【作者】 王莉；

【导师】 裘云庆；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2019， 博士

【摘要】 肝细胞肝癌是最为常见的肝脏原发肿瘤,每年约有74.55万人因肝癌死亡,具有发现迟,恶性程度高等特点,且肝癌目前是世界上癌症致死率最高的肿瘤之一。索拉非尼是目前临床用于肝癌治疗的唯一一线靶向分子药物,研究表明其能增加晚期肝癌患者的总体生存率,但存在价格昂贵、毒副作用发生频繁、个别患者发生耐药等情况。因此,研究具有自主知识产权的新型肝癌治疗药物迫在眉睫。BZG是一种化学结构与索拉非尼相似的多靶点酶抑制剂,我们前期药代/药动学研究表明,BZG可以快速而广泛地分布到动物脏器和组织,且BZG可以有效抑制多种肝癌细胞的生长。因此考虑到索拉非尼的毒副作用与耐药情况,本文拟探讨BZG、BZG与索拉非尼联合用药的体内外抗肝癌作用以及BZG抗肝癌的作用机制,为BZG以及BZG联合用药开发提供依据。关于BZG在体内的主要存在形式、代谢情况以及参与介导的代谢酶等目前尚未阐明,因此本文拟同时应用体内外代谢模型研究BZG在体内的代谢过程,分析其代谢产物的生物活性。方法:1.通过CCK-8法和细胞克隆形成实验观察BZG及BZG与索拉非尼联合用药对肝癌细胞Hep3B、SMMC-7721、Hep G2、Huh-7存活率的影响。分别通过Hoechst33342染色法、流式细胞术检测药物作用后细胞凋亡情况。通过流式细胞术检测药物对细胞周期的影响。通过Western blot法分别检测药物作用于Hep3B、SMMC-7721、Hep G2、Huh-7细胞后细胞内蛋白AKT、p-AKT、ERK、p-ERK、PI3K、m TOR、p-m TOR、RAF1及VEGFR-3的表达情况。2.建立Huh-7细胞裸鼠皮下异种移植瘤模型,观察BZG及BZG与索拉非尼联用后对裸鼠肿瘤体积、重量的影响。通过HE染色和TUNEL法观察组织坏死和肿瘤细胞凋亡等情况。3.采用UPLC-QTOF MS,应用体内动物模型、体外人肝微粒体、以及重组酶等代谢模型分析BZG的代谢情况,鉴定参与介导的代谢酶。4.利用分子对接软件e Hi Ts对BZG及其代谢产物与分子靶点VEGFR-2的亲和力进行分析,推测代谢产物可能的生物活性。结果:1.BZG可以明显降低肝癌细胞Hep3B、SMMC-7721、Hep G2、Huh-7的存活率,且呈一定的时间、剂量依赖性,另外高浓度BZG抑制Hep G2细胞存活的作用明显优于其他3种肝癌细胞。BZG与索拉非尼联合用药效果明显比BZG或索拉非尼单用效果好。2.BZG可以有效促进Hep3B、SMMC-7721、Hep G2、Huh-7细胞发生凋亡,除SMMC-7721细胞外,BZG对其他三种肝癌细胞促凋亡作用呈剂量依赖性,且BZG和索拉非尼联用后能够更有效促进人肝癌细胞的凋亡。3.单药BZG与索拉非尼在不同类型肝癌细胞中抑制蛋白表达作用不同,两药联合后可以更有效地抑制p-AKT的表达。也可不同程度抑制PI3K、p-m TOR、RAF1蛋白的表达。4.在荷Huh-7裸鼠异种移植模型中,随着BZG浓度的增高,荷瘤裸鼠的肿瘤重量和体积均下降,且BZG与索拉非尼联合应用效果明显比单独使用BZG或索拉非尼更优。在肿瘤病理组织中还可以观察到组织坏死和大量凋亡细胞。5.BZG发生了广泛的I相和II相代谢反应,共生成11种代谢产物(M1-M11),其中BZG在h CYP1A2,h CYP2B6,h CY2C19和h CYP2C8作用下发生了羟基化反应,在h UGT1A9作用下发生了葡萄糖醛酸化反应。6.BZG代谢产物M2-3和M9-11与VEGFR-2的亲和力比索拉非尼高。结论:1.BZG能够有效抑制人肝癌细胞Hep3B、SMMC-7721、Hep G2、Huh-7的细胞增殖,也能够促进肝癌细胞发生凋亡,且能够有效增加索拉非尼的抗肿瘤效果。BZG与索拉非尼联用后通过AKT/m TOR信号通路促进肿瘤细胞发生凋亡。2.通过BZG的I相和II相代谢反应产物,推测BZG的主要代谢途径包括羟基化、葡糖醛酸化、乙酰化、磺化和降解反应,其中h CYP1A2,h CYP2B6,h CY2C19和h CYP2C8参与了BZG的羟基化反应,h UGT1A9参与了BZG的葡萄糖醛酸化反应。通过计算机拟合技术发现BZG代谢产物M2、M3和M9、M10、M11比母体BZG更能有效抑制VEGFR-2。更多还原

