The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding
The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding
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DOI：
10.1021/bi800730q
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发表时间：
2008-07-29
期刊：
影响因子：
2.9
通讯作者：
Chakrabarti, Gopal
中科院分区：
文献类型：
--
作者：
Acharya, Bipul R.;Bhattacharyya, Bhabatarak;Chakrabarti, Gopal
白花丹素（5-羟基-2-甲基-1，4-萘醌）是从白花丹科植物的根部分离的萘醌，对几种肿瘤类型具有潜在的抗增殖活性。我们研究了白花丹素对离体细胞微管的影响，以及其与纯化的微管蛋白和微管在体外的结合。使用人非小肺上皮癌细胞（A549）的细胞存活力实验表明白花丹素的IC 50值为14.6 μ M。使用抗微管蛋白FITC偶联抗体的免疫荧光研究表明，以剂量依赖性方式的间期微管网络的显着扰动。纯化的微管蛋白在体外聚合成微管被白花丹素抑制，IC 50值为38 +/-0.5 μ M。其与微管蛋白的结合以时间和浓度依赖性方式淬灭蛋白质色氨酸荧光。白花丹素与微管蛋白的结合是缓慢的，在25 ℃下需要60分钟平衡。缔合反应动力学本质上是双相的，在25 ℃时，快相和慢相的缔合速率常数分别为235.12 +/-36 M-1 s（-1）和11.63 +/-11 M-1 s（-1）。白花丹素与微管蛋白结合的化学计量比为1：1（摩尔：摩尔），在25 ℃下解离常数为0.936 +/-0.71 μ M。白花丹素竞争秋水仙碱结合位点，如由改良的狄克逊图测定的Ki为7.5 μ M。基于这些数据，我们得出结论，白花丹素识别秋水仙素结合位点的微管蛋白。进一步的研究是必要的，以定位连接的抑制剂的微管蛋白的秋水仙素结合位点的药效学点。
Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC50 value for plumbagin is 14.6 mu M. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC50 value of 38 +/- 0.5 mu M. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M-1 s(-1)and 11.63 +/- 11 M-1 s(-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1: 1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 mu M at 25 degrees C. Plumbagin competes for the colchicine binding site with a K-i of 7.5 mu M as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.