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OBJECTIVE Na+/K+-ATPase(NKA)is a large membrane protein expressed universally in all cells.It is indispensable for the maintenance of ionic gradient.We previously reported that the dysfunction of this pump in neurons and astrocytes contributes to stroke and neurodegenerative diseases,respectively.However,its roles in the microglia and stress-related diseases are still unclear.METHODS Two classical models,chronic restraint stress(CRS)model and electronic foot shock(ES)model,were used to study the pathogenesis of anxi-ety in either NKAα1 global knockout(NKAα1 GKO)mice or NKA α1 conditional knockout(NKAα1 CKO)mice.Behavioral tests like open-field test,elevated plus maze,Morris water maze,novel object recognition test and gait imaging test were performed.A variety of molecular bio-logical methods were employed,including RNA sequenc-ing(RNA-seq)analyses,immunofluorescence and elec-trophysiological recordings etc.RESULTS NKAα1 defi-ciency had a broad impact on physical stress-induced anxiety-like behavior,but failed to exacerbate CRS induced memory deficits.Electrophysiology experiment showed that NKAα1 GKO and NKAα1 CKO mice exhibit-ed neuronal hyperexcitability under chronic stress.The underlying mechanisms may involve neuroinflammation,as NKAα1 deficiency exacerbated stress-induced microg-lia activation in vivo.Similarly,inhibition or downregula-tion of NKA α 1 aggravated LPS + ATP-induced inflam-mation in vitro.DR5-12D,a monoclonal antibody against the DR-region of NKAa1,improved stress-induced anxiety-like behavior through amelioration of the neuronal hyper-excitability and neurogenesis deficit in the ventral hippo-campus of mice.CONCLUSION NKA is closely related to neuroinflammation in microglia and DR-region of NKA a1 subunit may serve as a novel target to treat stress-induced anxiety.
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Objective: To investigate the effects of xanthotoxin from Apiaceae medicinal plants on cell proliferation and apoptosis, and explore its mechanism of action against human gastric carcinoma SGC-7901 cells in vitro. Methods: SGC-7901, HepG-2, MCF-7, and A549 cells were treated with different concentrations of xanthotoxin (10, 20, 60, 80, 100, 120, 140, and 160 µg/mL) for 48 h, and the cell viability (IC50) was determined by MTT assay; Xanthotoxin-induced apoptosis in cells was observed by using Hoechst 33258 Staining Kit and Annexin V-FITC Apoptosis Detection Kit; Flow cytometry was used to detect apoptosis related proteins of Fas/FasL, Bid, and DR5/TRAIL proteins in human gastric carcinoma SGC-7901 cells after being treated by xanthotoxin; The influence of xanthotoxin on Caspase-8 protein expression in the cells was determined by Flouormetric Assay Kit. Results: Xanthotoxin obviously inhibited SGC-7901, HepG-2, MCF-7, and A549 cells proliferation, and its inhibition was in a concentration-dependent manner; flow cytometry results showed that in a certain concentration range, xanthotoxin can increase the expression levels of Fas/FasL and DR5/TRAIL proteins in a concentration-dependence manner. The content of Bid protein in cells was increased, and it showed concentration-dependence. Conclusion: Xanthotoxin may induce SGC-7901 cells apoptosis in a certain concentration range through the Fas/FasL protein mediated death receptor pathway, or by DR5/TRAIL mediated death receptor pathway, and increase the expression level of death receptor protein, activation Caspase-8, activating downstream effect factor, inducing cell apoptosis, or activate Caspase-8 cutting activate protein Bid, and then enter the mitochondrial pathway, induction of apoptosis.
