Huang Mi, Li Duanzhuo, Xia Zhengzhen, Liao Shengjie, Si Wenxia, Yuan Chao, Liao Yanli, Wu Weibin, Jiang Minshu, Yu Xin, Quan Yi
Department of Scientific Research and Experiment Center, Zhaoqing Medical College, Guangdong, People's Republic of China.
Department of Oncology, The First People's Hospital of Zhaoqing Affiliated to Zhaoqing Medical College, Guangdong, People's Republic of China.
PLoS One. 2025 May 22;20(5):e0322746. doi: 10.1371/journal.pone.0322746. eCollection 2025.
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, with high mortality rates partially due to limited therapeutic options and drug resistance. Arsenic trioxide (ATO), a compound clinically proven for acute promyelocytic leukemia (APL), has garnered attention for its emerging efficacy in solid tumors, including HCC. However, the molecular mechanisms driving ATO's antitumor activity in HCC remain incompletely understood. In this study, we aimed to elucidate the ferroptosis-dependent effects of ATO on HCC and and propose a potential therapeutic strategy.
The response of HCC cells to ATO was evaluated using cell viability, wound healing, colony formation, Transwell migration assays, and cell cycle analysis. ATO-induced ferroptosis was assessed by measuring lipid peroxidation (via C11-BODIPY staining), intracellular iron levels, and malondialdehyde (MDA) production. Western blotting was performed to quantify protein levels of NRF2, HO-1, SLC7A11, and GPX4; immunofluorescence staining was employed to determine NRF2 subcellular localization.
ATO exhibited significant cytotoxicity and inhibited the progression of HCC cells. Treatment with ATO resulted in a notable increase in lipid ROS and MDA levels, which were subsequently reversed by the ferroptosis inhibitors Fer-1 and DFO. Mechanistically, ATO induced ferroptosis by inhibiting GPX4. Furthermore, NRF2 and its downstream targets, HO-1 and SLC7A11, were upregulated during ferroptosis. NRF2 knockdown enhanced lipid peroxidation and ATO-induced cell death.
ATO significantly promoted ferroptosis in HCC cells, and NRF2 knockdown enhanced the cytotoxic effects of ATO.
肝细胞癌（HCC）是全球癌症相关死亡的主要原因，其高死亡率部分归因于治疗选择有限和耐药性。三氧化二砷（ATO）是一种已在临床上被证明对急性早幼粒细胞白血病（APL）有效的化合物，因其在包括HCC在内的实体瘤中显示出的新疗效而受到关注。然而，ATO在HCC中发挥抗肿瘤活性的分子机制仍未完全阐明。在本研究中，我们旨在阐明ATO对HCC的铁死亡依赖性作用，并提出一种潜在的治疗策略。
使用细胞活力、伤口愈合、集落形成、Transwell迁移试验和细胞周期分析评估HCC细胞对ATO的反应。通过测量脂质过氧化（通过C11-硼二吡咯染色）、细胞内铁水平和丙二醛（MDA）生成来评估ATO诱导的铁死亡。进行蛋白质免疫印迹法以定量NRF2、HO-1、SLC7A11和GPX4的蛋白质水平；采用免疫荧光染色来确定NRF2的亚细胞定位。
ATO表现出显著的细胞毒性并抑制HCC细胞的进展。ATO处理导致脂质ROS和MDA水平显著增加，随后被铁死亡抑制剂Fer-1和DFO逆转。机制上，ATO通过抑制GPX4诱导铁死亡。此外，在铁死亡过程中NRF2及其下游靶点HO-1和SLC7A11上调。NRF2基因敲低增强了脂质过氧化和ATO诱导的细胞死亡。
ATO显著促进HCC细胞中的铁死亡，而NRF2基因敲低增强了ATO的细胞毒性作用。
