Hyperresistance to photosensitized lipid peroxidation and apoptotic killing in 5-aminolevulinate-treated tumor cells overexpressing mitochondrial GPX4

Antitumor photodynamic therapy (PDT) with administered 5-aminolevulinic acid (ALA) is based on metabolism of ALA to protoporphyrin IX (PpIX), which acts as a sensitizer of photo-oxidative damage leading to apoptotic or necrotic cell death. An initial goal of this study was to ascertain how the PpIX-sensitized death mechanism for a breast tumor line (COH-BR1 cells) might be influenced by the conditions of ALA exposure in vitro. Two different treatment protocols were developed for addressing this question: (i) continuous incubation with 1 mM ALA for 90 min; and, (ii) discontinuous incubation, i.e., 15 min with 1 mM ALA followed by 225 min without it. Following exposure to 2 J/cm2 of visible light, cell viability, death mechanism, and lipid hydroperoxide (LOOH) level were evaluated for each protocol using thiazolyl blue, Hoechst staining, and HPLC with electrochemical detection assays, respectively. PpIX was found to sensitize apoptosis when it existed mainly in mitochondria (protocol-1), but necrosis when it diffused to other sites, including plasma membrane (protocol-2). Experiments with a transfectant clone, 7G4, exhibiting approximately 85 times greater activity of the LOOH-detoxifying selenoenzyme GPX4 than parental cells, provided additional information about death mechanism. Located predominantly in mitochondria of 7G4 cells, GPX4 strongly inhibited both LOOH accumulation and apoptosis under protocol-1 conditions, but had no significant effect under protocol-2 conditions. These findings support the hypothesis that LOOHs produced by attack of photogenerated singlet oxygen on mitochondrial membrane lipids play an important early role in the apoptotic death cascade.