Photoactivation of a cationic zinc(II) phthalocyanine alters actin cytoskeleton and inhibits melanoma B16F0 cells migration

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

Pisa

Congreso; 17th Congress of the European Society for Photobiology; 2017

European Society for Photobiology

Malignant melanoma is the most aggressive form of skin carcinoma, which possesses fast proliferation rate and highly invasive characteristics. Phthalocyanines (Pc) are synthetic photosensitizers with potential application in photodynamic therapy. In order to investigate the effect of a sulfur-linked cationic zinc(II) phthalocyanine named Pc13 on melanoma B16F0 cell migration, an essential event for tumor metastasis, we first evaluated Pc13 phototoxic effect. While no cytotoxicity was observed with Pc13 in the dark, cell viability diminished in a concentration-dependent manner upon exposure to a light dose of 9.2 Jcm-2 (IC50= 0.19±0.09 µM). Since 90 % of cell viability was obtained at 0.12 µM, this was the maximum concentration employed to evaluate cell migration by wound healing assay, with minimum phototoxic effect. After 15 h post irradiation (p.i.), cells incubated in the absence of Pc13 (control), migrated 68±4 %. A similar level of migration was observed in B16F0 cells incubated with Pc13 in the dark. When cells were treated with 0.09 or 0.12 M of Pc13 and irradiated, migration decreased to 37.6±8 % or 24.9±6 %, respectively (p< 0.0001). In the presence of 5 mM of the antioxidant trolox, cell migration was restored, suggesting that reactive oxygen species mediated the inhibition of cell motility. In order to study the effect of Pc13 treatment on cell morphology, tetramethylrhodamine (TRITC)-phalloidin staining was performed. Morphological changes in actin cytoskeleton were observed by fluorescence microscopy. The organization of microfilaments as fibers observed in control cells was partially altered immediately after irradiation and completely lost 1 h p.i., when cells turned rounded and showed a marked retraction. The level of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor mediated signaling, was also investigated. A significant reduction in the amount of FAK relative to control was shown in treated cells by western blot assay. Furthermore, lower levels of metalloproteinases-2 (MMP-2), a protease that contributes to cancer cell migration, were observed after Pc13 irradiation.In conclusion, oxidative response triggered by irradiation of Pc13-treated cells was involved in cytoskeletal disorganization and the decrease of FAK and MMP-2 levels, which finally reduced cell migration. The inhibition of cell motility by non-cytotoxic concentrations of Pc13 could be considered an advantage for in vivo treatments, since a reduced cell spreading would be expected in tumor regions less accessible to light or drug delivery.