ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas

BACKGROUND: Inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All IAPs have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. IAPs physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. This allows them to function as sensors and inhibitors of death signals that emanate from a variety of pathways. RESULTS: Here we report the characterization of ML-IAP, a novel human IAP that contains a single BIR and RING finger motif. ML-IAP is a powerful inhibitor of apoptosis induced by death receptors and chemotherapeutic agents, probably functioning as a direct inhibitor of downstream effector caspases. Modeling studies of the structure of the BIR domain revealed it to closely resemble the fold determined for the BIR2 domain of X-IAP. Deletion and mutational analysis demonstrated that integrity of the BIR domain was required for anti-apoptotic function. Tissue survey analysis showed expression in a number of embryonic tissues and tumor cell lines. In particular, the majority of melanoma cell lines expressed high levels of ML-IAP in contrast to primary melanocytes, which expressed undetectable levels. These melanoma cells were significantly more resistant to drug-induced apoptosis. CONCLUSIONS: ML-IAP, a novel human IAP, inhibits apoptosis induced by death receptors and chemotherapeutic agents. The BIR of ML-IAP possesses an evolutionarily conserved fold that is necessary for anti-apoptotic activity. Elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the pathogenesis of this malignancy.     

Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension

The Bcl-2 family of proteins plays a central regulatory role in apoptosis. We have identified a novel, widely expressed Bcl-2 member which we have named Bcl-rambo. Bcl-rambo shows overall structural homology to the anti-apoptotic Bcl-2 members containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike Bcl-2, however, the C-terminal membrane anchor region is preceded by a unique 250 amino acid insertion containing two tandem repeats. No interaction of Bcl-rambo with either anti-apoptotic (Bcl-2, Bcl-x(L), Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) or pro-apoptotic (Bax, Bak, Bik, Bid, Bim, and Bad) members of the Bcl-2 family was observed. In mammalian cells, Bcl-rambo was localized to mitochondria, and its overexpression induces apoptosis that is specifically blocked by the caspase inhibitors, IAPs, whereas inhibitors controlling upstream events of either the 'death receptor' (FLIP, FADD-DN) or the 'mitochondrial' pro-apoptotic pathway (Bcl-x(L)) had no effect. Surprisingly, the Bcl-rambo cell death activity was induced by its membrane-anchored C-terminal domain and not by the Bcl-2 homology region. Thus, Bcl-rambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs.     

Nonlinear regulation of commitment to apoptosis by simultaneous inhibition of Bcl-2 and XIAP in leukemia and lymphoma cells

Apoptosis is a complex pathway regulated by the concerted action of multiple pro- and anti-apoptotic molecules. The intrinsic (mitochondrial) pathway of apoptosis is governed up-stream of mitochondria, by the family of Bcl-2 proteins, and down-stream of mitochondria, by low-probability events, such as apoptosome formation, and by feedback circuits involving caspases and inhibitor of apoptosis proteins (IAPs), such as XIAP. All these regulatory mechanisms ensure that cells only commit to death once a threshold of damage has been reached and the anti-apoptotic reserve of the cell is overcome. As cancer cells are invariably exposed to strong intracellular and extracellular stress stimuli, they are particularly reliant on the expression of anti-apoptotic proteins. Hence, many cancer cells undergo apoptosis when exposed to agents that inhibit anti-apoptotic Bcl-2 molecules, such as BH3 mimetics, while normal cells remain relatively insensitive to single agent treatments with the same class of molecules. Targeting different proteins within the apoptotic network with combinatorial treatment approaches often achieves even greater specificity. This led us to investigate the sensitivity of leukemia and lymphoma cells to a pro-apoptotic action of a BH3 mimetic combined with a small molecule inhibitor of XIAP. Using the computational probabilistic model of the apoptotic pathway, verified by experimental results from human leukemia and lymphoma cell lines, we show that inhibition of XIAP has a non-linear effect on sensitization towards apoptosis induced by the BH3 mimetic HA14-1. This study justifies further ex vivo and animal studies on the potential of the treatment of leukemia and lymphoma with a combination of BH3 mimetics and XIAP inhibitors.     

