Proteomics of the Effect of Gemcitabine with Intravenous Omega-3 Fish Oil Infusion in Patients with Unresectable Pancreatic Adenocarcinoma

Background: Pancreatic cancer carries the poorest prognosis of all solid organ tumours. Administration of intravenous omega-3 fatty acid (n-3FA) in advanced pancreatic adenocarcinoma patients receiving gemcitabine chemotherapy in our institute shows disease stabilisation and improved progression free survival. Uncovering the underlying biological mechanisms that are responsible for these clinical effects will be investigated using high definition plasma proteomics.

Methods: Plasma from patients with histologically confirmed un-resectable pancreatic adenocarcinoma, collected at baseline before treatment (Baseline group), after one-month treatment with intravenous gemcitabine and n-3FA (Treatment group) and intravenous gemcitabine only (Control group). Plasma was 99% immuno-depleted using Seppro IgY14 + Supermix columns, reduced, alkylated and tryptically digested. Two arms studies comparing Baseline vs Treatment groups and Treatment vs Control groups performed. Samples labelled with TMT-6plex, with a Quality Control sample comprising pooled samples. Combined TMT-labelled samples underwent high-pH reversed-phase fractionation. Fractions were injected into a QExactive-Orbitrap LC-MS/MS in triplicate and analysed on Proteome Discoverer 2.1 and Scaffold 4.7. Bioinformatic analysis was performed on Protein Centre for Gene Ontology Biological Process (GO-BP) enrichment analysis (p <0.05, Bonferroni corrected), Cytoscape for visualisation and KEGG pathway analysis. Selective Reaction Monitoring (SRM) analysis for verification were performed on selected peptides.

Results: 3476 proteins identified. 125 proteins were significant markers of pancreatic cancer, including REG1A, LVVE and TFF. Anti-inflammatory markers (CRP, Haptoglobin and Serum Amyloid-A1) were reduced in the treatment group confirming the anti-inflammatory effects of n-3FA. GO-BP enrichment analysis showed angiogenesis downregulation, complement immune systems upregulation and epigenetic modifications on histones. KEGG pathway analysis identified direct action via the Pi3K-AKT pathway, with decreased HSP90 and increased inhibitory protein 14-3-3. Serum Amyloid-A1 significantly reduced (p<0.01) as a potential biomarker of efficacy for n-3FA.

Conclusion: Administration of n-3FA and gemcitabine has anti-inflammatory, anti-angiogenic and pro-apoptotic effect via direct mechanism on cancer signalling pathways in patients with advanced pancreatic adenocarcinoma.