X Chromosome-linked Inhibitor Of Apoptosis Protein Expression Research Articles

Apoptosis-related factors are relevant to progression of pancreatic neuroendocrine tumors

BackgroundMultidisciplinary therapy centered on antitumor drugs is indicated in patients with unresectable pancreatic neuroendocrine tumors (PanNET). However, the criteria for selection of optimal therapeutic agents is controversial. The aim of this study was to assess the malignancy of PanNET for optimal therapeutic drug selection.MethodsForty-seven patients with PanNET who underwent surgery were reviewed retrospectively, and immunohistochemical characteristics, including expression of GLUT1, SSTR2a, SSTR5, Survivin, X-chromosome-linked inhibitor of apoptosis protein (XIAP), and Caspase3 in the resected specimens, were investigated. Relapse-free survival (RFS) and overall survival (OS) were evaluated with regard to the characteristics using the Kaplan–Meier method and compared with the log-rank test.ResultsGLUT1 expression showed significant correlation with sex (p = 0.036) and mitotic rate (p = 0.048). Survivin and XIAP expression showed significant correlation with T-stage (p = 0.014 and 0.009), p-Stage (p = 0.028 and 0.045), and mitotic rate (p = 0.023 and 0.007). XIAP expression also significantly influenced OS (p = 0.044).ConclusionsSurvivin and XIAP correlated with grade of malignancy, and expression of XIAP in particular was associated with a poor prognosis. Expression of these proteins may be a useful indicator to select optimal therapeutic agents in PanNET.

Targeting X chromosome-linked inhibitor of apoptosis protein in mucoepidermoid carcinoma of the head and neck: A novel therapeutic strategy using nitidine chloride.

Nitidine chloride (NC) was recently reported to exhibit a wide range of pharmacological properties for several diseases, including cancer. Here we report for the first time that NC is a potential therapeutic agent for mucoepidermoid carcinoma (MEC) occurring in the head and neck because it suppresses X chromosome-linked inhibitor of apoptosis protein (XIAP) in human MEC in vitro and in vivo. The antitumor effects of NC were evaluated by trypan blue exclusion assay, western blotting, live/dead assay, 4',6-diamidino-2-phenylindole (DAPI) staining, human apoptosis antibody array, immunofluorescence staining, immunohistochemistry, small interfering RNA assay, transient transfection of XIAP overexpression vector, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and histopathological examination of organs. NC inhibited cell viability and induced caspase-dependent apoptosis in vitro. A human apoptosis antibody array assay showed that XIAP is suppressed by NC treatment. XIAP was overexpressed in oral squamous cell carcinoma (OSCC) tissues that arose from the head and neck, and high XIAP expression was correlated with poor prognosis in OSCC patients. XIAP depletion significantly increased apoptosis, and ectopic XIAP overexpression attenuated the apoptosis induced by NC treatment. NC suppressed tumor growth in vivo at a dosage of 5 mg/kg/day. The number of TUNEL-positive cells increased and the protein expression of XIAP was consistently downregulated in NC-treated tumor tissues. In addition, NC caused no histopathological changes in the liver or kidney. These findings provide new insights into the mechanism of action underlying the anticancer effects of NC and demonstrate that NC is a promising therapeutic agent for the treatment of human MEC of the head and neck. KEY MESSAGES: • Nitidine chloride induces caspase-dependent apoptosis in MEC of the head and neck. • High XIAP expression correlates with poor prognosis of OSCC patients. • Nitidine chloride suppresses tumor growth in vivo without any systemic toxicities. • Targeting XIAP is a novel chemotherapeutic strategy for MEC of the head and neck.

Significance and expression of X chromosome linked inhibitor of apoptosis protein, B-cell lymphoma-2 and Survivin in thymoma

