Bcl-2 and caspase inhibition cooperate to inhibit tumor necrosis factor-alpha-induced cell death in a Bcl-2 cleavage-independent fashion.

The ability of proteins of the Bcl-2 family to either induce or inhibit apoptosis is dependent on both cell type and the apoptotic stimulus. We have shown in the murine pro-B cell line FL5.12 that Bcl-2 is incapable of inhibiting tumor necrosis factor alpha (TNFalpha)-induced cell death and is cleaved during this process. One potential explanation for this observation is that caspase activation directly or indirectly inhibits Bcl-2 function. It has been suggested that caspase cleavage of Bcl-2 is responsible for its inability to block certain cell deaths. Consistent with Bcl-2 cleavage being a caspase-mediated event, this cleavage is inhibitable by 50 microM CBZ-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Furthermore, Bcl-2 can cooperate with the caspase inhibitor zVAD-fmk in a dose-dependent manner to block TNFalpha-induced cell death. Overexpression of Bcl-2 results in a 10-fold decrease in the amount of zVAD-fmk required to inhibit TNFalpha-induced apoptosis. However, cleavage-defective mutants (D31A and D34A) show no enhanced viability relative to wild-type Bcl-2 in response to TNFalpha-induced cell death and also show the same cooperativity with zVAD-fmk. These results suggest that Bcl-2 cleavage is not important for the inhibition of TNFalpha-induced cell death but do not preclude an involvement in a post-commitment phase of apoptosis.     

Anti-autophagic Bcl-2

Bcl-2, originally identified as a universal inhibitor of apoptotic cell death, has since been implicated in suppressing autophagy, the cell’s quality control mechanism. Our recent study demonstrates that the anti-autophagic aspect of Bcl-2 can function as a promoter of oncogenic growth, independently of its role in apoptosis signaling. It is likely that the increase in Bcl-2 often seen in breast and other cancers might render cells error-prone by blunting autophagy, while concomitantly keeping damaged cells alive. The outcome of such a ‘double hit’ of Bcl-2 may synergistically promote tumor growth and increase the chance of cancer development and drug resistance.     

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2.

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.     

Curcumin sensitizes non-small cell lung cancer cell anoikis through reactive oxygen species-mediated Bcl-2 downregulation

Anoikis, an apoptosis triggered by loss of cell anchorage, has been shown to be a principal mechanism of inhibition of tumor metastasis. Recently, anti-apoptotic Bcl-2 and Cav-1 proteins have been demonstrated to be highly associated with tumor metastasis and apoptosis resistance. Curcumin, a major active component of turmeric, Curcuma longa, has been shown to inhibit neoplastic evolution and tumor progression; however, the underlying mechanisms are unclear. In this study, we investigated the effect of curcumin on cell anoikis as a possible mechanism of anti-tumorigenic action of curcumin, and evaluated the potential role of Bcl-2 and Cav-1 in this process. Our results showed that ectopic expression of either Bcl-2 or Cav-1 induced anoikis resistance of lung carcinoma H460 cells. Curcumin downregulated Bcl-2 protein during anoikis and sensitized the cells to detachment-induced apoptosis, whereas it had no significant effect on Cav-1 protein expression. Bcl-2 down-regulation as well as anoikis enhancement by curcumin were inhibited by superoxide anion scavenger, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride, but were unaffected by other ROS scavengers including catalase and deferoxamine, suggesting that superoxide anion is a key player in the downregulation of Bcl-2 by curcumin. Furthermore, we provided evidence that curcumin decreased Bcl-2 level through ubiquitin-proteasomal degradation which sensitized cells to detachment-induced apoptosis. These findings indicate a novel pathway for curcumin regulation of Bcl-2 and provide a key mechanism of anoikis regulation that may be exploited for metastatic cancer treatment.     

Transient expression of wild-type or mitochondrially targeted Bcl-2 induces apoptosis, whereas transient expression of endoplasmic reticulum-targeted Bcl-2 is protective against Bax-induced cell death

