从肝素酶-硫酸肝素-FGF探讨益气活血解毒中药干预乳腺癌生长、转移的机制

BACKGROUND: GUBUYILIU is one of the successful prescription based on YIQIHUOXUEJIEDU principle for tumor treatment in China. Glycans including glycosaminoglycans (GAGs), from Traditional Chinese Medicine (TCM), were widely used for kinds of tumor clinic treatment, and have been proved to can inhibit tumor proliferation and metastasis, alleviate side-effects of chemotherapy/radiotherapy. Some polysaccharides are found to be mimetics of GAGs. Heparanase and heparan sulfate have recently been reported to be new targets for cancer therapy, as heparanase often is expressed at high levels in the tumors. This project is based on the results of preliminary studies that we have found certain TCM can inhibit tumor growth in clinic, animal and cellular models. We are particularly interested in polysaccharides, extracted from the 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 GUBENYILIU can modulate the activity, expression, and structure of endogenous heparan sulfate, by thus change heparanase activity and FGF signal passthway. OBJECTIVE: To investigate the anti-tumor activity of active component alignments isolated from the prescription by different heparanase level 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 TCM on endogenous heparan sulfate and heparanase activity and expression, to explore FGF and cell death-apoptosis and autophagy in breast tumor. 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 heparan sulfate and heparanase. 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.

益气活血解毒是针对肿瘤虚瘀毒证候要点的重要治疗法则，固本抑瘤II号是郁仁存多年经验总结的该法则有效代表方药。前期研究发现该方抑制乳腺癌细胞增殖、转移与mTOR通路细胞自噬、caspase途径凋亡有关；而mTOR和caspase通路都受肝素酶(Hpa)、硫酸肝素(HS)及HS-FGF-FGFR信号转导复合体调控。肿瘤细胞Hpa表达增高，引起HS结构变化，有利于HS-FGF-FGFR形成，促进肿瘤细胞增殖、转移。因此我们推测该方抗肿瘤作用与Hpa、HS及 HS-FGF-FGFR有关。本课题以全方及其益气、活血、解毒组分为工具，以不同Hpa/HS水平的小鼠乳腺癌移植瘤和癌细胞株为研究对象，研究不同治则中药是否通过影响①Hpa；②HS；③HS-FGF-FGFR而干预乳腺癌细胞自噬/凋亡性死亡，抑制肿瘤生长、转移；从肿瘤微环境角度诠释益气活血解毒法组方配伍科学原理。

益气活血解毒是针对肿瘤虚瘀毒证候要点的重要治疗法则，固本抑瘤II号(GY)是郁仁存多年经验总结的该法则有效代表方药。前期研究发现GY抑制乳腺癌细胞增殖、转移与mTOR通路细胞自噬、caspase途径凋亡有关；而mTOR和caspase通路都受肝素酶(Hpa)、硫酸肝素(HS)及HS-FGF-FGFR信号转导复合体调控。肿瘤细胞Hpa表达增高，引起HS结构变化，有利于HS-FGF-FGFR形成，促进肿瘤细胞增殖、转移。因此我们推测GY抗肿瘤作用与Hpa、及及下游FGF、Mtor信号通路有关。提出研究假说：益气活血解毒中药GY干预乳腺癌生长及转移的原因可能在于作为外源性硫酸肝素结构类似物，该方通过影响肿瘤组织内源性肝素酶及硫酸肝素的功能，进而影响FGF信号转导，从而表现出前期研究中发现的诱导肿瘤细胞自噬、凋亡，抗肿瘤增殖、转移的作用。通过以GY及益气、活血、解毒组中药为工具，分别以不同肝素酶表达水平的小鼠移植性乳腺癌，不同肝素酶表达水平的乳腺癌细胞株为研究对象，观察各组中药对不同肝素酶水平的乳腺癌荷瘤小鼠肿瘤生长、转移、荷瘤生存时间的影响；对WT小鼠乳腺癌肿瘤生长、转移过程中肝素酶、FGF、自噬及凋亡的干预作用；对不同肝素酶表达水平的多种乳腺癌细胞生长、迁移能力的影响；对乳腺癌细胞肝素酶、硫酸肝素、FGF、自噬及凋亡的影响；中药体外对肝素酶活性的直接作用；比较不同治则指导下外源性中药干预肝素酶、硫酸肝素、FGF三方面作用的差异；并选择GY中含有的单体漆黄素，探讨其对乳腺癌细胞增殖、凋亡及Mtor信号通路的影响。通过我们的实验研究及进行的meta和TCGA数据库分析，我们证实了肝素酶对肿瘤增殖、转移的促进作用；证实益气活血解毒中药抗肿瘤作用，与抑制肝素酶表达，进而影响FGF信号转导，诱导肿瘤细胞死亡有关。肝素酶具有成为肿瘤进展及恶性程度的评价指标的潜在价值，值得进一步深入研究。