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Objective To observe the expression of DR5 in the ocular tissues of uveitis rats induced by endotoxin,and study the relationship between the apoptosis of inflammatory cells and expression of TRAIL / DR5.Methods SD rats were randomly divided into 3 groups:Blank control group,normal saline injection group and endotoxin injection group.The endotoxin injection group was injected with lipopolysaccharide into the rat posterior foot pad to make endotoxin-induced uveitis animal model.There were no operations in the blank control group,and the subgroups were divided into 6 hours,12 hours,24 hours and 48 hours groups according to the time of injection.The ultrastructural changes of inflammatory cells and endothelial cells in iris capillaries were observed by transmission electron microscopy (TEM).The expression of DtR5 protein on inflammatory cells at different time after endotoxin induction was detected by SABC method.Results TEM showed that the microvilli of the capillary endothelial cells in the iris tissue of the blank control group and saline injection group had more obvious vesicles with no obvious abnormal structure and shape.The number of swallowed vesicles in the capillary endothelial cells injected with endotoxin was decreased at 6 hours group,and the number of vesicles in the infiltrating neutrophils and lymphocytes decreased.Neutrophils and lymphocytes appear chromatin condensation,vacuolar changes in the expression of apoptosis.Immunohistochemistry showed that the DR5 protein was negative in the iridocular epithelium of the blank control group and saline injection group.In the endotoxin injection group,the DR5 protein was weakly colored in the iris pigment epithelium and appeared on the inflammatory cells.The number of staining and the intensity of coloring in the 24 hours group were significantly higher than those in the 6 hours group,and the color density was 0.085 9 ± 0.019 6,there were statistical differences compared with 6 hours group,12 hours group and 48 hours group (all P ＜ 0.05).Conclusion TRAIL and its receptor DR5 may be involved in the apoptosis of inflammatory cells in endotoxin-induced uveitis.
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MDL-12330A is a widely used adenylyl cyclase (AC) inhibitor that blocks AC/cAMP signaling. In this study, we demonstrated a novel antitumor activity of this drug in gastric carcinoma (GC) cell lines. In these GC cells, MDL-12330A reduced cell viability and induced cell death in a concentration-dependent manner. At a moderate concentration (~20 µM), MDL-12330A mainly induced apoptotic death whereas at concentrations greater than 20 µM, it increased non-apoptotic cell death. The induction of apoptosis was at least partially regulated by CHOP-mediated DR5 upregulation, as detected by immunoblotting and gene interference assays. More importantly, low concentrations of MDL-12330A effectively enhanced recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (rhTRAIL)-induced apoptosis and clonogenicity in these gastric cancer cells. This study demonstrates a possible role of MDL-12330A as a potential sensitizer to TRAIL, and suggests a novel therapeutic strategy targeting gastric cancer cells.
Subject(s)
Humans , Adenylyl Cyclases , Apoptosis , Cell Death , Cell Line , Cell Survival , Immunoblotting , Stomach Neoplasms , Tumor Necrosis Factor-alpha , Up-RegulationABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent.However,emergence of drug resistance limits its potential use.Plumbagin is a natural quinonoid compound isolated from plant.In this study,induced apoptosis effect of the combined treatment with plumbagin and TRAIL on human melanoma A375 cell line was examined and possible mechanism was investigated.The cells were divided into four groups:control group,plumbagin group (plumbagin,5 or 10 μmol/L),TRAIL group (TRAIL,30 ng/mL) and plumbagin+TRAIL group (combined treatment group).The apoptosis,and the expression of DR4 and DR5 were detected by flow cytometry.The activities of caspase-8 and caspase-3 were determined by colorimetric assay.The results showed that the apoptosis rate was 8.3% in TRAIL group,10.35%-16.94% in plumbagin group and 52.39%-55.39% in combined treatment group,respectively,with the difference being significant between combined treatment group and plumbagin or TRAIL group (P＜0.05 for each).Moreover,plumbagin alone could markedly up-regulate DR5 mRNA and protein expression,and slightly increase DR4 mRNA and protein expression.Treatment of human melanoma A375 cells with plumbagin resulted in the activation of Caspase-3,but not Caspase-8.These results suggest that plumbagin might be useful for TRAIL-based treatment for melanoma.