Bcl-2 protein family members function either to promote or inhibit programmed cell death. Bcl-2, typically an inhibitor of apoptosis, has also been demonstrated to have pro-apoptotic activity (Cheng, E. H., Kirsch, D. G., Clem, R. J., et al. (1997) Science 278, 1966-1968). The pro-apoptotic activity has been attributed to the cleavage of Bcl-2 by caspase-3, which converts Bcl-2 to a pro-apoptotic molecule. Bcl-2 is a membrane protein that is localized in the endoplasmic reticulum (ER) membrane, the outer mitochondrial membrane, and the nuclear envelope. Here, we demonstrate that transient expression of Bcl-2 at levels comparable to those found in stably transfected cells induces apoptosis in human embryonic kidney 293 cells and in the human breast cell line MDA-MB-468 cells. Furthermore, we have targeted Bcl-2 specifically to either the ER or the outer mitochondrial membrane to test whether induction of apoptosis by Bcl-2 is dependent upon its localization within either of these membranes. Our findings indicate that Bcl-2 specifically targeted to the mitochondria induces cell death, whereas Bcl-2 that is targeted to the ER does not. The expression of Bcl-2 does result in its cleavage to a 20-kDa protein; however, mutation of the caspase-3 cleavage site (D34A) does not inhibit its ability to induce cell death. Additionally, we find that transiently expressed ER-targeted Bcl-2 inhibits cell death induced by Bax overexpression. In conclusion, the ability of Bcl-2 to promote apoptosis is associated with its localization at the mitochondria. Furthermore, the ability of ER-targeted Bcl-2 to protect against Bax-induced apoptosis suggests that the ER localization of Bcl-2 may play an important role in its protective function.     

Anti-autophagic Bcl-2: Not just an innocent bystander

Bcl-2, originally identified as a universal inhibitor of apoptotic cell death, has since been implicated in suppressing autophagy, the cell's quality control mechanism. Our recent study demonstrates that the anti-autophagic aspect of Bcl-2 can function as a promoter of oncogenic growth, independently of its role in apoptosis signaling. It is likely that the increase in Bcl-2 often seen in breast and other cancers might render cells error-prone by blunting autophagy, while concomitantly keeping damaged cells alive. The outcome of such a 'double hit' of Bcl-2 may synergistically promote tumor growth and increase the chance of cancer development and drug resistance.     

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.     

A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis.

Resting thymocytes undergoing apoptosis in response to specific stimuli degrade the cdk inhibitor p27(Kip1) and upregulate Cdk2 kinase activity. Inhibition of Cdk2 kinase activity efficiently blocks cell death via certain apoptosis pathways whereas overexpression of Cdk2 accelerates such cell death, suggesting its involvement in the signal transduction pathways activated by certain apoptotic stimuli. We found that Cdk2 activation during thymocyte apoptosis can be regulated by p53, Bax and Bcl-2. The highly elevated Cdk2 kinase activity in the apoptosing thymocytes is not associated with its canonical cyclins, cyclin E and cyclin A, and requires de novo synthesis of proteins for activation to take place. We therefore propose Cdk2 activation to be a crucial event in distinct pathways of apoptosis and the point at which the cell cycle and cell death pathways interact.     

Bcl-2 blocks p53-dependent apoptosis.

Adenovirus E1A expression recruits primary rodent cells into proliferation but fails to transform them because of the induction of programmed cell death (apoptosis). The adenovirus E1B 19,000-molecular-weight protein (19K protein), the E1B 55K protein, and the human Bcl-2 protein each cause high-frequency transformation when coexpressed with E1A by inhibiting apoptosis. Thus, transformation of primary rodent cells by E1A requires deregulation of cell growth to be coupled to suppression of apoptosis. The product of the p53 tumor suppressor gene induces apoptosis in transformed cells and is required for induction of apoptosis by E1A. The ability of Bcl-2 to suppress apoptosis induced by E1A suggested that Bcl-2 may function by inhibition of p53. Rodent cells transformed with E1A plus the p53(Val-135) temperature-sensitive mutant are transformed at the restrictive temperature and undergo rapid and complete apoptosis at the permissive temperature when p53 adopts the wild-type conformation. Human Bcl-2 expression completely prevented p53-mediated apoptosis at the permissive temperature and caused cells to remain in a predominantly growth-arrested state. Growth arrest was leaky, occurred at multiple points in the cell cycle, and was reversible. Bcl-2 did not affect the ability of p53 to localize to the nucleus, nor were the levels of the p53 protein altered. Thus, Bcl-2 diverts the activity of p53 from induction of apoptosis to induction of growth arrest, and it is thereby identified as a modifier of p53 function. The ability of Bcl-2 to bypass induction of apoptosis by p53 may contribute to its oncogenic and antiapoptotic activity.     

