怒江藤黄中 Nujiangexanthone A 抗肿瘤作用机制研究

Nujiangexanthone A (NJXA) is a novel compound isolated from Garcinia nujiangensis. Our previous study indicated that NJXA could induce G0/G1 cell cycle arrest in HeLa cell. Using two-dimensional gel electrophoresis, it was found that the expression of hnRNPK was down regulated in HeLa cells. HnRNPK is overexpressed in many human cancers and has been associated with a worse prognosis for patients. However, until now there is little report about the effect and mechanism of natural compounds on hnRNPK protein. In this proposal, we will study whether the anti-tumor effect of NJXA was induced by down regulation of hnRNPK based on cell death, cell cycle and other related biological methods; the mechanism of how NJXA down regulated the expression of hnRNPK protein will also be studied; at last, the in vivo experiment will be used to further elucidate how NJXA showed anti-tumor effect by down regulation of hnRNPK protein. The creativity of the researches can provide theoretical guidance for the comprehensive utilization of the compound, as well as lay a solid foundation for the development of new drugs with intellectual properties.

Nujiangexanthone A (NJXA)为本课题组首次从怒江藤黄中分离的新化合物，前期研究发现NJXA能诱导HeLa细胞G0/G1周期阻滞，并下调hnRNPK(heterogeneous nuclear ribonucleoprotein K)蛋白的表达。文献报道hnRNPK蛋白与细胞周期密切相关，且其在多种肿瘤患者体内表达升高。为了阐明NJXA下调hnRNPK蛋白与抗肿瘤之间的关系，本项目计划通过CRISPR/Cas9介导的基因敲除及质粒过表达等技术，考察NJXA是否下调hnRNPK蛋白而发挥作用；进一步通过RT-PCR， Western blot等分子技术阐明NJXA下调该蛋白的作用机制；最后通过动物实验，验证NJXA下调hnRNPK蛋白抗肿瘤作用的机制。目前关于天然药物对hnRNPK蛋白的作用及机制罕有报道，本研究为以该蛋白为靶标的抗肿瘤中药及其活性成分的研究开拓了新领域。

为了阐明NJXA下调hnRNPK蛋白与抗肿瘤之间的关系，本项目首先系统考察了化合物对相关周期蛋白的影响。Western blot实验表明NJXA能浓度和时间依赖性下调cyclins B1及E1，同时下调CDKs 2、4和 6。该结果显示NJXA降低hnRNPK蛋白的表达，进一步通过下调c-Myc-Cyclins/Cdks-Rb-E2F1通路，从而诱导细胞周期阻滞而发挥抗肿瘤作用。.分别利用CRISPR/Cas9及siRNA技术敲除细胞中hnRNPK蛋白，结果表明两种方法均能有效敲低该蛋白。细胞计数实验结果显示，当细胞中hnRNPK蛋白敲低后，细胞的生长被极大的抑制，同时化合物对CDK-6和cyclin E1抑制作用被增强。过表达实验显示，hnRNPK蛋白的过表达诱导了c-Myc和cyclin E1的表达。NJXA对hnRNPK、c-Myc及cyclin E1表达的抑制作用在过表达后也被降低。以上结果表明hnRNPK蛋白是介导宫颈细胞生长的关键蛋白，NJXA可能通过降低hnRNPK的表达来抑制细胞生长。.利用RT-PCR及Western blot考察化合物对hnRNPK蛋白合成与降解的影响。结果显示，NJXA处理后，细胞中hnRNPK mRNA水平保持不变，表明NJXA不影响hnRNPK的转录或mRNA的稳定性。而给药后hnRNPK蛋白的半衰期显著减少，同时泛素化水平显著提高。免疫共沉淀实验进一步证明，hnRNPK可以与泛素相结合，从而阐明NJXA通过加速蛋白降解而抑制了其表达。.采用裸鼠移植瘤模型，通过体重、瘤重、瘤体积及脏器器官形态综合评价NJXA抗肿瘤疗效。结果显示，给药组能显著降低裸鼠的瘤重及瘤体积，且对裸鼠的体重无影响。主要脏器未见细胞形态变化或出血。免疫组化方法结果表明化合物能下调肿瘤组织中Ki-67和hnRNPK蛋白的水平，CDK-6、cycline E1和c-Myc的表达水平亦被抑制。体内实验显示，化合物在裸鼠体内通过抑制hnRNPK蛋白的表达发挥抗宫颈癌的作用，并且对裸鼠没有明显的毒副作用。.本项目基本阐明了NJXA通过下调hnRNPK蛋白而发挥抗肿瘤作用的机制，相关研究成果发表SCI论文4篇，中文期刊论文1篇，出版学术专著 1 本，申请中国专利1项。为以hnRNPK蛋白为靶标的抗肿瘤天然药物研发提供理论依据，同时为怒江藤黄的综合应用和开发奠定基础。