Proteomic Approaches for Identifying New Mechanisms of Action and Pharmacodynamic Biomarkers of Resveratrol in Colorectal Cancer

The dietary constituent resveratrol (trans-3,5,4’-trihydroxystilbene) has received a great deal of scientific attention and publicity due to its broad ranging pharmacological effects in preclinical systems, including cancer chemoprevention. Depending on the disease studied and the models used there have been a huge number of proposed ‘targets’ of resveratrol. Nevertheless, the key targets which may mediate its anticancer activity in humans have not yet been identified. To fully understand how resveratrol exerts its effects it is important to elucidate the direct molecular interactions and how they affect function and downstream networks in healthy and diseased cells. An affinity purification platform was developed to identify direct protein binding partners of resveratrol using resveratrol-derivatised affinity beads to identify the interacting proteins from human-derived colon adenocarcinoma (HCA7) or adenoma cells (AAC1), followed by LC-MS/MS. Resveratrol affinity beads were designed and synthesised. Proteins that were reliably pulled from biological and technical repeats and were not present in the controls were designated hits and considered for validation via Western blot prior to functional assay development to ascertain the consequence of the resveratrol-protein interaction. Following biological repeats, 980 proteins were identified by this method of which 29 proteins were validated and achieved 97% correlation between the two detection techniques. In a separate approach, plasma proteomics of ApcMin mice was conducted to compare the effect of high-fat diet with/without clinically relevant resveratrol doses. The proteins whose expression was significantly altered between the treatment groups were shortlisted and cross-referenced with the resveratrol-binders from the pull-down list to identify interactomes. In conclusion, the protein pull-down assay combined with plasma proteomics described here may help discover mechanism-related protein biomarkers that can be used to monitor the efficacy of multi-targeted cancer chemopreventive agents in chemoprevention trials and begin to shed light on the direct mechanisms of action of resveratrol.