金银花杂合黄酮通过调控NRF2介导的铁死亡逆转肝癌索拉非尼耐药的作用及机制研究

Sorafenib is the standard first-line theatment for patients with advanced hepatocellular carcinoma (HCC). However, the emergence of drug resistance in HCC makes the clinical therapeutic effect of sorafenib is not ideal, and it is very urgent to develop new drugs to reverse sorafenib resistance in HCC. Inhibition of ferroptosis is closely related to the development of sorafenib resistance in HCC. Considering the important role of NRF2 in the regulation of ferroptosis, using NRF2 as a target molecule to study the reversal of sorafenib resistance in HCC has a broad application prospect. In our previous work, a series of flavonoids, including six new compounds, were isolated and identified from Lonicera japonica flower buds. In addition, we found that L. japonica flower buds flavonoids had significant inhibitory activity against sorafenib-resistant HCC cells for the first time, and confirmed that it may play a role in enhancing the ferroptosis sensitivity of sorafenib-resistant HCC cells by inhibiting the activity of NRF2, but its mechanism and structure-activity relationship remain to be further explored. This project intends to further isolate and identify the flavonoids from L. japonica flower buds, evaluate its inhibitory activity against sorafenib-resistant HCC cells, study its regulatory mechanism and structure-activity relationship of NRF2, and elucidate the mechanism of L. japonica flower buds flavonoids regulate the ferroptosis sensitivity of sorafenib-resistant HCC cells by inhibiting NRF2, to provide theoretical support for the development of the new drugs to reverse sorafenib resistance in HCC.

索拉非尼是进展期肝癌患者的标准一线疗法，但肝癌耐药的出现致使其临床效果并不理想，研制克服肝癌索拉菲尼耐药药物已十分迫切。铁死亡被抑制与肝癌对索拉非尼耐药的产生有着十分密切的关系，鉴于NRF2在铁死亡调控中的重要作用，将NRF2作为靶标分子研究逆转肝癌索拉非尼耐药具有广阔的应用前景。本课题组在前期工作中从金银花中分离并鉴定了包括6个新化合物在内的一系列黄酮成分并进行了初步生物活性研究，首次发现金银花杂合黄酮对肝癌索拉非尼耐药细胞具有显著抑制活性，并证实其可能是通过抑制NRF2活性从而增强耐药细胞铁死亡敏感性发挥作用，但其作用机制和构效关系尚有待继续探索。本项目拟继续对金银花中杂合黄酮成分进行分离与富集，评价其对肝癌索拉非尼耐药细胞的抑制活性，研究其对NRF2的调控机制及构效关系，并阐明该类成分通过抑制NRF2调控耐药细胞铁死亡敏感性的作用机制，为研发新型克服肝癌索拉非尼耐药药物提供理论支撑。

索拉非尼是进展期肝癌患者的标准一线疗法，但肝癌耐药的出现致使其临床效果并不理想，因此研制新型抗肿瘤药物已十分迫切。本项目进一步对金银花的化学成分进行了系统分离和鉴定，共鉴定了102个化合物，包括27个黄酮化合物，38个咖啡酰奎宁酸类化合物，24个萜类化合物以及其他化合物13个。其中新化合物有21个。此外，根据生活活性主导，以金银花穗花杉双黄酮起始原料，合成了48个含氮的新颖穗花杉双黄酮系列衍生物。在此基础上，评价其抗体外肿瘤及促进铁死亡活性，筛选出7个强活性化合物，继而阐述了其作用机制：（1）JFA能通过竞争性结合Keap1-p62相互作用靶点，使得较多的Keap1蛋白和NRF2结合，从而抑制NRF2活性，影响下游GPX4和SLC7A11表达，进而调控ROS产物或细胞内铁的摄取，从而促进肝癌细胞发生铁死亡。（2）JFB能够激活p53，调控下游GSDME介导的肿瘤细胞焦亡及caspase3介导的肿瘤细胞凋亡，还能通过GPX4促进铁死亡，显著增强抗肿瘤作用。（3）JFD能够诱导SMMC-7721细胞凋亡，机制与内源性线粒体途径有关。分子对接分析表明，JFD可能通过调控NRF2，造成下游ROS减少，从而促进肝癌细胞死亡。（4）穗花杉双黄酮衍生物F24可促进阿霉素诱导的肿瘤细胞自噬-焦亡，显著增强抗肿瘤作用。机制研究发现F24联合阿霉素可以激活PKR，NF90的磷酸化及IFN-β转录。（5）GK在4 μM浓度下，可以达到对肝癌细胞50%的杀伤活性，进一步的分子实验提示GK可能诱导肝癌细胞发生了铁死亡。（6）ISO在体外能有效抑制HCC细胞的增殖和迁移，在体内能抑制HepG2细胞以及DEN+CCL4诱导的肿瘤形成。机制研究证实ISO可通过直接靶向CDK6抑制GLUT1表达，随后激活AMPK-ULK1通路，并导致肝细胞癌中的自噬细胞死亡。（7）3,4-CQIE通过调控YAP-CTGF通路抑制肝癌细胞的增殖和迁移。