scholarly journals OS6.4 The hypoxia-induced glioma derived exosome miRNA-199a-3p promotes glioma cells proliferation and increased ischemic injury of the para-tumor neurons by inhibiting mTOR pathway— A pivotal ischemic mechanism in the proliferation and growth of glioma

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii13-iii13
Author(s):  
J Zhao ◽  
J Hu ◽  
R Xie
Keyword(s):  
Cell Lines ◽  
Microarray Analysis ◽  
Culture Medium ◽  
Glioma Cells ◽  
Ischemic Injury ◽  
Signal Pathway ◽  
Mtor Pathway ◽  
C6 Cells ◽  
Mirna Microarray ◽  
Normal Culture

Abstract BACKGROUND Growth of glioma cells can be promoted by hypoxia, but its underlying molecular mechanisms are not clear. Exosomes and miRNAs were reported to play crucial roles in tumor progression. Effects of exosomes and exosomal miRNAs, induced by hypoxia, on glioma cells were still unclear. MATERIAL AND METHODS Glioma samples were analyzed by HE and HIF-1α staining. Image data of these patients were also retrospectively analyzed. HT22 and C6 cell lines were co-cultured in both direct and indirect system. Hypoxia (1% oxygen) and oxygen and glucose deprivation (OGD) were applied to evaluate hypoxia effects on the growth and proliferation of cell lines, and such effects were assessed by C6/HT22 ratio, MTT and LDH assay. Hypoxia-induced glioma derived exosomes (HIGDE) and non-HIGDE (NHIGDE) were isolated and were administrated to normal culture medium to evaluate their effects on cell growing. The target miRNA was selected by performing miRNA microarray analysis. MicroRNA mimics and shRNA were constructed to overexpress or inhibit the microRNA expression. MTOR signal pathway was activated by utilizing phosphatidic acid. The RNAs expression were detected by RT-qPCR and the proteins expression was evaluated by western blotting. RESULTS Para-tumor hypoxia area shared a same region with cytotoxic edema around the glioma lesion and can be easily detected by PET/CT. The density of positive HIF-1αstaining was higher in tumor area than that in para-tumor and normal parenchyma area. In hypoxia direct co-culture system, the cell number ratio of C6/HT22 was significantly higher than that without hypoxia pretreatment; while in hypoxia mono-culture and indirect co-culture systems, the proliferation ability of HT22 was statistical lower than C6. After applying OGD, neuron cells cultured with HIGDE showed a statistical higher LDH release level than with NHIGDE or normal culture medium. The miRNA microarray analysis revealed that miRNA-199a-3p was the highest expressed in HIGDE than in NHIGDE (p < 0.05; Fold Change > 2). Transfected with mimics or shRNA, it was indicated that upregulation of miR-199a-3p aggravated HIGDE-induced OGD injury in HT22 cells. Moreover, we interfered mTOR signal pathway and the expression of HIF-1αin C6 cells. We found that miRNA-199a-3p aggravated HIGDE-neuron cell injury via suppressing mTOR signal pathway, and hypoxia related upregulation of miRNA-199a-3p in HIGDE was induced by the activation of HIF-1α in C6 cells. CONCLUSION The Hypoxia-Induced Glioma Derived Exosome miRNA-199a-3p can be upregulated by the activation of HIF-1α, and is able to promote glioma cells proliferation and increase ischemic injury of the para-tumor neurons via inhibiting mTOR pathway.

Arctigenin upregulates apoptosis through the AKT/MTOR pathway, inhibiting the proliferation of glioma

2019 ◽  
Author(s):  
jiang yongan ◽  
Liu Jia yu ◽  
Hong Wangwang ◽  
Fei Xiaowei ◽  
Liu ru'en
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Glioma Cells ◽  
Mtor Pathway ◽  
Migration And Invasion ◽  
Glioblastoma Cell ◽  
Caspase 9 ◽  
Arctium Lappa ◽  
Dose Dependent ◽  
Glioblastoma Cell Lines

