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1.
Article in English | WPRIM | ID: wpr-728019

ABSTRACT

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca²⁺ homeostasis. The store-operated calcium (SOC) channel is the primary Ca²⁺ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca²⁺ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca²⁺ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.


Subject(s)
Blotting, Western , Calcium , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Endothelial Cells , Homeostasis , Human Umbilical Vein Endothelial Cells , Tunicamycin , Unfolded Protein Response
2.
Article in English | WPRIM | ID: wpr-763301

ABSTRACT

OBJECTIVES: Endoplasmic reticulum (ER) stress is known to be associated with inflammatory airway diseases, and three major transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6) play important roles in ER stress-related proinflammatory signaling. However, the effects of ER stress and these three major signaling pathways on the regulation of the production of airway mucins in human nasal airway epithelial cells have not been elucidated. METHODS: In primary human nasal epithelial cells, the effect of tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, ER stress inhibitor) on the expression of MUC5AC and MUC5B was investigated by reverse transcriptasepolymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis. Small interfering RNA (siRNA) transfection was used to identify the mechanisms involved. RESULTS: Tunicamycin increased the expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules, including spliced X-box binding protein 1 (XBP-1), transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP), and ATF6. In addition, 4-PBA attenuated the tunicamycin-induced expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules. Furthermore, siRNA knockdowns of XBP-1, CHOP, and ATF6 blocked the tunicamycin-induced mRNA expressions and glycoprotein productions of MUC5AC and MUC5B. CONCLUSION.: These results demonstrate that ER stress plays an important role in the regulation of MUC5AC and MUC5B via the activations of XBP-1, CHOP, and ATF6 in human nasal airway epithelial cells.


Subject(s)
Activating Transcription Factor 6 , Carrier Proteins , CCAAT-Enhancer-Binding Proteins , Endoplasmic Reticulum Stress , Endoplasmic Reticulum , Epithelial Cells , Glycoproteins , Humans , Immunoenzyme Techniques , Inositol , Mucins , Phosphotransferases , Real-Time Polymerase Chain Reaction , RNA, Messenger , RNA, Small Interfering , Transcription Factor CHOP , Transcription Factors , Transfection , Tunicamycin
3.
Acta Physiologica Sinica ; (6): 733-739, 2016.
Article in Chinese | WPRIM | ID: wpr-331609

ABSTRACT

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on the activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms of macrophage apoptosis. RAW264.7 macrophages were treated with AGE-alb (2, 4 and 6 g/L), control albumin (C-alb, 4 g/L), tunicamycin (TM, 4 mg/L), or pretreated with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then treated with AGE-alb (4 g/L). After incubation for 24 h, the cell viability and apoptosis were determined by using MTT assay and TUNEL detection kit, respectively. Lactate dehydrogenase (LDH) activity in media was determined by using an assay kit. The protein levels of caspase-12 were examined by Western blot analysis. The results showed that like TM (an ERS inducer), incubation with AGE-alb led to significant decrease in viability and increase in LDH activity in media and apoptotic rate in a dose-dependent manner. In addition, AGE-alb induced activation of caspase-12 especially at the concentration of 4 and 6 g/L (P < 0.01), which was similar to TM. However, PBA (an ERS inhibitor) protected RAW264.7 macrophages from AGE-alb-induced decrease in viability and increases in LDH activity and apoptosis. Moreover, PBA also inhibited the caspase-12 activation induced by AGE-alb (P < 0.05). These results suggest that AGE-alb may induce apoptosis in RAW 264.7 macrophages, and the mechanism may be related to the activation of ERS-associated apoptotic pathway mediated by caspase-12.


Subject(s)
Animals , Apoptosis , Caspase 12 , Cell Line, Tumor , Cell Survival , Endoplasmic Reticulum Stress , Macrophages , Mice , Phenylbutyrates , Serum Albumin , Tunicamycin
4.
Article in English | WPRIM | ID: wpr-160779

ABSTRACT

PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.


