Unveiling novel targets in melanoma under melanogenesis stimulation and photodynamic therapy by redox proteomics

Abstract

Melanogenesis- stimulated B16-F10 cells enter in a quiescent state, present inhib-ited mitochondrial respiration and increased reactive oxygen species levels. Thesealterations suggest that these cells may be under redox signaling, allowing tumorsurvival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy(RB-PDT). A redox proteomics label-free approach based on the biotin switchassay technique with biotin-HPDP and N-ethylmaleimide was used to assess thethiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451,citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2)was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimula-tion. After RB- PDT, only guanine nucleotide-binding protein subunit beta- 2- like1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation fol-lowed by RB- PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153and Cys- 249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh)presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) wasoxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1)was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation fol-lowed by RB-PDT. The redox alterations observed in murine melanoma cells andidentification of possible target proteins are of great importance to further under-stand tumor resistance mechanisms.