PHOTODYNAMIC THERAPY IN MELANOMA: ENDOCYTIC UPTAKE OF A CATIONIC ZN(II) PHTHALOCYANINE AND ROLE OF MELANOSOMES

FEDERICO VALLI; GARCÍA VIOR M. CECILIA; LEONOR P ROGUIN; JULIETA MARINO

Mar del Plata

Congreso; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2019

Sociedad Argentina de Investigación Clínica (SAIC)

Melanoma is an aggressive form of skin carcinoma, highly resistant to traditional therapies. Photodynamic therapy (PDT) is an alternative treatment modality, which combines a photosensitizer, visible light and molecular oxygen to produce reactive oxygen species that selectively destroy target cells. We have previously demonstrated that the cationic zinc (II) phthalocyanine Pc13 is a potent photosensitizer that localizes in mitochondria and lysosomes and promotes melanoma cell death after irradiation. In order to elucidate the mechanism underlying Pc13 phototoxicity, we first studied the internalization pathway of this phthalocyanine. Incubation of B16F0 cells at 4°C significantly reduced Pc13 uptake, suggesting the participation of an endocytic mechanism. When cells were preincubated with specific inhibitors of dynamin (Dynasore) or caveolae (Nystatin), cell death was partially prevented after PDT, as determined by hexosaminidase method. Similar results were obtained when cells were transfected with dominant negative mutants of dynamin and caveolin. Conversely, blockage of clathrin pathway did not affect Pc13 phototoxicity. Since melanosomes are lysosome-related organelles, we further examined Pc13 localization in these vesicles. By confocal microscopy we demonstrated that Pc13 targets melanosomes and damage in these structures was observed by TEM 1 h after irradiation. Therefore, the role of melanin in Pc13 phototoxicity was studied employing phenylthiourea (PTU), a melanin synthesis inhibitor. While short preincubation with PTU (3 h) produced a 23 % increase in cell viability after PDT, longer periods of inhibition (48 h) led to cell depigmentation and enhanced cell death. In conclusion, we demonstrated that Pc13 is internalized by a dynamin and caveolin-dependent endocytic mechanism. Melanosomes represent one of the primary sites of photodamage. Modulation of PDT sensitivity by inhibition of melanogenesis could have clinical relevance for melanoma treatment.