Subject(s)
Anti-Bacterial Agents/pharmacology , Apoptosis , Blotting, Western , C-Reactive Protein/biosynthesis , Cells, Cultured , Endoplasmic Reticulum Stress/drug effects , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation , Humans , Polymerase Chain Reaction , RNA, Messenger/genetics , Retinal Pigment Epithelium/metabolism , Serum Amyloid P-Component/biosynthesis , Tunicamycin/pharmacology
5.
Article in Chinese | WPRIM | ID: wpr-815081

ABSTRACT

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.


Subject(s)
Adenine , Pharmacology , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Autophagy , Beclin-1 , Carcinoma , Cell Line, Tumor , 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
6.
Article in Chinese | WPRIM | ID: wpr-239214

ABSTRACT

<p><b>OBJECTIVE</b>To explore the effect of up-regulation of KA1 subunit of the kainate receptor on endoplasmic reticulum stress (ERS)-induced excitotoxic neurodegeneration in mouse hippocampus.</p><p><b>METHODS</b>Seventy 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.</p><p><b>RESULTS</b>At 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).</p><p><b>CONCLUSION</b>Microinjection 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.</p>


Subject(s)
Animals , Apoptosis , Endoplasmic Reticulum Stress , Hippocampus , Pathology , Kainic Acid , Pharmacology , Male , Mice , Neurons , Pathology , Receptors, Kainic Acid , Metabolism , Tunicamycin , Pharmacology , Up-Regulation
7.
Article in English | WPRIM | ID: wpr-30196

ABSTRACT

BACKGROUND: Selenoprotein P (SEPP1) and fetuin-A, both circulating liver-derived glycoproteins, are novel biomarkers for insulin resistance and nonalcoholic fatty liver disease. However, the effect of exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, on the expression of hepatokines, SEPP1, and fetuin-A, is unknown. METHODS: The human hepatoma cell line HepG2 was treated with palmitic acid (PA; 0.4 mM) and tunicamycin (tuni; 2ug/ml) with or without exendin-4 (100 nM) for 24 hours. The change in expression of PA-induced SEPP1, fetuin-A, and endoplasmic reticulum (ER) stress markers by exendin-4 treatment were evaluated using quantitative real-time reverse transcription polymerase chain reaction and Western blotting. Transfection of cells with AMP-activated protein kinase (AMPK) small interfering RNA (siRNA) was performed to establish the effect of exendin-4-mediated AMPK in the regulation of SEPP1 and fetuin-A expression. RESULTS: Exendin-4 reduced the expression of SEPP1, fetuin-A, and ER stress markers including PKR-like ER kinase, inositol-requiring kinase 1alpha, activating transcription factor 6, and C/EBP homologous protein in HepG2 cells. Exendin-4 also reduced the expression of SEPP1 and fetuin-A in cells treated with tunicamycin, an ER stress inducer. In cells treated with the AMPK activator 5-aminoidazole-4-carboxamide ribonucleotide (AICAR), the expression of hepatic SEPP1 and fetuin-A were negatively related by AMPK, which is the target of exendin-4. In addition, exendin-4 treatment did not decrease SEPP1 and fetuin-A expression in cells transfected with AMPK siRNA. CONCLUSION: These data suggest that exendin-4 can attenuate the expression of hepatic SEPP1 and fetuin-A via improvement of PA-induced ER stress by AMPK.


Subject(s)
Activating Transcription Factor 6 , alpha-2-HS-Glycoprotein , AMP-Activated Protein Kinases , Blotting, Western , Carcinoma, Hepatocellular , Cell Line , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Fatty Liver , Glucagon-Like Peptide 1 , Glycoproteins , Hep G2 Cells , Humans , Insulin Resistance , Palmitic Acid , Phosphotransferases , Polymerase Chain Reaction , Reverse Transcription , RNA, Small Interfering , Selenoprotein P , Transfection , Tunicamycin , Biomarkers , Glucagon-Like Peptide-1 Receptor
8.
Acta Physiologica Sinica ; (6): 47-54, 2013.
Article in Chinese | WPRIM | ID: wpr-333136

ABSTRACT

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.


