基于功能蛋白组学和超滤质谱技术研究石蒜抗肝癌的作用机制与化学物质基础

Hepatocarcinoma is one of the most frequent malignant tumors. Although numerous synthetic medicines have been approved in clinical use for the treatment of hepatocarcinoma, most of them may lead to serious side effects. While the traditional Chinese medicines (TCMs) have significant efficacy for the treatment of hepatocarcinoma, and thus attracted considerable interest nowadays. Our recent studies provide evidences that Lycoris radiata showed good activity against hepatocarcinoma. However, its overall anti-tumor mechanisms of action and the responsible compounds in Lycoris radiata have remained elusive, and poorly studied due to its complex multi-component , multi-target feature and lack of effective strategies to date. In this context, we propose to develop and apply a new strategy for exploring the overall anti-hepatocarcinoma mechanisms of action and active compounds in Lycoris radiata based on functional proteomics and ultra-filtration LC-MS. Firstly, we prepare different effective fractions in Lycoris radiata and evaluate their inhibitory activity against Hep G2 cells. Secondly, we select the hepatocarcinoma animal models to investigate the effects of Lycoris radiata on overall proteomic changes using deuterium isobaric amine reactive tag (DiART) labeling based quantitative proteomics and subsequent bioinformatics analysis, in this way we could identify and predict multiple molecular targets related to hepatocarcinogenesis or affected by the treatment with Lycoris radiata. we then further develop ultra-filtration liquid chromatography-mass spectrometry assay to screen for the active compounds in Lycoris radiata using the protein targets verified in the aforementioned proteomic study. Finally, the interaction networks between the active compounds and their respective targets could be constructed, thus the mechanisms of action and active compounds in Lycoris radiata for the treatment of hepatocarcinoma could be elucidated.

原发性肝癌是一种严重威胁人类健康的恶性肿瘤。尽管已有不少合成药物用于治疗肝癌，但多数药物导致明显的毒副作用。近年来，传统中药治疗肝癌疗效显著并引起广泛关注。我们最近研究发现石蒜有显著的抗肝癌活性，然而目前对其抗肝癌的整体作用机制和化学物质基础的研究仍十分薄弱。本项目拟发展基于功能蛋白质组学及亲和超滤质谱技术的新方法用于中药石蒜抗肝癌的作用机理和物质基础的研究。首先，利用植物化学方法结合体外肝癌细胞活性评价，获得石蒜抗肝癌的有效组分；其次，应用定量蛋白质组学方法，分析肝癌造模前后及石蒜有效组分干预肝癌动物模型前后表达差异的蛋白，鉴定肝癌发病相关蛋白及石蒜抗肝癌的关键目标蛋白；在此基础上，采用超滤质谱技术，以前述的目标蛋白为靶点，筛选并鉴定石蒜抗肝癌的活性化合物。最后，通过揭示化合物作用的蛋白靶点，构建石蒜抗肝癌活性化合物的靶点蛋白网络，阐明中药石蒜抗肝癌的化学物质基础和作用机制。

原发性肝癌是一种严重威胁人类健康的恶性肿瘤，中药石蒜对其有显著疗效。该项目针对中药石蒜抗肝癌的整体作用机制和化学物质基础的研究仍十分薄弱的难题，发展了基于功能蛋白质组学及亲和超滤质谱技术等方法用于解决这一挑战。首先，利用植物化学方法结合体外细胞活性评价，中药石蒜经过MCX固相萃取柱制备的总生物碱对肝癌细胞有最高的抑制率为84.91%，加兰他敏型、石蒜碱型和高石蒜碱型生物碱，为石蒜总生物碱中最主要的3种类型生物碱，分别为占总生物碱的40.1% 、 26.9%和20.0%；其次，应用定量蛋白质组学方法，对石蒜生物碱处理前后的实验组和对照组样本中进行了功能蛋白组学研究，共鉴定到位于3804个蛋白上的13411个磷酸化位点，其中3366个蛋白的11317个磷酸化修饰位点包含定量信息，以实验组/对照组的差异变化超过2或小于1/2作为显著上调或下调阈值，并同时符合p 小于0.05的蛋白，得到上调的蛋白249个，下调的蛋白93个，随后对这些显著差异蛋白进行进一步基因注释和信号通路分析，最终筛选出了44个候选蛋白；并发现石蒜生物碱可通过调控肝癌细胞P38-MAPK、GSK3B、AMPK-TSC1/2-ATK1S1-mTOR-GRB10和DNA TOP1等多个信号通路以及细胞周期相关蛋白CDC25B/CDK7/CDK1等的磷酸化水平而发挥其抑制肝癌细胞增殖的作用。在此基础上，以其中一个目标蛋白TOP1为靶点采用超滤质谱技术筛选并鉴定出石蒜抗肝癌的活性化合物11个，其中一个高活性生物碱化合物小星蒜碱未经修饰下表现出与临床抗癌药物喜树碱相当的活性，据此推断其可能是中药石蒜抗肝癌的最主要活性成分之一。上述筛选结果也说明中药石蒜是通过多个生物碱组分调控上述多个信号通路发挥其抗肝癌作用，并具有开发抗肝癌中药新药巨大潜力。