蛴螬多糖以半乳凝素-1为靶点抑制肿瘤血管生成的结构基础与构效关系

The efficacy of polysaccharides, as an important water-soluble components of Chinese medicine, on the treatment of cancer and other diseases has been proved over the last three decades. However, because of the complexity of the structures and mechanisms of polysaccharides, the relationship between structures and anti-tumor activities remains unclear. Recent studies showed that some kinds of saccharides regulated the tumor angiogenesis by inhibiting Galectin-1 (Gal-1), which attracted our great attention. These results may help to find the effective polysaccharides from traditional Chinese medicine. Our previous studies showed that the polysaccharides are the main anti-tumor substances of Holotrichia diomphalia Bates. We had isolated five homo-polysaccharides from Holotrichia diomphalia larvae and found that the anti-tumor activities were closely related to their structures. Among these homo-polysaccharides, two acidic polysaccharides could significantly combine to Gal-1 and down-regulate the expression of Gal-1, and further inhibit the tumor angiogenesis. These results suggest that there are some important recognition units for Gal-1 in the polysaccharides isolated from H. diomphalia. To elucidate anti-tumor structure-activity relationship, and further to demonstrate the Gal-1 binding domain of H. diomphalia polysaccharide, we will analysis the primary structures of the polysaccharides from H. diomphalia Bates using a combination of instrumental and chemical analysis, including methylation analysis, GC-MS, IR, 2D NMR spectroscopy (C-H COSY, HSQC, HMBC, TOCSY and NOESY), etc. After the polysaccharides are degraded by specific enzymes and purified by gel columns, their affinities to Gal-1 will be measured using the solid phase binding assay and surface plasmon resonance analysis. Anti-angiogenesis effects will be determined using the tube formation assay and migration assay in vitro, and in nude mice xenografted cancer cells. The structure of anti-tumor polysaccharide fragments will be investigated using the chemical and instrumental methods as mentioned above. In addition, the binding constants between polysaccharide fragments and Gal-1 will be determined using 15N-1H-HSQC NMR and calculated with molecular dynamics simulation. From above, we will elucidate the anti-tumor structure-activity relationship of the H. diomphalia polysaccharides. These results might be helpful in understanding the anti-tumor mechanism of polysaccharides, and developing a novel anti-tumor agent.

多糖是中药重要的水溶性成分，对肿瘤有显著的治疗作用，但结构复杂，构效研究缓慢，是亟待解决的制约多糖应用与发展的科学问题。近年，具有糖结合活性的半乳凝素-1（Gal-1）调控肿瘤血管生成关键作用的发现，为多糖构效研究提供了新思路。我们前期研究发现：多糖是中药蛴螬抗肿瘤的主要药效物质，其活性与结构密切相关；且多种蛴螬均一性多糖中的2种可与Gal-1特异结合，显著抑制肿瘤血管生成。提示：蛴螬多糖糖链中存在活性结构域，可特异性拮抗Gal-1而产生抗肿瘤作用。基于此，本研究拟采用化学和波谱法解析蛴螬多糖的一级结构；酶法降解获得多糖片段，表面等离子体共振及固相结合法筛选结合Gal-1的多糖片段，进而综合评价其抑制血管生成的作用，解析活性片段结构；通过N-H-HSQC分析和分子动力学模拟研究多糖阻断Gal-1抑制肿瘤血管生成的构效关系。为揭示蛴螬抗肿瘤作用的物质基础，丰富活性多糖结构数据库提供科学依据。

多糖是中药蛴螬抑制肿瘤血管生成的主要药效物质，但其结构和作用机制复杂，构效研究进展缓慢。本课题对蛴螬多糖进行了系统地提取、分离，一级结构解析；用酶法和酸法降解多糖，并以半乳糖凝集素-1（Gal-1）为作用靶点筛选活性多糖及片段；进一步通过多糖片段的结构分析和体外抑制内皮细胞迁移、侵袭和微管形成作用的研究，揭示蛴螬多糖抑制肿瘤血管生成的药效基础。本项目共提取、分离得到重均分子量为3.8×105，8.2×103，1.7×104，1.9×104 Da的4种均一性蛴螬多糖，结构解析研究发现其中3种是新结构；明确了蛴螬多糖抑制肿瘤血管生成的多糖是HDPS-2II，其结构为(1,4)-2-去氧葡萄糖-(1,6)-2-去氧-半乳杂聚糖，以(1,2)-甘露糖为分枝点，支链含(1,4)-木糖；HDPS-2II的活性结构域为(1,4)-2-去氧葡萄糖-(1,6)-2-去氧-半乳杂聚糖主链，与人Gal-1的亲和力为13.1±0.3 μM；HDPS-2II可通过结合Gal-1从而阻断内皮细胞迁移、侵袭和微管形成，抑制肿瘤血管生成，产生明显的体内抗肿瘤作用。此外，本课题还解析了另两种与Gal-1有一定亲和力的多糖结构，分别为以→2)-Manp-(1→5)-Araf-(1→3)-Galp-(1→4)- GalA-(1→和→1)-Araf-(3→1)-Glcp-(3→为主链的杂多糖。通过目前得到的实验结果，确定的蛴螬多糖抑制肿瘤血管生成的初步构效关系为：糖链中Ara、Man、Glc、Fuc、Xyl的含量对Gal-1的亲和力无显著贡献；1,3-Galp、α-D-1-GalpA、1,6-Galp残基的存在可明显增加与Gal-1的亲和作用；(1,4)-2-去氧葡萄糖-(1,6)-2-去氧-半乳杂聚糖具有显著的Gal-1抑制作用，此作用与(1,6)-2-去氧-半乳糖摩尔比含量正相关，与多糖的高级结构无关；糖残基的硫酸酯化和羧甲基化修饰对Gal-1的抑制无增强作用。本研究首次发现了新结构蛴螬多糖，初步阐明蛴螬多糖抑制肿瘤血管生成的结构基础，为多糖药物的研发提供了实验依据。