agar and Glioma

NDRG1 has been reported to exhibit relatively low expression levels in glioma tissues compared with adjacent brain tissues. Additionally, NDRG1 is reported to be a tumor suppressor with the potential to suppress the proliferation, invasion, and migration of cancer cells. However, its exact roles in GBM are still unknown.. Gene Expression Profiling Interactive Analysis (GEPIA) was employed to evaluate the expression level of NDRG1 in GBM. After the introduction of NDRG1, proliferation, analyses of colony formation, migration, and invasion capacities were performed. A luciferase reporter assay was performed to detect the effect of NDRG1 on the vascular endothelial growth factor A (VEGFA) promoter.. In this study, data from GBM and healthy individuals were retrospectively collected by employing GBM, and VEGFA was found to be differentially expressed in GBM tissues compared with adjacent brain tissues. Furthermore, NDRG1 expression is positively correlated with VEGFA expression, but not expression of the other two VEGF isoforms, VEGFB and VEGFC. In the glioma cell lines U87MG and U118, overexpression of NDRG1 significantly upregulated VEGFA. By performing a dual-luciferase reporter assay, it was observed that overexpressed NDRG1 transcriptionally activated VEGFA. Expectedly, overexpression of NDRG1 decreased cell viability by blocking cell cycle phases at G1 phase. Additionally, overexpression of NDRG1 inhibited invasion, colony formation, and tumor formation in soft agar. Remarkably, VEGFA silencing or blockade of VEGF receptor 2 (VEGFR2) further inhibited malignant behaviors in soft agar, including proliferation, invasion, colony formation, and tumor formation.. NDRG1-induced VEGFA exerts protective effects in GBM via the VEGFA/VEGFR2 pathway. Therefore, targeting both NDRG1 and VEGFA may represent a novel therapy for the treatment of GBM.

Topics: Agar; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Intracellular Signaling Peptides and Proteins; Retrospective Studies; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

3D collagen scaffold culture is a good tool to study glioma metastasis and recurrence in vitro.. The effect of 3D collagen culture on the colony formation, the sphere formation, and drug sensitivity of glioma cells was observed by soft-agar colony formation assays, sphere formation assays, and CCK-8 assays, respectively. 3D-glioma-drug genes were identified by previous results and online databases. Gene enrichment and PPI analyses were performed by R software and Metacsape. Hub 3D-glioma-drug genes were screened by STRING and Cytoscape. TCGA and CGGA databases and R software were used to analyze the distribution of hub genes in glioma and their effects on the prognosis. Western Blot was used to verify the effect of 3D collagen culture on the expression of hub genes. miRNAs targeting hub genes were predicted by ENCORI.. 3D collagen scaffold culture promoted colony formation, sphere formation, and drug resistance of glioma cells. There were 77 3D-glioma-drug genes screened, and the pathways enriched in the protein interaction network mainly included responses to stressors, DNA damage and repair, and drug metabolism. Hub 3D-glioma-drug genes were AKT1, ATM, CASP3, CCND1, EGFR, PARP1, and TP53. These genes and predicted miRNAs were expressed differentially in glioma samples and partially affected the prognosis of patients with glioma. These findings suggested these hub genes and miRNAs may play a key role in the effects generated by the 3D culture model and become new markers for glioma diagnosis and treatment.

Topics: Agar; Caspase 3; Collagen; Drug Resistance; ErbB Receptors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Humans; MicroRNAs; Sincalide

The susceptibility gradients generated by super-paramagnetic iron oxide (SPIO) nanoparticles make them an ideal contrast agent in magnetic resonance imaging. Traditional quantification methods for SPIO nanoparticle-based contrast agents rely on either mapping T₂* values within a region or by modeling the magnetic field inhomogeneities generated by the contrast agent. In this study, a new model-based SPIO quantification method is introduced. The proposed method models magnetic field inhomogeneities by approximating regions containing SPIOs as ensembles of magnetic dipoles, referred to as the finite perturber method. The proposed method was verified using data acquired from a phantom and in vivo mouse models. The phantom consisted of an agar solution with four embedded vials, each vial containing known but different concentrations of SPIO nanoparticles. Gaussian noise was also added to the phantom data to test performance of the proposed method. The in vivo dataset was acquired using five mice, each of which was subcutaneously implanted in the flanks with 1 × 10(5) labeled and 1 × 10(6) unlabeled C6 glioma cells. For the phantom data set, the proposed algorithm was generate accurate estimations of the concentration of SPIOs. For the in vivo dataset, the method was able to give estimations of the concentration within SPIO-labeled tumors that are reasonably close to the known concentration.

