去甲基斑蝥素酸镁抗肝癌活性的分子机制研究

Metal-based anticancer drugs play a crucial role in the chemoprevention of cancer. The findings of our previous research showed that magnesium significantly improved the antitumor activity of cantharidin from Mylabris. As an extention of our previous research, we use norcantharidin with far below toxic side effects than cantharidin as the parent structure to synthesize magnesium norcantharidate (M-NCTD).We found M-NCTD showed better antitumor activity than cantharidin.However, the reasons for M-NCTD’s enhancement in anti-cancer activity were not clear. On this basis,this study attempt to further investigate the antitumor activity of M-NCTD in vitro and in vivo and compare it with other norcantharidin metal-derivatives. On the one hand,we intent to identify the effect of M-NCTD on the protein phosphatase 2A (PP2A) from the perspective of ability to inhibit PP2A and molecular dynamics simulation. On the other hand, we will use digital gene expression profile to look for differentially expressed genes in liver cancer before and after treatment by M-NCTD, while make use of cell-based high content screening(HCS) to abtain the key response gene for hepatocarcinoma cell proliferation before and after candidate gene function interference. Finally, functional verification of these key response genes will be tested at cell and animal level.In a word, our study will not only provide a theoretical rationale for anti-hepatoma activity of M-NCTD, but also provide examples for the subsequent development of magnesium-based anti-cancer drugs which are very rare in market.

金属抗肿瘤药物在肿瘤的化学预防中发挥着至关重要的作用。我们的前期研究发现镁元素可显著提升昆虫斑蝥体内斑蝥素的抗癌活性。受此启发，我们以毒性远低于斑蝥素的去甲基斑蝥素为母核，合成了去甲基斑蝥素酸镁，发现其抗癌活性较去甲基斑蝥素显著增强，但详细分子机制不明。本研究拟在此基础上进一步探讨去甲基斑蝥素酸镁体内体外抗肝癌效应并与其它去甲基斑蝥素的金属衍生物进行比较；一方面从磷酸酶PP2A活性抑制和分子模拟动力学角度，考察去甲基斑蝥素酸镁对PP2A的影响；另一方面利用数字化基因表达谱测序技术寻找肝癌细胞在去甲基斑蝥素酸镁处理前后差异表达的基因，并利用高内涵筛选的方法，挖掘候选功能基因干扰前后对肝癌细胞增殖明显变化的关键应答基因，并将关键应答基因进行细胞和动物水平的相关功能学验证。本研究将为阐明去甲基斑蝥素酸镁的抗肝癌科学内涵提供理论依据，同时也为市场上鲜见的含“镁”元素的抗癌药物研发提供范例。

金属抗肿瘤药物在肿瘤的化学预防中发挥着至关重要的作用。我们前期研究发现镁元素在抗癌中药成分斑蝥素的功能活性方面扮演着重要角色。以此为线索，课题组合成了活性更强、毒副作用更低的去甲基斑蝥素酸镁，并挖掘其抗肝癌活性的关键应答基因。通过构建DEN诱导的小鼠原发性肝癌模型，在进一步明确去甲斑蝥素酸镁具有体内抗肝癌活性的基础上，课题组利用基因芯片技术对去甲基斑蝥素酸镁干预肝癌SMMC-7721细胞前后基因的差异表达进行了研究。然后利用生物信息学结合文献调研的方法筛选出20个备选基因；紧接着构建了用于沉默这20个基因的mix慢病毒；最后利用这20个病毒去开展高内涵功能基因筛选工作；最终得到了一个关键应答基因DDX23。将DDX23敲减后，肝癌细胞SMMC-7721增殖速度变慢，凋亡数显著增多，细胞克隆形成的能力明显减弱，说明为功能基因。对DDX23在不同肝癌细胞株中的表达进行了检测，结果显示丰度都比较高，具有一定的普遍性。在对 DDX23 的肝癌临床相关性进行了分析后发现 DDX23 在肝癌样本中高表达，且高表达 DDX23 的患者生存期明显小于低/中表达的患者，说明该基因具有一定的临床相关性和潜在的临床价值。此外，课题组还发现去甲基斑蝥素酸镁可以通过激活FOXO1转录因子抑制肝癌细胞的侵袭和转移；并发现去甲基斑蝥素酸镁对原发性肝癌的一线治疗药物索拉菲尼具有增敏的作用。本研究将为阐明M-NCTD的抗肝癌科学内涵提供理论依据，同时也为M-NCTD后期的新药开发打下坚实的基础。