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

Abstract

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.

Keywords: Apoptosis; C6 cell; Caspase 3; Cytochrome c; Eslicarbazepine; G2/M cell cycle arrest; Glioblastoma; P53; p21.

Fig. 1

ESL suppressed cell viability, proliferation, colony formation, and migration of the C6 cells. MTT assay, using different concentrations of ESL (0-200 µM), was performed at 48 h (A) and 72 h (B). By increasing ESL concentration, the percentage of the viable C6 cells was significantly decreased at both time points (p < 0.001). The IC₅₀ of ESL was 1.63 and 1.37 µM at 48 h (C) and 72 h (D), respectively. E) Giemsa staining showed the morphology of the C6 cells under different concentrations of ESL (0. 25 and 50 µM). F & G) Soft-agar colony formation assay showed an inverse correlation of colony number with ESL concentration. Increased concentration of ESL indicated a more significant reduction in colony formation of the C6 cells (p < 0.001). H & I) wound-healing assay demonstrated that the treated cells had significantly less migration at the higher concentration of ESL (p < 0.001). Scale bar: 200 μm

Fig. 2

ESL increased the apoptosis rate in the c6 cells. (A & B) C6 cells under various concentrations of ESL (0, 10, 25, and 50 µM) were stained with AO/EB. ESL significantly increased the apoptosis rate at 24 and 48 h (green dots are viable cells and red dots are apoptotic cells). (C & D) The C6 cells treated with ESL (25 µM) were stained by Annexin V-PI and analyzed using flow cytometry. ESL significantly increased the rate of apoptotic cells at 24 h (p < 0.05) and 48 h (p < 0.01) compared to control cells. Moreover, the number of live cells decreased in the C6 cells treated with ESL (24 h, p < 0.001; 48 h, p < 0.001). (E) The expression level of the genes involved in apoptosis was determined at the RNA level in the C6 cells treated with 25 µM of ESL, using qRT-PCR. Bax/Bcl2 ratio, the expression of Cycs, Tp53 (p < 0.05), and Casp3 (p < 0.01) significantly increased in treated cells compared to control cells. (F) The expression of the candidate genes was also measured at the protein level using a Western blot test. The expression of BAX, P53, cleaved CASP3, and CYCS increased while the expression of BCL2 and p-CASP3 decreased in the C6 cells treated with 25 µM of ESL compared to control cells. Scale bar: 200 μm. The original and uncropped blot images are presented in supplementary Figure XA

Fig. 3

ESL modulates the expression of the G2/M cell cycle regulatory proteins. (A) PI staining showed that treatment of the C6 cells with 25 µM ESL arrested the cell cycle in the G2/M phase at 24 and 48 h. (B) qRT-PCR from cells treated with ESL (25 µM for 48 h) were analyzed for the expression of Ccnd1, Cdk1, and Cdkn1a. (C) Western blot of lysates prepared from cells treated with ESL (25 µM for 48 h) was analyzed for the expression of CCNB1, CDK1, and CDKN1A proteins. The expression of p21, as a negative regulator, was increased, while the expression of CDK1 and CCNB1, as positive regulators, was decreased in treated cells compared to control cells. The original and uncropped blot images are presented in supplementary Figure XB

Fig. 4

ESL significantly suppressed tumor growth in rat C6 glioma model. MRI images of rat C6 glioma model and tumor samples in VEH (A and B) and ESL-treated rats (C and D). Body weight differences between the VEH and ESL groups did not show significant differences (E). Analysis of tumor size data using MRI (F) and caliper (G) indicated that tumor growth was significantly suppressed in the ESL group. (H) The tumor weight in the ESL-treated animals was remarkably lower compared to the VEH-treated counterparts