土茯苓分离产物单体Aiphanol 通过不同靶点发挥抗肿瘤作用的机制研究

High degree of heterogeneity of the tumor and the tendency to develop resistance during chemotherapy make the development of more effective anti-tumor drugs an urgent need for clinical practice. Chinese herbal medicine is not only an important source of new anti-tumor compounds, but also a hot spot for the development of novel anti-cancer agents. Aiphanol is a phenolic natural monomer compound isolated from the anti-tumor components of the traditional Chinese herbal medicine Sarsaparilla (Smilax Glabra Rhizome). Through a series of studies, we found that Aiphanol can inhibit tumor cell growth by promoting autophagy and apoptosis. It can also inhibit the motility of tumor cells. Mechanistically, Aiphanol down-regulates the expression of various growth factors, meanwhile inhibits phosphorylations of EGFR and its downstream molecules in tumor cells. Additionally, Aiphanol inhibits enzymatic activity of COX and reduces the expression of COX2. These results suggest that Aiphanol may exert anti-tumor effect by targeting multiple factors, including EGFR and COX2. On the basis of the above work, this project will conduct in-depth study by using diverse types of systems, including molecular, cellular, and animal models, to characterize EGFR, COX, or other factors as the possible targets of Aiphanol and to determine the underlying molecular mechanisms. Results of this project will provide a solid basis for the development of novel multi-targeting anti-tumor therapeutic drugs with independent intellectual property rights.

肿瘤的高度异质性及在药物治疗中易于产生的耐药性，使得研发更有效的抗肿瘤新药成为临床的迫切需求。中药植物药是获得新型抗肿瘤化合物的重要来源，也是抗肿瘤药物的研发热点。Aiphanol是我们从中药土茯苓的抗肿瘤有效组分中分离获得的酚类天然单体化合物，结构新颖。经一系列研究发现， Aiphanol能够通过促进肿瘤细胞自噬和凋亡发挥明显的抗肿瘤作用，并能抑制肿瘤细胞的运动；它可以下调多种生长因子的表达、抑制肿瘤细胞的EGFR磷酸化及下游相关分子的活化，同时能抑制COX酶活性并抑制COX2的表达，提示Aiphanol 有可能通过EGFR、COX2等不同靶点发挥抗肿瘤作用。本项目拟在上述工作基础上，在分子、细胞和动物的不同层面，围绕Aiphanol的可能作用靶点EGFR、COX2和其它可能的潜在靶点及相关分子机制，进行系统的深入研究和验证，以期为研发具有自主知识产权的、全新的多靶点抗肿瘤药物奠定基础。

本项目对土茯苓来源的天然产物Aiphanol的抗肿瘤生长转移活性、抗血管生成活性、抗淋巴管生成活性、安全性等进行了系统性的评估，并阐明了相关机制。. 我们通过软琼脂集落形成、荷瘤裸鼠模型表明Aiphanol能显著抑制结肠癌的生长，其抑制效应与Aiphanol下调泛酪氨酸激酶和泛丝/苏氨酸激酶活性、促进肿瘤凋亡有关。Aiphanol可以逆转肿瘤细胞中EMT的发生，并干扰微丝的聚合，小鼠模型进一步验证了Aiphanol能抑制黑色素瘤细胞和结肠癌细胞的肺转移。Aiphanol能显著阻断鸡胚绒毛尿囊膜和小鼠主动脉弓等模型中新生血管的形成，通过激酶活性筛选实验，我们鉴定出Aiphanol对VEGFR3和VEGFR2具有最强的抑制作用，Aiphanol可以直接结合VEGFR2和 COX2, 通过VEGFR2或COX2或二者联合敲低能部分抵消Aiphanol对体外血管生成的抑制作用，耐药细胞株荷瘤生长模型证实了Aiphanol通过抑制VEGFR2和COX2-PGE2-VEGF途径阻断血管生成，进而抑制肿瘤生长。此外，Aiphanol可直接结合VEGFR3且以依赖VEGFR3的方式抑制体外的淋巴管生成，小鼠淋巴结发芽实验、胶栓实验和肿瘤细胞在小鼠体内的淋巴道转移实验验证了Aiphanol能拮抗淋巴管生成和肿瘤的淋巴道转移，并延长荷瘤小鼠的生存期。其机制与Aiphanol抑制VEGFR3活性、下调COX2介导的PGE2和VEGF-C分泌及减少巨噬细胞浸润有关，即通过协同调控肿瘤微环境的多细胞、多分子相互作用发挥抗肿瘤淋巴道转移的效应。在急毒实验中，与对照组相比，Aiphanol并未造成小鼠体重、血生化指标和主要脏器的异常，提示Aphanol用药安全。. 本项目通过体内外实验证实了小分子多激酶抑制剂Aiphanol除杀伤肿瘤细胞外，能有效阻断肿瘤微环境中的血管生成和淋巴管生成，拮抗肿瘤的生长和转移，可作为研发抗肿瘤新药的先导化合物。.