Anti-microtubule activity of tubeimoside I and its colchicine binding site of tubulin.
BACKGROUND
Tubeimoside I (TBMS1) was isolated from the tubers of Bolbostemma paniculatum (Maxim.) Franquet. TBMS1 shows potent anti-tumor activity. The present study was conducted to investigate the anti-microtubule role of TBMS1 and its binding site of tubulin.


METHODS
Cell growth inhibition was measured by MTT after treatment with TBMS1. Uptake kinetics of TBMS1 by human nasopharyngeal carcinoma CNE-2Z cell line (CNE-2Z) was assayed by HPLC. Microtubule protein (MTP) was prepared from porcine brain through two cycles of polymerization-depolymerization in a high molarity buffer. Inhibition of MTP polymerization induced by TBMS1 was determined by a turbidity measurement and a sedimentation assay; the interactions of TBMS1 with tubulin within CNE-2Z cells were investigated by immunofluorescence microscopy and immunoblotting. TBMS1 was tested for its ability to inhibit binding of known tubulin ligands through competitive binding assay.


RESULTS
TBMS1 displayed growth inhibitory activity against CNE-2Z cells with IC(50) value of 16.7 microM for 72 h. HPLC analysis of TBMS1 uptake by CNE-2Z cells displayed the initial slow TBMS1 uptake and then gradually reaching an maximum uptake near 18 h. CNE-2Z cells treated with TBMS1 (25 microM, 3 h) were sufficient to cause the microtubular network disruption. Immunoblot analysis showed that the proportion of cytosolic tubulin of cells treated with TBMS1 increased in a time- and concentration-dependent manner. TBMS1 did not inhibit the binding of vinblastine to tubulin. Colchicine binding to tubulin was inhibited in the presence of TBMS1.


CONCLUSIONS
TBMS1 is an anti-microtubule agent, and its binding site of tubulin is the colchicine binding site of tubulin.
Ma
Rundi
R
Key Laboratory of Marine Materia Medica, Guangdong Ocean University, Jiefang East Avenue 40, Xiashan, Zhanjiang, 524025, China.
Song
Gang
G
You
Wenbing
W
Yu
Lijian
L
Su
Weiming
W
Liao
Mingneng
M
Zhang
Yongping
Y
Huang
Laizhen
L
Zhang
Xiaoyu
X
Yu
Tingxi
T
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D002277:Carcinoma / Q000188:drug therapy / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003078:Colchicine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008868:Microtubule Proteins / Q000187:drug effects / Q000378:metabolism; D009303:Nasopharyngeal Neoplasms / Q000188:drug therapy / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D012503:Saponins / Q000378:metabolism / Q000494:pharmacology*; D013552:Swine; D014315:Triterpenes / Q000378:metabolism / Q000494:pharmacology*; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D050257:Tubulin Modulators / Q000378:metabolism / Q000494:pharmacology*
D000972:Antineoplastic Agents, Phytogenic; D004365:Drugs, Chinese Herbal; D008868:Microtubule Proteins; D011108:Polymers; D012503:Saponins; D014315:Triterpenes; D014404:Tubulin; D050257:Tubulin Modulators; C051084:tubeimoside I; D003078:Colchicine