Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, West Bengal, India.
Biochimie. 2013 Jun;95(6):1297-309. doi: 10.1016/j.biochi.2013.02.010. Epub 2013 Feb 26.
Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC50 value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC50 value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10(-7) M(-1) s(-1) and 4.0 × 10(-9) M(-1) s(-1) for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin-apigenin binding was 1:1 and binding the binding constant (Kd) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G2/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α-β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.
芹菜素是一种天然黄酮类化合物，存在于许多植物中，对肺癌上皮细胞（A549）有诱导凋亡和细胞死亡作用，其在 48 小时的治疗中对细胞的半抑制浓度（IC50）为 93.7 ± 3.7 μM。用 A549 细胞和无细胞体系进行的靶标鉴定研究表明，芹菜素使微管解聚，并抑制 A549 细胞中冷解聚微管的重新组装。此外，芹菜素以 79.8 ± 2.4 μM 的 IC50 值抑制纯化微管蛋白的聚合。它在无细胞体系中与微管蛋白结合，并以浓度和时间依赖的方式猝灭微管蛋白的本征荧光。这种相互作用依赖于温度，结合动力学呈两相性，在 37°C 时，快相和慢相的结合速率常数分别为 11.5×10(-7)M(-1)s(-1)和 4.0×10(-9)M(-1)s(-1)。微管蛋白-芹菜素结合的化学计量比为 1:1，结合常数（Kd）为 6.08±0.096 μM。有趣的是，芹菜素与另一种天然抗微管蛋白药物姜黄素表现出协同抗癌作用。芹菜素和姜黄素协同诱导 A549 细胞死亡和凋亡，并阻断细胞周期在 G2/M 期的进展。当联合使用时，芹菜素和姜黄素在体外微管上的强烈解聚作用和对冷解聚微管的重新组装的抑制作用也表明这些配体结合在微管蛋白的不同部位。计算机模拟表明，芹菜素结合在微管蛋白的α-β-亚基的界面处。结合位点距离之前预测的姜黄素结合位点有 19 Å 的距离。用纯化蛋白进行的结合研究也表明，芹菜素和姜黄素都可以同时与纯化的微管蛋白结合。了解芹菜素和姜黄素协同作用的机制，可以帮助从廉价易得的天然产物中开发抗癌联合药物。
