硫酸肝素寡糖类FGF-2抑制剂的构效关系及其对肿瘤糖酵解影响的研究

FGF-2 can promote the proliferation of various tumor cells, thus, is an important target for tumor treatment. Both former researches and our previous studies have shown that heparin oligosaccharide analogues attained good inhibition on FGF-2. Besides, the analogues can restrain expression of glucose transferase and pyruvate kinase M2, which are important enzymes in Warburg effect. However, the analogue structure with highest inhibition activity was unknown to date. The capsular polysaccharide derived from Escherichia coli K5 strain is very similar to heparin structure, which is a biosynthetic precursor of heparin..In this project, heparin oligosaccharide analogues with specific structures were enzymatic synthesized using E. coli K5 capsular polysaccharide as template. Oligosaccharide microarray and competitive ELISA will be assayed to screen the highest inhibitory analogue toward FGF-2. Structure-activity relationship of the inhibitors and FGF-2 activity will be studied on cells in vitro. For an unknown mechanism, altered glycolytic enzyme levels will also be determined in inhibitors-FGF-2 treated tumor cells, including glucose transferase, lactate dehydrogenase, pyruvate dehydrogenase and pyruvate kinase M2. We aim to explore the glycolytic molecular mechanism of inhibitors in FGF-2 treated tumor cells. This project is of great significance not only for a better understanding of Warburg effect in tumor cells, but also for treatment of diseases associated with FGF-2 overexpression.

FGF-2能促进多种肿瘤增殖，是肿瘤治疗的重要靶点。文献报道及笔者前期研究结果显示，硫酸肝素(HS)寡糖类似物对FGF-2有抑制作用，且能抑制FGF-2介导的肿瘤细胞内糖酵解相关酶的表达，但最佳HS寡糖类FGF-2抑制剂的结构不明。大肠杆菌K5菌的荚膜多糖与HS结构类似，是合成HS的前体化合物。.本项目以K5菌发酵的荚膜多糖为模板，酶法制备特定结构的HS寡糖类似物，采用糖芯片技术和竞争ELISA，筛选FGF-2的最佳HS寡糖类抑制剂。并通过体外细胞平台，研究HS寡糖类抑制剂的结构与FGF-2活性的构效关系。此外，进一步研究HS寡糖类抑制剂对FGF-2介导的肿瘤细胞内葡萄糖转移酶、乳酸脱氢酶、丙酮酸脱氢酶和丙酮酸激酶M2表达量的影响，探讨HS寡糖类抑制剂对肿瘤细胞糖酵解影响的分子机制。本研究不仅对进一步理解肿瘤细胞中瓦氏效应具有重要意义，还为治疗与FGF-2过度表达相关的疾病提供了新思路。

FGF-2能够促进血管生成、肿瘤增殖和转移，是肿瘤治疗的重要靶点。前人研究发现细胞外被糖萼上的硫酸肝素能够招募FGF-2，并将其呈递给FGFR，进而传递促增殖信号。本项目通过“一釜二酶”法成功制备了硫酸肝素类似物——2-脱乙酰基-3-O-硫酸化肝素（DSH），并发现DSH可以特异性抑制癌症细胞如A549、HCT-116、HepG2的增殖，而对正常细胞L02和HELF无毒性。通过相差倒立显微镜观察和DAPI染色观察DSH处理的A549细胞形态和结构的变化，发现细胞发生凋亡，典型特征包括细胞收缩变圆，呈现膜不对称性，且细胞粘附力丧失，漂浮在培养液中；细胞中的染色质压缩、核固缩以及凋亡小体和片段化DNA的形成。流式细胞仪检测发现凋亡细胞比例与DSH浓度呈正相关。此外，荧光定量分析和蛋白印迹结果均提示凋亡与Bax蛋白水平升高，Bcl-2、procaspase-3和procaspase-9蛋白水平降低有关。进一步通过流式细胞仪和免疫荧光分析发现，DSH抑制了FGFR1和FGFR3的表达，且Western Blot结果显示，MAPK/JNK的上调和MAPK/ERK和p38表达的下调可能与DSH诱导的A549细胞的凋亡相关。进一步通过JNK抑制剂SP600125证实MAPK通路参与DSH诱导的A549细胞凋亡。综上，本项目首次利用3-OST-1和AST-IV进行“一釜二酶”法合成DSH，得到以GlcA-GlcN3S为二糖单元的多糖。DSH能够特异性抑制肿瘤细胞的增殖，但对正常细胞的生长无影响。且进一步研究发现DSH通过抑制肺癌细胞A549中FGF2-FGFR1/FGFR3的表达，并激活下游的MAPK/JNK信号通路，从而启动A549细胞凋亡。合成DSH有望作为一种新型抗肿瘤药物的载体。

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