Lysosomal membrane permeabilization during apoptosis ‐ involvement of Bax?

Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.     

Release of Cytochrome c, Bax Migration, Bid Cleavage, and Activation of Caspases 2, 3, 6, 7, 8, and 9 during Endothelial Cell Apoptosis

Although the executioner phase of apoptosis has been well defined in many cell types, the subcellular events leading to apoptosis in endothelial cells remain undefined. In the current study, apoptosis was induced in primary human umbilical venous endothelial cells by the photosensitizer verteporfin and light. Release of mitochondrial cytochrome c into the cytosol was detectable immediately and accumulated over 2 hours after treatment while cytosolic levels of the proapoptotic Bcl-2 family member, Bax, decreased reciprocally over the same time period. Cleavage of another proapoptotic Bcl-2 family member, Bid, was observed by 2 hours after treatment. Although Bid cleavage has been shown to occur as an upstream event responsible for inducing cytochrome c release, we demonstrate that Bid cleavage can also occur after cytochrome c release. Activation of caspases 2, 3, 6, 7, 8, and 9 occurred following the release of cytochrome c, and cleavage of downstream substrates was observed. In summary, endothelial cell death involves the cellular redistribution of Bax and cytochrome c, followed by the activation of multiple caspases which manifest the apoptotic phenotype.     

Microinjection of cathepsin d induces caspase-dependent apoptosis in fibroblasts

Recent reports have indicated that enzymes such as cathepsins D and B are translocated from lysosomal compartments to the cytosol early during apoptosis. We have previously noted that a translocation of cathepsins D and B occur before cytochrome c release and caspase activation in cardiomyocytes and human fibroblasts during oxidative stress-induced apoptosis. In the present report, we use a microinjection technique to investigate if cytosolic location of the cathepsins D and B are important for induction of apoptosis. We found that microinjection of cathepsin D into the cytosol of human fibroblasts caused apoptosis, which was detected as changes in distribution of cytochrome c, cell shrinkage, activation of caspases, chromatin condensation, and formation of pycnotic nuclei. No apoptosis was, however, induced by microinjection of cathepsin B. Moreover, apoptosis was prevented in fibroblasts pretreated with a caspase-3-like inhibitor, and also when microinjected with cathepsin D mixed with the cathepsin D inhibitor, pepstatin A. These results show that cytosolic cathepsin D can act as a proapoptotic mediator upstream of cytochrome c release and caspase activation in human fibroblasts.     

Relocalization of cathepsin D and cytochrome c early in apoptosis revealed by immunoelectron microscopy

Cathepsin D was translocated from lysosomal structures to the cytosol in primary cultures of neonatal rat cardiomyocytes exposed to oxidative stress, and these cells underwent apoptotic death during subsequent incubation. Temporal aspects of cathepsin D relocalization, cytochrome c release, and decrease in mitochondrial transmembrane potential (delta psi(m)) were studied in myocytes exposed to the redox-cycling xenobiotic naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Immunofluorescence labeling revealed that cathepsin D was translocated to the cytosol after 30 minutes of naphthazarin treatment, and cytochrome c was released from mitochondria to the cytosol after 2 hours. Western blotting and immunoelectron microscopy indicated a minor release of cytochrome c after only 30 minutes and 1 hour, respectively. Thereafter, a decrease in delta psi(m) was detected using the delta psi(m)sensitive dye JC-1 and confocal microscopy, and ultrastructural analysis indicated apoptotic morphology. Pretreatment of the cultures with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria and maintained the delta psi(m). Moreover, ultrastructural examination showed no apoptotic morphology. These findings suggest that lysosomal destabilization (detected as the release of cathepsin D) and release of cytochrome c from mitochondria take place early in apoptosis. Also, the former event probably occurs before the latter during apoptosis induced by oxidative stress because pretreatment with pepstatin A prevented release of cytochrome c and loss of delta psi(m) in cardiomyocytes exposed to naphthazarin.     

Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization

Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. Human neutrophils are highly granulated phagocytes, which respond to pathogens by exhibiting increased production of reactive oxygen species (ROS) and lysosomal degranulation. In a previous study, we observed that intracellular, nonphagosomal generation of ROS triggered by adherent bacteria induced ROS-dependent neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no effect. In the present study, we measured lysosomal membrane stability and leakage in human neutrophils and found that adherent, noningested, Type 1-fimbriated Escherichia coli bacteria induced LMP rapidly in neutrophils. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium markedly blocked the early LMP and apoptosis in neutrophils stimulated with Type 1-fimbriated bacteria but had no effect on the late LMP seen in spontaneously apoptotic neutrophils. The induced lysosomal destabilization triggered cleavage of the proapoptotic Bcl-2 protein Bid, followed by a decrease in the antiapoptotic protein Mcl-1. Involvement of LMP in initiation of apoptosis is supported by the following observations: Bid cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine-cathepsins but not caspases, and the differential effects of inhibitors of cysteine-cathepsins and cathepsin D on apoptosis coincided with their ability to inhibit Bid cleavage in activated neutrophils. Together, these results indicate that in microbe-induced apoptosis in neutrophils, ROS-dependent LMP represents an early event in initiation of the intrinsic apoptotic pathway, which is followed by Bid cleavage, mitochondrial damage, and caspase activation.     

TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO.

Recent reports suggest that a cross-talk exists between apoptosis pathways mediated by mitochondria and cell death receptors. In the present study, we report that mitochondrial events are required for apoptosis induced by the cell death ligand TRAIL (TNF-related apoptosis-inducing ligand) in human cancer cells. We show that the Bax null cancer cells are resistant to TRAIL-induced apoptosis. Bax deficiency has no effect on TRAIL-induced caspase-8 activation and subsequent cleavage of Bid; however, it results in an incomplete caspase-3 processing because of inhibition by XIAP. Release of Smac/DIABLO from mitochondria through the TRAIL-caspase-8-tBid-Bax cascade is required to remove the inhibitory effect of XIAP and allow apoptosis to proceed. Inhibition of caspase-9 activity has no effect on TRAIL-induced caspase-3 activation and cell death, whereas expression of the active form of Smac/DIABLO in the cytosol is sufficient to reconstitute TRAIL sensitivity in Bax-deficient cells. Our results show for the first time that Bax-dependent release of Smac/DIABLO, not cytochrome c, from mitochondria mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis.     

The modulation of inter-organelle cross-talk to control apoptosis

Mitochondria fulfill a wide array of functions dedicated to the energetic metabolism as well as the control of cell death. These functions imply that mitochondria can be activated by a variety of signals and can integrate them to trigger a process called mitochondrial membrane permeabilization (MMP), which induces the ultimate events of apoptosis. MMP consists in a sudden increase in the permeability of mitochondrial membrane that results in the release of critical proapoptotic intermembrane space effectors into the cytosol such as cytochrome c, apoptosis-inducing factor (AIF), Smac/Diablo, Endo G, and pro-caspases. In many models of apoptosis, mitochondrial translocation of proteins and/or lipids concomitantly with alterations of the intracellular milieu has been shown to activate MMP. This applies to tumor suppressors of the Bax/Bcl-2 family (Bax, Bad, Bid, Bim), several protein kinases (Akt, ASK1, hexokinase), p53, NF-kappaB, and nuclear orphan receptors such as TR3/Nur77. After mitochondrial membrane association, these proteins target constitutive mitochondrial proteins including the permeability transition pore complex (PTPC), Bcl-X(L), HSP70, and/or the lipid interphase. Subsequently, they switch their vital function into a lethal function to promote membrane permeabilization and protein release. In this review, we will describe some general rules of inter-organelle cross-talk activating MMP and will review selected examples of pro-apoptotic protein translocation. Finally, we will propose new pharmacological strategies to modulate this process in a therapeutic perspective.     

Differential involvement of p38 MAP kinase pathway and Bax translocation in the mitochondria-mediated cell death in TCR- and dexamethasone-stimulated thymocytes

Mitochondria play a central role in many apoptotic reactions. Although mitochondrial apoptotic changes and caspase activation have been demonstrated in the apoptotic thymocytes, cell death signal through mitochondria in TCR-stimulated thymocytes has not been fully understood. In this study, we show that TCR stimulation induced disruption of mitochondrial transmembrane potential (Delta Psi(m)), the cytochrome c release from mitochondira, capase-3 activation, and the cell death of thymocytes. Bongkrekic acid, an inhibitor of Delta Psi(m) disruption, blocked the cytochrome c release from mitochondria and the following caspase-3-mediated cell death. Furthermore, a pro-apoptotic Bcl-2 family protein, Bax, but not Bad or Bid, was translocated from cytosol to mitochondria in TCR-stimulated thymocytes. This translocation and the following apoptotic changes were inhibited by SB203580, a p38 kinase inhibitor, in a specific manner. These results suggest that activated p38 kinase pathway by TCR stimulation induces translocation of Bax to mitochondria, causing Delta Psi(m) disruption, and the release of cytochrome c, which finally induces caspase-3-mediated apoptosis in thymocytes.     

