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The growth of many breast tumors is stimulated by estradiol (E2), which activates a classic mechanism of regulation of gene expression and signal transduction pathways inducing cell proliferation. Polyphenols of natural origin with chemical similarity to estrogen have been shown to interfere with tumor cell proliferation. The aim of this study was to investigate whether hydroxytyrosol (HT) and oleuropein (OL), two polyphenols contained in extra‐virgin olive oil, can affect breast cancer cell proliferation interfering with E2‐induced molecular mechanisms. Both HT and OL inhibited proliferation of MCF‐7 breast cancer cells. Luciferase gene reporter experiments, using a construct containing estrogen responsive elements able to bind estrogen receptor alpha (ERα) and the study of the effects of HT or OL on ERα expression, demonstrated that HT and OL are not involved in ERα‐mediated regulation of gene expression …