Search

X-linked inhibitor of apoptosis protein (XIAP), survivin, and BRUCE are members of the inhibitor-of-apoptosis protein (IAP) family known for their inhibitory effects on caspase activity and dysregulation of these molecules has widely been shown to cause embryonic defects and to promote tumorigenesis in human. Besides the anti-apoptotic functions, recent discoveries have revealed that XIAP, survivin, and BRUCE also exhibit regulatory functions for autophagy in cells. As the role of autophagy in human diseases has already been discussed extensively in different reviews; in this review, we will discuss the emerging autophagic role of XIAP, survivin, and BRUCE in cancer cells. We also provide an update on the anti-apoptotic functions and the roles in maintaining DNA integrity of these molecules. Second mitochondria-derived activator of caspases (Smac) is a pro-apoptotic protein and IAPs are the molecular targets of various Smac mimetics currently under clinical trials. Better understanding on the functions of XIAP, survivin, and BRUCE can enable us to predict possible side effects of these drugs and to design a more "patient-specific" clinical trial for Smac mimetics in the future.

Background: X-linked inhibitor of apoptosis (XIAP) is a vital factor in the anti-apoptosis mechanism of tumors and is highly expressed in renal cell carcinoma (RCC). However, the mechanism through which XIAP regulates DNA damage repair is unknown. This study investigated the regulatory mechanism of XIAP in etoposide-induced apoptosis in two Caki-1 cell lines with high or low XIAP expression., Methods: The two cell lines were established using RNA interference technology. The differentially expressed proteins in the two cell lines were globally analyzed through an isobaric tags for relative and absolute quantitation-based quantitative proteomics approach. Proteomic analysis revealed 255, 375, 362, and 5 differentially expressed proteins after 0, 0.5, 3, and 12 h of drug stimulation, respectively, between the two cell lines. The identified differentially expressed proteins were involved in numerous biological processes. In addition, the expression of histone proteins (H1.4, H2AX, H3.1, H3.2, and H3.3) was drastically altered, and the effects of XIAP silencing were accompanied by the marked downregulation of H2AX. Protein-protein interactions were assessed and confirmed through immunofluorescence and Western blot analyses., Results: The results suggested that XIAP may act as a vital cell signal regulator that regulates the expression of DNA repair-related proteins, such as H2AX, and influences the DNA repair process., Conclusions: Given these functions, XIAP may be the decisive factor in determining the sensitivity of RCC cell apoptosis induction in response to chemotherapeutic agents.

The E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) acts as a molecular rheostat for the immune deficiency (IMD) pathway of the tick Ixodes scapularis How XIAP activates the IMD pathway in response to microbial infection remains ill defined. Here, we identified the XIAP enzymatic substrate p47 as a positive regulator of the I. scapularis IMD network. XIAP polyubiquitylates p47 in a lysine 63-dependent manner and interacts with the p47 ubiquitin-like (UBX) module. p47 also binds to Kenny (IKKγ/NEMO), the regulatory subunit of the inhibitor of nuclear factor (NF)- κB kinase complex. Replacement of the amino acid lysine to arginine within the p47 linker region completely abrogated molecular interactions with Kenny. Furthermore, mitigation of p47 transcription levels through RNA interference in I. scapularis limited Kenny accumulation, reduced phosphorylation of IKKβ (IRD5), and impaired cleavage of the NF-κB molecule Relish. Accordingly, disruption of p47 expression increased microbial colonization by the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agent Anaplasma phagocytophilum Collectively, we highlight the importance of ticks for the elucidation of paradigms in arthropod immunology. Manipulating immune signaling cascades within I. scapularis may lead to innovative approaches to reducing the burden of tick-borne diseases., Competing Interests: The authors declare no conflict of interest.

Glioblastoma (GBM) is still one of the most lethal forms of brain tumor despite of the improvements in treatments. TRAIL (TNF-related apoptosis-inducing ligand) is a promising anticancer agent that can be potentially used as an alternative or complementary therapy because of its specific antitumor activity. To define the novel pathways that regulate susceptibility to TRAIL in GBM cells, we performed a genome-wide expression profiling of microRNAs in GBM cell lines with the distinct sensitivity to TRAIL-induced apoptosis. We found that the expression pattern of miR-7 is closely correlated with sensitivity of GBM cells to TRAIL. Furthermore, our gain and loss of function experiments showed that miR-7 is a potential sensitizer for TRAIL-induced apoptosis in GBM cells. In the mechanistic study, we identified XIAP is a direct downstream gene of miR-7. Additionally, this regulatory axis could also exert in other types of tumor cells like hepatocellular carcinoma cells. More importantly, in the xenograft model, enforced expression of miR-7 in TRAIL-overexpressed mesenchymal stem cells increased apoptosis and suppressed tumor growth in an exosome dependent manner. In conclusion, we identify that miR-7 is a critical sensitizer for TRAIL-induced apoptosis, thus making it as a promising therapeutic candidate for TRAIL resistance in GBM cells., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The X-linked inhibitor of apoptosis protein (XIAP) is a well-known potent apoptosis suppressor and also participates in cancer cell biological behaviors, therefore attracting great attentions as a potential antineoplastic therapeutic target for past years. Anti-IAP therapy is reported to be closely related to epidermal growth factor receptor (EGFR) expression level. However, whether and how XIAP modulates EGFR expression remains largely unknown., Methods: Human XIAP was knockdown with short-hairpin RNA in two different bladder cancer cell lines, T24T and UMUC3. Two XIAP mutants, XIAP ∆BIR (deletion of N-terminal three BIR domains) and XIAP ∆RING (deletion of C-terminal RING domain and keeping the function of BIR domains), were generated to determine which domain is involved in regulating EGFR., Results: We found here that lacking of XIAP expression resulted in a remarkable suppression of EGFR expression, consequently leading to the deficiency of anchorage-independent cell growth. Further study demonstrated that BIR domain of XIAP was crucial for regulating the EGFR translation by suppressing the transcription and expression of miR-200a. Mechanistic studies indicated that BIR domain activated the protein phosphatase 2 (PP2A) activity by decreasing the phosphorylation of PP2A at Tyr307 in its catalytic subunit, PP2A-C. Such activated PP2A prevented the deviant phosphorylation and activation of MAPK kinases/MAPKs, their downstream effector c-Jun, and in turn inhibiting transcription of c-Jun-regulated the miR-200a., Conclusions: Our study uncovered a novel function of BIR domain of XIAP in regulating the EGFR translation, providing significant insight into the understanding of the XIAP overexpression in the cancer development and progression, further offering a new theoretical support for using XIAP BIR domain and EGFR as targets for cancer therapy.

