scholarly journals β-Arrestin2 Inhibits Expression of Inflammatory Cytokines in BEAS-2B Lung Epithelial Cells Treated with Cigarette Smoke Condensate via Inhibition of Autophagy

2018 ◽  
Vol 50 (4) ◽  
pp. 1270-1285 ◽  
Author(s):  
Yanjun Wu ◽  
Yunxiao Li ◽  
Bo Wu ◽  
Chunting Tan ◽  
Xin He ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Cigarette Smoke ◽  
Inflammatory Cytokines ◽  
Bronchial Epithelial Cell ◽  
Mtor Signaling ◽  
Epithelial Cell Line ◽  
Mtor Signaling Pathway ◽  
Cigarette Smoke Condensate ◽  
Bronchial Epithelial

Background/Aims: β-arrestin2 has been shown to have a role in human inflammatory disease. However, the role of β-arrestin2 in cigarette smoke-induced inflammation in the lung remains unknown. The aims of this study were to investigate the effects of β-arrestin2 on cigarette smoke condensate (CSC)-induced expression of inflammatory cytokines in the BEAS-2B human bronchial epithelial cell line in vitro, and the mechanisms involved. Methods: The MTT assay determined cell viability of cultured BEAS-2B cells. Autophagy was assessed by western blot, adenoviral mRFP-GFP-LC3 transfection, and immunofluorescence. The effects of β-arrestin2 shRNA knockdown were studied by western blot and real-time reverse transcription-polymerase chain reaction (RT-PCR). Western blot evaluated the AMPK/mTOR signaling pathway. Levels of inflammatory cytokines, interleukin (IL)-6, IL-8, and MCP-1 were measured in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Results: CSC suppressed expression of β-arrestin2 in BEAS-2B cells, activated the AMPK/mTOR signaling pathway, increased cell autophagy and the expression of IL-6, IL-8, and MCP-1,pretreatment with the β-arrestin2 biased ligands, propranolol, and ICI118551 reversed these changes. Inhibition of autophagy reduced the expression of inflammatory cytokines following CSC. Conclusion: In the human bronchial epithelial cell line, BEAS-2B, β-arrestin2 reduced the expression of CSC-induced inflammatory cytokines by inhibiting autophagy, most likely via the AMPK/mTOR signaling pathway.

scholarly journals FH535 Potentiation of Cigarette Smoke Condensate Cytotoxicity is Associated With Changes in β-Catenin and EGR-1 Signaling

2012 ◽  
Vol 31 (4) ◽  
pp. 380-389 ◽  
Author(s):  
William W. Polk
Keyword(s):  
Cigarette Smoke ◽  
Epithelial To Mesenchymal Transition ◽  
Target Genes ◽  
Protein Localization ◽  
Bronchial Epithelial Cell ◽  
Nuclear Accumulation ◽  
Epithelial Cell Line ◽  
Cigarette Smoke Condensate ◽  
Bronchial Epithelial ◽  
Mesenchymal Transition

Cigarette smoke condensate (CSC) has been reported to elicit morphological and transcriptional changes that suggest epithelial-to-mesenchymal transition (EMT) in cultured bronchial epithelial cells. The transdifferentiation potential of acute and prolonged CSC exposure alone or in combination with the β-catenin inhibitor, FH535, was investigated in the bronchial epithelial cell line, BEAS-2B, through assessment of cell morphology, transcript expression, protein expression, and protein localization. Changes in morphology, β-catenin translocation, E-cadherin expression, metalloproteinase expression, and fibronectin could be demonstrated independent of molecular or physiological evidence of EMT. FH535 was shown to increase CSC-induced cytotoxicity and depress β-catenin expression. However, FH535 effects were not limited to the β-catenin pathway as it also blocked the expression of early growth responsive protein 1 (EGR-1) target genes, fibronectin and phosphatase and tensin homologue, without affecting EGR-1 nuclear accumulation.

scholarly journals M2 macrophage-derived exosomal microRNAs inhibit cell migration and invasion in gliomas through PI3K/AKT/mTOR signaling pathway

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jie Yao ◽  
Zefen Wang ◽  
Yong Cheng ◽  
Chao Ma ◽  
Yahua Zhong ◽  
...  
Keyword(s):  
Cell Migration ◽  
Western Blot ◽  
Signaling Pathway ◽  
Target Gene ◽  
Glioma Cells ◽  
Mtor Signaling ◽  
Migration And Invasion ◽  
M2 Macrophage ◽  
Mtor Signaling Pathway ◽  
Cell Migration And Invasion

