Cardiolipin acts as a mitochondrial signalling platform to launch apoptosis

Cardiolipin (CL) is a unique anionic phospholipid specific to the mitochondria. CL influences the activity of electron transport chain enzyme complexes as well as members of the Bcl-2 family. Interactions between Bcl-2 family members and other pro-apoptotic enzymes have been shown to be crucial for the transduction of the apoptotic signalling cascades during programmed cell death. Targeting of tBid to the mitochondria, which is necessary for Bax/Bak oligomerization and cristae remodelling, is dependent on the exposure of CL at contact sites between the inner and outer mitochondrial membranes. Also, the mobilization of cytochrome c, another key apoptotic event, is tightly regulated by the oxidative state of cardiolipin. Moreover, CL has been shown to be essential for translocation and autoprocessing of caspase-8 on the mitochondria after death receptor stimulation. Deficiencies in CL inhibit the formation of tBid and prevent apoptosis by removing an essential activation platform for the autoprocessing of caspase-8. It is now apparent that CL acts as a crucial signalling platform from which it orchestrates apoptosis by integrating signals from a variety of death inducing proteins.     

Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.     

Essential roles of the Bcl-2 family of proteins in caspase-2-induced apoptosis

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Recent studies suggest that it can mediate the death function of tumor suppressor p53 and is activated by a multimeric protein complex, PIDDosome. However, it is not clear how caspase-2 exerts its apoptotic function in cells and whether its enzymatic activity is required for the apoptotic function. In this study, we used both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses to investigate the mechanism by which caspase-2 induces apoptosis. We show that active caspase-2, but neither a catalytically mutated caspase-2 nor active caspase-2 with its inhibitor, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid(-/-) background, and the release could be restored by addition of the wild-type Bid protein, but not by Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax(-/-)Bak(-/-) mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak is essential for apoptosis triggered by caspase-2.     

Caspase-2 triggers Bax-Bak-dependent and -independent cell death in colon cancer cells treated with resveratrol

Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.     

Hepatitis C virus core protein modulates TRAIL-mediated apoptosis by enhancing Bid cleavage and activation of mitochondria apoptosis signaling pathway

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease, which leads to cirrhosis of liver and hepatocellular carcinoma. The HCV core protein, a viral nucleocapsid, has been shown to affect various intracellular events, including cell proliferation and apoptosis. However, the precise mechanisms of the effects are not fully understood. In this study, we show that HCV core protein sensitizes human hepatocellular carcinoma cell line, Huh7, conferred sensitivity to TRAIL-, but not Fas ligand-mediated apoptosis. Huh7 cells are resistant to TRAIL, despite the induction of caspase-8 after TRAIL engagement. However, HCV core protein induces TRAIL apoptosis signaling via sequential induction of caspase-8, Bid cleavage, activation of mitochondrial pathway, and effector caspase-3. HCV core protein also induces activation of caspase-9 after TRAIL engagement, and the induction of TRAIL sensitivity by HCV core protein could be reversed by caspase-9 inhibitor. Therefore, the HCV core protein-induced TRAIL-mediated apoptosis is dependent upon activation of caspase-8 downstream pathway to convey the death signal to mitochondria, leading to activation of mitochondrial signaling pathway and breaking the apoptosis resistance. These results combined indicate that the HCV core protein enhances TRAIL-, but not Fas ligand-mediated apoptotic cell death in Huh7 cells via a mechanism dependent on the activation of mitochondria apoptosis signaling pathway. These results suggest that HCV core protein may have a role in immune-mediated liver cell injury by modulation of TRAIL-induced apoptosis.     

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.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line

We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, down-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.     

Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells

Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.     

Bax targeting to mitochondria occurs via both tail anchor-dependent and -independent mechanisms

Bax is a member of the Bcl-2 family that, together with Bak, is required for permeabilisation of the outer mitochondrial membrane (OMM). Bax differs from Bak in that it is predominantly cytosolic in healthy cells and only associates with the OMM after an apoptotic signal. How Bax is targeted to the OMM is still a matter of debate, with both a C-terminal tail anchor and an N-terminal pre-sequence being implicated. We now show definitively that Bax does not contain an N-terminal import sequence, but does have a C-terminal anchor. The isolated N terminus of Bax cannot target a heterologous protein to the OMM, whereas the C terminus can. Furthermore, if the C terminus is blocked, Bax fails to target to mitochondria upon receipt of an apoptotic stimulus. Zebra fish Bax, which shows a high degree of amino-acid homology with mammalian Bax within the C terminus, but not in the N terminus, can rescue the defective cell-death phenotype of Bax/Bak-deficient cells. Interestingly, we find that Bax mutants, which themselves cannot target mitochondria or induce apoptosis, are recruited to clusters of activated wild-type Bax on the OMM of apoptotic cells. This appears to be an amplification of Bax activation during cell death that is independent of the normal tail anchor-mediated targeting.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase-3 activation, which is required for apoptosis as confirmed using the pan caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role of caspase-8 in curcumin-induced caspase-3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase-3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of proapoptotic proteins such as Bax, Bak, Bid, and Bim. Crosslinking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid, and Bim causes Bax-channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation, and apoptosis. Importantly, p21-deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement of p21 in Apaf-1 dependent caspase activation and apoptosis. Together, our findings demonstrate that Apaf-1, Bax, and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.     