The capacity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to selectively induce cell death in malignant cells triggered numerous attempts for therapeutic exploitation. In clinical trials, however, TRAIL did not live up to the expectations, as tumors exhibit high rates of TRAIL resistance in vivo. Response to anti-cancer therapy is determined not only by cancer cell intrinsic factors (e.g. oncogenic mutations), but also modulated by extrinsic factors such as the hypoxic tumor microenvironment.Here, we address the effect of hypoxia on pro-apoptotic TRAIL signaling in colorectal cancer cells. We show that oxygen levels modulate susceptibility to TRAIL-induced cell death, which is severely impaired under hypoxia (0.5% O2). Mechanistically, this is attributable to hypoxia-induced mitochondrial autophagy. Loss of mitochondria under hypoxia restricts the availability of mitochondria-derived pro-apoptotic molecules such as second mitochondria-derived activator of caspase (SMAC), thereby disrupting amplification of the apoptotic signal emanating from the TRAIL death receptors and efficiently blocking cell death in type-II cells. Moreover, we identify strategies to overcome TRAIL resistance in low oxygen environments. Counteracting hypoxia-induced loss of endogenous SMAC by exogenous substitution of SMAC mimetics fully restores TRAIL sensitivity in colorectal cancer cells. Alternatively, enforcing a mitochondria-independent type-I mode of cell death by targeting the type-II phenotype gatekeeper X-linked inhibitor of apoptosis protein (XIAP) is equally effective.Together, our results indicate that tumor hypoxia impairs TRAIL efficacy but this limitation can be overcome by combining TRAIL with SMAC mimetics or XIAP-targeting drugs. Our findings may help to exploit the potential of TRAIL in cancer therapy., Competing Interests: The authors declare no competing financial interests.

Context: Papillary thyroid cancer (PTC) is the second most common cancer in females in Saudi Arabia. However, the pathogenesis of PTC is still not fully elucidated., Objective: To identify potential genes that play important role in progression of PTC, we studied the role of X-linked inhibitor of apoptosis protein (XIAP) as a potential prognostic marker and therapeutic target in a large cohort of PTC samples and cell lines., Design: A DNA microarray chip was used to screen for gene copy number. XIAP expression was assessed by immunohistochemistry in a tissue microarray format on a cohort of 1022 clinical samples. In vitro and in vivo studies were performed using Embelin and/or LY294002 on PTC cell lines., Results: XIAP was found to be amplified in 14 of 29 and overexpressed in 48.8% of PTC cases. XIAP overexpression was significantly associated with old age, extrathyroidal extension, tumor size, nodal involvement, tall-cell variant, advanced stage disease, and significantly poor disease-free survival (P = .0341). XIAP was also significantly associated with phosphorylated AKT (P < .0001), Bcl-Xl (P < .0001), and Ki67 (P = .0006) proteins. Embelin treatment caused growth inhibition and apoptosis in PTC cell lines and induced tumor regression in PTC xenograft in nude mice. Finally, the combination of suboptimal doses of Embelin and LY294002 induced a synergistic apoptotic response in PTC cells., Conclusion: XIAP dysregulation in PTC confers an aggressive phenotype with poor outcome. In vitro and in vivo studies using an XIAP inhibitor suggest that this subgroup of PTC with overexpression of XIAP can be therapeutically targeted, either alone or in combination, to induce efficient apoptosis in these cancers.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization., (© 2014 The Authors.)

Release of mitochondrial contents often triggers inflammation and cell death, and modulating this process can be advantageous to invading pathogens. In this issue of The EMBO Journal, Andree and colleagues reveal new findings that an intracellular bacterial pathogen exploits apoptotic machinery to suppress host immune signaling, yet avoids cell death. This study emphasizes the need to expand our understanding of the roles played by pro‐apoptotic proteins in non‐death scenarios.

Unlabelled: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner., Conclusion: The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis., (© 2014 by the American Association for the Study of Liver Diseases.)

X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap(-/-) mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Loss of inhibitor of apoptosis proteins (IAPs), particularly cIAP1, can promote production of tumor necrosis factor (TNF) and sensitize cancer cell lines to TNF-induced necroptosis by promoting formation of a death-inducing signaling complex containing receptor-interacting serine/threonine-protein kinase (RIPK) 1 and 3. To define the role of IAPs in myelopoiesis, we generated a mouse with cIAP1, cIAP2, and XIAP deleted in the myeloid lineage. Loss of cIAPs and XIAP in the myeloid lineage caused overproduction of many proinflammatory cytokines, resulting in granulocytosis and severe sterile inflammation. In vitro differentiation of macrophages from bone marrow in the absence of cIAPs and XIAP led to detectable levels of TNF and resulted in reduced numbers of mature macrophages. The cytokine production and consequent cell death caused by IAP depletion was attenuated by loss or inhibition of TNF or TNF receptor 1. The loss of RIPK1 or RIPK3, but not the RIPK3 substrate mixed lineage kinase domain-like protein, attenuated TNF secretion and thereby prevented apoptotic cell death and not necrosis. Our results demonstrate that cIAPs and XIAP together restrain RIPK1- and RIPK3-dependent cytokine production in myeloid cells to critically regulate myeloid homeostasis.

