Phytoestrogenic activity of blackcurrant (Ribes nigrum) anthocyanins is mediated through estrogen receptor alpha

Scope: Blackcurrants (Ribes nigrum L., Grossulariaceae) contain high amounts of anthocyanin polyphenols, which have antioxidant and anti-carcinogenic health benefits. This study analyzed the potential phytoestrogenic effects of blackcurrant extract (BCE) in breast cancer (MCF-7) and human endometrial cancer (Ishikawa) cell lines that over-express estrogen receptor alpha (ER alpha), as well as in immature female rats. Methods and results: Microarray analysis and Ingenuity (R) Pathway Analysis showed that BCE activated the ER alpha pathway, whereas quantitative-PCR confirmed that BCE and four types of anthocyanins up-regulated genes downstream of ER alpha. BCE (0.1-1.0 mu g/mL) and anthocyanins (0.1-10 mu M) induced MCF-7 cell proliferation; however, this effect was blocked by ER antagonist fulvestrant. Flow cytometry showed that anthocyanins reduced and increased the number of MCF-7 cells in the G0/G1 and G2/M phases, respectively. Anthocyanins stimulated ER alpha transcriptional activity in human ER alpha reporter assays and induced alkaline phosphatase activity in Ishikawa cells. Competition assays and in silico analysis indicated that anthocyanins bind to ER alpha. Finally, BCE focally induced stratification of columnar epithelial cells in the rat uterus and increased cytoplasmic mucin levels in these cells. Conclusion: These results suggest that blackcurrant anthocyanins act as phytoestrogens in vitro and in vivo.