硫酸高乌甲素诱导肿瘤细胞凋亡的分子机制研究

It is an important approach to inhibit and destroy cancer by induction of tumor cell apoptosis. Lappaconitine which extracted from the root of Aconitum sinomontanum is a monoester-diterpenoid alkaloid and non-addiction analgesic new drug researched and developed by Northwest Normal University. The tablets and injection of lappaconitine hydrobromide are used clinically. Our studies demonstrated that the water solubility and analgesic effect of lappaconitine sulfate are better than that of lappaconitine hydrobromide. More meaningfully, the applicant found that lappaconitine sulfate could inhibit the growth of tumor cells, change the distribution of cell cycles, induce their apoptosis, and its antitumor activity in vitro is better than that of lappaconitine hydrobromide, but the studies of the molecular mechanisms for human tumor cell apoptosis induced by lappaconitine sulfate is still in blank all over the world. With the aid of the methods and techniques of modern cell biology and molecular biology in this project, the tumor cells with the strongest growth inhibition effect will be used as the research object based on our laboratory screening tests. Targeting on the PI3K/AKT/GSK3β, MAPK signal transduction pathways and mitochondria-mediated pathway, the influence on the signal transduction pathways and cell cycle checkpoint induced by lappaconitine sulfate will be researched so as to found the key protein factors or enzymes. The aim of the project is to reveal the molecular mechanisms of tumor cell cycle arrest and apoptosis which are induced by lappaconitine sulfate at cellular and molecular level. The tumor-burdened experimental animal model of mice will be established so as to study the antitumor mechanisms in vivo subsequently. Through the implementation of this project, we hope to provide an experimental foundation and theoretical basis for the development and ultilization of lappaconitine sulfate antitumor drugs.

通过诱发肿瘤细胞凋亡来抑制、消灭肿瘤已成为肿瘤治疗的一个重要策略。单酯型二萜类生物碱高乌甲素为非成瘾性镇痛新药，临床上用其氢溴酸盐。项目组研究发现硫酸高乌甲素（LS）的水溶性和镇痛效果优于氢溴酸高乌甲素，更有意义的是申请者初步研究发现LS可诱导肿瘤细胞凋亡，且其体外抗肿瘤活性优于临床用氢溴酸高乌甲素，但有关其作用机制的研究尚属空白。本项目拟在体外抗增殖和细胞毒性筛选的基础上，以LS对生长抑制最强的肿瘤细胞为研究对象，以PI3K/AKT/GSK3β和MAPK通路以及线粒体途径为靶点，运用现代细胞生物学和分子生物学的方法、技术和手段，研究LS对凋亡信号传导通路、细胞周期关卡等的影响，找到起关键作用的蛋白质因子或酶，期望从细胞和分子水平探究并揭示LS诱导肿瘤细胞周期阻滞和细胞凋亡的可能分子机制，并建立荷瘤小鼠动物模型开展在体抗肿瘤实验研究，以期为LS抗肿瘤药物的开发及应用提供实验依据和理论基础。

通过诱发肿瘤细胞凋亡来抑制、消灭肿瘤已成为肿瘤治疗的一个重要策略。单酯型二萜类生物碱高乌甲素为非成瘾性镇痛新药，临床上用其氢溴酸盐。项目组研究发现硫酸高乌甲素（LS）和盐酸高乌甲素（LH）的水溶性和镇痛效果优于氢溴酸高乌甲素，更有意义的是申请者初步研究发现LS和LH可诱导肿瘤细胞凋亡，且其体外抗肿瘤活性优于临床用氢溴酸高乌甲素，但有关其作用机制的研究尚属空白。本项目在体外抗增殖和细胞毒性筛选的基础上，以LS和LH对生长抑制最强的肿瘤细胞为研究对象，以PI3K/AKT/GSK3β和MAPK通路以及线粒体途径为靶点，运用现代细胞生物学和分子生物学的方法、技术和手段，研究了LS和LH对凋亡信号传导通路、细胞周期关卡等的影响，找到了起关键作用的蛋白质因子，从细胞和分子水平上探究并揭示了LS和LH诱导肿瘤细胞周期阻滞和细胞凋亡的分子机制，并建立荷瘤小鼠动物模型开展了在体抗肿瘤实验研究。研究结果表明，在体外和体内，LS通过PI3K/AKT/GSK3β信号通路诱导A549和HT-29细胞凋亡和G0/G1细胞周期阻滞，通过ROS的产生诱导HepG2细胞和异种移植模型动物的线粒体途径介导的细胞凋亡。LH可能通过诱导pRb通路介导Hela细胞发生G0/G1期周期阻滞，从而抑制Hela细胞的增殖，促进细胞凋亡；LH不仅能显著抑制HCT-116和HepG2细胞的生长，诱导细胞S期周期阻滞并通过线粒体和MAPK通路在诱导HCT-116和HepG2细胞凋亡中发挥重要作用。研究结果提示LS和LH可能是治疗人类结肠癌、肺癌、肝癌、宫颈癌的潜在候选药物，这些发现为LS和LH抗肿瘤药物的开发及应用提供了实验依据和理论基础。为了探究LS和LH的抗肿瘤效果及其机制，相关研究还需要进一步深化。