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Objective: To synthesize DR5-derived complementary peptide(s) that can influence the function of TRAIL and to observe its(their) inhibitory effect on TRAIL-mediated cell death. Methods: A software was designed to identify the matching domains of two known peptide sequences according to the theory of Proteomic Code. The antisense homology box sequences between DR5 and its ligand TRAIL were searched by the designed software. Possibly active peptide(s) was/were selected and synthesized based on the structural information of TRAIL-DR5 complex. The binding between TRAIL molecule and the selected peptide(s) was examined by ELISA; the inhibitory effect of the selected peptide(s) against TRAIL-induced cell death was studied by cellular experiments. Results: Ten complementary peptide sequences were obtained based on software selection and structural analyzing. One active DR5-derived peptide FR-11 was selected with ligand-binding analysis using ELISA; FR-11 showed specific binding to TRAIL in a dose-dependant manner and blocked the binding of TRAIL with DR5. The matching rate between FR-11 and TRAIL 97-103aa was higher than 50%. Computer analysis showed that FR-11 corresponded to the extracellular domain of DRS, a key binding site to TRAIL. Further study indicated that FR-11 inhibited TRAIL-induced apoptosis of SW1990 cells and L929 cells in a dose-dependant manner; the inhibitory effect of FR-11 was more potent when the concentration of TRAIL was low, suggesting that it inhibited the binding between TRAIL and death receptor. Conclusion: The software designed based on the theory of Proteomic Code can be used to select complementary peptides which influence the interaction between ligand and receptor. The selected peptide FR-11 can bind to the ligand TRAIL and subsequently inhibit the biological function of the ligand.
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Objective:To study the effects of Anti-DR5 mAb on inducing synovial cells of adjuvant arthritis (AA) rats and its mechanisms.Methods:AA was induced by CFA in rats.The synovial cells of rats were separated and cultured in vitro system.The apoptosis induced by anti-DR5 mAb on synovial cells was measured by MTT analysis,DNA fragmentation and flow cytometry.Caspase-3 and Bcl-2 expression in synovial cells were detected by Western blot.The involvement of the apoptotic pathway was further proved by a caspase inhibition assay.Results:MTT result showed that Anti-DR5 mAb could inhibit synovial cells growth.And flow cytometry suggested that the cell death mode was apoptosis.The protein level of caspase-3 in synovial cells treated with anti-DR5 mAb was raised,while Bcl-2 level declined.When the caspase inhibitor was added to the synovial cells treated with anti-DR5 mAb,it was showed in a dose-dependence inhibition effect,indicating that anti-DR5 mAb inducing apoptosis might be through the caspase pathway.Conclusion:Anti-DR5 mAb could induce synovial cell apoptosis through caspase pathway.And this effect may be related to the protein level of Caspase-3 and Bcl-2.
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BACKGROUND: TRAIL is a promising anticancer agent which induces selective tumor cell death due to a unique receptor system that includes death receptors and decoy receptors. DR5 TRAIL receptor is an originally identified p53-regulated death receptor gene that was induced, by doxorubicine, only in cells with a wild-type p53 status. We investigated that focused on the correlation between the DR5 and p53 expressions in non-small cell lung cancer (NSCLC). METHODS: Immunohistochemical analysis, with using avidin-biotinylated horseradish peroxidase complex, was carried out in 89 surgically resected NSCLC formalin-fixed paraffin-embedded tissue sections. As primary antibodies, we used anti-DR5 polyclonal antibody and anti-p53 monoclonal antibody. A negative control was processed with each slide. The positive tumor cells were quantified twice and these values were expressed as percentage of the total number of tumor cells, and the intensity of immunostaining was expressed. The analysis of the DR5 expression was done separately in tumor area and in a nearby region of normal tissue. RESULTS: The DR5 expression was high in the bronchial epithelium (89% of cases) but this was almost absent in type I & II pneumocytes, lymphocytes and smooth muscle cells. High DR5 expression rate in tumor was seen in 28% (15/53) of squamous cell carcinomas, in 47% (15/32) of adenocarcinomas and, in 50% (2/4) of large cell carcinomas. The DR5 expression did not show any statistical significance relationship with the T stage, N stage, or survival. However, the DR5 expression showed significant inverse correlation with the p53 expression. (p<0.01). CONCLUSION: We demonstrated that the DR5 expression in NSCLC via immunohistochemical analysis is relatively tumor-specific except for that in the normal bronchial epithelium and it is significantly dependent on the p53 status. This might be in vivo evidence for the significance of the DR5 gene as a p53 downstream gene.
