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To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe ( Cur-P ) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin  in situ . iTRAQ TM  quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway Analysis TM  (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death.

In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line