Curcumin is a polyphenol compound derived from Curcuma longa with an aromatic ring structure connected by two α,β-unsaturated carbonyl groups. It is well-known for its anti-cancer properties, although its signaling pathways remain elusive. Sathe et al. (2016) used stable isotope labeling with amino acids in cell culture (SILAC) to investigate tyrosine signaling in CAL 27 cells in response to curcumin.1
The researchers obtained CAL 27 cells, a head and neck squamous cell carcinoma cell line, from the American Type Culture Collection. They cultured the cells in Dulbecco’s Modified Eagle Medium (DMEM) containing SILAC media with heavy stable isotopic forms of lysine and arginine until they reached 70% confluence, at which point the investigators starved them for eight hours. As a control, they also cultured cells in regular DMEM. Sathe et al. then treated cells cultured in SILAC media with dimethyl sulfoxide for four hours and the cells cultured in regular DMEM with curcumin. To analyze the peptides they used an Orbitrap Fusion Tribrid mass spectrometer coupled with an EASY-nLC II nano-flow liquid chromatography system (both Thermo Scientific).
The team found that curcumin significantly reduced the cells’ ability to form colonies and that they had decreased invasive capacity. They suggest that this indicates that curcumin in CAL 27 is able to inhibit cellular proliferation and metastatic potential in cells. Searching mass spectrometry–liquid chromatography data using Mascot and SEQUEST search algorithms, they identified 5,368 phosphopeptide-spectral matches. A phosphoRS probability cutoff of 75% resulted in identifying 672 unique phosphopeptides corresponding to 627 phosphorylation sites mapping to 359 proteins. Using a 1.5-fold cutoff for hyperphosphorylation and a 0.67-fold cutoff for hypophosphorylation, they identified 265 hyperphosphorylated and 40 hypophosphorylated phosphopeptides as a result of curcumin treatment. These corresponded to 187 proteins.
Sathe et al. compared their data to the Gene Ontology database and found that curcumin-regulated proteins mostly localized in the cytoplasm (43%), followed by the plasma membrane (24%), nucleus (19%) and cytoskeleton (5%). Furthermore, the majority of proteins regulated by curcumin participate in cell communication (44%), followed by cell growth (18%) and metabolic processes (12%). They correlate most significantly with biological networks associated with FAK and PI3K signaling pathways. The team notably also identified a number of kinases not previously reported in association with curcumin activity, such as pseudopodium-enriched atypical kinase 1 (PEAK1), a member of the new kinase family three (NKF3) family, which is involved in cell migration, proliferation and cancer metastasis.
In summary, Sathe et al. identified known curcumin-regulated phosphosites as well as several sites not previously reported. They suggest that the tyrosine kinases they identified could be potential future targets for cancer treatments. Their study is the first phosphotyrosine proteome analysis of curcumin signaling.
1. Sathe, G., et al. (2016) “Phosphotyrosine profiling of curcumin‑induced signaling,” Clinical Proteomics, 13(13).
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