Cyclopia Extracts Act as ERα Antagonists and ERβ Agonists, In Vitro and In Vivo Koch Visser, Morné Mortimer, Ann Louw * Department of Biochemistry, University of Stellenbosch, Matieland, Stellenbosch, Republic of South Africa Abstract Hormone replacement therapy associated risks, and the concomitant reluctance of usage, has instigated the search for new generations of estrogen analogues that would maintain estrogen benefits without associated risks. Furthermore, if these analogues display chemo-preventative properties in breast and endometrial tissues it would be of great value. Both the selective estrogen receptor modulators as well as the selective estrogen receptor subtype modulators have been proposed as estrogen analogues with improved risk profiles. Phytoestrogen containing extracts of Cyclopia, an indigenous South African fynbos plant used to prepare Honeybush tea may serve as a source of new estrogen analogues. In this study three extracts, P104, SM6Met, and cup-of-tea, from two species of Cyclopia, C. genistoides and C. subternata, were evaluated for ER subtype specific agonism and antagonism both in transactivation and transrepression. For transactivation, the Cyclopia extracts displayed ERα antagonism and ERβ agonism when ER subtypes were expressed separately, however, when co-expressed only agonism was uniformly observed. In contrast, for transrepression, this uniform behavior was lost, with some extracts (P104) displaying uniform agonism, while others (SM6Met) displayed antagonism when subtypes were expressed separately and agonism when co-expressed. In addition, breast cancer cell proliferation assays indicate that extracts antagonize cell proliferation in the presence of estrogen at lower concentrations than that required for proliferation. Furthermore, lack of uterine growth and delayed vaginal opening in an immature rat uterotrophic model validates the ERα antagonism of extracts observed in vitro and supports the potential of the Cyclopia extracts as a source of estrogen analogues with a reduced risk profile. Citation: Visser K, Mortimer M, Louw A (2013) Cyclopia Extracts Act as ERα Antagonists and ERβ Agonists, In Vitro and In Vivo. PLoS ONE 8(11): e79223. doi:10.1371/journal.pone.0079223 Editor: Wei Xu, University of Wisconsin - Madison, United States of America Received July 31, 2013; Accepted September 20, 2013; Published November 4, 2013 Copyright: © 2013 Visser et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors would like to thank the Medical Research Council (MRC) and the Cancer Association of South Africa (CANSA) for financial support to A. L. (grant for projects entitled “Cyclopia Phytoestrogens”and “Cyclopia and breast cancer”) and for financial support to K. V. and M. M. the National Research Foundation (NRF) and the Harry Crossley foundation is acknowledged. Any opinion, findings and conclusions or recommendations expressed in this material are those of the author(s) and therefore the funders do not accept any liability in regard thereto. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: al@sun.ac.za Introduction Hormone replacement therapy (HRT), estrogens alone or in combination with progestins, is traditionally prescribed to women undergoing menopausal transition to alleviate symptoms associated with menopause [1], such as hot flashes, night sweats, sleeping problems, vaginal dryness, and osteoporosis [2-4]. However, a number of side effects have been associated with the use of HRT, for example, an increased occurrence of breast cancer [5,6], vaginal bleeding [7], and heart disease or strokes [6,8]. These side effects have led to reluctance among concerned consumers to use HRT and instigated a search for new estrogen analogues with an improved risk profile. Furthermore, it would be of great value if these analogues should also display chemo-preventative properties in breast tissue [9,10]. Estrogens elicit their biological effects by binding to transcription factors called estrogen receptors (ERs) in the target organ/tissue (uterus, ovary, vagina, liver, bone, and breast) [11-13]. The ER exists as two subtypes, namely ERα and ERβ [14]. Current estrogens in HRT activate both subtypes of ER in all tissues [14-19]. This attribute is beneficial in bone [18,20,21] and for hot flashes [18,21], but detrimental in the breast [6,21,22] and uterus [21,23] as it increases the risk of tumorigenesis. In contrast, the selective estrogen receptor modulators (SERMs), although not ER subtype specific [24,25], act as agonists in certain tissues, such as bone [26-28], and as antagonists in others, such as breast [9,10,29]. Although, the well-known SERMs, raloxifene and tamoxifen [30], have been shown to decrease the risk of breast cancer [18,31,32] and increase bone mineral density [26-28,33], they have also been linked to an increased risk of venous thromboembolism and PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e79223