Hydrogen Sulfide in the context of 5-aminolevulinic acid based Photodynamic Therapy: modulation of the response in a mice breast tumor cell line model

MARIELA CESPEDES; ADRIANA CASAS; GUSTAVO CALVO; GABRIELA DI VENOSA; ROBERTO SEBASTIÁN TOMÁS; DANIEL SÁENZ

Barcelona

Congreso; 17th Congress of the International Union of Photobiology and 18th Congress of the European Society for Photobiology; 2019

European Society for Photobiology

Introduction: In 5-aminolevulinc acid based-PDT (ALA-PDT), ALA leads to the synthesis of Protoporphyrin IX (PpIX). Hydrogen sulfide (H2S) is a gas that belongs to the gasotransmitter family (together with nitric oxide and carbon monoxide), which can diffuse through biological membranes and have relevant physiological effects[1]. is involved in cardiovascular functions, vasodilatation, inflammation, cell cycle and neuromodulation[2]. It was also proposed to have cytoprotective effects[3]. Our aim was to study the effect of H2S on ALA-PDT in the LM2 cell line.Materials and Methods: LM2 cell line (mammary adenocarcinoma murine tumor) was employed. NaSH was employed as source of H2S. The light source consisted in a bank of fluorescent tubes. Cell survival was quantified by the MTT method. The intracellular reduced glutathione (GSH) was determined using the Ellman´s reagent. PpIX was visualized by fluorescence microscopy and ulterior image analysis. The levels of oxidized proteins were quantified by the 2,4-dinitrophenylhydrazine spectrophotometric assay [4]. Intracellular ROS formation after ALA-PDT was estimated employing 2,7- dichlorofluorescein diacetate by fluorescence microscopy. The capacity of the H2S to scavenge singlet oxygen, was assessed using the Singlet Oxygen Sensor Green probe®.Results: Cells exposed to ALA-PDT with different concentration of NaSH (0-10 mM) exhibited an increased survival to ALA-PDT treatment in a dose- dependent manner. Light dose 50 (LD50) of the different treatments were calculated.H2S was added at different stages of ALA-PDT treatment: i) 24 h before irradiation, ii) co-incubated with 1 mM ALA; iii) during irradiation; iv) post-PDT, and v) the combination of the three former conditions.Calculated LD50:s were as follows: Control in the absence of H2S: 114 mJ/cm2; Treatments: i) 340 mJ/cm2; ii) 304 mJ/cm2; iii) 116 mJ/cm2; iv) LD50 152 mJ/cm2 and v) LD50 was not achieved at the highest dose used.Several parameters were related to H2S abrogation of ALA-PDT response: a) a slight but significant increase in the levels of GSH in cells incubated with 10 mM H2S (84 ± 1 nmol/106 cells) compared to control cells (73 ±4), b) PpIX accumulation from ALA suffered a dose-dependant reduction after H2S (0-10 mM) exposure, c) the levels of oxidized proteins 4 h after ALA-PDT with NaSH (0.1-10mM) decreased compared to the treatment without H2S in a dose dependent manner , d) intracellular ROS after ALA-PDT was diminished after NaSH treatment, e) NaSH decreased the levels of singlet oxygen during an in vitro assay in the absence of cells.Conclusions: These results suggest that the H2S has a role in modulating the redox state of the cells, and thus decreasing the response to ALA-PDT through different pathways.