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Rat C6 glioma cell growth is related to glucose transport and metabolism.

ABSTRACT: In order to establish whether growth of glioma cells is associated with glucose transport and metabolism, we investigated expression of the glucose transporter and hexokinase, as well as glucose transport and glucose phosphorylation in rat C6 glioma cells growing at different rates. Rat C6 glioma cells were subcloned to produce four different cell lines (CL1, CL2, CL3 and CL4) differing in growth, differentiation and morphology: CL1 cells were slow-growing with an astrocytic appearance whereas CL4 cells grew rapidly and were small and spindle-shaped. Immunocytochemical analysis using glial fibrillary acidic protein and galactocerebroside antibodies revealed that CL1 and CL4 cells differentiate to astrocytes and oligodendrocytes respectively. Both of these cell lines expressed GLUT1 mRNA predominantly, whereas little GLUT3 mRNA was evident by Northern-blot analysis. The GLUT1 mRNA level was much higher in CL4 than in CL1 cells, and the uptake of 2-deoxy-D-glucose and 3-O-methyl-D-glucose by CL4 cells was markedly higher than that by CL1 cells, indicating a correlation between the growth rate, glucose transporter (GLUT1) level and glucose-transport rate of C6 glioma cells. We then studied glucose metabolism by CL1 and CL4 cells by measuring their hexokinase activities and intracellular concentrations of glucose and ATP. The mitochondrial hexokinase activity of CL4 cells was about three times higher than that of CL1 cells, whereas the cytosolic hexokinase activity of CL4 cells was only about half that of CL1 cells. As the total amount of cellular hexokinase protein in CL4 cells was only slightly higher (about 20%) than that in CL1 cells, the hexokinase protein of CL4 cells was considered to have moved from the cytosol to the mitochondrial membranes. Consistent with the increased mitochondrial hexokinase activity of CL4 cells, the intracellular glucose concentration was undetectable, and the ATP concentration was higher than that of CL1 cells, suggesting that glucose transport is the rate-limiting factor for overall glucose metabolism is rapidly growing C6 cells. Therefore the present data demonstrate that glioma cell growth is related to glucose transport, which is closely associated with glucose metabolism.

SUBMITTER: Nagamatsu S

PROVIDER: S-EPMC1217793 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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Project description:BackgroundGlioblastoma multiforme (GBM) is the most malignant brain tumor, with a poor prognosis and life expectancy of 14-16 months after diagnosis. The standard treatment for GBM consists of surgery, radiotherapy, and chemotherapy with temozolomide. Most patients become resistant to treatment after some time, and the tumor recurs. Therefore, there is a need for new drugs to manage GBM. Eslicarbazepine (ESL) is a well-known antiepileptic drug belonging to the dibenzazepine group with anticancer potentials. In this study, for the first time, we evaluated the potential effects of ESL on C6 cell growth, both in vitro and in vivo, and examined its molecular effects.MethodsTo determine the effect of ESL on the c6 cell line, cell viability, proliferation, and migration were evaluated by MTT assay, colony formation, and wound healing assay. Also, apoptosis and cell cycle were examined by flow cytometry, qRT-PCR, and western blotting. In addition, an intracranial model in Wistar rats was used to investigate the effect of ESL in vivo, and the tumor size was measured using both Caliper and MRI.ResultsThe obtained results are extremely consistent and highly encouraging. C6 cell viability, proliferation, and migration were significantly suppressed in ESL-treated C6 cells (p < 0.001), as determined by cell-based assays. ESL treatment led to significant enhancement of apoptosis (p < 0.01), as determined by flow cytometry, and upregulation of genes involved in cell apoptosis, such as the Bax/Bcl2 ratio at RNA (p < 0.05) and protein levels (5.37-fold). Flow cytometric analysis of ESL-treated cells revealed G2/M phase cell cycle arrest. ESL-treated cells demonstrated 2.49-fold upregulation of p21 alongside, 0.22-fold downregulation of cyclin B1, and 0.34-fold downregulation of cyclin-dependent kinase-1 at the protein level. Administration of ESL (30 mg/kg) to male rats bearing C6 intracranial tumors also suppressed the tumor volume and weight (p < 0.01).ConclusionsBased on these novel findings, ESL has the potential for further experimental and clinical studies in glioblastoma.

Project description:Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.

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Rat C6 glioma cell growth is related to glucose transport and metabolism.

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