【Abstract】 Background and Objectives Hepatocellular carcinoma(HCC)is the most common primary tumor of the liver and also one of the most deadly cancers in the world.Moreover,liver cancer is highly malignant and difficult to detect.Currently,sorafenib is the only targeted molecular drug for the first-line treatment of advanced hepatocellular carcinoma.Though it increases the hope of patients with advanced hepatocellular carcinoma,it’s overall survival rate is still low and does have some severe side effects.Therefore,it is very critical to improve new therapeutic drugs for hepatocellular carcinoma.BZG is a multi-targeted enzyme inhibitor with a similar chemical structure to sorafenib,and our previous pharmacokinetic studies have shown that BZG can be rapidly and widely distributed to organs and tissues of animal models.Besides,BZG could inhibit a panel of human cancer cells’ proliferation.Therefore,this research intends to explore the mechanism of BZG alone and also BZG with sorafenib on cell proliferation and cell apoptosis in vitro and in vivo.In addition,to study the process of drug metabolisms of BZG and analyze its active metabolites from the metabolic model in vivo and in vitro.Methods 1.CCK-8 assay and cell cloning formation were performed to evaluate the cell viabilities of hepatocarcinoma cells Hep3 B,SMMC-7721,Hep G2 and Huh-7 after treatment of BZG alone and BZG with sorafenib.Cell apoptosis were detected by Hoechst 33342 staining and flow cytometry.The effects of BZG and sorafenib with BZG’s co-treatment were also done by flow cytometry.The expressions of AKT,p-AKT,ERK,p-ERK,PI3 K,m TOR,p-m TOR,RAF1 and VEGFR-3 were detected by western blot.2.Huh-7 cell xenografts were used to assess the effects of BZG and BZG with sorafenib’s co-treatment in vivo.Cell apoptosis and tissue necrosis of the tumors were observed by HE staining and TUNEL separately.3.UPLC-QTOF MS was used to analyze metabolites of BZG through the metabolic model in vivo,and human liver microsomes and recombinant enzymes in vitro.4.Molecular docking software e Hi Ts was used to analyze the affinity of BZG and its metabolites to the molecular target VEGFR-2.Results 1.BZG can significantly reduce the viability rate of Hep3 B,SMMC-7721,Hep G2 and Huh-7 in a time and dose-dependent manner.Besides,the high concentration of BZG could dramatically inhibit the survival of Hep G2 cells compared to the other three kinds of HCC cells.BZG combined with sorafenib has significantly better efficacy than BZG or sorafenib alone.2.BZG can effectively promote the apoptosis of Hep3 B,SMMC-7721,Hep G2 and Huh-7 cells.However,BZG has a dose-dependent manner on the other three kinds of liver cancer cells except for SMMC-7721 cells,and moreover,BZG combined with sorafenib could more effectively promote the apoptosis of human liver cancer cells compared with BZG or sorafenib alone.3.BZG and sorafenib have different inhibitory effects on protein expression of Raf/ ERK and PI3K/AKT/m TOR pathways in four types of liver cancer cells.The combination of BZG and sorafenib could inhibit the expression of p-AKT more effectively and can inhibit the expression of PI3 K,p-m TOR,Raf1 and p-ERK to some extent.4.With the increasing concentration of BZG,the weights and volumes of the tumors of Huh-7 xenografts were significantly decreased.Besides,the anti-tumor outcomes of BZG and sorafenib’s co-treatment were significantly better than that of BZG or sorafenib alone.Tissue necrosis and a large number of apoptotic cells were also observed in the tumors.5.BZG generated a total of 11 metabolites(M1-M11)through phase I and phases II metabolic reactions in vivo and in vitro.A screening of Cytochrome P450(CYP)enzymes confirmed that h CYP1A2,h CYP2B6,h CY2C19 and h CYP2C8 exhibited hydroxylation activity and h UGT1A9 exhibited glucuronidation activity.6.The affinities of BZG’s metabolites M2,M3,M9,M10 and M11 to VEGFR-2 was higher than sorafenib.Conclusion 1.BZG can effectively inhibit the proliferation of human hepatocellular carcinoma cells such as Hep3 B,SMMC-7721,Hep G2 and Huh-7,and could promote the apoptosis of hepatocellular carcinoma cells.Besides,BZG can effectively enhance the anti-tumor effects of sorafenib.Through the AKT/m TOR signaling pathway,BZG and sorafenib’s co-treatment promotes the apoptosis of hepatocellular carcinoma cells.2.Through the analysis of the BZG’s metabolites of phase I and phase II,it is speculated that the major metabolic pathways of BZG may include hydroxylation,gluconate acidification,acetylation,sulfonation and degradation.And h CYP1A2,h CYP2B6,h CY2C19 and h CYP2C8 exhibited hydroxylation activity and h UGT1A9 exhibited glucuronidation activity.The metabolites M2,M3,M9,M10 and M11 are more effective in inhibiting VEGFR-2 than their parent BZG.更多还原