依托泊苷通过线粒体信号通路释放细胞色素C诱导Jurkat白血病细胞凋亡

目的：研究在人类Jurkat白血病细胞株中依托泊苷诱导凋亡的分子机制，揭示由依托泊苷启动的凋亡信号通路。 方法：分别用annexin V-FITC和碘化丙啶（PI）染色，通过流式细胞仪测定annexin V阳性和出现亚二倍体DNA的凋亡细胞。以3,3＇-dihexyloxyacarbocyanine iodide ［DiOC6(3)］为染色剂，采用流式细胞术检测细胞线粒体膜电位的变化。采用离心技术分离细胞的胞浆与线粒体。细胞色素c从线粒体转入胞浆，caspase-3的激活，多聚二磷酸腺苷核糖聚合酶（PARP）的切割等蛋白质的表达由免疫印迹技术（Western blotting）检测。 结果：依托泊苷诱导Jurkat白血病细胞凋亡，细胞凋亡与依托泊苷的作用时间呈线性关系。广谱的caspase抑制剂zVAD.fmk可抑制依托泊苷诱导的DNA片段化和磷脂酰丝氨酸外翻。依托泊苷引起的线粒体膜电位下降早于DNA片段化和磷脂酰丝氨酸外翻，形成明显对照的是zVAD.fmk不能阻断依托泊苷诱导的线粒体膜电位的下降。依托泊苷介导细胞色素c从线粒体释放到胞浆，激活caspase-3，caspase-3的底物PARP被切割。 结论：依托泊苷诱导Jurkat白血病细胞株凋亡的机制是降低线粒体膜电位和释放细胞色素c到细胞浆启动线粒体信号转导通路, 最终激活caspase而导致细胞凋亡。     

Bid, a critical mediator for apoptosis induced by the activation of Fas/TNF-R1 death receptors in hepatocytes

The Bcl-2 family proteins consist of both antiapoptosis and pro-apoptosis members that regulate apoptosis typically at the mitochondrial level, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by Caspase 8 in response to Fas/TNF-R1 death receptor activation. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates the downstream caspases. This Bid-mediated pathway is critical in hepatocyte apoptosis induced by Fas/TNF-R1 engagement, where direct activation of cytosolic caspase cascade seems inefficient. The dependence on Bid, and thus on the mitochondrial cytochrome c release, of hepatocyte apoptosis induced by the death receptors also renders it sensitive to the inhibitory regulation by the anti-apoptosis members of the Bcl-2 family proteins, such as Bcl-2 and Bcl-xL. Moreover, the revealing of this death pathway in hepatocytes is important to the understanding of the pathogenesis of a number of hepatic diseases such as hepatitis or endotoxemia-related hepatic failure.     

Lipopolysaccharide protects primary B lymphocytes from apoptosis by preventing mitochondrial dysfunction and bax translocation to mitochondria

Mature B lymphocytes undergo apoptosis when they are cultured in the absence of survival factors. Gram-negative bacterial lipopolysaccharide (LPS) prevents this spontaneous apoptosis. This study aimed to better define the signaling pathway(s) involved in the antiapoptotic activity of this endotoxin. We report here that, in addition to its effects on spontaneous apoptosis, LPS protects B cells from apoptosis induced by the broad-spectrum protein kinase inhibitor staurosporine. LPS increased cell viability and concomitantly maintained the mitochondrial transmembrane potential (DeltaPsim) and high glutathione levels. Moreover, LPS inhibited cytosolic cytochrome c release and decreased caspase-9 activation. Unlike staurosporine, LPS induced the retention of Bax, a proapoptotic protein of the Bcl-2 family, in the cytosol by preventing its translocation to mitochondria. These results suggest that Bax relocalization from the cytosol to the mitochondria is an important step of mature B-cell apoptosis and that the antiapoptotic activity of LPS occurs upstream of mitochondrial events.     

Thimerosal induces neuronal cell apoptosis by causing cytochrome c and apoptosis-inducing factor release from mitochondria

There is a worldwide increasing concern over the neurological risks of thimerosal (ethylmercury thiosalicylate) which is an organic mercury compound that is commonly used as an antimicrobial preservative. In this study, we show that thimerosal, at nanomolar concentrations, induces neuronal cell death through the mitochondrial pathway. Thimerosal, in a concentration- and time-dependent manner, decreased cell viability as assessed by calcein-ethidium staining and caused apoptosis detected by Hoechst 33258 dye. Thimerosal-induced apoptosis was associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, and release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol. Although thimerosal did not affect cellular expression of Bax at the protein level, we observed translocation of Bax from cytosol to mitochondria. Finally, caspase-9 and caspase-3 were activated in the absence of caspase-8 activation. Our data suggest that thimerosal causes apoptosis in neuroblastoma cells by changing the mitochondrial microenvironment.     

