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Photo-Induced Necrosis on Oropharyngeal Carcinoma (HEp-2) Cells Mediated by the Xanthene Erythrosine.

Journal of nanoscience and nanotechnology (2020-05-10)
Maria J Bistaffa, Mirella B Kobal, Priscila S S Souza, Karina A Toledo, Sabrina A Camacho, Pedro H B Aoki
ABSTRACT

The photodynamic therapy (PDT) has been outstanding as a promising alternative for treating different carcinomas. However, the lack of detailed knowledge on the mechanisms of action prevents exploitation of the therapy full potential. Herein we shall evaluate not only the photodynamic efficiency but the mechanism of cell death triggered by the photoactivated erythrosine in oropharyngeal cancer cells (HEp-2). Cytotoxic assays were performed by MTT at distinct concentrations (10-3 to 10-6 mol/L) and incubation time (3, 24 and 48 h) of erythrosine in HEp-2 in vitro culture. In addition to the cytotoxic effect, the mechanisms of cell death were evaluated by flow cytometry following the annexin V/propidium iodide double staining protocol. Erythrosine was incorporated by HEp-2 cells in a dose- and time-dependent pathway. The incubation of erythrosine in dark has not shown any significant effect over the culture until 24 h and 1.25×10-6 mol/L concentration, from which a small portion (<25% and statistically significant) of the cell population have undergone apoptosis. On the other hand, 50% of cell viability is reduced mainly by necrosis when 10, 3.75 and 1.9×10-6 mol/L of erythrosine concentrations at 3, 24 and 48 h of incubation are photoactivated, respectively. Bioinspired models of tumor membrane based on Langmuir monolayers of 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) mixture reveled that electrostatic interactions with the lipid head groups are the main driving forces allowing the erythrosine adsorption. Furthermore, light-induced hydroperoxidation significantly increased the surface area of the monolayers, which might be the origin of the necrotic pathway triggered in HEp-2 cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
MTT Formazan, powder
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With L-glutamine and 15 mM HEPES, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
Erythrosin B, Dye content ≥95 %
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With L-glutamine and 15 mM HEPES, without sodium bicarbonate and phenol red, powder, suitable for cell culture