Subject(s)
Activating Transcription Factor 6 , Metabolism , Animals , Apoptosis , Cell Survival , Endoplasmic Reticulum Stress , Macrophages , Cell Biology , Mice , Quercetin , Pharmacology , Transcription Factor CHOP , Metabolism , Tunicamycin , Pharmacology
9.
Rio de Janeiro; s.n; 2013. 110 p. ilus.
Thesis in Portuguese | LILACS | ID: lil-716903

ABSTRACT

O câncer colorretal representa uma das maiores causas de morbidade e mortalidade relacionadas ao câncer. No Brasil, é o terceiro tipo de câncer mais frequente em homens e mulheres. Muitos estudos estão sendo desenvolvidos no sentido de esclarecer os diversos aspectos moleculares que regulam as alterações fenotípicas exibidas pelas células que constituem o câncer colorretal, no entanto, comparativamente, ainda são poucos os que são dedicados a investigar o papel de modificações co- e pós-traducionais neste processo. Entre os vários tipos destas modificações que ocorrem em proteínas, a glicosilação é a mais comum. Cogita-se que aproximadamente cinquenta por cento de todas as proteínas são glicosiladas. Durante a transformação maligna, mudanças no perfil de expressão de glicanos (carboidratos covalentemente ligados a proteínas ou lipídios) estão envolvidas em uma variedade de mecanismos celulares, tais como: perda da adesão célula-célula e célula matriz, migração, invasão e evasão da apoptose. Neste estudo, foi investigada a atividade antitumoral de inibidores da biossíntese de N-glicanos, swainsonina e tunicamicina, em células derivadas de câncer colorretal (Caco-2, HCT-116 e HT-29). Os resultados obtidos mostram que o tratamento das células HCT-116 com tunicamicina inibe mecanismos celulares relacionados ao fenótipo maligno, como formação de colônia dependente e independente de ancoragem, migração e invasão. Estes resultados sugerem que modulação da biossíntese de N-glicanos parece ser uma potencial ferramenta terapêutica para o tratamento do câncer colorretal. Em outra etapa do trabalho, foram avaliados também o impacto da estimulação com insulina e IGF-1 na expressão N-glicanos bissectados em células tumorais MDA-MB-435. Os resultados obtidos confirmaram também a existência de uma relação entre a estimulação dos receptores de insulina e IGF-1 e a regulação da expressão de N-glicanos bissectados em células tumorais MDA-MB-435, fornecendo assim informações ...


Colorectal cancer is a major cause of cancer-related morbidity and mortality. In Brazil, it is the third most common cancer. Many studies have been developed to clarify several molecular features which regulate phenotypic changes exhibited by cells that constitute colorectal cancer, however, comparatively, there are few studies dedicated to investigate co- and post-translational modifications of proteins in this process. Glycosylation is the most common post-translational modification of proteins. Approximately fifty percent of all proteins are glycosylated. During malignant transformation, changes in the expression profile of glycans (carbohydrates covalently bound to proteins or lipids) may be involved in a variety of events, including the loss of cell–cell and cell–matrix adhesion, migration, invasion, and evasion of apoptosis. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors, swainsonine and tunicamycin, in cells derived from colorectal cancer (Caco-2, HCT-116 e HT-29). Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in HCT-116 colon cancer cells. Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for CRC treatment. Moreover, we confirmthe existence of an interplay between stimulation with insulin and IGF-1 and bisecting GlcNAc N-glycans expression in MDA-MB435 cancer cells, providing new insights into the role that Insulin/IGF-I signaling play during carcinoma progression


Subject(s)
Humans , Glycosylation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Cadherins , Disease Progression , Polysaccharides/biosynthesis , Polysaccharides/metabolism , Receptor, IGF Type 1 , Receptor, Insulin , Swainsonine/pharmacology , Tunicamycin/pharmacology
10.
Article in Chinese | WPRIM | ID: wpr-269001

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Nasopharyngeal 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.</p><p><b>RESULTS</b>Treatment 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.</p><p><b>CONCLUSION</b>Tunicamycin 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.</p>