An insight into the mechanism of cytotoxicity of ricin to hepatoma cell: roles of Bcl-2 family proteins, caspases, Ca(2+)-dependent proteases and protein kinase C

The ability of ricin, a type II ribosome-inactivating protein, to induce hepatoma cell (BEL7404) to apoptosis in vitro was examined by fluorescence microscopy, flow cytometry, and DNA fragmentation assay. As a Bcl-2 lacking model, BEL7404 bore unique advantage to study the effect of over-expressing Bcl-2 on the apoptosis induced by the inhibitor of protein synthesis. By establishing a Bcl-2 over-expressing cell line (BEL7404/ Bcl-2), we found that Bcl-2 could promote the survival of the hepatoma cell against ricin insult. The ricin-induced apoptosis of BEL7404 was accompanied by increased expression of Bak and decreased levels of Bcl-xl and Bax. Caspases and PARP cleavage activity were found to be implicated in the death process. Through the inhibitor tests, our results excluded the participation of calcium-dependent proteases or protein kinase C in the apoptotic process induced by ricin, though an elevation of intracellular calcium did occur as an immediate response to ricin treatment. Cycloheximide, another protein synthesis inhibitor, did synergistically enhance rather than inhibit the cytotoxicity of ricin to hepatoma cell BEL7404. Actually, cycloheximide alone was able to induce hepatoma cell BEL7404 to death that could also be inhibited by over-expressing Bcl-2. The elevation of apoptotic protein Bak was discussed to challenge the notion that ricin exerted its cytotoxicity through nonspecific inhibition of all the de novo protein synthesis.     

Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells

Etoposide (VP-16) a topoisomerase II inhibitor induces apoptosis of tumor cells. The present study was designed to elucidate the mechanisms of etoposide-induced apoptosis in C6 glioma cells. Etoposide induced increased formation of ceramide from sphingomyelin and release of mitochondrial cytochrome c followed by activation of caspase-9 and caspase-3, but not caspase-1. In addition, exposure of cells to etoposide resulted in decreased expression of Bcl-2 with reciprocal increase in Bax protein. z-VAD.FMK, a broad spectrum caspase inhibitor, failed to suppress the etoposide-induced ceramide formation and change of the Bax/Bcl-2 ratio, although it did inhibit etoposide-induced death of C6 cells. Reduced glutathione or N-acetylcysteine, which could reduce ceramide formation by inhibiting sphingomyelinase activity, prevented C6 cells from etoposide-induced apoptosis through blockage of caspase-3 activation and change of the Bax/Bcl-2 ratio. In contrast, the increase in ceramide level by an inhibitor of ceramide glucosyltransferase-1, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol caused elevation of the Bax/Bcl-2 ratio and potentiation of caspase-3 activation, thereby resulting in enhancement of etoposide-induced apoptosis. Furthermore, cell-permeable exogenous ceramides (C2- and C6-ceramide) induced downregulation of Bcl-2, leading to an increase in the Bax/Bcl-2 ratio and subsequent activation of caspases-9 and -3. Taken together, these results suggest that ceramide may function as a mediator of etoposide-induced apoptosis of C6 glioma cells, which induces increase in the Bax/Bcl-2 ratio followed by release of cytochrome c leading to caspases-9 and -3 activation.     

Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.     

Anti-apoptotic activity of Bcl-2 is enhanced by its interaction with RTN3

Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. It can interact with many members of the Bcl-2 family, influence mitochondrial membrane permeability and modulate cell apoptosis. RTN3, a member of the reticulon (RTN) family, was predominantly localized on the endoplasmic reticulum (ER). Its N- and C-termini, both facing the cytoplasm, can recruit some proteins to the ER to modulate some physiological functions. We found that RTN3, which does not belong to the Bcl-2 family, can interact with Bcl-2 on the ER. In normal HeLa cells, ectopic overexpressed Bcl-2 could reduce the cell apoptosis induced by overexpressed RTN3. When the HeLa cells stably expressing Bcl-2 were treated with tunicamycin, endogenous RTN3 increased in the cell microsomal fraction. This change increased the Bcl-2 in microsomal fractions and also in the mitochondrial fractions where the anti-apoptotic activity of Bcl-2 mainly acts. These results suggest that RTN3 could bind with Bcl-2 and mediate its accumulation in mitochondria, which modulate the anti-apoptotic activity of Bcl-2.     

Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway

Cytotoxic T lymphocytes kill virus-infected and tumor cell targets through the concerted action of proteins contained in cytolytic granules, primarily granzyme B and perforin. Granzyme B, a serine proteinase with substrate specificity similar to the caspase family of apoptotic cysteine proteinases, is capable of cleaving and activating a number of death proteins in target cells. Despite the ability to engage the death pathway at multiple entry points, the preferred mechanism for rapid induction of apoptosis by granzyme B has yet to be clearly established. Here we use time lapse confocal microscopy to demonstrate that mitochondrial cytochrome c release is the primary mode of granzyme B-induced apoptosis and that Bcl-2 is a potent inhibitor of this pivotal event. Caspase activation is not required for cytochrome c release, an activity that correlates with cleavage and activation of Bid, which we have found to be cleaved more readily by granzyme B than either caspase-3 or caspase-8. Bcl-2 blocks the rapid destruction of targets by granzyme B by blocking mitochondrial involvement in the process.     

