Chromosome-linked Inhibitor Of Apoptosis Research Articles

Long Non-coding RNA Peg13 Alleviates Hypoxic-Ischemic Brain Damage in Neonatal Mice via miR-20a-5p/XIAP Axis.

Long noncoding RNA (LncRNA) Peg13 has been demonstrated to protect against neurological diseases. However, its underlying mechanism in the progression of hypoxic-ischemic brain damage (HIBD) has not been well investigated. The expression of target genes was determined in neonatal mice with HIBD and in mouse hippocampal neurons during oxygen-glucose deprivation (OGD) using quantitative real-time PCR (qRT-PCR) and immunoblotting. Functional assays, including CCK-8 cell viability and apoptotic cell detection using TdT mediated dUTP nick ending labeling (TUNEL) assay were used to examine the neuroprotective role of Peg13 in mouse hippocampal neurons. Luciferase assays were performed to determine the regulatory mechanism of Peg13 in OGD-induced neuronal apoptosis. Peg13 was reduced in HIBD mice and OGD-treated mouse hippocampal neurons. Altered Peg13 expression relieved OGD-induced neuronal apoptosis. Mechanistically, Peg13 may serve as a sponge for miR-20a-5p to increase the expression of X chromosome-linked inhibitor of apoptosis (XIAP), a downstream target of miR-20a-5p. Our study showed that Peg13 fulfilled its anti-apoptotic function in neurons through suppressing XIAP expression by sponging miR-20a-5p. Together, Peg13 binds to miR-20a-5p to upregulate XIAP and alleviate HIBD in neonatal mice. The Peg13/miR-20a-5p/XIAP competing endogenous RNA (ceRNA) axis could be a potential therapeutic target for HIBD.

In-cell destabilization of a homodimeric protein complex detected by DEER spectroscopy

The complexity of the cellular medium can affect proteins' properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer-monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.

Hsa_circ_0067997 promotes the progression of gastric cancer by inhibition of miR-515-5p and activation of X chromosome-linked inhibitor of apoptosis (XIAP)

Circular RNAs (circRNAs) have been revealed to play vital roles in modulating gene expression and participate in several pathological responses including gastric cancer (GC). However, the larger numbers of the circRNAs in GC progression remain undetermined. In the present study, four GC related circRNAs expression profiles from the Gene Expression Omnibus (GEO) database were enrolled. We identified hsa_ circRNA_00067997 (circ_0067997) as an oncogene in GC. qRT-PCR was used to validate the expression of circ_0067997 in GC tissues and cell lines. The results revealed that circ_0067997 was upregulated in GC, and high circ_0067997 expression was associated with the poor overall survival rate of GC patients. Knockdown of circ_0067997 significantly reduced cell viability, inhibited colony formation, and attenuated invasive ability, whereas overexpression of circ_0067997 exhibited opposed effects. Circ_0067997 was identified to be a sponge for miR-515-5p directly. Moreover, XIAP was demonstrated to be targeted and regulated by miR-515-5p. In conclusion, circ_0067997 was identified to be an oncogene in GC by regulating miR-515-5p/XIAP axis.

The Critical Role of X Chromosome-Linked Inhibitor of Apoptosis (XIAP) in Differential Synergism Induced by Pentachlorophenol and Copper-1,10-Phenanthroline Complex in Normal and Cancer Liver Cells.

