Development And Validation Of A Global Proteomics Approach For Identifying Novel Binding Partners Of Resveratrol

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 in a variety of rodent carcinogenesis models. It has been hypothesized that resveratrol exerts its effects by interacting directly with specific proteins such as cyclooxygenase 2 (COX2) and NAD(P)H dehydrogenase quinone 2 (NQO2). Nevertheless the key targets which may mediate its anticancer activity in humans have not yet been identified. An affinity purification (pull-down) assay coupled with global LC-MS/MS analysis was developed to identify proteins that bind directly to resveratrol. Resveratrol was synthetically linked via one of its phenol moieties to agarose beads using a short amine linker, generating two resveratrol bead species. Resveratrol beads were incubated with cell lysate from human-derived colon adenocarcinoma (HCA7) or adenoma cells (AAC1) and then washed to remove unbound proteins. Resveratrol-bound proteins were eluted from the beads by heat denaturation in SDS buffer. Control beads end-capped with ethanolamine were used to identify non-specific protein binding. Proteins were eluted from the beads and identified in trypsinised gel slices by PAGE coupled with either LC-MS/MS to identify all proteins bound to resveratrol or immunoblotting to identify known target proteins. More than 30 proteins were identified by LC-MS/MS that were reproducibly ‘pulled’ from cell lysates by resveratrol beads. The proteins identified fall into five main groups, including enzymes and proteins associated with nuclear transport, apoptosis, DNA repair, prostaglandin and fatty acid synthesis. Following MS/MS, 30 proteins were confirmed as selective binding partners of resveratrol by Western blotting, with 97% agreement between the two detection methods. Among the proteins identified were COX2, NQO2, fatty acid synthase (FAS) and Exportin 1 (CRM1). The first two are known resveratrol targets, which supports the validity of the method. Resveratrol at clinically achievable concentrations (0.001-10 μM) did not change the expression of the proteins identified in the pull-down assay. It is conceivable that resveratrol modulates the function of the proteins to which it binds. To test this hypothesis the effects of resveratrol on FAS activity and on Exportin 1 and its cargoes is currently under investigation. In conclusion, the protein pull-down assay described here may help discover mechanism-related protein biomarkers that can be used to monitor the efficacy of multi-targeted cancer chemopreventive diet constituents in individuals in chemoprevention trials. Citation Format: Christina J. Kovoor, Robert G. Britton, Emma Horner-Glister, Catherine Andreadi, Raj Singh, Rebekah Jukes-Jones, Kelvin Cain, William P. Steward, Andreas Gescher, Karen Brown. Development and validation of a global proteomics approach for identifying novel binding partners of resveratrol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1246. doi:10.1158/1538-7445.AM2014-1246