Yang Lu, Pi Peixian, Zhang Mingzhu, Jiang Yutong, Wu Tiantian, Qing Lijuan, Wang Haibo, Ma Ming, Zhang Zhiyu, Yang Hao, Tian Qiwei, Lu Xiuhong, Huang Gang, Zhao Jian, Liang Beibei
Shanghai Key Laboratory of Molecular Imaging, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences, Shanghai 201318, China; Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Shanghai Key Laboratory of Molecular Imaging, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences, Shanghai 201318, China.
J Adv Res. 2025 Sep 1. doi: 10.1016/j.jare.2025.08.066.
Lenvatinib resistance remains a major challenge in hepatocellular carcinoma (HCC) treatment, highlighting the need for novel therapeutic strategies. Cuproptosis, a copper-dependent form of cell death, has been increasingly implicated in cancer progression. Quercetin, a naturally occurring bioactive flavonol known to modulate mitochondrial metabolism and multiple oncogenic signaling pathways, has emerged as a potential sensitizer.
This study aimed to elucidate the molecular mechanisms by which lenvatinib-resistant HCC cells evade copper-induced cell death and to evaluate whether quercetin enhances ES-Cu-induced cuproptosis by targeting FDX1 and reprogramming mitochondrial metabolism.
Integrated methodologies including bioinformatics analysis, clinical specimen profiling, qRT-PCR, cell proliferation assays, intracellular copper quantification, and Western blot were employed to investigate (1) the insensitivityof lenvatinib-resistant hepatocellular carcinoma (HCC) to cuproptosis but target FDX1 can reverse it(2) the potential of quercetin-ES-Cu combinatorial treatment to reverse drug resistance. Furthermore, subcutaneous xenograft models, mitochondrial OCR measurements, mitochondrial enzyme activity/functional assays, and bio-quercetin administration analyses (immunofluorescence, co-immunoprecipitation) were systematically implemented in both in vitro and in vivo settings to delineate the FDX1-mediated cuproptosis activation mechanism and validate therapeutic efficacy.
Lenvatinib-resistant HCC cells displayed downregulation of cuproptosis-related genes (FDX1, DLAT) and impaired copper accumulation. Quercetin bind with FDX1, enhanced mitochondrial OCR, and synergistically increased intracellular copper accumulation with ES-Cu, leading to lipoylated protein aggregation, mitochondrial dysfunction, and copper-induced cell death. In vivo, quercetin plus ES-Cu significantly suppressed tumor growth without evident toxicity.
This study elucidates the mechanisms by which lenvatinib-resistant HCC cells evade cuproptosis, highlighting the therapeutic potential of quercetin and ES-Cu combination treatment as a novel strategy to overcome lenvatinib resistance in HCC.
乐伐替尼耐药仍然是肝细胞癌（HCC）治疗中的一个主要挑战，这凸显了对新型治疗策略的需求。铜死亡是一种依赖铜的细胞死亡形式，越来越多地与癌症进展相关。槲皮素是一种天然存在的生物活性黄酮醇，已知可调节线粒体代谢和多种致癌信号通路，已成为一种潜在的增敏剂。
本研究旨在阐明乐伐替尼耐药的肝癌细胞逃避铜诱导的细胞死亡的分子机制，并评估槲皮素是否通过靶向FDX1和重新编程线粒体代谢来增强ES-Cu诱导的铜死亡。
采用包括生物信息学分析、临床标本分析、qRT-PCR、细胞增殖试验、细胞内铜定量和蛋白质印迹在内的综合方法，以研究（1）乐伐替尼耐药的肝细胞癌（HCC）对铜死亡不敏感，但靶向FDX1可逆转这种情况；（2）槲皮素-ES-Cu联合治疗逆转耐药的潜力。此外，在体外和体内系统地实施皮下异种移植模型、线粒体氧消耗率测量、线粒体酶活性/功能测定以及生物槲皮素给药分析（免疫荧光、免疫共沉淀），以描绘FDX1介导的铜死亡激活机制并验证治疗效果。
乐伐替尼耐药的肝癌细胞显示铜死亡相关基因（FDX1、DLAT）下调且铜积累受损。槲皮素与FDX1结合，增强线粒体氧消耗率，并与ES-Cu协同增加细胞内铜积累，导致脂酰化蛋白聚集、线粒体功能障碍和铜诱导的细胞死亡。在体内，槲皮素加ES-Cu显著抑制肿瘤生长且无明显毒性。
本研究阐明了乐伐替尼耐药的肝癌细胞逃避铜死亡的机制，突出了槲皮素和ES-Cu联合治疗作为克服肝癌乐伐替尼耐药的新策略的治疗潜力。
