CONICET | Buscador de Institutos y Recursos Humanos

Riboflavin (RF) is an efficient photosensitizer that can act by types I and II photochemical mechanisms. Considering that riboflavin 2´, 3´, 4´, 5´- tetraacetate (RFTA) has a higher tissue uptake than RF, the aim of the present study was to compare the efficacy of RFTA and RF as photosensitizers in photodynamic therapy (PDT) to induce cell death in a cell line of human squamous carcinoma (SCC-13). The cells were incubated with RF or RFTA (50μM) for 3h and irradiated with 444nm λ (4.5, 9 and 18 J/cm2). Firstly, it was shown that the photosensitizers in darkness are not cytotoxic and their photoactivation generate decrease in cell viability and proliferation of SCC-13 in a light dose-dependent manner, with RFTA being more effective than RF. Cell cycle variations were observed in cells after PDT with an increment in S and G2/M phases vs. control. The ultrastructural studies showed that cells subjected to PDT exhibit chromatin condensation and apoptotic bodies. Apoptotic death was confirmed by flow cytometry with AnnexinV/7AAD and TUNEL assay. Cells treated with RFTA presented cleavage of PARP and decrease in procaspase 3, suggesting that apoptosis is induced by a caspasa dependent mechanism. Moreover, it was determined that RFTA is photo-stable and has the ability to generate singlet oxygen using 9, 10- dimethylanthracene as a probe. The cytotoxicity of PDT depend on the increased of ROS intracellular levels that was detected by 2´-7´-dichlorofluorescein diacetate. In summary, these results suggest that RFTA is a photo-stable compound and its photo-activation generates oxidative damage, leading to cell death by apoptosis. Therefore RFTA might be an effective photosensitizer in PDT and a good alternative for treating squamous carcinoma in situ, as well as in other epidermic pathologies.