Inhibitor of apoptosis (IAP) proteins, such as XIAP or cIAP1/2, are important regulators of apoptosis in cancer cells, and IAP antagonists are currently evaluated as antitumor agents. Beyond their function in cancer cells, this study demonstrates a novel role of IAPs as regulators of vascular endothelial permeability. Two structurally different IAP antagonists, ABT and Smac085, as well as silencing of IAPs, reduced the thrombin receptor-activating peptide (TRAP)-induced barrier dysfunction in human endothelial cells as assessed by measuring macromolecular permeability or transendothelial electrical resistance. ABT diminished thrombin-evoked stress fiber formation, activation of myosin light chain 2, and disassembly of adherens junctions independent of calcium signaling, protein kinase C, and mitogen-activated protein kinases. Interestingly, ABT and silencing of IAPs, in particular XIAP, reduced the TRAP-evoked RhoA activation, whereas Rac1 was not affected. XIAP and, to a lesser extent, cIAP1 were found to directly interact with RhoA independently of the RhoA activation status. Under cell-free conditions, XIAP did not induce an ubiquitination of RhoA. In summary, our work discloses IAPs as crucial regulators of endothelial permeability and suggests IAP inhibition as interesting approach for the prevention of endothelial barrier dysfunction.

The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.

Although there is no standard treatment protocol for inflammatory breast cancer (IBC), multi-modality treatment has improved survival. In this study we profiled the NCI approved oncology drug set in a qHTS format to identify those that are efficacious in basal type and ErbB2 overexpressing IBC models. Further, we characterized the sensitivity of an acquired therapeutic resistance model to the oncology drugs. We observed that lapatinib-induced acquired resistance in SUM149 cells led to cross-resistance to other targeted- and chemotherapeutic drugs. Removal of the primary drug to which the model was developed led to re-sensitization to multiple drugs to a degree comparable to the parental cell line; this coincided with the cells regaining the ability to accumulate ROS and reduced expression of anti-apoptotic factors and the antioxidant SOD2. We suggest that our findings provide a unique IBC model system for gaining an understanding of acquired therapeutic resistance and the effect of redox adaptation on anti-cancer drug efficacy., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

X-linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro-inflammatory signalling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Mutations in XIAP cause X-linked lymphoproliferative syndrome type-2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2-BIR2 mutations severely impair NOD1/2-dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines. XLP2-BIR2 mutations abolish the XIAP-RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2-complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM-binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2-RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling., (© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Background and Purpose: In the present study, we aimed to investigate the effect of single and double knockdown of the inhibitor of apoptosis proteins (IAP) Survivin and X-linked IAP (XIAP) on three-dimensional (3D) clonogenic survival, migration capacity and underlying signaling pathways., Materials and Methods: Colorectal cancer cell lines (HCT-15, SW48, SW480, SW620) were subjected to siRNA-mediated single or Survivin/XIAP double knockdown followed by 3D colony forming assays, cell cycle analysis, Caspase activity assays, migration assays, matrigel transmigration assays and Western blotting (Survivin, XIAP, Focal adhesion kinase (FAK), p-FAK Y397, Akt1, p-Akt1 S473, Extracellular signal-regulated kinase (ERK1/2), p-ERK1/2 T202/Y204, Glycogen synthase kinase (GSK)3β, p-GSK3β S9, nuclear factor (NF)-κB p65)., Results: While basal cell survival was altered cell line-dependently, Survivin or XIAP single and Survivin/XIAP double knockdown enhanced cellular radiosensitivity of all tested cancer cell lines grown in 3D. Particularly double knockdown conditions revealed accumulation of cells in G2/M, increased subG1 fraction, elevated Caspase 3/7 activity, and reduced migration. Intracellular signaling showed dephosphorylation of FAK and Akt1 upon Survivin and/or Survivin/XIAP silencing., Conclusions: Our results strengthen the notion of Survivin and XIAP to act as radiation resistance factors and further indicate that these apoptosis-regulating proteins are also functioning in cell cycling and cell migration., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.

X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis protein family, is involved in regulating a number of functions including receptor-mediated intracellular signalling and early development. Its role as an endogenous caspase inhibitor, however, is the most highly characterised. Consequently, this protein has been implicated as an anti-apoptotic factor in the ovary. In vitro and in vivo studies have begun dissecting the stimuli and signalling networks that lead to XIAP upregulation in granulosa cells. The objective of this review is to briefly summarise the current knowledge concerning XIAP and its interactions with different caspases. Furthermore, XIAP's emerging role in the mammalian ovary will be explored and comparison is made with its functions in the mammary gland. Finally, the idea that XIAP may act as a molecular signalling switch in granulosa cells following detachment from underlying layers to promote follicular atresia will be introduced.

In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfering RNA (siRNA) restores p53 functionality and sensitizes the HPV16-positive cervical cancer cell line SiHa to apoptosis by cisplatin, irradiation, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), or agonistic anti-Fas antibody. E6 siRNA resulted in decreased E6 mRNA levels and enhanced p53 and p21 expression, demonstrating the restoration of p53 functionality in SiHa cells, without inducing high levels of apoptosis (<10%). Cell surface expression of the proapoptotic death receptors (DRs) DR4, DR5, and Fas was not affected by E6 suppression. E6 suppression conferred susceptibility to cisplatin-induced apoptosis but not to irradiation-, rhTRAIL-, or anti-Fas antibody-induced apoptosis. Combining cisplatin with rhTRAIL or anti-Fas antibody induced even higher apoptosis levels in E6-suppressed cells. At the molecular level, cisplatin treatment resulted in elevated p53 levels, enhanced caspase-3 activation, and reduced p21 levels in E6-suppressed cells. Cisplatin in combination with death receptor ligands enhanced caspase-8 and caspase-3 activation and reduced X-linked inhibitor-of-apoptosis protein (XIAP) levels in these cells. We showed using siRNA that the enhanced apoptosis in E6-supressed cells was related to reduced XIAP levels and not due to reduced p21 levels. In conclusion, targeting E6 or XIAP in combination with cisplatin can efficiently potentiate rhTRAIL-induced apoptosis in HPV-positive cervical cancer cells.

