OBJECTIVETumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance greatly limits the clinical therapeutic efficacy of TRAIL. Elucidating the molecular mechanism underlying TRAIL resistance will be fundamental to resolving this problem.

METHODSNuclear and cytoplasmic protein extraction and immuno？uorescence (IF) assay were used to detect changes in heterogeneous nuclear ribonucleoprotein K (hnRNPK) localization in H1299 cells. The evaluation of cell apoptosis in cells transfected with GFP-hnRNPK, GFP-hnRNPK S284/353A, or GFP-hnRNPK S284/353D mutant was performed using cleaved caspase-3 antibody. The gene expression of XIAP was tested by quantitative RT-PCR.

RESULTSPreviously, we reported that hnRNPK antagonized TRAIL-induced apoptosis through inhibition of PKC-mediated GSK3β phosphorylation. In this study, we further demonstrate that TRAIL treatment induces cytoplasmic accumulation of hnRNPK in H1299 cells. The hnRNPK localized in the cytoplasm has a higher capacity to antagonize TRAIL-induced apoptosis. Both ERK1/2 signaling inhibitor U0126 and ERK-phosphoacceptor-site mutant (GFP-hnRNPK S284/353A) diminish cytoplasmic accumulation of hnRNPK induced by TRAIL. Moreover, we show that XIAP is involved in hnRNPK-mediated TRAIL resistance in H1299 cells.

CONCLUSIONTaken together, these results give new insights into the understanding of the molecular mechanism associated with TRAIL resistance in lung adenocarcinoma.

Apoptosis ; physiology ; Cell Line, Tumor ; Gene Expression Regulation ; physiology ; Heterogeneous-Nuclear Ribonucleoprotein K ; genetics ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Up-Regulation ; physiology ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism

OBJECTIVE: To investigate the effects of LCL161, a Smac mimetic, on the proliferation and apoptosis in hepatocellular carcinoma cells and the underlying mechanisms. 
 METHODS: The effect of LCL161 on the cell viability of HepG2 and SMMC7721 cells was measured by MTT assay. The effect of LCL161 at lower concentrations on the proliferation in hepatocellular carcinoma (HCC) cells was detected by colony formation assay. Apoptosis was assessed by flow cytometry with PI staining. The mitochondrial membrane potential was measured by JC-1 staining. The expression of PARP, p-Akt, cIAP1 and XIAP protein was analyzed by Western blot.
 RESULTS: LCL161 displayed notable antiproliferative activity on HCC cells at the concentrations of 1-16 μmol/L (P<0.01), with IC50 values of 4.3 and 4.9 μmol/L for HepG2 and SMMC7721 cells, respectively, after treatment for 48 h. LCL161 at lower concentrations obviously inhibited the colony formation of HCC cells. LCL161 induced significant apoptosis in HCC cells (P<0.01), and resulted in the apoptotic rate at (1.5±0.8)% or (1.8±0.6)% , (15.2±2.8)% or (12.2±2.4)%, (28.7±3.0)% or (22.4±2.7)%, (34.6±2.3)% or (30.2±2.4)% for HepG2 cells or SMMC7721 cells at the concentration of 0, 2, 4 or 8 μmol/L, respectively. The result of JC-1 staining indicated that the mitochondrial membrane potential of HCC cells was reduced by LCL161. In addition, LCL161 promoted the cleavage of PARP, down-regulated the protein expression of p-Akt, and degraded cIAP1.
 CONCLUSION LCL161 possesses significant anti-proliferative activity and pro-apoptotic action in HepG2 and SMMC7721 cells, which might be correlated with reduction in mitochondrial membrane potential, down-regulation of p-Akt and degradation of cIAP1.
Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; Down-Regulation ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Liver Neoplasms ; Membrane Potential, Mitochondrial ; drug effects ; Proto-Oncogene Proteins c-akt ; genetics ; Thiazoles ; pharmacology ; Ubiquitin-Protein Ligases ; metabolism ; X-Linked Inhibitor of Apoptosis Protein

