A Proteomic Investigation of the Inhibition of Pancreatic Cancer Cell Growth by Polyphenol-Rich Pomegranate Extract
Authors:Michael Boone, Michael Boone
- Melissa Rowland-Goldsmith, Assistant Professor of Biology, Chapman University
- Craig Adams, Research Associate Professor, Keck Graduate Institute
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the U.S. with an overall five year survival rate of approximately 3-5%. Recent studies have shown that the antioxidant activity of polyphenols in pomegranate juice extract (PJE) can reduce the metastasizing effect of various cancers by promoting apoptosis and cell-cell adhesion and by reducing cell migration, angiogenesis, and inflammatory properties. Our lab initially used microarray technology to study the regulation of PJE treatment on human PDAC. The goal of this joint research project was to use proteomic methods to identify specific proteins or post-translational modifications associated with inhibiting pancreatic cancer cell growth. We hoped to identify similar proteins that were found to be transcriptionally regulated in addition to new proteins involved in slowing cancer cell growth. COLO-357 pancreatic cancer cells were treated with PJE at different concentrations ranging from 0 to 100 ug/ml and incubated for 48 hours at 37C. The cells were then lysed and the resulting protein extract was purified and labeled with fluorescent Cyanine dyes. Through 2-D difference gel electrophoresis (DIGE), the proteins were separated by charge via isoelectric focusing and by size via SDS polyacrylamide gel electrophoresis (SDS-PAGE). The overlay image of the treatment (Cy5) and untreated cancer control protein (Cy3) helped determine key spots of regulation. These spots were trypsinized and separated by a C-18 column; the resulting peptide fragments were further split and identified by their mass to charge ratio (m/z) using tandem mass spectrometry (MS/MS). The mass spectrometry results were compared to peptide sequences of protein databases using SEQUEST software, yielding several proteins of interest, including Zinc finger domains and BAG family molecular chaperone regulators. Additional trials of the experiment will be conducted to discover a potential link between polyphenol-rich PJE and its inhibitory proteomic effects on PDAC.