2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.

Abstract

A metabolite of estradiol, 2-methoxyestradiol (2ME), inhibits angiogenesis In the chicken embryo chorioallantoic membrane assay. Since 2ME causes mitotic perturbalions, we ea ined Its interactions with tubulin. In our standard 1.0 M glutamate system (plus 1.0 mM MgCl2 at 3rC), superstoichiometric concentrations (relative to tubulin) of 2ME inhibited the nucleation and propagation phases of tubulin assembly but did not affect the reaction extent. Although polymer formed In the presence of 2ME was more cold-stable than control polymer, morphology was little changed. Under suboptimal reaction conditions (0.8 M glutamate/no MgCl2 at 26WC), substoichiometric 2ME totally inhibited polymerization. No other estrogenic compound was as effective as 2ME as an inhibitor of polymerization or of the binding of colchicine to tubulin. Inhibition ofcolchicine binding was competitive (KM,22 aM). Thus, a m an metabolite of estradiol binds to the colchicine site of tubulin and, depending on reaction conditions, either inhibits assembly or seems to be incorporated into a polymer with altered stability properties.There is growing evidence that estrogenic compounds affect cell division and act directly on microtubules by interacting with tubulin. The most extensively studied of these compounds is the synthetic analog diethylstilbestrol (DES; Fig. 1, compound 1) and related agents (1-3). These drugs cause disturbances in mitosis, inhibit microtubule assembly at relatively high concentrations, and inhibit the binding of colchicine to tubulin. Estradiol (compound 2), the major estrogenic hormone of human beings, also causes disturbances ofmitosis in cultured cells (1,4,5). These perturbations include aneuploidy, multinucleation, and mitotic arrest, and estradiol has been reported to inhibit the polymerization of rat brain tubulin (6). Seegers et al. (5) found that 2-methoxyestradiol (2ME; compound 3), a metabolite of both estradiol (7) and the oral contraceptive agent 17-ethynylestradiol (compound 4), was more potent than estradiol in producing mitotic perturbations and proposed that it was 2ME rather than estradiol that caused the observed disturbances. Although 2-methoxyestrogens are extremely weak in binding to cytosol estrogen receptors (8), 2ME is found in blood and urine after sequential hepatic hydroxylation and methylation ofestradiol (7).We recently found that 2ME inhibited angiogenesis in the chicken embryo chorioallantoic membrane assay of Crum et al. (9). Disks of2ME (100 Mg) produced large avascular zones 48 hr after implantation on a 6-day embryo, similar to in vitro results of others (10). In attempting to define the mechanistic basis, we observed that 2ME inhibited microtubule assembly. This finding led us to study the interactions of 2ME with purified tubulin.MATERIALS AND METHODS Materials. Purified bovine brain tubulin and H2-CSA4 were prepared as described (11,12). Monosodium glutamate (2.0 M) was adjusted to pH 6.6 with HCl. 2ME, 2-fluoroestradiol, 2-methoxy-17-ethynylestradiol, 2-methoxyestradiol 3-0-meth...

In vitro and in vivo antitumor effect of 2‐methoxyestradiol on human melanoma

“…Several reports demonstrated that 2ME 2 inhibits tubulin polymerization, 22,25 but generally at much higher concentrations than necessary for growth arrest. 23 Others have found stabilization of microtubules, a mechanism resembling that of other compounds affecting microtubule dynamics, like Taxol.…”

“…Its antiproliferative activity has been attributed to the disruption of microtubule function through its effects on tubulin polymerization or depolymerization, or altering microtubule stability. [22][23][24][25] It has also been suggested that 2ME 2 regulates apoptosis by influencing caspase activation, upregulation of the death receptor 5 protein or p53, phosphorylation and inactivation of Bcl-2, or increasing superoxide production. 10,11,16,24,26 -29 In the case of human melanoma, the in vitro apoptosis-inducing effect of 2ME 2 has been reported on 1 cell line, 29 while its in vivo antitumor or antimetastatic effect remained to be determined.…”

In vitro effects of 2‐methoxyestradiol on MCF‐12A and MCF‐7 cell growth, morphology and mitotic spindle formation

“…4,6,11,17,18 Thus, the effects of 2ME was investigated on mitotic spindle formation by means of immunofluorescent detection of ß-tubulin.…”

“…Several possible mechanisms have been suggested including the upregulation of death receptor 5, 13 induction of p53-dependent or p53-independent apoptosis, 4,14-16 interaction with tubulin resulting in faulty spindle formation and cell cycle arrest, 4,6,11,17,18 upregulation of cyclindependent kinase 2 (Cdk2) activity, 19 overexpression of cyclin B1 20 and increased cell division µg/ml insulin and 500 ng/ml hydrocortisone, while the MCF-7 cells were grown in DMEM. The growth media were supplemented with 10% heat-inactivated FCS, penicillin (100 µg/l), streptomycin (100 µg/l) and fungizone (250 µg/l).…”

Inhibition of MDA-MB-231 cell cycle progression and cell proliferation by C-2-substituted oestradiolmono- andbis-3-O-sulphamates

“…[12][13][14] Although the exact mechanism by which 2-MeOE2 induces its antiangiogenic and antitumour effects is not known, several cellular responses, such as G2-M cell cycle arrest, caspase-3 activation, BCL-2 phosphorylation, increased expression of FAS and p53, downregulation of HIF-1, mitochondrial release of cytochrome c, and inhibition of tubulin polymerisation, have been reported. 4,5,9,10,[15][16][17][18][19][20][21] However, it has been shown that its antimitotic effects are mediated via inhibition of tubulin polymerisation by binding to the colchicine binding site on tubulin. 19,20 Because of the antiproliferative properties associated with 2-MeOE2, several analogues of 2-MeOE2 have been synthesised and tested for various biological activities.…”