| Objective:Increasing evidence from epidemiological, clinical and animal xenograft investigations suggested that dietary anthocyanins can prevent chronic diseases, including cancers. It is noteworthy that HER-2(human epidermal growth factor receptor-2) protein overexpression or HER-2gene amplification has been included as an indicator for higher risk of metastasis and recurrence for breast cancer patients. Black rice anthocyanins (BRACs) is a kind of anthocyanins exists in aleurone layer of black rice. Our previous studies have proved that BRACs can decrease the phosphorylation level of HER-2, inhibit downstream signal transduction in cancer prevention and treatment, whereas the interaction between BRACs main constituents and HER-2is still unknown.(1) In this study, we predicted the interaction site and mechanism between anthocyanins components and HER-2by molecular docking firstly.(2) Furthermore, we studied the metastatic inhibition by BRACs in HER-2positive breast cancer cells.(3) Finally, as PI3K signaling pathway is a major downstream pathway mediated by HER-2receptor, we investigated the role of PI3K pathway in this metastatic inhibition process.Methods:(1) Firstly, according to the crystal structure of HER-2kinase domain (HER2TK) and12main antitumor constituents of anthocyanins as well as ATP, a molecular docking study was performed by MVD (Molecular Virtual Docker5.5) program to prediect the interaction site and phosphorylation suppression mechanism.(2) Moreover, taken MDA-MB-453positive breast cancer cells as major study subject, after treated with BRACs, flow cytometry was performed to detect live cells percent, adhesion and wound healing assay were applied to evaluate adhesion and migration abilities; Millicell and cell invasion assay were used to analyze cell motility and invasion abilities.(3) Fianlly, u-PA and MMP-2/-9activity were analyzed by using colorimetry and gelatin-zymography separately; protein expression and molecular interaction were detected by performing western blot and co-immunoprecipitation.Results:(1) All12compounds can bind to the same cavity of HER-2kinase domain by high affinity (MolDock Score<-105kJ/mol for anthocyanidins,<-130kJ/mol for anthocyanidins-glc), in which, hydrophobic force and hydrogen bond played key roles, and anthocyanins showed higher affinity than anthocyanidins. It is presumed that the binding site of anthocyanins in HER-2was decided by parent nucleus, and glycon can increase the binding affinity. Additionally, the cavity of anthocyanins binding to HER-2overlapped with ATP binding domain, which had a lower binding energy (MolDock Score=-161kJ/mol). We presumed that anthocyanins may competitively bind to ATP binding site in HER-2kinase domain by interfering the H bond formation between ATP and HER-2, therefore suppressed HER-2activation and downstream signaling cascade to prevent and cure cancers.(2) The IC50values over24hours were354.95μg and652.57μg of BRACs/mL for MDA-MB-453and MCF-7cells, respectively. Interestingly, BRACs barely affected the untransformed MCF-10A breast cells; this may due parly to the lower affinity between HER-2and BRACs compared to ATP. The adhesion, migration, motility and ECM invasion abilities of breast cancer cells MDA-MB-453decreased siginificantly (P<0.05) after BRACs treatment.(3) Furthermore, the activity of metastatic promoting factors u-PA and MMP-2/-9lowered, accompanying with MMP-2accumulation reduced in cytoplasm by BRACs. BRACs decreased the phosphorylation level of Akt, mTOR, p70S6K and eIF4E, as well as the interaction of Akt/mTOR/p70S6K and PDK/mTOR/p70S6K.Conclusion:All together, here we proved that BRACs can decrease HER-2phosphorylation level by competitive inhibition of ATP binding to HER-2kinase domain, then lowered the activities of metastatic factors, thus weaken the metastatic ability of HER-2positive cells. The decreasing of phosphorylation and molecular interaction of PI3K-Akt-mTOR signaling pathway may involve in this metastatic inhibition by BRACs. |
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