Journal of Steroid Biochemistry & Molecular Biology 120 (2010) 184–191 Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb Estrogenic properties of naturally occurring prenylated isoflavones in U2OS human osteosarcoma cells: Structure–activity relationships Sefirin Djiogue a,b,c , Dieudonné Njamen a,c , Maria Halabalaki b , Georg Kretzschmar a , Antje Beyer a , Jean-Claude Mbanya d , Alexios-Leandros Skaltsounis b , Günter Vollmer a,∗ a Molekulare Zellphysiologie & Endokrinologie, Institut für Zoologie, Technische Universität Dresden, 01062 Dresden, Germany b School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece c Laboratory of Animal Physiology, Department of Animal Biology and Physiology, University of Yaoundé 1, P.O. Box 812, Yaoundé, Cameroon d Department of Internal Medicine, Faculty of Medicine and Biomedical Sciences, University of Yaoundé 1, Yaoundé, Cameroon article info Article history: Received 22 December 2009 Received in revised form 15 April 2010 Accepted 17 April 2010 Keywords: Estrogenic activity Erythrina poeppigiana Leguminosae Prenylated isoflavones Natural genistein derivatives Human osteosarcoma cell line (U2OS) abstract Eight isoflavones derivatives, with isoprenyl and/or 7-methoxy substitution, isolated from Erythrina poep- pigiana (Fabaceae) have been investigated for their estrogenic properties in receptor subtype-specific reporter gene assays. First we focused on their estrogen receptor alpha and beta (ER and ER) selectiv- ity, second we addressed structure–activity relationships, using bone-derived human osteosarcoma cell line (U2OS cells) stably expressing ER or transiently expressing ER. Our results show that a substitu- tion at position 3 ′ together with a 7-methoxy substitution on the genistein skeleton is associated with a statistically significant activation of the ER- and ER-dependent reporter gene expression in U2OS cells starting from 0.1 nM. Particularly, the 7-methoxy-3 ′ -isoprenyl (1) and the 7-methoxy-3 ′ -(3-methyl- 2-hydroxybuten-3-yl) (3) derivatives of genistein induces an ER- and ER-coupled luciferase activity at a concentration ten times lower than that of genistein, for which a statistically significant effect was observable at 1 nM. On the other hand, isoprenyl substitution at position 6 of the A ring, compound 5, seems to have very little impact on the genistein ability to induce ER-coupled luciferase activity in U2OS cells, while a double prenylation at positions 8 and 3 ′ , compound 7, is associated with an almost complete loss of function on the reporter gene activation in U2OS-ER, but in ER expressing system the effec- tiveness remains on a statistically significant level, demonstrating an “exclusive ER-selectivity” in U2OS human osteosarcoma cells, and therefore 7 can be considered as an isotype-selective ER ligand. Finally all the tested isoflavones derivatives appear to exhibit a slightly pronounced ER preference, depending upon the position and the nature of the substituent moiety on the isoflavone skeleton. The estrogen-like effect of these prenylated isoflavone derivatives could be inhibited by the pure ER antagonist ICI 182 780, indicating that these effects were primarily mediated through ERs. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Erythrina poeppigiana (Walp.) O.F. Cook [1] is a large tree dis- tributed in South America, Africa, and Asia. Like other Erythrina species, this plant has been reported as being of use in the folk empirical system of medicine as an abortive agent and for the treat- ment of various diseases, including amenorrhea [2]. We recently reported the isolation and characterization of novel and known isoflavonoids from E. poeppigiana, among which eight appear to be structurally related to genistein with isoprenyl and/or 7-methoxy substitution. In addition, significant binding affinities for the two isotypes of the estrogen receptor ER and ER were demonstrated (additional table) [3]. ∗ Corresponding author. Tel.: +49 351 463 31922; fax: +49 351 463 31923. E-mail address: Guenter.Vollmer@tu-dresden.de (G. Vollmer). Estrogens play an important role in growth, differentiation and function of many typical tissues of the female reproduc- tive tract as well as in bone maintenance [4,5]. Cessation of ovarian estrogen production occurring in menopausal and post- menopausal women is accompanied with a variety of symptoms, among them are, hot flushes, anxiety, depressions, vaginal atro- phy, and postmenopausal osteoporosis. As a therapeutic strategy, hormone replacement therapy (HRT) has been used for decades to treat estrogen deficiency symptoms [6], and to prevent long- term development of postmenopausal osteoporosis [7,8]. Recent data from the estrogen/progesterone combination in the Million Women Study [9] have once again opened up the discussion about the benefit/risk ratio of HRT and alternative strategies for the treat- ment of menopausal and postmenopausal disorders. Besides the employment of selective estrogen receptor modulators (SERMs) as therapeutic agents [10], the use of natural occurring compounds with estrogenic activity and specially ER subtype-specific ligands 0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2010.04.014