Objective To explore significance and expression of X chromosome linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-2 (bcl-2) and survivin in thymoma. Methods The expressions of XIAP, bcl-2 and Survivin in 19 cases of thymic hyperplasia, 45 cases of thymoma and 13 cases of thymic adenocarcinoma were detected by immunohistochemical method, and the relationship between the expression of XIAP, bcl-2 and Survivin and age, sex, histological type, clinical stage, ymph node metastasis and myasthenia gravis was analyzed. Results The positive rate of XIAP in normal thymic tissue (2/19), thymoma (26/45) and thymic adenocarcinoma (10/13) increased in turn, and the difference was statistically significant (P<0.05). The positive rates of bcl-2 in normal thymic tissue (2/19), thymoma (29/45) and thymic adenocarcinoma (11/13) increased in turn (P<0.05). The positive rates of Survivin in normal thymic tissue (1/19), thymoma (23/45) and thymic adenocarcinoma (11/13) increased in turn (P<0.05). The positive rates of XIAP, bcl-2 and Survivin in thymoma tissues were correlated with histological type, clinical stage, ymph node metastasis, and was not correlated with age, sex and myasthenia gravis. The expressions of XIAP was correlated wih the expression of bcl-2 (r=0.568, P<0.01), the expression of XIAP was correlated with the expression of the expression of Survivin (r=0.671, P<0.01), the expression of bcl-2 was correlated with Survivin in thymoma tissues (r=0.529, P<0.01). Conclusion XIAP, bcl-2 and Survivin were highly expressed in thymoma tissues, which could be used as predictors of the occurrence and malignancy of thymoma. Key words: X chromosome linked inhibitor of apoptosis protein; B-cell lymphoma-2; Survivin; Thymoma

Sevoflurane prevents miR-181a-induced cerebral ischemia/reperfusion injury

BackgroundSevoflurane (sevo) has been reported to be an effective neuroprotective agent in cerebral ischemia/reperfusion injury (CIRI). However, the precise molecular mechanism underlying sevo preconditioning in CIRI remains largely unknown. MethodsA middle cerebral artery occlusion (MCAO) rat model and primary cortical neurons after oxygen-glucose deprivation and reoxygenation (OGDR) were used as the in vivo and in vitro models of CIRI. The expression profiles of miR-181a and X chromosome-linked inhibitor-of-apoptosis protein (XIAP) in the cerebral cortex of rats and in cortical neurons were examined by qRT-PCR and Western blot, respectively. The infarct volumes were measured by TTC staining and neurological deficits in rats was determined by Zea-Longa scoring criteria. The cell viability, lactate dehydrogenase (LDH) release and apoptotic rate were detected in cortical neurons by MTT assay, LDH analysis and flow cytometry. Western blot analysis was performed to assess the expression of apoptosis-related protein. Luciferase reporter assay was used to confirm the interaction between miR-181a and XIAP. ResultsmiR-181a was upregulated and XIAP was downregulated in rats after MCAO. Sevo preconditioning attenuated miR-181a expression and promoted XIAP level in a rat model of CIRI. Sevo preconditioning ameliorated anti-miR-181a-mediated protective effects on cerebral ischemia in rat model of CIRI, presented as the decrease of infarct volume, neurological deficit and apoptosis. Moreover, sevo pretreatment abated miR-181a-induced cellular injury in primary cortical neurons after OGD, embodied by the increase of cell viability, the reduction of LDH release and the decline of apoptosis. Furthermore, miR-181a suppressed XIAP expression by binding to its 3′UTR in cortical neurons, and sevo-mediated increase on XIAP expression was counteracted by miR-181 overexpression in OGDR-treated neurons. ConclusionSevo preconditioning protected against CIRI in vitro and in vivo possibly by inhibiting miR-181a and facilitating XIAP.

Significance and expression of X chromosome linked inhibitor of apoptosis protein、CD44v6 and matrix metalloproteinase-2 in gastric cancer

Objective To explore significance and expression of X chromosome linked inhibitor of apoptosis protein (XIAP), CD44v6 and matrix metalloproteinase-2 (MMP-2) in gastric cancer. Methods The expression of XIAP, CD44v6 and MMP-2 in 82 cases of gastric cancer and adjacent normal tissues was detected by immunohistochemical method The relationship between XIAP, CD44v6 and MMP-2 and age, sex, tumor size and histological grade, clinical stage, lymph node metastasis was analyzed. Statisticalanalysis of data using the statistical product and service solutions 17.0 software. Results The positive rate of XIAP in gastric cancer (54/82, 65.85%) was higher than that in adjacent normal tissues (16/82, 19.51%) (χ2=32.083, P<0.01). The positive rate of CD44v6 in gastric cancer (72/82, 87.80%) was higher than that in adjacent normal tissues (18/82, 21.95%) (χ2=71.805, P<0.01). The positive rate of MMP-2 in gastric cancer (61/82, 74.39%) was higher than that in adjacent normal tissues (13/82, 15.85%) (χ2=56.735, P<0.01). The positive expression rate of XIAP, CD44v6 and MMP-2 in gastric cancer was related to clinical stage and lymph node metastasis (P<0.05), was not related to age, sex, tumor size and histological grade. The expression of XIAP was positively correlated with the expression of CD44v6 (r=0.652, P<0.05), the expression XIAP was positively correlated with the expression of MMP-2 (r=0.613, P<0.05), the expression CD44v6 was positively correlated with the expression of MMP-2 (r=0.729, P<0.05). Conclusion XIAP, CD44v6 and MMP-2 were highly expressed in gastric cancer tissues and were closely related to the occurrence, development and metastasis of breast cancer. Key words: Gastric cancer; X chromosome linked inhibitor of apoptosis protein; CD44v6; Matrix metalloproteinase-2; Expression; Significance