Subject(s)
Apoptosis , Carcinoma , Caspase 3 , Metabolism , Cell Line, Tumor , Cell Proliferation , Cisplatin , Pharmacology , Endoplasmic Reticulum Stress , Heat-Shock Proteins , Metabolism , Humans , Nasopharyngeal Neoplasms , Metabolism , Pathology , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Tunicamycin , Pharmacology , bcl-2-Associated X Protein , Metabolism
11.
Experimental Neurobiology ; : 94-100, 2012.
Article in English | WPRIM | ID: wpr-192762

ABSTRACT

The teleost fish has been widely used in creating neurodegenerative models. Here we describe the teleost medaka fish Parkinson's disease (PD) models we developed using toxin treatment and genetic engineering. 1-Methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors and tunicamycin treatment in our model fish replicated some salient features of PD: selective dopamine cell loss and reduced spontaneous movement with the last three toxins producing inclusion bodies ubiquitously in the brain. Despite the ubiquitous distribution of the inclusion bodies, the middle diencephalic dopaminergic neurons were particularly vulnerable to these toxins, supporting the idea that this dopamine cluster is similar to the human substantia nigra. PTEN-induced putative kinase 1 (PINK1) homozygous mutants also showed reduced spontaneous swimming movements. These data indicate that medaka fish can serve as a new model animal of PD. In this review we summarize our previous data and discuss future prospects.


Subject(s)
Animals , Brain , Dopamine , Dopaminergic Neurons , Genetic Engineering , Humans , Inclusion Bodies , Lysosomes , Oryzias , Oxidopamine , Parkinson Disease , Phosphotransferases , Proteasome Inhibitors , Protein Kinases , Substantia Nigra , Swimming , Tunicamycin
12.
Chinese Journal of Oncology ; (12): 494-498, 2011.
Article in Chinese | WPRIM | ID: wpr-320187

ABSTRACT

<p><b>OBJECTIVE</b>To establish a model of ER stress-induced apoptosis with tunicamycin and to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under endoplasmic reticulum (ER) stress.</p><p><b>METHODS</b>A model of ER stress-induced apoptosis was established with tunicamycin. Apoptotic cells were quantitated using the annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm the apoptotic cell death. Western blotting was used to measure the activation of caspase-3 and -9, and the expression of Bim, GRP78, CHOP, and Foxo1 at the protein level. The expression of Bim, CHOP and Foxo1 at the mRNA level was quantitated by qPCR. The siRNA technique was used to inhibit the expression of Bim.</p><p><b>RESULTS</b>Treatment of the melanoma cells with tunicamycin did not induce significant apoptosis and activation of caspase cascade, whereas it caused marked activation of caspase-3 and -9, and apoptosis in HEK293 cells which were used as a control. With exposure to tunicamycin (3 µmol/L) for 12, 24, 36 hours the Bim protein levels were not increased in Mel-RM and MM200 cells. Its mRNA levels were 0.37 ± 0.05, 0.13 ± 0.02 and 0.02 ± 0.01 in Mel-RM cells, while 0.41 ± 0.06, 0.16 ± 0.04 and 0.21 ± 0.03 in MM200 cells, respectively. The expression of Bim mRNA was significantly reduced compared with that in the control groups of the two cell lines (P < 0.01). siRNA knockdown of Bim protected HEK293 cells against activation of caspase-3. The cell apoptosis of Bim siRNA group was (5.69 ± 0.38)%, significantly lower than that of the siRNA control group (40.32 ± 1.64)% and blank control group (35.46 ± 2.01)% (P < 0.01). In the melanoma cells after exposure to tunicamycin (3 µmol/L) for 6, 12, 24, and 36 hours the transcription factor CHOP at mRNA level were significantly increased and the expressions at protein level were also up-regulated. The expressions of another transcription factor Foxo1 at mRNA level significantly decreased and the expressions at protein level were down-regulated, too.</p><p><b>CONCLUSIONS</b>The lack of Bim up-regulation contributes to the resistance of melanoma cells to ER stress-induced apoptosis and may be a mechanism by which melanoma cells adapt to ER stress conditions. Transcription factors CHOP and Foxo1 may be responsible for the dysregulation of Bim in melanoma cells upon ER stress.</p>


