Modification of the response to ALA-PDT by hydrogen sulfide (H2S)

CALVO, G; DI VENOSA, G; VALLECORSA, P; CERVINI BOHM G; ORLANDO, C; CASAS, A; SAENZ D

Villa Carlos Paz, Cordoba

Congreso; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología; XIII ELAFOT.; 2017

Grupo Argentino de Fotobiología

Photodynamic Therapy (PDT) is atreatment for cancer, that combines a photosensitizer (PS) and light. When the light reaches the PS,  it is excited, and triggers a series ofreactions that generate reactive oxygen species (ROS), killing the cells[1]. Our group has been focused on the studyof the pro-PS, 5-aminolevulinic acid (ALA),that leads to the synthesis of the PS Protoporphyrin IX (PpIX), inside the cell. PpIXis accumulated preferentially in cancer cells, and exerts its cytotoxic effect uponillumination. Hydrogen sulfide (H2S) is amolecule that belongs to the gasotransmitters family, along with the nitricoxide (NO) and carbon monoxide (CO). These molecules can freely diffuse through biological membranes andact as messengers in signaling pathways[2]. H2S is produced endogenously by 3 enzymes (CBS, CGL, CAT/3MST),and it is involved in a variety of physiological processes, such as vasodilatation,inflammation, cellular cycle and neuromodulation[3]. Especially, it is supposed to have a cytoprotectiveeffect[4], although the mechanisms are almost unknown. The aim of this work is to studythe effect of the H2S on ALA-PDT treatment.  The LM2 cell line (mammary murine tumor, RoffoInstitute, Argentina) wastreated with ALA-PDT and H2S wasadded at different time points of the treatment (24 h before irradiation, coincubatedwith  1 mM ALA; during irradiation; post-PDT, and thecombination of the three conditions). WithoutH2S, lethal light dose 50 (LD50) was 114 mJ/cm2; with H2S during irradiation, LD50 was 116 mJ/cm2; with H2S post PDT, LD50 was 152 mJ/cm2; with H2S coincubated with ALA, LD50 was 304 mJ/cm2 and withH2S at every point of PDT, LD50 was not achieved even at thehighest dose applied.Those results show that H2S  induced adecrease in cells sensitivity to PDT. Parallel experiements employing ovary and macrophage cell lines  (SKOV-3, IGROV-1 and Raw 264.7) were carried outand the same effect was observed.PpIX was extracted from cellsafter incubation with ALA or ALA+H2S, and then measured fluorometrically.In all cell lines, PpIX synthesis was inhibited by 10 mM H2S, 53% in LM2; 55,6%in SKOV-3; 81,2% in IGROV-1 and 32,8% in Raw 264.7.The amount of glutathione(GSH) was measured in LM2 cells after exposure to 10 mM H2S (2 and 24 hbefore the measurement) and a slight but significant increase in GSH levels wasobserved in cells that have been exposed to H2S (control:73 and treatment:84 nmoles/106 cells).  The direct effect of H2S as ROS scavengerwas also measured, and it was found that ROS levels decreased when H2S concentration wasincreased.The activity of 2 enzymesinvolved in PpIX biosynthesis, ALA-dehydrase (ALA-D) and porphobilinogen deaminase(PBG-D), in fresh blood erythrocytes, was measured after incubation withdifferent concentrations of H2S. ALA-D activity decreased 38% with the lowest H2S concentration (10mM)and PBG-D activity was not modified by H2S at any concentration (up to 10mM). Our results show that H2S  produces a protective effect on the PDTtreatment and it could be due to many factors: the increase of antioxidantstate of the cell, the direct scavenging of ROS, and the decrease of the PpIXbiosynthesis. These results should be taken into account since several tissuesproduce high levels of H2S and they could have a differential response to PDT,and in addition,  these findings wouldhelp improve the PDT efficiency. [1] P. Agostinis, K. Berg, et al. CA Cancer J Clin. 2011 61:250.; [2] P. Bindu, S. Solomon. Trends biochem Sci. 2015 40(11): 687.; [3] W. Hua, et al. Chin Med J. 2013 126(7): 1360.; [4] B. Fox et al. J Cell Mol Med 2012 16:896-910.