Estrogenic effects of resveratrol in breast cancer cells expressing mutant and wild-type estrogen receptors: Role of AF-1 and AF-2

Resveratrol, a hydroxystilbene found in grapes and wine, has previously been shown to be a non-flavonoid phytoestrogen, and to act as an estrogen receptor (ER) superagonist in MCF-7 cells transiently transfected with estrogen-responsive reporter constructs. Several additional hydroxystilbenes, including diethylstilbestrol (DES) and piceatannol, were tested, and all showed ER agonism or partial agonism, but superagonism was specific to resveratrol. Moreover, superagonism was observed in cells carrying a stably integrated reporter gene, indicating that this phenomenon is not a result of transient transfection. To examine the role of the transcriptional activation function (AF) domains of ERα in resveratrol agonism, we compared the effects of resveratrol and estradiol (E2) on expression of exogenous reporter genes and an endogenous estrogen-regulated gene (TGFα) in MDA-MB-231 cells stably transfected with wild-type (wt) ERα or mutants with deleted or mutated AF domains. In reporter gene assays, cells expressing wtERα showed a superagonistic response to resveratrol. Deletion of AF-1 or mutation of AF-2 attenuated the effect of resveratrol disproportionately compared to that of E2, while deletion of AF-2 abrogated the response to both ligands. In TGFα expression assays, resveratrol acted as a full agonist in cells expressing wtERα. Deletion of AF-1 attenuated stimulation by E2 more severely than that by resveratrol, as did deletion of AF-2. In contrast, mutation of AF-2 left both ligands with a limited ability to induced TGFα expression. In summary, the effect of modifying or deleting AF domains depends strongly on the ligand and the target gene.