School of Biological Sciences, UM-DAE Centre for Excellence in Basic Sciences, University of Mumbai, Vidyanagari, Mumbai, 400098, India.
Department of Biotechnology and Bioinformatics, Sambalpur University, Sambalpur, 768019, Odisha, India.
Comput Biol Med. 2022 Aug;147:105789. doi: 10.1016/j.compbiomed.2022.105789. Epub 2022 Jun 30.
Shikonin (SK), a naphthoquinone compound from the purple gromwell, Lithospermum erythrorhizon, possesses a considerable antiproliferative potential. By using a combination of biophysical techniques, cellular assays, immunofluorescence imaging, and molecular dynamic simulation, we identified a possible mechanism of action of SK. SK inhibited the viability of the triple negative breast cancer cells MDA-MB-231 (IC of 1 ± 0.1 μM), and its inhibitory effect was irreversible. It strongly suppressed the clonogenic and migratory potential of the cells. Although SK did not show any phase-specific inhibition of cell cycle progression, it induced apoptosis as confirmed by annexin-V-based flow cytometry and Western immunoblotting of PARP1. Probing further into its mechanism using a tryptophan-quenching assay, it was found that SK binds the microtubule-building protein tubulin with a dissociation constant (K) of 8 ± 2.7 μM, without grossly damaging the tertiary structure of the protein. The drug-bound tubulin could not assemble microtubules properly in vitro as confirmed by polymer mass analysis, turbidimetry analysis, and transmission electron microscopy, and in cells, as visualized by immunofluorescence imaging. In cells, SK also suppressed the dynamicity of microtubules as indicated by considerable acetylation of the cellular microtubules. The fine details of tubulin-SK interactions were then elucidated using molecular docking and molecular dynamic simulation. The free energy change of the interaction (ΔG,) was found to be -14.60 kcal/mol and the binding involved both the intermolecular van der Waals (ΔE) and the electrostatic (ΔE) interactions. Taken together, our data provide evidence for a possible mechanism of action of SK as a tubulin-targeted anticancer agent.
紫草素（SK）是来自紫草的萘醌类化合物，具有相当的抗增殖潜力。通过结合生物物理技术、细胞测定、免疫荧光成像和分子动力学模拟，我们确定了 SK 的一种可能作用机制。SK 抑制三阴性乳腺癌细胞 MDA-MB-231 的活力（IC 为 1 ± 0.1 μM），且其抑制作用是不可逆的。它强烈抑制细胞的集落形成和迁移能力。虽然 SK 对细胞周期进程没有表现出任何时相特异性抑制作用，但通过 Annexin-V 流式细胞术和 PARP1 的 Western 免疫印迹证实其诱导了细胞凋亡。通过色氨酸猝灭实验进一步探究其机制，发现 SK 与微管蛋白结合，解离常数（K）为 8 ± 2.7 μM，而不会严重破坏蛋白质的三级结构。药物结合的微管蛋白在体外不能正确组装微管，这一点通过聚合物质量分析、浊度分析和透射电子显微镜得到了证实，在细胞中，通过免疫荧光成像也可以观察到。在细胞中，SK 还通过细胞微管的大量乙酰化来抑制微管的动态性。然后使用分子对接和分子动力学模拟来阐明微管蛋白-SK 相互作用的细节。相互作用的自由能变化（ΔG）为-14.60 kcal/mol，结合涉及分子间范德华（ΔE）和静电（ΔE）相互作用。综上所述，我们的数据为 SK 作为一种微管靶向抗癌药物的可能作用机制提供了证据。