Chemical pollutants often co-occur and can interact to cause unexpected combined toxic effects. Both pentachlorophenol (PCP) and copper-1,10-phenanthroline [Cu(OP)2], used as wood preservatives, coexist in fluids and tissues of ordinary population. Our previous studies demonstrate that a combination of subtoxic PCP and Cu(OP)2 causes synergistic toxicity on Escherichia coli and hepatocarcinoma cells. However, it is not clear whether this effect also occurs in normal hepatocytes; and if so, what are the differences as compared with the hepatocarcinoma cells. We demonstrate that the combination of low-toxic PCP and Cu(OP)2 (0-1.6 µM; PCP/Cu(OP)2 molar ratio: 2:1) induces a concentration-dependent intracellular copper accumulation, apoptosis, caspase-3/9 activation, depolarization of mitochondrial membrane potential, and oxidative stress (reactive oxygen species increasing and glutathione/oxidized glutathione ratio decreasing) in both normal hepatocytes HL-7702 and hepatocarcinoma HepG2 cells. However, HepG2 cells are more susceptible to the above molecular events as compared with HL-7702 cells. Further data reveal that PCP/Cu(OP)2 markedly decreases X chromosome-linked inhibitor of apoptosis (XIAP), p-ERK-1/2, and p-JNK protein expression in HepG2, but not HL-7702. Overexpression of XIAP gene in HepG2 significantly blocks PCP/Cu(OP)2-induced cytotoxicity, caspase activity, apoptosis, ROS accumulation, and antioxidant genes expression. These results suggest that the combination of low-toxic PCP and Cu(OP)2 preferentially induce synergistic cytotoxicity in human hepatocarcinoma cells by XIAP-ROS-apoptosis pathway, compared with the normal hepatocytes. The present data not only confirm the synergistic toxicity of PCP/Cu(OP)2 combination in normal liver cells, but also suggest a possible opportunity in developing new therapeutic approaches for liver cancer by sensitizing cancer cells to chemotherapy.

Polymethoxyflavones prevent benzo[a]pyrene/dextran sodium sulfate-induced colorectal carcinogenesis through modulating xenobiotic metabolism in ICR mice

Toona sinensis leaf extract (TSL) has been shown to have anti-tumor effects on cancer cell lines. This study aimed to investigate the chemopreventive potential and the underlying mechanism of TSL during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis.One hundred hamsters were divided into control (n = 30), carcinogenic (n = 20), preventive (n = 42), and therapeutic (n = 8) groups. The animals in carcinogenic and preventive groups were administered reverse osmosis water (carcinogenic group) or TSL (1 g/kg bw) (preventive group) by gavage daily for 4 weeks, and their bilateral pouches were painted with a 0.5% DMBA solution for 4, 9, and 12 weeks. The animals in the therapeutic group were treated with DMBA for 12 weeks prior to TSL administration for 4 weeks. Expression levels of survivin, X chromosome-linked inhibitor of apoptosis (XIAP), proliferating cell nuclear antigen (PCNA), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) proteins were analyzed by immunohistochemistry. Apoptotic activity was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, cytochrome C, and poly (ADP-ribose) polymerase (PARP).In the preventive group, the results showed significant decreases not only in the incidences of squamous cell carcinoma (SCC) (50%) and epithelial dysplasia (62.5%) but also in the tumor number, tumor volume, tumor burden, and the severity of dysplastic lesions. The down-regulation of survivin, XIAP, PCNA, iNOS, and COX-2 proteins and the increased apoptotic activity indicated anti-proliferative and apoptosis-inducing abilities of TSL on DMBA-induced HBP carcinogenesis.The results suggested that TSL might be a promising candidate for the prevention of oral cancer.

How genetic testing can lead to targeted management of XIAP deficiency-related inflammatory bowel disease.

