The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth Mary Ann Jordan, 1 Kathryn Kamath, 1 Tapas Manna, 1 Tatiana Okouneva, 1 Herbert P. Miller, 1 Celia Davis, 1 Bruce A. Littlefield, 2 and Leslie Wilson 1 1 University of California Santa Barbara, Santa Barbara, California and 2 Eisai Research Institute, Andover, Massachusetts Abstract E7389, which is in phase I and II clinical trials, is a synthetic macrocyclic ketone analogue of the marine sponge natural product halichondrin B. Whereas its mechanism of action has not been fully elucidated, its main target seems to be tubulin and/or the microtubules responsible for the construction and proper function of the mitotic spindle. Like most microtubule-targeted antitumor drugs, it inhibits tumor cell proliferation in association with G 2 -M arrest. It binds to tubulin and inhibits microtubule polymerization. We examined the mechanism of action of E7389 with purified micro- tubules and in living cells and found that, unlike antimitotic drugs including vinblastine and paclitaxel that suppress both the shortening and growth phases of microtubule dynamic instability, E7389 seems to work by an end-poisoning mechanism that results predomi- nantly in inhibition of microtubule growth, but not shortening, in association with sequestration of tubulin into aggregates. In living MCF7 cells at the concentration that half-maximally blocked cell proliferation and mitosis (1 nmol/L), E7389 did not affect the shortening events of microtubule dynamic instability nor the catastrophe or rescue frequencies, but it significantly suppressed the rate and extent of microtubule growth. Vinblastine, but not E7389, inhibited the dilution-induced microtubule disas- sembly rate. The results suggest that, at its lowest effective concentrations, E7389 may suppress mitosis by directly binding to microtubule ends as unliganded E7389 or by competition of E7389-induced tubulin aggregates with unliganded soluble tubulin for addition to growing microtubule ends. The result is formation of abnormal mitotic spindles that cannot pass the metaphase/ anaphase checkpoint. [Mol Cancer Ther 2005;4(7): 1086 – 95] Introduction Halichondrin B (Fig. 1) is a large polyether macrolide found in a number of marine sponges. It was shown to inhibit the proliferation of tumor cells with high potency, and strong evidence indicates that it acts by blocking cell cycle progression at G 2 -M by an action on tubulin or microtubules (1). In vitro , halichondrin B noncompetitively inhibits the binding of vinblastine to tubulin, indicating that it may bind to tubulin in the Vinca binding domain (2), and it inhibits the polymerization of tubulin into microtubules. E7389, currently in phase I and II clinical trials, is a synthetic derivative of halichondrin B that is simpler in structure than the parent compound but retains the potency of halichondrin B (Fig. 1). The complete chemical synthesis of E7389 represents a major advance, eliminating the economic and ecological problems inherent in obtaining ample quantities of potential drugs from natural sources. E7389 strongly inhibits growth of a number of human tumor xenografts in mice, and like halichondrin B, it induces G 2 -M cell cycle arrest, disrupts mitotic spindle organization, and induces apoptosis in tumor cells (3, 4). Recent evidence indicates that E7389 binds to tubulin and inhibits the polymerization of bovine brain tubulin into microtubules at micromolar concentrations (3). However, when compared with other microtubule-targeted classes of drugs, the halichondrins exhibit a unique constellation of effects on the conformation of tubulin as indicated by their effects on tubulin alkylation, on the chemical cross-linking of tubulin, and on the binding to tubulin of the hydrophobic probe, bis- 8-anilinonaphthalene sulfate (5). Such data have led to the idea that the halichondrins interact with tubulin in a manner distinct from that of other microtubule-targeted drugs and, thus, that they might possess unique antitumor activities (3). Whereas the target for the halichondrins in general, and for E7389 in particular, seems to be tubulin and/or microtubules, the way in which these compounds perturb microtubule polymerization and/or dynamics both in cell- free systems and in cells has not been determined. Antimitotic drugs can interact with tubulin and micro- tubules in a large number of distinct ways to disrupt microtubule polymerization and dynamics, and their distinct mechanisms may be important determinants of their specific anticancer activities (6). Microtubules are highly dynamic polymers and their dynamics, which are critically important for many cellular processes, are tightly regulated both spatially and temporally (6, 7). In one form of microtubule dynamics, called ‘‘dynamic instability,’’ the Received 12/21/ 04; revised 4/1/ 05; accepted 5/4/ 05. Grant support: Eisai Research Institute and NIH grants CA 57291 and NS13560. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Mary Ann Jordan, Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA 93106-9610. Phone: 805-893-5317; Fax: 805-893-4724. E-mail: jordan@lifesci.ucsb.edu Copyright C 2005 American Association for Cancer Research. 1086 Mol Cancer Ther 2005;4(7). July 2005 Downloaded from http://aacrjournals.org/mct/article-pdf/4/7/1086/1870674/1086-1095.pdf by guest on 11 June 2022