BACKGROUND/AIMS: The aim of this study was to identify the profile of rare variants associated with Crohn's disease (CD) using whole exome sequencing (WES) analysis of Korean children with CD and to evaluate whether genetic profiles could provide information during medical decision making. METHODS: DNA samples from 18 control individuals and 22 patients with infantile, very-early and early onset CD of severe phenotype were used for WES. Genes were filtered using panels of inflammatory bowel disease (IBD)-associated genes and genes of primary immunodeficiency (PID) and monogenic IBD. RESULTS: Eighty-one IBD-associated variants and 35 variants in PID genes were revealed by WES. The most frequently occurring variants were carried by nine (41%) and four (18.2%) CD probands and were ATG16L2 (rs11235604) and IL17REL (rs142430606), respectively. Twenty-four IBD-associated variants and 10 PID variants were predicted to be deleterious and were identified in the heterozygous state. However, their functions were unknown with the exception of a novel p.Q111X variant in XIAP (X chromosome) of a male proband. CONCLUSIONS: The presence of many rare variants of unknown significance limits the clinical applicability of WES for individual CD patients. However, WES in children may be beneficial for distinguishing CD secondary to PID.
Asian Continental Ancestry Group/genetics ; Carrier Proteins/genetics ; Child ; Child, Preschool ; Crohn Disease/*genetics ; *Exome ; Female ; Genetic Predisposition to Disease ; *Genetic Variation ; Humans ; Immunologic Deficiency Syndromes/genetics ; Infant ; Male ; Phenotype ; Receptors, Interleukin-17/genetics ; Republic of Korea ; Sequence Analysis, DNA/*methods ; X-Linked Inhibitor of Apoptosis Protein/genetics

OBJECTIVETo explore the effect of silence of Pin1 expression on hyperoxia-induced apoptosis in alveolar epithelial cells A549.

METHODSA549 cells were divided into four groups: control, hyperoxia, negative lentivirus and Pin1-shRNA hyperoxia. The hyperoxia group was exposed to a mixture of 95%O2 and 5%CO2 for 10 minutes. Then cells were cultured in a closed environment. After 24 hours, the changes of morphology were observed under an inverted microscope. Cell apoptosis was detected by flow cytometry (FCM). The expression of X-linked inhibitor of apoptosis protein (XIAP) and Caspase-9 were detected by immunohistochemistry. The production of reactive oxygen species (ROS) and cellular mitochondria membrane potential (△Ψm) were determined by fluorescence microscopy.

RESULTSUnder the inverted microscope, the A549 cells grew slowly and the changes in morphology of the cells were most obvious in the hyperoxia and negative lentivirus groups. The changes in morphology of A549 cells were obviously improved in the Pin1-shRNA hyperoxia group. The FCM results showed that the apoptosis rate of A549 cells increased, Caspase-9 expression increased, XIAP expression decreased, mitochondrial ROS production increased and mitochondrial membrane potential decreased in the hyperoxia and negative lentivirus groups compared with the control group (P<0.05). Compared with the hyperoxia and negative lentivirus groups, the apoptosis rate of A549 cells decreased, Caspase-9 expression decreased, XIAP expression increased, mitochondrial ROS production decreased and mitochondrial membrane potential increased in the Pin1-shRNA hyperoxia group (P<0.05), although the levels of the indexes did not reach to those of the control group.

CONCLUSIONSSilencing of Pin1 could suppress hyperoxia-induced apoptosis of A549 cells.

Apoptosis ; Caspase 9 ; genetics ; Humans ; Hyperoxia ; pathology ; Membrane Potential, Mitochondrial ; NIMA-Interacting Peptidylprolyl Isomerase ; Peptidylprolyl Isomerase ; physiology ; Reactive Oxygen Species ; metabolism ; X-Linked Inhibitor of Apoptosis Protein ; genetics

OBJECTIVETo investigate the prevalence of mutations and sequence variations in X-linked inhibitor of apoptosis (XIAP) gene among Chinese pediatric patients with hemophagocytic lymphohistiocytosis (HLH).

METHODSSixty-five children who were diagnosed with HLH between January 2009 and December 2012 (case group), as well as 70 healthy children (control group), were enrolled in the study. The exons of XIAP gene (1-1, 1-2, 2-6) were amplified by PCR and directly sequenced. The genotypic and allelic frequencies of single nucleotide polymorphism (SNP) were analyzed.

RESULTSNone of the HLH patients showed mutations in these exons of XIAP gene. Only one nonsynonymous SNP, rs5956583 located in exon 5, was observed, but there were no significant differences in the genotypic and allelic frequencies of this SNP between the case and control groups (P>0.05).

CONCLUSIONSHLH caused by XIAP mutations may be rare in children. SNP rs5956583 of XIAP gene may have little contribution to the development of childhood HLH.

