Proteomic Analysis of Combined Gemcitabine and Birinapant in Pancreatic Cancer Cells

Abstract

Pancreatic cancer is characterized by mutated signaling pathways and a high incidence of drug resistance. Comprehensive, large-scale proteomic analysis can provide a system-wide view of signaling networks, assist in understanding drug mechanisms of action and interactions, and serve as a useful tool for pancreatic cancer research. In this study, liquid chromatography-mass spectrometry-based proteomic analysis was applied to characterize the combination of gemcitabine and birinapant in pancreatic cancer cells, which was shown previously to be synergistic. A total of 4069 drug-responsive proteins were identified and quantified in a time-series proteome analysis. This rich dataset provides broad views and accurate quantification of signaling pathways. Pathways relating to DNA damage response regulations, DNA repair, anti-apoptosis, pro-migration/invasion were implicated as underlying mechanisms for gemcitabine resistance and for the beneficial effects of the drug combination. Promising drug targets were identified for future investigation. This study also provides a database for systems mathematical modeling to relate drug effects and interactions in various signaling pathways in pancreatic cancer cells.

Integrative proteomic and functional analyses provide novel insights into the action of the repurposed drug candidate nitroxoline in AsPC-1 cells

“…Moreover, the PI3K/AKT pathway that was recently shown to be downregulated by nitroxoline www.nature.com/scientificreports www.nature.com/scientificreports/ in prostate cancer cells 12 emerged as one of the Canonical Pathways highly enriched by IPA in our proteomic analysis. Intriguingly, sirtuin and mTOR were previously reported to be upregulated in gemcitabine-resistant PC cells 35 . In addition, inhibition of PI3K, an enzyme downregulated by nitroxoline in our study, was previously shown to sensitize to gemcitabine 36 .…”

Multi‐Scale Network Model Supported by Proteomics for Analysis of Combined Gemcitabine and Birinapant Effects in Pancreatic Cancer Cells

“…8 Liquid chromatographymass spectrometry (LC-MS) analysis of total extracted proteins used a nano-flow liquid chromatography (LC; Eksigent, Dublin, CA), an Orbitrap Fusion mass spectrometry (MS) analyzer (ThermoFisher, Waltham, MA), and the Ionstar workflow to provide accurate, sensitive, and highthroughput identification and quantification of proteins, as described previously. [9][10][11][12][13] Analysis of the same protein samples by Western blotting provided supporting information and validation. Figure 1 shows a schematic of the QSP model.…”

“…Our proteomic analysis identified and quantified 1,481 proteins that were changed significantly by gemcitabine and birinapant. 9 Together with published literature, they provide data for key signaling pathways relevant to drug mechanisms of action and interactions. Mechanistic information for pathways regulating cell cycle progression, DNA damage response (DDR), DNA repair, mitogen-activated protein kinase (MAPK)-p38, nuclear factor-kappa β (NFκβ), and intrinsic and extrinsic apoptosis was summarized to support the development of the system network model ( Figure 1).…”

“…The RFC1 protein was selected as a marker for DNA repair because its profile followed the general pattern of other DNA repair proteins ( Figure S3 in Appendix S2). 9,39 Binding of TNFα to CV%, coefficients of variation; IAP, inhibitor of apoptosis protein; NFκβ, nuclear factor-kappa β; TNFα, tumor necrosis factor α. a Parameters were estimated based on data digitized from Benetatos et al 16 For details refer to Appendix S1. b Parameters were fixed based on Zhu et al 8 Table 1 Continued www.psp-journal.com TNFR1, mediated by TAK1, or DNA damage protein TAO and binding of FasL, mediated by ASK1, were assumed to induce the phosphorylation of p38.…”

“…www.psp-journal.com and birinapant displayed synergistic interactions, 8 and we used proteomic analysis to characterize numerous signaling and functional pathways that might underlie the improved efficacy. 9 This report describes a QSP approach to assess quantitatively the biological status of signaling networks, as well as drug actions that perturb those systems. This study utilizes comprehensive, quantitative proteomic data to support the protein interaction relationships and constrain parameter values in the development of a network model.…”