Photosensitization of human skin cell lines by ATMPn [9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene] in vitro: mechanism of action

Abstract

9-Acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) is a promising new photosensitizer characterized by high absorption around 640 nm and high singlet oxygen yield. To study the mechanism of action in vitro we have investigated uptake, intracellular localization, cell survival and ultrastructural changes following photodynamic treatment in human cell lines derived from the skin (SCL1 and SCL2, squamous cell carcinoma; HaCaT keratinocytes; N1 fibroblasts). Using flow cytometry we have determined the cellular fluorescence as a marker for the uptake of ATMPn after incubation for 60 min. Co-staining with ATMPn and fluorescent dyes specific for cell organelles reveals an intracellular localization of ATMPn in lysosomes. Following irradiation using an incoherent light source (580-740 nm) and a light fluence of 24 J cm-2, phototoxicity is determined by means of the 3-4.5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assay. For all cell lines ATMPn concentrations above 15 nM yield a significant phototoxic effect. The 50% effective concentration, EC50, for SCL1 cells is 11.2 +/- 2.9 nM ATMPn. ATMPn uptake and phototoxicity are more effective for HaCaT and SCL1 as compared to SCL2 and N1 cells. Growth curves confirmed the results of the MTT assay. Because of the high lysosomal accumulation of ATMPn, already low photosensitizer concentrations without dark toxicity yield a high photodynamic effect. Immunofluorescence and electron microscopy reveal damage to tonofilaments, plasma membrane and mitochondria, indicating a mechanism unrelated to apoptosis. A dose yielding complete cell killing, as needed for oncological indications, might lead to necrosis, whereas lower sub-lethal doses result in induction of apoptosis.