Estrogen-like activities of saikosaponin-d in vitro: A pilot study

Abstract

Saikosaponin-d (SSd), a saponin derivative with a similar structure to estradiol, was extracted from Bupleurum falcatum L. (Umbelliferae). It was found that SSd stimulated the proliferation of MCF-7 cells by using MCF-7 cell proliferation assay. Cell-cycle analysis revealed that the proliferation-stimulating effect was associated with a marked increase in the number of MCF-7 cells in S phase. These actions of SSd were dose-dependent at doses ranging from 10 nM to 10 µM and could be significantly inhibited by the specific estrogen receptor (ER) antagonist ICI-182780. Co-incubation of MCF-7 cells with 1 μM of ER antagonist ICI-182780 abolished the inductive effects of SSd on estrogen response element (ERE)-luciferase activity, suggesting that the estrogenic effects of SSd were mediated through the estrogen receptors. To evaluate the relative involvement of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) in mediating the actions of SSd, ER-negative human cervical carcinoma (HeLa) cells were cotransfected with the ERE-luciferase reporter construct and either ERα or ERβ construct. The results showed that SSd could activate ERE-luciferase activity via the ERα-mediated pathway in a dose-dependent manner (10 nM to 10 µM); whereas, the activation of ERβ-mediated ERE-luciferase activity by SSd only occurred at a high concentration (10 µM). Furthermore, the ERα protein and mRNA levels were increased by treatment with SSd within 24 h. These data support our hypothesis that SSd acts as a weak phytoestrogen. Presumably, the estrogenic effect of SSd is mediated by the estrogen receptor.

Introduction

Radix Bupleuri (Chinese name: Chaihu) has been used for over 2000 years for the treatment of common cold with fever, hepatitis, kidney syndrome, inflammatory diseases, menoxenia, etc. (Yang et al., 2007, Zhao et al., 1996). It is believed that saikosaponins are responsible for part of the pharmaceutical properties of Radix Bupleuri (Zhu et al., 2006). In particular, saikosaponin-d (SSd) is especially known for its pharmacological activities such as anticancer, anti-inflammation, and liver protection, as well as corticosterone-like activity (Aoyagi et al., 2001, Hsu et al., 2004, Leung et al., 2005, Morinaga et al., 2006). Furthermore, Xiao-Chai-Hu-Tang (Japanese name: Sho-Saiko-To), a Bupleurum soup for oral administration, the main active constituent of which is SSd, has achieved great success in the treatment of menoxenia, menopausal symptoms and hepatic fibrosis (Ikegami et al., 2003). Various studies have indicated that Bupleurum has estrogenic activity, although the detailed mechanism of action, the effective dosages as well as its efficacy in different clinical conditions are not clear. We assumed that, as the main constituent of Bupleurum soup, saikosaponin-d might be a new type of phytoestrogen.

Many flavone glycosides, including isoflavones such as daidzein and genistein, are structurally similar to estrogens and are believed to possess the estrogenic activities through ER-dependent pathway and ER-independent pathway (Singhal et al., 2009). The estrogenic activities of phytoestrogens are closely associated with their structures. The number and position of hydroxyl groups in the molecular structure are other important factors which contribute to strong binding to estrogen receptors and the resulting estrogenic activity (Ding et al., 2005). Interestingly, it seems that SSd has similar structure with estrogen mimetics (Fig. 1).

There are two major types of estrogen receptors, ERα and ERβ, which differ in their ligand specificities and physiological functions. Certain natural hormones or phytochemicals are being shown to possess differential affinity for and transactivation of ERα versus ERβ. For example, the natural hormone estradiol binds with equal affinity to ERα and ERβ, while the well-known phytoestrogens genistein and coumestrol, preferentially interact with and activate ERβ to mediate estrogenic effects in vitro assays (Brigitte and Johannes, 2001). Thus, one of our aims in the present study is to observe whether SSd activates ER isoforms α and β differentially.

In this study, by using the estrogen-dependent human breast cancer cell line MCF-7 and the ER-negative human cervical carcinoma cell line HeLa as model systems, we investigated the estrogenic-like activity of SSd as well as the associated mechanisms.