OBJECTIVE: To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP). METHODS: Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay. RESULTS: Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP. CONCLUSION IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.
Animals ; Autophagy ; Calcium/metabolism* ; Chondrocytes ; Endoplasmic Reticulum/metabolism* ; Endoribonucleases/pharmacology* ; Homeostasis ; Inositol ; Mice ; Mice, Knockout ; Protein Serine-Threonine Kinases ; RNA, Messenger/metabolism* ; Sirolimus/pharmacology* ; Tunicamycin/pharmacology*

OBJECTIVETo explore the effect of up-regulation of KA1 subunit of the kainate receptor on endoplasmic reticulum stress (ERS)-induced excitotoxic neurodegeneration in mouse hippocampus.

METHODSSeventy adult male KM mice were subjected to microinjections into the hippocampus of kainic acid (KA) or 500, 1000, or 2000 µg/ml tunicamycin (TM). At 1, 2, 3, 4, 5, 8, and 12 h after the injections, the mice were assessed for Bederson scores and sacrificed for FJB staining and immunofluorescence observation of the brain slices.

RESULTSAt 3, 4, 5, and 8 h after KA injection and at 4 and 5 h after of 2000 µg/ml TM injection, the mice showed severe central nervous system dysfunction, and FJB staining revealed increased cell death in the hippocampus, where up-regulated expressions of KA1 receptor and ERS marker P-eIF2α were found by immunofluorescence staining (P<0.05).

CONCLUSIONMicroinjection of KA or TM into the hippocampus causes neuronal death and ERS with up-regulated expression of KA1. In this process of neuronal apoptosis, the membrane receptor KA1 receives the apoptosis signal and transfers it to the inside of the cells to cause cell endoplasmic reticulum dysfunction and ERS response, which ultimately leads to neuronal death.

Animals ; Apoptosis ; Endoplasmic Reticulum Stress ; Hippocampus ; pathology ; Kainic Acid ; pharmacology ; Male ; Mice ; Neurons ; pathology ; Receptors, Kainic Acid ; metabolism ; Tunicamycin ; pharmacology ; Up-Regulation

The purposes of the present study were to investigate the inhibitory effect of quercetin (QUE) preconditioning on endoplasmic reticulum stress (ERS) inducer tunicamycin (TM)-induced apoptosis in RAW264.7 macrophages and the underlying molecular mechanisms. RAW264.7 cells were pretreated with different concentrations (20, 40, and 80 μmol/L) of QUE for 30 min and then treated with TM (5 mg/L) for 12 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC apoptosis detection kit, respectively. The nuclear translocation of activating transcription factor 6 (ATF6) in cells was detected by immunofluorescence analysis and Western blot. Protein and mRNA expressions of C/EBP homologous protein (CHOP) and Bcl-2 were examined by Western blot and real-time PCR, respectively. The results showed that TM reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of TM were significantly inhibited by QUE pretreatment at the concentrations of 40 and 80 μmol/L. Interestingly, we found that QUE also significantly suppressed the TM-induced translocation of ATF6, an ERS sensor, from the cytoplasm to the nucleus. In addition, exposure of RAW264.7 macrophages to TM resulted in a significant increase of the expression of CHOP, a transcription factor regulated by ATF6 under conditions of ERS, as well as a decrease of Bcl-2 at transcript and protein levels. QUE blocked these effects in a dose-dependent manner. These data indicate that QUE can protect RAW264.7 cells from TM-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ATF6-CHOP signaling pathway.
Activating Transcription Factor 6 ; metabolism ; Animals ; Apoptosis ; Cell Survival ; Endoplasmic Reticulum Stress ; Macrophages ; cytology ; drug effects ; Mice ; Quercetin ; pharmacology ; Transcription Factor CHOP ; metabolism ; Tunicamycin ; pharmacology

