中药P-糖蛋白抑制剂的筛选及协同抗肿瘤机制的细胞代谢组学研究

That P-glycoprotein (P-gp) mediated drug efflux is the important reason which the tumor cells produce multi-drug resistance. The multidrug resistance is closely related to the development, invasion and metastasis of tumor. How to find reversal agents against tumor drug resistance, which have synergistic action with chemotherapy drugs on anti-tumor development and transfer, is very import to improve the curative effect of chemotherapy. In the project, the high-throughput screening method and verification mode of P - glycoprotein inhibitors will be established and they will be used for the screening and verification of P-gp inhibitors from Chinese medicine extracts/compounds. The mechanism of these new P-gp inhibitors will be clarified. Further, based on the tumor cell models, targeting metabonomics method will be used to investigate the anti-tumor mechanism of these P-gp inhibitors from Chinese medicine. The target molecules in targeting metabonomics analysis are related biomarkers with tumor signal pathways. Moreover, the activities of these P-gp inhibitors on anti-angiogenesis and invasion and metastasis of tumor will be studied using the co-culture cell model and the real-time analysis by MD-UPLC-MS/MS, and cellular metabolomics. Their inhibitory activities on related enzymes with glycolysis pathway will be screened. Based on the above research, the synergistic action mechanism of the P–gp inhibitors from Chinese medicine with chemotherapy drugs on anti-tumor will be clarified. Combined with the development of three-dimensional cell model, the anti-invasion and anti-transfer abilities will be investigated. These results will provide the scientific basis for research and development of new anti-tumor drugs with multiple targets and multiple ways.

P-糖蛋白介导的化疗药物外排是肿瘤细胞产生多药耐药性的重要原因。而多药耐药性与肿瘤的发展、侵袭转移密切相关。如何发现具有逆转肿瘤耐药性及协同抗肿瘤发展、转移多重作用的化合物，对于改善化疗疗效至关重要。本项目利用建立的P-糖蛋白抑制剂的高通量筛选及活性验证的细胞模型，从中药提取物/单体成分中获得机制明确的P-糖蛋白抑制剂，进一步以乳腺癌敏感及耐药细胞为模型，以肿瘤相关信号通路的生物标志物为靶分子，通过P-糖蛋白抑制剂的靶向代谢组学研究，以及与肿瘤血管生成、侵袭转移相关的二维细胞共培养模型，靶向代谢物的在线微透析-液/质联用实时分析及细胞相互作用的代谢组学研究，结合它们对糖酵解途径相关限速酶的酶抑制活性筛选，明确具有抑制P-糖蛋白表达作用的中药活性成分的协同抗肿瘤发展机制，结合三维肿瘤多细胞球体浸润模型，探讨其抗肿瘤侵袭转移的能力，为具有多靶点、多途径抗肿瘤作用的新药研发提供科学依据。

肿瘤多药耐药和侵袭转移是肿瘤化学治疗失败和高死亡率的主要原因。本项目以代谢组学和脂质组学分析方法为主要研究手段，在分子水平以及细胞水平上对MDR的发生机制进行深入研究，发现与MDR相关的17个潜在生物标志物，涉及甘油磷脂以及鞘脂代谢通路、花生四烯酸代谢通路以及抗氧化通路；发现MCF-7/ADR细胞拥有更为刚性的细胞膜、异常的调节细胞生长的信号转导通路、更强的抗氧化能力以及更快的能量代谢速率。针对糖酵解过程中的关键限速酶-乳酸脱氢酶（LDH）在许多肿瘤细胞中高表达，以LDH为靶分子，采用固定化酶方法从中药虎杖和大黄中筛选得到10余种LDH的抑制剂。基于MDCK-MDR1细胞模型，进行P-糖蛋白（P-gp）介导的MDR逆转剂的筛选，筛选得到具有1种中药活性提取物，15种活性单体。基于代谢组学和细胞生物学相结合的研究策略对活性成分-芦荟大黄素、熊果酸的逆转机制及抑制肿瘤侵袭转移作用机制进行深入研究。推测芦荟大黄素通过抑制P-gp的外排功能来发挥逆转作用。芦荟大黄素与阿霉素联用可抑制糖酵解、TCA循环、谷氨酰胺代谢及相关氨基酸合成途径。还可以导致MCF-7/ADR细胞G2/M期阻滞并通过DNA损伤、ROS生成、caspase-3激活诱导细胞凋亡。熊果酸通过作为P-gp的底物抑制P-gp介导的药物外排，同时抑制能量代谢途径以及相关氨基酸的合成，且联用效果优于阿霉素单用。 采用细胞共培养模型研究芦荟大黄素、大黄素、熊果酸对MCF-7细胞侵袭转移的抑制作用。发现芦荟大黄素可以抑制MCF-7细胞的黏附、侵袭及血管生成，生物标志物涉及多胺代谢、维生素B6代谢、甲硫氨酸循环、TCA循环、谷胱甘肽代谢、嘌呤代谢和天冬氨酸合成等通路。熊果酸能够抑制MCF-7/ADR的黏附、迁移以及浸润能力，推测熊果酸可能通过多胺代谢途径逆转MDR以及抑制耐药细胞的侵袭转移能力。本项目发表SCI 论文7 篇；获得吉林省科技进步一等奖1项；培养博士生3名，硕士生1名。