BACKGROUND: beta-cell death due to endoplasmic reticulum (ER) stress has been regarded as an important pathogenic component of type 2 diabetes. The possibility has been suggested that sulfonylurea, currently being used as one of the main oral hypoglycemic agents of type 2 diabetes, increases ER stress, which could lead to sulfonylurea failure. The authors of the present study examined ER stress of beta-cells in a glucolipotoxic condition using glyburide (GB) in an environment mimicking type 2 diabetes. METHODS: Apoptosis was induced by adding various concentrations of GB (0.001 to 200 microM) to a glucolipotoxic condition using 33 mM glucose, and the effects of varied concentrations of palmitate were evaluated via annexin V staining. The markers of ER stress and pro-apoptotic markers were assessed by Western blotting and semi-quantitative reverse transcription-polymerase chain reaction. Additionally, the anti-apoptotic markers were evaluated. RESULTS: Addition of any concentration of GB in 150 microM palmitate and 33 mM glucose did not increase apoptosis. The expression of phosphorylated eukaryotic initiation factor (eIF-2alpha) was increased and cleaved caspase 3 was decreased by adding GB to a glucolipotoxic condition. However, other ER stress-associated markers such as Bip-1, X-box binding protein-1, ATF-4 and C/EBP-homologous protein transcription factor and anti-apoptotic markers phosphor-p85 phosphatidylinositol 3-kinase and phosphorylation of Akt did not change significantly. CONCLUSION: GB did not show further deleterious effects on the degree of apoptosis or ER stress of INS-1 cells in a glucolipotoxic condition. Increased phosphorylation of eIF-2alpha may attenuate ER stress for adaptation to increased ER protein load.
Annexin A5 ; Apoptosis ; Blotting, Western ; Caspase 3 ; Endoplasmic Reticulum ; Endoplasmic Reticulum Stress ; Eukaryotic Initiation Factor-2 ; Glucose ; Glyburide ; Hypoglycemic Agents ; Insulin-Secreting Cells ; Peptide Initiation Factors ; Phosphatidylinositol 3-Kinase ; Phosphorylation ; Transcription Factors

OBJECTIVETo investigate the inhibitory effect of elemene on laryngeal carcinoma (Hep-2 cells) xenograft growth in nude mice and its mechanisms, and to explore the relationship between the expression of eukaryotic initiation factor families (eIF4E & eIF4G) and angiogenesis factors (bFGF & VEGF) after the administration of elemene.

METHODSHuman laryngeal carcinoma cells from Hep-2 cell strain were transplanted subcutaneously to BALB/c-nu/nu nude mice to produce tumors (42 nude mice were separated into seven groups to be treated by intraperitoneal injection). The tumor volume, tumor weight and tumor inhibition rate were evaluated, the expression of eIF4E, eIF4G, bFGF, VEGF and microvessel density were estimated by paraffin-embedded sections of seven groups' tumor samples analyzed utilizing immunohistochemical streptavidin peroxidase technique.

RESULTSElemene could inhibit the tumor growth in vivo. A significant suppression of tumor growth was observed when the dosage was increased. The tumor inhibition rates (IR) of elemene 50 mg/kg, 100 mg/kg and 200 mg/kg treated group were 5.2% , 41.7% and 50. 5% respectively. The IR of 100 mg/kg elemene (41.7%) was not significantly different with that of 3 mg/kg cisplatin (44.6%), and the IR of the drug combination (100 mg/kg elemene + 3 mg/kg cisplatin) was 51.2%. Compared with control groups the protein expression of eIF4E, eIF4G, bFGF and VEGF were significantly inhibited by elemene (P < 0.05), and the microvessel density in elemene treated groups decreased (P < 0.05). The tumor inhibition rate of combined elemene 100 mg/kg and cisplatin 3 mg/kg was 51.2%.

CONCLUSIONSElemene could inhibit the subcutaneous plantation of human laryngeal carcinoma in nude mice and its mechanism may be associated with inhibited expression of eIF families and angiogenesis factors. The combination of elemene and cisplatin could promote the synergistic effect on chemotherapy in the target tumor cells.

Animals ; Carcinoma, Squamous Cell ; blood supply ; pathology ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Eukaryotic Initiation Factor-4E ; metabolism ; Eukaryotic Initiation Factor-4G ; metabolism ; Fibroblast Growth Factor 2 ; metabolism ; Humans ; Laryngeal Neoplasms ; blood supply ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Sesquiterpenes ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism ; Xenograft Model Antitumor Assays

Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. I/R injury induces serious hepatic dysfunction and failure. In this study, we identified proteins that were differentially expressed between sham and I/R injured livers. Animals were subjected to hepatic ischemia for 1 hr and were sacrificed at 3hr after reperfusion. Serum ALT and AST levels were significantly increased in I/R-operated animals compared to those of sham-operated animals. Ischemic hepatic lobes of I/R-operated animals showed the hepatic lesion with unclear condensation and sinusoidal congestion. Proteins from hepatic tissue were separated using two dimensional gel electrophosresis. Protein spots with a greater than 2.5-fold change in intensity were identified by mass spectrometry. Among these proteins, glutaredoxin-3, peroxiredoxin-3, glyoxalase I, spermidine synthase, dynamin-1-like protein, annexin A4, eukaryotic initiation factor 3, eukaryotic initiation factor 4A-I, 26S proteasome, proteasome alpha 1, and proteasome beta 4 levels were significantly decreased in I/R-operated animals compared to those of sham-operated animals. These proteins are related to protein synthesis, cellular growth and stabilization, anti-oxidant action. Moreover, Western blot analysis confirmed that dynamin-1-like protein levels were decreased in I/R-operated animals. Our results suggest that hepatic I/R induces the hepatic cells damage by regulation of several proteins.
Animals ; Annexin A4 ; Blotting, Western ; Estrogens, Conjugated (USP) ; Eukaryotic Initiation Factor-3 ; Hepatocytes ; Ischemia ; Lactoylglutathione Lyase ; Liver ; Mass Spectrometry ; Mice ; Peptide Initiation Factors ; Proteasome Endopeptidase Complex ; Proteins ; Reperfusion ; Reperfusion Injury ; Salicylamides ; Spermidine Synthase

OBJECTIVE: To investigate the effect of eukaryotic translation initiation factor 4E(eIF4E) on the autophagy of CD138 plasma cells in multiple myeloma(MM). METHODS: Multiple myeloma CD138 plasma cells were treated with eIF4E inhibitor 4EGI, the changes of autophagy-related factors LC3-II and Beclin1 were detected by fluorescent quantitative PCR and Western blot, the changes of cell proliferation inhibition were detected by MTT assay, and cell apoptosis was detected by flow cytometry. RESULTS: Quantitative fluorescence PCR showed that after treatment of myeloma cells with 4EGI, the expression levels of LC3-II and Beclin1 mRNA gradually increased with the enhancomer of 4EGI concentration and the prolongation of action time, and the differences were statistically significant (48 h: LC3-Ⅱ,r=0.942, Beclin1,r=0.952; 80 μg/ml: LC3-Ⅱ,r=0.966, Beclin1,r=0.998）; Western blot showed that with the enhancement of 4EGI concentration, the expression of LC3-II and Beclin1 protein gradually increased（LC3-Ⅱ,r=0.923, Beclin1,r=0.977）; CCK-8 showed that the inhibition rate of cells gradually increased （r=0.996）; the apoptotic rate of 4EGI-treated groups (23.23±4.47, 7.59±1.67, 2.03±0.19) was significantly different from that of control group (0.03±0.04) (P<0.05). CONCLUSION The inhibition of eIF4E can activate the autophagy of CD138 plasma cells in multiple myeloma and induce the death of myeloma cells.
Autophagy ; Beclin-1 ; Cell Line, Tumor ; Eukaryotic Initiation Factor-4E ; Humans ; Multiple Myeloma

Evidences show that eukaryotic mRNAs can perform protein translation through internal ribosome entry sites (IRES). 5'-Untranslated region of the mRNA encoding apoptotic protease-activating factor 1 (Apaf-1) contains IRES, and, thus, can be translated in a cap-independent manner. Effects of changes in protein translation pattern through rapamycin pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-butanone(NNK, tobacco-specific lung carcinogen)-induced apoptosis in human bronchial epithelial cells were examined by caspase assay, FACS analysis, Western blotting, and transient transfection. Results showed that NNK induced apoptosis in concentration- and time-dependent manners. NNK-induced apoptosis occurred initially through cap-independent protein translation, which during later stage was replaced by cap-dependent protein translation. Our data may be pplicable as the mechanical basis of lung cancer treatment.
Antibiotics, Antineoplastic/pharmacology ; Apoptosis/*drug effects ; Apoptotic Protease-Activating Factor 1 ; BH3 Interacting Domain Death Agonist Protein ; Blotting, Western ; Bronchi/metabolism/*pathology ; Carcinogens/*pharmacology ; Carrier Proteins/metabolism ; Caspases/metabolism ; Cytochromes c/metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells/metabolism/*pathology ; Eukaryotic Initiation Factor-4E/metabolism ; Flow Cytometry ; Humans ; Nitrosamines/*pharmacology ; Protein Biosynthesis ; Proteins/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA Cap-Binding Proteins/*physiology ; Sirolimus/pharmacology ; Time Factors ; bcl-2-Associated X Protein

OBJECTIVETo analyze the interaction between the microRNA-338 and its targeting proteins during the cerebral ischemia and reperfusion injury.

METHODSTargetScan was used to predict the targets of microRNA-338. The potential targeting proteins were then selected according to their secondary structures using RNA structure 4.6 software and their involvement in cerebral ischemia and reperfusion injury was studied. Dual-luciferase reporter assay was used to testify whether microRNA-338 can recognize the 3'UTR of target protein. Western blot was applied to analyze the expression of eiF4E3 in both experimental group and control group.