Subject(s)
Apoptosis , Apoptosis Regulatory Proteins , Genetics , Metabolism , Bcl-2-Like Protein 11 , Caspase 3 , Metabolism , Caspase 9 , Metabolism , Cell Line, Tumor , Endoplasmic Reticulum Stress , Forkhead Box Protein O1 , Forkhead Transcription Factors , Genetics , Metabolism , HEK293 Cells , Heat-Shock Proteins , Metabolism , Humans , Melanoma , Genetics , Metabolism , Pathology , Membrane Proteins , Genetics , Metabolism , Proto-Oncogene Proteins , Genetics , Metabolism , RNA, Messenger , Metabolism , RNA, Small Interfering , Genetics , Transcription Factor CHOP , Genetics , Metabolism , Tunicamycin , Pharmacology
13.
Chinese Medical Journal ; (24): 3560-3567, 2011.
Article in English | WPRIM | ID: wpr-336527

ABSTRACT

<p><b>BACKGROUND</b>Although 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>We 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.</p><p><b>CONCLUSION</b>Our results suggested that C6ORF120 could induce apoptosis of CD4(+) T cells, at least in part, mediated with endoplasmic reticulum stress.</p>


Subject(s)
Antiviral Agents , Pharmacology , Apoptosis , Blotting, Western , CD4-Positive T-Lymphocytes , Metabolism , CD8-Positive T-Lymphocytes , Cell Cycle , Cells, Cultured , Endoplasmic Reticulum Stress , Female , HIV Infections , Allergy and Immunology , Humans , Immunohistochemistry , Male , Microscopy, Confocal , Proteins , Genetics , Metabolism , Tunicamycin , Pharmacology
14.
Chinese Journal of Hematology ; (12): 675-679, 2010.
Article in Chinese | WPRIM | ID: wpr-353566

ABSTRACT

<p><b>OBJECTIVE</b>To explore the molecular mechanism of myeloma cell differentiation induced by low dose tunicamycin.</p><p><b>METHOD</b>U266 and RPMI8226 cells were incubated with low dose tunicamycin for 72h. Surface CD49e expression was assayed by flow cytometer (FCM), light chain protein in the cell culture supernatant by ELISA, the unfolded protein response (UPR) related gene GRP78 and GRP94 by real time PCR, and XBP1u and XBP1s transcription and translation changes by real time PCR and Western blot. After XBP1u gene was interfered with small RNA, and constructed plasmid was transfected into myeloma cells to up-regulated gene XBP-1u and XBP-1s reseparately, the differentiation of myeloma cells was observed again.</p><p><b>RESULTS</b>Small dose tunicamycin could induce both U266 and RPMI8226 myeloma cells differentiation. Compared with the control group, cell morphology changed to mature feature, the nucleo- cytoplasm ratio decreased and nucleolus reduced or disappearance, CD49e expression increased the light chain protein concentration of cell culture supernatant was up-regulated and UPR related gene GRP78 and GRP94 were up-regulated during the differentiation. XBP-1u was up-regulated at both transcription and translation level, while XBP-1s down-regulated. After XBP1u gene expression interfered with small RNA, cell differentiation was disturbed. Cell differentiation was induced while XBP-1u gene was up-regulated by plasmid transfection.</p><p><b>CONCLUSION</b>Low dosage of tunicamycin could induce myeloma cell UPR and differentiation, while XBP-1u a key role during the process.</p>


Subject(s)
Cell Differentiation , Cell Line, Tumor , DNA-Binding Proteins , Genetics , Humans , Multiple Myeloma , Metabolism , Transcription Factors , Genetics , Tunicamycin , Unfolded Protein Response
15.
Article in English | WPRIM | ID: wpr-344950

ABSTRACT

<p><b>OBJECTIVE</b>Changes 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.</p><p><b>METHODS</b>Primarily 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.</p><p><b>RESULTS</b>Protein 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.</p><p><b>CONCLUSION</b>Tiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.</p>


