Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700019, India.
Biochemistry. 2010 Mar 2;49(8):1702-12. doi: 10.1021/bi901760d.
Genistein (4',5,7-trihydroxyisoflavone), an isoflavone, is a major constituent of soyfoods. It has potential antiproliferative activity against several tumor types. We have examined the effect of genistein on cellular microtubules as well as its binding with purified tubulin in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC(50) value for genistein is 72 microM. Flow cytometry experiments demonstrated that genistein arrested cell cycle progression at the G(2)/M phase, but mitotic index data showed that genistein did not arrest cell cycle progression at mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody demonstrated a significant depolymerization of the interphase microtubules in a dose-dependent manner, and this was confirmed by the Western blot experiment using genistein-treated A549 cells. In vitro polymerization of purified tubulin into microtubules was inhibited by genistein with an IC(50) value of 87 microM. Genistein binding to tubulin quenched protein tryptophan fluorescence in a time- and concentration-dependent manner. Binding of genistein to tubulin was slow, taking approximately 45 min for equilibration at 37 degrees C. The association rate constant was 104.64 +/- 20.63 M(-1) s(-1) at 37 degrees C. The stoichiometry of genistein binding to tubulin was nearly 1:1 (molar ratio) with a dissociation constant of 15 microM at 37 degrees C. It was interesting to note that genistein did not recognize either the colchicine site or the vinblastine binding site of tubulin. Surprisingly, genistein inhibited ANS binding and competed for its binding site of tubulin with a K(i) of 20 microM as determined from a modified Dixon plot. Hence, we conclude that one of the mechanisms of antiproliferative activity of genistein is depolymerization of microtubules through binding of tubulin.
染料木黄酮（4'，5，7-三羟基异黄酮），一种异黄酮，是大豆食品的主要成分。它对几种肿瘤类型具有潜在的抗增殖活性。我们已经研究了染料木黄酮对细胞微管的影响以及它在体外与纯化微管蛋白的结合。用人非小细胞肺癌细胞（A549）进行的细胞活力实验表明，染料木黄酮的 IC50 值为 72μM。流式细胞术实验表明，染料木黄酮将细胞周期阻滞在 G2/M 期，但有丝分裂指数数据表明，染料木黄酮并未在有丝分裂时阻滞细胞周期进程。用抗α-微管蛋白抗体进行的免疫荧光研究表明，微管蛋白的间期微管在剂量依赖性方式下显著解聚，并且使用用染料木黄酮处理的 A549 细胞进行的 Western blot 实验证实了这一点。体外纯化微管蛋白聚合为微管被染料木黄酮抑制，IC50 值为 87μM。染料木黄酮与微管蛋白的结合以时间和浓度依赖的方式猝灭蛋白质色氨酸荧光。染料木黄酮与微管蛋白的结合缓慢，在 37°C 下平衡约需 45 分钟。在 37°C 下，缔合速率常数为 104.64±20.63M-1s-1。染料木黄酮与微管蛋白的结合几乎是 1:1（摩尔比），在 37°C 时解离常数为 15μM。有趣的是，染料木黄酮既不识别秋水仙碱结合位点，也不识别长春花碱结合位点。令人惊讶的是，染料木黄酮抑制 ANS 结合，并通过结合微管蛋白的结合位点与 K i为 20μM 竞争，这是从修改的 Dixon 图确定的。因此，我们得出结论，染料木黄酮抗增殖活性的机制之一是通过结合微管蛋白解聚微管。
