NM23-H2 involves in negative regulation of Diva and Bcl2L10 in apoptosis signaling

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. Diva (Boo), an ortholog of Bcl2L10 or Bcl-B, is a member of the Bcl-2 family that has contradictory functions in apoptosis. To understand the signaling mechanisms of Diva, we searched for proteins that interact with Diva using the yeast two-hybrid system. We identified a nucleoside diphosphate kinase isoform, NM23-H2. Here, we show that Diva bound to NM23-H2 in cells in which the transmembrane domain of Diva was required, and both proteins were colocalized in cytoplasm. Of interest, Diva protein level was significantly down-regulated by NM23-H2 as knock down of NM23-H2 restored Diva expression. Overexpression of NM23-H2 induced apoptosis, and the depletion of NM23-H2 led to the increase of Diva's apoptotic activity. Thus, these results indicate the existence of a previously undiscovered mechanism by which NM23-H2 involves in the regulation of Diva-mediated apoptosis.     

Sequence-specific 1H, 13C, and 15N resonance assignments of Diva (Boo), an apoptosis regulator of the Bcl-2 family

Diva, also known as Boo, is a member of the Bcl-2 family that regulates apoptosis by interacting with other Bcl-2 proteins and with the key component of the apoptosome Apaf-1. The function of Diva is puzzling as it apparently can both promote and inhibit apoptosis depending on the cellular context. The structural characterization of Diva will likely help to understand this dual behavior in programmed cell death. To this aim we report here the NMR resonance assignments of residues 1–160 of mouse Diva (lacking the predicted transmembrane domain, which spans residues 161–191). These data are used to obtain information on Diva’s secondary structure and provide the basis for further structural studies.     

Bcl-B, a novel Bcl-2 family member that differentially binds and regulates Bax and Bak

A novel human member of the Bcl-2 family was identified, Bcl-B, which is closest in amino acid sequence homology to the Boo (Diva) protein. The Bcl-B protein contains four Bcl-2 homology (BH) domains (BH1, BH2, BH3, BH4) and a predicted carboxyl-terminal transmembrane (TM) domain. The BCL-B mRNA is widely expressed in adult human tissues. The Bcl-B protein binds Bcl-2, Bcl-X(L), and Bax but not Bak. In transient transfection assays, Bcl-B suppresses apoptosis induced by Bax but not Bak. Deletion of the TM domain of Bcl-B impairs its association with intracellular organelles and diminishes its anti-apoptotic function. Bcl-B thus displays a unique pattern of selectivity for binding and regulating the function of other members of the Bcl-2 family.     

Characterization of NR13-related human cell death regulator, Boo/Diva, in normal and cancer tissues

Mouse Boo/Diva is an ovary-specific member of the Bcl-2 family identified through homology with the avian cell death antagonist NR13. We identified a human orthologue of Boo/Diva, which is highly conserved between mouse and human and related to avian NR13. Human Boo/Diva is also expressed in human liver and kidney in addition to the ovary. We found that green fluorescence protein (EGFP)-tagged Boo/Diva was not exclusively localized to mitochondria before the induction of apoptosis. However, EGFP-Boo/Diva translocated to mitochondria in the process of apoptosis induced by vincristine, a microtubule-interfering agent. Overexpression of human Boo/Diva promoted cell death in HeLa and 293 cells. The cell death antagonist Bcl-XL interacts with Boo, but is unable to protect 293 cells from Boo/Diva-induced cell death. Finally, we mapped human Boo/Diva to chromosome 15q21, a locus known to be related to human cervical cancer. Moreover, we found that genomic DNAs of three of 24 human cervical cancer samples display deletions within their Boo/Diva genes. This result suggests a role for human Boo/Diva in the pathogenesis of cervical cancer.     

Murine ovarian development is not affected by inactivation of the bcl-2 family member diva

Diva (also called Boo/Bcl-B) is a member of the Bcl-2 gene family and most likely functions during apoptosis. Diva is highly expressed in the ovary, and both pro- and antiapoptotic functions have been ascribed to this protein. To determine the role of Diva during murine development, we used gene targeting to inactivate DIVA: The Diva-null mice are born at the expected ratios, are fertile, and have no obvious histological abnormalities, and long-term survival did not differ from littermate controls. Additionally, Diva was not required for apoptosis occurring from genotoxic insult in the ovaries or other organs. Thus, Diva is not critical for the normal development of the ovaries, or in its absence its function is subserved by another protein.     

Characterization of a novel interaction between Bcl-2 members Diva and Harakiri

Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13%) α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding.     

Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,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 signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Demethoxycurcumin induces Bcl-2 mediated G2/M arrest and apoptosis in human glioma U87 cells

Docking analysis of curcumin (C1), demethoxycurcumin (C2) and bisdemethoxycurcumin (C3) with Bcl-2 illustrated that among the three curcuminoids, C2 binds more efficiently into its putative active site. C1, C2 and C3 were purified from turmeric rhizomes to demonstrate the molecular mechanism of their anticancer activity on human glioma U87 cells. Human glioma U87 cells treated with curcuminoids resulted in activation of Bcl-2 mediated G2 checkpoint, which was associated with the induction of G2/M arrest and apoptosis. The binding of C1, C2 and C3 with Bcl-2 protein was confirmed with circular dichroism (CD) spectroscopy. Present work revealed that C2 induced Bcl-2 mediated G2/M arrest and apoptosis most effectively.     

Bcl-2 attenuates anticancer agents-induced apoptosis by sustained activation of Akt/protein kinase B in U937 cells

Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family contributes to resistance to anticancer therapeutic drugs. Thus, this protein represent attractive target for novel anticancer agents. In the present study, we determined the effect of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-γ1 degradation and Akt activation during the various anticancer agents-induced apoptosis. Treatment with chrysin for 12 h produced morphological features of apoptosis in U937 cells, which was associated with caspase-3 activation and PLC-γ1 degradation. Induction of apoptosis was also accompanied by down-regulation of XIAP and inactivation of Akt. Chrysin-induced caspase-3 activation, PLC-γ1 degradation and apoptosis were significantly attenuated in Bcl-2 overexpressing U937/Bcl-2 cells. Ectopic expression of Bcl-2 appeared to inhibit ceramide-, and Akt specific inhibitor (SH-6)-induced apoptosis by sustained Akt activation. Thus, our findings imply that some of the biological functions of Bcl-2 may be attributed to their ability to inhibit anticancer agents-induced apoptosis through the sustained Akt activation.     

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (ΔΨm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-X_L, Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-X_L sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.     

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.     

Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.     

Glycogen synthase kinase 3alpha and 3beta mediate a glucose-sensitive antiapoptotic signaling pathway to stabilize Mcl-1

Glucose uptake and utilization are growth factor-stimulated processes that are frequently upregulated in cancer cells and that correlate with enhanced cell survival. The mechanism of metabolic protection from apoptosis, however, has been unclear. Here we identify a novel signaling pathway initiated by glucose catabolism that inhibited apoptotic death of growth factor-deprived cells. We show that increased glucose metabolism protected cells against the proapoptotic Bcl-2 family protein Bim and attenuated degradation of the antiapoptotic Bcl-2 family protein Mcl-1. Maintenance of Mcl-1 was critical for this protection, as glucose metabolism failed to protect Mcl-1-deficient cells from apoptosis. Increased glucose metabolism stabilized Mcl-1 in both cell lines and primary lymphocytes via inhibitory phosphorylation of glycogen synthase kinase 3alpha and 3beta (GSK-3alpha/beta), which otherwise promoted Mcl-1 degradation. While a number of kinases can phosphorylate and inhibit GSK-3alpha/beta, we provide evidence that protein kinase C may be stimulated by glucose-induced alterations in diacylglycerol levels or distribution to phosphorylate GSK-3alpha/beta, maintain Mcl-1 levels, and inhibit cell death. These data provide a novel nutrient-sensitive mechanism linking glucose metabolism and Bcl-2 family proteins via GSK-3 that may promote survival of cells with high rates of glucose utilization, such as growth factor-stimulated or cancerous cells.     

Caspase cleavage of Mcl-1 impairs its anti-apoptotic activity and proteasomal degradation in non-small lung cancer cells

Global cleavage of cellular proteins by activated caspases is a hallmark of apoptosis, which causes biochemical collapse of the cell. Recent studies suggest that, rather than completely destroying a protein, caspase cleavage can confer novel characteristics or functions. In this respect, the post-caspase role of Bcl-2 family proteins remains uncharacterized. Here, we showed that Mcl-1, a pro-survival member of the Bcl-2 family, was cleaved by caspase-3 in non-small cell lung cancer (NSCLC) cells undergoing chemotherapeutic agent-triggered apoptosis. Caspase cleavage partially impaired the anti-apoptotic activity of Mcl-1 by reducing its mitochondrial localization and impeding its association with the permeability transition pore-forming protein Bak. However, the stability of cleaved Mcl-1 was markedly enhanced because it was more refractory to ubiquitination-dependent proteasomal degradation, thereby improving cell viability to a greater extent than full-length Mcl-1 when transiently expressed in NSCLC cells. These findings shed new light on the role of Mcl-1 in apoptosis and suggest potential novel targets for optimizing the tumoricidal capacity of chemotherapy.     