Child ; Child, Preschool ; Female ; Humans ; Lymphohistiocytosis, Hemophagocytic ; genetics ; Male ; Mutation ; Polymorphism, Single Nucleotide ; X-Linked Inhibitor of Apoptosis Protein ; genetics

Adenocarcinoma ; metabolism ; pathology ; Apoptosis ; drug effects ; Baculoviral IAP Repeat-Containing 3 Protein ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Morpholines ; pharmacology ; Ovarian Neoplasms ; metabolism ; pathology ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; Ubiquitin-Protein Ligases ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism

Amino Acid Sequence ; Base Sequence ; Child ; Flow Cytometry ; methods ; Genes, X-Linked ; Hematopoietic Stem Cell Transplantation ; Humans ; Lymphocytes ; metabolism ; Lymphohistiocytosis, Hemophagocytic ; diagnosis ; genetics ; therapy ; Lymphoproliferative Disorders ; diagnosis ; genetics ; therapy ; Mutation ; Transplantation, Homologous ; X-Linked Inhibitor of Apoptosis Protein ; deficiency ; genetics ; metabolism

OBJECTIVE: To investigate the simultaneous inhibition of X-linked inhibitor of apoptosis protein (XIAP) and survivin expression on epithelial-mesenchymal transition (EMT) and invasiion of pancreatic cancer cells Panc-1, and its mechanism. METHODS: On the established human pancreatic cancer cells Panc-1-XS, the expression of XIAP and survivin was inhibited simultaneously. Cell invasion and migration were detected by Transwell chamber experiments and scratch test, and the expression of epithelial marker E-cadherin, mesenchymal markers Slug, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and P-Akt protein was determined by Western blot. RESULTS: Cell invasion and migration of Panc-1-XS cells decreased significantly, accompanied by significantly upregulated protein expression of E-cadherin, and significantly declined protein expression of the Slug, indicating increased mesenchymal-epithelial conversion (MET); and increased protein expression of PTEN, and declined protein expression of P-Akt. CONCLUSION Simultaneously inhibiting the expression of XIAP and survivin can partially reverse EMT phenotype of pancreatic cancer Panc-1 cells, which then significantly reduces the cell invasion and migration of Panc-1 cell lines. This process may be regulated by PTEN/PI3K/Akt signaling pathway.
Antigens, CD ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Neoplasm Invasiveness ; PTEN Phosphohydrolase ; metabolism ; Pancreatic Neoplasms ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Snail Family Transcription Factors ; Survivin ; Transcription Factors ; metabolism ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism

This study was purpose to investigate the expression of XIAP mRNA in chronic myeloid leukemia (CML) and to explore its significance in the advance and prognosis of CML. The chromosomal karyotype analysis and detection of XIAP mRNA were performed by the technique of chromosomal R banding and real time PCR in 71 patients with CML and 10 healthy controls. The results showed that there was a significant increase of XIAP mRNA expression in accelerated and blastic phase of the CML, compared with the patients in chronic phase (t = 9.10, 9.30, p < 0.01). Moreover, the difference of XIAP mRNA expression level was not statistically significant in different karyotype groups. It is concluded that the XIAP gene expression in accelerated and blastic phases of CML patients obviously increases, XIAP gene is closely related to the advance of CML.
Adult ; Case-Control Studies ; Female ; Gene Expression ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; pathology ; Male ; Prognosis ; X-Linked Inhibitor of Apoptosis Protein ; genetics

To study the expression of X-linked inhibitor of apoptosis protein (XIAP) and cell apoptosis in vitro model of acute pancreatitis (AP), we carried out experiments to stimulate AR42J cell line with caerulein (10(-8) mol/L) for 12 hours, then collected cells at various time points (0 h, 4 h, 8 h, 12 h, and 24 h, respectively). We then observed the morphologic changes of AR42J cells with the stimulation of caerulein with electronic microscope. The gene expression of XIAP, caspase-3 and caspase-9 was detected using real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), and the protein expression of XIAP was assessed by western blot. The activation of nuclear factor-kappa B (NF-kappaB) was measured by flow cytometry (FCM). With the stimulation of caerulein, the expression of XIAP and the NF-kappaB activation could first decrease and then increase, but the change of caspase-3 and caspase-9 expressions were opposite. XIAP may inhibit the cell apoptosis in rat pancreatic acinus AR42J cell lines at first with the stimulation of caerulein, then NF-kappaB can upgrade the expression of XIAP and increase the cell apoptosis.
Acinar Cells ; cytology ; metabolism ; Animals ; Apoptosis ; physiology ; Cell Line ; Ceruletide ; pharmacology ; NF-kappa B ; metabolism ; Pancreas ; cytology ; metabolism ; Pancreatitis ; metabolism ; Rats ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism