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

GUSTAVO CALVO,GABRIELA DI VENOSA, PABLO VALLECORSA, GABRIELA CERVINI, CRISTAN ORLANDO, ADRIANA CASAS, DANIEL SÁENZ

Encuentro;  XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología; XIII ELAFOT; 2017

Photodynamic Therapy (PDT) is a treatment for cancer, that combines a photosensitizer (PS) and light. When the light reaches the PS, it is excited, and triggers a series of reactions that generate reactive oxygen species (ROS), killing the cells[1]. Our group has been focused on the study of the pro-PS, 5-aminolevulinic acid (ALA), that leads to the synthesis of the PS Protoporphyrin IX (PpIX), inside the cell. PpIX is accumulated preferentially in cancer cells, and exerts its cytotoxic effect upon illumination. Hydrogen sulfide (H2S) is a molecule that belongs to the gasotransmitters family, along with the nitric oxide (NO) and carbon monoxide (CO). These molecules can freely diffuse through biological membranes and act 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 cytoprotective effect[4], although the mechanisms are almost unknown.The aim of this work is to study the effect of the H2S on ALA-PDT treatment.The LM2 cell line (mammary murine tumor, Roffo Institute, Argentina) was treated with ALA-PDT and H2S was added at different time points of the treatment (24 h before irradiation, coincubated with 1 mM ALA; during irradiation; post-PDT, and the combination of the three conditions).Without H2S, 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 with H2S at every point of PDT, LD50 was not achieved even at the highest dose applied.Those results show that H2S induced a decrease in cells sensitivity to PDT. Parallel experiements employing ovary and macrophage cell lines (SKOV-3, IGROV-1 and Raw 264.7) were carried out and the same effect was observed.PpIX was extracted from cells after 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 h before the measurement) and a slight but significant increase in GSH levels was observed in cells that have been exposed to H2S (control:73 and treatment:84 nmoles/106 cells).The direct effect of H2S as ROS scavenger was also measured, and it was found that ROS levels decreased when H2S concentration was increased.The activity of 2 enzymes involved in PpIX biosynthesis, ALA-dehydrase (ALA-D) and porphobilinogen deaminase (PBG-D), in fresh blood erythrocytes, was measured after incubation with different concentrations of H2S. ALA-D activity decreased 38% with the lowest H2S concentration (10 mM) and PBG-D activity was not modified by H2S at any concentration (up to 10 mM).Our results show that H2S produces a protective effect on the PDT treatment and it could be due to many factors: the increase of antioxidant state of the cell, the direct scavenging of ROS, and the decrease of the PpIX biosynthesis. These results should be taken into account since several tissues produce high levels of H2S and they could have a differential response to PDT, and in addition, these findings would help 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.