Garcinoic acid promotes ferroptosis-associated growth inhibition in esophageal squamous cell carcinoma through an MDM2-associated mechanism

Background: Garcinoic acid (GA), a plant metabolite, has been shown to exhibit anti-inflammatory and antioxidant activities; however, its mechanism of action against esophageal squamous cell carcinoma (ESCC) remains unclear. This study aimed to investigate the effects of GA on ESCC and to elucidate its underlying molecular mechanisms.

Methods: The anti-ESCC effects of GA were evaluated using cell counting kit-8 (CCK-8) assays, 5-ethynyl-2'-deoxyuridine (EdU) staining, acridine orange/ethidium bromide (AO/EB) staining, and Transwell assays. Ferroptosis-associated changes were assessed using C11-BODIPY staining, reactive oxygen species (ROS), Fe²⁺, malondialdehyde (MDA), and glutathione (GSH) assays, transmission electron microscopy, and Western blotting of ferroptosis-associated proteins. Network pharmacology and Mendelian randomization were employed to prioritize candidate mediators linking GA, ESCC, and ferroptosis. Molecular docking and molecular dynamics simulations were performed to predict potential GA-mouse double minute 2 (MDM2) interactions and assess complex stability. Rescue experiments were conducted to evaluate the functional relevance of MDM2.

Results: GA inhibited ESCC cell proliferation, induced apoptosis, and reduced cell migration. GA increased levels of MDA and Fe²⁺, and induced GSH depletion. Pretreatment with Ferrostatin-1 attenuated GA-induced inhibition of cell viability. MDM2 was prioritized as a candidate mediator linking GA, ESCC, and ferroptosis. Molecular docking predicted a favorable binding affinity between GA and MDM2, with a binding energy of −9.1 kcal/mol. Molecular dynamics simulations suggested that the predicted GA-MDM2 complex remained stable under the simulation conditions. Rescue experiments demonstrated that MDM2 may mediate GA-associated ferroptosis and growth inhibition in ESCC cells.

Conclusions: These findings suggest that GA induces ferroptosis-associated growth inhibition in ESCC cells, potentially through an MDM2-associated mechanism, and provide a rationale for further investigation of GA-related ferroptosis modulation in ESCC.