scholarly journals NOS2 inhibitor 1400W Induces Autophagic Flux and Influences Extracellular Vesicle Profile in Human Glioblastoma U87MG Cell Line

2019 ◽  
Vol 20 (12) ◽  
pp. 3010 ◽  
Author(s):  
Paola Palumbo ◽  
Francesca Lombardi ◽  
Francesca Rosaria Augello ◽  
Ilaria Giusti ◽  
Sabino Luzzi ◽  
...  
Keyword(s):  
Extracellular Vesicle ◽  
Acid Sphingomyelinase ◽  
Cell Counting ◽  
Autophagic Flux ◽  
U87mg Cell ◽  
Cell Growth And Migration ◽  
Acridine Orange Staining ◽  
Transmission Electron ◽  
And Migration ◽  
Nitric Oxide Synthase 2

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.

scholarly journals Inhibition of Osteoblast Proliferation and Migration by Exogenous and Endogenous Formaldehyde

2019 ◽  
Author(s):  
Xu Teng ◽  
Wei Huang ◽  
Hefeng Yu ◽  
Pei Wang ◽  
Weishi Li ◽  
...  
Keyword(s):  
Cell Growth ◽  
Potential Role ◽  
Expression Patterns ◽  
Cell Counting ◽  
Rna Seq ◽  
Cell Growth And Migration ◽  
Naturally Occurring ◽  
Significant Enrichment ◽  
And Migration ◽  
Proliferation And Migration

AbstractExogenous and endogenous formaldehyde (FA) plays an important role in cell growth and migration; however, its potential role in osteoblasts remains largely unclear. Cell counting kit-8 (CCK-8) and wound healing assays revealed that FA exposure at naturally occurring concentrations inhibited the proliferation and migration of mouse preosteoblast MC3T3-E1 cells. Moreover, RNA sequencing (RNA-seq) analysis revealed that FoxO1 signaling pathway components displayed distinct expression patterns upon FA exposure, reflected through significant enrichment of cell migration. In particular, FoxO1 Sirt1 and FA-induced related protein expression which were closely with cell proliferation and migration were confirmed by western blotting. The present results indicate that the FoxO1 pathway is involved in FA-induced inhibition of cell growth and migration.

scholarly journals High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry

2019 ◽  
Author(s):  
Gopika SenthilKumar ◽  
Justin H. Skiba ◽  
Randall J. Kimple
Keyword(s):  
Acridine Orange ◽  
High Throughput ◽  
Late Stage ◽  
Image Cytometry ◽  
Alternative Methods ◽  
Quantitative Detection ◽  
Autophagic Flux ◽  
Objective Method ◽  
Acridine Orange Staining ◽  
Transmission Electron

AbstractQuantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevent accurate quantification. We report a high-throughput, inexpensive, reliable, and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.Methods summaryA high-throughput, inexpensive, reliable, and objective method for studying both basal autophagy and autophagic flux is reported. This approach uses acridine orange staining of late-stage autophagy and image cytometry to quantify autophagy.

scholarly journals The Potential Role of Cycloastragenol in Promoting Diabetic Wound Repair In Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Yi Cao ◽  
Li Xu ◽  
Xiaohong Yang ◽  
Yuan Dong ◽  
Hongbin Luo ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Repair ◽  
Cell Counting ◽  
Migration Ability ◽  
Cell Growth And Migration ◽  
Cell Based Therapy ◽  
And Migration ◽  
Proliferation And Migration

