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Objective:To evaluate the role of orphan nuclear receptor Nur77 in tunicamycin(TM)-induced injury to hippocampal neurons and the relationship with endoplasmic reticulum stress in mice.Methods:Mouse HT22 cells were divided into 4 groups ( n=10 each) using a random number table method: control group (C group), Nur77 specific agonist Csn-B group (Csn-B group), endoplasmic reticulum stress inducer TM group (TM group), and TM+ Csn-B group. Cells in C group were cultured for 24 h under normal condition. In Csn-B group, Csn-B at a final concentration of 10 μg/ml was added to the culture medium, and the cells were incubated for 24 h. In TM group, TM at a final concentration of 200 ng/ml was added to the culture medium and the cells were incubated for 24 h to induce cell endoplasmic reticulum stress injury. Cells in TM+ Csn-B group were pretreated with Csn-B at a final concentration of 10 μg/ml for 15 min, then TM at a final concentration of 200 ng/ml was added, and the cells were co-incubated for 24 h. The cell viability was examined by CCK-8 assay kit after treatment in each group. The expression of endoplasmic reticulum stress-related protein CCAAT/enhancer-binding protein homologous protein (CHOP), glucose regulated protein 78 (GRP78)and apoptosis-associated protein Bcl-2, Bax, caspase-3 and cleaved-caspase-3 was detected by Western blot. Results:Compared with C group, the cell viability was significantly decreased, and the expression of CHOP, GRP78, Bax and cleaved-caspase-3 was up-regulated, and the expression of Bcl-2 and caspase-3 was down-regulated in TM group ( P<0.05 or 0.01). Compared with TM group, the cell viability was significantly increased, the expression of CHOP, GRP78, Bax and cleaved-caspase-3 was down-regulated, and the expression of Bcl-2 and caspase-3 was up-regulated in TM+ Csn-B group ( P<0.05 or 0.01). Conclusions:Orphan nuclear receptor Nur77 is involved in TM-induced injury to hippocampal neurons, which is related to activation of the endoplasmic reticulum stress in mice.
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AIM:To evaluate the morphological and functional changes of retinas induced by treatment of tunicamycin with optical coherence tomography (OCT) in rats.METHODS:Totally 60 SD rats were randomly divided into 3 groups (20 in each group), 0.5mg/kg (in low dose group), 1.5mg/kg (in high dose group) tunicamycin were injected into vitreous cavity and saline (9g/L NaCl) were injected in the same dose as a control group.Changes of retinas were observed by OCT on the 1,7 and 14d after treatment of tunicamycin.Then the rats were sacrificed, retinas were taken out and embedded by the paraffin, tissue sections and the HE staining were performed.RESULTS:OCT results suggested that tunicamycin played damage effects on retinal morphology and structure which appeared a time-and dose-dependent.Fundus photography results suggested that 2wk after tunicamycin treatments, with the gradually changing of tunicamycin concentration, peripheral retinal and macular region became pale color gradually, edema occurred in optic disk, retinal vessels appeared thinner in the high dose group, optic nerve came out atrophy.Fluorescein angiography confirmed that tunicamycin injection in vitreous cavity 2wk later, retinal vessels injury occurred, resulted in leaking of intravascular contrast agent from peripheral to the central part of the retinas.Electrophysiological data showed that retinal electrogram occurred disorder induced by tunicamycin, such as the amplitude of a wave, b wave decreased gradually, even closed to zero, which was very different from control significantly (P<0.05).HE staining of paraffin sections showed that retina injuries induced by tunicamycin were in dose-time dependent, which was consistent with the results of OCT.CONCLUSION: Clinical retinal diseases could be simulated by retinal damage animal model induced by tunicamycin treatment.OCT detection offered real-time images of the retinal cross-section, which provided a helpful non-invasive method for detecting and evaluating the retinal damages.
