基于益气活血解毒法的中药多糖通过糖胺聚糖影响乳腺癌生长、转移的机理研究

Scientific background: YIQIHUOXUEJIEDU is the main principle for tumor treatment in China, while GUBUYILIU is one of the successful prescription based on this principle.Glycans including glycosaminoglycans (GAGs), from Traditional Chinese Medicine (TCM), widely used for clinic treatment in China, have been proved to have effects on inhibiting tumor proliferation and metastasis, and alleviating side-effects induced by chemotherapy/radiotherapy. Some polysaccharides are found to be mimetics of GAGs. While how they inhibit tumor proliferation/metastasis and their implication in the regulation of the endogenous GAGs still have not been considered. Heparanase and heparan sulfate have recently been reported to be a new target for cancer therapy. In cancer patients, heparanase is expressed at high level..Description of the project: This project is based on the results of preliminary studies that we have found certain polysaccharides extracted from TCM can inhibit tumor growth both in clinic, animal and cellular experiments. We are particularly interested in some polysaccharides and GAGs, extracted from a typical prescription (GUBENYILIU) of Beijing Hospital of Traditional Chinese Medicine. These extracts can be regarded as extrinsic GAGs. We postulate that some active components from these extrinsic GAGs can modulate the activity of endogenous GAGs. Thus, we aim to identify the compound that plays the key role and its effects on endogenous GAGs and heparanase expression, and how endogenous GAGs and heparanase change during breast cancer proliferation and metastasis..Objective: To investigate the anti-tumor activity of active component alignments isolated from the prescription by 4T1 mouse breast tumor transplantation models and cell lines (4T1,MCF7,MAB231), to investigate its mechanisms of inhibiting tumor proliferation and metastasis, to explore the extrinsic GAGs on endogenous GAGs and heparanase, and to research roles of endogenous GAGs and heparanase in carcinoma proliferations and metastasis in breast tumor. .Methods: Active polysaccharide extracts from the prescription will be isolated further by chemical methods. Transplantation tumor models, human and mouse cell lines will be used to explore the anti-tumor activity of these components. Heparanase and endogenous GAGs will be investigated by cell biology..Expected results: The aim of this project is to compare the different components based on different treatment principle,to find the most active components or compound from some TCM, and to make clear of its mechanisms,expecially by its effects on endogeous GAGs. The expected result of this project is to provide a new therapeutic strategy or preclinical component from herbal medicine for breast tumor. Articles published in journals and patents will be the direct results of this project.

虚瘀毒是肿瘤的症候要点，益气活血解毒是治疗肿瘤的重要法则。肿瘤细胞胞外基质ECM（主成分糖胺聚糖GAGs）和基底膜BM的降解是癌细胞穿透组织屏障发生转移的重要步骤，干预GAGs可起到抗肿瘤作用。肝素酶在易发生转移的肿瘤中高表达，通过降解GAGs（主要为硫酸乙酰肝素HSPG）而降解 ECM和BM，促进肿瘤转移。中药多糖影响内源性GAGs及肝素酶而抑制肿瘤生长转移、调节免疫功能成为中医药抗肿瘤研究新方向。基于益气活血解毒法的固本抑瘤II号是郁仁存教授多年经验总结，方中含GAGs类似物，在前期工作基础上，本课题进一步研究中药多糖①对生物发光标记的小鼠乳腺癌生长、转移影响；②对肿瘤生长转移过程中GAGs、肝素酶及T细胞免疫的干预作用及机制，从中药多糖对内源性GAGs、肝素酶及免疫功能影响角度诠释益气活血解毒法的科学原理，为中药多糖在GAGs这一肿瘤研究新热点领域的作用提供实验依据。

乳腺癌的病因病机可归因为“虚”、“瘀”、“毒”，国家级名老中医郁仁存在多年临床经验基础上提出“益气活血解毒法”治疗中晚期恶性肿瘤的主要法则，并创制了基于该法则的固本抑瘤II号，已作为院内制剂大量应用。“益气活血解毒法”与汤钊猷院士的肿瘤治疗要“消灭与改造并举”理论不谋而合，强调了对机体、癌、癌的微环境的干预。本项目在以往研究 “益气活血解毒法”代表方固本抑瘤II号及拆方抗肿瘤作用的基础上，基于该方的主要成分之一为多糖，中药多糖具有广泛的生物学活性，且固本抑瘤II 号中含有糖胺聚糖GAGs 的结构类似物，提出假说：外源性益气活血解毒中药多糖通过干预乳腺癌肿瘤组织的内源性糖胺聚糖GAGs，而抑制乳腺癌生长及转移。.本项目以固本抑瘤II号方及其拆方益气、活血、解毒中药多糖为工具，以生物发光标记的高转移小鼠乳腺癌4T1 移植瘤及细胞株为研究对象，观察了各组中药多糖：①对乳腺癌荷瘤小鼠肿瘤细胞生长、转移、荷瘤生存时间的影响；②对肿瘤细胞外基质中糖胺聚糖（GAGs）及肝素酶的干预作用；③对小鼠T 细胞免疫的影响；④对体外培养乳腺癌细胞生长、迁移能力的影响；⑤对细胞内GAGs及肝素酶的影响。.通过本项目研究，我们发现：①乳垫原位接种造模，切除原位瘤后仍有80%以上肺转移发生，模拟了临床乳腺癌患者手术切除后转移复发的特点，为较好的乳腺癌肺转移模型。②体外实验中活血中药具有较好的抑制肿瘤增殖、转移的作用，与诱导细胞凋亡有关；体内实验中单独应用活血组中药具有一定的促进肿瘤转移的作用，但与“益气”、“解毒”配伍后的全方却可以抑制肿瘤增殖及转移，提示临床应慎用活血中药；③解毒组中药具有抑制肿瘤增殖、转移、侵袭血管内皮细胞的作用，并在体内实验中表现出较好的抑瘤作用，为全方抗肿瘤的主要有效组分；但与表阿霉素联合应用，并未表现出化疗增效或相加作用；④解毒组中药多糖体内外的抗肿瘤作用与抑制肝素酶、总糖胺聚糖、FGF2，诱导肿瘤细胞凋亡有关。⑤小分子量多糖衍生物在细胞受损后可以进入细胞浆，而在正常情况下则被阻断在细胞膜外，可能通过细胞外基质或细胞表面的硫酸肝素链等结构粘附在细胞膜。实验结果从中药多糖对GAGs、肝素酶作用角度诠释“益气活血解毒”法的科学原理，验证了本项目所提出的科学假说，为临床配伍应用该法则提供了实验依据。