Background. Refractory wound healing is a severe complication of diabetes with a significant socioeconomic burden. Whereas current therapies are insufficient to accelerate repair, stem cell-based therapy is increasingly recognized as an alternative that improves healing outcomes. The aim of the present study is to explore the role of cycloastragenol (CAG), a naturally occurring compound in Astragali Radix, in ameliorating refractory cutaneous wound healing in vitro, which may provide a new insight into therapeutic strategy for diabetic wounds. Methods. Human epidermal stem cells (EpSCs) obtained from nine patients were exposed to CAG, with or without DKK1 (a Wnt signaling inhibitor). A lentiviral short hairpin RNA (shRNA) system was used to establish the telomerase reverse transcriptase (TERT) and β-catenin knockdown cell line. Cell counting kit-8, scratch wound healing, and transwell migration assay were used to determine the effects of CAG in cell growth and migration. The activation of TERT, β-catenin, and c-Myc was determined using real-time qPCR and western blot analysis. Chromatin immunoprecipitation (ChIP) was performed to evaluate the associations among CAG, TERT, and Wnt/β-catenin signals. Results. CAG not only promoted the proliferation and migration ability of EpSCs but also increased the expression levels of TERT, β-catenin, c-Myc. These effects of CAG were most pronounced at a dose of 0.3 μM. Notably, the CAG-promoted proliferative and migratory abilities of EpSCs were abrogated in TERT and β-catenin-silenced cells. In addition, the ChIP results strongly suggested that CAG-modulated TERT was closely associated with the activation of Wnt/β-catenin signaling. Conclusion. Our data indicate that CAG is a TERT activator of EpSCs and is associated with their proliferation and migration, a role it may play through the activation of Wnt/β-catenin signaling.

scholarly journals Inhibition of osteoblast proliferation and migration by exogenous and endogenous formaldehyde

2020 ◽  
pp. 096032712097512
Author(s):  
Xu Teng ◽  
Pei Wang ◽  
Tianshu Yang ◽  
Wei Huang ◽  
Hefeng Yu ◽  
...  
Keyword(s):  
Cell Growth ◽  
Potential Role ◽  
Expression Patterns ◽  
Cell Counting ◽  
Rna Seq ◽  
Cell Growth And Migration ◽  
Naturally Occurring ◽  
Significant Enrichment ◽  
And Migration ◽  
Proliferation And Migration

Exogenous and endogenous formaldehyde (FA) both play an important role in cell growth and migration; however, their potential role in osteoblasts remains largely unclear. Cell counting kit-8 (CCK-8) and wound-healing assays revealed that FA exposure at naturally occurring concentrations inhibited the proliferation and migration of mouse preosteoblast MC3T3-E1 cells. Moreover, RNA sequencing (RNA-seq) analysis revealed that FoxO1 signaling pathway components displayed distinct expression patterns upon FA exposure, reflected through significant enrichment of cell migration. In particular, FoxO1-, Sirt1-, and FA-induced protein expression, which was closely associated with cell proliferation and migration, was confirmed by western blotting. The results obtained indicated that the FoxO1 pathway is involved in FA-induced inhibition of cell growth and migration.

miR-338-5p inhibits cell growth and migration via inhibition of the METTL3/m6A/c-Myc pathway in lung cancer

2020 ◽  
Author(s):  
Hongyu Wu ◽  
Fangjuan Li ◽  
Ren Zhu
Keyword(s):  
Lung Cancer ◽  
Cell Growth ◽  
Cancer Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Lung Cancer Cells ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Cell Growth And Migration ◽  
Public Health Burden ◽  
And Migration

Abstract Lung cancer is a common type of cancer that causes a very large public health burden worldwide. Achieving a better understanding of the molecular mechanism underlying the progression of lung cancer is of benefit for the diagnosis, prognosis, and treatment of lung cancer. Here, we first identified dramatically decreased expression of miR-338-5p in lung cancer tissues and cells using quantitative polymerase chain reaction (qPCR) analysis. We then revealed that miR-338-5p inhibited the cell growth and migration of lung cancer cells using cell counting kit 8 (CCK8), EdU, and Transwell analysis. Furthermore, we demonstrated that miR-338-5p inhibited METTL3 expression by qPCR, western blot analysis, and luciferase reporter assay, while upregulation of METTL3 alleviated the role of miR-338-5p in lung cancer cells. We also showed that METTL3 promoted c-Myc expression by increasing the m6A modification of c-Myc, and overexpression of c-Myc restored the inhibition of cell growth and migration of lung cancer cells induced by METTL3 silencing. Ultimately, this research illustrated that modification of the miR-338-5p/METTL3/c-Myc pathway affected cellular progression in lung cancer cells. Collectively, our study revealed the underlying mechanism of miR-338-5p in lung cancer, providing a novel regulatory pathway in lung cancer. There is potential for this pathway to serve as a diagnostic, prognostic, and therapeutic biomarker for lung cancer.

