Abstract

Background Clear cell renal cell carcinoma (ccRCC) exhibits profound metabolic reprogramming and exquisite dependence on redox homeostasis, rendering it particularly susceptible to ferroptosis. Embelin, a natural benzoquinone isolated from Embelia ribes, demonstrates antitumor potential but is limited by poor bioavailability. Its structural derivative, 5-O-methylembelin (5-O-ME), may offer improved pharmacological properties, yet its mechanism in ccRCC remains undefined. Purpose This study aimed to investigate the antitumor efficacy of 5-O-ME against ccRCC and elucidate the underlying molecular mechanisms, particularly focusing on metabolic reprogramming and ferroptosis induction. Methods Cell proliferation, migration, and invasion were assessed using CCK-8, colony formation, transwell, and wound healing assays. Ferroptosis was evaluated by measuring lipid peroxidation (MDA, BODIPY 581/591 C11), reactive oxygen species (ROS), glutathione (GSH), intracellular labile iron (Fe 2+ ), and rescue experiments with ferrostatin-1 and deferoxamine (DFO). Direct target identification was performed using limited proteolysis–mass spectrometry (LiP-MS). Protein-protein interactions were validated by co-immunoprecipitation (Co-IP) and immunofluorescence colocalization. ACO1 function was assessed through both overexpression and siRNA-mediated knockdown rescue experiments. Intracellular levels of G6P, pyruvate, and the NADPH/NADP+ ratio were determined using enzymatic colorimetric assays. In vivo antitumor activity and ferroptosis verification (GPX4, SLC7A11 IHC) were evaluated using a 786-O xenograft mouse model. Results 5-O-ME markedly suppressed ccRCC cell proliferation, migration, and invasion while inducing lipid peroxidation and ferroptotic cell death. LiP-MS identified ACO1 as a candidate protein exhibiting conformational alterations upon 5-O-ME treatment, suggesting a potential direct or indirect interaction. Functional analyses revealed that ACO1 enhances ferroptosis sensi