c-Myc augments the apoptotic activity of cytosolic death receptor signaling proteins by engaging the mitochondrial apoptotic pathway

Activation of c-Myc sensitizes cells to apoptosis induction by ligand-activated death receptors. Such sensitization to death receptors by oncogenes may well be the mechanism underlying tumor cell sensitivity to tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (TRAIL). The mechanism by which this c-Myc-induced sensitization occurs is unclear but could involve modulation of expression of death receptors or their ligands or potentiation of the sensitivity of mitochondria to release pro-apoptotic effectors such as holocytochrome c. Here, we show that ectopic expression of the death receptor signaling protein RIP (receptor-interactive protein) triggers apoptosis via a FAS-associated death domain protein (FADD) and caspase 8-dependent pathway. Induction of apoptosis by this intracellular activation of the death receptor signaling pathway is significantly augmented by c-Myc expression. Moreover, c-Myc expression strongly promotes the potential of RIP to induce cytochrome c release from mitochondria. This implicates the mitochondrial apoptotic pathway in this synergy, a notion confirmed by the inability of c-Myc to sensitize to RIP killing in cells lacking the obligate mitochondrial apoptotic effectors Bax and Bak. We conclude that the lethality of the RIP-activated cytosolic caspase 8 pathway is augmented by c-Myc priming mitochondria to release cytochrome c. This places the intersection of apoptotic synergy between c-Myc and death receptor signaling downstream of the death receptors.     

N-acetylphytosphingosine-induced apoptosis of Jurkat cells is mediated by the conformational change in Bak

N-acetylphytosphingosine (NAPS), a sphingolipid derivative, is one of the well-known signal molecules that mediates various cellular functions, including cell growth, differentiation, and apoptosis. In this study, we demonstrated that NAPS induces apoptosis of Jurkat cells by activating Bak, but not Bax, which are both members of a proapoptotic subfamily of the Bcl-2 proteins. NAPS activated caspase-8 in a FADD-independent manner, but the lack of caspase-8 did not suppress the activation of caspase-3 and -9 and cell death, indicating that caspase-8 activation does not play an important role in NAPS-induced cell death. The overexpression of Bcl-x_L, an anti-apoptotic protein, completely inhibited the activation of the caspases and apoptosis, assuming that NAPS-induced apoptosis was initiated by the mitochondria. The expression levels of pro- and anti-apoptotic Bcl-2 family members were not changed by the NAPS treatment. However, Bad was translocated from the cytosol into the mitochondria, where it bound to Bcl-x_L, and Bak was dissociated from Bcl-x_L and conformationally changed. Taken together, these findings indicate that NAPS induced apoptosis of Jurkat cells in a mitochondria-dependent manner that was controlled by the translocation of Bad and the conformational change in Bak. These authors contributed equally to this paper     

Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces programmed cell death through the caspase activation cascade and translocation of cleaved Bid (tBid) by the apical caspase-8 to mitochondria to induce oligomerization of multidomain Bax and Bak. However, the roles of prosurvival Bcl-2 family proteins in TRAIL apoptosis remain elusive. Here we showed that, besides the specific cleavage and activation of Bid by caspase-8 and caspase-3, TRAIL-induced apoptosis in Jurkat T cells required the specific cleavage of Mcl-1 at Asp-127 and Asp-157 by caspase-3, while other prototypic antiapoptotic factors such as Bcl-2 or Bcl-X(L) seemed not to be affected. Mutation at Asp-127 and Asp-157 of Mcl-1 led to cellular resistance to TRAIL-induced apoptosis. In sharp contrast to cycloheximide-induced Mcl-1 dilapidation, TRAIL did not activate proteasomal degradation of Mcl-1 in Jurkat cells. We further established for the first time that the C-terminal domain of Mcl-1 became proapoptotic as a result of caspase-3 cleavage, and its physical interaction and cooperation with tBid, Bak, and voltage-dependent anion-selective channel 1 promoted mitochondrial apoptosis. These results suggested that removal of N-terminal domains of Bid by caspase-8 and Mcl-1 by caspase-3 enabled the maximal mitochondrial perturbation that potentiated TRAIL-induced apoptosis.     

Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis

Although tissue transglutaminase (tTG) has been recognized as a mediator of apoptosis in various experimental models, little is currently known about the molecular mechanisms by which this protein modulates cell death. Recent work from our laboratory has shown that activation of tTG in cells exposed to the apoptotic inducer calphostin C triggers the crosslinking of dual leucine zipper-bearing kinase (DLK), a proapoptotic kinase acting as an essential component of the c-Jun amino-terminal kinase (JNK) signaling pathway. As a consequence of this observation, we have undertaken experiments to investigate the functional relevance of DLK oligomerization in tTG-mediated apoptosis. Our results indicate that, in cells undergoing calphostin C-induced apoptosis, tTG-dependent DLK oligomerization occurs early in the apoptotic response. Both immunocomplex kinase assays and immunoblotting with phosphospecific antibodies revealed that oligomer formation by tTG-mediated crosslinking reactions significantly enhanced the kinase activity of DLK and its ability to activate the JNK pathway. Moreover, functional studies demonstrate that tTG-mediated oligomerization of wild-type DLK sensitizes cells to calphostin C-induced apoptosis, while crosslinking of a kinase-inactive variant of DLK does not. Collectively, these data strongly suggest that tTG facilitates apoptosis, at least partly, by oligomerization and activation of the proapoptotic kinase DLK.     

Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis

Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level.     

p38 MAPK and MSK1 mediate caspase-8 activation in manganese-induced mitochondria-dependent cell death

Heavy metals are important regulators of cell apoptosis. Manganese (Mn(2+)) is a potent inducer of apoptosis in different cell types, but the precise mechanisms that mediate such effects are not well defined. We previously reported that Mn(2+) was a potent apoptotic agent in human B cells, including lymphoma B cell lines. We show here that Mn(2+)-induced cell death in human B cells is associated with caspase-8-dependent mitochondrial activation leading to caspase-3 activity and apoptosis. We used specific caspase-8 interfering shRNAs to reduce caspase-8 expression, and this also reduced Mn(2+)-induced caspase-3 activation and apoptosis. Mn(2+)-triggered caspase-8 activation is associated with a specific pathway, which is independent of Fas-associated death domain protein, and dependent on the sequential activation of p38-mitogen-activated protein kinase (p38 MAPK) and mitogen- and stress-response kinase 1 (MSK1). Inhibition of p38 activity using either pharmacological inhibitors or dominant-negative mutant forms of p38 blocked Mn(2+)-mediated phosphorylation of MSK1 and blocked subsequent caspase-8 activation. However, specific inhibitors and the expression of a dominant-interfering mutant of MSK1 only inhibited caspase-8 activation, but not p38 activity. These findings suggest a novel model for the regulation of caspase-8 during Mn(2+)-induced apoptosis based on the sequential activation of p38 MAPK, MSK1, caspase-8 and mitochondria, respectively.     

p38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.     

Bax-dependent regulation of Bak by voltage-dependent anion channel 2

Many studies have demonstrated a critical role of Bax in mediating apoptosis, but the role of Bak in regulating cancer cell apoptotic sensitivities in the presence or absence of Bax remains incompletely understood. Using isogenic cells with defined genetic deficiencies, here we show that in response to intrinsic, extrinsic, and endoplasmic reticulum stress stimuli, HCT116 cells show clear-cut apoptotic sensitivities in the order of Bax+/Bak+ > Bax+/Bak- > Bax-/Bak+ > Bax-/Bak-. Small interference RNA-mediated knockdown of Bak in Bax-deficient cells renders HCT116 cells completely resistant to apoptosis induction. Surprisingly, however, Bak knockdown in Bax-expressing cells only slightly affects the apoptotic sensitivities. Bak, like Bax, undergoes the N terminus exposure upon apoptotic stimulation in both Bax-expressing and Bax-deficient cells. Gel filtration, chemical cross-linking, and co-immunoprecipitation experiments reveal that different from Bax, which normally exists as monomers in unstimulated cells and is oligomerized by apoptotic stimulation, most Bak in unstimulated HCT116 cells exists in two distinct protein complexes, one of which contains voltage-dependent anion channel (VDAC) 2. During apoptosis, Bak and Bax form both homo- and hetero-oligomeric complexes that still retain some VDAC-2. However, the oligomeric VDAC-2 complexes are diminished, and Bak does not interact with VDAC-2 in Bax-deficient HCT116 cells. These results highlight VDAC-2 as a critical inhibitor of Bak-mediated apoptotic responses.     

c-Myc primed mitochondria determine cellular sensitivity to TRAIL-induced apoptosis

Oncogenic c-Myc renders cells sensitive to TRAIL-induced apoptosis, and existing data suggest that c-Myc sensitizes cells to apoptosis by promoting activation of the mitochondrial apoptosis pathway. However, the molecular mechanisms linking the mitochondrial effects of c-Myc to the c-Myc-dependent sensitization to TRAIL have remained unresolved. Here, we show that TRAIL induces a weak activation of procaspase-8 but fails to activate mitochondrial proapoptotic effectors Bax and Bak, cytochrome c release or downstream effector caspase-3 in non-transformed human fibroblasts or mammary epithelial cells. Our data is consistent with the model that activation of oncogenic c-Myc primes mitochondria through a mechanism involving activation of Bak and this priming enables weak TRAIL-induced caspase-8 signals to activate Bax. This results in cytochrome c release, activation of downstream caspases and postmitochondrial death-inducing signaling complex -independent augmentation of caspase-8-Bid activity. In conclusion, c-Myc-dependent priming of the mitochondrial pathway is critical for the capacity of TRAIL-induced caspase-8 signals to activate effector caspases and for the establishment of lethal caspase feedback amplification loop in human cells.