Abstract Background Glioma, the most common primary brain tumor, account Preparing figures for 30 to 40% of all intracranial tumors. Herein, we aimed to study the effects of M2 macrophage-derived exosomal microRNAs (miRNAs) on glioma cells. Methods First, we identified seven differentially expressed miRNAs in infiltrating macrophages and detected the expression of these seven miRNAs in M2 macrophages. We then selected hsa-miR-15a-5p (miR-15a) and hsa-miR-92a-3p (miR-92a) for follow-up studies, and confirmed that miR-15a and miR-92a were under-expressed in M2 macrophage exosomes. Subsequently, we demonstrated that M2 macrophage-derived exosomes promoted migration and invasion of glioma cells, while exosomal miR-15a and miR-92a had the opposite effects on glioma cells. Next, we performed the target gene prediction in four databases and conducted target gene validation by qRT-PCR, western blot and dual luciferase reporter gene assays. Results The results revealed that miR-15a and miR-92a were bound to CCND1 and RAP1B, respectively. Western blot assays demonstrated that interference with the expression of CCND1 or RAP1B reduced the phosphorylation level of AKT and mTOR, indicating that both CCND1 and RAP1B can activate the PI3K/AKT/mTOR signaling pathway. Conclusion Collectively, these findings indicate that M2 macrophage-derived exosomal miR-15a and miR-92a inhibit cell migration and invasion of glioma cells through PI3K/AKT/mTOR signaling pathway.

SIK2 Promotes Cisplatin Resistance Induced by Aerobic Glycolysis in Breast Cancer Cells through PI3K/AKT/mTOR Signaling Pathway

2020 ◽  
Author(s):  
Shoukai Zong ◽  
Wei Dai ◽  
Wencheng Fang ◽  
Xiangting Guo ◽  
Kai Wang
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Breast Cancer Cells ◽  
Cisplatin Resistance ◽  
Aerobic Glycolysis ◽  
Mtor Signaling ◽  
Mtor Signaling Pathway ◽  
Protein Levels

Abstract Objective This study aimed to investigate the effect of SIK2 on cisplatin resistance induced by aerobic glycolysis in breast cancer cells and its potential mechanism. Methods qRT-PCR and Western blot were used to detect SIK2 mRNA and protein levels. Cisplatin (DDP) resistant cell lines of breast cancer cells were established, CCK-8 was used to measure and evaluate the viability, and Transwell was used to evaluate the cell invasion capability. Flow cytometry was adopted to evaluate the apoptosis rate. The glycolysis level was evaluated by measuring glucose consumption and lactic acid production. The protein levels of p-PI3K, p- protein kinase B (Akt) and p-mTOR were determined by western blot. Results SIK2 is highly expressed in breast cancer tissues and cells compared with adjacent tissues and normal human breast epithelial cells, and has higher diagnostic value for breast cancer. Silencing SIK2 expression can inhibit proliferation and invasion of breast cancer cells and induce their apoptosis. In addition, SIK2 knockdown inhibits glycolysis, reverses the resistance of drug-resistant cells to cisplatin, and inhibits PI3K/AKT/mTOR signaling pathway. When LY294002 is used to inhibit PI3K/AKT/mTOR signaling pathway, the effect of Sh-SIK2 on aerobic glycolysis of breast cancer cells can be reversed. Conclusion SIK2 can promote cisplatin resistance caused by aerobic glycolysis of breast cancer cells through PI3K/AKT/mTOR signaling pathway, which may be a new target to improve cisplatin resistance of breast cancer cells.

scholarly journals Ginsenoside Rg3 Suppresses Proliferation and Induces Apoptosis in Human Osteosarcoma

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Yi Li ◽  
Jinying Lu ◽  
Furong Bai ◽  
Yanan Xiao ◽  
Yiran Guo ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Mtor Signaling ◽  
Assay Method ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Ginsenoside Rg3 ◽  
Mtor Signaling Pathway ◽  
Human Osteosarcoma ◽  
Human Osteosarcoma Cells

Osteosarcoma is the most common primary malignancy of bone in children and the elderly. Recently, more and more researches have demonstrated that Ginsenoside Rg3 (Rg3) is involved in chemotherapy resistance in many cancer, making it a promising Chinese herbal monomer for oncotherapy. In this study, we investigated the efficacy of Rg3 in human osteosarcoma cell lines (MG-63, U-2OS, and SaOS-2). Cell proliferation was measured by CCK8 assay. The migration of cells was examined using the scratch assay method. Quantification of apoptosis was assessed further by flow cytometry. In addition, the expression of apoptosis-related genes (caspase9, caspase3, Bcl2, and Bax) were investigated using RT-PCR. We further investigated the protein level expression of Bcl 2, cleaved-caspase3, and PI3K/AKT/mTOR signaling pathway factors by Western blot assay. Our results revealed that Rg3 inhibited the proliferation and migration of human osteosarcoma cells and induced apoptosis in a concentration- and time-dependent manner. Western blot results showed that Rg3 reduced the protein expression of Bcl2 and PI3K/AKT/mTORbut increased the levels of cleaved-caspase3. Therefore, we hypothesized Rg3 inhibits the proliferation of osteosarcoma cell line and induces their apoptosis by affecting apoptosis-related genes (Bcl2, caspase3) as well as the PI3K/AKT/mTOR signaling pathway. To conclude, Rg3 is a new therapeutic agent against osteosarcoma.