Age-related activation of mitochondrial caspase-independent apoptotic signaling in rat gastrocnemius muscle

Mitochondria-mediated apoptosis represents a central process driving age-related muscle loss. However, the temporal relation between mitochondrial apoptotic signaling and sarcopenia as well as the regulation of release of pro-apoptotic factors from the mitochondria has not been elucidated. In this study, we investigated mitochondrial apoptotic signaling in skeletal muscle of rats across a wide age range. We also investigated whether mitochondrial-driven apoptosis was accompanied by changes in the expression of Bcl-2 proteins and components of the mitochondrial permeability transition pore (mPTP). Analyses were performed on gastrocnemius muscle of 8-, 18-, 29- and 37-month-old male Fischer344 x Brown Norway rats (9 per group). Muscle weight declined progressively with advancing age, concomitant with increased apoptotic DNA fragmentation. Cytosolic and nuclear levels of apoptosis inducing factor (AIF) and endonuclease G (EndoG) increased in old and senescent animals. In contrast, cytosolic levels of cytochrome c were unchanged with age. Mitochondrial Bcl-2, Bax and Bid increased dramatically in 37-month-old rats, with no changes in the Bax/Bcl-2 ratio in any of the age groups. Finally, expression of cyclophilin D (CyPD) was enhanced at very old age. Our findings indicate that the mitochondrial caspase-independent apoptotic pathway may play a more prominent role in skeletal muscle loss than caspase-mediated apoptosis.     

c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release

Expression of c-Myc sensitizes cells to a wide range of pro-apoptotic stimuli. We here show that this pro-apoptotic effect is mediated through release of mitochondrial holocytochrome c into the cytosol. First, activation of c-Myc triggers release of cytochrome c from mitochondria. This release is caspase-independent and blocked by the survival factor IGF-1. Second, c-Myc-induced apoptosis is blocked by microinjection of anticytochrome c antibody. In addition, we show that microinjection of holocytochrome c mimics the effect of c-Myc activation, sensitizing cells to DNA damage and to the CD95 pathway. Both p53 and CD95/Fas signaling have been implicated in c-Myc-induced apoptosis but neither was required for c-Myc-induced cytochrome c release. Nonetheless, inhibition of CD95 signaling in fibroblasts did prevent c-Myc-induced apoptosis, apparently by obstructing the ability of cytosolic cytochrome c to activate caspases. We conclude that c-Myc promotes apoptosis by causing the release of cytochrome c, but the ability of cytochrome c to activate apoptosis is critically dependent upon other signals.     

Baicalein induces apoptosis through ROS-mediated mitochondrial dysfunction pathway in HL-60 cells

Baicalein is one component of the dried root of Scutellaria Baicalensis Georgi. (Huang Qin) which is widely used in the traditional Chinese herbal medicine. In this study, we report that baicalein was able to induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by poly-(ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation. The efficacious induction of apoptosis was observed at 100 microM for 6 h. Mechanistic analysis demonstrated that baicalein induced the cleavage of Bid protein, cytochrome c release from mitochondria into cytosol, and activation of caspase-3, -8 and -9. Moreover, baicalein caused elevation of intracellular hydrogen peroxide level. Catalase could effectively block baicalein-induced DNA fragmentation. These data indicate that baicalein may trigger an apoptotic death program through reactive oxygen species (ROS)-mediated mitochondrial dysfunction pathway. The findings enhance our understanding of anticancer function of baicalein in herbal medicine.     

Bax-inhibiting peptide protects glutamate-induced cerebellar granule cell death by blocking Bax translocation

Glutamate-induced excitotoxicity has been implicated in the pathogenesis of various neurological damages and disorders. In the brain damage of immature animals such as neonatal hypoxic-ischemic brain injury, the excitotoxicity appears to be more intimately involved through apoptosis. Bax, a member of the Bcl-2 family proteins, plays a key role in the promotion of apoptosis by translocation from the cytosol to the mitochondria and the release of apoptogenic factors such as cytochrome c. Recently, Bax-inhibiting peptide (BIP), a novel membrane-permeable peptide which can bind Bax in the cytosol and inhibit its translocation to the mitochondria, was developed. To investigate the possibility of a new neuroprotection strategy targeting Bax translocation in glutamate-induced neuronal cell death, cerebellar granule neurons (CGNs) were exposed to glutamate with or without BIP. Pretreatment of CGNs with BIP elicited a dose-dependent reduction of glutamate-induced neuronal cell death as measured by MTT assay. BIP significantly suppressed both the number of TUNEL-positive cells and the increase in caspases 3 and 9 activities induced by glutamate. In addition, immunoblotting after subcellular fractionation revealed that BIP prevented the glutamate-induced Bax translocation to the mitochondria and the release of cytochrome c from the mitochondria. These results suggest that agents capable of inhibiting Bax activity such as BIP might lead to new drugs for glutamate-related diseases in the future.     

