The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

The Bcl-2 Homology Domain 3 (BH3)-only Proteins Bim and Bid Are Functionally Active and Restrained by Anti-apoptotic Bcl-2 Family Proteins in Healthy Liver

An intrinsic pathway of apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) family proteins. We previously reported that a fine rheostatic balance between the anti- and pro-apoptotic multidomain Bcl-2 family proteins controls hepatocyte apoptosis in the healthy liver. The Bcl-2 homology domain 3 (BH3)-only proteins set this rheostatic balance toward apoptosis upon activation in the diseased liver. However, their involvement in healthy Bcl-2 rheostasis remains unknown. In the present study, we focused on two BH3-only proteins, Bim and Bid, and we clarified the Bcl-2 network that governs hepatocyte life and death in the healthy liver. We generated hepatocyte-specific Bcl-xL- or Mcl-1-knock-out mice, with or without disrupting Bim and/or Bid, and we examined hepatocyte apoptosis under physiological conditions. We also examined the effect of both Bid and Bim disruption on the hepatocyte apoptosis caused by the inhibition of Bcl-xL and Mcl-1. Spontaneous hepatocyte apoptosis in Bcl-xL- or Mcl-1-knock-out mice was significantly ameliorated by Bim deletion. The disruption of both Bim and Bid completely prevented hepatocyte apoptosis in Bcl-xL-knock-out mice and weakened massive hepatocyte apoptosis via the additional in vivo knockdown of mcl-1 in these mice. Finally, the hepatocyte apoptosis caused by ABT-737, which is a Bcl-xL/Bcl-2/Bcl-w inhibitor, was completely prevented in Bim/Bid double knock-out mice. The BH3-only proteins Bim and Bid are functionally active but are restrained by the anti-apoptotic Bcl-2 family proteins under physiological conditions. Hepatocyte integrity is maintained by the dynamic and well orchestrated Bcl-2 network in the healthy liver.     

Simultaneous knock-down of Bcl-xL and Mcl-1 induces apoptosis through Bax activation in pancreatic cancer cells

Anti-apoptotic Bcl-2 family proteins have been reported to play an important role in apoptotic cell death of human malignancies. The aim of this study was to delineate the mechanism of anti-apoptotic Bcl-2 family proteins in pancreatic cancer (PaCa) cell survival. We first analyzed the endogenous expression and subcellular localization of anti-apoptotic Bcl-2 family proteins in six PaCa cell lines by Western blot. To delineate the functional role of Bcl-2 family proteins, siRNA-mediated knock-down of protein expression was used. Apoptosis was measured by Cell Death ELISA and Hoechst 33258 staining. In the results, the expression of anti-apoptotic Bcl-2 family proteins varied between PaCa cell lines. Mcl-1 knock-down resulted in marked cleavage of PARP and induction of apoptosis. Down-regulation of Bcl-2 or Bcl-xL had a much weaker effect. Simultaneous knock-down of Bcl-xL and Mcl-1 strongly induced apoptosis, but simultaneous knock-down of Bcl-xL/Bcl-2 or Mcl-1/Bcl-2 had no additive effect. The apoptosis-inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 was associated with translocation of Bax from the cytosol to the mitochondrial membrane, cytochrome c release, and caspase activation. These results demonstrated that Bcl-xL and Mcl-1 play an important role in pancreatic cancer cell survival. Targeting both Bcl-xL and Mcl-1 may be an intriguing therapeutic strategy in PaCa.     

Differential Dependence on Beclin 1 for the Regulation of Pro-Survival Autophagy by Bcl-2 and Bcl-xL in HCT116 Colorectal Cancer Cells

Autophagy is described to be involved in homeostasis, development and disease, both as a survival and a death process. Its involvement in cell death proceeds from interrelationships with the apoptotic pathway. We focused on survival autophagy and investigated its interplays with the apoptotic machinery. We found that while Mcl-1 remained ineffective, Bcl-2 and Bcl-xL were required for starved cells to display a fully functional autophagic pathway as shown by proteolysis activity and detection of autophagic vesicles. Such pro-autophagic functions of Bcl-2 and Bcl-xL were independent of Bax. However they appeared to operate through non redundant mechanisms as Bcl-xL wielded a tighter control than Bcl-2 over the regulation of autophagy: unlike Bcl-2, Bcl-xL and Atg7 manipulation yielded identical phenotypes suggesting they could be components of the same signalling pathway; Bcl-xL subcellular localisation was modified upon starvation, and importantly Bcl-xL acted independently of Beclin 1. Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway. This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival. Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.     