Subject(s)
Adenocarcinoma , Antibodies , Carcinoma, Large Cell , Carcinoma, Non-Small-Cell Lung , Carcinoma, Squamous Cell , Cell Death , Doxorubicin , Epithelium , Horseradish Peroxidase , Lymphocytes , Myocytes, Smooth Muscle , Alveolar Epithelial Cells , Receptors, Death DomainABSTRACT
Objective:The purpose of this study is to evaluate the effects of a single-chain antibody against death receptor 5 (ZF1) on tumor growth and survival in murine H22 hepatocellular carcinoma tumor model.Methods:Killing effect of ZF1 on H22 cells was analyzed by MTT assay in vitro. The apoptosis rate of H22 cells induced by ZF1 was detected using Flow Cytometry assay. The transplanted model of H22 tumor was developed in mice. The mice were randomly divided into four groups, PBS group, ZF1 group, EPI group and combined treatment group of ZF1/EPI. Tumor growth and body weight changes were observed. After treatment over 13 days, the tumor tissue for HE staining and TUNNEL assay was performed to detect apoptosis.Results:The results showed that ZF1 could inhibit growth of H22 cells in a dose dependent manner. The growth inhibition rate was up to 84.5%. The results showed that ZF1 alone or in combination with ZF1/EPI, the tumor growth was significantly inhibited. HE staining and TUNNEL analysis showed that ZF1 could effectively induce apoptosis of tumor cells without toxic effects, especially in ZF1/EPI combined treatment group.Conclusion:It is showed that ZF1 induces a good inhibition on the proliferation of H22 cell, especially in combined treatment group of ZF1/EPI.
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Objective:To study the role of DR5 in TRAIL apoptotic signal transduction. Methods:BALB/C mice were immunized with recombinant DR5 that contains the full-length extracellular domain of the human DR5. Anti-DR5 mAb was generated by hybridoma. The level of DR5 expression on Jurkat cell line was examined by flow cytometry. The rates of TRAIL-induced apoptosis and anti-DR5 mAb blocking on Jurkat cells were tested by flow cytometry with TRAIL apoptosis kit.Results:The percentage of DR5 expression on Jurkat cells was 94 83%. TRAIL and anti-TRAIL mAb could kill Jurkat cells on dose-dependent, and the killing rate was 90% in the concentration of 50～100 ng/ml. The killing role of TRAIL could be blocked on Jurkat cells pretreated with anti-DR5 mAb. The average percentage of blocking was 90 49%.Conclusion:DR5 plays a very key role in TRAIL induced apoptosis in Jurkat cells.
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Objective:To determine the sensitivity of SMMC-7721 cells to mDRA-6,the synergistic damage effect of mDRA-6 and adriamycin on human hepatocellular carcimoma cell lines and its possible mechanism.Methods:SMMC-7721 cells were cultured with RPMI1640 medium in regular condition.The morphology was observed by microscope.Cytotoxicity was examined by MTT assay. Apoptosis were detected by flow cytometry.Results:(1)The apoptosis of SMMC-7721 cells could be induced by mDRA-6,1.89 ?g/ml of mDRA-6 cound kill 36% of the cells;(2)Concentration-dependent cytotoxicity of adriamycin was exhibited in SMMC-7721 cells;(3)The combination of mDRA-6 and adriamycin exhibited synergistic effect on SMMC-7721 cells.2 ?g/ml of mDRA6 and 40 ng/ml of adriamycin killed 60%SMMC-7721.Conclusion:MDRA-6 can induce SMMC-7721 cell apoptosis.The combination of mDRA-6 and adriamycin exhibit synergistic effect on SMMC-7721 cells.