Overcoming resistance of cancer cells to apoptosis

Discovery of the B cell lymphoma gene 2 (Bcl-2 gene) led to the concept that development of cancers required the simultaneous acquisition, not only of deregulated cell division, but also of resistance to programmed cell death or apoptosis. Apoptosis is arguably the common pathway to cell death resulting from a range of therapeutic initiatives, so that understanding the basis for the resistance of cancer cells to apoptosis may hold the key to development of new treatment initiatives. Much has already been learnt about the apoptotic pathways in cancer cells and proteins regulating these pathways. In most cells, apoptosis is dependent on the mitochondrial dependent pathway. This pathway is regulated by pro- and anti-apoptotic members of the Bcl-2 family, and manipulation of these proteins offers scope for a number of treatment initiatives. Effector caspases activated by the mitochondrial pathway or from death receptor signaling are under the control of the inhibitor of apoptosis protein (IAP) family. Certain proteins from mitochondrial can, however, competitively inhibit their binding to effector caspases. Information about the structure of these proteins has led to initiatives to develop therapeutic agents to block the IAP family. In addition to development of selective agents based on these two (Bcl-2 and IAP) protein families, much has been learnt about signal pathways that may regulate their activity. These in turn might provide additional approaches based on selective regulators of the signal pathways.     

Cyclin-dependent kinase inhibitors,roscovitine and purvalanol,induce apoptosis and autophagy related to unfolded protein response in HeLa cervical cancer cells

Roscovitine (Rosc) and purvalanol (Pur) are competitive inhibitors of cyclin-dependent kinases (CDKs) by targeting their ATP-binding pockets. Both drugs are shown to be effective to decrease cell viability and dysregulate the ratio of pro- and anti-apoptotic Bcl-2 family members, which finally led to apoptotic cell death in different cancer cell lines in vitro. It was well established that Bcl-2 family members have distinct roles in the regulation of other cellular processes such as endoplasmic reticulum (ER) stress. The induction of ER stress has been shown to play critical role in cell death/survival decision via autophagy or apoptosis. In this study, our aim was to investigate the molecular targets of CDK inhibitors on ER stress mechanism related to distinct cell death types in time-dependent manner in HeLa cervical cancer cells. Our results showed that Rosc and Pur decreased the cell viability, cell growth and colony formation, induced ER stress-mediated autophagy or apoptosis in time-dependent manner. Thus, we conclude that exposure of cells to CDK inhibitors induces unfolded protein response and ER stress leading to autophagy and apoptosis processes in HeLa cervical cancer cells.     

Understanding IAP function and regulation: a view from Drosophila

Apoptosis is an active form of cell suicide that results in the orderly death and phagocytosis of cells during normal development and in the adult. Many death signals lead to the activation of members of a family of cysteine proteases known as caspases. These proteins act to transduce death signals from different cellular compartments and they cleave a number of cellular proteins, leading ultimately to many of the biochemical and morphological events associated with death. Many mechanisms act to inhibit cell death upstream of caspase activation. However, only one family of cellular proteins, the inhibitors of apoptosis (IAPs), has been identified that inhibit caspase activation and/or activity. The observations that IAP function is essential for cell survival in Drosophila, and that IAP expression is deregulated in many forms of cancer in humans, argue that IAPs are important cell death inhibitors and that deregulation of their function is likely to be important in human disease. Here we review IAP function, with particular reference to insights that study of the Drosophila IAPs has provided. We also discuss some directions for future study.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Stress-induced apoptosis: Toward a symmetry with receptor-mediated cell death

Apoptosis is a form of programmed cell death executed by caspases activated along signalling pathways initiated by ligation of cell-surface death receptors ( extrinsic pathway ) or by perturbation of the mithocondrial membrane promoted by physical or chemical stress agents ( intrinsic pathway ). In metazoans, this evolutionary conserved, genetically controlled process has a role in a variety of physiological settings, as development, homeostasis of tissues and maintenance of the organism integrity. When deranged by impaired regulation or inappropriate activation apoptosis contributes to the pathogenesis of diseases as autoimmunity, cancer, restenosis, ischaemia, heart failure and neurodegenerative disorders. In this review we will present a survey of the stress-induced intrinsic, mithochondrial, pathway and, based on recent experimental data, we will propose a view compatible with an emergent conceptual symmetry between the two apoptogenic extrinsic and intrinsic pathways. Elements of symmetry present in both the apoptogenic signalling pathways include: early activation of initiator caspases (feed-forwarded by a direct or post-mitocondrial effector caspase-mediated amplification loop in some cell types) and mitochondrial membrane permeabilization with required release of antagonists of active caspase inhibitors (IAPs) in high-level IAPs-expressing cells and apoptosome-mediated amplification of the caspase cascade more or less needed in different cell types.     

Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins

Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1/2 → Bcl/Bcl-xL pathway and may have important clinical implications.     

UV-radiation, apoptosis and skin

Apoptosis or programmed cell death is a key function in regulating skin development, homeostasis and tumorigenesis. The epidermis is exposed to various external stimuli and one of the most important is UV radiation. The UVA and UVB spectra differ in their biological effects and in their depth of penetration through the skin layers. UVB rays are absorbed directly by DNA which results in its damage. UVA can also cause DNA damage but primarily by the generation of reactive oxygen species. By eliminating photodamaged cells, apoptosis has an important function in the prevention of epidermal carcinogenesis. UV-induced apoptosis is a complex event involving different pathways. These include: 1. activation of the tumour suppressor gene p53; 2. triggering of cell death receptors directly by UV or by autocrine release of death ligands; 3. mitochondrial damage and cytochrome C release. The extrinsic pathway through death receptors such as fibroblast-associated, tumour necrosis factor receptor and TNF related apoptosis inducing ligand receptor activate caspase cascade. The intrinsic or mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins, anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w) and the pro-apoptotic (Bax, Bak, Bid). The balance between the pro-apoptotic and anti-apoptotic proteins determines cell survival or death. We discuss recent findings in the molecular mechanisms of UV induced apoptosis.     

Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori

We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis.     

Expression of inhibitor of apoptosis proteins in small- and non-small-cell lung carcinoma cells

Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells both initiate apoptotic signaling, resulting in caspase activation, after treatment with anti-cancer agents. However, in contrast to SCLC cells, NSCLC cells do not fully execute apoptosis. The apoptotic process in NSCLC cells seems to be blocked downstream of caspase activation, thus the failure of NSCLC cells to execute apoptosis could result from inhibition of active caspases by inhibitor of apoptosis proteins (IAPs). Here we investigate the mRNA and protein expression of IAPs in a panel of SCLC and NSCLC cell lines. The NSCLC cell lines had a stronger cIAP-2 expression at both mRNA and protein levels, while the SCLC cell lines had a higher level of XIAP protein. Expression of cIAP-1, cIAP-2, and XIAP, the most potent caspase inhibitors, was further investigated in three lung carcinoma cell lines after treatment with 8 Gy of ionizing radiation or etoposide (VP16). In response to treatment, the level of IAPs was not altered in a way that explained the differences in cellular chemo- and radiosensitivity. The intracellular localization of IAPs was analyzed in untreated and treated lung cancer cells. Surprisingly, we found that cIAP-2 was mainly detected in the mitochondrial fraction, although the function of this protein in mitochondria is unknown. No major relocalization of IAPs was observed after treatment. Taken together, these results indicate that IAPs alone are not the main factor responsible for the resistance of NSCLC cells to treatment.     