TNF-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising cancer therapy that preferentially induces apoptosis in cancer cells, but not most normal tissues. However, many cancers are resistant to TRAIL by mechanisms that are poorly understood. In this study, we showed that tunicamycin, a naturally occurring antibiotic, was a potent enhancer of TRAIL-induced apoptosis through downregulation of survivin. The tunicamycin-mediated sensitization to TRAIL was efficiently reduced by forced expression of survivin, suggesting that the sensitization was mediated at least in part through inhibition of survivin expression. Tunicamycin also repressed expression of cyclin D1, a cell cycle regulator commonly overexpressed in thyroid carcinoma. Furthermore, silencing cyclin D1 by RNA interference reduced survivin expression and sensitized thyroid cancer cells to TRAIL; in contrast, forced expression of cyclin D1 attenuated tunicamycin-potentiated TRAIL-induced apoptosis via over-riding downregulation of survivin. Collectively, our results demonstrated that tunicamycin promoted TRAIL-induced apoptosis, at least in part, by inhibiting the expression of cyclin D1 and subsequent survivin. Of note, tunicamycin did not sensitize the differentiated thyroid epithelial cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may offer an attractive strategy for safely treating resistant thyroid cancers.
Anti-Bacterial Agents/*pharmacology ; *Apoptosis ; Cell Line, Tumor ; Cyclin D1/*antagonists & inhibitors/metabolism ; *Down-Regulation ; Humans ; Microtubule-Associated Proteins/*genetics/metabolism ; TNF-Related Apoptosis-Inducing Ligand/*metabolism ; Tunicamycin/*pharmacology

TNF-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising cancer therapy that preferentially induces apoptosis in cancer cells, but not most normal tissues. However, many cancers are resistant to TRAIL by mechanisms that are poorly understood. In this study, we showed that tunicamycin, a naturally occurring antibiotic, was a potent enhancer of TRAIL-induced apoptosis through downregulation of survivin. The tunicamycin-mediated sensitization to TRAIL was efficiently reduced by forced expression of survivin, suggesting that the sensitization was mediated at least in part through inhibition of survivin expression. Tunicamycin also repressed expression of cyclin D1, a cell cycle regulator commonly overexpressed in thyroid carcinoma. Furthermore, silencing cyclin D1 by RNA interference reduced survivin expression and sensitized thyroid cancer cells to TRAIL; in contrast, forced expression of cyclin D1 attenuated tunicamycin-potentiated TRAIL-induced apoptosis via over-riding downregulation of survivin. Collectively, our results demonstrated that tunicamycin promoted TRAIL-induced apoptosis, at least in part, by inhibiting the expression of cyclin D1 and subsequent survivin. Of note, tunicamycin did not sensitize the differentiated thyroid epithelial cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may offer an attractive strategy for safely treating resistant thyroid cancers.
Anti-Bacterial Agents/*pharmacology ; *Apoptosis ; Cell Line, Tumor ; Cyclin D1/*antagonists & inhibitors/metabolism ; *Down-Regulation ; Humans ; Microtubule-Associated Proteins/*genetics/metabolism ; TNF-Related Apoptosis-Inducing Ligand/*metabolism ; Tunicamycin/*pharmacology

BACKGROUNDAlthough CD4(+) T cell apoptosis and CD8(+) T cell responses have been extensively studied during HIV infection, how apoptosis signals being initiated in CD4(+) T cells still need to be elucidated. The present study was designed to characterize the function-unknown gene, C6orf120, and elucidates its primary role in tunicamycin-induced CD4(+) T apoptosis.

METHODSThe C6orf120 coding sequence was amplified from peripheral blood mononuclear cells (PBMCs) total RNA of AIDS patients. The DNA fragment was inserted into the pET-32a expression system, transformed into Escherichia coli, and preparation of C6ORF120 recombinant protein. The magnetic cell separation technology was used to prepare primary CD4(+) T cells and CD8(+) T cells. The primary T cells were cultured at 1 × 10(6) cells/ml, treated with 0, 0.1, 1, 10, 100, and 200 ng/ml of C6orf120 recombinant protein for 48 hours, then harvested for cell cycle and apoptosis analysis. Tunicamycin (0.5 µmol/L) was used to induce endoplasmic reticulum stress in Jurkat cells. The biomarker 78 KDa glucose-regulated protein (GRp78) and growth arrest and DNA damage (GADD) were used to evaluate endoplasmic reticulum stress of Jurkat cells.

