PHOTODYNAMIC THERAPY INDUCED OXIDATIVE STRESS IN OCULAR MELANOMA CELLS

Abstract

Purpose: To assess the oxidative stress induced by photodynamic therapy (PDT) using new photosensitizers the dihydroxymethyl ring-fused chlorin (PS1) and dihydroxymethyl-Pt(II) ring-fused chlorin (PS2), in ocular melanoma cells. Methods: Ocular melanoma cell line MP-41 was cultured and incubated with 100 and 500 nM concentrations of the photosensitizers for 24 hours. After renewing the culture medium, the cells were irradiated with visible light (λ>570nm, 10J). To study the role of singlet oxygen and hydroxyl radicals, the cells were treated with PDT in the presence of sodium azide (5mM) and D-mannitol (40mM). Intracellular superoxide and peroxide levels were measured using DHE (5μM) and DCF-HA (5μM), respectively, followed by spectrophotometric and fluorimetric analyses. Results: Inhibition of singlet oxygen increased metabolic activity in cells treated with 100 nM PS1 and PS2 from 48.4±13.6 to 118.8±4.7 and from 56.4±16.8 to 102.0±3.6, respectively. With 500 nM PS1 and PS2 from 0,77±0,2 to 8,3±3,29 and from 2,3±1,7 to 12,2±1,6, respectively. The presence of a superoxide radical scavenger led to metabolic activities with 100 nM PS1 and PS2 from 48,4±13,6 to 67,3±19,5 and from 56,4±15,6 to 75,5±13,2, respectively. With 500 nM PS1 and PS2 from 0,77±0,2 to 6,3±4,5 and from 2,3±1,7 to 17,4±8,3, respectively. Conclusions: Reactive Oxygen Species (ROS) generation is key in PDT-induced cell death, with singlet oxygen playing a predominant role in the type II photodynamic process, as demonstrated by the chlorins' efficacy in ocular melanoma cells. The reduced peroxide and superoxide levels suggest singlet oxygen's significant generation, though type I reactions may also be involved. Key-words: ocular melanoma, PDT, photosensitizers, ROS.