A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing β-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Evasion of apoptosis is one of the crucial acquired capabilities used by cancer cells to fend off anticancer therapies. Inhibitor of apoptosis (IAP) proteins exert a range of biological activities that promote cancer cell survival and proliferation. X chromosome-linked IAP is a direct inhibitor of caspases - pro-apoptotic executioner proteases - whereas cellular IAP proteins block the assembly of pro-apoptotic protein signalling complexes and mediate the expression of anti-apoptotic molecules. Furthermore, mutations, amplifications and chromosomal translocations of IAP genes are associated with various malignancies. Among the therapeutic strategies that have been designed to target IAP proteins, the most widely used approach is based on mimicking the IAP-binding motif of second mitochondria-derived activator of caspase (SMAC), which functions as an endogenous IAP antagonist. Alternative strategies include transcriptional repression and the use of antisense oligonucleotides. This Review provides an update on IAP protein biology as well as current and future perspectives on targeting IAP proteins for therapeutic intervention in human malignancies.

The function of IAP has long been limited to an inhibition of apoptosis through their capacity to bind some caspases. Since the expression of these proteins is altered in some tumor samples, IAPs are targets for anticancer therapy and many small molecules have been designed for their capacity to inhibit IAP-caspase interaction. Unexpectedly, these molecules appeared to significantly affect NF-κB activation. In this review, we will discuss the central role of cIAP1, cIAP2 and XIAP in the regulation of NF-κB activating signaling pathways., (© 2012 médecine/sciences – Inserm / SRMS.)

Unlabelled: Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage., Conclusion: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid., (Copyright © 2010 American Association for the Study of Liver Diseases.)

The X-linked inhibitor of apoptosis (XIAP) is the only cellular protein that has evolved to potently inhibit the enzymatic activity of mammalian caspases and promotes resistance to apoptosis. Given its role in apoptosis and its frequently elevated expression in malignant cells, XIAP has garnered the most attention as a promising therapeutic target in cancer to overcome drug resistance. Accordingly, XIAP is thought to render tumor cells resistant to chemotherapy through its ability to inhibit caspases, and it is on this basis that XIAP has been proposed as an important adverse biomarker for chemoresistance in cancer patients. Here, the current understanding of the role of XIAP in cancer is reviewed. Further, the notion is explored that the elevated XIAP expression frequently observed in malignant tissues is, at least, not exclusively responsible for the resistance of tumor cells to conventional therapeutic treatment; rather, the function of XIAP seems to be conducive to the process of malignant transformation and/or progression., (©2010 AACR.)

This study explored the effects of nuclear factor-kappa B (NF-κB) inhibitor Bay 11-7082 on Fas/FasL system and Fas-mediated apoptosis in cell line HL-60 cells. The mRNA and protein levels of Fas, FasL, and X-linked inhibitor of apoptosis protein (XIAP) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM); the level of sFasL was evaluated by enzyme-linked immunosorbent assay (ELISA); and apoptosis was determined by FCM. After treatment with Bay 11-7082, the mRNA and protein levels of FasL and XIAP in HL-60 cells were significantly lower than in the controls (P<0.05), but the mRNA and protein levels of Fas and sFasL did not change significantly (P>0.05). Apoptotic rate of HL-60 cells treated with Bay 11-7082 was significantly higher than in the controls (P<0.05). Therefore, we conclude that Bay 11-7082 can enhance Fas-mediated apoptosis in HL-60 cells by downregulating FasL and XIAP levels.

Background: Cadmium has been classified as a human carcinogen, affecting health through occupational and environmental exposure. Cadmium has a long biological half-life (>25 years), due to the flat kinetics of its excretion. The prostate is one of the organs with highest levels of cadmium accumulation. Importantly, patients with prostate cancer appear to have higher levels of cadmium both in the circulation and in prostatic tissues., Results: In the current report, we demonstrate for the first time that cadmium down-regulates expression of the X-linked inhibitor of apoptosis protein (XIAP) in prostate cancer cells. Cadmium-mediated XIAP depletion occurs at the post-transcriptional level via an NF-kappaB-independent, proteasome-mediated mechanism and coincides with an increased sensitivity of prostate cancer cells to TNF-alpha-mediated apoptosis. Prolonged treatment with cadmium results in selection of prostate cancer cells with apoptosis-resistant phenotype. Development of apoptosis-resistance coincides with restoration of XIAP expression in cadmium-selected PC-3 cells., Conclusions: Selection of cadmium-resistant cells could represent an adaptive survival mechanism that may contribute to progression of prostatic malignancies.

Apoptosis of cochlear cells plays a significant role in age-related hearing loss or presbycusis. In this study, we evaluated whether over-expression of the anti-apoptotic protein known as X-linked Inhibitor of Apoptosis Protein (XIAP) slows the development of presbycusis. We compared the age-related hearing loss between transgenic (TG) mice that over-express human XIAP tagged with 6-Myc (Myc-XIAP) on a pure C57BL/6J genetic background with wild-type (WT) littermates by measuring auditory brainstem responses. The result showed that TG mice developed hearing loss considerably more slowly than WT littermates, primarily within the high-frequency range. The average total hair cell loss was significantly less in TG mice than WT littermates. Although levels of Myc-XIAP in the ear remained constant at 2 and 14 months, there was a marked increase in the amount of endogenous XIAP from 2 to 14 months in the cochlea, but not in the brain, in both genotypes. These results suggest that XIAP over-expression reduces age-related hearing loss and hair cell death in the cochlea., (Copyright 2008 Elsevier Inc. All rights reserved.)

Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression. ErbB2 targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small-molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. In the present study, using a clonal population of GW583340 (lapatinib analogue, ErbB1/2 inhibitor)-resistant IBC cells, we identified the overexpression of an antiapoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), in acquired resistance to GW583340 in both ErbB2-overexpressing SUM190 and ErbB1-activated SUM149 cell lines derived from primary IBC tumors. A marked decrease in p-ErbB2, p-ErbB1, and downstream signaling was evident in the GW583340-resistant cells (rSUM190 and rSUM149) similar to parental counterparts treated with the drug, suggesting that the primary mechanism of action of GW583340 was not compromised in resistant cells. However, rSUM190 and rSUM149 cells growing in GW583340 had significant XIAP overexpression and resistance to GW583340-mediated apoptosis. Additionally, stable XIAP overexpression using a lentiviral system reversed sensitivity to GW583340 in parental cells. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. XIAP downregulation in rSUM190 and rSUM149 cells using a small-molecule inhibitor (embelin), which abrogates the XIAP/procaspase-9 interaction, resulted in decreased viability, showing that XIAP is required for survival of cells with acquired resistance to GW583340. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to ErbB2-targeting agents.

Berbamine is an herbal compound derived from Berberis amurensis, which is used in Chinese traditional medicine. However, few studies have investigated this anti-tumor effect or the underlying mechanisms of berbamine on lymphoma cells. We investigate the effect, as well as the mechanism of action, of 4-chlorobenzoyl berbamine (BBD9) on Raji, L428, Namalwa and Jurkat lymphoma cells lines. Our findings show that BBD9 inhibits cell proliferation and induces cell apoptosis in lymphoma cell lines as well as G2/M cell cycle arrest through PI3K/Akt and NF-kappaB signaling pathways in a caspase-dependent manner. These results may provide new insights into the treatment of lymphoma.

Clinical pancreatic islet transplantation has great promise as a treatment for type 1 diabetes but despite recent advances, it is still limited by the need for lifelong immunosuppression, restricted availability of donor islets, and uncertainty regarding long-term graft survival. Using a syngeneic, suboptimal islet transplantation model, we asked whether adenoviral overexpression of an anti-apoptotic protein, the X-linked inhibitor of apoptosis protein (XIAP) would protect transplanted islet cells from death and reduce the number of islets required for successful transplantation. Transplantation of 100 XIAP-expressing islets into the kidney capsule of syngeneic Balb/c mice restored euglycemia in 86% of recipients, where transplantation of 100 islets transduced with a control adenovirus expressing LacZ restored euglycemia in only 27% of recipients. Analysis of islet grafts by insulin/TUNEL double immunostaining revealed fewer apoptotic beta-cells in recipients of XIAP- compared with LacZ-expressing grafts (0.8±0.5 vs. 2.4±0.8 double-positive cells/graft), suggesting that XIAP enhances graft success by inhibiting β-cell apoptosis in the immediate post-transplant period. In summary, XIAP overexpression inhibits beta cell apoptosis in syngeneic islet transplants, thereby reducing the number of islets and decreasing the number of days required to restore euglycemia. These data raise the possibility that ex vivo XIAP gene transfer in islets prior to transplantation has the potential to increase the number of donor islets available for transplantation and may enhance graft function and long-term transplant success.

Galectin-3 is known to modulate cell proliferation and apoptosis and is highly expressed in human cancers, but its function in gastric cancer is still controversial. Here, we examined the role of galectin-3 in gastric cancer cells by silencing it with synthetic double-stranded siRNA. After silencing of galectin-3, cell numbers decreased and cell shape changed. Galectin-3 siRNA treatment also induced G(1) arrest. DNA microarray analysis was used to assess changes in gene expression following galectin-3 silencing. We found that silencing of galectin-3 caused changes in gene expression. RT-PCR and real-time PCR were utilized for validation of the changes found in microarray studies. Western blot analysis confirmed changes in the expression of proteins of interest: cyclin D1, survivin, XIAP, XAF, PUMA, and GADD45alpha. Generally, it tended to increase the expression of several pro-apoptotic genes, and to decrease the expression of cell cycle progressive genes. We also confirmed that changes in the expression of these genes were caused by galectin-3 overexpression. Finally, we demonstrated that silencing of galectin-3 enhanced apoptosis induction with chemotherapeutic agents by further reducing the expression of anti-apoptotic and/or cell survival molecules such as survivin, cyclin D1, and XIAP, and increasing the expression of pro-apoptotic XAF-1. We conclude that galectin-3 is involved in cancer progression and malignancy by modulating the expression of several relevant genes, and inhibition of galectin-3 may be an approach to improve chemotherapy of gastric cancers.

The ability of the vertebrate X-linked inhibitor of apoptosis (XIAP) protein to directly suppress apoptotic cell death pathways has been the subject of much research. Studies of this broadly expressed protein have largely focused on the unique interactions between XIAP and caspases - proteases that conduct and participate in the ordered disassembly of the cell during apoptosis. However, relatively less attention has been given to the RING domain of XIAP, which functions as an E3 ligase to catalyze the ubiquitination of substrate proteins. Here, we discuss the evidence implicating the RING domain of XIAP in the ubiquitin-mediated regulation of three, somewhat arbitrarily divided, categories of substrate: XIAP itself, XIAP-interacting proteins involved in apoptosis, and other targets whose physiological roles likely extend beyond cell death. Collectively, these multiple activities of XIAP show that this enigmatic protein participates in a range of cellular activities beyond apoptotic suppression.

Nearly 25 years after the seminal publication of John Foxton Kerr that first described apoptosis, the process of regulated cell death, our understanding of this basic physiological phenomenon is far from complete [39]. From cardiovascular disease to cancer, apoptosis has assumed a central role with broad ranging therapeutic implications that depend on a complete understanding of this process, yet have also identified an incredibly complex regulatory system that is critical for development and is at the core of many diseases, challenging scientist and clinicians to step into its molecular realm and modulate its circuitry for therapeutic purposes. This chapter will review our understanding of the molecular circuitry that controls apoptosis in leukemia and the pharmacological manipulations of this pathway that may yield therapeutic benefit.

