氧化亚铜纳米粒抑制恶性黑色素瘤转移的分子机制研究

Melanoma is highly aggressive, leaving untreated patients with a median survival of less than 12 months, which is difficult to cure by conventional therapy methods. In recent years, the incidence rate of melanoma has been increasing rapidly, and more than 20,000 new patients are diagnosed with melanoma every year in China. It is greatly necessary to develop new methods and new drugs. With the fast development of nanoscience and nanotechnology, many nanomedicines show anti-tumor pharmacological effects, which can target the tumor tissue and show low systemic toxicity. Nanomedicine has been emerging as one of these new treatment options. Combining the nanoscience and traditional Chinese medicine, our previous research firstly discovers that cuprous oxide nanoparticles (CONPs) show anti-melanoma pharmacological effects obviously. CONPs can inhibit the metastasis and invasion of melanoma in vitro and in vivo, which is a significant pharmacological character for the therapy of melanoma, and it is of great meaning to explore the anti-metastasis mechanism of CONPs. As we all know, tumor angiogenesis, expression of matrix metalloproteinase，circulating tumor cells, and epithelial to mesenchymal transition (EMT) are all involved with the progression of tumor metastasis. In order to explore the mechanism of anti-metastasis effects, the subcutaneous melanoma and metastatic lung tumors mice models are built. After treated with CONPs, subcutaneous melanoma, metastases on the lung, and the circulating tumor cells, which are enriched and separated from peripheral blood by the single cell extraction system, are collected for further research. Single cell qRT-PCR, western blot and some other advanced methods are used to explore the influence of CONPs on the molecules expression, which are associated with tumor angiogenesis, expression of matrix metalloproteinase, and EMT regulatory network. Furthermore, in order to discover the key molecules in the upstream of EMT regulatory network and reveal the signaling pathway related with the metastasis inhibition, the normal transcriptome sequencing and single-cell transcriptome sequencing are performed. Finally, operation specimens of human melanoma will be collected to further validate the anti-metastasis effects of CONPs and the key signaling pathway associated with metastasis inhibition. In this research, we will reveal the effects of CONPs on the whole progression of melanoma metastasis and discover the significant signaling pathways associated with anti-metastasis pharmacological effects, providing meaningful example for new inorganic nanomedicine research and development, and pushing forward the mechanism study on the anti-tumor properties of CONPs.

黑色素瘤高度恶性，具有极强的增殖和转移能力，常规治疗方法的效果均不理想。纳米抗肿瘤药物具有肿瘤靶向性等优点，为肿瘤治疗提供了新的思路。本课题组将中医学与纳米科学有机地结合起来，首次报道氧化亚铜纳米粒（Cuprous oxide nanoparticles，CONPs）可显著抑制黑色素瘤转移，并启动线粒体凋亡通路选择性诱导肿瘤细胞凋亡等原创性成果，揭示了CONPs在黑色素瘤治疗中良好的应用前景。在前期研究的基础之上，本项目将利用循环肿瘤细胞提取系统、单细胞转录组测序等先进方法对肿瘤原发灶、循环肿瘤细胞、肺转移灶相关生物学指标进行检测，探讨CONPs对肿瘤新生血管及上皮/间充质转化调控网络的干预作用，并发现CONPs作用的关键基因，以阐明CONPs抑制黑色素瘤转移的分子机制和作用的关键信号通路，为深入了解无机纳米药物抗肿瘤转移的机制及开发同类纳米抗肿瘤药物提供有意义的参考。

黑色素瘤高度恶性，具有极强的增殖和转移能力，常规治疗方法的效果均不理想。纳米抗肿瘤药物具有肿瘤靶向性等优点，为肿瘤治疗提供了新的思路。本项目从临床实际需要出发，选择氧化亚铜纳米粒（Cuprous oxide nanoparticles， CONPs）为研究对象，利用体外培养细胞、荷瘤小鼠等模型，系统的开展了其药效学、药代学、毒理学、药理学等方面的研究。.研究发现CONPs可以通过抑制黑色素瘤细胞EMT进程以及黑色素瘤干细胞的干性，抑制黑色素瘤的增殖以及转移过程，同时发现CONPs可以通过网格蛋白介导的胞吞进入细胞，并且表现出CD271阳性黑色素瘤瘤干细胞的选择性。由于无机纳米药物理化性质独特，非特异作用因素复杂，这就导致无机纳米药物的作用机制研究困难。黑色素瘤、肾癌等多种肿瘤的铜元素代谢转运发生紊乱，CONPs作为含亚铜原子的无机纳米材料，可能会参与铜元素代谢及转运的调控。本项目研究发现CONPs可同时诱发内质网应激以及线粒体细胞凋亡，结合该无机纳米药物含有铜元素的结构基础和表达谱测序，发现CONPs可通过干扰铜元素伴侣蛋白ATOX1和CCS对铜元素的转运，同时构建ATOX1-CCS敲低的工具肿瘤细胞系，发现CONPs对铜元素代谢非依赖的细胞系的杀伤作用显著降低，这就暗示了CONPs通过干扰ATOX1和CCS介导的铜元素转运起到肿瘤杀伤作用的。本研究完整地发现了CONPs干预细胞内铜元素转运，进而诱导线粒体及内质网的含铜元素蛋白的合成障碍和未成熟蛋白的堆积，进而激活相关凋亡信号通路的完整机制，为拓展无机纳米药物作用机制研究领域提供新视角；研究也表明无机纳米药物具有特异性的生物学作用机制，为丰富对无机纳米药物的理解提供重要支撑。