RESULTSWe prepared C6ORF120 recombinant protein and its polyclonal antibody. Immunohistochemical analysis showed that C6orf120 mainly expressed in hepatocytes and cells in germinal center of lymph node. At concentration of 0.1, 1, 10, 100, and 200 ng/ml, C6orf120 recombinant protein could induce apoptosis of Jurkat cells and primary CD4(+) T cells, and promoting G2 phase of its cell cycle. Western blotting analysis showed that C6ORF120 recombinant protein increased the expression of GRp78 and GADD in Jurkat cells in vitro.

CONCLUSIONOur results suggested that C6ORF120 could induce apoptosis of CD4(+) T cells, at least in part, mediated with endoplasmic reticulum stress.

Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; CD8-Positive T-Lymphocytes ; Cell Cycle ; Cells, Cultured ; Endoplasmic Reticulum Stress ; Female ; HIV Infections ; immunology ; Humans ; Immunohistochemistry ; Male ; Microscopy, Confocal ; Proteins ; genetics ; metabolism ; Tunicamycin ; pharmacology

OBJECTIVETo detect the effect of tunicamycin on Fas protein expression and Fas monoclonal antibody (FasMcAb)-induced apoptosis of fibroblast from hypertrophic scar and keloid.

METHODSThe expression of Fas protein was detected by immunostaining in 5 cases of keloid, 5 cases of hypertrophic scar and 5 cases of normal skin as control. The fibroblasts were cultured and treated with tunicamycin. The Fas protein expression and the fibroblast apoptosis rate were assessed by Western Blot and flow cytometry.

RESULTSIt revealed that Fas protein was detectable in all the three groups. The Fas glycosylation level was highest in hypertrophic scar, but lowest in normal skin. The FasMcAb-induced apoptosis had a positive relationship with the Fas glycosylation. Tunicamycin had a significant inhibitory effect on the Fas glycosylation in keloid and hypertrophic scar, but not in normal skin.

CONCLUSIONSThe FasMcAb-induced apoptosis has a positive relationship with the Fas glycosylation. Tunicamycin has a significant inhibitory effect on the Fas glycosylation in keloid and hypertrophic scar.

Adolescent ; Adult ; Antibodies, Monoclonal ; pharmacology ; Apoptosis ; Child ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Female ; Fibroblasts ; metabolism ; Glycosylation ; Humans ; Keloid ; metabolism ; pathology ; Male ; Signal Transduction ; Tunicamycin ; pharmacology ; Young Adult ; fas Receptor ; metabolism

OBJECTIVE: To explore the effects of 3-methyladenine (3-MA, an autophagy inhibitor) on sensitivities of nasopharyngeal carcinoma cells to radiotherapy and chemotherapy and the underlying mechanisms.
 METHODS: Cell proliferation was examined by MTT and colony formation assay, while cell apoptosis was evaluated by annexin V/PI double staining and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. Mitochondrial membrane potential was measured by commercial kit (JC-1). The expression of endoplasmic reticulum stress (ERS)-related protein, glucose-regulated protein 78 (GRP78) and autophagy-related protein beclin1, microtubule-associated protein 1 light chain 3 (LC3) were examined by Western blot.
 RESULTS: Cisplatin (DDP), ionizing radiation (IR) or tunicamycin (TM) treatment obviously inhibited the proliferation of HONE-1 cells in a concentration-dependent and time-dependent manner. Compared with control group, pretreatment with 1 mmol/L of 3-MA significantly 
reduced cell viability and enhanced the apoptosis in the DDP (6.00 μmol/L), 4.00 Gy IR or TM (1.00 μmol/L) groups. There was no significant difference in the apoptosis between the DDP (5.8%) and 4Gy IR (6.7%) groups. Compared with the control group, protein levels of GRP78, beclin1 and lipid-conjugated membrane-bound form (LC3-II) were significantly increased after the treatment of DDP, 4.00 Gy IR or TM, which were inhibited by pretreatment of 3-MA.
 CONCLUSION 3-MA can sensitize HONE-1 cells to chemotherapy and radiotherapy, which is related to prevention of endoplasmic reticulum stress-induced autophagy in nasopharyngeal carcinoma cells.
Adenine ; analogs & derivatives ; pharmacology ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Carcinoma ; Cell Line, Tumor ; drug effects ; radiation effects ; Cell Proliferation ; Cell Survival ; Cisplatin ; pharmacology ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; pathology ; Radiation, Ionizing ; Radiation-Sensitizing Agents ; pharmacology ; Tunicamycin ; pharmacology

