线粒体靶向性萜类SIRT3调节剂的制备及其选择性抗NSCLC作用研究

The differences of mitochondria between tumor cells and normal cells are very good points to design selective anti-tumor drugs. Histone deacetylase SIRT3 in mitochondria, an important regulator of mitochondrial function, is closely related to tumor pathology. Recent study suggested that the regulation effect of SIRT3 on mitochondrial function is a new anti-tumor breakthrough. In non-small cell lung cancer (NSCLC) cell, SIRT3 acts as a tumor promotor, molecules that could down regulate SIRT3 level show anti-NSCLC effect. Our previous research showed that three natural anti-tumor terpenoids (triptolide, betulin and AKBA) could down regulate SIRT3 level in NSCLC cell. However, the anti-NSCLC potential of these compounds was limited by their selectivity, toxicity problems, etc. Based on the higher mitochondrial membrane potential of tumor cells, this project will firstly modify the structures of these natural compounds using mitochondrial targeting carrier; study systematically the mitochondrial distribution, cytotoxicity and selectivity to tumor cells of these derivatives to select strong anti-NSCLC compounds with good selectivity; then, study their anti-tumor mechanism based on the SIRT3 regulation on mitochondrial function; at last evaluate their anti-tumor activity and toxicity with in vivo animal experiments. This study will lay the foundation for developing effective and selective anti-NSCLC drugs from natural products, and also contribute to the knowledge of SIRT3 function, promoting greatly the application of SIRT3 in anti-tumor field.

利用肿瘤细胞与正常细胞线粒体的差异可设计选择性抗肿瘤药物。新近研究表明线粒体去乙酰化酶SIRT3是调控线粒体功能的关键酶，其在非小细胞肺癌（NSCLC）中显现肿瘤促进作用，下调SIRT3可发挥抗NSCLC作用。申请者前期研究发现具有抗NSCLC活性的萜类天然产物雷公藤甲素、白桦脂醇与乳香酸类AKBA可下调NSCLC细胞的SIRT3水平，却受限于选择性差、毒性大等问题。本课题拟利用肿瘤细胞线粒体膜电势较正常细胞高的特点，开展三个活性萜类化合物的线粒体靶向性结构修饰；通过系统分析衍生物的细胞毒性和在线粒体中的分布特点，获取抗NSCLC活性强、选择性高的化合物；基于SIRT3对线粒体功能调控机理揭示有效化合物抗NSCLC作用机制，并评价其体内抗肿瘤有效性与毒性。研究结果将为基于天然产物研发抗NSCLC活性强、毒副作用低的有效药物奠定理论基础，对推动SIRT3在抗肿瘤研究的应用有重要意义。

针对天然产物受限于选择性差、毒性大或作用强度不足等问题，本项目在前期研究基础上，继续利用肿瘤细胞线粒体膜电势明显高于正常细胞的特点，采用线粒体靶向载体对具有抗非小细胞肺癌（NSCLC）活性的萜类天然产物雷公藤甲素（TP）、白桦脂醇（BN）、乳香酸类AKBA、和厚朴酚进行修饰，设计并合成了11个系列70个不同结构类型的线粒体靶向性衍生物，通过系统分析衍生物的肿瘤细胞细胞毒性、选择性和在线粒体中的分布特点，优选出5个活性与选择性优于原天然产物的衍生物（Mito-TP-2、F9、P-5、6b和E-3-F16）进行了抗肿瘤作用机制探索，探究了其SIRT3调节作用；对潜力化合物利用体内模型进行了抗肿瘤活性与毒性评价。结果发现这些衍生物相比原天然产物，较好地积聚于线粒体，细胞水平很好的保持或提升了化合物的抗肿瘤活性（IC50从几十μM降至μM或nM级），并且相对正常细胞具有了一定的对肿瘤细胞的选择性，如F9、P-5、E-3-F16相对于正常细胞株Beas-2B或HBE，对一些NSCLC细胞株具有更强的细胞毒作用，选择指数为几倍或几十倍。这些衍生物可引起肿瘤细胞ROS升高、线粒体膜电势降低、线粒体MPTP孔道异常开放等线粒体紊乱现象。P-5与6b还引起肿瘤细胞空泡化现象，其诱导的细胞死亡与凋亡关系不密切，其可下调组蛋白去乙酰化酶SIRT3的表达水平，SIRT3是线粒体中的主要脱乙酰酶，在调节ROS和细胞氧化损伤方面起着至关重要的作用，SIRT3活性对维持线粒体稳态至关重要。斑马鱼体内实验模型进一步证实其抗肿瘤活性，雷公藤甲素衍生物Mito-TP-2与F9在斑马鱼模型实验中毒性比雷公藤甲素本身降低。本项目为基于天然产物的药物开发提供了研究基础与思路，同时推动了SIRT3调节剂抗肿瘤作用研究。