Analysis in vitro of photocytotoxic effects on cell cultures system during 5-aminolevulinic acid PDT

Photodynamic therapy (PDT) is based on the uptake of a photosensitizer followed by its photosensitization through a selective irradiation, which induce oxidative stress and cytotoxicity. In this work the effect of 5-aminolevulinic acid (5-ALA) was evaluated during photodynamic treatment. We have used as experimental system four cell lines: COLO 829 (primary melanoma), A2058 (metastatic melanoma), BJ-5ta (human immortalized fibroblasts), SH-SY5Y (human neuroblastoma). 5-ALA is the precursor of protoporphyrin IX (PpIX) in the biosynthetic pathway of heme. PpIX is a potent photosensitizer, thus leading to an ALA-based PDT. When exogenous 5-ALA is added to culture medium, the production of cellular PpIX can be induced. We have verified the intracellular production of PpIX in the above mentioned cell lines after treatment with different concentration of 5-ALA for various times in the dark. Fluorimetric analysis has shown a dose-dependent increase of fluorescence intensity up to 0.3 mM 5-ALA after 6 hours of incubation. Hence, to evaluate the 5-ALA photocytotoxicity effect, the cells were incubated with 0.3 mM 5-ALA for 6 hours in the dark. After incubation, the cells were irradiated with different light doses using a halogen lamp with a 520 nm long pass filter and a fluence rate of 2.6 mW/cm2. After this exposure, the cells were incubated for additional 24 hours and then the photocytotoxicity effect was evaluated. The results showed that the cytotoxic effect was proportional to the light dose and, interestingly, the metastatic melanoma cell line A2058, is more responsive than the other cell lines used. Furthermore, analysis of expression profiles of some antioxidant enzymes, after 5-ALA-based-PDT lead to a reduction of SOD2 protein levels in cellular extracts of A2058 cell. These results may provide further insights for clinical applications of PDT in cancers particularly resistant to conventional therapies