Abstract Arctigenin (ARG) is a natural lignan compound extracted from arctium lappa and has displayed anticancer functions and effective treatments in a variety of cancers.Studies had shown that Arctigenin(ARG) inhibits tumors through the AKT/MTOR pathway and mediates autophagy.However,the role in glioma cellshave not still fully understood.This study was designed to investigate whether Arctigenin(ARG) can mediateAKT/mTOR pathway in glioma to regulate autophagy,and affected glioma cells growth and survival.We found that the dose-dependent downregulation of Arctigenin(ARG),reducing cell proliferation,migration and invasion in two human glioblastoma cell lines (U87, T98G),These phenomena were reversed after the administration of the AKT agonist (SC79). Arctigenin(ARG) also affected other autophagy markers such as p62, LC3B.In addition, the apoptotic molecules cleaved-PARP,caspase-9, and cleaved-caspase3 were also dose-dependently altered.

scholarly journals Hypoxia-Induced Glioma-Derived Exosomal miRNA-199a-3p Promotes Ischemic Injury of Peritumoral Neurons by Inhibiting the mTOR Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Jian-Lan Zhao ◽  
Bo Tan ◽  
Gong Chen ◽  
Xiao-Ming Che ◽  
Zhuo-Ying Du ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Glioma Cells ◽  
Ischemic Injury ◽  
Neuronal Injury ◽  
Mtor Pathway ◽  
Mtor Signaling ◽  
C6 Glioma Cells ◽  
Mtor Signaling Pathway ◽  
Exosomal Mirna

The underlying molecular mechanisms that the hypoxic microenvironment could aggravate neuronal injury are still not clear. In this study, we hypothesized that the exosomes, exosomal miRNAs, and the mTOR signaling pathway might be involved in hypoxic peritumoral neuronal injury in glioma. Multimodal radiological images, HE, and HIF-1α staining of high-grade glioma (HGG) samples revealed that the peritumoral hypoxic area overlapped with the cytotoxic edema region and directly contacted with normal neurons. In either direct or indirect coculture system, hypoxia could promote normal mouse hippocampal neuronal cell (HT22) injury, and the growth of HT22 cells was suppressed by C6 glioma cells under hypoxic condition. For administrating hypoxia-induced glioma-derived exosomes (HIGDE) that could aggravate oxygen-glucose deprivation (OGD)/reperfusion neuronal injury, we identified that exosomes may be the communication medium between glioma cells and peritumoral neurons, and we furtherly found that exosomal miR-199a-3p mediated the OGD/reperfusion neuronal injury process by suppressing the mTOR signaling pathway. Moreover, the upregulation of miRNA-199a-3p in exosomes from glioma cells was induced by hypoxia-related HIF-1α activation. To sum up, hypoxia-induced glioma-derived exosomal miRNA-199a-3p can be upregulated by the activation of HIF-1α and is able to increase the ischemic injury of peritumoral neurons by inhibiting the mTOR pathway.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

2021 ◽  
pp. 096032712110237
Author(s):  
L Zhou ◽  
S Li ◽  
J Sun
Keyword(s):  
Endometrial Cancer ◽  
B Cells ◽  
Western Blot ◽  
Signal Pathway ◽  
Mtor Pathway ◽  
Related Protein ◽  
Western Blot Assay ◽  
Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

scholarly journals α-Conotoxins and α-Cobratoxin Promote, while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells

Marine Drugs ◽  
2021 ◽  
Vol 19 (2) ◽  
pp. 118
Author(s):  
Tatiana I. Terpinskaya ◽  
Alexey V. Osipov ◽  
Elena V. Kryukova ◽  
Denis S. Kudryavtsev ◽  
Nina V. Kopylova ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Glioma Cells ◽  
Nordihydroguaiaretic Acid ◽  
C6 Glioma ◽  
Cyclooxygenase Inhibitors ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Lipoxygenase Inhibitor ◽  
Glioma C6

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, β2 and β4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.

Phorbol Esters Stimulate Phospholipase D Activity in C-6 Glioma Cells

1989 ◽  
Vol 16 (3) ◽  
pp. 257-262
Author(s):  
Lena Gustavsson ◽  
Christofer Lundqvist ◽  
Christer Ailing
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Culture Medium ◽  
Phorbol Esters ◽  
Glioma Cells

The effects of phorbol esters on phospholipase D activity were studied in C-6 glioma cells. The cell lipids were prelabelled with [3H]-glycerol or [14C]-arachidonic acid. Phosphatidylethanol was formed during stimulation with 100nM 12-0-tetradecanoylphorbol-13-acetate (TPA), when ethanol was present in the culture medium. After 30 minutes of stimulation, phosphatidylethanol constituted 2.6% of the [3H]-glycerol-labelled lipids. Stimulating the cells with TPA in the absence of ethanol caused a significant increase in labelled phosphatidic acid. This increase was inhibited by ethanol. The present findings demonstrate that TPA stimulates phospholipase D activity in cultured C-6 glioma cells.

