ABSTRACT
ObjectiveTo observe the repair effect of Dahuanglingxian prescription （DHLX） on bile duct epithelial cells of rats. To explore whether its mechanism of action is to adjust the mutual binding of transforming growth factor -β （TGF-β） activated kinase 1（TAK1） and tumor necrosis factor receptor-associated factor 6 （TRAF6）， and regulate the activation of the nuclear transcription factor -κB （NF-κB）/mitogen-activated protein kinase （MAPK） signaling pathway. MethodThe 20 SD rats were randomly divided into normal group and DHLX group， 10 rats in each group， were given saline and DHLX （320 mg·kg-1·d-1） for 8 days， to prepare normal serum and DHLX serum. Biliary epithelial cells were extracted from normal SD rats and divided into 9 groups： Normal group， model group （20 mg·L-1）， LPS+DHLX group （20 mg·L-1+10% DHLX）， LPS+PDTC group （20 mg·L-1+200 μmol·L-1）， LPS+SB203580 group （20 mg·L-1+0.5 μmol·L-1）， LPS+PDTC+SB203580 group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1）， LPS+PDTC+DHLX group （20 mg·L-1+200 μmol·L-1+10% DHLX serum）， LPS+SB203580+DHLX group （20 mg·L-1+0.5 μmol·L-1+10% DHLX serum）， LPS+PDTC+SB203580 +DHLX group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1+10% DHLX serum）. The microscopic observation of morphological changes in each group of cells after drug intervention. Enzyme-linked immunosorbent assay（ELISA） was used to detect the expression of （IL）-1β and IL-6 in each group of cells. Western blot detected the expression levels of TAK1 and TRAF6 proteins in each group of cells， Co-IP detected the interaction between TAK1 and TRAF6， and further observed the distribution and co-localization of TAK1 and TRAF6 using Laser confocal microscope. ResultAfter the action of LPS， the cell synapses are reduced， the cell body becomes significantly rounded and smaller， but the cell morphology of each group tends to be normal after medication. Compared with normal group， the expression levels of IL-1β and IL-6 in model group were significantly increased （P<0.05）， while the expression level of TAK1 was decreased while the expression level of TRAF6 was increased （P<0.05）. The content of TAK1-TRAF6 protein complex showed a decreasing trend， and the two proteins co-located in the cytoplasm. Compared with model group， the expression levels of IL-1β and IL-6 in LPS+DHLX group were significantly decreased （P<0.05）， the expression level of TAK1 was increased and the expression level of TRAF6 was decreased （P<0.05）， the content of TAK1-TRAF6 protein complex was significantly increased （P<0.01）， and the two proteins were significantly co-located in cytoplasm. Compared with LPS+DHLX group， the expression levels of IL-1β and IL-6 in other groups were significantly decreased （P<0.05，P<0.01）. TAK1-TRAF6 protein complex content in each group was significantly decreased after pathway blocker intervention （P<0.05）， while TAK1-TRAF6 protein complex content in each group was significantly increased after pathway blocker combined with DHLX intervention （P<0.05）. Co-localization of the TAK1-TRAF6 in cytoplasm was not obvious. ConclusionIn the LPS-induced inflammatory response of bile duct cells， the binding of TAK1 and TRAF6 showed a weakening trend， but DHLX could reverse the phenomenon， we think the mechanism of action may be related to promoting the mutual binding of TAK1 and TARF6 to inhibit the activation of the NF-κB/MAPK signaling pathway.
ABSTRACT
Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aβ plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.
ABSTRACT
Objective@#Antimony (Sb) has recently been identified as a novel nerve poison, although the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. This study aimed to assess the effects of the nuclear factor kappa B (NF-κB) signaling pathway on antimony-induced astrocyte activation.@*Methods@#Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of p65. The expression of protein in brain tissue sections was detected by immunohistochemistry. The levels of mRNAs were detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR).@*Results@#Antimony exposure triggered astrocyte proliferation and increased the expression of two critical protein markers of reactive astrogliosis, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), indicating that antimony induced astrocyte activation @*Conclusion@#Antimony activated astrocytes by activating the NF-κB signaling pathway.
Subject(s)
Animals , Male , Rats , Antimony/toxicity , Astrocytes/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Glial Fibrillary Acidic Protein/metabolism , MAP Kinase Kinase Kinases , Mice, Inbred ICR , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Signal Transduction/drug effectsABSTRACT
Objective The biological function of E3 ubiquitin-protein ligase RNF19A in lung cancer is yet clear. This study was to investigate the effects of RNF19A on the proliferation, invasion and migration of lung cancer cells and its underlying mechanisms. Methods A549 cells were transfected with control siRNA or RNF19A siRNA for 48 hours. Then, the viability and proliferation of the cells were measured by CCK8 and BrdU incorporation assay, respectively. Immunoprecipitation and Western immunoblotting were used to detect the effect of RNF19A-TAK1 interaction on the ubiquitination of TAK1 and the effects of TAK1 and NF-κB inhibitors on the proliferation, invasion and migration of the Flag-RNF19A-mediated A549 cells. Results After 48 hours of transfection, the viability and proliferation of the A549 cells were significantly decreased in the RNF19A siRNA group as compared with the control group (P < 0.001), and so were the numbers of migrating (441.0 ± 18.63 vs 960.6 ± 37.82, P < 0.05) and invading cells (488.2 ± 26.06 vs 1120 ± 58.96, P < 0.05) and the level of TAK1 ubiquitination in the A549 cells (0.425 ± 0.01 vs 0.656 ± 0.012, P < 0.05). Over-expressed Flag-RNF19A markedly enhanced the proliferation of the cells in comparison with that of the control group (P < 0.05), and increased the numbers of migrating (1032 ± 38.86 vs 721.7 ± 26.60, P < 0.05) and invading cells (657.7 ± 13.74 vs 355.7 ± 15.51, P < 0.05), but showed no statistically significantly difference from the control in the proliferation of the cells with the addition of TAK1 and NF-κB inhibitors (P > 0.05). Conclusion RNF19A can increasing the proliferation, migration and invasion of A549 lung cancer cells, probably by enhancing TAK1 ubiquitination and NF-κB activation.
