Abstract：
Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.
摘要：
肌动蛋白细胞骨架的重排是癌细胞发展细胞突起的先决条件，这是迁移所必需的，入侵,和转移。Fascin是参与肌动蛋白捆绑的关键蛋白，在侵袭性和侵袭性癌中表达。此外,fascin似乎参与微管蛋白结合和微管重排。进行了基于药物的计算机筛选，以鉴定具有比migrastatin更好的fascin抑制特性的化合物，金标准Fascin抑制剂.我们假设monastrol在结直肠癌细胞系中通过fascin阻断表现出抗迁移和抗侵袭特性。生物物理（热荧光和配体滴定，然后荧光光谱），生物化学（NMR），和细胞测定(MTT，侵入人体组织)，以及动物模型研究（斑马鱼入侵）进行了表征monastrol对fascin活性的抑制作用。计算机分析显示，monastrol是一种潜在的fascin结合化合物。生物物理和生化测定表明，monastrol与fascin结合并干扰其肌动蛋白捆绑活性。细胞培养研究,包括3D人类肌瘤椎间盘模型，表明monastrol抑制fascin驱动的细胞质突起以及入侵。在硅片中，共聚焦显微镜，和免疫沉淀试验表明，monastrol破坏了fascin-tubulin相互作用。这些抗侵入作用在体内得到证实。进行了计算机共聚焦显微镜和免疫沉淀测定，以测试monastrol是否破坏了fascin-tubulin的相互作用。这项研究报告，第一次,monastrol在结直肠肿瘤细胞中的体外和体内抗侵袭特性。相互作用的数量和类型表明monastrol在肌动蛋白和Fascin上的微管蛋白位点之间的潜在结合，这对抗肿瘤疗法的发展可能是有价值的。
