Flow cytometry study of the role of superoxide anion and hydrogen peroxide in cellular photodestruction with 5‐aminolevulinic acid‐induced protoporphyrin IX

Abstract

Flow cytometry was used to investigate the participation of reactive oxygen species, other than singlet oxygen, in the cytotoxic effect of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in vitro in A-431 squamous cell carcinoma (SCC) cells and human skin fibroblasts (HSF). We used propidium iodide to determine cellular cytotoxicity, hydroethidine to measure intracellular superoxide anion (O2-) and dihydrorhodamine 123 to assess intracellular hydrogen peroxide (H2O2) content. Our data support the importance of the incubation time with ALA in the selectivity of PDT with ALA against SCC cells, inducing minimum damage on normal HSF. Photoradiation mortality curves of the response of these cell lines to ALA-induced PpIX photosensitization correlated with the extent of photosensitizer accumulation. Intracellular O2- production correlated with cell death, increasing both in a light dose-dependent fashion in ALA treated cells. This correlation was not observed with H2O2-intracellular production. These results suggest the effectiveness of PDT with ALA in vitro in SCC, the significant participation of O2- in its phototoxic mechanism, and the usefulness of flow cytometry in the study of the cytotoxic effect of ALA-induced PpIX PDT.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

“…photodynamic cell killing in a manner that does not agree with previous investigations [7,16,65,66,68,69]. Additional data (Fig.…”

“…Compared to doxorubicin and vincristine resistant lines, parent LBR cells exhibited the lowest level of mitochondrial dysfunction posttreatment, despite generating the highest level of ROS. In contrast, the efficacy of PpIX-dependent photodynamic treatments has been shown to correlate with photo-generated ROS, oxidative damage and cell death when the cells concerned are phenotypically similar [68,69]. Under hyperoxia, cells expressed significantly higher levels of genes coding for proteins linked to maintaining cellular redox homeostasis, including several key antioxidants and the oxidative stress-activated transcription factor Nrf2 (Fig.…”

Antitumor Effects of Photodynamic Therapy Are Potentiated by 2-Methoxyestradiol

“…Intracellular O 2 . production correlates with cell death in 5-aminolevulinic acid-and zinc(II) phthalocyanine-mediated PDT (16,17). Cells scavenge O 2 .…”

Fluorescence-based selection of retrovirally transduced cells in congenital erythropoietic porphyria: direct selection based on the expression of the therapeutic gene

“…ALA undergoes enolization and iron catalysed‐oxidation, leading to the formation of superoxide radical (O), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (HO . )19, 22–24. Uroporphyrin and protoporphyrin, in the presence of H 2 O 2 , can potentiate the peroxidase‐catalysed oxidation of NADPH25.…”