Anti-tumor effects of perampanel in malignant glioma cells

Abstract

Glioblastoma has a poor prognosis even after multimodal treatment, such as surgery, chemotherapy and radiation therapy. Patients with glioblastoma frequently develop epileptic seizures during the clinical course of the disease and often require antiepileptic drugs. Therefore, agents with both antiepileptic and antitumoral effects may be very useful for glioblastoma treatment. Perampanel, an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonist, is an antiepileptic drug that is widely used for intractable epilepsy. The present study aimed to assess the potential antitumoral effects of perampanel using malignant glioma cell lines. The cell proliferation inhibitory effect was evaluated using six malignant glioma cell lines (A-172, AM-38, T98G, U-138MG, U-251MG and YH-13). A dose-dependent inhibitory effect of perampanel on cell viability was demonstrated; however, the sensitivity of cells to perampanel varied and further antitumoral effects were demonstrated in combination with temozolomide (TMZ) in certain malignant glioma cells. Furthermore, cell cycle distribution and apoptosis induction analyses were performed in T98G and U-251MG cells using a fluorescence activated cell sorter (FACS) and the expression levels of apoptosis-related proteins were evaluated using western blotting. No significant change was demonstrated in the proportions of cells in the G0/G1, S and G2/M phases under 1.0 µM perampanel treatment, whereas induction of apoptosis was demonstrated using FACS at 10 µM perampanel and western blotting at 1.0 µM perampanel in both glioma cell lines. Overexpression of SERPINE1 may be related to poor prognosis in patients with gliomas. The combination of 1.0 µM perampanel and 5.0 µM tiplaxtinin, a SERPINE1 inhibitor, demonstrated further reduced cell viability in perampanel-resistant U-138MG cells, which have high expression levels of SERPINE1. These results indicated that the antitumor effect of perampanel may not be expected for malignant gliomas with higher expression levels of SERPINE1. The findings of the present study suggested that the antiepileptic drug perampanel may also have an antitumor effect through the induction of apoptosis, which is increased when combined with TMZ in certain malignant glioma cells. These findings also suggested that SERPINE1 expression may be involved in perampanel susceptibility. These results may lead to new therapeutic strategies for malignant glioma.

Keywords: antiepileptic drug; glioblastoma; perampanel; temozolomide; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid.

Figure 1.

Inhibitory effect of perampanel on cell viability in six human glioma cell lines. Perampanel exhibited inhibitory effects on cell viability in a dose-dependent manner against all cell lines at 72 h. Significantly reduced cell numbers were observed in five cell lines, all except U-138MG, from treatment with 1.0 µM perampanel, which is the blood concentration achieved by oral administration as an antiepileptic drug. The IC₅₀ of perampanel was ≤10 µM for U-251MG and >10 µM for the other five cell lines. Data are presented as the mean ± SEM. ^*P<0.05 vs. 0 µM.

Figure 2.

Cell cycle distribution analysis of T98G and U-251MG cells treated with perampanel. Untreated cells without perampanel served as the control. Fluorescence activated cell sorter data did not demonstrate any significant increase in the populations of cells in the G0/G1, S or G2/M phases following 1.0 µM perampanel treatment for 6, 24 and 48 h compared with the respective control. The results are presented as the mean ± SEM.

Figure 3.

Induction of apoptosis by perampanel in T98G and U-251MG cells assessed using a fluorescence activated cell sorter. Proportions of apoptotic cells (Annexin V-Alexa Fluor 488-positive, early-stage apoptosis; Annexin V-Alexa Fluor 488-positive/PI-positive, late-stage apoptosis) were increased after 48 h of treatment with 10 µM perampanel. The results are presented as the mean ± SEM. ^*P<0.05 vs. 0 µM. PI, propidium iodide.

Figure 4.

Western blotting of apoptosis-associated proteins in T98G and U-251MG cells. The protein expression levels of cleaved caspase-3 were markedly greater after treatment with 1.0 µM perampanel for 24 h compared to the control in both T98G and U-251MG glioma cells.

Figure 5.

Inhibitory effect of a combination of perampanel and TMZ on cell viability in six glioma cell lines. Cells were cultured in media containing perampanel (0, 0.01, 0.1, 1, 10 and 100 µM) with or without 10 µM TMZ for 72 h. The combination of TMZ and perampanel demonstrated significant additive inhibitory effects on cell viability in the A-172, T98G and U-251MG cell lines, but not in the AM-38, U-138MG and YH-13 cell lines. Data are presented as the mean ± SEM. ^*P<0.05 vs. respective perampanel without TMZ. TMZ, temozolomide.

Figure 6.

Inhibitory effect of a combination of perampanel and tiplaxtinin on cell viability in U-138MG cells. U-138MG cells were exposed to media containing 1.0 µM perampanel, 5.0 µM tiplaxtinin (an inhibitor of SERPINE1) or both perampanel and tiplaxtinin for 72 h. The combination of perampanel and tiplaxtinin demonstrated a significant additive inhibitory effect on cell viability, whereas perampanel or tiplaxtinin treatment alone demonstrated no inhibitory effect on viability. Data are presented as the mean ± SEM. ^*P<0.05. n.s., not significant.