抗明胶酶单链抗体与力达霉素融合蛋白抗肿瘤作用机制及疗效研究  

Antitumor mechanism and efficacy of the fusion protein consisting of lidamycin and anti-gelatinases single-chain Fv antibody

作　　者：高瑞娟[1] 尚伯杨[1] 盛唯瑾[1] 赵春燕[1] 李电东[1] 甄永苏[1] 

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所肿瘤室,北京100050

出　　处：《中国医药生物技术》2014年第4期250-259,共10页Chinese Medicinal Biotechnology

基　　金："重大新药创制"国家科技重大专项(2009ZX09103-136;2013ZX09102064);国家自然科学基金(81001021)

摘　　要：目的探讨抗明胶酶单链抗体Fv与力达霉素(LDM)融合蛋白的抗肿瘤作用机制及疗效。方法采用三联径向加压C4柱制备LDM发色团AE,体外分子重建将其装入融合蛋白Fv-LDP中,Superdex 75层析获得Fv-LDP-AE。反相HPLC测定纯度或相对含量。流式细胞术测定DNA含量;采用SA-β-gal染色、Western blot和细胞伤口愈合实验分别检测药物对细胞衰老、蛋白表达和迁移的影响;通过动物模型评价药物疗效。结果 Fv-LDP-AE承袭了LDM诱导肿瘤细胞周期阻滞、凋亡和裂亡的作用特点,并显示出更强的诱导衰老和抗迁移作用。Fv-LDP-AE诱导凋亡和抑制迁移依次与caspases通路激活和MMP-2降调节有关。Fv-LDP-AE可抑制小鼠和裸鼠移植瘤生长,抑制率分别可达到86.6%和82.5%。结论 Fv-LDP-AE对小鼠和裸鼠移植瘤有效,可能与其对细胞周期阻滞、凋亡、裂亡和衰老的诱导以及迁移的抑制有关。Objective To investigate the antitumor mechanism and efficacy of the fusion protein Fv-LDP-AE composed of lidamycin （LDM） and anti-gelatinases single-chain Fv antibody. Methods Free AE （the active enediyne chromophore of LDM） prepared using the triple radial pressure C4 column was reassembled into fusion protein Fv-LDP by in vitro molecular reconstitution. The rebuilt fusion protein Fv-LDP-AE was obtained by Superdex 75 chromatography to remove the free AE. Reverse phase HPLC was used to determine the purity or the relative content of the drug components. DNA content was measured by flow cytometry. SA-β-gal staining, Western blot and cell wound healing assay were exploited to detect the effect of tested drugs on cell senescence, protein expression and cell migration, respectively. Antitumor efficacies of tested drugs were evaluated by different animal models. Results Fv-LDP-AE inherited the action characteristics derived from LDM including the induction of cell cycle arrest, apoptosis and mitotic cell death in human hepatoma BEL-7402 and lung cancer PG-BE1 cells. Furthermore, Fv-LDP-AE also showed more potent activity than LDM in triggering cellular senescence and inhibiting cell migration in human cancer cells. The induction of cell apoptosis was related to the activation of caspases signaling pathway and the inhibition of migration was associated with the down-regulation of MMP-2 in Fv-LDP-AE-treated BEL-7402 cells. In vivo, Fv-LDP-AE also suppressed the growth of murine tansplantable hepatoma 22 （H22） and human hepatoma BEL-7402 xenograft in athymic mice with the inhibition rate of 86.6%and 82.5%, respectively. Conclusion Fv-LDP-AE showes antitumor effects against murine transplantable tumor and cancer xenograft in athymic mice, which may be related to the induction of cell cycle arrest, death and senescence as well as the inhibition of cell migration by Fv-LDP-AE in tumor cells.

关 键 词：明胶酶类 单链抗体 肿瘤微环境 力达霉素 

分 类 号：R965[医药卫生—药理学]