Porphyrin Biosynthesis and Photosensitization in Human and Murine Lung Adenocarcinoma Cell Lines

CASSINELLI J,; TEIJO MJ,; ROSSETTI MV,; BATLLE A,; FUKUDA H.

Lucerna

Congreso; International Congress of Porphyrins and Porphyrias; 2013

Swiss Medical Association

Endogenously synthesized porphyrins have photodynamic properties which are used to especifically destroy malignant tissues in the so called photodynamic therapy (PDT). This is a cancer treatment receiving increased attention, involving the combination of visible light and a photosensitizer (PS). Each component is harmless by itself, but, in combination with molecular oxygen, lead to the generation of singlet oxygen ( 1 O 2 ) and other reactive oxygen species (ROS), oxidative cell damage and cell death. The administration of the biological precursor of tetrapyrroles, 5-aminolevulinic acid (ALA), induces the accumulation of Protoporphyrin IX (PpIX), a powerful PS, through the haem biosynthetic pathway. In recent years, ALA-mediated PDT (ALA-PDT) has become one of the most promising fields in cancer research. We investigate the synthesis of PpXI in two lung adenocarcinoma cell lines: murine LP07, (Instituto AH Roffo, Argentina) and human A549. Different ALA concentrations ranging from 0.05 to 5 mM and different incubation times (1 to 24 h) were tested. Intracellular accumulation of PpIX increased almost linearly with ALA incubation time. The amount of porphyrins synthesized in the cells was markedly higher in the A549 cell line, reaching a plateau with 1mM ALA at 3 h incubation: A549: 28.44 ± 0.4 ng porph/10 5 cells; LP07: 15.15 ± 0.2 ng porph/10 5 cells. At 6 h incubation, the amount of PpIX increased to 107.1 ± 1.6 ng porph/10 5 (A549) and 24.1 ± 0.3 ng porph/10 5 cells (LP07). ALA does not affect cell viability up to 1mM in the absence of light after 3 h incubation; however, upon illumination a decrease in cell survival (measured by the MTT assay), thatdepends on the drug concentration and light dose were observed. The lethal luminic dose causing the 50 % mortality of cells (DL 50 ) was 7 min for A549 cells, and 10 min for the LP07 cells. Morphological studies with Hoechst staining, showed rounded cells with a markedly size reduction, nuclear condensation and chromatin in the periphery of the cells; these changes were more pronounced in the A549 cells. Results show a strong correlation between the amount of porphyrins and cell death, reinforcing the photodynamic properties of endogenously synthetized porphyrins, which are of relevance in the photosensitization of the porphyrias