Photodynamic therapy in lung adenocarcinoma (NSCLC) cells. Reactive Oxygen Species Induction and Apoptotic death

TEIJO M J; DIEZ B; BATLLE A; FUKUDA H

CABA

Congreso; 4th Latin American Conference on Lung Cancer; 2010

4ht Latin American Lung Cancer Association

Photodynamic therapy (PDT) is a relatively new modality for the treatment of obstructive or microinvasive lung cancer. It involves the administration of a photosensitising drug (PS) which selectively accumulates in tumor tissue. Bronchoscopic irradiation with an appropriate wavelength light triggers photochemical reactions inducing  Reactive Oxygen Species (ROS) production with the consequent cellular damage which ultimately lead to cell death. Porphyrins are the only PSs endogenously synthesized by means of incubation with the biological precursor, 5- aminolevulinic acid (ALA). ALA-PDT was performed on A549  human lung adenocarcinoma cells,  by incubation with 1mM ALA for 3h, followed by irradiation with a bank of two fluorescent lamps (Osram L36W/10) for 10 min, with or without 1h preincubation with several ROS scavenger agents: reduced Glutathion (GSH), Mannitol, Tryptophan, Ascorbate and Trolox.  Scavengers were previously tested for possible toxicity or photoactivity per se, in concentrations ranging from 0,01 to 20 mM. Cell viability was determined by the MTT assay, and protection grade (PG) was calculated as the ratio between  cell survival after ALA-PDT in the presence and in the absence of the scavenger agent. The scavenger concentration showing no toxicity or photoactivity per se and providing the highest PG (GSH: 1mM, PG: 2,1±0,3; Mannitol: 10mM, PG: 1,1±0,1; Tryptophan: 1mM, PG: 1,7±0,1; Ascorbate: 1mM, PG: 2,6±0,1; Trolox: 1 mM, 3,5±0,1) was used in the subsecuent protection experiments. ALA-PDT alone induced a high percentage of apoptotic cell death as revealed 2h after the treatment, by morphological analysis with Hoechst staining, acridine orange and ethidium bromide staining (control: 10,16±1,1% ; ALA-PDT: 98,4±3,5%) and propidium iodide - Annexin V-FITC labelling (control: 2,2±0,1%; ALA.PDT: 26,8±4,2%). Preincubation with the scavengers at the highest PG concentrations significantly increased cell viability after PDT. Ascorbate and Trolox showed the highest protection, reverting the apoptotic death in percentages between 23,6±4,4% and 50,1±6,2%, for Annexin and morphological staining respectively. Reduction of apoptosis was also observed with other scavengers, as analyzed by morphological staining (GSH: 23,5±6,6%; Mannitol: 34,1±5,1%; Tryptophan: 23,8±6,1%) and Annexin labelling as well (GSH: 8,5±6,3%; Mannitol: 16,8±8,6; Tryptophan: 23,3±4,4) In order to investigate direct participation of ROS in early cell death induction after ALA-PDT, cells were loaded with fluorescent probes for detection of superoxide anion (hydroethidine-HE) and peroxides (dichlorofluorescein diacetate-DCFDA) and analyzed by flow cytometry. It was observed that ROS production increased with irradiation time: HE positive cells: control: 1,1±0,1 %; 10 min-PDT: 27,9±0,3 %; 15 min-PDT: 31,5±0,7 %; DCFDA positive cells: control: 1,1±0,1 %; 10min-PDT: 44,4±0,1 %; 15min-PDT: 54,5±0,1 %. Preincubation with Ascorbate only prevented peroxide formation after 15 min-PDT (DCFDA positive cells: 10,3±0,2%); whereas Trolox preincubation prevented peroxide formation at both irradiation times (DCFDA positive cells: 10min-PDT: 18,1±0,1%; 15 min-PDT: 28,9±0,1%). Scavenger-mediated protection against PDT-induced cell death and direct detection of specific prooxidative agents, entail a strong involvement of ROS in PDT-mediated tumor eradication, suggesting that undesired photodamage to normal tissue might be attenuated by administration of antioxidant agents.