天然新型甲基化放线菌素D下调mtTFA诱导肝癌细胞凋亡的分子机制

Heptocellular carcinoma (HCC) is a kind of malignant tumor which severely threatens public health. However, the clinical outcomes of anti-HCC drugs currently available are not so satisfied. 2-aminomethyl-Actinomycin D (mAct D), a novel actinomycin D analog, was first purified from Ganoderma endophytic treptomyces in our previous study. mAct D exhibited a significant inhibitory effect on several human cancer cell lines and lower toxicity on normal cells. Meanwhile, mAct D could downregulate protein levels of mitochondrial transcription factor A (mtTFA)and induced cell apoptosis in HCC cells and in vivo. Overexpression of mtTFA decreased cell mortality induced by mAct D, indicating that mAct D induce cell apoptosis by downregulating mtTFA protein expression in the heptocellular carcinoma cells. To test this hypothesis, we investigate that mtTFA effect on the apoptosis of HCC cells induced by mAct D at cell cultured in vitro level combined with animal models. To definite the molecular mechanism of mAct D inducing HCC cells apoptosis by downregulating the expression of mtTFA, the expression of related protein in apoptosis signaling pathway and mitochondrial metabolic pathway and the change of mitochondrial functional structure was detected. In addition, the mode of action and expression regulation mechanism of mAct D downregulating of mtTFA was clarified from the biochemical mechanism. These studies will not only lay a solid pharmacology foundation for identifying new natural anti-cancer drugs mAct D targeting clear HCC cells, but also provide reliable experimental and theoretical basis for discovering new mAct D target.

肝癌严重威胁人类健康，但现有药物疗效并不理想。课题组首次从灵芝内生菌中发现抗癌药物放线菌素D的新型衍生物（mActD）；mActD具有高抗肿瘤活性、低细胞毒性等优点；在体外和裸鼠体内均显著下调线粒体转录因子（mtTFA）蛋白的表达，促进细胞凋亡；过表达mtTFA可削弱mActD对肝癌细胞存活的抑制作用，提示其可能通过下调mtTFA发挥促凋亡功能。为验证该假说并研究其分子机制，我们拟从细胞水平结合动物模型研究mtTFA对mActD诱导肝癌细胞凋亡的影响；同时检测凋亡通路、线粒体代谢通路相关因子的表达变化以及线粒体功能结构的变化，明确mActD下调mtTFA诱导肝癌细胞凋亡的作用机理；并从生化机制上阐明mActD下调mtTFA的作用方式及表达调控机制。本研究为明确新型天然抗癌药物mActD靶向清除肝癌细胞奠定坚实的药理基础，也为发现新的抗肿瘤靶标提供可靠地实验和理论依据。

肝癌严重威胁人类健康，但现有药物疗效并不理想。mAct D是课题组前期发现的一种天然新型甲基化放线菌素D衍生物，具有开发肝癌新药的潜力。本报告探讨线粒体转录子A（mtTFA）在mAct D诱导肝癌细胞过程中的作用及其分子机制，旨在系统研究mAct D诱导肝癌细胞的作用机理。通过构建高表达和低表达mtTFA的重组质粒筛选稳定表达的细胞株作为研究材料分析其功能，结果显示，mtTFA高表达在一定程度上可逆转mAct D的促凋亡作用，但是该逆转效果随着mAct D浓度的增加会逐渐降低，本报告中并没有筛选到稳定低表达mtTFA的细胞株，说明mtTFA本身的低表达对于肝癌细胞的增殖其重要作用；当低表达重组质粒和mAct D共同处理细胞是可协同促进肝癌细胞的凋亡。Western blot 和试剂盒检测发现mtTFA主要是通过调节线粒体内源性凋亡信号通路中的关键蛋白发挥作用，如：p53、Bax、PARP、Bcl-2、Caspase 9等；另外，mtTFA对于线粒体的功能的正常发挥有重要作用，主要是调控线粒体中ROS的产生以及线粒体膜电位的去极化等氧化应激方面发挥作用，将低表达mtTFA重组质粒VRS-3与mAct D共同作用HepG2细胞，发现mtTFA可协同mAct D增加线粒体中ROS的产生以及线粒体膜的通透性，最终导致细胞的凋亡。Western blot检测显示mtTFA可明显激活MAPK信号通路中ERK1/2、JNK1/2 及p38 的磷酸化参与氧化应激过程来调节mAct D诱导肝癌细胞凋亡的过程。本报告显示mtTFA在肝癌细胞的增殖及凋亡过程中具有重要的作用，通过调控线粒体内源性信号通路和MAPK信号通路调节mAct D的抗肝癌活性，可做为潜在的调节肝癌发生、发展的药物作用靶标分子。项目资助发表论文7篇，其中SCI论文3篇，待发表2篇。培养硕士研究生2名，其中1名已经取得硕士学位。项目投入17万元，支出12.2096万元，各项支出基本上与预算相符。剩余经费4.7904万元计划用于本项目的后续支出。