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

Abstract

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.

An experimental investigation of a novel iron chelating protoporphyrin IX prodrug for the enhancement of photodynamic therapy

“…This suggests that the iron chelator CP94 is most effective in the cancer cells where the haem biosynthesis pathway is more likely to be disrupted . These findings support several studies that found that the addition of an iron chelator to a PpIX precursor increased PpIX accumulation and therefore fluorescence before irradiation , with MAL + CP94 treated cells producing the greatest enhancement of PpIX fluorescence in tumor cells . This occurs because iron chelating agents reduce cellular levels of free iron, which is essential for haem formation to take place and thus this change in cellular environment, forces more PpIX to accumulate in the cell .…”

“…PpIX is produced in the mitochondria, but it can subsequently localize within cell membranes and the cytosol , leading to apoptosis or necrosis respectively . We previously found that cell death following PDT resulted from low amounts of apoptosis but high amounts of necrosis, possibly as a result of huge mitochondrial damage . However, the main type of cell death following PDT is still to be established and heavily depends on the treatment parameters employed .…”

“…We previously found that cell death following PDT resulted from low amounts of apoptosis but high amounts of necrosis, possibly as a result of huge mitochondrial damage . However, the main type of cell death following PDT is still to be established and heavily depends on the treatment parameters employed . For this study, the lactate dehydrogenase assay was used to measure cell death.…”

“…After absorbing energy from the light, the photosensitizer moves into higher energetic states and transfers this additional energy to molecular oxygen directly or indirectly via type II or type I photochemical reactions, respectively . These reactions form reactive oxygen species (ROS), which then damage cellular components like proteins, lipids, and DNA or indeed the photosensitizer itself, inducing photobleaching . The cellular cascades of ROS thus generated, overwhelm the cell's inherent antioxidant defense and ultimately lead to cell death via apoptosis and necrosis, or alternatively, a destructive form of autophagy .…”

“…In fact many adaptations to the standard treatment protocol have been considered to improve efficacy including skin pre‐treatment with the malignant cell differentiation potentiator dimethyl sulfoxide , light dose fractionation , low fluence rate light administration as well as combinations with other techniques (e.g., low dose Photofrin , hyperthermia , iontophoresis , and bioreductive drugs ). Concurrent administration of an iron chelator during PpIX‐PDT has also been demonstrated to increase cellular accumulation of PpIX . This approach works because once the PpIX has bound Fe 2+ to form haem, it is no longer active for PDT and so by adding iron chelators to trap iron ions in combination with PpIX precursors, this competition can be undermined elevating PpIX accumulation and thus increasing cell kill on subsequent irradiation .…”