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

CÉSPEDES, M; CASAS A; TOMÁS R; SAENZ D; CALVO G; DI VENOSA, G

Barcelona

Congreso; 17th International Congress on Photobiology & 18th Congress of the European Society for Photobiology; 2019

European Society for Photobiology and International Union of Photobiology

Hydrogen Sulfide in the context of5-aminolevulinic acid based Photodynamic Therapy: modulation of the response ina mice breast tumor cell line model.Gustavo Calvo, Mariela Céspedes, RobertoS. Tomás, Gabriela Di Venosa, Adriana Casas, Daniel SáenzCentro deInvestigaciones sobre Porfirinas y Porfirias, Hospital de Clínicas ?José de SanMartin?, UBA-CONICET.e-mail: gustavohcalvo@gmail.com Introduction: In 5-aminolevulinc acid based-PDT(ALA-PDT), ALA leads to the synthesis of Protoporphyrin IX (PpIX). Hydrogensulfide (H2S) is a gas that belongs to the gasotransmitter family (togetherwith nitric oxide and carbon monoxide), which can diffuse through biologicalmembranes and have relevant physiological effects[1]. is involved incardiovascular functions, vasodilatation,inflammation, cell cycle and neuromodulation[2]. It was also proposedto have cytoprotective effects[3]. Our aim was to study the effectof H2S on ALA-PDT in the LM2 cell line. Materials and Methods: LM2 cell line (mammaryadenocarcinoma murine tumor) was employed. NaSH was employed as source of H2S.The light source consisted in a bank of fluorescent tubes. Cell survival wasquantified by the MTT method. The intracellular reduced glutathione (GSH) wasdetermined using the Ellman´s reagent. PpIX was visualized by fluorescencemicroscopy and ulterior image analysis. The levels of oxidized proteins werequantified by the 2,4-dinitrophenylhydrazinespectrophotometric assay [4]. Intracellular ROS formationafter ALA-PDT was estimated employing 2,7- dichlorofluorescein diacetate byfluorescence microscopy. The capacity of the H2S to scavenge singletoxygen, was assessed using the Singlet Oxygen Sensor Green probe®. Results: Cells exposed to ALA-PDT with differentconcentration of NaSH (0-10 mM) exhibited an increased survival to ALA-PDT treatmentin a dose- dependent manner. Light dose 50 (LD50) of the different treatmentswere calculated.  H2S was added at different stages ofALA-PDT treatment: i) 24 h before irradiation, ii) co-incubated with 1 mM ALA; iii) duringirradiation; iv) post-PDT, and v) the combination of the three former conditions.Calculated LD50:s were as follows: Controlin the absence of H2S: 114 mJ/cm2; Treatments: i) 340mJ/cm2; ii) 304 mJ/cm2; iii) 116 mJ/cm2; iv) LD50152 mJ/cm2 and v) LD50 was not achieved at the highestdose used.  Several parameters were related to H2Sabrogation of ALA-PDT response: a) a slight but significant increase in thelevels of GSH in cells incubated with 10 mM H2S (84 ± 1 nmol/106cells) 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 withNaSH (0.1-10mM) decreased compared to the treatment without H2S in adose dependent manner , d) intracellular ROS after ALA-PDT was diminished afterNaSH treatment, e)  NaSH decreased thelevels of singlet oxygen during an in vitro assay in the absence of cells. Conclusions: These results suggest that the H2Shas a role in modulating the redox state of the cells, and thus decreasing theresponse to ALA-PDT through different pathways. References[1] Trends biochem Sci. 2015 40(11): 687[2] Chin Med J. 2013 126(7): 1360[3] J Cell Mol Med 2012 16:896-910[4] Anal Biochem. 2014 458: 69-71