Many viruses induce cell death and lysis as part of their replication and dissemination strategy, and in many cases features of apoptosis are observed. Attempts have been made to further increase productivity by prolonging cell survival via the over-expression of anti-apoptotic genes. Here, we extend the study to investigate the association between virus replication and apoptosis, pertinent to large-scale vector production for gene therapy. Infection of an HEK293 cell line with a replication defective type-5-adenovirus expressing a GFP reporter (Ad5GFP) resulted in rapid decline in viability associated with increased virus titer. The over-expression of bcl-2 resulted in improved cell resistance to apoptosis and prolonged culture duration, but reduced virus specific and total productivity. In contrast, the over-expression of pro-caspase-3 (Yama/CPP32/apopain) resulted in reduced cell survival but increased virus productivity. The treatment of infected cells with caspase inhibitors support the preposition that caspase-3 dependent apoptosis, and to a lesser degree caspase-9 dependent apoptosis, represent important steps in virus production, thus implicating the intrinsic apoptosis pathway in the production of adenovirus from HEK293 cells. The suppression of apoptosis by the over-expression of XIAP (inhibitors of caspase family cell death proteases) further shows that caspase-mediated activation plays an important role in virus infection and maturation., (Copyright 2009 Wiley Periodicals, Inc.)

Our previous studies and those of others have indicated that X-linked inhibitor of apoptosis protein (XIAP) holds promise as a target gene in colon cancer gene therapy. In this study, we constructed an adenoviral vector to deliver small hairpin RNA (shRNA) against XIAP (XIAP-shRNA) into colon cancer cells and tested its therapeutic efficacy in vitro and in vivo. We first confirmed an overexpression of XIAP in colon cancer cells and human cancer tissues. We then designed XIAP-small interfering RNA (siRNA) and confirmed the knockdown effect of these siRNAs in colon cancer cells. The sequences of the effective siRNAs were converted into shRNA and then packed into replication-deficient adenoviral vectors using BLOCK-iT Adenoviral RNAi Expression System to generate Adv-XIAP-shRNA. Infection of HT29 and HCT116 cells with Adv-XIAP-shRNA led to enhanced caspase-3 activity, which was associated with increased apoptosis and reduced cell proliferation. The therapeutic effect of Adv-XIAP-shRNA was then tested in xenograft tumors in nude mice. We showed that treatment of the xenograft tumors derived from HCT116 cells with Adv-XIAP-shRNA resulted in a retardation of tumor growth, which was associated with enhanced apoptosis, increased caspase-3 activity, and reduced expression of proliferating cell nuclear antigen in the tumor tissues. Treatment of xenograft tumors with Adv-XIAP-shRNA did not affect the expressions of inflammatory cytokines in tumor-bearing mice. Thus, Adv-XIAP-shRNA-mediated down-regulation of XIAP exerts a therapeutic effect in colon cancer by promoting apoptosis and inhibiting proliferation of colon cancer cells, and the antitumor effect of Adv-XIAP-shRNA was unlikely to be related to virus-induced immune response.

Background: During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-beta isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo., Methods: Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Pseudopregnancy was induced with sex steroids in ovariectomized rats and rats were killed at different days (days 1-9). Uteri were collected and either fixed for immunohistochemical staining (IHC) or processed for RT-PCR and Western analyses. For the in vitro part of the study, rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were purified, cultured and treated with different concentrations of TGF-beta isoforms., Results: Our results showed that all three TGF-beta isoforms are present, but are localized differently in the endometrium during the estrous cycle and their expression is regulated differently during pseudopregnancy. In cultured stromal cells, we found that TGF-beta3 isoform induced Smad2 phosphorylation, indicating that the Smad pathway is activated by TGF-beta3 in these cells. Furthermore, TGF-beta2 and TGF-beta3 induced a dose-dependant increase of apoptosis in cultured stromal cells, as demonstrated by Hoechst nuclear staining. Noteworthy, TGF-beta2 and TGF-beta3 reduced the level of the anti-apoptotic XIAP protein, as well as the level of phosphorylated/active Akt, a well known survival protein, in a dose-dependent manner., Conclusion: Those results suggest that TGF-beta might play an important role in the remodelling endometrium during the estrous cycle and in the regulation of apoptosis in rat decidual cells, in which inhibition of Akt survival pathway might be an important mechanism involved in the regulation of apoptosis.

XIAP is known as a potent inhibitor of apoptosis, but in addition is involved in cellular signalling, including the NFkappaB, JNK and TGFbeta pathways. Our search for XIAP-interacting partners led us to Siva1, a proapoptotic protein that is known to play a role in T-cell apoptosis through a caspase-dependent mitochondrial pathway. The interaction sites between XIAP and Siva1 were mapped to the RING domain of XIAP and the N-terminal, SAH-containing and death-homology-region-containing domains of Siva1. Co-immunoprecipitation experiments showed that XIAP, Siva1 and TAK1 form a ternary complex in Jurkat T cells. Reporter-gene analysis revealed that Siva1 inhibits XIAP- and TAK1-TAB1-mediated NFkappaB activation. By contrast, Siva1 increased XIAP- and TNFalpha-mediated AP1 activity and prolonged TNFalpha-induced JNK activation, whereas knock down of Siva1 resulted in reduced JNK activation. This suggests that Siva1 differentially modulates signalling by JNK and NFkappaB and shifts the balance between these pathways towards enhanced JNK activation, a situation that promotes apoptosis. Ectopically expressed Siva1 increased caspase-3 activity, which was inhibited by XIAP in a ubiquitin-ligase-dependent manner. In line with this, Siva1 was lysine-48-linked polyubiquitylated by XIAP. Our findings suggest that, via physical interaction with XIAP and TAK1, Siva1 diminishes NFkappaB and enhances JNK activity to favour apoptosis.

