Human lung adenocarcinoma cells sensitive and resistant to ALA-based Photodynamic Therapy

FUKUDA H.,; TEIJO MJ.,; BATLLE A.

Lima

Congreso; 6th LATIN AMERICAN CONFERENCE ON LUNG CANCER (LALCA).; 2014

International Association for the Study of Lung Cancer (IASLC)

Abstract Human lung adenocarcinoma cells sensitive and resistant to ALA-based Photodynamic Therapy H Fukuda1,2, M J1,2 Teijo, A Batlle2 1- Departamento de Química Biológica, FCEN, UBA. Argentina 2- Centro de Investigaciones sobre Porfirias y Porfirinas (CIPYP), CONICET, Hospital de Clínicas, UBA. Argentina Background Photodynamic therapy (PDT) is an approved anticancer treatment modality that eradicates tumor cells by the photochemical generation of reactive oxygen species (ROS) following absorption of visible light by a photosensitizer, which is selectively taken up by malignant cells. 5-Aminolevulinic acid (ALA) is the biological precursor of Protoporphyrin IX, the only endogenous photosensitizer. Generation of cells resistant to the treatment is an undesired effect. Methods ALA-PDT was performed in A549 human lung adenocarcinoma cells growing as monolayers. Cells were incubated with 1 mM ALA for 3 h followed by irradiation with a bank of two fluorescent lamps (Osram L36W/10) for different times, and within 1 h analyzed for PDT-induced events (MTT assay). The surviving cells were harvested 24 h after treatment and replaced. After 12 cycles of treatment, ALA-PDT resistant cells were obtained (C-12 cells). Endogenously generated porphyrins were extracted from the cells and quantified spectrophotometrically. Results After incubation with ALA, C-12 cells accumulated 50% less porphyrins than the parental control cells: 9,5±0,7 ng porph/105 cells in C-12; 19,9±1,2 ng porph/105 cells in control cells. Photosensitization with exogenous PpIX (5uM, 3 h incubation time) induced partial recovery of sensibility to PDT in the C-12 cells (surviving cells after 10 min irradiation: 39,5 % control cells with ALA-PDT; 27,5 % control cells with PpIX-PDT; 100 % C-12 cells with ALA-PDT; 75 % C-12 cells with PpIX-PDT). The low sensitivity of C-12 cells to PpIX-PDT was not due to PpIX level, as both cell types uptake similar amount of porphyrins (80,6±4,8 ng porph/105 C-12 cells; 77,1±,2,2ng porph/105 control cells). Moreover, both parental and resistant cells showed similar cell morphology (accomplished with toluidine blue and Hoechst staining) and ability to migrate (wound healing assay). In the resistant C-12 clone, as result of photodynamic treatment, mitochondrial depolarization without cytochrome c release and caspase-3 activation was observed, and expression of the anti-apoptotic protein Bcl-2 was detected by means of WB assays. The nuclear factor NFkB is a well known oxidative stress-induced transcription factor, so, it is relevant to surviving after PDT. We analyzed the expression of IkB and p65 subunit of NFkB in the cytosolic and nuclear fractions of parental and C-12 cells after ALA-PDT treatment. IkB was detected in the parental cells. In the resistant C-12 cells NFkB was observed after all the treatments, and the pattern of IkB expression was similar to the parental cells. Conclusions These results suggest that NFkB activation is an event related to a long term effect of ALA-PDT. When cells are subjected to a single PDT treatment, NFkB is activated only with subletal doses lower than DL50.