X-linked lymphoproliferative disease type 2 (XLP-2, OMIM 300635) is a primary immunodeficiency caused by the loss of X chromosome-linked inhibitor of apoptosis (XIAP), the X-linked inhibitor of apoptosis gene at Xq25. XLP-2 individuals are susceptible to several specific and potentially fatal infections, such as Epstein-Barr virus (EBV). Children with XIAP-related XLP-2 may present with either familial hemophagocytic lymphohistiocytosis, often triggered in response to EBV infection, or with a treatment-refractory severe pediatric form of inflammatory bowel disease (IBD) that might be diagnosed as Crohn disease. However, this monogenic cause of IBD is distinct from adult Crohn disease (a polygenic and multifactorial disease) in its etiology and responsiveness to therapy. XLP-2 and the associated IBD symptoms are managed by a reduced-intensity conditioning regimen with an allogeneic hematopoietic stem cell transplantation that causes resolution of gastrointestinal symptoms. Exome sequencing has enabled identification of XIAP-deficient diseased individuals and has altered their morbidity by providing potentially lifesaving strategies in a timely and effective manner. Here, we summarize XLP-2 IBD treatment history and patient morbidity/mortality since its original identification in 2006. Since XLP-2 is rare, cases are probably undergiagnosed or misdiagnosed. Consideration of XLP-2 in children with severe symptoms of IBD can prevent serious morbidities and mortality, avoid unnecessary procedures, and expedite specific targeted therapy.Genet Med 19 2, 133-143.

Chemopreventive effect of Toona sinensis leaf extract on 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch squamous cell carcinogenesis

ObjectiveToona sinensis leaf extract (TSL) has been shown to have anti-tumor effects on cancer cell lines. This study aimed to investigate the chemopreventive potential and the underlying mechanism of TSL during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. MethodsOne hundred hamsters were divided into control (n=30), carcinogenic (n=20), preventive (n=42), and therapeutic (n=8) groups. The animals in carcinogenic and preventive groups were administered reverse osmosis water (carcinogenic group) or TSL (1g/kg bw) (preventive group) by gavage daily for 4 weeks, and their bilateral pouches were painted with a 0.5% DMBA solution for 4, 9, and 12 weeks. The animals in the therapeutic group were treated with DMBA for 12 weeks prior to TSL administration for 4 weeks. Expression levels of survivin, X chromosome-linked inhibitor of apoptosis (XIAP), proliferating cell nuclear antigen (PCNA), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) proteins were analyzed by immunohistochemistry. Apoptotic activity was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, cytochrome C, and poly (ADP-ribose) polymerase (PARP). ResultsIn the preventive group, the results showed significant decreases not only in the incidences of squamous cell carcinoma (SCC) (50%) and epithelial dysplasia (62.5%) but also in the tumor number, tumor volume, tumor burden, and the severity of dysplastic lesions. The down-regulation of survivin, XIAP, PCNA, iNOS, and COX-2 proteins and the increased apoptotic activity indicated anti-proliferative and apoptosis-inducing abilities of TSL on DMBA-induced HBP carcinogenesis. ConclusionsThe results suggested that TSL might be a promising candidate for the prevention of oral cancer.

Discovery of Novel Second Mitochondria-Derived Activator of Caspase Mimetics as Selective Inhibitor of Apoptosis Protein Inhibitors

Inhibitor of apoptosis (IAP) proteins are widely considered as promising cancer drug targets, especially for drug-resistant tumors. Mimicking the IAP-binding motif of second mitochondria-derived activator of caspases (SMAC) is a rational strategy to design potential IAP inhibitors. In this report, we used the bioactive conformation of AVPI tetrapeptide in the N terminus of SMAC as a template and performed a shape-based virtual screening against a drug-like compound library to identify novel IAP inhibitors. Top hits were subsequently docked to available IAP crystal structures as a secondary screening followed by validation using in vitro biologic assays. Four novel hit compounds were identified to potently inhibit cell growth in two human melanoma (A375 and M14) and two human prostate (PC-3 and DU145) cancer cell lines. The best compound, UC-112 [5-((benzyloxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol], has IC50 values ranging from 0.7 to 3.4 µM. UC-112 also potently inhibits the growth of P-glycoprotein (P-gp)-overexpressed multidrug-resistant cancer cells, strongly activates caspase-3/7 and caspase-9 activities, and selectively downregulates survivin level at a concentration as low as 1 µM. Coincubation of UC-112 with a known proteasome inhibitor Z-Leu-Leu-Leu-CHO (MG-132) rescued survivin inhibition, consistent with the anticipated mechanism of action for UC-112. As a single agent, UC-112 strongly inhibits tumor growth and reduces both X chromosome-linked IAP and survivin levels in an A375 human melanoma xenograft model in vivo. Overall, our study identified novel scaffolds, especially UC-112, as new platforms on which potent and selective IAP antagonists can be developed.

