Yan Wei, Yang Tao, Yang Jianhong, Wang Taijin, Yu Yamei, Wang Yuxi, Chen Qiang, Bai Peng, Li Dan, Ye Haoyu, Qiu Qiang, Zhou Yongzhao, Hu Yiguo, Yang Shengyong, Wei Yuquan, Li Weimin, Chen Lijuan
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China.
Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, China.
Cell Physiol Biochem. 2018;47(2):489-504. doi: 10.1159/000489983. Epub 2018 May 22.
BACKGROUND/AIMS: Many tubulin inhibitors are in clinical use as anti-cancer drugs. In our previous study, a novel series of 4-substituted coumarins derivatives were identified as novel tubulin inhibitors. Here, we report the anti-cancer activity and underlying mechanism of one of the 4-substituted coumarins derivatives (SKLB060).
The anti-cancer activity of SKLB060 was tested on 13 different cancer cell lines and four xenograft cancer models. Immunofluorescence staining, cell cycle analysis, and tubulin polymerization assay were employed to study the inhibition of tubulin. N, N '-Ethylenebis(iodoacetamide) assay was used to measure binding to the colchicine site. Wound-healing migration and tube formation assays were performed on human umbilical vascular endothelial cells to study anti-vascular activity (the ability to inhibit blood vessel growth). Mitotic block reversibility and structural biology assays were used to investigate the SKLB060-tubulin bound model.
SKLB060 inhibited tubulin polymerization and subsequently induced G2/M cell cycle arrest and apoptosis in cancer cells. SKLB060 bound to the colchicine site of β-tubulin and showed antivascular activity in vitro. Moreover, SKLB060 induced reversible cell cycle arrest and reversible inhibition of tubulin polymerization. A mitotic block reversibility assay showed that the effects of SKLB060 have greater reversibility than those of colcemid (a reversible tubulin inhibitor), indicating that SKLB060 binds to tubulin in a totally reversible manner. The crystal structures of SKLB060-tubulin complexes confirmed that SKLB060 binds to the colchicine site, and the natural coumarin ring in SKLB060 enables reversible binding.
These results reveal that SKLB060 is a powerful and reversible microtubule inhibitor that binds to the colchicine site and is effective in multidrug-resistant cell lines.
背景/目的：许多微管蛋白抑制剂作为抗癌药物正在临床使用。在我们之前的研究中，一系列新型的4-取代香豆素衍生物被鉴定为新型微管蛋白抑制剂。在此，我们报告一种4-取代香豆素衍生物（SKLB060）的抗癌活性及其潜在机制。
在13种不同的癌细胞系和4种异种移植癌模型上测试SKLB060的抗癌活性。采用免疫荧光染色、细胞周期分析和微管蛋白聚合试验研究对微管蛋白的抑制作用。使用N，N'-亚乙基双（碘乙酰胺）试验测量与秋水仙碱位点的结合。对人脐静脉内皮细胞进行伤口愈合迁移和管形成试验，以研究抗血管活性（抑制血管生长的能力）。采用有丝分裂阻滞可逆性和结构生物学试验研究SKLB060-微管蛋白结合模型。
SKLB060抑制微管蛋白聚合，随后诱导癌细胞的G2/M期细胞周期阻滞和凋亡。SKLB060与β-微管蛋白的秋水仙碱位点结合，并在体外显示出抗血管活性。此外，SKLB060诱导可逆的细胞周期阻滞和微管蛋白聚合的可逆抑制。有丝分裂阻滞可逆性试验表明，SKLB060的作用比秋水仙酰胺（一种可逆的微管蛋白抑制剂）具有更大的可逆性，表明SKLB060以完全可逆的方式与微管蛋白结合。SKLB060-微管蛋白复合物的晶体结构证实SKLB060与秋水仙碱位点结合，并且SKLB060中的天然香豆素环实现可逆结合。
这些结果表明，SKLB060是一种强效且可逆的微管抑制剂，它与秋水仙碱位点结合，对多药耐药细胞系有效。