scholarly journals An E3 Ubiquitin Ligase RNF139 Serves as a Tumor-Suppressor in Glioma

2021 ◽  
Author(s):  
Xiaofeng Chen ◽  
Weiping Kuang ◽  
Yong Zhu ◽  
Bin Zhou ◽  
Xiaosong Li ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Glioma Cell ◽  
Protein Modification ◽  
E3 Ubiquitin Ligase ◽  
Ring Finger ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Tissue Samples

AbstractGlioma is highly lethal because of its high malignancy. Ubiquitination, a type of ubiquitin-dependent protein modification, has been reported to play an oncogenic or tumor-suppressive role in glioma development, depending on the targets. Ring finger protein 139 (RNF139) is a membrane-bound E3 ubiquitin ligase serving as a tumor suppressor by ubiquitylation-dependently suppressing cell growth. Herein, we firstly confirmed the abnormal downregulation of RNF139 in glioma tissues and cell lines. In glioma cells, ectopic RNF139 overexpression could inhibit, whereas RNF139 knockdown could aggravate the aggressive behaviors of glioma cells, including hyperproliferation, migration, and invasion. Moreover, in two glioma cell lines, RNF139 overexpression inhibited, whereas RNF139 knockdown enhanced the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and AKT serine/threonine kinase 1 (AKT). In a word, we demonstrate the aberration in RNF139 expression in glioma tissue samples and cell lines. RNF139 serves as a tumor-suppressor in glioma by inhibiting glioma cell proliferation, migration, and invasion and promoting glioma cell apoptosis through regulating PI3K/AKT signaling.

scholarly journals DDRE-03. IDH1-MUTANT GBM CELLS ARE HIGHLY SENSITIVE TO COMBINATION OF KDM6A/B AND HDAC INHIBITORS

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i6-i7
Author(s):  
Alişan Kayabölen ◽  
Gizem Nur Sahin ◽  
Fidan Seker ◽  
Ahmet Cingöz ◽  
Bekir Isik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Mutant Cell ◽  
Glioma Cells ◽  
Epigenetic Therapy ◽  
Low Grade ◽  
Orthotopic Model ◽  
Cycle Arrest ◽  
Mutant Cells

Abstract Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

On the Application of Cultured Neuronal Cell Lines in Neurotoxicological Studies: Cytotoxicity of Acrylamide

1983 ◽  
Vol 11 (3) ◽  
pp. 135-145
Author(s):  
Erik Walum
Keyword(s):  
Cell Lines ◽  
Culture Medium ◽  
Neuroblastoma Cells ◽  
Neuronal Cell ◽  
Leucine Incorporation ◽  
Biochemical Process ◽  
Neurite Retraction ◽  
Mouse Neuroblastoma ◽  
Neuronal Cell Lines

Summary Acrylamide, a well known neurotoxic compound, was used in a first evaluation of cultured mouse neuroblastoma cells as an alternative to animal models for neurotoxicological studies. Hence, the effects of acrylamide on the growth, size, morphology and leucine incorporation of three neuroblastoma (41A3, N18 and N1E115), one neuroblastoma x glioma hybrid (NG108CC15), two glioma (138MG and C6) and two fibroblast (RLF and RMC) cell lines were studied. It was found that the concentration of acrylamide needed to inhibit the growth by 50% in 24 hr was similar in all cell lines, i.e. around 2 x 10-4g/ml culture medium. In the two cell lines, N1E115 and NG108CC15, acrylamide at this concentration caused neurite retraction and at higher concentrations (5 x 10-4g/ml) a decrease in cell viability. In a concentration range of 5 x 10-5 - 5 x 10-4g/ml acrylamide did not affect cell size, or at 2 x 10-4g/ml incorporation of leucine into trichloroacetic acid precipitable material. It is suggested that acrylamide interferes with a biochemical process common to all the tested cells, but of greater importance in differentiated nerve cells than in others. Whether this process is consistent with the in vivo target for the neurotoxic action of acrylamide remains to be unravelled.

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