Benzimidazoles induce concurrent apoptosis and pyroptosis of human glioblastoma cells via arresting cell cycle

Abstract

Glioblastoma multiforme (GBM) is the most malignant and lethal primary brain tumor in adults accounting for about 50% of all gliomas. The only treatment available for GBM is the drug temozolomide, which unfortunately has frequent drug resistance issue. By analyzing the hub genes of GBM via weighted gene co-expression network analysis (WGCNA) of the cancer genome atlas (TCGA) dataset, and using the connectivity map (CMAP) platform for drug repurposing, we found that multiple azole compounds had potential anti-GBM activity. When their anti-GBM activity was examined, however, only three benzimidazole compounds, i.e. flubendazole, mebendazole and fenbendazole, potently and dose-dependently inhibited proliferation of U87 and U251 cells with IC₅₀ values below 0.26 μM. Benzimidazoles (0.125-0.5 μM) dose-dependently suppressed DNA synthesis, cell migration and invasion, and regulated the expression of key epithelial-mesenchymal transition (EMT) markers in U87 and U251 cells. Benzimidazoles treatment also dose-dependently induced the GBM cell cycle arrest at the G₂/M phase via the P53/P21/cyclin B1 pathway. Furthermore, the drugs triggered pyroptosis of GBM cells through the NF-κB/NLRP3/GSDMD pathway, and might also concurrently induced mitochondria-dependent apoptosis. In a nude mouse U87 cell xenograft model, administration of flubendazole (12.5, 25, and 50 mg · kg^-1 · d^-1, i.p, for 3 weeks) dose-dependently suppressed the tumor growth without obvious adverse effects. Taken together, our results demonstrated that benzimidazoles might be promising candidates for the treatment of GBM.

Keywords: apoptosis; benzimidazoles; cell cycle arrest; drug repurposing; glioblastoma; pyroptosis.

Fig. 1. Drug repurposing based on hub gene identification by WGCNA analysis on the CMAP platform.

a Volcano map of differentially expressed genes from analysis of GBM transcriptome data in TCGA. b Biological pathways and cellular component enrichment of the differential genes. c Cluster module of the WGCNA of GBM transcriptome data. d Relationship between the WGCNA module and clinical characteristics of GBM patients. e Hub genes from the module genes of WGCNA identified with the CytoHubba package. f Protein–protein interactions between the hub genes

Fig. 2. Benzimidazoles inhibited proliferation of GBM cells in a dose- and time-dependent manner.

a Structures of flubendazole, mebendazole and fenbendazole. Benzimidazoles’ effects on morphology of U87 cells after 24 h (b), and on morphology of U251 cells at 24 h (c). IC₅₀ values (CCK-8 assay) of benzimidazoles on U87 cells (d), and on U251 cells (e). Benzimidazoles inhibited DNA synthesis (EdU assay) in U87 cells (f) and in U251 cells (g) at 24 h. Scale bar = 100 μm

Fig. 3. Benzimidazoles inhibited migration and invasion of GBM cells in a dose-dependent manner.

Benzimidazoles inhibited the migration of U87 cells (a) and the invasion of U87 cells (b) at 24 h. Benzimidazoles inhibited the migration of U251 cells (c) and the invasion of U251 cells (d) at 24 h. Expression of EMT-related markers after flubendazole treatment in U87 cells (e) and in U251 cells (f) at 24 h

Fig. 4. Benzimidazoles arrested GBM cell cycle at G₂/M phase in a dose-dependent manner.

Benzimidazoles arrested the U87 cell cycle at G₂/M phase (a) and the U251 cell cycle at G₂/M phase (b). Flubendazole arrested the G₂/M cell cycle of U87 cells via the P53/P21/cyclin B1 pathway (c) and the G₂/M cell cycle of U251 cells via the P53/P21/cyclin B1 pathway (d)

Fig. 5. Benzimidazoles triggered pyroptosis in GBM cells.

U87 cells (a) and U251 cells (b) were treated with benzimidazoles and imaged. Arrows indicate ballooned cell membranes characteristic of pyroptotic cells (scale bar = 50 μm). (I) control, (II) flubendazole (0.5 μmol/L, 24 h), (III) mebendazole (0.5 μmol/L, 24 h), and (IV) fenbendazole (0.5 μmol/L, 24 h). Benzimidazoles induced release of LDH from U87 cells in a dose- and time-dependent manner (c) and from U251 cells (d). e, f Benzimidazoles triggered pyroptosis in GBM cells via the NF-κB/NLRP3/GSDMD pathway. The percentage of annexin V-PI double-positive cells (pyroptotic) is labeled in red. g Flubendazole significantly promoted the nuclear translocation of NF-κB. *P < 0.05, **P < 0.01, ***P < 0.001 vs control

Fig. 6. Benzimidazoles induced mitochondria-dependent apoptosis of GBM cells.

Flow cytometry using JC-1 staining showed that benzimidazoles reduced mitochondrial membrane potential in U87 cells (a) and U251 cells (b). Effect of flubendazole on expression of Bcl-2 family proteins in U87 cells (c) and U251 cells (d). Effect of flubendazole on the expression of caspase family proteins in U87 cells (e) and in U251 cells (f). **P < 0.01, ***P < 0.001 vs Control

Fig. 7. The effects of benzimidazoles were blocked by Z-VAD-FMK.

Flow cytometry analysis of benzimidazole-treated U87 cells stained with annexin V-FITC and PI (a) and U251 cells (b). The CCK-8 assay showed that Z-VAD-FMK prevented flubendazole cytotoxicity in U87 cells (c) and in U251 cells (d). e Z-VAD-FMK reversed flubendazole-mediated changes of cell morphology in U87 and U251 cells. f Z-VAD-FMK blocked flubendazole-induced apoptosis and pyroptosis in U87 and U251 cells. *P < 0.05, **P < 0.01 vs -Z-VAD-FMK

Fig. 8. Flubendazole suppressed the growth of GBM xenograft tumors.

a Schematic of xenograft tumor experiment for the antitumor effects of flubendazole in vivo. b Images of xenograft tumors. c Changes in tumor volumes during the flubendazole administration period. d Tumor weights at the end of the experiment. e Changes in body weight during the flubendazole administration period. f Relative organ weights at the end of the experiment. The data were presented as mean ± SD with **P < 0.01, ***P < 0.001 vs. vehicle group

Fig. 9. Schematic model for the mechanism of action of benzimidazoles.

Benzimidazoles induced concurrent apoptotic and pyroptotic cell death of glioblastoma cells by arresting the cell cycle