Mitochondrial apoptosis-induced channel (MAC) function triggers a Bax/Bak-dependent bystander effect

Collateral spread of apoptosis to nearby cells is referred to as the bystander effect, a process that is integral to tissue homeostasis and a challenge to anticancer therapies. In many systems, apoptosis relies on permeabilization of the mitochondrial outer membrane to factors such as cytochrome c and Smac/DIABLO. This permeabilization occurs via formation of a mitochondrial apoptosis-induced channel (MAC) and was mimicked here by single-cell microinjection of cytochrome c into Xenopus laevis embryos. Waves of apoptosis were observed in vivo from the injected to the neighboring cells. This finding indicates that a death signal generated downstream of cytochrome c release diffused to neighboring cells and ultimately killed the animals. The role of MAC in bystander effects was then assessed in mouse embryonic fibroblasts that did or did not express its main components, Bax and/or Bak. Exogenous expression of green fluorescent protein-Bax triggered permeabilization of the outer membrane and apoptosis in these cells. Time-lapse videos showed that neighboring cells also underwent apoptosis, but expression of Bax and/or Bak was essential to this effect, because no bystanders were observed in cells lacking both of these MAC components. These results may guide development of novel therapeutic strategies to selectively eliminate tumors or minimize the size of tissue injury in degenerative or traumatic cell death.     

Visna/maedi virus-induced apoptosis involves the intrinsic mitochondrial pathway

Summary. Visna/maedi virus (VMV) infection in sheep choroid plexus cells was associated with the appearance of apoptosis and the implication of a caspase-dependent mechanism. Sheep choroid plexus cells were mock-infected or infected with VMV to examine the time course of activation of the intrinsic pathway of apoptosis. The role of mitochondria and related apoptotic events were evaluated. A drop in mitochondrial potential was observed following mitochondrial membrane permeabilization using JC-1, a fluorescent probe, which shifted its fluorescence emission from green to red. Apoptosis Inducing Factor translocated to the nucleus of infected-cells and this translocation was concomitant with the release of cytochrome c in the cytosol of infected-cells and mitochondrial membrane permeabilization which seemed to be regulated by the p53 pathway. Following phosphorylated p53 induced downregulation of bcl-2. In addition, DNA flow cytometric analyses revealed a sub-G peak characteristic of an apoptotic population that gradually appeared as virus infection progressed. No cell cycle arrest was detected in infected cells while p21 expression increased. It was concluded that VMV apoptosis is mediated in part by the activation of p53 and the intrinsic mitochondrial apoptotic pathway.     

The combination of calmodulin antagonists and interferon-gamma induces apoptosis through caspase-dependent and -independent pathways in cholangiocarcinoma cells

Calmodulin (CaM) antagonists have been shown to inhibit tumor cell invasion and metastasis and to induce apoptosis in various tumor models, but the molecular mechanism of CaM antagonist-mediated apoptosis is poorly understood. Here, we demonstrate that interferon (IFN)-gamma induces susceptibility to CaM antagonist-mediated apoptosis in human cholangiocarcinoma cells weakly expressing Fas (Fas-low cells). During CaM antagonist-mediated apoptosis in IFN-gamma-pretreated Fas-low cells, cleavage of caspases-8, -9, and -3 and Bid, release of cytochrome c from the mitochondria and an increase in the free cytosolic calcium concentration were observed. CaM antagonists also caused depolarization of the mitochondrial membrane independent of caspase activation. Although a broad-range caspase inhibitor partially blocked CaM antagonist-mediated apoptosis, the neutralizing Fas antibody had no effect, suggesting that CaM antagonist-mediated apoptosis does not require interaction between CaM antagonists and surface Fas. CaM antagonists induce apoptosis via mechanisms other than inhibition of CaM-dependent protein kinase II and calcineurin, as their inhibitors, KN93 and cyclosporine A, had no effect on apoptosis. Taken together, these results indicate that CaM antagonists induce apoptosis in both caspase-dependent and -independent manners, and that susceptibility to CaM antagonists is modulated by IFN-gamma. The combination of IFN-gamma and CaM antagonists, including tamoxifen, may be a potential therapeutic modality for cholangiocarcinoma and possibly other malignancies.