β-III Tubulin Levels Determine the Neurotoxicity Induced by Colchicine-Site Binding Agent Indibulin

Indibulin, a microtubule-depolymerizing agent, produces minimal neurotoxicity in animals. It is also less cytotoxic toward differentiated neuronal cells than undifferentiated cells. We found that the levels of β-III tubulin, acetylated tubulin, and polyglutamylated tubulin were significantly increased in differentiated neuroblastoma cells (SH-SY5Y). Since neuronal cells express β-tubulin isotypes differently from other cell types, we explored the binding of indibulin to different β-tubulin isotypes. Our molecular docking analysis suggested that indibulin binds to β-III tubulin with lower affinity than to other β-tubulin isotypes. We therefore studied the implications of different β-tubulin isotypes on the cytotoxic effects of indibulin, colchicine, and vinblastine in differentiated SH-SY5Y cells. Upon depletion of β-III tubulin in the differentiated cells, the toxicity of indibulin and colchicine significantly increased, while sensitivity to vinblastine was unaffected. Using biochemical, bioinformatics, and fluorescence spectroscopic techniques, we have identified the binding site of indibulin on tubulin, which had not previously been established. Indibulin inhibited the binding of colchicine and C12 (a colchicine-site binder) to tubulin and also increased the dissociation constant of the interaction between tubulin and colchicine. Indibulin did not inhibit the binding of vinblastine or taxol to tubulin. Interestingly, indibulin antagonized colchicine treatment but synergized with vinblastine treatment in a combination study performed in MDA-MB-231 cells. The results indicate that indibulin is a colchicine-site binder and that the efficacy of colchicine-site binders is affected by the β-III tubulin levels in the cells.