Negative regulation of RNA-binding protein HuR by tumor-suppressor ECRG2.

Esophageal cancer-related gene 2 (ECRG2) is a newer tumor suppressor whose function in the regulation of cell growth and apoptosis remains to be elucidated. Here we show that ECRG2 expression was upregulated in response to DNA damage, and increased ECRG2 expression induced growth suppression in cancer cells but not in non-cancerous epithelial cells. ECRG2-mediated growth suppression was associated with activation of caspases and marked reduction in the levels of apoptosis inhibitor, X chromosome-linked inhibitor of apoptosis protein (XIAP). ECRG2, via RNA-binding protein human antigen R (HuR), regulated XIAP mRNA stability and expression. Furthermore, ECRG2 increased HuR ubiquitination and degradation but was unable to modulate the non-ubiquitinable mutant form of HuR. We also identified missense and frame-shift ECRG2 mutations in various human malignancies and noted that, unlike wild-type ECRG2, one cancer-derived ECRG2 mutant harboring glutamic acid instead of valine at position 30 (V30E) failed to induce cell death and activation of caspases. This naturally occurring V30E mutant also did not suppress XIAP and HuR. Importantly, the V30E mutant overexpressing cancer cells acquired resistance against multiple anticancer drugs, thus suggesting that ECRG2 mutations appear to have an important role in the acquisition of anticancer drug resistance in a subset of human malignancies.

Silencing XIAP suppresses osteosarcoma cell growth, and enhances the sensitivity of osteosarcoma cells to doxorubicin and cisplatin.

X-chromosome-linked inhibitor of apoptosis protein (XIAP) is an important member of the inhibitors of apoptosis (IAP) family. It has been shown that XIAP promotes the invasion, metastasis, growth and survival of malignant cells, and confers resistance to some chemotherapeutic drugs in various types of cancer. However, little is known regarding its detailed role in osteosarcoma (OS). In the present study, we first investigated the expression of XIAP in OS tissues, and an increased expression of XIAP in OS tissues compared to adjacent non-tumor tissue was identified. Additionally, its expression level correlated with key pathological characteristics including clinical stage, tumor size and metastasis. Subsequently, small interfering RNA (siRNA) was used to block XIAP expression to evaluate the effect of XIAP siRNA on cell proliferation, colony formation, cell cycle, apoptosis, tumorigenicity, and the combined effects of doxorubicin or cisplatin in OS cell lines (MG63 cells). Downregulation of XIAP expression using the RNA silencing approach efficiently decreased cell proliferation and colony formation, and induced cell apoptosis and cell cycle in the G0/G1 stage. In addition, this downregulation inhibited tumor growth in a nude murine model. The resuls also showed that treatment with XIAP-shRNA in combination with doxorubicin or cisplatin enhanced chemosensitivity. These results suggested that XIAP may aid the diagnosis of OS and may be an effective strategy for gene therapy of this disease.

Stabilization of XIAP mRNA through the RNA binding protein HuR regulated by cellular polyamines

The X chromosome-linked inhibitor of apoptosis protein (XIAP) is the most potent intrinsic caspase inhibitor and plays an important role in the maintenance of intestinal epithelial integrity. The RNA binding protein, HuR, regulates the stability and translation of many target transcripts. Here, we report that HuR associated with both the 3'-untranslated region and coding sequence of the mRNA encoding XIAP, stabilized the XIAP transcript and elevated its expression in intestinal epithelial cells. Ectopic HuR overexpression or elevated cytoplasmic levels of endogenous HuR by decreasing cellular polyamines increased [HuR/XIAP mRNA] complexes, in turn promoting XIAP mRNA stability and increasing XIAP protein abundance. Conversely, HuR silencing in normal and polyamine-deficient cells rendered the XIAP mRNA unstable, thus reducing the steady state levels of XIAP. Inhibition of XIAP expression by XIAP silencing or by HuR silencing reversed the resistance of polyamine-deficient cells to apoptosis. Our findings demonstrate that HuR regulates XIAP expression by stabilizing its mRNA and implicates HuR-mediated XIAP in the control of intestinal epithelial apoptosis.

