Microtubule‐targeting agents impair kinesin‐2‐dependent nuclear transport of β‐catenin: Evidence of inhibition of Wnt/β‐catenin signaling as an important antitumor mechanism of microtubule‐targeting agents

Abstract

An aberrant accumulation of nuclear β-catenin is closely associated with the augmentation of cancer malignancy. In this work, we report that several microtubule-targeting agents (MTAs) such as vinblastine, taxol, and C12 (combretastatin-2-aminoimidazole analog) inhibit Wnt/β-catenin signaling in oral squamous cell carcinoma (OSCC). We showed that the inhibition of microtubule dynamics by MTAs decreased the level of β-catenin by increasing Axin and adenomatous polyposis coli levels and reducing the level of dishevelled. Furthermore, MTAs strongly reduced the localization of β-catenin in the nucleus. The reduction in the level of nuclear β-catenin was neither due to the degradation of β-catenin in the nucleus nor due to an increase in the export of nuclear β-catenin from the nucleus. A motor protein kinesin-2 was found to assist the nuclear transportation of β-catenin. Interestingly, Wnt/β-catenin signaling antagonist treatment synergized with MTAs and the activators of Wnt/βcatenin signaling antagonized with the MTAs. C12 potently suppressed the growth of 4-Nitroquinoline 1-oxide-induced OSCC in the tongue of C57 black 6 mice and also abrogated Wnt/β-catenin signaling pathway in the tumor. Our results provide evidence that the decrease in Wnt/β-catenin signaling is an important antitumor effect of MTAs and the combined use of MTAs with Wnt/β-catenin signaling antagonists could be a promising strategy for cancer chemotherapy. K E Y W O R D S apoptosis, cell cycle, microtubule-targeting agents, oral squamous cell carcinoma, Wnt/β-catenin signaling 2 of 16 | KUMARI et Al.

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

“…Western blot was performed as described previously . Briefly, the cells were collected by trypsinization and centrifugation.…”

“…The combination index (CI) of two drugs was calculated by Chou-Talalay’s method. , Briefly, the median dose of the drugs was calculated by incubating the MDA-MB-231 cells with increasing concentrations of indibulin, colchicine, or vinblastine, followed by sulforhodamine B (SRB) assay . Then, MDA-MB-231 cells were treated with 100 nM indibulin for 2 h before adding colchicine or vinblastine and then incubated for another 36 h. The cells were then processed for SRB assay to calculate the percentage inhibition of cell proliferation.…”

“…Then, MDA-MB-231 cells were treated with 100 nM indibulin for 2 h before adding colchicine or vinblastine and then incubated for another 36 h. The cells were then processed for SRB assay to calculate the percentage inhibition of cell proliferation. The CI was calculated from the values of percentage inhibition of cell proliferation in different combinations of drugs …”

“…Western blot was performed as described previously. 55 Briefly, the cells were collected by trypsinization and centrifugation. The cells were then lysed using lysis buffer, followed by quantification of proteins in lysate by Bradford's assay.…”

“…The CI was calculated from the values of percentage inhibition of cell proliferation in different combinations of drugs. 55 Statistical Analysis. Data were analyzed using GraphPad Prism version 8.0.2 (GraphPad Software, Inc.).…”