Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells

• Published in: Food and chemical toxicology 2014-01, Vol.63, p.195-204
• Main Authors: da Silva Frozza, Caroline Olivieri, da Silva Ribeiro, Tanara, Gambato, Gabriela, Menti, Caroline, Moura, Sidnei, Pinto, Paulo Marcos, Staats, Charley Christian, Padilha, Francine Ferreira, Begnini, Karine Rech, Leon, Priscila Marques Moura de, Borsuk, Sibele, Savegnago, Lucielli, Dellagostin, Odir, Collares, Tiago, Seixas, Fabiana Kömmling, Henriques, João Antonio Pêgas, Roesch-Ely, Mariana
• Format: Article
• Language: English
• Subjects: Annexin-V; Apoptosis; Biological and medical sciences; Cell Line, Tumor; Chromatography, Liquid; Electrophoresis, Gel, Two-Dimensional; Flow Cytometry; Hep-2; Humans; In Situ Nick-End Labeling; Medical sciences; Neoplasm Proteins - metabolism; Propolis - pharmacology; Proteomics; Red propolis; Tandem Mass Spectrometry; Toxicology; TUNEL
• ISSN: 0278-6915; 1873-6351
• DOI: 10.1016/j.fct.2013.11.003

•First report with Brazilian red propolis in 2-DE protein profile of Hep-2 cells.•Five proteins were down-regulated in presence of 120μg/mL of red propolis extract.•Red propolis showed an increase in the mid- to late-stage apoptosis by TUNEL assay.•Red propolis presented increase of late apoptosis by in situ Annexin-V analysis. Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC–MS–MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed.