Ansamitocin P3 Depolymerizes Microtubules and Induces Apoptosis by Binding to Tubulin at the Vinblastine Site

Tilak Kumar Gupta,Ambarish Kunwar,Dulal Panda,Lukasz Kurgan,Paul J Verma,Jubina B Venghateri

doi:10.1371/journal.pone.0075182

Abstract

Maytansinoid conjugates are currently under different phases of clinical trials and have been showing promising activity for various types of cancers. In this study, we have elucidated the mechanism of action of ansamitocin P3, a structural analogue of maytansine for its anticancer activity. Ansamitocin P3 potently inhibited the proliferation of MCF-7, HeLa, EMT-6/AR1 and MDA-MB-231 cells in culture with a half-maximal inhibitory concentration of 20±3, 50±0.5, 140±17, and 150±1.1 pM, respectively. Ansamitocin P3 strongly depolymerized both interphase and mitotic microtubules and perturbed chromosome segregation at its proliferation inhibitory concentration range. Treatment of ansamitocin P3 activated spindle checkpoint surveillance proteins, Mad2 and BubR1 and blocked the cells in mitotic phase of the cell cycle. Subsequently, cells underwent apoptosis via p53 mediated apoptotic pathway. Further, ansamitocin P3 was found to bind to purified tubulin in vitro with a dissociation constant (Kd) of 1.3±0.7 µM. The binding of ansamitocin P3 induced conformational changes in tubulin. A docking analysis suggested that ansamitocin P3 may bind partially to vinblastine binding site on tubulin in two different positions. The analysis indicated that the binding of ansamitocin P3 to tubulin is stabilized by hydrogen bonds. In addition, weak interactions such as halogen-oxygen interactions may also contribute to the binding of ansamitocin P3 to tubulin. The study provided a significant insight in understanding the antiproliferative mechanism of action of ansamitocin P3.

Highlights

Potent cytotoxic agents like maytansine (Fig.1) are entering clinical trials with significant improvements mainly by conjugation with tumor specific antibodies [1,2]
Flow cytometric analysis of propidium iodide (PI)-stained cells suggested that ansamitocin P3 inhibited the cell cycle progression of MCF-7 cells in G2/M phase (Fig. 2B)
The results suggested that ansamitocin P3 treatment blocked cells in mitosis (Figure 2C)

Summary

Introduction

Potent cytotoxic agents like maytansine (Fig.1) are entering clinical trials with significant improvements mainly by conjugation with tumor specific antibodies [1,2]. Recent clinical trials of maytansine conjugated antibodies namely, trastuzumab emtansine (under Phase III), SAR3419 and BT062 (under Phase II), and several others such as BAY 94–9343, BIIB015, IMGN529, lorvotuzumab mertansine, SAR566658, IMGN529 under Phase I clinical trial have strengthened the hope of targeting tumor cells with highly potent cytotoxic agents that were previously withdrawn from cancer chemotherapeutic regimen [1]. Current progress in antibody drug conjugate mediated anti-cancer therapy has revived the interest in maytansine. Recent studies have shown that antibody maytansinoid conjugates block the cells at mitosis by suppressing the microtubule dynamics similar to the results obtained involving the unconjugated maytansine [9,10]

Methods

Results

Conclusion