Subject(s)
Animals , Anti-Bacterial Agents , Pharmacology , Cells, Cultured , Drug Antagonism , Drugs, Chinese Herbal , Pharmacology , Endoplasmic Reticulum , Metabolism , Gene Expression Regulation , Heat-Shock Proteins , Genetics , Metabolism , Male , Membrane Glycoproteins , Genetics , Metabolism , Mesenchymal Stem Cells , Metabolism , RNA , Rabbits , Rats , Rats, Sprague-Dawley , Stress, Physiological , Genetics , Tunicamycin , Pharmacology
16.
Korean Diabetes Journal ; : 312-319, 2010.
Article in English | WPRIM | ID: wpr-23770

ABSTRACT

BACKGROUND: The highly developed endoplasmic reticulum (ER) structure in pancreatic beta cells is heavily involved in insulin biosynthesis. Thus, any perturbation in ER function inevitably impacts insulin biosynthesis. Recent studies showed that the expression of tribbles-related protein 3 (TRB3), a mammalian homolog of Drosophilia tribbles, in various cell types is induced by ER stress. Here, we examined whether ER stress induces TRB3 expression in INS-1 cells and found that TRB3 mediates ER stress-induced suppression of insulin gene expression. METHODS: The effects of tunicamycin and thapsigargin on insulin and TRB3 expression in INS-1 cells were measured by Northern and Western blot analysis, respectively. The effects of adenovirus-mediated overexpression of TRB3 on insulin, PDX-1 and MafA gene expression in INS-1 cells were measured by Northern blot analysis. The effect of TRB3 on insulin promoter was measured by transient transfection study with constructs of human insulin promoter. RESULTS: The treatment of INS-1 cells with tunicamycin and thapsigargin decreased insulin mRNA expression, but increased TRB3 protein expression. Adenovirus-mediated overexpression of TRB3 decreased insulin gene expression in a dose-dependent manner. A transient transfection study showed that TRB3 inhibited insulin promoter activity, suggesting that TRB3 inhibited insulin gene expression at transcriptional level. Adenovirus-mediated overexpression of TRB3 also decreased PDX-1 mRNA expression, but did not influence MafA mRNA expression. CONCLUSIONS: This study showed that ER stress induced TRB3 expression, but decreased both insulin and PDX-1 gene expression in INS-1 cells. Our data suggest that TRB3 plays an important role in ER stress-induced beta cell dysfunction.


Subject(s)
Blotting, Northern , Blotting, Western , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Gene Expression , Humans , Insulin , Insulin-Secreting Cells , RNA, Messenger , Thapsigargin , Transfection , Tunicamycin
17.
Protein & Cell ; (12): 1093-1105, 2010.
Article in English | WPRIM | ID: wpr-757464

ABSTRACT

Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β-1″,11'-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunA-tunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.


Subject(s)
Actinobacteria , Genetics , Base Sequence , Biological Assay , Carbohydrate Sequence , Carbohydrates , Genetics , Cloning, Molecular , Gene Deletion , Gene Library , High-Throughput Screening Assays , Molecular Sequence Data , Multigene Family , Recombinant Proteins , Genetics , Sequence Analysis, DNA , Streptomyces , Genetics , Tunicamycin , Chemistry , Genetics
18.
Article in Chinese | WPRIM | ID: wpr-328699

ABSTRACT

<p><b>OBJECTIVE</b>To detect the effect of tunicamycin on Fas protein expression and Fas monoclonal antibody (FasMcAb)-induced apoptosis of fibroblast from hypertrophic scar and keloid.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>It 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.</p><p><b>CONCLUSIONS</b>The 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.</p>


Subject(s)
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
19.
Article in English | WPRIM | ID: wpr-136579

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Apoptosis , Cell Line, Tumor , Cyclin D1/antagonists & inhibitors , Down-Regulation , Humans , Microtubule-Associated Proteins/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tunicamycin/pharmacology
20.
Article in English | WPRIM | ID: wpr-136578

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Apoptosis , Cell Line, Tumor , Cyclin D1/antagonists & inhibitors , Down-Regulation , Humans , Microtubule-Associated Proteins/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tunicamycin/pharmacology
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