scholarly journals Long Noncoding RNA GATA3-AS1 Promotes Cell Proliferation and Metastasis in Hepatocellular Carcinoma by Suppression of PTEN, CDKN1A, and TP53

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Xuee Luo ◽  
Ning Zhou ◽  
Le Wang ◽  
Qinghua Zeng ◽  
Hongying Tang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Cell Counting ◽  
Cell Growth And Migration ◽  
Expression Levels ◽  
Normal Tissues ◽  
Proliferation And Metastasis ◽  
And Migration ◽  
Cell Proliferation And Metastasis

Background. Long noncoding RNAs (lncRNAs) have been known to play important roles in the progression of various types of human cancer. LncRNA GATA3 antisense RNA 1, GATA3-AS1, has been reported to be associated with T-cell development and differentiation. However, the expression pattern and function of GATA3-AS1 in hepatocellular carcinoma (HCC) remain unknown. Methods. Real-time quantitative PCR (RT-qPCR) assay was conducted to detect GATA3-AS1 expression levels in 80 cases of pairs HCC tissues and matched normal tissues. Chi-squared (χ2) test was used to analyze the correlation between GATA3-AS1 expression and clinicopathologic variables. Survival curves were plotted using the Kaplan–Meier method and were compared via the log-rank test. The cell counting kit-8 (CCK-8) and wound scratch assays were applied to detect the effect of GATA3-AS1 knockdown and overexpression on cell growth and migration of HCC. RT-qPCR was performed for the detection of the phosphatase and tensin homolog (PTEN), cyclin-dependent kinase inhibitor 1A (CDKN1A), and tumor protein p53 (TP53) expression in HCC cells after GATA3-AS1 knockdown and overexpression. Results. GATA3-AS1 was significantly upregulated in HCC tissues compared with matched normal tissues. The high expression of GATA3-AS1 was significantly correlated with larger tumor size, advanced TNM stage, and more lymph node metastasis. High GATA3-AS1 expression was markedly correlated with shorter overall survival times of HCC patients. Furthermore, knockdown of GATA3-AS1 obviously inhibited Hep3B and HCCLM3 cell growth and migration, whereas overexpression of GATA3-AS1 had the opposite effects. In addition, GATA3-AS1 knockdown resulted in upregulated expression levels of tumor-suppressive genes, PTEN, CDKN1A, and TP53, in Hep3B and HCCLM3 cells, while restoration of GATA3-AS1 decreased PTEN, CDKN1A, and TP53 expression levels. Conclusion. Our data suggested that GATA3-AS1 promotes cell proliferation and metastasis of HCC by suppression of PTEN, CDKN1A, and TP53.

Effects and Mechanisms of Anlotinib and Dihydroartemisinin Combination Therapy in Ameliorating Malignant Biological Behavior of Gastric Cancer Cells

2020 ◽  
Vol 21 ◽  
Author(s):  
Qiong Luo ◽  
Suyun Zhang ◽  
Donghuan Zhang ◽  
Rui Feng ◽  
Nan Li ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Molecular Mechanisms ◽  
Chorioallantoic Membrane ◽  
Cell Counting ◽  
Biological Behavior ◽  
Gastric Cancer Cells ◽  
Invasion And Migration ◽  
Apoptosis Related Protein ◽  
And Migration

Background: Gastric cancer(GC) is currently one of the major malignancies that threatens human lives and health. Anlotinib is a novel small-molecule that inhibits angiogenesis to exert anti-tumor effects. However, the function in gastric cancer is incompletely understood. Objective: The aim of the present study was to investigate the anti-tumor effects and molecular mechanisms of anlotinib combined with dihydroartemisinin (DHA) in SGC7901 gastric cancer cells. Method: Different concentrations of anlotinib and DHA were used to treat SGC7901 gastric cancer cells, after which cell proliferation was measured. Drug interactions of anlotinib and DHA were analyzed by the Chou-Talalay method with CompuSyn software. proliferation, apoptosis, invasion, migration, and angiogenesis were measured using the cell counting kit-8 (CCK8) assay, flow cytometry, Transwell invasion assays, scratch assays, and chicken chorioallantoic membrane (CAM) assays. proliferation-associated protein (Ki67), apoptosis-related protein (Bcl-2), and vascular endothelial growth factor A (VEGF-A) were quantified by Western bloting. Results: The combination of 2.5 μmol/L of anlotinib and 5 of μmol/L DHA was highly synergistic in inhibiting cell growth, significantly increased the apoptosis rate and suppressed obviously the invasion and migration capability and angiogenesis of gastric cancer cells. In addition, the expression levels of Ki67, Bcl-2, and VEGF-A, as well as angiogenesis, were significantly decreased in the Combination of drugs compared with in control and either drug alone. Conclusion: The combination of anlotinib and DHA showed synergistic antitumor activity, suggesting their potential in treating patients with gastric cancer.