The phosphorylation status and anti-apoptotic activity of Bcl-2 are regulated by ERK and protein phosphatase 2A on the mitochondria

Bcl-2 protein play important roles in the regulation of apoptosis. We previously reported that the phosphorylation of Bcl-2 was augmented by treatment with protein phosphatase 2A (PP2A) inhibitor; however, the kinase responsible for Bcl-2 phosphorylation had not yet been identified. In this study, we identified extracellular-signal-regulated kinase (ERK) as the responsible kinase for the phosphorylation of Bcl-2. We also found that the transmembrane region (TM) deleted form of Bcl-2 (Bcl-2DeltaTM), which was unable to localize on the mitochondria was constitutively phosphorylated, whereas wild-type Bcl-2 that localized on the mitochondria, was present in its hypophosphorylated form. The phosphorylation of Bcl-2DeltaTM was retarded by treatment with MAP kinase ERK kinase (MEK) inhibitor and PP2A did not bind to Bcl-2DeltaTM. These observations suggest that Bcl-2DeltaTM is constitutively phosphorylated by ERK, but is not dephosphorylated by PP2A in human tumor cell lines. The phosphorylation of Bcl-2 resulted in a reduction in anti-apoptotic function, implying that dephosphorylation promoted the anti-apoptotic activity of Bcl-2 protein in human tumor cell lines. Thus, the present findings suggest that ERK and PP2A are physiological regulators of Bcl-2 phosphorylation, and these enzymes exert an influence on the anti-apoptotic function of Bcl-2.     

Sensitization of prostate carcinoma cells to Apo2L/TRAIL by a Bcl-2 family protein inhibitor

Overexpression of anti-apoptotic Bcl-2 family proteins may play an important role in the aggressive behavior of prostate cancer cells and their resistance to therapy. The Bcl-2 homology 3 domain (BH3) is a uniquely important functional element within the pro-apoptotic class of the Bcl-2-related proteins, mediating their ability to dimerize with other Bcl-2-related proteins and promote apoptosis. The BH3 inhibitors (BH3Is) function by disrupting the interactions mediated by the BH3 domain between pro- and anti-apoptotic members of the Bcl-2 family and liberating more Bax/Bak to induce mitochondrial membrane permeabilization. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to non-tagged, human recombinant soluble Apo2 ligand [Apo2L, also Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL], a tumor specific drug that is now in clinical trials. However, when Apo2L/TRAIL was combined with the Bcl-xL inhibitor, BH3I-2′, it induced apoptosis synergistically through activation of Caspase-8 and the proapoptotic Bcl-2 family member Bid, resulting in the activation of effector Caspase-3 and proteolytic cleavage of Poly(ADP-ribose) polymerase, events that were blocked by the pan-caspase inhibitor zVAD-fmk. Our data indicate that, in combination with the BH3 mimetic, BH3I-2′, Apo2L/TRAIL synergistically induces apoptosis in C4-2 human prostate cancer cells through both the extrinsic and intrinsic apoptotic pathways.     

Suppression of transcription factor NF-kappaB activity by Bcl-2 protein in NIH3T3 cells: implication of a novel NF-kappaB p50-Bcl-2 complex for the anti-apoptotic function of Bcl-2

Bcl-2 can suppress apoptosis by controlling genes that encode proteins required for programmed cell death and by interference with peroxidative damage. Overexpression of Bcl-2 in NIH3T3 cells can prevent GSNO-induced (S-nitrosoglutathione-induced) apoptosis. The experimental results indicated that activation of NF-kappaB by GSNO is involved in inducing apoptosis. Surprisingly, we found that Bcl-2 delayed the release of IkB by formation of a Bcl-2-NF-kappaB complex (p50-p65-IkappaB) in the cytoplasm during cell apoptosis. Furthermore, a novel Bcl-2-p50 complex was found in the nucleus. These features were only observed in Bcl-2-transfected cells but not in the parental NIH3T3 cells. Overexpression of Bcl-2 suppressed the levels of c-myc, a target gene of NF-kappaB, and influenced the DNA-binding activity of NF-kappaB during GSNOinduced apoptosis. We suggest that the Bcl-2-p50 complex inhibits NF-kappaB DNA-binding activity by competing with the p65-p50 heterodimer for the DNA-binding site in the nucleus. Finally, it has been demonstrated that the anti-apoptotic potential of Bcl-2 may be attributed to its complexing with p50 in the nucleus that leads to blockage of nuclear gene expression.