Yuan Jie, Huang Wenjun, Lin Minxia, Sun Shishen, Zhong Fei, Ye Lei, Yin Hao, Ou Xiaowei, Zeng Zhiqiang
Department of General Surgery, Foshan Fosun Chancheng Hospital, Foshan, China.
Foshan Municipal Key Laboratory of Precision Oncology, Foshan Fosun Chancheng Hospital, Foshan, China.
Naunyn Schmiedebergs Arch Pharmacol. 2025 May 28. doi: 10.1007/s00210-025-04256-y.
The purpose of this study was to study the mechanism of the increased sensitivity of breast cancer tamoxifen-resistant strains by enhancing ferroptosis. CCK-8 and colony formation were used to detect cell proliferation. Ferroptosis indicator reactive oxygen species (ROS), glutathione (GSH) activity, ATP activity, Fe content, and GPX4 protein expression were detected using ROS assay kit, reduced GSH content assay kit, ATP content assay kit, ferrous ion content assay kit, and western blotting, respectively. The protein levels of HO-1 and Nrf2-central regulators of antioxidant defense and ferroptosis resistance-were assessed by western blot. Tumor changes were observed in nude mice with subcutaneous tumorigenesis. Tamoxifen treatment reduced cell proliferation and colony formation, decreased GSH levels, and downregulated the expression of GPX4, Nrf2, and HO-1. Conversely, it increased ROS fluorescence intensity and Fe⁺ accumulation and impaired the formation of MCF-7 organoids. Overexpression of Nrf2 reversed the effect of tamoxifen. Compared with MCF-7 treated with tamoxifen, in MCF-7 TAMR treated with tamoxifen, the cell proliferation, clone number, ATP activity, and the mRNA expression of Nrf2 and HO-1 significantly increased. The subcutaneous tumor formation experiment in nude mice confirmed that tumors simultaneously treated with ML385 and tamoxifen can further shrink the tumor. The expression of Nrf2 in clinical breast cancer and recurrent breast cancer tissues was significantly higher than that in paracancerous and primary breast cancer tissues, respectively. ML385, the inhibitor of Nrf2, increased ferroptosis via inhibiting Nrf2/HO-1 pathway to enhance the sensitivity of MCF-7 TAMR to tamoxifen.
本研究旨在通过增强铁死亡来探讨乳腺癌他莫昔芬耐药株敏感性增加的机制。采用CCK-8法和集落形成实验检测细胞增殖。分别使用活性氧（ROS）检测试剂盒、还原型谷胱甘肽（GSH）含量检测试剂盒、ATP含量检测试剂盒、亚铁离子含量检测试剂盒和蛋白质免疫印迹法检测铁死亡指标ROS、GSH活性、ATP活性、铁含量及谷胱甘肽过氧化物酶4（GPX4）蛋白表达。通过蛋白质免疫印迹法评估抗氧化防御和铁死亡抵抗的核心调节因子血红素加氧酶-1（HO-1）和核因子E2相关因子2（Nrf2）的蛋白水平。在皮下成瘤的裸鼠中观察肿瘤变化。他莫昔芬处理可降低细胞增殖和集落形成，降低GSH水平，并下调GPX4、Nrf2和HO-1的表达。相反，它增加了ROS荧光强度和Fe⁺积累，并损害了MCF-7类器官的形成。Nrf2的过表达逆转了他莫昔芬的作用。与用他莫昔芬处理的MCF-7相比，在用他莫昔芬处理的MCF-7他莫昔芬耐药株（MCF-7 TAMR）中，细胞增殖、克隆数、ATP活性以及Nrf2和HO-1的mRNA表达显著增加。裸鼠皮下成瘤实验证实，同时用ML385和他莫昔芬处理的肿瘤可进一步缩小。Nrf2在临床乳腺癌组织和复发性乳腺癌组织中的表达分别显著高于癌旁组织和原发性乳腺癌组织。Nrf2抑制剂ML385通过抑制Nrf2/HO-1通路增加铁死亡，从而增强MCF-7 TAMR对他莫昔芬的敏感性。
