Journal of Steroid Biochemistry & Molecular Biology 88 (2004) 223–234 Estrogenic effects of resveratrol in breast cancer cells expressing mutant and wild-type estrogen receptors: role of AF-1 and AF-2 Barry D. Gehm a,b,1 , Anait S. Levenson b , Hong Liu b , Eun-Jig Lee a,b , Beth M. Amundsen b , Mark Cushman c , V. Craig Jordan b , J. Larry Jameson a,b,∗ a Division of Endocrinology, Metabolism and Molecular Medicine, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611, USA b Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611, USA c Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907, USA Received 25 July 2003; accepted 4 December 2003 Abstract 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 ER in resveratrol agonism, we compared the effects of resveratrol and estradiol (E2) on expression of exogenous reporter genes and an endogenous estrogen-regulated gene (TGF) in MDA-MB-231 cells stably transfected with wild-type (wt) ER or mutants with deleted or mutated AF domains. In reporter gene assays, cells expressing wtER 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 TGF expression assays, resveratrol acted as a full agonist in cells expressing wtER. 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 TGF expression. In summary, the effect of modifying or deleting AF domains depends strongly on the ligand and the target gene. © 2004 Elsevier Ltd. All rights reserved. Keywords: Resveratrol; Phytoestrogens; Estrogen receptor; Activation function; Transforming growth factor ; Reporter gene assay 1. Introduction Resveratrol (trans-3,4 ′ ,5-trihydroxystilbene) was discov- ered by Michio Takaoka more than 60 years ago, in the resin of Veratrum grandiflorum (false hellebore) [1]. Resver- atrol attracted attention when it was found to be present in grapes and red wine [2]; its anti-oxidant, anti-inflammatory and anti-thrombotic properties have been suggested to play a 1 Present address: Lyon College, Science Division, 2300 Highland Road, Batesville, AR 72501, USA. Abbreviations: AF, activation function; E2, 17-estradiol; ER, estro- gen receptor; ERE, estrogen response element; MEM, minimal essential medium; PR, progesterone receptor; SERM, selective estrogen receptor modulator; TGF, transforming growth factor alpha; tk, thymidine ki- nase; wt, wild-type ∗ Corresponding author. Tel.: +1-312-503-0469; fax: +1-312-926-7260. E-mail address: ljameson@northwestern.edu (J.L. Jameson). role in the health benefits of wine and the “French paradox” [3,4]. In addition, it is present at substantial concentrations in some traditional Asian medicinal plants [5,6]. Interest in resveratrol was further heightened when Jang et al. [7] re- ported that it has anti-carcinogenic effects in a variety of models of tumor initiation, promotion and progression. Resveratrol has some structural similarity to the synthetic estrogen DES, which led us to consider the possibility that it would modulate the activity of ERs. As previously reported [8], we found that it acts as an ER agonist in human breast cancer cell lines. Specifically, we observed that it stimu- lates the growth of estrogen-dependent T47D cells; it also induces the expression of endogenous estrogen-regulated genes and exogenous estrogen-responsive reporter genes in MCF-7 cells. Reporter gene activation is also ob- served in ER-negative MDA-MB-231 breast cancer cells but only when cotransfected with a plasmid expressing 0960-0760/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2003.12.002