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Objective To observe the protective effects of roscovitine on the podocyte injury induced by endoplasmic reticulum stress (ERS) caused by tunicamycin. Methods The differentiated podocytes cultured at 37℃were randomly di-vided into:(1) Control group, DMSO group and tunicamycin group (TM, 1.0μmol/L). The treatment was given for 3, 6 and 12 hours in three groups. (2) For control group, tunicamycin group, tunicamycin+roscovitine group (20, 40μmol/L, TM+ROS), the treatment was given for 12 hours. The podocyte apoptosis was detected by flow cytometry and TUNEL method. The ex-pressions of Cdk5, GRP78, Caspase-12 and CHOP were detected by Western blot assay. Results (1) Compared with con-trol group and DMSO group, the podocyte apoptosis was increased significantly in a time dependent manner after tunicamy-cin treatment in TM group;the protein expressions of Cdk5, GRP78, Caspase-12 and CHOP were also up-regulated signifi-cantly in TM group (P<0.05). (2) Flow cytometry and TUNEL analysis showed that tunicamycin induced apoptosis in podo-cytes, which was significantly inhibited by roscovitine in a concentration dependent manner in TM+ROS group as compared to that of TM group (P<0.05). The protein expressions of GRP78, Caspase-12 and CHOP were also significantly decreased in a concentration dependent manner in TM+ROS group compared to those of TM group (P<0.05). Conclusion Roscovi-tine, the inhibitor of Cdk5, can reduce the podocyte apoptosis induced by tunicamycin. The protective effects of roscovitine on podocytes can be a novel approach of treating diabetic nephropathy.
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[ ABSTRACT] AIM:To explore the effect of sterol regulatory element-binding protein 2 ( SREBP-2) on tunicamy-cin-induced endoplasmic reticulum stress ( ERS) in chondrocytes.METHODS:After isolation of human normal chondro-cytes and osteoarthritis ( OA) chondrocytes, the normal cells were cultured and treated with tunicamycin and SREBP-2 siR-NA.After 24 h treatment, fluorescent quantitative RT-PCR ( RT-qPCR) was applied to quantify microRNA-185 ( miR-185) levels.The cell apoptotic rate was determined by flow cytometry.The expression of SREBP-2 and ERS-related pro-teins, C/EBP homologous protein (CHOP), phosphorylated eukaryotic initiation factor-2α(p-eIF2α) and activating tran-scription factor 4 (ATF4), and the expression of apoptosis-related proteins, Bcl-2, Bax and caspase-3, were determined by Western blot.The caspase-3 activity kit was used to determine the caspase-3 activity.RESULTS: Compared with hu-man normal chondrocytes, both SREBP-2 up-regulation and miR-185 down-regulation were observed in OA chondrocytes (P<0.05).SREBP-2 siRNA transfection enhanced tunicamycin-inhibited miR-185 level (P<0.05).miR-185 overex-pression reduced tunicamycin-induced SREBP-2 expression ( P <0.05 ) .OA control group and tunicamycin treatment group consistently resulted in ERS and cell apoptosis with concomitant enhancement of CHOP, p-eIF2αand ATF4 proteins, increases in Bax and caspase-3 proteins, and reduction of Bcl-2 (P<0.05).However, SREBP-2 silencing significantly re-versed these effects ( P<0.05) .The apoptotic rates were consistent with the expression tendency of apoptosis-related pro-teins (P<0.05).SREBP-2 siRNA transfection markedly down-regulated tunicamycin-induced caspase-3 activity, which was notably blocked by miR-185 inhibition (P<0.05).CONCLUSION:SREBP-2 silencing may inhibit tunicamycin-in-duced ERS and cell apoptosis via up-regulating miR-185 expression.
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OBJECTIVE: To investigate whether the proteasome inhibitor, gliotoxin, combined with the endoplasmic reticulum stress inducer, tunicamycin, could enhance the apoptotic death of tumor cells. METHODS: MTT assay was used to detect the cytotoxicity. The combined drug index (CDI) was used to evaluate the synergistic effect. Flow cytometry was used to analyze the apoptosis rate. The protein expression level was detected by Western blot. RESULTS: The IC50 of gliotoxin and tunicamycin is (1.44±0.23) and (26.14±6.14) μmol·L-1, respectively. In the effective concentration, gliotoxin combined with tunicamycin could significantly inhibit cell proliferation and induce typical apoptotic morphological changes. The combination was synergistic according to the result of MTT assay. As measured by flow cytometry, the combination remarkably increased the apoptosis rates of HT-1080 cells, especially for 0.2 μmol·L-1 gliotoxin combined with tunicamycin, the apoptosis rate was up to 66.6% and CDI was 0.649. The expression changes of apoptosis-related proteins such as caspase-8, caspase-3, PARP and NF-kB were detected when treated with the combination. CONCLUSION: Gliotoxin can improve the chemosensitivity of fibrosarcoma HT-1080 cells to tunicamycin and enhance the apoptosis of tumor cells induced by tunicamycin. Thus, our study may provide a new drug combination to antitumor therapy.