Bcl-2-related protein family gene expression during oligodendroglial differentiation

Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.     

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.     

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.     

Importance of Bcl-2-family proteins in murine hematopoietic progenitor and early B cells

Mitochondrial apoptosis regulates survival and development of hematopoietic cells. Prominent roles of some Bcl-2-family members in this regulation have been established, for instance for pro-apoptotic Bim and anti-apoptotic Mcl-1. Additional, mostly smaller roles are known for other Bcl-2-members but it has been extremely difficult to obtain a comprehensive picture of the regulation of mitochondrial apoptosis in hematopoietic cells by Bcl-2-family proteins. We here use a system of mouse ‘conditionally immortalized’ lymphoid-primed hematopoietic progenitor (LMPP) cells that can be differentiated in vitro to pro-B cells, to analyze the importance of these proteins in cell survival. We established cells deficient in Bim, Noxa, Bim/Noxa, Bim/Puma, Bim/Bmf, Bax, Bak or Bax/Bak and use specific inhibitors of Bcl-2, Bcl-X_L and Mcl-1 to assess their importance. In progenitor (LMPP) cells, we found an important role of Noxa, alone and together with Bim. Cell death induced by inhibition of Bcl-2 and Bcl-X_L entirely depended on Bim and could be implemented by Bax and by Bak. Inhibition of Mcl-1 caused apoptosis that was independent of Bim but strongly depended on Noxa and was completely prevented by the absence of Bax; small amounts of anti-apoptotic proteins were co-immunoprecipitated with Bim. During differentiation to pro-B cells, substantial changes in the expression of Bcl-2-family proteins were seen, and Bcl-2, Bcl-X_L and Mcl-1 were all partially in complexes with Bim. In differentiated cells, Noxa appeared to have lost all importance while the loss of Bim and Puma provided protection. The results strongly suggest that the main role of Bim in these hematopoietic cells is the neutralization of Mcl-1, identify a number of likely molecular events during the maintenance of survival and the induction of apoptosis in mouse hematopoietic progenitor cells, and provide data on the regulation of expression and importance of these proteins during differentiation along the B cell lineage.Subject terms: Apoptosis, Immune cell death     

Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.     

鱼类和哺乳类Bcl-2家族蛋白的研究进展

细胞凋亡, 即细胞程序性死亡, 在多细胞生物的发育和稳态调控过程中发挥关键作用。Bcl-2家族蛋白是凋亡过程中的主要调控因子, 关于Bcl-2家族蛋白在凋亡过程中的功能及其作用机制一直是研究的热点。已有研究显示Bcl-2家族蛋白不仅作用于线粒体引发凋亡, 并且参与了包括对细胞内质网Ca2+的调控、DNA损伤的修复及与自噬的相互作用等多种反应, 从多方面对细胞的生存状态进行调控。Bcl-2家族蛋白保守存在于脊椎动物和无脊椎动物中, 其功能在进化中存在异同。文章以高等脊椎动物(哺乳动物)和低等脊椎动物(硬骨鱼类)为代表, 总结了近年来Bcl-2家族蛋白在调控宿主凋亡与自噬、DNA损伤及新陈代谢等方面取得的最新进展。该研究为深入了解鱼类和哺乳类Bcl-2家族蛋白的功能和作用机制提供了重要参考。     

Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.     

Mcl-1 antagonizes Bax/Bak to promote effector CD4+ and CD8+ T-cell responses

Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear. Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses in vivo. We found, at the peak of the response to lymphocytic choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4⁺ and CD8⁺ T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death, whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4⁺ and CD8⁺ T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim.     

