2-Methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules

Abstract

2-Methoxyestradiol (2ME2), a metabolite of estradiol-17B, is a novel antimitotic and antiangiogenic drug candidate in phase I and II clinical trials for the treatment of a broad range of tumor types. 2ME2 binds to tubulin at or near the colchicine site and inhibits the polymerization of tubulin in vitro, suggesting that it may work by interfering with normal microtubule function. However, the role of microtubule depolymerization in its antitumor mechanism of action has been controversial. To determine the mechanism by which 2ME2 induces mitotic arrest, we analyzed its effects on microtubule polymerization in vitro and its effects on dynamic instability both in vitro and in living MCF7 cells. In vitro, 2ME2 (5 -100 Mmol/L) inhibited assembly of purified tubulin in a concentration-dependent manner, with maximal inhibition (60%) at 200 Mmol/L 2ME2. However, with microtubule-associated protein -containing microtubules, significantly higher 2ME2 concentrations were required to depolymerize microtubules, and polymer mass was reduced by only 13% at 500 Mmol/L 2ME2. In vitro, dynamic instability was inhibited at lower concentrations. Specifically, 4 Mmol/L 2ME2 reduced the mean growth rate by 17% and dynamicity by 27%. In living interphase MCF7 cells at the IC 50 for mitotic arrest (1.2 Mmol/L), 2ME2 significantly suppressed the mean microtubule growth rate, duration and length, and the overall dynamicity, consistent with its effects in vitro , and without any observable depolymerization of microtubules. Taken together, the results suggest that the major mechanism of mitotic arrest at the lowest effective concentrations of 2ME2 is suppression of microtubule dynamics rather than microtubule depolymerization per se.

Structure Activity Analysis of 2-Methoxyestradiol Analogues Reveals Targeting of Microtubules as the Major Mechanism of Antiproliferative and Proapoptotic Activity

“…At low therapeutic concentrations, 2ME2 inhibits microtubule dynamics, which leads to inhibition of the formation of the mitotic spindle apparatus, and consequently, to the arrest of cells in metaphase and induction of apoptosis (4,5,7). Therefore, the accumulation of cells in metaphase was measured by determining the mitotic index, measured as the percentage of cells with chromatin condensation.…”

“…2ME2 has been shown to inhibit the polymerization of tubulin in vitro, thus disrupting normal microtubule function (3). However, at therapeutic doses in vivo, the drug is likely to act by impairing microtubule dynamics (4,5). Given that rapid microtubule dynamics are essential for the correct assembly and function of the mitotic spindle, 2ME2 activates the spindle assembly checkpoint and causes metaphase arrest, and as a consequence, inhibition of cell proliferation and induction of cellular apoptosis (6,7).…”

Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

“…In contrast, at low concentrations, 2ME2-induced mitotic block involved kinetic stabilization of microtubule dynamics rather than alteration of microtubule polymerization (10,11). Recently, Kamath et al (12) showed that, at low concentrations of 2ME2, mitotic cell arrest was due to suppression of microtubule dynamics and not depolymerization of microtubules. However, the binding site of 2ME2 on h-tubulin is far from clear.…”

Novel in silico-designed estradiol analogues are cytotoxic to a multidrug-resistant cell line at nanomolar concentrations

“…It has undergone phase I and phase II clinical trials as an anti-mitotic and anti-angiogenic agent in various cancer types [3]. Kamath et al [4] demonstrated that 2ME suppressed microtubule dynamics at low concentrations, while higher doses resulted in microtubule Abstract Purpose 2-Methoxyestradiol (2ME) is a promising anticancer agent that disrupts the integrity and dynamics of the spindle network. In order to overcome the pharmacokinetic constraints of this compound, a panel of sulphamoylated estradiol analogues were in silico-designed by our laboratory.…”