The hydroxypyridinone iron chelator CP94 increases methyl-aminolevulinate-based photodynamic cell killing by increasing the generation of reactive oxygen species

Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT) is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX), have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431) occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin) and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time) the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.