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 microtubules 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 predominantly 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 disassembly 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]
Eribulin mesylate (E7389), a synthetic analog of the marine natural product halichondrin B, is in Phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening. Using [ 3 H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K d of 46 μM, but also showing a real or apparent very high affinity (K d , 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (K d , 3.5 μM), strongly suggesting the presence of a relatively high affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per 2 microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Pre-incubation of microtubules with 2 or 4 μM vinblastine induced additional lower affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability. Keywords eribulin; halichondrin; tubulin; microtubule; binding Eribulin mesylate is a tubulin/microtubule-targeting chemotherapeutic drug that inhibits the proliferation of multiple cancer cell types (1,2). It is a synthetic analog of the natural compound, halichondrin B (Fig. 1A), initially isolated from the sea sponge Halichondria okadai (3). Eribulin is currently in Phase III clinical trials for the treatment of metastatic breast cancer. Phase I and Phase II clinical trials have demonstrated that eribulin is active in heavily pretreated individuals while maintaining a tolerable therapeutic index, with the most frequent adverse effects being neutropenia and fatigue (4-6). Neuropathy, a common dose-limiting toxicity of other microtubule-targeting drugs like paclitaxel and some vinca alkaloids (7-9), has a low incidence in eribulin-treated patients, and no grade 4 neuropathy occurred (4)(5)(6) Eribulin exerts its anticancer properties through a novel action on tubulin and microtubules (1,(10)(11)(12). In MCF7 cells, eribulin inhibited microtubule dynamic instability at low concentrations and induced depolymerization of the microtubule network at high concentrations (10 × IC 50 for inhibition of cell proliferation) (11). At significantly lower eribulin concentrations, eribulin potently inhibited microtubule dynamics, resulting in prolonged mitotic arrest and subsequent apoptosis [for a review of microtubule structure and dynamic instability, see (13,14)]. Eribulin binds at or near the vinca domain, a region that is located at the interface of two tubulin heterodimers when arranged end-to-end and overlaps the ex...
E7389, a macrocyclic ketone analog of the marine natural product halichondrin B, currently is undergoing clinical trials for cancer. This fully synthetic agent exerts its highly potent in vitro and in vivo anticancer effects via tubulin-based antimitotic mechanisms, which are similar or identical to those of parental halichondrin B. In an attempt to understand the impressive potency of E7389 in animal models of human cancer, its ability to induce apoptosis following prolonged mitotic blockage was evaluated. Treatment of U937 human histiocytic lymphoma cells with E7389 led to time-dependent collection of cells in the G 2 -M phase of the cell cycle, beginning as early as 2 h and becoming maximal by 12 h. Increased numbers of hypodiploid events were seen beginning at 12 h, suggesting initiation of apoptosis after prolonged E7389-induced mitotic blockage. The identity of hypodiploid events as apoptotic cells under these conditions was confirmed by two additional morphologic criteria: green to orange/yellow shifts on acridine orange/ethidium bromide staining, and cell surface annexin V binding as assessed by flow cytometry. Several biochemical correlates of apoptosis also were seen following E7389 treatment, including phosphorylation of the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria, proteolytic activation of caspase-3 and -9, and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). In LNCaP human prostate cancer cells, treatment with E7389 also led to generation of hypodiploid cells, activation of caspase-3 and -9, and appearance of cleaved PARP, indicating that E7389 can activate cellular apoptosis pathways under anchorage-independent and -dependent cell culture conditions. These results show that prolonged mitotic blockage by E7389 can lead to apoptotic cell death of human cancer cells in vitro and can provide a mechanistic basis for the significant in vivo anticancer efficacy of E7389.
We have developed an estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line growing in 96-well microtiter plates. Alkaline phosphatase enzyme activity (AlkP) in these cells is markedly stimulated by estrogens, and this enzyme can be easily quantified in situ using a chromogenic substrate. These cells are very sensitive to estrogens; estradiol induces AlkP at levels as low as 10(-12) M. Antiestrogens completely block the action of estradiol. Various estrogens stimulate AlkP with potencies comparable to those achieved in vivo. The induction of AlkP is specific for estrogens; no other type of steroid, including androgens, progestins, mineralocorticoids, or glucocorticoids produce this effect. The stimulation of AlkP in Ishikawa cells is specific for estrogens, is highly reproducible and sensitive, and permits large numbers of samples to be assayed with ease. We have used this assay to investigate the estrogenic action of the adrenal delta 5-3 beta-hydroxysteroids. While pregnenolone is inactive, dehydroepiandrosterone and its sulfate ester induce AlkP slightly. However, the C19 steroid, 5-androstene-3 beta, 17 beta-diol is considerably more estrogenic in this assay, although it stimulates Ishikawa AlkP with a potency of 1/30,000 that of estradiol. The stimulation by 5-androstene-3 beta,17 beta-diol is inhibited by antiestrogens, but it is not blocked by the delta 5-3 beta-hydroxysteroid isomerase/dehydrogenase inhibitor, cyanoketone, or by the aromatase inhibitor, 4-hydroxy-androstenedione. Thus, neither conversion to a delta 4-3-ketone nor aromatization is required for the action of this unusual estrogen.
Eribulin (E7389), a synthetic analogue of halichondrin B in phase III clinical trials for breast cancer, binds to tubulin and microtubules. At low concentrations, it suppresses the growth phase of microtubule dynamic instability in interphase cells, arrests mitosis, and induces apoptosis, suggesting that suppression of spindle microtubule dynamics induces mitotic arrest. To further test this hypothesis, we measured the effects of eribulin on dynamics of centromeres and their attached kinetochore microtubules by time-lapse confocal microscopy in living mitotic U-2 OS human osteosarcoma cells. Green fluorescent proteinlabeled centromere-binding protein B marked centromeres and kinetochore-microtubule plus-ends. In control cells, sister chromatid centromere pairs alternated under tension between increasing and decreasing separation (stretching and relaxing). Eribulin suppressed centromere dynamics at concentrations that arrest mitosis. At 60 nmol/L eribulin (2 Â mitotic IC 50 ), the relaxation rate was suppressed 21%, the time spent paused increased 67%, and dynamicity decreased 35% (but without reduction in mean centromere separation), indicating that eribulin decreased normal microtubule-dependent spindle tension at the kinetochores, preventing the signal for mitotic checkpoint passage. We also examined a more potent, but in tumors less efficacious antiproliferative halichondrin derivative, ER-076349. At 2 Â IC 50 (4 nmol/L), mitotic arrest also occurred in concert with suppressed centromere dynamics. Although media IC 50 values differed 15-fold between the two compounds, the intracellular concentrations were similar, indicating more extensive relative uptake of ER-076349 into cells compared with eribulin. The strong correlation between suppression of kinetochore-microtubule dynamics and mitotic arrest indicates that the primary mechanism by which eribulin blocks mitosis is suppression of spindle microtubule dynamics.
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