5-O-Methylembelin disrupts the ACO1-TPI1 interaction to promote ferroptosis in clear cell renal cell carcinoma

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.

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.

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 (Fe²⁺), 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.

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 sensitivity, whereas its depletion attenuates 5-O-ME-induced cell death. Mechanistically, 5-O-ME potentiates the physical interaction between ACO1 and the glycolytic enzyme TPI1, leading to alterations in the intermediate metabolites of glycolysis, manifested as G6P depletion and pyruvate accumulation, ultimately leading to redox homeostasis disruption. In vivo, 5-O-ME significantly inhibited tumor growth without overt gross toxicity under the tested experimental conditions.

Our findings demonstrate that 5-O-ME induces ferroptosis in ccRCC through perturbation of the ACO1-TPI1 metabolic interaction, providing a preclinical rationale for exploiting this metabolic vulnerability and offering a promising natural product-inspired therapeutic strategy against renal cell carcinoma.