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.

Lead Affects Apoptosis and Related Gene XIAP and Smac Expression in the Hippocampus of Developing Rats

Lead (Pb) exposure poses devastating effects on central nervous system development of children. To replicate aspects of this neurotoxicity, we examined the effect of lead on the expression of apoptosis and of apoptosis-related genes, XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac (second mitochondrial activator of caspase), in the hippocampus of developing rats. A total of 48 rats (30-day old) were randomly divided into four groups for intragastrical perfusion of lead acetate [Pb(Ac)2]: untreated, low (2 mg/kg/d), medium (20 mg/kg/d), and high (200 mg/kg/d) dose groups. Pb content was determined in blood, and the apoptosis indexes and XIAP and Smac gene expression were analyzed in the hippocampus. There was a significant difference in apoptosis indexes (AI) between the exposed and control groups (p < 0.01). AI was highest in the high exposure group. XIAP gene expression was reduced in the exposed groups and the expression was negatively correlated with blood lead levels (BLLs) (p < 0.05). But the four groups did not differ in the expression of Smac (p > 0.05). Our data indicate that exposure to Pb(Ac)2 caused a dose-dependent and significant increase of apoptosis in the hippocampus of developing rats through depressing the expression of the XIAP but not the Smac genes.

Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma

Members of the inhibitor of apoptosis protein (IAP) family, survivin and X-chromosome-linked IAP (XIAP), contribute to apoptosis resistance of cancer cells, and an increase in their expression may elevate the apoptotic threshold of malignant tumours during their growth and progression. In the present study, we investigated the expression status of survivin and its interactants hepatitis B X-interacting protein (HBXIP) and XIAP in non-small cell lung carcinoma (NSCLC) cell lines and NSCLC tumours and matched lungs from surgically treated patients in relation to their clinicopathological data. The expression of survivin, HBXIP and XIAP mRNAs was quantitated by real-time RT-PCR. The expression of survivin and XIAP proteins was analysed by Western blotting and ELISA. Survivin mRNA and protein levels were highly upregulated in NSCLC cells and tissues as compared to the lungs. In fact, the levels of survivin mRNA and protein in the tumours were more than 10-fold higher in 96 (64%) and 72 (82%) of the 150 and 88 examined NSCLC patients, respectively. The expression of survivin mRNA was higher in squamous cell lung carcinomas than in lung adenocarcinomas (LACs; P=0.003) and in less-differentiated tumours than in well-differentiated ones (P=0.007). The level of survivin protein was higher in stage IB and stage II+III tumours (P=0.049 and P=0.044), than in stage IA tumours. The BIRC5 promoter polymorphism at nucleotide -31 did not influence the expression of survivin mRNA and protein in NSCLC cells and tumours. HBXIP mRNA was abundantly expressed in NSCLC cell lines and NSCLC tumours and lungs, while its level was comparable in the tumours and lungs. The expression of XIAP mRNA in NSCLC cell lines and NSCLC tumours and lungs was not significantly different. However, the expression of XIAP protein was higher in NSCLC tumours, particularly in LACs, as compared to the lungs (P=0.017 and P=0.004). In conclusion, the overexpression of survivin in the majority of NSCLCs together with the abundant or upregulated expression of HBXIP and XIAP suggest that tumours are endowed with resistance against a variety of apoptosis-inducing conditions.

Stabilization of XIAP mRNA through the RNA binding protein HuR regulated by cellular polyamines.