Selective involvement of BH3-only proteins and differential targets of Noxa in diverse apoptotic pathways

The BH3-only proteins of the Bcl-2 family are known to mediate mitochondrial dysfunction during apoptosis. However, the identity of the critical BH3-only proteins and the mechanism of their action following treatment by diverse apoptotic stimuli remain to be fully resolved. We therefore used RNAi to screen the entire Bcl-2 family for their involvement in three major apoptotic pathways in HeLa cells. We found that Bcl-xL and Mcl-1 are major inhibitors of apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL), endoplasmic reticulum (ER) stress, and proteasome inhibition. Among the 10 BH3-only proteins, Bid and Noxa were found to be critically involved in TRAIL-induced apoptosis, in which Noxa participates by constitutively binding to Mcl-1. Bim and Noxa were found to be necessary for ER stress-induced apoptosis, in which Noxa assisted Bim function by sequestering Mcl-1 and binding to Bcl-xL. As a critical BH3-only protein, Noxa was strongly upregulated and became associated with both Mcl-1 and Bcl-xL during apoptosis induced by proteasome inhibition. In addition, we found that Noxa became 'Mcl-1 free' following treatment by ER stress and proteasome inhibition, but not after TRAIL treatment. These results defined the critical Bcl-2 network during apoptosis and suggested that Noxa participated in triggering mitochondrial dysfunction in multiple apoptotic pathways through distinct mechanisms.     

Cafestol, a coffee-specific diterpene, induces apoptosis in renal carcinoma Caki cells through down-regulation of anti-apoptotic proteins and Akt phosphorylation

Cafestol, one of the major compounds in coffee beans, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity, although the mechanism of action is poorly understood. In the present study, we investigated the effect of cafestol on the apoptotic pathway in human renal Caki cells and other cancer cell lines. Cafestol treatment inhibited Caki cells viability a dose-dependent manner by inducing apoptosis, as evidenced by DNA fragmentation and the accumulation of sub-G1 phase. Cafestol-induced apoptosis is associated with the reduction of mitochondrial membrane potential (MMP), activation of caspase 3, cytochrome c release, and down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and cFLIP). Cafestol-induced apoptosis was blocked by pretreatment with broad caspase inhibitor z-VAD-fmk, showing its dependence on caspases. Ectopic expression of Bcl-2 or Mcl-1 in Caki cells attenuates cafestol-induced apoptosis. In addition, we have also shown that cafestol inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, and PI3K inhibitor LY29004 significantly increases cafestol-induced apoptosis in Caki cells. Taken together, our results show the activity of cafestol to modulate multiple components in apoptotic response of human renal Caki cells and a potential as a therapeutic agent for preventing cancers such as renal carcinoma.     

The anti-apoptotic Bcl-2 family member Mcl-1 promotes T lymphocyte survival at multiple stages

T lymphocyte development and function are tightly regulated by the intrinsic death pathway through members of the Bcl-2 family. Genetic studies have demonstrated that the Bcl-2 family member Mcl-1 is an important anti-apoptotic protein in the development of multiple cell types including T lymphocytes. However, the expression pattern and anti-apoptotic roles of Mcl-1 in T lymphocytes at different developmental stages remain to be fully determined. In this study, we examined the expression pattern of Mcl-1 in different populations of T cells at the single-cell level and found that Mcl-1 protein is constitutively expressed in all T cell populations and up-regulated upon TCR stimulation. We then investigated the role of Mcl-1 in the survival of these different populations by conditionally deleting Mcl-1 at various T cell stages. Our results show that Mcl-1 is required for the survival of double-negative and single-positive thymocytes as well as naive and activated T cells. Furthermore, we demonstrate that Mcl-1 functions together with Bcl-xL to promote double-positive thymocyte survival. Thus, Mcl-1 is a critical anti-apoptotic factor for the survival of T cells at multiple stages in vivo.     

