Characterization of Microtubule Destabilizing Drugs: A Quantitative Cell-Based Assay That Bridges the Gap between Tubulin Based- and Cytotoxicity Assays

Simple Summary: The characterization of new microtubule depolymerizing agents relies mainly on purified tubulin assays in vitro and on cytotoxicity tests. However, the relationship between the in vitro effects of drugs and their effect on cell viability may not be direct. Here, we have systematically compared the effect of four reference drugs on tubulin polymerization in vitro and in cells, using a recently-developed quantitative assay of the cellular microtubule content. By comparing these results with cell viability assays, we found that this new cellular microtubule content test better predicts cellular drug toxicity than the in vitro tubulin polymerization assay. This test can thus be easily implemented in the process of discovery and characterization of novel microtubule poisons.(1) Background: Microtubule depolymerizing agents (MDAs) are commonly used for cancer treatment. However, the therapeutic use of such microtubule inhibitors is limited by their toxicity and the emergence of resistance. Thus, there is still a sustained effort to develop new MDAs. During the characterization of such agents, mainly through in vitro analyses using purified tubulin and cytotoxicity assays, quantitative comparisons are mandatory. The relationship between the effect of the drugs on purified tubulin and on cell viability are not always direct. (2) Methods: We have recently developed a cell-based assay that quantifies the cellular microtubule content. In this study, we have conducted a systematic comparative analysis of the effect of four well-characterized MDAs on the kinetics of in vitro tubulin assembly, on the cellular microtubule content (using our recently developed assay) and on cell viability. (3) Conclusions: These assays gave complementary results. Additionally, we found that the drugs' effect on in vitro tubulin polymerization is not completely predictive of their relative cytotoxicity. Their effect on the cellular microtubule content, however, is closely related to their effect on cell viability. In conclusion, the assay we have recently developed can bridge the gap between in vitro tubulin assays and cell viability assays.

Automated summary

The study systematically compared the effects of four reference drugs on tubulin polymerization in vitro and in cells, using a new quantitative assay of cellular microtubule content. The new cellular microtubule content test was found to better predict cellular drug toxicity, bridging the gap between in vitro tubulin assays and cell viability assays in the discovery and characterization of novel microtubule poisons.