ESR evidence of superoxide anion, hydroxyl radical and singlet oxygen generation during photosensitization of PPME in HCT-116 cells

Pyropheophorbide-a methyl ester (PPME), a derivative of chlorophyll a, is a second-generation photosensitizer and is studying largely in vitro for nearly a decade on cancerous cells. It has been previously established on HCT-116 (human colon carcinoma cell line) that PPME is a molecule able to create apoptotic and necrotic death (Matroule et al). The cytotoxicity of PPME is presumed to be induced by reactive oxygen species (ROS) generated by the photoexcited molecule. Actually, to the best of our knowledge, no experimental evidence enables to confirm this supposition in an indubitable manner.

Electron spin resonance (ESR) associated with spin trapping technique is a powerful method to detect, quantify and identify the ROS produced after photoactivation of PPME.
Previous results indicate that PPME penetrates inside cells and localizes inside specific organelles (endothelial reticulum, Golgi apparatus and lysosome) (Matroule et al). The recent researches of Guelluy et al have also clearly demonstrated the presence of PPME inside mitochondrion. Consequently, ESR experiments were performed using an intracellular located spin trap, POBN (4-pyridyl 1-oxide-N-tert-butylnitrone), in order to detect in situ the ROS production.

It has been shown that PPME is able to generate superoxide anions and hydroxyl radicals. Irradiation of the dye in HCT-116 cells in the presence of POBN spin trap and ethanol scavenger (2%, a non-toxic concentration) leads to the apparition of the ESR spectrum characteristics of POBN/ethoxy adduct.
To assess the extent of contribution of ROS and to determine a possible reaction mechanism, competition experiments with specific quencher agents were carried out. Addition of catalase (CAT), a hydrogen peroxide quencher, or superoxide dismutase (SOD), a superoxide anion quencher, inhibits 30% of the signal. The parallel effect of SOD and CAT suggest that superoxide anion and hydrogen peroxide are involved in the generation of hydroxyl radicals via a Fenton reaction. This assertion is reinforced by the 20% reduction of signal intensity when adding desferroxamine, a Fe3+ chelator also implicated in Fenton reaction.
Addition of DABCO, a quencher of singlet oxygen, to cells reduces 70% of the POBN/ethoxy adduct signal intensity.