The role of hydrogen sulfide in the modulation of PDT mediated by ALA

CALVO G, CÉSPEDES M, TOMÁS R, DI VENOSA G, CASAS A, SAÉNZ D.

virtual

Congreso; Update PDT PDD 2020; 2020

Introduction: In 5-aminolevulinic acid based-PDT (ALA-PDT), ALA leads to the synthesis of Protoporphyrin IX (PpIX) that acts as a photosensitizer. Hydrogen sulfide (H2S) is a gas that belongs to the gasotransmitter family (together with NO and CO), which can diffuse through biological membranes and have relevant physiological effects[1]. Is involved in cardiovascular functions, vasodilatation, inflammation, cell cycle, and neuromodulation. It was also proposed to have cytoprotective effects[1] . 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 provided by Instituto Angel Roffo, CABA, Argentina) was employed. NaSH was employed as a source of H2S. The light source consisted of a bank of fluorescent tubes. Cell survival was quantified by the MTT method. The intracellular reduced glutathione (GSH) was determined using Ellman´s reagent. PpIX was visualized by fluorescence microscopy. Carbonyl in oxidized proteins were quantified by the 2,4-DNPH spectrophotometric assay[2] . 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 ( 1O2) was assessed using the Singlet Oxygen Sensor Green probe®. H2S production was measured by the formation of methylene blue[3] . Results: Cells exposed to ALA-PDT with different concentrations 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) coincubated with 1 mM ALA; iii) during irradiation; iv) post-PDT, and v) the combination of the three former conditions. Calculated LD50 (mJ/cm2 ) were as follows: Control without H2S: 338±21; Treatments: i) 976±27; ii) 960±29; iii) 362±23; iv) 513±28 and v) >1440 (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 (nmol/106 cells) in cells incubated with 10 mM NaSH (84±1 compared to control cells 73±4), b) PpIX accumulation from ALA suffered a dose-dependent reduction after NaSH (0-10 mM) exposure, c-d) After ALA-PDT, the levels carbonyls (4 h) and intracellular ROS (30 min) decreased compared to the treatment without NaSH (0.1-10mM) in a dose-dependent manner, e) NaSH decreased the levels of 1O2 during an in vitro assay in the absence of cells. LM2 cells produce H2S stimulated with 10 mM cysteine, and synthesis is inhibited by propargylglycine and aspartic acid. Similar cytoprotective effect against ALA-PDT was observed in other cancer cell lines: human ovarian cancer cells SKOV-3 and IGORV-1 and mice leukemia induced cell line of macrophages Raw264.7. 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. The inhibition of H2S improves ALA-PDT. We suggest that impairing H2S tissue production prior or concomitant to ALA-PDT will enhance the effect of the therapy and the malignant tissue elimination. This might be of particular interest in organs that produce or are exposed to high amounts of sulfide, such as colonic epithelial, colonic cancer, and ovarian cancer cells. Both more specific biochemical inhibitors of H2S synthesis or specific siRNA raise as possibilities.REFERENCES[1] C. Szabo, (2018) A timeline of hydrogen sulfide (H2S) research: From environmental toxin to biological mediator, Biochem. Pharmacol. 149:5-19. doi: 10.1016/j.bcp.2017.09.010. [2] Mesquita, C., Oliveria, R., Bento, F., Geraldo, D., Rodriguez, J.V. and Marcos, J.C. (2014) Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins, Anal biochem 458:69-71, doi: 10.1016/j.ab.2014.04.034 [3] Kartha, R.V., Zhou, J., Hovde, L.B., Cheung, B.W.Y. and Schröder, H. (2012) Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method, Anal Biochem 423(1): 102-108, doi: 10.1016/j.ab.2012.01.001