitochondrial membrane potential and release of cytochrome C. In addition, Bcl-2, inhibitor of Bax, rescues the disruption of mitochondrial membrane potential and DNA fragmentation induced by serum starvation in HepG2-X cells expressing HBx. We also found that HBx binds directly to Bax and interferes with the interaction between Bax and 14-3-3epsilon to enhance the translocation of Bax to mitochondria. Taken together, our data suggest that HBx induces apoptosis by interacting with Bax and enhancing its translocation to mitochondria.     

Cytoprotective membrane-permeable peptides designed from the Bax-binding domain of Ku70

Bax is a pro-apoptotic member of Bcl-2 family proteins and is central to mitochondria-dependent apoptosis. Bax resides in the cytosol as a quiescent protein and translocates into mitochondria after apoptotic stimuli. Ku70 is a 70K subunit of the Ku complex, which has an important role in DNA double-strand break (DSB) repair in the nucleus. In another article in this issue, we reported that Ku70 interacts with pro-apoptotic protein Bax in the cytosol and prevents its mitochondrial translocation, suggesting that Ku70 suppresses Bax-mediated apoptosis. Here, we describe the development of a new membrane-permeable peptide, Bax-inhibiting peptide (BIP) that inhibits Bax-mediated apoptosis, on the basis of the previous finding that showed an interaction between Ku70 and Bax. BIP is comprised of five amino acids designed from the Bax-binding domain of Ku70, and suppresses the mitochondrial translocation of Bax. BIP inhibited Bax-mediated apoptosis induced by staurosporine, UVC irradiation and anti-cancer drugs in several types of cells. BIP may provide valuable information in the development of therapeutics that control apoptosis-related diseases.     

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.     

Ethanol induces apoptosis in hepatocytes by a pathway involving novel protein kinase C isoforms

Ethanol abuse is one of the major etiologies of cirrhosis. Ethanol has been shown to induce apoptosis via activation of oxidative stress, mitogen-activated protein kinases (MAPK), and tyrosine kinases. However, there is a paucity of data that examine the interplay among these molecules. In the present study we have systematically elucidated the role of novel protein kinase C isoforms (nPKC; PKCdelta and PKCepsilon) in ethanol-induced apoptosis in hepatocytes. Ethanol enhanced membrane translocation of PKCdelta and PKCepsilon, which was associated with the phosphorylation of p38MAPK, p42/44MAPK and JNK1/2, and the nuclear translocation of NF-kappaB and AP-1. This resulted in increased apoptosis in primary rat hepatocytes. Inhibition of both PKCdelta and PKCepsilon resulted in a decreased MAPK activation, decreased nuclear translocation of NF-kappaB and AP-1, and inhibition of apoptosis. In addition, ethanol activated the tyrosine phosphorylation of PKCdelta via tyrosine kinase in hepatocytes. The tyrosine phosphorylated PKCdelta was cleaved by caspase-3 and these fragments were translocated to the nucleus. Inhibition of ethanol-induced oxidative stress blocked the membrane translocation of PKCdelta and PKCepsilon, and the tyrosine phosphorylation of PKCdelta in hepatocytes. Inhibition of oxidative stress, tyrosine kinase or caspase-3 activity caused a decreased nuclear translocation of PKCdelta in response to ethanol, and was associated with less apoptosis. Conclusion: These results provide a newly-described mechanism by which ethanol induces apoptosis via activation of nPKC isoforms in hepatocytes.     

Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria

Bax is a potent pro-apoptotic member of the Bcl-2 protein family that localizes to the mitochondrial membrane during apoptosis. Tauroursodeoxycholic acid (TUDCA) modulates the apoptotic threshold, in part, by preventing Bax translocation both in vitro and in vivo. The mechanisms by which Bax induces and TUDCA inhibits release of cytochrome c are unclear. We show here that recombinant Bax protein induced cytochrome c release in isolated mitochondria without detectable swelling. Co-incubation with TUDCA prevented efflux of mitochondrial factors and proteolytic processing of caspases in cytosolic extracts. Spectroscopic analyses of mitochondria exposed to Bax revealed increased polarity and fluidity of the membrane lipid core as well as altered protein order, indicative of Bax binding, together with loss of spin-label paramagnetism, characteristic of oxidative damage. TUDCA markedly abrogated the Bax-induced membrane perturbation. In conclusion, our results indicate that Bax protein directly induces cytochrome c release from mitochondria through a mechanism that does not require the permeability transition. Rather, it is accompanied by changes in the organization of membrane lipids and proteins. TUDCA is a potent inhibitor of Bax association with mitochondria. Thus, TUDCA modulates apoptosis by suppressing mitochondrial membrane perturbation through pathways that are also independent of the mitochondrial permeability transition.     

Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures

It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.     

Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis

The proapoptotic protein Bax plays an important role in cardiomyocytic cell death. Ablation of this protein has been shown to diminish cardiac damage in Bax-knockout mice during ischemia-reperfusion. Presently, studies of Bax-mediated cardiac cell death examined primarily the expression levels of Bax and its prosurvival factor Bcl-2 rather than the localization of this protein, which dictates its function. Using immunofluorescence labeling, we have shown that in neonatal rat cardiomyocytes and in H9c2 cardiomyoblasts, Bax translocates from cytosol to mitochondria upon the induction of apoptosis by hypoxia-reoxygenation-serum withdrawal and by the presence of the free-radical inducer menadione. Also, we found that Bax translocation to mitochondria was associated with the exposure of an NH2-terminal epitope, and that this translocation could be partially blocked by the prosurvival factors Bcl-2 and Bcl-XL. To visualize the translocation of Bax in living cells, we have developed an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This cell line has GFP-Bax localized primarily in the cytosol in the absence of apoptotic inducers. Upon induction of apoptosis by a number of stimuli, including menadione, staurosporine, sodium nitroprusside, and hypoxia-reoxygenation-serum withdrawal, we could observe the translocation of Bax from cytosol to mitochondria. This translocation was not affected by retinoic acid-induced differentiation of H9c2 cells. Additionally, this translocation was associated with loss of mitochondrial membrane potential, release of cytochrome c, and fragmentation of nuclei. Finally, using a tetramethylrhodamine-based dye, we have shown that a rapid screening process based on the loss of mitochondrial membrane potential could be developed to monitor GFP-Bax translocation to mitochondria. Overall, the GFP-Bax-stable H9c2 cell line that we have developed represents a unique tool for examining Bax-mediated apoptosis, and it could be of great importance in screening therapeutic compounds that could block Bax translocation to mitochondria to attenuate apoptosis.     

Protective effects of cilostazol on ethanol-induced damage in primary cultured hepatocytes

Alcoholic liver disease (ALD) caused by excessive alcohol consumption is associated with oxidative stress, mitochondrial dysfunction, and hepatocellular apoptosis. Cilostazol, a licensed clinical drug used to treat intermittent claudication, has been reported to act as a protective agent in a spectrum of diseases. However, little information regarding its role in ethanol-induced hepatocellular toxicity has been reported. In the current study, we investigated the protective effects and mechanisms of cilostazol on ethanol-induced hepatocytic injury. Rat primary hepatocytes were pretreated with cilostazol prior to ethanol treatment. MTT and LDH assay indicated that ethanol-induced cell death was ameliorated by cilostazol in a dose-dependent manner. Our results display that overproduction of intracellular reactive oxygen species (ROS) and 4-hydroxy-2-nonenal (4-HNE) induced by ethanol was attenuated by pretreatment with cilostazol. Furthermore, cilostazol significantly inhibited ethanol-induced generation of ROS in mitochondria. Importantly, it was shown that cilostazol could improve mitochondrial function in primary hepatocytes by restoring the levels of ATP and mitochondrial membrane potential (MMP). Additionally, cilostazol was found to reduce apoptosis induced by ethanol using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Mechanistically, we found that cilostazol prevented mitochondrial pathway-mediated apoptotic signals by reversing the expression of Bax and Bcl2, the level of cleaved caspase-3, and attenuating cytochrome C release. These findings suggest the possibility of novel ALD therapies using cilostazol.     

Role of mitochondrial oxidative stress in the apoptosis induced by diospyrin diethylether in human breast carcinoma (MCF-7) cells

Mitochondria and associated oxidative stress have been shown to play critical roles in apoptotic death induced by various stress agents. Previously, we reported the antitumor property of diospyrin (D1), a plant-derived bisnaphthoquinonoid, and its diethylether derivative (D7), which was found to cause apoptotic death in human cancer cell lines. The present study aims to explore the relevant mechanism of apoptosis involving generation of cellular reactive oxygen species (ROS) by D7 in human breast carcinoma (MCF-7) cells. It was found that while D7 inhibited the proliferation of tumor cells, the associated apoptosis induced by D7 was prevented by treating the cells with N-acetyl-L: -cysteine (NAC), an antioxidant, and cyclosporine A (CsA), an inhibitor of mitochondrial permeability transition (MPT). Experiments using suitable inhibitors also demonstrated that D7 could alter the electron flow in mitochondrial electron transport chain by affecting target(s) between complex I and complex III, and indicated the probable site of D7-induced generation of ROS. These results were further supported by confocal microscopic observation on changes in mitochondrial organization and shape in cells treated with D7. Taken together, the results of our study clearly suggested that the apoptosis induced by D7 would involve alteration of MPT, cardiolipin peroxidation, migration of Bax from cytosol to mitochondria, decreased expression of Bcl-2, and release of cytochrome c, indicating oxidative mechanism at the mitochondrial level in the tumor cells.     

Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition

Ursodeoxycholic acid (UDCA) has been shown to be a strong modulator of the apoptotic threshold in both hepatic and nonhepatic cells. 3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, appears to cause apoptotic neuronal cell death in the striatum, reminiscent of the neurochemical and anatomical changes associated with Huntington's disease (HD). This study was undertaken (a) to characterize further the mechanism by which 3-NP induces apoptosis in rat neuronal RN33B cells and (b) to determine if and how the taurine-conjugated UDCA, tauroursodeoxycholic acid (TUDCA), inhibits apoptosis induced by 3-NP. Our results indicate that coincubation of cells with TUDCA and 3-NP was associated with an approximately 80% reduction in apoptosis (p < 0.001), whereas neither taurine nor cyclosporin A, a potent inhibitor of the mitochondrial permeability transition (MPT), inhibited cell death. Moreover, TUDCA, as well as UDCA and its glycine-conjugated form, glycoursodeoxycholic acid, prevented mitochondrial release of cytochrome c (p < 0.001), which probably accounts for the observed inhibition of DEVD-specific caspase activity and poly(ADP-ribose) polymerase cleavage. 3-NP decreased mitochondrial transmembrane potential (p < 0.001) and increased mitochondrial-associated Bax protein levels (p < 0.001). Coincubation with TUDCA was associated with significant inhibition of these mitochondrial membrane alterations (p < 0.01). The results suggest that TUDCA inhibits 3-NP-induced apoptosis via direct inhibition of mitochondrial depolarization and outer membrane disruption, together with modulation of Bax translocation from cytosol to mitochondria. In addition, cell death by 3-NP apparently occurs through pathways that are independent of the MPT.     

TNF-alpha-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8

Alcoholic liver disease is associated with an increase in the number of necrotic and apoptotic liver parenchymal cells. Part of this injury is mediated by TNF-alpha. Ethanol exposure sensitizes cells to the cytotoxic effects of TNF-alpha. This may be due, in part, to the increased propensity of the mitochondria in ethanol-exposed cells to induction of mitochondrial permeability transition (MPT) by various agents, including the proapoptotic protein Bax. This idea is supported by the observation that increased cell death induced by TNF-alpha in ethanol-exposed cells was dependent on development of the MPT. In the present study, we elucidate the pathways through which ethanol exposure enhances TNF-alpha induction of the MPT and the resulting cytotoxicity. Specifically, ethanol-exposed cells display caspase-8- and Bid-independent cell killing during TNF-alpha treatment. Moreover, the ethanol-enhanced pathway is dependent on p38 MAPK signaling, which brings about caspase-3 activation, mitochondrial depolarization, accumulation of cytochrome c in the cytosol, and the translocation of Bax to the mitochondria. Additionally, ethanol-exposed cells display a blunting of TNF-alpha-induced Akt activation and Bcl-2 antagonist of cell death phosphorylation that may account, in part, for the increased sensitivity of the mitochondria to Bax-mediated damage.     

Bcl-X(L) and calyculin A prevent translocation of Bax to mitochondria during apoptosis

During many forms of apoptosis, Bax, a pro-apoptotic protein of the Bcl-2 family, translocates from the cytosol to the mitochondria and induces cytochrome c release, followed by caspase activation and DNA degradation. Both Bcl-X(L) and the protein phosphatase inhibitor calyculin A have been shown to prevent apoptosis, and here we investigated their impact on Bax translocation. ML-1 cells incubated with either anisomycin or staurosporine exhibited Bax translocation, cytochrome c release, caspase 8 activation, and Bid cleavage; only the latter two events were caspase-dependent, confirming that they are consequences in this apoptotic pathway. Both Bcl-X(L) and calyculin A prevented Bax translocation and cytochrome c release. Bcl-X(L) is generally thought to heterodimerize with Bax to prevent cytochrome c release and yet they remain in different cellular compartments, suggesting that their heterodimerization at the mitochondria is not the primary mechanism of Bcl-X(L)-mediated protection. Using chemical cross-linking agents, Bax appeared to exist as a monomer in undamaged cells. Upon induction of apoptosis, Bax formed homo-oligomers in the mitochondrial fraction with no evidence for cross-linking to Bcl-2 or Bcl-X(L). Considering that both Bcl-X(L) and calyculin A inhibit Bax translocation, we propose that Bcl-X(L) may regulate Bax translocation through modulation of protein phosphatase or kinase signaling.     

A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.