scholarly journals Classifying gastric cancer using FLORA reveals clinically relevant molecular subtypes and highlights LINC01614 as a biomarker for patient prognosis

Oncogene ◽  
2021 ◽  
Author(s):  
Yiyun Chen ◽  
Wing Yin Cheng ◽  
Hongyu Shi ◽  
Shengshuo Huang ◽  
Huarong Chen ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Noncoding Rna ◽  
Molecular Subtype ◽  
Sequencing Data ◽  
Cell Growth And Migration ◽  
Protein Coding ◽  
Over Expression ◽  
And Migration ◽  
Patient Prognosis ◽  
Subtype 3

AbstractMolecular-based classifications of gastric cancer (GC) were recently proposed, but few of them robustly predict clinical outcomes. While mutation and expression signature of protein-coding genes were used in previous molecular subtyping methods, the noncoding genome in GC remains largely unexplored. Here, we developed the fast long-noncoding RNA analysis (FLORA) method to study RNA sequencing data of GC cases, and prioritized tumor-specific long-noncoding RNAs (lncRNAs) by integrating clinical and multi-omic data. We uncovered 1235 tumor-specific lncRNAs, based on which three subtypes were identified. The lncRNA-based subtype 3 (L3) represented a subgroup of intestinal GC with worse survival, characterized by prevalent TP53 mutations, chromatin instability, hypomethylation, and over-expression of oncogenic lncRNAs. In contrast, the lncRNA-based subtype 1 (L1) has the best survival outcome, while LINC01614 expression further segregated a subgroup of L1 cases with worse survival and increased chance of developing distal metastasis. We demonstrated that LINC01614 over-expression is an independent prognostic factor in L1 and network-based functional prediction implicated its relevance to cell migration. Over-expression and CRISPR-Cas9-guided knockout experiments further validated the functions of LINC01614 in promoting GC cell growth and migration. Altogether, we proposed a lncRNA-based molecular subtype of GC that robustly predicts patient survival and validated LINC01614 as an oncogenic lncRNA that promotes GC proliferation and migration.

Knockout of Ajuba Attenuates the Growth and Migration of Hepatocellular Carcinoma Cells

2020 ◽  
Vol 160 (11-12) ◽  
pp. 650-658
Author(s):  
Yichen Le ◽  
Yi He ◽  
Meirong Bai ◽  
Ying Wang ◽  
Jiaxue Wu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Growth ◽  
Cancer Progression ◽  
Normal Liver ◽  
Cell Growth And Migration ◽  
And Migration ◽  
Hcc Cells

Ajuba has been found to be mutated or aberrantly regulated in several human cancers and plays important roles in cancer progression via different signaling pathways. However, little is known about the role of Ajuba in hepatocellular carcinoma (HCC). Here, we found an upregulation of Ajuba expression in HCC tissues compared with normal liver tissues, while a poor prognosis was observed in HCC patients with high Ajuba expression. Knockout of Ajuba in HCC cells inhibited cell growth in vitro and in vivo, suppressed cell migration, and enhanced the cell apoptosis under stress. Moreover, re-expression of Ajuba in Ajuba-deficient cells could restore the phenotype of Ajuba-deficient cells. In conclusion, these results indicate that Ajuba is upregulated in HCC and promotes cell growth and migration of HCC cells, suggesting that Ajuba could possibly be a new target for HCC diagnosis and treatment.

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