Abstract 3333: Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2.

Abstract A variety of Smac-mimetic drugs have been developed to target the inhibitor of apoptosis (IAP) proteins including X chromosome-linked IAP (XIAP), cellular IAP proteins (cIAP1 and cIAP2) and ML-IAP whose gene mutations, amplifications and chromosomal translocations have been implicated in various malignancies. The IAPs also play an important role in multiple signaling pathways including the TNFα-mediated NF-kB and MAPK pathways (inflammatory responses) and pattern recognition receptor signaling pathways (innate immunity). Smac-mimetics have been designed to mimic the IAP-binding motif of the second mitochondria-derived activator of caspase (Smac). The IAP-binding motif consists of four amino acids (AVPI) that serve as an endogenous IAP antagonist. Structurally different Smac-mimetic compounds have been published and showed different binding affinity to IAP proteins. These differences in structure and binding have important consequences in terms of biological activity. Here, we present evidence that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in their ability to inhibit NF-kB in cells stimulated with TNFα. Over 300 monovalent and 300 bivalent Smac-mimetics, including compounds whose structures have been published, were tested for ability to degrade cIAP1 and cIAP2, and inhibit NF-kB in cell-based assays. Inhibition of NF-kB by bivalent Smac-mimetics correlated (R²=0.78) with the degradation of cIAP1 over a range of four logs, while monovalent Smac-mimetics did not (R²=0.20). Furthermore, monovalent Smac-mimetics degraded TRAF2-bound cIAP1 and cIAP2 less effectively (1/10-1/100 fold) compared to bivalent Smac-mimetics. While bivalent Smac-mimetics effectively degraded cIAP1 and cIAP2, birinapant (TL32711), a bivalent Smac-mimetic currently in Phase 2 clinical trials, was unique in its ability to preferentially degrade TRAF2-bound cIAP1 and cIAP2. Inhibition of NF-kB by birinapant was further characterized by the ImageStream cytometry, which showed that the nuclear translocation of p65 in response to TNFα stimulation was blocked in both HeLa and HL-60 cells. These data demonstrate that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in degrading TRAF2-bound cIAPs, in inhibition of NF-kB and efficiently mediating cell death. Furthermore, the unique profile of birinapant versus other bivalent Smac-mimetics may explain its preclinical and clinical safety profile. Citation Format: Yasuhiro Mitsuuchi, Stephen M. Condon, Eric M. Neiman, Christopher A. Benetatos, Martin E. Seipel, Gurpreet Singh Kapoor, Angeline C. Mufalli, Guangyao Yu, Orla Maguire, Hans Minderman, Mark A. Mckinlay, Martin Graham, David Weng, Srinivas Chunduru. Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2013-3333

Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2

Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-x(L). We now show that the FGF-2-S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-x(L) messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-x(L) translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-x(L)) that mediate FGF-2-S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression.

Abstract B8: Targeting p21-activated kinases in human lung cancer

Abstract P21-activated kinases (PAKs) are important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAKs have been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion and actin cytoskeleton organization. To better understand the role of PAK1 in lung tumorigenesis, PAK1 genomic copy number and expression were determined for a panel of adenocarcinoma and squamous non-small cell lung cancers (NSCLC) and small cell lung cancers (SCLC). 64% of squamous NSCLC samples were positive for PAK1 expression and 52% of all cases showed staining of moderate (2+) or strong (3+) immunohistochemical intensity in the malignant cells. Nuclear localization of PAK1 was also evident in a significant proportion of squamous NSCLCs. Adjacent normal lung tissue did not express appreciable levels of PAK1. Selective PAK1 inhibition was associated with delayed cell cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small molecule inhibitors and several synergistic combination therapies, including combination with antagonists of inhibitor of apoptosis (IAP) proteins, were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked IAP (XIAP) efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together, our results provide evidence for dysregulation of PAK1 in NSCLC and a role for PAK1 in cellular survival and proliferation in this indication.