Cisplatin is a first-line chemotherapeutic agent and a powerful component of standard treatment regimens for several human malignancies including bladder cancer. DNA-Pt adducts produced by cisplatin are mainly responsible for cellular toxicity and induction of apoptosis. Identification of the mechanisms that control sensitivity to cisplatin is central to improving its therapeutic index and to successfully encountering the acquired resistance frequently emerging during therapy. In the present study, using MTT-based assays, Western blotting and semi-quantitative RT-PCR, we examined the apoptosis-related cellular responses to cisplatin exposure in two human urinary bladder cancer cell lines characterized by different malignancy grade and p53 genetic status. Both RT4 (grade I; wild-type p53) and T24 (grade III; mutant p53) cell types proved to be vulnerable to cisplatin apoptotic activity, albeit in a grade-dependent and drug dose-specific manner, as demonstrated by the proteolytic processing profiles of Caspase-8, Caspase-9, Caspase-3, and the Caspase repertoire characteristic substrates PARP and Lamin A/C, as well. The differential resistance of RT4 and T24 cells to cisplatin-induced apoptosis was associated with an RT4-specific phosphorylation (Ser15; Ser392) pattern of p53, together with structural amputations of the Akt and XIAP anti-apoptotic regulators. Furthermore, cisplatin administration resulted in a Granzyme B-mediated proteolytic cleavage of Hsp90 molecular chaperone, exclusively occurring in RT4 cells. To generate functional networks, expression analysis of a number of genes, including Bik, Bim, Bcl-2, FAP-1, Fas, FasL, TRAIL, Puma, Caspase-10, ATP7A, ATP7B and MRP1, was performed, strongly supporting the role of p53-dependent and p53-independent transcriptional responses in cisplatin-induced apoptosis of bladder cancer cells.

Drug resistance remains a major clinical challenge for cancer treatment. One mechanism by which tumor cells develop resistance to cytotoxic agents and radiation is related to resistance to apoptosis. Apoptosis is a well-organised process of cell death pre-programmed inside the cell. Apoptosis can be initiated either by activation of death receptors on the cell surface membranes (extrinsic pathway) or through a series of cellular events primarily processed at mitochondria (intrinsic pathway). Apoptosis has been shown to be important for tumorigenesis and cancer treatment. Defects in apoptosis can result in the expansion of a population of neoplastic cells. However, because the death of tumor cells induced by chemotherapy and radiotherapy is largely mediated by activation of apoptosis, inhibition of apoptosis will make tumor cells resistant to anti-tumor treatment. Herein, we will review the molecular changes that have the potential to cause apoptotic dysregulation, including activation of antiapoptotic factors (Bcl-2, BCLX(L), Bfl1/A1 etc.), inactivation of pro-apoptotic effectors (p53, p53 pathway), and /or reinforcement of survival signals (Survivin, FLIP, NF-kappaB etc). Furthermore, we will discuss therapeutic intervention and/or strategies that can lower the threshold for apoptosis of tumor cells that could became useful approaches to treat cancer with special emphasis placed on the important priority to develop new cancer therapeutics toward tumor stem cells.

Evasion of apoptosis is a hallmark of human cancers, for example in hematological malignancies. Apoptosis is an intrinsic cell death program that is crucial to maintain tissue homeostasis, for example in the hematopoietic system where there is a high turnover rate of cells. As a result, a decrease in the rate of apoptosis besides an increase in proliferation favors tumorigenesis as well as tumor progression. Further, the anti-leukemic action of current treatment approaches, including chemo-, radio- or immunotherapy, critically relies on intact cell death programs in cancer cells. Therefore, defects in apoptosis pathways are frequently associated with the resistance to anticancer therapies. In recent years, the identification and characterization of the molecules and pathways that are involved in the regulation and execution of cell death in leukemia and lymphoma cells have set the ground for the development of novel diagnostic tools and molecular therapeutics targeting apoptosis pathways in hematological malignancies.

Various genotoxic agents cause monoubiquitination of NEMO/IKKgamma-the regulatory subunit of IkappaB kinase (IKK) complex-in the nucleus. Ubiquitinated NEMO exits from the nucleus and forms a complex with the IKK catalytic subunits IKKalpha and IKKbeta, resulting in IKK activation and, ultimately, nuclear factor-kappaB (NF-kappaB) activation. Thus, NEMO ubiquitination is a prerequisite for IKK-dependent activation of NF-kappaB. However, the IKK activation mechanism is unknown and the NEMO-ubiquitinating E3 enzyme has not been identified. We found that inhibitors of apoptosis protein (IAP) regulate genotoxic stress-induced NF-kappaB activation at different levels. XIAP mediates activation of the upstream IKK kinase, TAK1, and couples activated TAK1 to the IKK complex. This XIAP-dependent event occurs in response to camptotechin or etoposide/VP16; however, XIAP is dispensable for activation of NF-kappaB by doxorubicin, which engages a MEK-ERK pathway to activate IKK. We also show that cIAP1 mediates NEMO ubiquitination and cIAP2 regulates an event downstream of NEMO ubiquitination. Our study highlights nonredundant cooperative contributions of IAPs to antiapoptotic NF-kappaB activation by genotoxic signals beyond their classic caspase inhibitory functions.

Pancreatic beta-cell apoptosis plays a critical role in the pathogenesis of type 1 diabetes mellitus. As death effector molecules, perforin, Fas ligand, tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1, interferon (IFN)-gamma, and nitric oxide have been claimed. Recently, combinations or synergisms between IFN-gamma and TNF-alpha or IL-1beta are being revisited as the death effectors, and signal transduction of such synergisms has been explored to find molecular mechanism of beta -cell death. Among the regulators of apoptosis, nuclear factor-kappaB (NF-kappaB) has emerged as a master switch of cytokine-induced beta -cell dysfunction and death. By employing TNF-alpha / IFN-gamma synergism model which causes beta -cell apoptosis, we found that the antiapoptotic X-linked inhibitor of apoptosis (XIAP) molecule is upregulated by NF-kappaB in response to TNF-alpha and XIAP induction was inhibited by IFN-gamma-induced signal transducer and activator of transcription-1 (STAT1) activation, which explains the death of beta -cells by TNF-alpha /IFN-gamma synergism.

XIAP (X chromosome-linked inhibitor of apoptosis) is a member of the anti-apoptotic IAP gene family and an inhibitor of caspase-3. We show here that loss of XIAP renders cells highly sensitive to oxidative stress. Stimulation of XIAP(-/-) MEF with hydrogen peroxide, or other agents that generate reactive oxygen species (ROS) results in increased apoptosis assessed by caspase-3 activity and PARP cleavage. Furthermore, we observed increased levels of ROS and diminished expression of antioxidative genes, e.g., SOD1, -2, NQO1, HO-1, and Txn2 in XIAP(-/-) cells. In addition, stimulation of XIAP(-/-) MEF with hydrogen peroxide resulted in enhanced phosphorylation of JNK. Our findings reveal that XIAP, in addition to its well described caspase-inhibitory function, prevents prolonged JNK activation and is critically involved in modulating ROS levels through regulation of antioxidative genes, thereby inhibiting ROS-induced apoptosis.