MicroRNA-1271-5p inhibits the tumorigenesis of ovarian cancer through targeting E2F5 and negatively regulates the mTOR signaling pathway

2020 ◽  
Author(s):  
Qin Li ◽  
Junyu Shi ◽  
Xiaoli Xu
Keyword(s):  
Ovarian Cancer ◽  
Western Blot ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Mtor Signaling ◽  
Migration And Invasion ◽  
Mtor Signaling Pathway ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Worse Prognosis

Abstract Background: MicroRNA-1271-5p (miR-1271-5p) has been reported to participate in the progression of many malignancies. However, the molecular mechanism of miR-1271-5p still remains vague in ovarian cancer (OC). Therefore, we explored the effect of miR-1271-5p in the development of OC in present study.Methods: We measured the miR-1271-5p expression via qRT-PCR assay. Western blot analysis was employed to examine protein expression. Then, the functional mechanism of miR-1271-5p was analyzed by MTT, Transwell and dual luciferase assays.Results: Downregulation of miR-1271-5p was found in OC, which can predict worse prognosis in OC patients. Further, miR-1271-5p directly targets E2F5 in OC. And miR-1271-5p restrained the proliferation, migration and invasion of OC cells via targeting E2F5. Additionally, upregulation of E2F5 was observed in OC, which predicted unfavorable prognosis in OC patients. Besides that, miR-1271-5p suppressed EMT and mTOR pathway in OC.Conclusion: MiR-1271-5p inhibited the tumorigenesis of OC through targeting E2F5 and negatively regulated the mTOR signaling pathway.

Kaempferol reversals retinal ischemia/reperfusion (IR) injury through activating of PI3K/Akt/mTOR signaling pathway

2021 ◽  
Author(s):  
Da Sun ◽  
Fusheng Shang ◽  
Dagui Chen ◽  
Wenwen Wang ◽  
lili lin
Keyword(s):  
Oxidative Damage ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Ischemia Reperfusion ◽  
Retinal Ischemia ◽  
Mtor Signaling ◽  
Cell Counting ◽  
Mtor Signaling Pathway ◽  
Autophagy Gene ◽  
Retinal Vascular Occlusion

Abstract Purpose Retinal ischemia/reperfusion (IR) injury is associated with many ocular diseases, including acute glaucoma, diabetic retinopathy, and retinal vascular occlusion. However, currently there are no effective medications to prevent the development ofretinal IR injury.Kaempferol is a kind of plant extract which has showed an excellent ability to inhibit the inflammation.. Materials and Methods In this study, both in vitro and in vivo retinaloxidative damage models were established.Cell viability was assessed by Cell Counting Kit-8 assay. Apoptosis was examined using flow cytometry analysis.Atherosclerotic lesion analysis was performed using hematoxylin-eosin staining,The expressions of Inflammatory cytokines were detected by quantitative real-time PCR and ELISA respectively.The effect of expression of apoptosis、utophagy and the PI3K/Akt/mTOR signaling pathway related pathway was evaluated by Western blot. Results We found kaempferol was able to protect the viability of ARPE-19 cells against oxidative damage by reducing its apoptosis. In addition, it also kept structurally complete epithelium, stroma and endothelium of cornea after oxidative damage. Moreover, it also able to reduce the expression of inflammatory cytokines and increased the expression of anti-inflammatory cytokines.Kaempferol was able to enhanced the expression of anti-apoptotic genes BCL-2, and reduced the expression of autophagy gene Beclin 1 and increased the expression of anti-autophagy gene LC-3,was also able to enhance the expression PI3K and the phosphorylation ofAkt andmTOR. Conclusion Kaempferolreversals retinal ischemia/reperfusion (IR) injury through activating of PI3K/Akt/mTOR signaling pathway.

