Two Antagonistic Microtubule Targeting Drugs Act Synergistically to Kill Cancer Cells

Abstract

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

The microtubule cytoskeleton: An old validated target for novel therapeutic drugs

“…They found that low non-saturating concentrations of a MT depolymerizing agent, which enhances catastrophes, promote taxane binding to growing MT tips ( Rai et al, 2020 ). In collaboration with this team, we found that such a mechanism is at work with Carba1, thus explaining the observed synergy ( Peronne et al, 2020 ).…”

“…Interestingly, the synergistic effect of Carba1 with PTX on tumor cell viability was also observed in vivo in xenografted mice ( Peronne et al, 2020 ).…”

“…To that aim, we have screened an original collection of several thousand compounds using a cytotoxicity assay and selected a derivative of the carbazolone series (Carba1) able to sensitize cells to a low, non-toxic dose of PTX. While searching for the Carba1 mechanism of action, we were surprised to discover that this compound was able to bind tubulin at the colchicine site and, in vitro , to induce a dose-dependent decrease of the rate of tubulin polymerization ( Peronne et al, 2020 ). How is it, then, that a MT depolymerizing agent synergizes with a MT stabilizing agent?…”

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

“…In addition, some drugs, such as our recently described compound Carba1, with low affinity for tubulin, may show a visible depolymerizing effect on tubulin assembly kinetics, but no detectable effect on the microtubular network of interphase cells [30]. The cell-based assay described here is thus complementary to in vitro assays on purified tubulin.…”

“…The process of characterizing new MTAs is commonly based on the in vitro analysis of their binding to tubulin (binding assays, competition assays with known agents, affinity measurements [24][25][26][27][28]) as well as on the analysis of their effect on the polymerization of tubulin into microtubules (spectrometric monitoring of the kinetic of tubulin assembly [29,30] or, more recently, tracking of the growth of individual microtubules reconstituted in vitro, by time-lapse fluorescence microscopy [31]). The compounds' characterization is then complemented using tests on cells, such as immunofluorescence analysis of the effect of the compounds on the microtubule network, FACS analysis of their ability to interfere with the cell cycle, and finally analysis of their cytotoxicity.…”