We investigated whether the anticancer effect of a combination of XIAP down-regulation and PPAR gamma activation on colon cancer is PPARgamma receptor dependent. HCT116-XIAP(+/+) cells and HCT116-XIAP(-/-) cells were treated with troglitazone or 15-deoxy-Delta(12,14)-prostaglandin J2 (15-PGJ2) with or without prior exposure to PPARgamma inhibitor GW9662. Cell proliferation and apoptosis was evaluated. Athymic mice carrying HCT116-XIAP(-/-) cells-derived tumors were treated with troglitazone in the presence or absence of GW9662. Inhibition of cell proliferation and induction of apoptosis by troglitazone and 15-PGJ2 were more prominent in HCT116-XIAP(-/-) cells. PPARgamma ligand-induced growth inhibition, apoptosis, caspase and PARP cleavage could not be blocked by GW9662. Troglitazone significantly retarded growth of xenograft tumors and this effect was not blocked by GW9662. Marked apoptosis and an up-regulation of E-cadherin were observed in xenograft tumor tissues, and GW9662 did not affect these effects. Thus, a combination of XIAP down-regulation and PPARgamma ligands exert a significant anticancer effect in colon cancer via a PPARgamma independent pathway.

Inhibitor of Apoptosis Proteins (IAPs) can bind to and inhibit caspases, the key executioners of apoptosis. Because IAPs are frequently overexpressed in human tumors, they have become major pharmacological targets for developing new cancer therapeutics. However, the precise physiological function of individual mammalian IAPs and their role as E3 ubiquitin-ligases in situ remain largely obscure. Here, we investigated the function of XIAP ubiquitin-ligase activity by inactivating the RING motif via gene targeting in the mouse. Removing the RING stabilized XIAP in apoptotic thymocytes, demonstrating that XIAP ubiquitin-ligase activity is a major determinant of XIAP protein stability. Surprisingly, the increased amounts of "XIAP-BIR-only" protein did not lead to attenuated but rather increased caspase activity and apoptosis. DeltaRING embryonic stem cells and fibroblasts had elevated caspase-3 enzyme activity, and XIAP DeltaRING embryonic fibroblasts were strongly sensitized to TNF-alpha-induced apoptosis. Similar results were obtained with XIAP deficient mice. Furthermore, deletion of the RING also improved the survival of mice in the Emu-Myc lymphoma model. This demonstrates a physiological requirement of XIAP ubiquitin-ligase activity for the inhibition of caspases and for tumor suppression in vivo.

The inhibitors of apoptosis (IAPs) are emerging as key proteins in the control of cell death. In this study, we evaluated the expression and subcellular distribution of the antiapoptotic protein X-linked IAP (XIAP), and its interactions with the XIAP-associated factor 1 (XAF1) in neonatal rat brain following hypoxia-ischemia (HI). HI triggered the mitochondrial release of cytochrome c, Smac/DIABLO, and caspase 3 activation. Confocal microscopy detected XIAP-specific immunofluorescence in the cytoplasm under normal condition, which exhibited a diffuse distribution at 6 h post-HI and by 12 h the majority of XIAP was redistributed into the nucleus. XIAP nuclear translocation was confirmed by subcellular fractionations and by expressing FLAG-tagged XIAP in primary cortical neurons. Over-expression of XIAP significantly reduced, whereas XIAP gene silencing further enhanced cell death, demonstrating a specific requirement of cytoplasmic XIAP for cell survival. An elevated level of cytosolic XIAP was also evident under the conditions of neuroprotection by fibroblast growth factor-1. XAF1 expression was increased temporally and there was increased nuclear co-localization with XIAP in hypoxic-ischemic cells. XIAP co-immunoprecipitated > 9-fold XAF1 protein concurrent with decreased association with caspases 9 and 3. This is evidenced by the enhanced caspase 3 activity and neuronal death. Our findings implicate XIAP nuclear translocation in neuronal death and point to a novel mechanism in the regulation of hypoxic-ischemic brain injury.

Endometrial carcinomas are often chemoresistant. TNFalpha shows potent antitumor activity against various cancers, and if it demonstrates good antitumor activity against endometrial cancer, the cytokine could represent a valuable alternative therapeutic approach. We have tested the ability of TNFalpha to induce apoptosis in endometrial carcinoma cells, and examined a putative role for X-linked inhibitor of apoptosis protein (XIAP) in regulating cellular sensitivity to the cytokine. Exposure to TNFalpha triggered TNF-R1-dependent activation of caspases-8, -9, and -3, down-regulated Akt and XIAP proteins and induced dose-dependent and time-dependent apoptosis in Ishikawa cells. On the opposite, TNFalpha up-regulated XIAP in Hec-1A cells; in these cells, the cytokine induced delayed TNF-R1-dependent activation of caspase-8, and failed to activate caspases -9 and -3 and to induce apoptosis. However, XIAP small interfering RNA restored TNFalpha-induced caspase signaling and apoptosis in Hec-1A cells; XIAP small interfering RNA also increased TNFalpha-induced apoptosis in Ishikawa cells. In addition, inhibition of protein kinase C activity enhanced TNFalpha-induced down-regulation of XIAP and potentiated apoptosis induction, in both Ishikawa and Hec-1A cells. Finally, we found XIAP immunoreactivity in epithelial cells from a large number of human endometrial tumor tissue samples, indicating that XIAP is produced by endometrial tumor cells in vivo. This could allow XIAP to play a putative in vivo role in counteracting TNFalpha-induced apoptosis in endometrial tumor cells; in this case, direct or indirect targeting of XIAP should potentiate the antitumor effect of TNFalpha.