Glioblastoma multiforme (GBM) is one of the most aggressive primary brain tumors in adults with insufficient current treatment options and very poor prognosis. Aggressive behavior of GBM cells and their therapeutic resistance are based on complex biological features including changes in the microtubule cytoskeleton. Microtubule targeting belongs to one of the fundamental approaches to cancer treatment, and previously described overexpression of certain tubulin isoforms suggests possible benefit in the use of microtubule targeting agents, such as the repurposed compound flubendazole (FLU). This benzimidazole was previously shown to inhibit a range of solid tumor and leukemic cells with described effect on tubulin structure and polymerization. The aim of this study was to investigate the effect of FLU on microtubule polymerization in two GBM cell lines (A172 and T98G) and to examine the changes in microtubule appearance and posttranslational modifications after FLU treatment.
In FLU-exposed cells growing microtubule ends were detected via EB1/EB3-specific immunofluorescence staining. The changes in microtubules and their modifications were studied with fluorescent microscopy with subsequent image processing and analysis (ImageJ). The relative quantification of tubulin modifications was performed by Western blotting.
FLU treatment reduced the abundance of EB1/EB3-labeled microtubule ends indicating inhibition of microtubule polymerization in a concentration-dependent manner. FLU also caused significant changes in the organization of microtubules and their posttranslational modifications.
Our results demonstrate that FLU exerts its effect on GBM cells by inhibiting microtubule polymerization, as well as inducing significant changes in the organization of microtubule network. Targeting the cytoskeleton with FLU could offer a strategic advantage for improving the outcome of GBM therapy and should be further investigated.
The authors.
This study was supported by Ministry of Health, Czech Republic, project No. NU20-03-00360.
All authors have declared no conflicts of interest.