Topics: Agar; Algorithms; Animals; Ferric Compounds; Glioma; Magnetic Resonance Imaging; Mice; Models, Statistical; Models, Theoretical; Nanoparticles; Phantoms, Imaging; Sensitivity and Specificity

The leucine-rich, glioma-inactivated (LGI1) gene, located in 10q24, was originally identified because it was interrupted and inactivated by a reciprocal chromosome translocation in the T98G glioma cell line. Loss of LGI1 expression in high-grade brain tumors is correlated with the frequent loss of chromosome 10 during progression of gliomas. To investigate whether this gene can suppress the malignant phenotype in glioma cells, we introduced the LGI1 gene into cells that do (U87) and do not (T98G and A172) express LGI1 endogenously. A172 and T98G cells showed a significant reduction in cell proliferation potential as a result of re-expression of LGI1, whereas U87 cells did not. Using BD matrigel matrix chamber assays we were also able to show that the migration ability of the reconstituted A172 and T98G cells was also reduced considerably. Finally, these reconstituted T98G and A172 cells showed a significant reduction in the ability to form colonies in soft agar compared with the parental cells. This analysis clearly demonstrates that re-expression of the LGI1 gene in glioma cells that were null for its activity can greatly reduce their malignant potential. These observations provide the opportunity to investigate the role of LGI1 in gliomagenesis and, since LGI1 is predicted to be a membrane-bound protein, potentially provides the opportunity to develop novel treatment strategies for malignant gliomas.

Topics: Agar; Blotting, Western; Cell Division; Cell Membrane; Chromosomes, Human, Pair 10; Collagen; Drug Combinations; Genetic Vectors; Glioma; Humans; Intracellular Signaling Peptides and Proteins; Laminin; Leucine; Neoplasm Invasiveness; Phenotype; Proteins; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transfection; Tumor Cells, Cultured

Strategies that antagonize growth factor signaling are attractive candidates for the biological therapy of brain tumors. HGF/NK2 is a secreted truncated splicing variant and potential antagonist of scatter factor/hepatocyte growth factor (SF/HGF), a multifunctional cytokine involved in the malignant progression of solid tumors including glioblastoma. U87 human malignant glioma cells that express an autocrine SF/HGF stimulatory loop were transfected with the human HGF/NK2 cDNA and clonal cell lines that secrete high levels of HGF/NK2 protein (U87-NK2) were isolated. The effects of HGF/NK2 gene transfer on the U87 malignant phenotype were examined. HGF/NK2 gene transfer had no effect on 2-dimensional anchorage-dependent cell growth. In contrast, U87-NK2 cell lines were approximately 20-fold less clonogenic in soft agar and approximately 4-fold less migratory than control-transfected cell lines. Intracranial tumor xenografts derived from U87-NK2 cells grew much slower than controls. U87-NK2 tumors were approximately 50-fold smaller than controls at 21 days post-implantation and HGF/NK2 gene transfer resulted in a trend toward diminished tumorigenicity. This report shows that the predominant effect of transgenic HGF/NK2 overexpression by glioma cells that are autocrine for SF/HGF stimulation is to inhibit their malignant phenotype.

Topics: Agar; Alternative Splicing; Animals; Autocrine Communication; Brain Neoplasms; Cell Adhesion; Cell Division; Cell Movement; Cell Transformation, Neoplastic; Gene Transfer Techniques; Glioma; Hepatocyte Growth Factor; Humans; Mice; Mice, SCID; Molecular Weight; Neoplasm Transplantation; Phenotype; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

N-(4-hydroxyphenyl)retinamide (fenretinide) is a synthetic retinoid with anticancer properties. We investigated the effects of fenretinide on the growth of glioma cells. Four glioma cell lines (C6, 9L, Med3 and U87) were treated with fenretinide. Cell viability and independent growth was determined by MTS assay and soft agar assay, respectively. The induction of apoptosis was evaluated by microscopic examination, flow cytometric DNA content analysis, and in situ TdT methods. Fenretinide markedly reduced cell viability of all the glioma cell lines examined at a range of concentrations from 1 to 10 microM. In all cell lines examined, fenretinide also induced morphological changes consistent with apoptosis, including cellular shrinkage, chromatin condensation, and nuclear fragmentation. Flow cytometric analysis also revealed an apoptotic pattern of the DNA content, and in situ detection of apoptosis showed increased incorporation of digoxigenin-nucleotide triphosphate in fenretinide-treated glioma cells. These findings indicate that fenretinide inhibits the growth of glioma cells via the induction of apoptosis, suggesting potential clinical use of fenretinide for treatment of glioma patients.

Topics: Agar; Animals; Antineoplastic Agents; Apoptosis; Drug Screening Assays, Antitumor; Fenretinide; Flow Cytometry; Glioma; Humans; Rats; Tumor Cells, Cultured

The effect of concentrated conditioned medium from each of eight human malignant glioma cell lines on the growth of indicator cells (normal rat kidney fibroblasts (NRK), clone 14) was determined in monolayer and in soft agar assay systems. The conditioned medium from all cell lines was mitogenic in the monolayer assay, but only SF-210, U-343 MG-A, and U-251 MG produced soluble factors that caused NRK cells to grow in soft agar. The soluble growth-promoting factors from these three cell lines were acid- and heat-stable (60 degrees C for 30 minutes) but were inactivated by trypsin (100 microns/ml) and dithiothreitol (50 microM). The growth factors from SF-210 and U-343 MG-A were further purified by molecular-sieve chromatography. The partially purified growth factor from U-343 MG-A retained transforming growth factor (TGF)-like activity, had a molecular weight of 9 kD, was potentiated by TGF-beta in the soft agar assay, competed effectively with 125I-epidermal growth factor (EGF) radiolabeled for the EGF receptor on A 431 epidermoid carcinoma cells, and was completely inhibited by monoclonal antibodies to TGF-alpha. The partially purified growth factor from SF-210 had a molecular weight of 17 kD, was not inhibited by monoclonal antibodies to platelet-derived growth factor (PDGF) or TGF-alpha, and did not bind to a heparin-Sepharose column. These results imply that U-343 MG-A secretes a growth factor with TGF-alpha-like activity, and SF-210 secretes a TGF with neither TGF-alpha nor TGF-beta activity.

Topics: Agar; Animals; Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Culture Media; ErbB Receptors; Fibroblasts; Glioma; Humans; Kidney; Transforming Growth Factors; Tumor Cells, Cultured

The soft agar technique for culturing human clonogenic tumor cells has been usefully applied for predicting individual clinical responses to chemotherapy, for screening of new antineoplastic drugs, and in basic biological research. The counting of colonies formed by clonogenic cells is, however, a rather time consuming and inaccurate procedure. We here report a method to combine the easy and precise registration of DNA-synthesis by 3H-thymidine incorporation with the ability of soft agar to permit proliferation of clonogenic cells and inhibit proliferation of non-neoplastic cells. The glioma cell lines U 251 MG and T-MG 1, the benignant glia cells T-BG 1, T-BG 2, T-BG 3 and fibroblasts were cultured in Furcellaran gel. Twenty hours before harvesting 3H-thymidine was added. The Furcellaran gel was resolved by 50 mM LiI. The cells were trapped on glass fiber filters and incorporated radioactivity was measured. 3H-thymidine incorporation in malignant cells increased exponentially with time, while 3H-thymidine incorporation in the benignant glia cells and fibroblasts was inhibited. The correlation between number of colonies counted after 16 days and 3H-thymidine incorporation registered after different culture times was very good. The correlation was best when the cultures were harvested after 8 days (r = 0.95), indicating that it is possible to reduce the assay time. The five glioma biopsies tested grew well with a mean plating efficiency of 0.4% (range 0.02-1.8%). The most intense proliferation seemed to take place during the first week in culture. The good correlation between 3H-thymidine incorporation on day 7 and colony number on day 14 (r = 0.93), indicate that reduction of assay time is possible also for the glioma biopsies.

Topics: Agar; Brain Neoplasms; Cell Division; Cell Line; Cells, Cultured; Clone Cells; Culture Techniques; DNA Replication; Glioma; Humans; Kinetics; Tritium

Cellular growth interactions were studied between neonatal human lung fibroblasts (NLF-13) and human tumor lines derived from carcinomas of the prostate (PC-3, DU145), bladder (J82), and endometrium (HEC-1A) and from a glioma (Hs 683t). NLF-13 were interacted with tumor cells in soft agar or agarose media using two experimental protocols. In one system, NLF-13 cells were grown as anchored monolayers proliferating under the tumor cell layer. In the second, NLF-13 were embedded directly (nonanchored) into the agar or agarose layer with the tumor cells. The results from both interaction systems were similar for all five tumor lines. Anchored NLF-13 caused a dose-dependent inhibition of tumor growth, whereas nonanchored cells produced a dose-dependent growth stimulation. A time exposure experiment indicated that tumor stimulation and inhibition were biphasic responses to NLF-13. It was concluded that low concentrations of a diffusible NLF-13 product(s) accelerated tumor growth, whereas high concentrations were inhibitory. Further, the production of the active NLF-13 substance(s) was positively correlated with NLF-13 growth rate. Tumor cell inhibition was irreversible after a 5-day exposure to proliferating NLF-13 cells. Another line of normal neonatal human lung fibroblasts (NLF-147) showed inhibitory properties similar to those described for NLF-13. However, preliminary studies with fibroblasts from the skin of a Down's syndrome neonate (DS-172) and from a human kidney tumor (KTF-130) have shown both these fibroblast types to have a reduced ability to inhibit tumor cell cultures (J82) compared to the neonatal lung fibroblasts (NLF-13 and NLF-147).

Topics: Agar; Cell Communication; Cell Division; Cell Line; Female; Fibroblasts; Glioma; Humans; Infant, Newborn; Kinetics; Lung; Male; Prostatic Neoplasms; Urinary Bladder Neoplasms; Uterine Neoplasms

Topics: Agar; Animals; Cell Line; Chick Embryo; Cricetinae; Culture Techniques; Ducks; Glioma; Heart; HeLa Cells; Humans; Hydrogen-Ion Concentration; Immune Sera; Kidney; L Cells; Methylcellulose; Polysaccharides; Rabbits; Rats; Simplexvirus; Skin; Species Specificity; Viral Plaque Assay