Targeting IAP proteins for therapeutic intervention in cancer

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.

IAPs targeting RAC1

The RAC1 GTPase can promote elongated cell morphology during cell migration. Here, Oberoi et al. find that the inhibitor of apoptosis (IAP) proteins can directly target RAC1 for proteasomal degradation and that their loss results in increased RAC1 levels, cell elongation and enhanced directed migration. Consistent with an interaction previously observed between RAC1 and a Drosophila melanogaster IAP, they show here that both X chromosome-linked IAP (XIAP) and cellular IAP1 (cIAP1) act directly as E3 ligases for RAC1. This control mechanism appears to be physiologically relevant during development of the zebrafish hindbrain.

XAF1 as a prognostic biomarker and therapeutic target in pancreatic cancer

XAF1 (X chromosome-linked inhibitor of apoptosis [XIAP]-associated factor 1) is a novel XIAP modulator that negatively regulates the anti-apoptotic effects of XIAP and sensitizes cells to other cell death triggers. It has been reported to be downregulated in a variety of human cancer cell lines. However, the role of XAF1 in pancreatic carcinogenesis remains unclear. In the present study, we investigated the prognostic values of XAF1 expression and its regulation in cancer cell growth and apoptosis both in vitro and in vivo. From the immunohistochemistry staining of tissue microarray, 40 of 89 (44.9%) pancreatic specimens showed low levels of XAF1 expression. Statistical analysis suggested the downregulation of XAF1 was significantly correlated with tumor staging (P = 0.047) and those patients with low XAF1 levels had shorter survival times (P = 0.0162). Multivariate analysis indicated that XAF1 expression was an independent prognostic indicator of the survival of patients with pancreatic cancer (P = 0.007). Furthermore, we found that restoration of XAF1 expression mediated by Ad5/F35 virus suppressed cell proliferation and induced cell cycle arrest and apoptosis, accompanied by the activation of caspases 3, 8, and 9 and poly(ADP-ribose) polymerase as well as increased level of cytochrome c and Bid cleavage. Notably, XAF1 restoration robustly decreased survivin expression rather than XIAP. In addition, in vivo s.c. xenografts from Ad5/F35-XAF1 treatment, which showed less cellular proliferation and enhanced apoptosis, were significantly smaller than those from control groups. Our findings document that XAF1 is a valuable prognostic marker in pancreatic cancer and could be a potential candidate for cancer gene therapy.

X Chromosome-linked Inhibitor of Apoptosis Regulates Cell Death Induction by Proapoptotic Receptor Agonists

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-alpha signaling. By contrast, blockade of tumor necrosis factor-alpha does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.

Cardioprotection against myocardial infarction with PTD-BIR3/RING, a XIAP mimicking protein

The purpose of the present study was to investigate the potential cardioprotective effects of an original approach based on the properties of the X chromosome-linked Inhibitor of Apoptosis (XIAP), the most effective endogenous inhibitor of apoptosis. For this purpose, the C-terminal part of XIAP (BIR3 and RING domains) was fused to the protein transduction domain (PTD) of the HIV1 transactivator of transcription, which confers to fused protein the ability to cross cell membranes. This protein, so-called PTD-BIR3/RING, was administered intravenously in C57BL/6J mice subjected to 30 min coronary artery occlusion and 24 h of reperfusion. Administration of PTD-BIR3/RING at 5 min before and 30 min after the onset of reperfusion reduced infarct size vs control (23 ± 2% vs 41 ± 4% and 27 ± 4% vs 41 ± 3%, respectively, p < 0.05). Similar reduction in infarct size was observed when PTD-BIR3/RING was administered prior to ischemia (28 ± 1% vs 44 ± 3%). In addition to inhibition of caspase-3 and -9 activities, PTD-BIR3/RING induced an inhibition of caspase-8 and several other actors of the apoptotic pathways. In conclusion, this study demonstrates that the administration of PTD-BIR3/RING reduces myocardial infarct size even when injected during reperfusion through interruption of caspase activation by pharmacologically mimicking endogenous XIAP.

