解淀粉芽孢杆菌Q-426 Bacillomycin D的分离纯化及其抗肿瘤活性

Isolation, purification and antitumor activity of Bacillomycin D from Bacillus amyloliquefaciems Q-426

摘要:

环脂肽化合物因其独特的结构特点和生物活性在医药领域具有广泛的应用前景。本研究旨在从解淀粉芽孢杆菌Q-426发酵液中分离纯化获得高纯度的环脂肽单体，并对分离得到的环脂肽C-15 Bacillomycin D和C-16 Bacillomycin D的抗肿瘤活性及机制进行初步研究。首先，联合使用酸沉淀和双树脂层析去除大量杂质，然后通过制备型HPLC分离得到纯度较高的两种环脂肽，经ESI-MS/MS对其进行结构鉴定，分别确定为C-15 Bacillomycin D与C-16 Bacillomycin D。其次，将两种环脂肽单体及其1：1混合液 (摩尔比) 按不同浓度梯度作用于人癌细胞 (Hela、MG、Hep-G2、HT-29)，结果表明环脂肽对Hela与MG细胞增殖的抑制作用呈浓度依赖性，长链环脂肽的抑制率较短链环脂肽略高。最后，采用细胞划痕实验和PI染色法考察了C-16 Bacillomycin对细胞侵袭与迁移能力、细胞凋亡和周期的影响，结果显示，C-16 Bacillomycin D可有效影响细胞的迁移能力，诱导细胞凋亡呈浓度依赖性，且阻滞细胞G0G1期。

Abstract:

Cyclic lipopeptide has extensive application prospect in the field of medicine due to its unique chemical structure and biological activity. This study aims to obtain high purity of cyclic lipopeptide monomer from Bacillus amyloliquefaciems strain Q-426, and illuminate preliminary antitumor mechanism of C-15 Bacillomycin D and C-16 Bacillomycin D. Firstly, crude cyclic lipopeptide solution was prepared by two-steps purification of acid precipitation and double-resins chromatography. In order to obtain purer product preparative HPLC was utilized to separate and purify cyclic lipopeptide. Component 1 and component 2 were detected as C-15 Bacillomycin D and C-16 Bacillomycin D by HPLC-MS and ESI-MS/MS. Secondly, the effect of C-15 Bacillomycin D, C-16 Bacillomycin D and their mixture (1:1, mol:mol) on cell proliferation was measured using human cancer cells (Hela, MG, Hep-G2 and HT-29). The cyclic peptide showed a dose dependent manner on the cell proliferation inhibition of Hela and MG cells. Finally, the results of the scratch wound healing assay and FACS analysis revealed that C-16 Bacillomycin D can effectively influence the cells migration and the cells treated with C-16 Bacillomycin D showed typical apoptotic morphology with the increase of drug concentration in the early apoptosis, late apoptosis percentage increased, and G0G1 arrest was induced significantly.