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

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

Villa Carlos Paz

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

light. When the light reaches the PS, it is excited, and trigger a series of reactions that generate reactive oxygen species (ROS), killing the cell[1]. Our group focalises the pro-PS, 5-aminolevulinic acid (ALA), that leads to the synthesis of the PS, Protoporphyrin IX (PpIX), inside the cell. The PpIX is accumulated preferentially in cancer cells, and exerts its cytotoxic effect upon encountering the light. Hydrogen sulfide (H2S) is a molecule that belongs to the gasotransmitter 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, neuromodulation[3]. Especially, it is supposed to have a cytoprotective effect[4], although the mechanisms are partially or totally unknown. The aim of this work is to study the effect of the H2S on ALA-PDT treatment. LM2 cell line (mammary spontaneous tumor, Roffo Institute, Argentina) was treated with ALA-PDT and H2S was added at different points of the treatment (24 h before irradiation, coincubated with 1mM ALA; during irradiation; post-PDT, and the combination of all those conditions). Without H2S, 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 reached even with 456 mJ/cm2 .Those results show that H2S causes cells less sensitive to PDT. Doing the same proceeding with other cell lines (SKOV-3, IGROV-1 and Raw 264.7) 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 ROS levels decrease when H2S concentration increases.The activity of 2 enzymes involved in PpIX biosynthesis, ALA-dehydrase (ALA-D) and porphobilinogen deaminase (PBG-D), taken from fresh blood erythrocytes, were measured after incubation with different concentrations of H2S, ALA-D activity decreased 38% with the lowest H2S concentration (10 M) 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 PDT treatment and it could be due to many contributions: 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 because of their presence, and also this 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.