Induction of morphological and biochemical apoptosis following prolonged mitotic blockage by halichondrin B macrocyclic ketone analog E7389
Induction of morphological and biochemical apoptosis following prolonged mitotic blockage by halichondrin B macrocyclic ketone analog E7389
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DOI：
10.1158/0008-5472.can-04-1169
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发表时间：
2004-08-15
期刊：
影响因子：
11.2
通讯作者：
Littlefield, BA
中科院分区：
文献类型：
--
作者：
Kuznetsov, G;Towle, MJ;Littlefield, BA
E7389是海洋天然产物软海绵素B的大环酮类似物，目前正在进行癌症临床试验。这种完全合成的药物通过基于微管蛋白的抗有丝分裂机制发挥其高度有效的体外和体内抗癌作用，这些机制与这些机制相似或相同。亲本软海绵素B。为了了解E7389在人类癌症动物模型中令人印象深刻的效力，评估了其在长时间有丝分裂阻断后诱导细胞凋亡的能力。用E7389处理U937人组织细胞性淋巴瘤细胞导致细胞周期的G（2）-M期细胞的时间依赖性收集，早在2小时开始，到12小时达到最大。亚二倍体事件的数量增加，看到在12小时开始，这表明启动细胞凋亡后，延长E7389诱导的有丝分裂阻滞。通过两个额外的形态学标准确认在这些条件下亚二倍体事件作为凋亡细胞的身份：吖啶橙子/溴化乙锭染色上的绿色至橙子/黄色移位，以及通过流式细胞术评估的细胞表面膜联蛋白V结合。在E7389处理后还观察到细胞凋亡的几种生化相关性，包括抗凋亡蛋白Bcl-2的磷酸化、线粒体释放细胞色素c、半胱天冬酶-3和-9的蛋白水解活化以及半胱天冬酶-3底物聚（ADP-核糖）聚合酶（PARP）的裂解。在LNCaP人前列腺癌细胞中，用E7389处理也导致亚二倍体细胞的产生、半胱天冬酶-3和-9的活化以及裂解的PARP的出现，表明E7389可以在锚定非依赖性和依赖性细胞培养条件下激活细胞凋亡途径。这些结果表明，E7389延长的有丝分裂阻断可导致体外人癌细胞的凋亡性细胞死亡，并可为E7389的显著体内抗癌功效提供机制基础。
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.