Eslicarbazepine induces apoptosis and cell cycle arrest in C6 glioma cells in vitro and suppresses tumor growth in an intracranial rat model.

BACKGROUND: Glioblastoma 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. METHODS: To 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. RESULTS: The 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). CONCLUSIONS: Based on these novel findings, ESL has the potential for further experimental and clinical studies in glioblastoma.

CLEC19A overexpression inhibits tumor cell proliferation/migration and promotes apoptosis concomitant suppression of PI3K/AKT/NF-κB signaling pathway in glioblastoma multiforme.

BACKGROUND: GBM is the most frequent malignant primary brain tumor in humans. The CLEC19A is a member of the C-type lectin family, which has a high expression in brain tissue. Herein, we sought to carry out an in-depth analysis to pinpoint the role of CLEC19A expression in GBM. METHODS: To determine the localization of CLEC19A, this protein was detected using Western blot, Immunocytochemistry/Immunofluorescence, and confocal microscopy imaging. CLEC19A expression in glioma cells and tissues was evaluated by qRT-PCR. Cell viability, proliferation, migration, and apoptosis were examined through MTT assay, CFSE assay, colony formation, wound healing assay, transwell test, and flow cytometry respectively after CLEC19A overexpression. The effect of CLEC19A overexpression on the PI3K/AKT/NF-κB signaling pathway was investigated using Western blot. An in vivo experiment substantiated the in vitro results using the glioblastoma rat models. RESULTS: Our in-silico analysis using TCGA data and measuring CLEC19A expression level by qRT-PCR determined significantly lower expression of CLEC19A in human glioma tissues compared to healthy brain tissues. By employment of ICC/IF, confocal microscopy imaging, and Western blot we could show that CLEC19A is plausibly a secreted protein. Results obtained from several in vitro readouts showed that CLEC19A overexpression in U87 and C6 glioma cell lines is associated with the inhibition of cell proliferation, viability, and migration. Further, qRT-PCR and Western blot analysis showed CLEC19A overexpression could reduce the expression levels of PI3K, VEGFα, MMP2, and NF-κB and increase PTEN, TIMP3, RECK, and PDCD4 expression levels in glioma cell lines. Furthermore, flow cytometry results revealed that CLEC19A overexpression was associated with significant cell cycle arrest and promotion of apoptosis in glioma cell lines. Interestingly, using a glioma rat model we could substantiate that CLEC19A overexpression suppresses glioma tumor growth. CONCLUSIONS: To our knowledge, this is the first report providing in-silico, molecular, cellular, and in vivo evidences on the role of CLEC19A as a putative tumor suppressor gene in GBM. These results enhance our understanding of the role of CLEC19A in glioma and warrant further exploration of CLEC19A as a potential therapeutic target for GBM.

In vivo C6 glioma models: an update and a guide toward a more effective preclinical evaluation of potential anti-glioblastoma drugs.

Glioblastoma multiform (GBM) is the most common primary brain tumor with a poor prognosis and few therapeutic choices. In vivo, tumor models are useful for enhancing knowledge of underlying GBM pathology and developing more effective therapies/agents at the preclinical level, as they recapitulate human brain tumors. The C6 glioma cell line has been one of the most widely used cell lines in neuro-oncology research as they produce tumors that share the most similarities with human GBM regarding genetic, invasion, and expansion profiles and characteristics. This review provides an overview of the distinctive features and the different animal models produced by the C6 cell line. We also highlight specific applications of various C6 in vivo models according to the purpose of the study and offer some technical notes for more convenient/repeatable modeling. This work also includes novel findings discovered in our laboratory, which would further enhance the feasibility of the model in preclinical GBM investigations.