The incidence of breast cancer has been increasing in China and the age of breast cancer onset is earlier compared with Western countries. Compounds commonly used in Traditional Chinese Medicine (TCM) are an important source of anticancer drugs. Ginseng is one of the most common medicines used in TCM. Ginsenosides, which are saponins found in the ginseng plant, are the major active components responsible for the chemopreventive effects of ginseng in cancer. However, the mechanisms by which ginsenosides exert their anticancer effects remain elusive. The current study combined tandem mass tag (TMT)-based quantification with titanium dioxide-based phosphopeptide enrichment to quantitatively analyze the changes in phosphoproteomes in breast cancer MDA-MB-231 cells that occur following treatment with the ginsenoside Rg3. A total of 5,140 phosphorylation sites on 2,041 phosphoproteins were quantified and it was demonstrated that the phosphorylation status of 13 sites were altered in MDA-MB-231 cells following treatment with Rg3. The perturbed phosphoproteins were: Cleavage and polyadenylation specificity factor subunit 7, elongation factor 2 (EEF2), HIRA-interacting protein 3, melanoma-associated antigen D2, myosin phosphatase Rho-interacting protein, probable E3 ubiquitin-protein ligase MYCBP2, PRKC apoptosis WT1 regulator protein, protein phosphatase 1 regulatory subunit 12A, E3 SUMO-protein ligase RanBP2, Septin-9, thymopoietin, and E3 UFM1-protein ligase 1. Western blotting confirmed that Rg3 increased the phosphorylation of EEF2 on Thr57 but did not alter the protein expression of EEF2 in MDA-MB-231 and HCC1143 cells. These ginsenoside Rg3-regulated proteins are involved in various biological processes, including protein synthesis, cell division and the inhibition of nuclear factor-κB signaling. The results of the present study revealed that Rg3 exerts its anticancer effects via a combination of different signaling pathways.
Keywords: elongation factor 2; ginsenoside Rg3; quantitative phosphoproteomics; tandem mass tag.