Inhibitor of Apoptosis (IAP) Proteins–Modulators of Cell Death and Inflammation

Misregulated innate immune signaling and cell death form the basis of much human disease pathogenesis. Inhibitor of apoptosis (IAP) protein family members are frequently overexpressed in cancer and contribute to tumor cell survival, chemo-resistance, disease progression, and poor prognosis. Although best known for their ability to regulate caspases, IAPs also influence ubiquitin (Ub)-dependent pathways that modulate innate immune signaling via activation of nuclear factor κB (NF-κB). Recent research into IAP biology has unearthed unexpected roles for this group of proteins. In addition, the advances in our understanding of the molecular mechanisms that IAPs use to regulate cell death and innate immune responses have provided new insights into disease states and suggested novel intervention strategies. Here we review the functions assigned to those IAP proteins that act at the intersection of cell death regulation and inflammatory signaling.Apoptosis represents a fundamental biological process that relies on the activation of caspases. Inhibitor of apoptosis (IAP) proteins represent a group of negative regulators of both caspases and cell death. Although best known for their ability to regulate caspases and cell death, it is now clear that they function as arbiters of diverse biological processes (Gyrd-Hansen and Meier 2010). Most prominently, IAPs control ubiquitin (Ub)-dependent signaling events that regulate activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways that in turn drive expression of genes important for inflammation, immunity, cell migration, and cell survival. IAPs thereby function as E3 Ub ligases, mediating the transfer of Ub from E2s to target substrates. This in turn modulates the signaling process through regulating protein stability as well as via nondegradative means (see below for details). Many of the cellular processes controlled by IAPs are frequently deregulated in cancer and, directly or indirectly, contribute to disease initiation, tumor maintenance, and/or progression, making IAPs obvious targets for anticancer therapy (LaCasse et al. 2008). Accordingly, small pharmacological inhibitors of IAPs, frequently referred to as Smac-mimetics (SM), were developed and are currently undergoing clinical trials for the treatment of cancer (LaCasse et al. 2008). The use of SMs in preclinical tumor models and clinical trials has provided compelling evidence for the therapeutic benefit of IAP inhibition.     

Modulation of mitochondrial apoptosis by PI3K inhibitors

Most anticancer therapies exert their action by triggering programmed cell death (apoptosis) in cancer cells. The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization, leading to the release of apoptogenic factors such as cytochrome c or Smac from the mitochondrial intermembrane space into the cytosol. Mitochondrial outer membrane permeabilization is tightly controlled, for example by pro- and anti-apoptotic proteins of the Bcl-2 family. Recent evidence indicates that inhibition of the PI3K/Akt/mTOR pathway by small-molecule PI3K inhibitors primes cancer cells to mitochondrial apoptosis by tipping the balance towards pro-apoptotic Bcl-2 proteins, resulting in increased mitochondrial outer membrane permeabilization. Thus, mitochondrial apoptotic events play an important role in PI3K inhibitor-mediated sensitization for apoptosis.     

Up-regulation of IAPs by PI-3K: a cell survival signal-mediated anti-apoptotic mechanism

Under normal cell physiology, a balance between cell survival and apoptosis is crucial for homeostasis. Many studies have demonstrated that apoptosis is modulated by cell survival stimuli. Active Akt, a common mediator of cell survival signals, has been shown to inhibit apoptosis by attenuating activity of pro-apoptotic factors Bad and caspase-9. However, the anti-apoptotic mechanisms mediated by various cell survival signals are poorly understood. Human prostate cancer LNCaP cells, known to contain constitutively activated Akt as a result of a frame-shift mutation in PTEN, an inhibitor of PI-3K/Akt pathway, were observed to be completely resistant to TRAIL-induced apoptosis. In agreement with the known action of Akt, blockade of the PI-3K/Akt pathway rendered LNCaP cells highly susceptible to TRAIL. Importantly, active PI-3K/Akt prevented processing/activation of caspase-3, a phenomenon associated with the function of inhibitor of apoptosis proteins (IAPs). In fact, inhibition of PI-3K activity using Wortmannin significantly decreased the protein levels of IAPs, concomitantly promoting processing/activation of caspase-3 and TRAIL-induced apoptosis. My data indicate that in addition to blocking Bad and caspase-9 through Akt, PI-3K also inhibits caspase-3 through up-regulating IAPs, thereby attenuates apoptosis.     

The mitochondrial antiviral signaling protein, MAVS, is cleaved during apoptosis

Apoptosis of virus-infected cells is one important host strategy used to limit viral infection. Recently a member of the innate immune signaling pathway, MAVS, was localized to mitochondria, an organelle important for apoptosis regulation. Here we investigate what role MAVS may play in apoptosis. Induction of cell death led to the rapid cleavage of MAVS, resulting in its release from the outer mitochondrial membrane. This cleavage is blocked in cells incubated with proteasome or caspase inhibitors. Transfection of synthetic viral dsRNA and dsDNA also led to cleavage of MAVS, indicating that this process may be important during infection. Preventing apoptosis by over-expression of anti-apoptotic Bcl-xL blocks MAVS cleavage, placing this process downstream of caspase activation in the apoptotic program.