Delphinidin, a major anthocyanin in pigmented fruits and vegetables is a potent inhibitor of epidermal growth factor receptor and its downstream signaling pathway

2266 Abnormalities in the expression and signaling pathways downstream of the epidermal growth factor receptor (EGFR) contribute to progression, invasion, and maintenance of the malignant phenotype in breast cancer. EGFR is expressed at high levels in at least 30% of breast cancers and is associated with poor prognosis. Many synthetic inhibitors of EGFR are known, but their use is limited because of their unacceptable cytotoxic effects on normal cells. Therefore, identification of a natural, nontoxic agent(s) as an inhibitor of EGFR is of utmost importance. Delphinidin, a major anthocyanin known to be present in pigmented fruits and vegetables (such as pomegranate, berries, dark grapes, egg plant, tomato, carrot and red onion) possesses potent antioxidant and antiproliferative properties. In this study, employing EGFR positive breast cancer cell lines, we evaluated the effect of delphinidin on EGFR and its downstream signaling pathways. Delphinidin (5-40 μM; 3h) treatment to EGFR positive breast cancer cells AU-565 and MCF-10A was found to result in a dose-dependent decrease in the phosphorylation of specific tyrosine residues of EGFR at 1068, 1045 and 845 sites. Delphinidin was also found to inhibit phosphorylation of EGFR in other EGFR positive cancer cells such as A431 and A549. The signaling pathways induced by activated EGFR include the PI3K/AKT and MAPK, both of which play a significant role in the mitogenic and cell survival responses mediated by EGFR. Therefore, we analyzed the expression of these proteins and found that delphinidin treatment of AU-565 and MCF-10A cells inhibited the (i) activation of PI3K, (ii) phosphorylation of AKT, and (iii) phosphorylation of MAPK in a dose-dependent manner. In additional experiments, serum starved AU-565 cells were treated with delphinidin (5-40 μM; 3 h) and then incubated without or with EGF (50 ng/ml) for 15 minutes. We found that delphinidin treatment of AU-565 cells inhibited EGF-induced phosphorylation of EGFR, AKT and MAPK, and activation of PI3K in a dose-dependent manner. We next investigated whether blockage of EGFR signaling pathways by delphinidin would inhibit cell growth and induce apoptosis. Treatment of AU-565 and MCF-10A cells with delphindin (5-40 μM; 48 h) inhibited cell growth and induced apoptosis in both these cell lines as determined by MTT assay, confocal microscopy and TUNEL assay. Delphinidin treatment of AU-565 and MCF-10A cells also resulted in cleavage of PARP protein, activation of caspase-3, downregulation of Bcl-2 and increased expression of Bax protein. In summary this study identifies an abundant fruits and vegetables based anthocyanin delphinidin as an effective blocker of EGFR signaling at least in breast cancer cells. Delphinidin could be developed as an agent for the management of EGFR positive human cancers.