A peroxisome proliferator-activated receptor-gamma agonist, troglitazone, facilitates caspase-8 and -9 activities by increasing the enzymatic activity of protein-tyrosine phosphatase-1B on human glioma cells

Despite dramatic advances in adjuvant therapies, patients with malignant glioma face a bleak prognosis. Because many adjuvant therapies seek to induce glioma apoptosis, strategies that lower thresholds for the induction of apoptosis may improve patient outcomes. Therefore, elucidation of the biological mechanisms that underlie resistance to current therapies is needed to develop new therapeutic strategies. Here we proposed a novel mechanism of proapoptotic effect induced by a pharmacological peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, troglitazone, that facilitates caspase signaling in human glioma cells. Troglitazone activates protein-tyrosine phosphatase (PTP)-1B, which subsequently reduces phosphotyrosine 705 STAT3 (pY705-STAT3) via a PPARgamma-independent pathway. Reduction of pY705-STAT3 in glioma cells caused down-regulation of FLIP (FADD-like IL-1beta-converting enzyme-inhibitory protein) and Bcl-2. Furthermore, troglitazone induced Ser-392 phosphorylation of p53 via a PPARgamma-dependent pathway and up-regulation of Bax in a p53 wild-type glioma. When given with tumor necrosis factor-related apoptosis-inducing ligand or caspase-dependent chemotherapeutic agents, such as etoposide and paclitaxel, troglitazone exhibited a synergistic effect by facilitating caspase-8/9 activities. A PPARgamma antagonist, GW9662, did not block this effect, although a PTP inhibitor abrogated it. Knockdown of STAT3 by STAT3-small interfering RNA negated the inhibitory effect of PTP inhibitor on troglitazone, indicating that troglitazone uses a STAT3 inactivation mechanism that makes caspase-8/9 activities susceptible to cytotoxic agents in glioma cells and that PTP1B plays a critical role in the down-regulation of activated STAT3, as well as FLIP and Bcl-2. When taken with caspase-dependent anti-neoplastic agents, troglitazone may be a promising drug for use against malignant gliomas because it facilitates the caspase cascade, thereby lowering thresholds for the apoptosis induction of glioma cells.     

Homology modeling and docking studies of human Bcl-2L10 protein

Cancer, an unrestrained proliferation of cells, is one of the lead cause of death. Nearly 12.5 million people are diagnosed with cancer worldwide, 7.5 million people die of which 2.5 million cases are from India. Major cause for cancer is restriction of programmed cell death (apoptosis). Multiple signaling pathways regulate apoptosis. Bcl-2 (B - Cell Lymphomas-2) family proteins play a vital role as central regulators of apoptosis. Bcl-2L10, a novel anti-apoptotic protein, blocks apoptosis by mitochondrial dependent mechanism. The present study evaluates the 3D structure of Bcl-2L10 protein using homology modeling and aims to understand plausible functional and binding interactions between Bcl-2L10 with BH3 domain of BAX using protein - protein docking. The docking studies show binding of BH3 domain at Lys 110, Trp-111, Pro-115, Glu-119 and Asp-127 in the groove of BH 1, 2 and 3 domains of Bcl-2L10. Heterodimerization of anti-apoptotic Bcl-2 and BH3 domain of pro-apoptotic Bcl-2 proteins instigates apoptosis. Profound understanding of Bcl-2 pathway may prove useful in identification of future therapeutic targets for cancer.     

APAF-1-ALT,a novel alternative splicing form of APAF-1, potentially causes impeded ability of undergoing DNA damage-induced apoptosis in the LNCaP human prostate cancer cell line

We have found a novel alternative splicing product of the apoptotic protease activating factor 1 (APAF-1), termed APAF-1-ALT, in the LNCaP human prostate cancer cell line. APAF-1-ALT harbors the caspase recruitment domain and an incomplete CED-4 like/ATPase domain, but lacks the WD-40 repeat units. The LNCaP cell expressed the full-length APAF-1 weakly and APAF-1-ALT rather abundantly, especially after mycoplasma infection. LNCaP underwent a retarded DNA damage-induced apoptosis, which was independent of caspase 9 activity. APAF-1-ALT functioned less effectively in inducing apoptosis than did APAF-1-XL, the full-length APAF-1, in transient transfection. Moreover, APAF-1-ALT interfered with APAF-1-XL's ability to induce apoptosis in transient double transfection experiment. These results indicate that APAF-1-ALT is a molecule that is deficient and impeded for mediating apoptosis and that it may contribute to the resistance to DNA damage-induced treatment observed in LNCaP.