水翁花中查耳酮DMC靶向Nrf2通路发挥逆转肿瘤细胞耐药的作用机制

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) isolated from the buds of Cleistocalyx operculatus has been shown to have antitumor effects on human hepatocellular carcinoma in vitro and in vivo. Our previous studies indicated that DMC reduced multidrug resistance (MDR) in chemo-resistant cancer cells through inhibiting MRP1 and GST-π as well as regulating Nrf2. It was evidenced that Nrf2 activation played a pivotal role in enhancing cell survival and resistance to anticancer drugs in many tumors. However, it is remains unclear whether Nrf2 signaling pathway has relation with the reversal effect of DMC on MRP1- and GSH- mediated MDR in hepatocellular carcinoma cells. In this study, we aim at investigating the effect of DMC on Nrf2-ARE signaling pathways and the downstream modulation, as well as the up-regulators as PKC, MAPK and PI3K/Akt involved in the regulation of Nrf2 activation and related genes expression after gaining access to the nucleus. Further insights into the crosstalk of DMC-induced apoptosis and Nrf2 signaling will deepen our understanding of its target molecular and specific pathways. Taken together, we intend to elucidate the detailed molecular mechanisms of Nrf2 pathway in reversing multidrug resistance by DMC. The completion of this project would promote the exploit for medicinal application of myrtaceae plants, which is in line with national strategy for the requirements of Chinese medicine development (2016-2030).

2′,4′-二羟基-6′-甲氧基-3′,5′-二甲基查耳酮（DMC）是桃金娘科植物水翁花花蕾中的主要活性成分。课题组前期研究发现DMC具有显著的肝癌多药耐药逆转活性，是通过抑制MRP1的表达和GST-π的转录来起效的，且对核转录因子Nrf2有靶向调控作用。文献报道Nrf2信号途径往往与MRP1和GST-π的转录表达相关，那么DMC通过Nrf2信号途径逆转肝癌多药耐药的分子机制是什么？本课题拟进一步分析DMC对Nrf2/Keap1/ARE的影响及Nrf2的核易位作用；探讨DMC对Nrf2途径上下游关键信号通路的作用；考察DMC影响肿瘤细胞凋亡途径的靶点与Nrf2信号途径的交叉作用；阐明DMC如何通过Nrf2信号途径逆转细胞耐药的作用机制。本课题的完成对促进桃金娘科药用植物资源的开发利用具有重要意义，符合国家中医药发展战略规划纲要（2016-2030年）。

2',4'-二羟基-6'-甲氧基-3',5'-二甲基查耳酮（DMC）是桃金娘科植物水翁[Cleistocalyx operculatus（Roxb.）Merr. et Perry] 花蕾中的一种天然化合物。前期研究发现，DMC能在细胞实验及裸鼠移植瘤实验中，显著逆转肝癌多药耐药性。本项目对DMC逆转肝癌耐药的机制进行探究。.考察了DMC对BEL-7402/5-FU细胞中Nrf2/ARE的影响。EMSA结果表明DMC削弱了核转录因子Nrf2与抗氧化元件（antioxidant responsive element，ARE）的结合活性，10 μM DMC作用2 h使两者结合活性降低了36%；免疫荧光化学结果证明了DMC能抑制Nrf2核易位，减少Nrf2在细胞核中积累。用10 μM DMC处理8 h，BEL-7402/5-FU细胞中的Nrf2核质分布情况与敏感株相似。此外，Western blot结果还表明DMC下调了BEL-7402/5-FU细胞内Nrf2的表达，同时上调了Nrf2的负调节蛋白Keap1（Kelch-like ECH-associated protein 1）的表达。结果阐明了DMC通过抑制Nrf2表达及核易位，削弱Nrf2与ARE的结合活性，从而抑制Nrf2/ARE。.本项目进一步通过RNAi技术，验证DMC是否通过Nrf2/ARE，对BEL-7402/5-FU细胞中耐药相关蛋白（Ⅱ相解毒酶、MRP1等蛋白）产生影响。结果表明10 μM DMC作用使细胞中谷氨酸半胱氨酸连接酶GCLC和GCLM的表达分别下调了85% 和58%，并减弱MRP1在细胞膜上的定位。而通过RNAi技术敲减Nrf2表达后，DMC不再能对GCLC，GCLM与MRP1产生明显影响。证明了DMC确实是通过抑制Nrf2/ARE，下调GCLC和GCLM的表达使GSH合成减少，并减弱MRP1在细胞膜上的定位，从而抑制药物外排。.综上所述，DMC可能是通过抑制Nrf2/ARE信号通路，减少GSH合成及MRP1介导的药物外排，实现逆转肝癌耐药性，为其进一步开发应用提供了理论依据。