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...

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.

Sulforaphane (SFN), a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables. SFN arrests mitosis, possibly by affecting spindle microtubule function. A critical property of microtubules is their rapid and time-sensitive growth and shortening dynamics (dynamic instability), and suppression of dynamics by antimitotic anticancer drugs (e.g. taxanes and the vinca alkaloids) is central to the anticancer mechanisms of such drugs. We found that at concentrations that inhibited proliferation and mitosis of MCF7-green fluorescent protein-alpha-tubulin breast tumor cells by approximately 50% (~15 microM), SFN significantly modified microtubule organization in arrested spindles without modulating the spindle microtubule mass, in a manner similar to that of much more powerful antimitotic drugs. By using quantitative fluorescence video microscopy, we determined that at its mitotic concentration required to inhibit mitosis by 50%, SFN suppressed the dynamic instability of the interphase microtubules in these cells, strongly reducing the rate and extent of growth and shortening and decreasing microtubule turnover, without affecting the polymer mass. SFN suppressed the dynamics of purified microtubules in a similar fashion at concentrations well below those required to depolymerize microtubules, indicating that the suppression of dynamic instability by SFN in cells is due to a direct effect on the microtubules. The results indicate that SFN arrests proliferation and mitosis by stabilizing microtubules in a manner weaker than but similar to more powerful clinically used antimitotic anticancer drugs and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for SFN's chemopreventive activity.

Cabazitaxel, a novel chemotherapeutic taxane, is effective against docetaxel-resistant cells and tumors. It is approved for treatment of metastatic hormone-refractory prostate cancer in patients pretreated with docetaxel. Objective responses have been observed in many other cancers, including pretreated metastatic breast cancer. Cabazitaxel and docetaxel share a high degree of structural similarity. The basis for cabazitaxel's efficacy is unclear, and its mechanism has not been described. We compared the effects of cabazitaxel and docetaxel on MCF7 human breast cancer cells expressing fluorescent tubulin. Both drugs inhibited cell proliferation (IC 50s , cabazitaxel, 0.4 AE 0.1 nmol/L, docetaxel, 2.5 AE 0.5 nmol/L) and arrested cells in metaphase by inducing mitotic spindle abnormalities. Drug concentrations required for halfmaximal mitotic arrest at 24 hours were similar (1.9 nmol/L cabazitaxel and 2.2 nmol/L docetaxel). Cabazitaxel suppressed microtubule dynamic instability significantly more potently than docetaxel. In particular, cabazitaxel (2 nmol/L) suppressed the microtubule shortening rate by 59% (compared with 49% for 2 nmol/L docetaxel), the growing rate by 33% (vs. 19%), and overall dynamicity by 83% (vs. 64%). Cabazitaxel was taken up into cells significantly faster than docetaxel, attaining an intracellular concentration of 25 mmol/L within 1 hour, compared with 10 hours for docetaxel. Importantly, after washing, the intracellular cabazitaxel concentration remained high, whereas the docetaxel concentration was significantly reduced. The data indicate that the potency of cabazitaxel in docetaxel-resistant tumors is due to stronger suppression of microtubule dynamics, faster drug uptake, and better intracellular retention than occurs with docetaxel.

Consumption of cruciferous vegetables is associated with reduced risk of various types of cancer. Isothiocyanates including sulforaphane and erucin are believed to be responsible for this activity. Erucin [1-isothiocyanato-4-(methylthio)butane], which is metabolically and structurally related to sulforaphane, is present in large quantities in arugula (Eruca sativa, Mill.), kohlrabi and Chinese cabbage. However, its cancer preventive mechanisms remain poorly understood. We found that erucin inhibits proliferation of MCF7 breast cancer cells (IC50 = 28 µM) in parallel with cell cycle arrest at mitosis (IC50 = 13 µM) and apoptosis, by a mechanism consistent with impairment of microtubule dynamics. Concentrations of 5–15 µM erucin suppressed the dynamic instability of microtubules during interphase in the cells. Most dynamic instability parameters were inhibited, including the rates and extents of growing and shortening, the switching frequencies between growing and shortening, and the overall dynamicity. Much higher erucin concentrations were required to reduce the microtubule polymer mass. In addition, erucin suppressed dynamic instability of microtubules reassembled from purified tubulin in similar fashion. The effects of erucin on microtubule dynamics, like those of sulforaphane, are similar qualitatively to those of much more powerful clinically-used microtubule-targeting anticancer drugs, including taxanes and the vinca alkaloids. The results suggest that suppression of microtubule dynamics by erucin and the resulting impairment of critically important microtubule-dependent cell functions such as mitosis, cell migration and microtubule-based transport may be important in its cancer preventive activities.

Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the guanosine-5'-triphosphate composition of the growing and shortening microtubule tips. It is the main objective of this article to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed.

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Eribulin mesylate (Halaven) is a microtubule-targeted anticancer drug used to treat patients with metastatic breast cancer who have previously received a taxane and an anthracycline. It binds at the plus ends of microtubules and has been shown to suppress plus end growth selectively. Because the class III β tubulin isotype is associated with resistance to microtubule targeting drugs, we sought to determine how βIII tubulin might mechanistically influence the effects of eribulin on microtubules. We found that while [(3)H]eribulin bound to bovine brain soluble tubulin depleted of βIII tubulin in a manner similar to that of unfractionated tubulin, it bound to plus ends of microtubules that were depleted of βIII-depleted tubulin with a maximal stoichiometry (20 ± 3 molecules per microtubule) higher than that of unfractionated microtubules (9 ± 2 molecules per microtubule). In addition, eribulin suppressed the dynamic instability behavior of βIII-depleted microtubules more strongly than and in a manner different from that of microtubules containing βIII tubulin. Specifically, with βIII tubulin present in the microtubules, 100 nM eribulin suppressed the growth rate by 32% and marginally reduced the catastrophe frequency (by 17%) but did not modulate the rescue frequency. However, in the absence of βIII tubulin, eribulin not only reduced the growth rate but also strongly reduced the shortening rate (by 43%) and the catastrophe and the rescue frequencies (by 49 and 32%, respectively). Thus, when present in microtubules, βIII tubulin substantially weakens the effects of eribulin.