5-Aminolevulinic acid-mediated photodynamic therapy: an alternative treatment in multidrug resistant leukemia cells.

DIEZ B; FUKUDA H; GARCÍA MG; CORDO RUSSO R; HAJOS S; BATTLE A

Brixen/Bressanone, Italia.

Simposio; 7º Simposio Internacional de Terapia Fotodinámica y Fotodiagnóstico en Prácticas Clínicas.; 2008

Photosensitizing properties of porphyrins accumulated after the exogenous administration of 5-aminolevulinic acid (ALA) have been successfully used to visualize and destroy malignant cells. We evaluated the effect of ALA-based Photodynamic therapy (ALA-PDT) in three leukemia murine cell lines: LBR- (sensitive to antineoplastic drugs), LBR-D (resistant to doxorubicin) and LBR-V (resistant to vincristine). Protoporphyrin IX (PpIX) synthesis was measured after incubation with 0.1 mM  ALA during different times. Porphyrin synthesis (pmoles porphyrins/106 cells) increases with incubation time, and accumulates at the highest levels in LBR-V160 cell line. (LBR-: 3 h, 15.2 ± 0.6; 4 h, 22.5 ± 1.1;  5 h, 34.5 ± 0.9; 6 h, 35.4 ± 1.3. LBR-D160: 3 h, 22.2 ± 0.8; 4 h, 27.5 ± 1.2; 5 h, 36.6 ± 1.3; 6 h, 35.4 ± 0.8 LBR-V160: 3h, 25.2 ± 1.6; 4 h, 51.6 ± 0.7; 5 h, 66.1 ± 1.5; 6 h, 75.4 ± 1.1). We also evaluated intracellular and extracellular PpIX concentration. At short times (2-3 h) of incubation with ALA, the proportion of externalized PpIX is higher than the intracellular content. As the incubation time increases both values become similar, and after 20 h incubation again the extracellular porphyrin content increases. (% of extracellular PpIX: LBR-: 3 h, 71.2 ± 2.1; 4 h, 51.2 ± 1; 5 h, 55.2 ± 1 ; 20 h, 78 ± 4.95. LBR-D160: 3h, 63.4 ± 1.47; 4h, 36.3 ± 1.62; 5 h, 43.4 ± 0.3; 20 h, 77.2 ± 8.05. LBR-V160: 3 h, 69.3± 2.1; 4 h, 50.8 ± 1.3; 5 h, 55.1 ± 2.4; 6 h, 66.7 ± 1.1; 20 h, 81.9 ± 0.6). PDT phototoxicity assessed by the MTT assay showed that the viability decreased as irradiation time increases (% viability: LBR-: 10 min, 69 ± 0.9; 20 min, 40.9 ± 0.65; 30 min, 16.9 ± 0.4. LBR-D160: 10 min, 49.2 ± 2.8; 20 min, 48.6 ± 1.5; 30 min, 29.9 ± 4.8. LBR-V160: 10 min, 50.5 ± 3.9; 20 min, 34.9 ± 1.6; 30 min, 28.4 ± 1.9). The cellular localization of photosensitizers during PDT plays a major role in the cell destruction; therefore, after incubation with 0.1 mM ALA, cells were examined using fluorescence and confocal microscopy to visualize the PpIX accumulation, using. LysoTracker Green and MitoTracker Green for lysosome and mitochondria identification respectively. The granular pattern and fluorescence distribution were similar in the three lines. At short times of incubation PpIX shows a high concentration in mitochondria and adyacent areas, and as the incubation time increases the fluorescence pattern diffuses to all the cytoplasm. PpIX fluorescence was also observed in lysosomes but to a lesser extent than in mitochondria, however, it increases at longer times of incubation (20 h). The mitochondrial localization supports the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which was confirmed by preliminary studies using acridine orange/ethidium bromide (AO/EB) staining, to evaluate the fragmentation of DNA after PDT. Our results show that ALA-PDT could overcome multidrug resistance (MDR) when it is applied to eradicate minimal residual disease in patients with leukemia.