Mitochondrial dysfunction promotes aquaporin expression that controls hydrogen peroxide permeability and ferroptosis

Abstract

Most anti-cancer agents and radiotherapy exert their therapeutic effects via the production of free radicals. Ferroptosis is a recently described cell death process that is accompanied by iron-dependent lipid peroxidation. Hydrogen peroxide (H₂O₂) has been reported to induce cell death. However, it remains controversial whether H₂O₂-induced cell death is ferroptosis. In the present study, we aimed to elucidate the involvement of mitochondria in H₂O₂-induced ferroptosis and examined the molecules that regulate ferroptosis. We found that one mechanism underlying H₂O₂-induced cell death is ferroptosis, which occurs soon after H₂O₂ treatment (within 3 h after H₂O₂ treatment). We also investigated the involvement of mitochondria in H₂O₂-induced ferroptosis using mitochondrial DNA-depleted ρ⁰ cells because ρ⁰ cells produce more lipid peroxidation, hydroxyl radicals (^•OH), and are more sensitive to H₂O₂ treatment. We found that ρ⁰ cells contain high Fe²⁺ levels that lead to ^•OH production by H₂O₂. Further, we observed that aquaporin (AQP) 3, 5, and 8 bind nicotinamide-adenine dinucleotide phosphate oxidase 2 and regulate the permeability of extracellular H₂O₂, thereby contributing to ferroptosis. Additionally, the role of mitochondria in ferroptosis was investigated using mitochondrial transfer in ρ⁰ cells. When mitochondria were transferred into ρ⁰ cells, the cells exhibited no sensitivity to H₂O₂-induced cytotoxicity because of decreased Fe²⁺ levels. Moreover, mitochondrial transfer upregulated the mitochondrial quality control protein prohibitin 2 (PHB2), which contributes to reduced AQP expression. Our findings also revealed the involvement of AQP and PHB2 in ferroptosis. Our results indicate that H₂O₂ treatment enhances AQP expression, Fe²⁺ level, and lipid peroxidation, and decrease mitochondrial function by downregulating PHB2, and thus, is a promising modality for effective cancer treatment.

Keywords: Aquaporin; Fe(2+); Ferroptosis; Hydrogen peroxide; Mitochondria.