Okouneva Tatiana, Azarenko Olga, Wilson Leslie, Littlefield Bruce A, Jordan Mary Ann
Department of Molecular, Cellular, and Developmental Biology and Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, CA 93106, USA.
Mol Cancer Ther. 2008 Jul;7(7):2003-11. doi: 10.1158/1535-7163.MCT-08-0095.
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 protein-labeled 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 x 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 x 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.
艾瑞布林（E7389）是一种正在进行乳腺癌III期临床试验的海兔毒素B的合成类似物，它能与微管蛋白和微管结合。在低浓度时，它抑制间期细胞中微管动态不稳定性的生长阶段，阻止有丝分裂，并诱导细胞凋亡，这表明纺锤体微管动力学的抑制会导致有丝分裂停滞。为了进一步验证这一假设，我们通过延时共聚焦显微镜观察活的有丝分裂U-2 OS人骨肉瘤细胞，测量了艾瑞布林对着丝粒及其附着的动粒微管动力学的影响。绿色荧光蛋白标记的着丝粒结合蛋白B标记着丝粒和动粒微管的正端。在对照细胞中，姐妹染色单体着丝粒对在张力作用下在分离增加和减少（伸展和松弛）之间交替。艾瑞布林在导致有丝分裂停滞的浓度下抑制着丝粒动力学。在60 nmol/L艾瑞布林（2倍有丝分裂IC50）时，松弛率被抑制21%，暂停时间增加67%，动态性降低35%（但平均着丝粒分离没有减少），这表明艾瑞布林降低了动粒处正常的微管依赖性纺锤体张力，阻止了有丝分裂检查点通过的信号。我们还研究了一种更有效的但在肿瘤中抗增殖效果较差的海兔毒素衍生物ER-076349。在2倍IC50（4 nmol/L）时，有丝分裂停滞也与着丝粒动力学的抑制同时发生。尽管两种化合物的培养基IC50值相差15倍，但细胞内浓度相似，这表明与艾瑞布林相比，ER-076349在细胞中的相对摄取更广泛。动粒微管动力学抑制与有丝分裂停滞之间的强相关性表明，艾瑞布林阻断有丝分裂的主要机制是纺锤体微管动力学的抑制。