The X chromosome-linked inhibitor of apoptosis protein (XIAP) is the most potent intrinsic caspase inhibitor and plays an important role in the maintenance of intestinal epithelial integrity. The RNA binding protein, HuR, regulates the stability and translation of many target transcripts. Here, we report that HuR associated with both the 3′-untranslated region and coding sequence of the mRNA encoding XIAP, stabilized the XIAP transcript and elevated its expression in intestinal epithelial cells. Ectopic HuR overexpression or elevated cytoplasmic levels of endogenous HuR by decreasing cellular polyamines increased [HuR/XIAP mRNA] complexes, in turn promoting XIAP mRNA stability and increasing XIAP protein abundance. Conversely, HuR silencing in normal and polyamine-deficient cells rendered the XIAP mRNA unstable, thus reducing the steady state levels of XIAP. Inhibition of XIAP expression by XIAP silencing or by HuR silencing reversed the resistance of polyamine-deficient cells to apoptosis. Our findings demonstrate that HuR regulates XIAP expression by stabilizing its mRNA and implicates HuR-mediated XIAP in the control of intestinal epithelial apoptosis.

Extrinsic pathway- and cathepsin-dependent induction of mitochondrial dysfunction are essential for synergistic flavopiridol and vorinostat lethality in breast cancer cells

The present studies have determined whether interactions between the cyclin-dependent kinase inhibitor flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat; Zolinza) occur in breast cancer cells. MDA-MB-231 and MCF7 cells were treated with flavopiridol (25-100 nmol/L) and vorinostat (125-500 nmol/L) in vitro, and mechanisms of cell killing were determined. Concurrent treatment of cells with flavopiridol and vorinostat or treatment of cells with flavopiridol followed by vorinostat promoted cell killing in a greater than additive fashion. Similar data were obtained with the CDK inhibitor roscovitine. Flavopiridol suppressed c-FLIP-l/s and BCL-xL expression, whereas vorinostat reduced expression of BCL-xL, and combined exposure to flavopiridol and vorinostat reduced MCL-1 and X-chromosome-linked inhibitor of apoptosis protein (XIAP) levels. Pharmacologic or genetic inhibition of caspase-8 reduced flavopiridol toxicity, but abolished killing by vorinostat and cell death caused by the vorinostat/flavopiridol regimen. Loss of BAX/BAK function or loss of BID function modestly reduced flavopiridol toxicity, but abolished vorinostat-mediated potentiation of flavopiridol toxicity, as did inhibition of caspase-9. Inhibition and/or deletion of cathepsin B function significantly attenuated vorinostat/flavopiridol lethality. Flavopiridol suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT activity and expression of activated forms of AKT and mitogen-activated protein/ERK kinase 1 maintained c-FLIP-l/s, BCL-xL, and XIAP expression and protected cells against flavopiridol/vorinostat lethality. Overexpression of c-FLIP-s and BCL-xL abolished the lethality of flavopiridol/vorinostat. Collectively, these data argue that flavopiridol enhances the lethality of vorinostat in breast cancer cells in part through the inhibition of AKT and ERK1/2 function, leading to reduced expression of multiple inhibitors of the extrinsic and intrinsic apoptosis pathways, as well as activation of cathepsin protease-dependent pathways.

Activation of Caspase 8 in the Pituitaries of Streptozotocin-Induced Diabetic Rats: Implication in Increased Apoptosis of Lactotrophs

Lactotroph cell death is increased in streptozotocin-induced diabetic rats. To determine the mechanism involved, cell death proteins were accessed in pituitaries of diabetic (streptozotocin at 65 mg/kg, 2 months evolution) and control male rats by Western blot analysis and double immunohistochemistry. The intact and cleaved forms of caspase 9 were increased in diabetic rat pituitaries compared with controls. Although the proforms of caspases 3, 6, and 7 were increased in diabetic rat pituitaries, their activated forms were either unchanged or decreased. Activation of these effector caspases may be blocked by the increased expression of X-chromosome-linked inhibitor of apoptosis protein (XIAP) in diabetic rat pituitaries. However, in diabetic rats, XIAP expression in lactotrophs was decreased, suggesting that this cell type is not protected. Caspase 8, p53, and nuclear factor kappaB were more highly activated in diabetic rat pituitaries, with caspase 8 colocalization in lactotrophs being increased. These results suggest that, in the pituitaries of diabetic rats, the cascades of normal cell turnover are partially inhibited, possibly via XIAP, and this may be cell specific. Furthermore, activation of the extrinsic cell-death pathway, including activation of caspase 8, may underlie the diabetes-associated increase in lactotroph death.

