METHYL GALLATE TRIGGERS OXIDATIVE STRESS–MEDIATED FERROPTOSIS VIA KEAP1/NRF2 PATHWAY IN BREAST CANCER CELLS

Introduction Breast cancer remains one of the most prevalent malignancies worldwide, characterized by complex pathogenesis and heterogeneous clinical manifestations that hinder effective treatments and prevention [1]. Natural compounds derived from plants, marine organisms, and microorganisms have attracted increasing attention due to their multitarget anticancer properties [2]. Among these, methyl gallate, a plant-derived polyphenolic compound, has demonstrated promising biological activities, including antitumor, antioxidant, and anti-inflammatory effects [3-4]. Materials and Methods MDA-MB-231 breast cancer cell line was treated with increasing concentrations of methyl gallate. Cell viability was assessed using the MTT assay and the IC₅₀ value was calculated. To investigate the potential involvement of oxidative stress, cells were pre-incubated with N-acetylcysteine (NAC), and antioxidant signaling pathways were analyzed, such as Keap1/Nrf2. Ferroptosis was evaluated using the specific inhibitor ferrostatin-1 and by western blot analyses of the main markers associated with iron- dependent cell death (GPX4; FTH1). Reactive oxygen species (ROS) levels, intracellular glutathione (GSH) content, and lipid peroxidation were assessed by fluorescence microscopy and flow cytometry. Total and ferrous iron levels were quantified using a colorimetric assay. Results Methyl gallate reduced cell viability in MDA-MB-231 cells in a dose-dependent manner, with calculated IC₅₀ value indicating a remarkable cytotoxic activity. Pre-incubation with NAC prevented methyl gallate effect suggesting the involvement of oxidative stress that was confirmed by the production of intracellular ROS, the decrease in GSH, and the lipid peroxidation. Indeed, the phytocompound also triggered the activation of Keap1/Nrf2 pathway and promoted a remarkable iron overload with changes in ferroptosis- related proteins. Consistent with ferroptosis, the methyl gallate effect was counteracted by ferrostatin- 1, a specific inhibitor of iron-dependent cell death. Conclusions These findings suggest that methyl gallate induces breast cancer cell death through oxidative stress and iron-dependent mechanisms consistent with ferroptosis.