XIAP regulates bi-phasic NF-κB induction involving physical interaction and ubiquitination of MEKK2

The transcription factor NF-κB is transiently activated by a wide variety of stress signals, including pro-inflammatory mediators, and regulates the expression of genes with e.g., immune, inflammatory, and anti-apoptotic functions. The strength and kinetics of its induction, as well as its ultimate down-regulation is subject to multiple levels of regulation. One such regulatory protein is X chromosome-linked inhibitor of apoptosis (XIAP) that, besides its anti-apoptotic properties, has been shown to enhance NF-κB activity, however, the underlying molecular mechanism has remained elusive. We show here that following TNFα stimulation XIAP regulates a second wave of NF-κB activation. XIAP interacts with and ubiquitinates MEKK2, a kinase that has previously been associated with bi-phasic NF-κB activation. We conclude that, through interaction with MEKK2, XIAP functions in an ubiquitin ligase dependent manner to evoke a second wave of NF-κB activation, resulting in the modulation of NF-κB target gene expression.

XIAP regulates intracellular ROS by enhancing antioxidant gene expression

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.

Mitochondrial Extrusion through the Cytoplasmic Vacuoles during Cell Death

Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.

Morin (3,5,7,2′,4′-Pentahydroxyflavone) Abolishes Nuclear Factor-κB Activation Induced by Various Carcinogens and Inflammatory Stimuli, Leading to Suppression of Nuclear Factor-κB–Regulated Gene Expression and Up-regulation of Apoptosis

Morin is a flavone that exhibits antiproliferative, antitumor, and anti-inflammatory effects through a mechanism that is not well understood. Because of the role of transcription factor nuclear factor-kappaB (NF-kappaB) in the control of cell survival, proliferation, tumorigenesis, and inflammation, we postulated that morin mediates its effects by modulating NF-kappaB activation. We investigated the effect of morin on NF-kappaB pathway activated by inflammatory agents, carcinogens, and tumor promoters. The effect of this flavone on expression of NF-kappaB-regulated gene products involved in cell survival, proliferation, and invasion was also examined. We showed by DNA-binding assay that NF-kappaB activation induced by tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, lipopolysaccharide, ceramide, interleukin-1, and H(2)O(2) was suppressed by morin; the suppression was not cell type specific. The suppression of NF-kappaB by morin was mediated through inhibition of IkappaBalpha (inhibitory subunit of NF-kappaB) kinase, leading to suppression of phosphorylation and degradation of IkappaBalpha and consequent p65 nuclear translocation. Morin also inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNF receptor (TNFR) 1, TNFR1-associated death domain, TNFR-associated factor 2, NF-kappaB-inducing kinase, IkappaB kinase, and the p65 subunit of NF-kappaB. NF-kappaB-regulated gene products involved in cell survival [inhibitor of apoptosis (IAP) 1, IAP2, X chromosome-linked IAP, Bcl-xL, and survivin], proliferation (cyclin D1 and cyclooxygenase-2), and invasion (matrix metalloproteinase-9) were down-regulated by morin. These effects correlated with enhancement of apoptosis induced by TNF and chemotherapeutic agents. Overall, our results indicate that morin suppresses the activation of NF-kappaB and NF-kappaB-regulated gene expression, leading to enhancement of apoptosis. This may provide the molecular basis for the ability of morin to act as an anticancer and anti-inflammatory agent.