Response of XIAP, ARC, and FLIP apoptotic suppressors to 8 wk of treadmill running in rat heart and skeletal muscle

Although it has been demonstrated that exercise training has an antiapoptotic effect on postmitotic myocytes, the mechanisms responsible for this effect are still largely unclear. Because the antiapoptotic effect of exercise training in postmitotic myocytes could be possibly mediated by the upregulation of apoptotic suppressors, this study examined the effect of endurance training on endogenous apoptotic suppressors including X-chromosome-linked inhibitor of apoptosis protein (XIAP), apoptosis repressor with caspases recruitment domain protein (ARC), and FADD-like inhibitor protein (FLIP) in skeletal and cardiac muscles. Eight adult Sprague-Dawley rats were trained 5 days weekly for 8 wk on treadmill, and eight sedentary rats served as controls. Soleus and ventricle muscles were dissected 2 days after the last training session. The mRNA content of XIAP, ARC, and FLIP was estimated by RT-PCR with ribosomal 18S RNA used as an internal control. The protein expression of XIAP, ARC, FLIP(S), and FLIP(alpha) was assessed by Western immunoblot. After training, mRNA content of ARC and FLIP was not different between the control and trained animals, whereas XIAP mRNA content was elevated by 22 and 14% in the trained soleus and cardiac muscles, respectively, relative to the control samples. No difference was found in the protein content of FLIP(S) and FLIP(alpha) between control and trained muscles, whereas XIAP and ARC protein content was increased by 18 and 38%, respectively, in the soleus muscle of trained animals. Furthermore, negative relationships were found between XIAP and apoptotic DNA fragmentation as well as ARC and caspase-3 activity. These findings are consistent with the hypothesis that the modulation of apoptotic suppressors is involved in training-induced attenuation of apoptosis in skeletal and cardiac muscles.

Regulation of XIAP and Smac/DIABLO in the rat hippocampus following transient forebrain ischemia

We investigated the expression of XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac/DIABLO, a newly identified mitochondrila apoptogenig molecule in the hippocampus following transient global ischemia. Transient global ischemia produced by two-vessel occlusion triggers the delayed neuronal death of CA1 neurons in the hippocampus. We demonstrate that CA1 neuronal loss induced by ischemia (10 min) is preceded by a selective and marked elevation of catalytically active caspase-3 in these neurons, indicative of apoptosis. XIAP (X chromosome-linked inhibitor of apoptosis protein) is a member of the inhibitor of apoptosis (IAP) gene family that, in addition to suppressing cell death by inhibition of caspases, is involved in an increasing number of signalling cascades. The present study shows alterations in the levels of XIAP and of Smac/DIABLO (second mitochondrial activator of caspase) after cerebral ischemia. The protein levels of XIAP and the number of XIAP-positive cells were regulated by cerebral ischemia in a strictly time and region dependent manner. The largest change in XIAP-IR was observed in the CA1 sub field, which is the most vulnerable area of hippocampus. The mitochondrial expression level of Smac/DIABLO increased during reperfusion. Smac/DIABLO expression was associated with alteration of the XIAP levels and the appearance of activated form of caspase-3 within the hippocampus during reperfusion in spatial and temporal manners.

Oxidative stress is associated with XIAP and Smac/DIABLO signaling pathways in mouse brains after transient focal cerebral ischemia.

The interaction of X chromosome-linked inhibitor-of-apoptosis protein (XIAP) with second mitochondria-derived activator of caspase (Smac)/direct inhibitor-of-apoptosis protein-binding protein with low pI (DIABLO) contributes to regulation of apoptosis after a variety of cell death stimuli, and in our reported in vivo transient focal cerebral ischemia (tFCI) model. We have also reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduces apoptotic cell death after tFCI. Our present study was designed to clarify the relationship between the XIAP signaling pathway and oxidative stress in the regulation of apoptosis after tFCI. We used a tFCI model of SOD1 transgenic mice and wild-type littermates to examine the expression of XIAP and Smac/DIABLO by Western blotting and the interaction of XIAP with Smac/DIABLO (XIAP/Smac) or caspase-9 (XIAP/caspase-9) by coimmunoprecipitation. The direct oxidation of carbonyl groups, an indication of oxidative injury to total and individual proteins caused by tFCI, was examined using a 2,4-dinitrophenylhydrazone reaction assay. Direct oxidative injury to cytosolic and mitochondrial proteins was reduced by SOD1 after tFCI. The individual oxidized carbonyls in XIAP, mitochondrial Smac/DIABLO, and caspase-9 were also reduced by SOD1. Expression of XIAP and XIAP/caspase-9 was promoted, whereas translocation of Smac/DIABLO and XIAP/Smac was reduced, by SOD1 after tFCI. These results suggest that overexpression of SOD1 may affect the XIAP pathway after tFCI by reducing the direct oxidative reaction to XIAP regulators after reperfusion injury.

