海洋中药毛蚶新多肽H3在乏氧微环境下抗肝癌作用及其机制研究

Hypoxic microenvironment plays a prominent role in the evolution of hepatic carcinoma. Developing new drugs with anti-tumor activity against hepatic carcinoma in hypoxic microenvironment becomes a critical strategy in the therapy of hepatic carcinoma. Arca subcrenata Lischke, a marine Traditional Chinese Medicine,, has many efficacies including blood enriching, strengthening and so on. H3 is a new polypeptide isolated from Arca subcrenata Lischke through modern chromatographic techniques in the preliminary stage of investigation by our team. In the recent study, H3 was testified to exhibit good stability and anti-proliferation on HepG2 cells with low toxicity on normal L02 hepatic cells. In hypoxic environment, the effect of H3 was markedly enhanced in the following aspects: ①anti-proliferation on HepG2 cancer cells (IC50 value decreased to 0.57μg/mL from 5μg/mL), ②induction of apoptosis (apoptotic rate increased to 34.3% from 16.7%), ③down-regulation of the expression of hypoxic induction factor-1α (HIF-1α) and suppression of the activation of AKT and ERK1/2. Based on the knowledge of that the signal pathways of PI3K/AKT and MEK/ERK are both important for the evolution of hepatic carcinoma in hypoxic environment, it is therefore speculated that H3 probably executed the resistance of hepatic carcinoma by regulating HIF-1α and the pathways of PI3K/AKT and MEK/ERK under hypoxic condition. This project aims to investigate the anti-hepatic carcinoma activity of H3 in hypoxic microenvironment and the underlying mechanism on the basis of our preliminary research. The project pioneers in the investigation of anti-hepatic carcinoma activity and the underlying mechanism of the polypeptide component of Arca subcrenata, which would provide scientific evidence for research in clinical applications.

乏氧在肝癌恶性发展中发挥关键作用，寻找乏氧下能特异抗肝癌的药物已成肝癌治疗的重要策略。毛蚶为传统海洋中药，具补血、温中等功效。课题组前期采用现代色谱技术从毛蚶中分离获得一个新多肽H3，并发现：H3具有较好稳定性及抗肝癌HepG2细胞增殖活性且对正常肝L02细胞毒较小；乏氧下H3抗HepG2增殖(IC50由5μg/mL降至0.57μg/mL)、诱导凋亡(凋亡率由16.7%增至34.3%)、抑制乏氧诱导因子HIF-1α表达及AKT、ERK1/2活化的作用显著增强。乏氧下PI3K/AKT与MEK/ERK通路对肝癌的发展起关键作用。基于此，我们推测H3可能在乏氧下调控HIF-1α及PI3K/AKT与MEK/ERK通路发挥抗肝癌作用。本项目拟在前期基础上研究H3在乏氧下抗肝癌作用及其机制。本项目系首次研究海洋动物类中药毛蚶之多肽成分抗肝癌及作用机制，结果可为毛蚶多肽抗肝癌研究与临床应用提供基础资料。

肝癌（ HCC）是致死率仅次于肺癌和胃癌的5大常见癌症之一， 严重威胁着人类生命健康。我国是肝癌重发区，全球每年新生肝癌病例有一半以上在中国，且仍呈上升趋势。乏氧是肝癌的一种重要特征， HIF-1 是介导肝癌乏氧反应的关键转录因子， 其在肝癌恶性侵袭转移过程中发挥关键作用。毛蚶是我国重要海洋贝类生物，性温味甘，归脾、肝经，具温中健胃、滋阴补血之功效，主治贫血、体虚等症。P2系于荣敏教授团队从毛蚶分离获得的抗癌多肽组分，研究显示P2具有良好抗肿瘤活性。H3系从P2中分离得到的新多肽。本研究表明：H3可显著抑制肝癌细胞生长，低氧下H3对肝癌中HIF-1的抑制作用更为显著；H3诱导肝癌凋亡及S和G2/M期阻滞，但对正常肝细胞毒性小；H3显著抑制肝癌AKT/mTOR通路，降低ATP生成；H3显著抑制VEGF诱导HepG2和HUVEC的细胞侵袭、转移及管腔形成；免疫荧光/共沉淀/RNAi/蛋白互助等实验表明VEGFR2为H3作用靶点；裸鼠移植瘤实验表明H3可抑制肝癌体内生长；血道转移模型表明H3能抑制鼠源肝癌H22血道转移、斑马鱼胚胎生成实验表明H3显著抑制血管生成。综上：H3具有良好抗肝癌生长、迁移、侵袭、转移及血管新生作用，VEGFR2可能是作用靶点，H3与VEGFR2结合，调控下游MAPK/PI3K/MMP2/MMP9信号通路，抑制肝癌生长。在本项目经费的资助下，已发表文章4篇，其中SCI文章3篇；在培养人才及青年科技人员方面取得良好成绩，申请人由中级职称晋升为高级职称，培养了1位博士和1位硕士研究生，均已顺利毕业。本项目研究结果不仅对从海洋中药中寻找抗肝癌药物，明确其抗肝癌作用机制及作用靶点有重要意义，而且能够为最终开发具有自主知识产权的高效、低毒、价廉的抗肝癌新药奠定重要的科学基础，以及为毛蚶的开发和应用提供重要的基础参考资料，为H3作为肝癌治疗药物的研究和开发提供重要基础研究资料。本研究可为毛蚶的研究与开发提供重要科学研究资料，有助于促进毛蚶的推广和应用，产生巨大的经济价值；H3抗肝癌药物的开发可为患者提供一种高效、低毒、机制与靶点明确的多肽类抗肝癌药物，可望带来巨大的经济价值和社会价值。