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.