ABSTRACT
Aim We used bone marrow-derived macro-phages ( BMMs ) , to explore the mechanism of macro-phage activation and the effect of TGF-β activated ki-nase-1 ( TAK1 ) inhibitor 5 Z-7-oxozeaenol on it under AGEs conditions. Methods The BMMs were obtained from C57 mice, and purity of BMMs was detected by flow cytometry. Cell viability was tested after treatment with different concentrations of TAK1 inhibitors. Laser confocal microscopy was used to detect macrophage M1 subtype . Flow cytometry was used to analyse the macro-phage activated by AGEs. TNF-α and MCP-1 mRNA levels were evaluated by qRT-PCR. Western blot was used to detect the expression levels of TAK1 signal pathway protein. Results AGEs stimulation could in-crese the activity of M1 macrophages,and 5Z-7-oxoze-aenol could inhibit the differentiation of BMMs. Com-pared with control group, AGEs increased the expres-sion of MCP-1 and TNF-α mRNA(P NF-κBp65 proteins ( P <0. 05 ) . Conclusions AGEs can induce BMMs to M1 phenotypic polarization. 5Z-7-oxozeaenol reduces the expression of inflammatory cyto-kine via inhibiting TAK1/MAPKs, MAPKs/NF-κB pathways.
ABSTRACT
Objective To investigate the expression levels of TAK1 and p38 genes among different subtypes of acute myeloid leukemia (AML) patients,and to analyze the clinical characteristics of patients with different expression levels of TAK1 and p38 genes.Methods GAPDH was made as an internal reference,14 healthy people as control group.The quantitative real-time PCR was used to detect the expression of TAK1 and p38 in bone marrow samples of 87 AML patients,and the results were analyzed statistically.Results The expression levels of TAK1 and p38 in experiment group were higher than those in control group (0.194± 0.125 vs 0.015±0.008,0.233±0.140 vs 0.010±0.005,P ＜ 0.001).TAK1 expression in M4 was higher than that in M2,M3 and M5 (P =0.005,0.000,0.002),TAK1 expression in M3 was lower than that in M2 (P =0.022).p38 expression in M4 was higher than that in M1,M2,M3 and M5 (P =0.013,0.035,0.000,0.045),as it was higher in M2 and M5 than that in M3 (P =0.001,0.012).The CD56 positive rate cells and the number of peripheral blood leukocytes of the TAK1 high expression group were higher than those of the TAK1 low expression group,the CD19 positive rate of the p38 low expression group was higher than that of the p38 high expression group.Conclusion The expression levels of TAK1 and p38 genes are elevated in AML patients,and the up-regulation may play an important role in the pathogenesis of AML.
ABSTRACT
Objective To examine the expression of transforming growth factor β(TGF-β) activated kinase 1 (TAK1) in esophageal organization and the impact of TAK1 expression on clinicopathologic data. Methods Specimens from 80 patients with esophageal adenocarcinoma managed in our hospital were included in this study. Immunohistochemical staining was used to detect the expression of TAK1 in 80 cases of esophageal squamous cell carcinoma tissues and 80 cases of normal esophageal mucosal tissues. All the specimens were confirmed by pathology for esophageal squamous cell carcinoma or esophageal normal tissues. Results The positive expression rate of TAK1 in esophageal cancer organizations and normal esophageal mucosal tissues were 80% and 11.25%, respectively. The positive expression rate of TAK1 in esophageal cancer were significantly higher than those in normal esophageal mucosal tissue (P 0.05), but with lymph node metastasis and clinical stage (P<0.05). Patients with positive TAK1 expression had significantly lower five-year survival rate than those with tumors having positive TAK1 expression Conclusion TAK1 played an important role in the pathology and development of squamous cell carcinoma , and could be an important therapeutic target in the treatment of esophageal cancer.
ABSTRACT
Objective: To investigate the effect of miR-143 (microRNA-143) on biological activity of pancreatic cancer PANC-1 cells. Methods: MiR-143 mimics were chemically synthesized and transfected transiently into PANC-1 cells by LipofectAMINE™ 2000. The expression of miR-143 in pancreatic cancer cells was examined by real-time fluorescence quantitative PCR. The cell proliferation was measured by MTT method. The apoptosis was detected by flow cytometry. Transwell and wound healing assays were performed to examine the migration ability of PANC-1 cells. The dual luciferase reporter vectors containing 3'-UTR (3'-untranslated region) with miR-143 binding site of wild type or mutant TAK 1 (transforming growth factor-β activated kinase 1) were constructed by using Dual-Luciferase® Reporter Assay System, and then the relative activity of Renilla reniformis luciferase was detected to confirm the binding site of miR-143 on TAK1's mRNA. Results: The expression of miR-143 in miR-143 mimicstransfected PANC-1 cells was significantly up-regulated. The transfection of miR-143 failed to influence the activities of proliferation and apoptosis of PANC-1 cells (P > 0.05), and however, the migration ability of PANC-1 cells was reduced (P < 0.05). Compared with negative control or wild type TAK 1 or mutant TAK 1, cotransfection with miR-143 and wild type TAK 1 3'-UTR could significantly decrease the relative activity of Renilla reniformis luciferase. Conclusion: miR-143 can restrain the migration ability of PANC-1 cells in vitro , in which TAK 1 may be one of the target genes. Copyright © 2012 by TUMOR.