OBJECTIVEChanges of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum (ER). This ER function disorder is called endoplasmic reticulum stress (ERS). Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease and so on. Therefore, the protection effect of the traditional Chinese drug-Tiantai No. 1 (1) on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug.

METHODSPrimarily cultured marrow mesenchymal stem cells (MSCs) of rats were treated by tunicamycin (TM) in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 (which would assist cells to resist cellular stress injury), and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively.

RESULTSProtein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs (P<0.05). All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group (P<0.05 or P<0.01) after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group (P<0.05 or P<0.01). This effect showed a dose dependent manner.

CONCLUSIONTiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.

Animals ; Anti-Bacterial Agents ; antagonists & inhibitors ; pharmacology ; Cells, Cultured ; Drug Antagonism ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Heat-Shock Proteins ; genetics ; metabolism ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; RNA ; analysis ; drug effects ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological ; drug effects ; genetics ; Tunicamycin ; antagonists & inhibitors ; pharmacology

OBJECTIVETo study the effects of tunicamycin (a glycosylation inhibitor) combined with cisplatin on the proliferation and apoptosis of human nasopharyngeal carcinoma cells and explore the molecular mechanism.

METHODSNasopharyngeal carcinoma CNE-1 and CNE-2 cells cultured in vitro were treated with different concentrations of tunicamycin with or without cisplatin. The inhibition of cell proliferation was examined using MTT assay and colony formation assay, and the cell apoptosis was analyzed using flow cytometry with propidium iodide staining. The expressions of Bax, Bcl-2, and GRP78 in cells treated with 3 µmol/L tunicamycin with or without 6.00 µmol/L cisplatin were measured with Western blotting.

RESULTSTreatment with tunicamycin or cisplatin obviously inhibited the proliferation of CNE-1 and CNE-2 cells. Treatment with 3 µmol/L tunicamycin for 24, 36 and 48 h resulted in a viability of 72.13%, 51.97%, and 37.56% in CNE-1 cells and 85.61%, 56.95%, and 43.66% in CNE-2 cells, respectively, and the combined treatment with 6 µmol/L cisplatin lowered the cell viability to 67.97%, 47.76%, and 34.68% in CNE-1 cells and 56.89%, 37.05%, and 29.30% in CNE-2 cells, respectively. Tunicamycin at 0.3 µmol/L combined with 0.6 µmol/L cisplatin showed an obviously enhanced inhibitory effect on colony formation of CNE-1 and CNE-2 cells. Tunicamycin treatment (3 µmol/L) of CNE-1 and CNE-2 cells for 48 h induced an apoptosis rate of only 8.89% and 8.67%, but when combined with 6 µmol/L cisplatin, the cell apoptosis rate increased to 37.02% and 32.25%, significantly higher than that in cells with cisplatin treatment alone (7.25% and 6.36%, respectively). Compared with tunicamycin and cisplatin alone, the combined treatment significantly increased Bax expression and decreased Bcl-2 expression in the cells; tunicamycin up-regulated the expression of GRP-78 and enhanced the activity of caspase-3.

CONCLUSIONTunicamycin can inhibit the proliferation of CNE-1 and CNE-2 cells and enhance cisplatin-induced cell death, the mechanism of which may involve excessive endoplasmic reticulum stress response and increased activity of caspase-3.

Apoptosis ; drug effects ; Carcinoma ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Humans ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tunicamycin ; pharmacology ; bcl-2-Associated X Protein ; metabolism