Assessment of microtubule stabilizers by semiautomated in vitro microtubule protein polymerization and mitotic block assays

Cancer Chemotherapy and Pharmacology
N M LopesB K Bhuyan

Abstract

Paclitaxel (Taxol) a clinically active anticancer agent, exerts its cytotoxicity by inducing tubulin polymerization, leading to cellular mitotic block. In contrast, other antimitotic drugs, such as colchicine, podophyllotoxin, and vinblastine, act by depolymerizing microtubules. We report here (a) a semiautomated assay which measures the tubulin-polymerizing activity of paclitaxel analogs and (b) a cellular assay to measure the potential of these compounds to block cells in mitosis. The microtubule-polymerizing assay measured the turbidity of bovine brain microtubule protein (MTP) polymerized by the test compound in a 96-well plate. We maximized the sensitivity of this assay by conducting the polymerization reaction at 20 degrees C, at which temperature the baseline reaction, i.e. the basic ability of the untreated MTP control to polymerize, was minimal. At 20 degrees C, the effect of 0.05 microg/ml of paclitaxel on MTP could be detected, whereas at 37 degrees C, > 1 microg/ml of paclitaxel was required to detect a significant effect relative to untreated MTP. We describe the analysis of the complex curves of MTP polymerization with varying concentrations of test compounds. The polymerization of microtubules leads to cells bein...Continue Reading

Citations

Jun 14, 2005·Cancer Investigation·Federico Pellegrini, Daniel R Budman
Nov 10, 2007·Chemical Biology & Drug Design·Marina N SemenovaVictor V Semenov
Dec 4, 2003·Expert Opinion on Pharmacotherapy·Marko B Lens, Tim G Eisen
Jun 3, 2016·Analytical Biochemistry·Veronica M GarciaKevin A Morano
Nov 20, 2018·Chemical Communications : Chem Comm·Akinobu Z SuzukiToshiaki Furuta

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