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Objective To investigate the effect of endoplasmic reticulum stress (ER stress) in cisplatin-induced apoptosis of human cervical cancer HeLa cells. Methods HeLa cells were used as the study object which were divided into four groups: TUNI (5mg/L) group, cisplatin (6mg/L) group, TUNI(5mg/L)+cisplatin(6mg/L) group, and negative control group (no drug treatment). MTT assay was employed to examine the growth status of the cells. Hoechst staining was used to observe the morphological change in the nucleus. Immunoblotting was used to detect the activation of apoptotic proteins, caspase-3 and caspase-4. Indirect immunofluorescence was used to assess the expression of the protein disulfide isomerase (PDI) and phosphorylated histone H2AX (γ-H2AX). Results MTT assay showed that the growth inhibition rates were 2.65%±2.71%, 19.60%±4.34%, 44.69%±7.07% and 0% in TUNI group, cisplatin group, TUNI+cisplatin group and control group, respectively (P<0.05). Cisplatin showed a significant inhibitory effect on the growth of HeLa cells, and TUNI enhanced the effect of cisplatin. Statistical significance was found between TUNI+cisplatin group and cisplatin group (P<0.05). Hoechst staining showed that the fluorescence of the nucleus in control group was weak and well-distributed. At 12h after treatment, the nuclei in some HeLa cells in cisplatin group and TUNI+cisplatin group diminished in size, thus showing dense hyperfluorescence, and some of them were broken. The proportion of karyorrhexis cells in TUNI+cisplatin group (44.5%±5.1%) was significantly higher than that in cisplatin group (22.7%±3.9%, P<0.05). Immunoblotting showed the expressions of activated caspase-3 and caspase-4 were up-regulated obviously in cisplatin group. Compared to cisplatin group, the expressions of those proteins significantly increased in TUNI+cisplatin group (P<0.05). Indirect immunofluorescence staining showed no PDI expression and weak fluorescence was found in control group. PDI proteins presented in granular form, distributing around the nuclei with strong fluorescence were found in TUNI group and cisplatin group. PDI proteins showed obviously stronger fluorescence in a large proportion of cells in TUNI+cisplatin group, and the fluorescence intensity was obviously higher than that in TUNI group and cisplatin group. No expression of γ-H2AX protein was found in the nucleus in either control group or TUNI group. However, obvious green fluorescence was observed in nuclei of a part of cells in cisplatin group and TUNI+cisplatin group, no obvious difference existed between the two groups. Conclusion Heightened ER stress by tunicamycin may increase the apoptosis of HeLa cells induced by cisplatin.
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Objective: To observe the induced effect of low dose of tunicamycin on endoplasmic reticulum stress (ERS) of A549 human lung adenocarcinoma cells in vitro and the change of Bcl-2 associated athanogene 1 (BAG-1) protein expression, as well as the changes of cisplatin (DDP) sensitivity and the BAG-1 protein expression. Methods: The expressions of GRP78 (a marker protein in ERS) and BAG-1 proteins in A549 cells treated with low dose of tunicamycin were detected by Western blotting. The change of half inhibitory concentration (IC50) of DDP for A549 cells after pretreatment with tunicamycin was measured by MTT method, and the DDP sensitivity was determined by flow cytometry (FCM). The Western blotting was carried out to detect the expressions of BAG-1 and procaspase-12 proteins in A549 cells induced by DDP after pretreatment with tunicamycin. Results: The ERS in A549 cells could be induced by pretreatment with 1.25 μg/mL tunicamycin for 8 h, which displayed that the expression levels of GRP78 and BAG-1 proteins were both up-regulated (P <0.05). The ERS could increase the DDP sensitivity in the A549 cells (P <0.05). Before ERS occurred, all of three doses of DDP (1.25, 2.5 and 5 μg/mL) could up-regulate the expression level of BAG-1 protein (P <0.05), and this effect was more obvious in 1.25 μg/mL DDP-treated A549 cells. When ERS occurred, the expression levels of BAG-1 protein in all of three doses of DDP (1.25, 2.5 and 5 μg/mL)-treated A549 cells were down-regulated in a dosedependent manner as compared with that in the A549 cells without DDP treatment (P <0.05). The DDP dose of 2.5 and 5 μg/mL could induce apoptosis by ERS pathway, and the expression levels of BAG-1 and procaspase-12 proteins were both down-regulated during this process (P <0.05). Conclusion: BAG-1 may be one of the important regulatory factors in both pathways of ERS- and cisplatin-induced apoptosis of lung cancer cells, and ERS-related apoptotic pathway may be one of the important pathways of cisplatininduced apoptosis of lung cancer cells. Copyright © 2012 by TUMOR.
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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)
Humans , Anti-Bacterial Agents/pharmacology , Apoptosis , Cell Line, Tumor , Cyclin D1/antagonists & inhibitors , Down-Regulation , Microtubule-Associated Proteins/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tunicamycin/pharmacologyABSTRACT
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.