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

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 (H(2)O(2)) has been reported to induce cell death. However, it remains controversial whether H(2)O(2)-induced cell death is ferroptosis. In the present study, we aimed to elucidate the involvement of mitochondria in H(2)O(2)-induced ferroptosis and examined the molecules that regulate ferroptosis. We found that one mechanism underlying H(2)O(2)-induced cell death is ferroptosis, which occurs soon after H(2)O(2) treatment (within 3 h after H(2)O(2) treatment). We also investigated the involvement of mitochondria in H(2)O(2)-induced ferroptosis using mitochondrial DNA-depleted ρ(0) cells because ρ(0) cells produce more lipid peroxidation, hydroxyl radicals ((•)OH), and are more sensitive to H(2)O(2) treatment. We found that ρ(0) cells contain high Fe(2+) levels that lead to (•)OH production by H(2)O(2). Further, we observed that aquaporin (AQP) 3, 5, and 8 bind nicotinamide-adenine dinucleotide phosphate oxidase 2 and regulate the permeability of extracellular H(2)O(2), thereby contributing to ferroptosis. Additionally, the role of mitochondria in ferroptosis was investigated using mitochondrial transfer in ρ(0) cells. When mitochondria were transferred into ρ(0) cells, the cells exhibited no sensitivity to H(2)O(2)-induced cytotoxicity because of decreased Fe(2+) 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 H(2)O(2) treatment enhances AQP expression, Fe(2+) level, and lipid peroxidation, and decrease mitochondrial function by downregulating PHB2, and thus, is a promising modality for effective cancer treatment.