从抑制HIF-1α介导糖酵解角度探讨疏花蛇菰三萜组分抗肝癌作用及机制

Balanophora laxiflora is used to treat primary hepatocellular carcinoma clinically. Previous research found that Balanophora laxiflora triterpene constituents regulated the balance of oxidative phosphorylation/glycolysis of hepatoma carcinoma cells, decreased the hepatoma cells proliferation by inhibiting HIF-1α protein expression. We speculated that Balanophora laxiflora triterpenoid constituents inhibited hepatocellular carcinoma by inhibiting HIF-1α-mediated tumor cell glycolysis. The program focuses on HIF-1α signaling and degradation, and try to build up DEN-induced rat liver cancer model and use hepatocellular carcinoma cell lines under hypoxia. MRI, MicroPET, siRNA, CHIP, and Luciferase Reporter Assay will be applied to reveal the characteristic of protein expression of glycolysis of liver cancer cells and displayed the association between “HIF-1α—glycolysis—DEN induced- liver cancer progression”. The differentially expressed key targets were screened by suspension microarray technology. To explore the effect that Balanophora laxiflora exerted on degradation of HIF-1α and glycolysis and clarify its anti-liver cancer molecule mechanism. The present project could provide support for the clinical use of Balanophora laxiflora as well as a new perspective for the treatment of liver cancer.

中药疏花蛇菰主要用于临床原发性肝癌的治疗。前期研究表明疏花蛇菰三萜组分调节肝癌细胞能量代谢方式--氧化磷酸化/糖酵解的平衡，并通过抑制HIF-1α蛋白表达阻滞肝癌细胞增殖。我们推测：疏花蛇菰三萜组分可能通过干预HIF-1α通路介导的糖酵解抑制肝癌病理进程。本研究围绕HIF-1α信号转导和降解途径，拟采用化学诱导的大鼠肝癌模型以及低氧环境下的肝癌细胞株，结合MRI、MicroPET、siRNA干扰、CHIP、荧光素酶报告基因等方法，揭示三萜组分干预肝癌细胞糖酵解相关蛋白的改变特征，挖掘“HIF-1α、糖酵解、化学诱导的肝癌病理进程”三者关联性；采用悬浮芯片技术筛选差异表达的关键靶标，深入探索三萜组分对HIF-1α通路及糖酵解的干预作用，阐明三萜组分治疗肝癌的作用机制，为疏花蛇菰的临床应用提供支撑，为肝癌治疗提供新的视角。

本项目基于肿瘤细胞特有的Warburg效应及低氧诱导因子1α（hypoxia-inducible factor1α，HIF-1α）对糖酵解的调控机制，重点围绕肝癌HIF-1α信号转导和降解途径，研究疏花蛇菰三萜通过HIF-1α影响肝癌细胞的糖代谢及关键糖代谢因子的作用机制。首先我们利用大孔树脂富集疏花蛇菰总三萜，液相色谱/质谱(Q-TOF/MS)鉴定其主要成分。建立DEN诱导的大鼠原位肝癌模型，通过核磁共振和病理分析证实其发病进程符合原发性肝癌的临床特征（如胆管扩张、腺样结构、肿瘤坏死和出血等）。Spearman相关性分析显示大鼠肝癌结节数量、HIF-1α、HKII三者有相关性。疏花蛇菰三萜对DEN诱导的肝癌有抑制作用，同时减少肝脏组织中HIF-1α、糖酵解关键酶GLUT1和HKII的表达。体外实验中疏花蛇菰三萜显著抑制肝癌细胞葡萄糖摄取、乳酸生成、ECAR（胞外酸化率）。缺氧条件下HepG2细胞HIF-1α、LDHA、HKII上调，HIF-OH下调，疏花蛇菰三萜调控上述变化，抑制HIF-1α的核转位及其与VEGF启动子HRE的结合。siRNA干扰、质粒转染、EMSA、免疫共沉淀等实验证实疏花蛇菰三萜可能通过PHD-VHL-HIF-1α途径和调控线粒体中乙酰化酶SIRT3、SOD2诱导HIF-1α降解，继而抑制下游靶基因糖酵解关键酶LDHA、HKII，导致糖酵解作用被抑制。此外，疏花蛇菰三萜中活性成分羽扇豆醇和蛇菰素B具有较强的体内外抗肿瘤作用，蛇菰素B可能通过泛素-蛋白酶体途径诱导HIF-1α降解抑制HepG2细胞增殖与糖酵解。本项目对于中药组分的临床应用与进一步开发具有一定的积极意义，有望开发成为肝癌治疗的药物；也为筛选靶向肿瘤能量代谢的天然产物研究提供有益尝试。