RESULTEiF4E3 was the most likely potential targeting protein of microRNA-338. The secondary structure of local region of eiF4E3 recognizing microRNA-338 was conservative. The ratio of firefly to renilla luciferase activity in the experimental group was much higher than that of control group. However, there was no significant difference in the expression of eiF4E3 between these two groups.

CONCLUSIONMicroRNA-338 can recognize the 3'UTR of eiF4E3 while it has no significant effect on the expression of eiF4E3. The post-target-recognizing regulation for miRNA do exist and this mechanism is possibly related to the tertiary structure of target mRNA.

3' Untranslated Regions ; genetics ; Animals ; Eukaryotic Initiation Factor-4E ; genetics ; Gene Expression Regulation ; MicroRNAs ; genetics ; PC12 Cells ; Protein Structure, Secondary ; Protein Transport ; genetics ; RNA, Messenger ; genetics ; Rats

Intramuscular hemangioma originated in chest wall is a rare benign tumor, with no relevant reports in Korea. In most cases, the tumor is discovered before the age of 30 years and it is reported that trauma operates as the initiation factor. It is essential to concern the clinical suspicion and conduct a CT scan for diagnosis. The principle of treatment is surgical excision with clear resection margin. The authors of this study report a case of surgical excision for post-traumatic intramuscular hemangioma of the chest wall with review of literature.
Hemangioma ; Korea ; Peptide Initiation Factors ; Thoracic Wall ; Thorax

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.
Animals ; Connectin/genetics* ; Cricetinae ; Eukaryotic Initiation Factor-4G/genetics* ; Gene Expression Regulation/genetics* ; Gene Silencing ; Growth Hormone/genetics* ; Hepatitis B Surface Antigens/genetics* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/virology* ; Humans ; Hydro-Lyases/metabolism* ; Male ; Pregnancy-Specific beta 1-Glycoproteins/genetics* ; RNA, Viral/analysis* ; Spermatozoa/virology* ; Trans-Activators/genetics* ; Transcription, Genetic ; Transfection ; Viral Regulatory and Accessory Proteins

Eukaryotic translation initiation factor 4G (eIF4G) is a scaffold component of eukaryotic translation initiation factor 4F (eIF4F) complex, which takes principal part in the initiating of protein synthesis. Both two subtypes (eIF4G1 and eIF4G2) of eIF4G were found to be closely related with various tumors. The eIF4G1 expression is significantly up-regulated in breast cancer, cervical cancer, nasopharyngeal carcinoma, lung squamous cell carcinoma, prostatic carcinoma and other malignant tumors, compared with those in adjacent tissues; and the eIF4G2 is obviously over-expressed in diffuse large B cell lymphoma and acute myeloid leukemia, but low-expressed in bladder transitional cell carcinoma. This paper reviews the progress in the study of the role of eIF4G in tumor genesis, development, diagnosis and prognosis.
Eukaryotic Initiation Factor-4G ; Humans ; Neoplasms ; Protein Biosynthesis ; Up-Regulation

Cyclooxygenase-2 (COX-2) is an enzyme induced by various proinflammatory and mitogenic stimuli. Celecoxib is a selective inhibitor of COX-2 that have been shown to affect cell growth and apoptosis. Lung cancer cells expressing COX-2 is able to be a target of celecoxib, this study focuses on investigating that celecoxib induces apoptosis via endoplasmic reticulum (ER) stress on lung cancer cells. We investigated whether celecoxib induced apoptosis on non-small cell lung cancer cell line, A549 and H460. The 50 µM of celecoxib increased apoptotic cells and 100 µM of celecoxib significantly induced apoptosis. To check involvement of caspase cascade, pretreatment of z-VAD-fmk blocked celecoxib-induced apoptosis. However, caspase-3, -8, and -9 were not activated, but cleavage of non-classical caspase-4 was detected using western blot. As checking ER stress associated molecules, celecoxib did not increase expressions of growth arrest and DNA damage inducible protein 34, activating transcription factor 4, and spliced X-box binding protiens-1, but increase of both glucose-regulated protein 78 (GRP78) and C/EBP homologous transcription factor were detected. Salubrinal, inhibitor of eIF2 and siRNA for IRE1 did not alter celecoxib-induced apoptosis. Instead, celecoxib-induced apoptosis might be deeply associated with ER stress depending on GRP78 because siRNA for GRP78 enhanced apoptosis. Taken together, celecoxib triggered ER stress on lung cancer cells and celecoxib-induced apoptosis might be involved in both non-classical caspase-4 and GRP78.
Activating Transcription Factor 4 ; Apoptosis ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung* ; Caspase 3 ; Celecoxib* ; Cell Death* ; Cell Line ; Cyclooxygenase 2 ; DNA Damage ; Endoplasmic Reticulum Stress* ; Endoplasmic Reticulum* ; Eukaryotic Initiation Factor-2 ; Lung Neoplasms ; RNA, Small Interfering ; Transcription Factors