基于肿瘤炎性微环境调控抗肿瘤的甘草黄酮类成分及分子机制研究

Functional polarization of macrophages into M2 cells and their secretion of inflammatory mediators are key events in inflammation-associated tumorigenesis. Identification of novel targets and plant active ingredients targeting the tumor inflammatory microenvironment is a hot topic of current research. Our previous work demonstrated that new compound A, B and isoliquiritigenin, from licorice significantly inhibit proliferation of tumour cells in vitro through inhibition of TLR4-NF-kappa B activation and macrophage polarization. Herein, bioassay-guided isolation of flavonoids with antitumor activities from licorice will be further investigated by using the dual luciferase reporter system and macrophage model. In addition, the three types of novel derivatives with isoliquritlgenin as a lead compound will be designed and synthesized through the esterification reaction with acyl chlorides, Mannish reaction with methanal and amines, or etherification reaction with isopropanolamines, and structure-activity relationships will be studied. In a rat lung cancer model induced by 3-methylcholanthrene and a mouse model of colitis-associated colon cancer, the inhibitory activity of the screened compounds for phenotypic transformation of macrophages in the tumor inflammatory microenvironment will be dynamically studied. The effects of the compounds will be further validated in a human tumor xenograft nude mouse model. The effects of the compounds on signaling networks involved in the polarization of macrophages to the M2 phenotype will be finally studied in vitro. Taken together, licorice flavonoids have the potent anti-tumor effect through inhibition of polarization of macrophages and their secretion of inflammatory mediators in the tumor microenvironment via downregulating the cascade of events triggered by TLR4-NF-κB signal pathway. These new concepts have wide-ranging implications for identifyication of promising therapeutic targets and drug candidates, and thus have important theoretical significance and clinical value.

肿瘤炎性微环境中巨噬细胞表型转化及炎症介质释放是炎症相关肿瘤发生发展的关键环节。寻找靶向肿瘤炎性微环境抗肿瘤的植物活性成分是当前研究热点。本项目是在我们前期从甘草中分离出可靶向巨噬细胞激活抗肿瘤的黄酮类化合物（新化合物A、B和异甘草素）的基础上，通过TLR4-NF-κB双荧光素酶报告系统和巨噬细胞药筛模型采用活性追踪分离法对甘草中的黄酮类活性成分进一步提取分离；随后通过对酰氯酯化，或引入Mannish碱和异丙醇胺类基团，合成三系列异甘草素衍生物，研究其构效关系。最后采用大鼠诱癌模型、小鼠结肠炎相关性结肠癌模型、裸鼠移植瘤模型和体外试验对其作用和机制进行研究。有望从TLR4-NF-κB信号→花生四烯酸代谢酶→炎症介质→巨噬细胞向M2型转化→肿瘤炎性微环境形成角度阐明甘草中抗肿瘤的黄酮类成分及网络调控机制，可为肿瘤治疗提供新的靶点和候选药物，因而具有重要的理论意义和临床价值。

肿瘤炎性微环境中巨噬细胞表型转化及炎症介质释放是炎症相关肿瘤发生发展的关键环节。寻找靶向肿瘤炎性微环境抗肿瘤的植物活性成分是当前肿瘤治疗研究的热点。本项目首先通过TLR4-NF-κB双荧光素酶报告系统和巨噬细胞药筛模型筛选得到具有显著抑制巨噬细胞TLR4-NF-κB活性的8种黄酮类化合物，其中FLA-16、新化合物A和异甘草素处理肿瘤相关巨噬细胞制备的条件培养液对肿瘤细胞的增殖具有更显著的抑制作用。另外，通过设计合成18种异甘草素衍生物，对其构效关系进行了研究，结果发现在2’位酚羟基对NF-κB活性影响不大，A环引入强供电基团后，显示出较强的抗NF-κB活性。在偶氮甲烷/右旋葡聚糖苷钠诱导小鼠结肠炎相关结肠癌中，研究发现异甘草素可通过阻断PGE2/PPARδ和IL-6/STAT3信号的交互作用抑制肿瘤相关巨噬细胞M2极化从而预防结肠炎相关结肠癌发生。在三甲基胆蒽诱发的大鼠肺癌模型中研究发现，异甘草素可通过抑制COX-2/PGE2和CYP4A/20-HETE介导的巨噬细胞M2极化从而预防肺癌发生。在MDA-MB-231人乳腺癌裸鼠移植瘤模型中发现，异甘草素高剂量（50和100 mg/kg）可显著抑制人乳腺癌生长；尽管异甘草素低剂量（10和20 mg/kg）对MDA-MB-231人乳腺癌的生长无抑制作用，但可通过下调PI3K/Akt信号抑制巨噬细胞M2极化从而显著降低乳腺癌自发性肺转移和实验性肺转移。最后，在大鼠C6胶质瘤模型、小鼠GL261胶质瘤模型和裸鼠U87胶质瘤模型中研究发现，FLA-16可正常化肿瘤血管，并显著延长荷胶质瘤鼠的生存期。综上所述，本项目在国内外首次从TLR4/NF-κB/STAT3信号→花生四烯酸代谢酶(COX-2、LOX和CYP4A)表达→炎症介质释放→巨噬细胞M2极化→肿瘤炎性微环境形成角度阐明了甘草黄酮类化合物（FLA-16、新化合物A和异甘草素等）的抗肿瘤作用及网络调控机制，为肿瘤治疗提供了新的靶点和候选药物，因而具有重要的理论意义和潜在临床应用价值。