Inhibition of Glycogen Phosphorylation Induces Changes in Cellular Proteome and Signaling Pathways in MIA Pancreatic Cancer Cells


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Abstract

ObjectivesNovel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells.MethodsWe performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (5-chloro-1H-indole-2-carboxylic acid [1-(4-fuorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2 oxoethyl] amide) (25, 50, and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC (stable isotope labeling with amino acids in cell culture) method.ResultsA total of 22 protein spots and 4 phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of mitogen-activated protein kinase/extracellular signal–regulated kinase and tumor necrosis factor α/nuclear factor κB.ConclusionsSignaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through 1 pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown, and posttranslational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways.AbbreviationsMALDI - matrix-assisted laser desorption ionization, TOF/TOF MS - time-of-flight/time-of-flight mass spectrometry, 2-DE - 2-dimensional electrophoresis, PMF - peptide mass fingerprinting, CP-320626 - 5-chloro-1H-indole-2-carboxylic acid [1-(4-fuorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2 oxoethyl]amide

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