ZBP-89-induced apoptosis is p53-independent and requires JNK

ZBP-89 induces apoptosis in human gastrointestinal cancer cells through a p53-independent mechanism. To understand the apoptotic pathway regulated by ZBP-89, we identified downstream signal transduction targets. Ectopic expression of ZBP-89 induced apoptosis through the mitochondrial pathway and was accompanied by activation of all three MAP kinase subfamilies: JNK1/2, ERK1/2 and p38 MAP kinase. ZBP-89-induced apoptosis was markedly enhanced by ERK inhibition with U0126. In contrast, inhibiting JNK with a JNK1-specific peptide inhibitor or dominant-negative JNK2 expression abrogated ZBP-89-mediated apoptosis. The p38 inhibitor SB202190 had no effect on ZBP-89-induced cell death. Protein dephosphorylation assays revealed that ZBP-89 activates JNK via repression of JNK dephosphorylation. Oligonucleotide microarray analyses revealed that ectopic expression of ZBP-89 downregulated expression of the dual-specificity phosphatase MKP6. Overexpression of MKP6 blocked ZBP-89-induced JNK phosphorylation and PARP cleavage. In addition, ectopic expression of ZBP-89 repressed Bcl-xL and Mcl-1 expression, but had no effect on Bcl-2. Silencing ZBP-89 with small interfering RNA enhanced both Bcl-xL and Mcl-1 expression. Taken together, ZBP-89-mediated apoptosis occurs via a p53-independent mechanism that requires JNK activation.     

Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1, or Bcl-B

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.     

Gamma-secretase inhibition attenuates oxaliplatin-induced apoptosis through increased Mcl-1 and/or Bcl-xL in human colon cancer cells

The Notch signaling pathway plays a significant role in differentiation, proliferation, apoptosis, and stem cell processes. It is essential for maintenance of the normal colon crypt and has been implicated in colorectal cancer oncogenesis. Downregulation of the Notch pathway through gamma-secretase inhibitors (GSIs) has been shown to induce apoptosis and enhance response to chemotherapy in a variety of malignancies. In this study, we analyzed the effect of MRK-003 (Merck), a potent inhibitor of gamma-secretase, on oxaliplatin-induced apoptosis in colon cancer. Unexpectedly, gamma-secretase inhibition reduced oxaliplatin-induced apoptosis while GSI treatment alone was shown to have no effect on growth or apoptosis. We determined that the underlying mechanism of action involved an increase in protein levels of the anti-apoptotic Bcl-2 family members Mcl-1 and/or Bcl-xL which resulted in reduced Bax and Bak activation. Blocking of Mcl-1 and/or Bcl-xL through siRNA or the small molecule inhibitor obatoclax restored the apoptotic potential of cells treated with both oxaliplatin and MRK-003. Moreover, obatoclax synergized with MRK-003 alone to induce apoptosis. Our findings warrant caution when treating colon cancer with the combination of GSIs and chemotherapy, whereas other drug combinations, such as GSIs plus obatoclax, should be explored.     

Caspase-9-induced mitochondrial disruption through cleavage of anti-apoptotic BCL-2 family members

Mitochondrial disruption during apoptosis results in the release of cytochrome c that forms apoptosomes with Apaf-1 and caspase-9. Activation of caspase-9 by dimerization in apoptosomes then triggers a caspase signaling cascade. In addition, other apoptosis signaling molecules released from the mitochondrion, such as apoptosis-inducing factor and endonuclease G, may induce caspase-9-independent apoptosis. To determine the signaling events induced by caspase-9, we used chemically induced dimerization for specific activation of caspase-9. We observed that caspase-9 dimerization resulted in the loss of mitochondrial membrane potential and the cleavage of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1. Moreover, cleavage-resistant Bcl-2, Bcl-xL, or Mcl-1 potently inhibited caspase-9-dependent loss of mitochondrial membrane potential and the release of cytochrome c. Our data suggest that a caspase-9 signaling cascade induces feedback disruption of the mitochondrion through cleavage of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1.     

Exposure to the viral by-product dsRNA or Coxsackievirus B5 triggers pancreatic beta cell apoptosis via a Bim / Mcl-1 imbalance

The rise in type 1 diabetes (T1D) incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA) and a diabetogenic enterovirus (Coxsackievirus B5). Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR) promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.