In vitro photodynamic therapy of nasopharyngeal carcinoma using 5-aminolevulinic acid

Abstract

The purpose of this study was to investigate the potential use of 5-aminolevulinic acid (5-ALA, 5-amino-4-oxovaleric acid) induced protoporphyrin IX (PPIX) for photodynamic therapy (PDT) of nasopharyngeal carcinoma (NPC) and its related mechanisms of inducing cell death. PPIX biosynthesis at 1 to 8 h after incubation of a cultured NPC cell line (HNE1) with 5-ALA (10-5000 ώg ml⁻¹) was determined via fluorescence analysis. HNE1 cells were irradiated at 4 h after incubation with 5-ALA (10-200 ώg ml⁻¹) by diode laser (λ = 630 nm) at various energy levels (1-50 J cm⁻²). The survival rates at 6, 12, 24 and 48 h after PDT were determined by MTT assay. Mechanisms of PDT-induced cell death were investigated via Annexin-V/propidium iodide staining and DNA electrophoresis. After incubation with 5-ALA, a time- and dose-dependent increase of cellular PPIX-fluorescence was recorded up to a threshold concentration of 1000 ώg ml⁻¹ 5-ALA, above which a decline of fluorescence intensities occurred. Similar values of PPIX-fluorescence were found at 100-1000 ώg ml⁻¹ of 5-ALA. Unlike sole incubation with 5-ALA or sole laser irradiation, the combination of both factors lead to a significant, concentration-, energy- and time-dependent increase of cell death (p < 0.01). At 100 ώg ml⁻¹ ALA and 10 J cm⁻² laser irradiation, cellular survival was <5% after 48 h. More than 80% of induced cell deaths thereby occurred via apoptosis within the first 12 h following irradiation; necrosis was accountable for less than 20%. High level induction of apoptosis by 5-ALA-PDT was confirmed by DNA electrophoresis. Our investigations show promising results of 5-ALA based PDT of nasopharyngeal carcinoma in vitro and set the basis for future studies in tumor models or humans, respectively.