MicroRNA-34a Promotes Apoptosis of Rheumatoid Arthritis Fbroblast-Like Synoviocyte via AMPK/Akt/mTOR Signaling Pathway

2020 ◽  
Vol 10 (8) ◽  
pp. 1176-1183
Author(s):  
Juan Ni ◽  
Zhe Hao
Keyword(s):  
Rheumatoid Arthritis ◽  
Western Blot ◽  
Signaling Pathway ◽  
Opposite Effect ◽  
Life Quality ◽  
Tunel Assay ◽  
Mtor Signaling ◽  
Mrna Levels ◽  
Mtor Signaling Pathway ◽  
Rt Pcr

Background and Objectives: Rheumatoid arthritis (RA) is an autoimmune arthropathy characterised by chronic synovitis, joint cartilage breakdown and bone erosions. The life quality of RA patients has been substantially effected by the disease and it is of great significance to search for more efficacious and novel therapeutic agents. Methods: In this study, mRNA levels of miR-34a in HFLS and RA-FLS were examined by RT-PCR. CCK8 assay was applied to detect the viability of RA-FLS transfected with miR-34a mimic or inhibitor. Furthermore, TUNEL assay and Hoechest-33342 assay was applied to detect the apoptosis of RA-FLS transfected with miR-34a mimic or inhibitor. The expressions of proteins related to AMPK/Akt/mTOR and autophagy were also detected by western blot. Results: The RT-PCR result showed that miR-34a mRNA levels was markedly downregulated in RA-FLS compared to HFLS. CCK8 assay results demonstrated that miR-34a overexpression significantly suppressed RA-FLS proliferation and miR-34a interference had the opposite effect. TUNEL assay and Hoechest-33342 assay demonstrated that miR-34a overexpression significantly promoted RA-FLS apoptosis and miR-34a interference had the opposite effect. The western blot results revealed that miR-34a can affect autophagy via AMPK/Akt/mTOR signaling pathway. Conclusion: This study suggested that miR-34a can regulate proliferation and apoptosis in RA-FLS by affecting autophagy through AMPK/Akt/mTOR signaling pathway.

scholarly journals Hypoxia-Related Gene FUT11 Promotes Pancreatic Cancer Progression by Maintaining the Stability of PDK1

2021 ◽  
Vol 11 ◽  
Author(s):  
Wenpeng Cao ◽  
Zhirui Zeng ◽  
Runsang Pan ◽  
Hao Wu ◽  
Xiangyan Zhang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Western Blot ◽  
Signaling Pathway ◽  
Mtor Signaling ◽  
Mtor Signaling Pathway ◽  
Cell Growth And Migration ◽  
And Migration ◽  
The Stability

BackgroundHypoxia is associated with the development of pancreatic cancer (PC). However, genes associated with hypoxia response and their regulatory mechanism in PC cells were unclear. The current study aims to investigate the role of the hypoxia associated gene fucosyltransferase 11 (FUT11) in the progression of PC.MethodsIn the preliminary study, bioinformatics analysis predicted FUT11 as a key hypoxia associated gene in PC. The expression of FUT11 in PC was evaluated using quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. The effects of FUT11 on PC cells proliferation and migration under normoxia and hypoxia were evaluated using Cell Counting Kit 8, 5-ethynyl-2’-deoxyuridine (EDU) assay, colony formation assay and transwell assay. The effects of FUT11 in vivo was examined in mouse tumor models of liver metastasis and subcutaneous xenograft. Furthermore, Western blot, luciferase assay and immunoprecipitation were performed to explore the regulatory relationship among FUT11, hypoxia-inducible factor 1α (HIF1α) and pyruvate dehydrogenase kinase 1 (PDK1) in PC.ResultsFUT11 was markedly increased of PC cells with hypoxia, upregulated in the PC clinical tissues, and predicted a poor outcome of PC patients. Inhibition of FUT11 reduced PC cell growth and migratory ability of PC cells under normoxia and hypoxia conditions in vitro, and growth and tumor cell metastasis in vivo. FUT11 bound to PDK1 and regulated the expression PDK1 under normoxia and hypoxia. FUT11 interacted with PDK1 and decreased the ubiquitination of PDK1, lead to the activation of AKT/mTOR signaling pathway. FUT11 knockdown significantly increased the degradation of PDK1 under hypoxia, while treatment with MG132 can relieve the degradation of PDK1 induced by FUT11 knockdown. Overexpression of PDK1 in PC cells under hypoxia conditions reversed the suppressive impacts of FUT11 knockdown on PC cell growth and migration. In addition, HIF1α bound to the promoter of FUT11 and increased its expression, as well as co-expressed with FUT11 in PC tissues. Furthermore, overexpression of FUT11 partially rescued the suppressive effects of HIF1α knockdown on PC cell growth and migration in hypoxia condition.ConclusionOur data implicate that hypoxia-induced FUT11 contributes to proliferation and metastasis of PC by maintaining the stability of PDK1, thus mediating activation of AKT/mTOR signaling pathway, and suggest that FUT11 could be a novel and effective target for the treatment of pancreatic cancer.

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