Cisplatin inhibits the expression of X-chromosome-linked inhibitor of apoptosis protein in an oral carcinoma cell line.

Cisplatin is known to activate caspase-3 through the mitogen-activated kinase (MAPK) pathway. We found that X-chromosome-linked inhibitor of apoptosis protein (XIAP), a direct inhibitor of caspase-3, 7, and 9, is constitutively expressed in a cell line of oral squamous cell carcinoma, KOSC-2. Cisplatin treatment of the cells inhibited the expression of XIAP, and this was associated with DNA fragmentation. Overexpression of XIAP, by a transfection experiment, inhibited the cisplatin-induced apoptosis. We conclude that cisplatin induces apoptosis mediated not only by the activation of MAPK pathway but by the inhibition of XIAP.

Expression and Function of X Chromosome-Linked Inhibitor of Apoptosis Protein in Sjögren's Syndrome

Apoptotic cell death in acinar and ductal epithelial cells is thought to play an important role in the development of salivary gland dysfunction in patients with Sjögren's syndrome (SS). We examined the expression of anti-apoptotic molecules in salivary glands from patients with SS. The labial salivary glands from six human T-cell leukemia virus (HTLV)–I–seronegative and eleven HTLV-I–seropositive SS patients were analyzed by immunohistochemistry. In vitro experiments were performed with a human salivary gland cell line (HSG cells). Immunohistologic analyses revealed that Bcl-2 and Bcl-x were preferentially expressed in salivary infiltrating mononuclear cells more than acinar and ductal epithelial cells. In contrast, strong X chromosome-linked inhibitor of apoptosis protein (XIAP) expression was evident in both acinar and ductal epithelial cells. The pattern of expression of these anti-apoptotic molecules was similar in both HTLV-I–seropositive and HTLV-I–seronegative SS patients. Western blot analysis confirmed expression of XIAP in cultured HSG cells. The expression of XIAP in HSG cells was increased by IL-1β, TGF-β1, or IL-10. However, XIAP expression was down-regulated by TNF-α, which induced apoptotic cell death of HSG cells with an increase in caspase-3 activity. These effects of TNF-α in HSG cells were antagonized by IL-1β, TGF-β1, or IL-10. Our results suggest that XIAP is important in regulating apoptotic cell death of acinar and ductal epithelial cells in patients with SS.

Nuclear factor-kappaB and caspases co-operatively regulate the activation and apoptosis of human macrophages.

Accumulating evidence suggests that macrophages function as major effector cells in the pathological process of various human diseases. We examined here the role of nuclear factor-kappaB (NF-kappaB) and caspases in the regulation of activation and apoptosis of macrophages. Activation of the human monoblastic leukaemia cell line, U937, by phorbol 12-myristate 13-acetate (PMA) increased the expression of CD14/CD86, and cytokine production. PMA stimulation also increased the expression of both pro-caspase-8 and pro-caspase-3 in U937, but not apoptosis or intracellular caspase-3 activity. PMA also increased the expression of X-chromosome-linked inhibitor of apoptosis protein (XIAP) in U937, suggesting an inhibitory action for XIAP on the caspase cascade in PMA-stimulated U937. Electrophoretic mobility shift assay (EMSA) showed a significant increase of nuclear NF-kappaB activity in PMA-stimulated U937. When a potent NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was added to U937 cell culture in the presence of PMA, apoptosis was triggered by activation of caspase-3, which was induced by caspase-8 activation. XIAP expression was markedly suppressed in PMA-treated U937 in the presence of PDTC. The inhibitors of caspase-8 and caspase-3 mostly inhibited apoptosis of U937 treated with PMA in the presence of PDTC. Furthermore, a phenotype of U937 treated with PMA and PDTC in the presence of caspase inhibitor was almost identical to that of unstimulated U937. Our results suggest that the signalling pathways involved in the activation and apoptosis of human macrophages could be co-operatively regulated by the use of NF-kappaB and caspase inhibitors, thus enabling the control of macrophage function and number.