Endocrine pharmacology
Egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae promote the biosynthesis of estrogen by aromatase

https://doi.org/10.1016/j.ejphar.2012.07.005Get rights and content

Abstract

Estrogen deficiency is associated with a variety of diseases, including osteoporosis, atherosclerosis, and Alzheimer's disease. Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyze the biosynthesis of estrogens from androgens. Inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer. However, small molecular agonists of aromatase, which would be useful to locally promote estrogen biosynthesis for the prevention of estrogen deficiency-induced diseases, are rarely reported. In this study, we established a nonradioactive assay for measuring aromatase activity by using human ovarian granulosa KGN cells and identified two estrogen biosynthesis-promoting compounds, egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae. The compounds also promoted estrogen biosynthesis in 3T3-L1 preadipocyte cells. Further study showed that neither compound affected the transcriptional and translational expression of aromatase in KGN cells, but that both significantly promoted the in vitro enzyme activity of recombinant expressed aromatase. Egonol gentiotrioside was also found to increase the serum estrogen level in ovariectomized rats. These results suggest that these two compounds may promote estrogen biosynthesis through the allosterical regulation of aromatase activity. Egonol gentiobioside and egonol gentiotrioside are, therefore, valuable targets for structural modification and warrant further investigation for their potential as novel pharmaceutical tools for the prevention of estrogen deficiency-induced diseases.

Introduction

Estrogens are steroid hormones that regulate growth, differentiation, and function in a broad range of target tissues in the body. The actions of estrogen are mediated by the estrogen receptors, which are expressed in a variety of cells and function through genomic or nongenomic actions on target genes (Heldring et al., 2007). Estrogen biosynthesis is catalyzed by aromatase (CYP19A1), which is responsible for binding of the C19 androgenic steroid substrate and catalyzing the necessary reactions to form the phenolic A ring characteristic of estrogens (Simpson et al., 2002). In humans, the expression of aromatase at the various sites is regulated by tissue-specific promoters through the use of alternative splicing mechanisms (Simpson, 2004). In the ovary and testes, aromatase expression is mediated by promoter II, which binds the transcription factors cAMP-response element binding protein (CREB) and steroidogenic factor-1. Aromatase expression in the gonads is thus regulated by gonadotropins through the stimulation of cAMP generation (Michael et al., 1995). In adipose cells and osteoblasts, aromatase expression is driven by promoter I.4, which is regulated by class I cytokines (Zhao et al., 1995, Shozu and Simpson, 1998). Thus, the regulation of estrogen biosynthesis in each tissue site of expression is unique, and switches in promoter usage have been found to be associated with the incidence of breast cancer (Simpson et al., 1997).

Aromatase inhibitors, such as anastrozole, letrozole, and exemestane, have been developed for the treatment of hormone-dependent breast cancer in postmenopausal women since they show superiority over conventional anti-estrogen receptor drugs such as tamoxifen (Johnston and Dowsett, 2003). In comparison with the extensive number of studies dedicated to discovering new aromatase inhibitors, the search for small molecular aromatase agonists that promote estrogen biosynthesis has been small. Although some herbicides, fungicides, and insecticides have been found to promote aromatase expression in a cAMP-dependent or -independent manner (Sanderson et al., 2000, You et al., 2001, Morinaga et al., 2004), more work is needed to find potent aromatase agonists that produce less side effects. Epidemiological studies have suggested the involvement of estrogen insufficiency in osteoporosis, neurodegenerative diseases, and cardiovascular diseases (Deroo and Korach, 2006). Estrogen therapy is an established regimen for the prevention of these diseases, but recent evidence indicates that its long-term use is accompanied by side effects such as increased risks of breast, ovarian, and endometrial cancer (Davison and Davis, 2003). Thus, alternative methods that improve the therapeutic efficacy and safety by locally promoting estrogen biosynthesis should be developed for the prevention and treatment of these diseases caused by estrogen deficiency.

In this study, we established a nonradioactive assay for measuring aromatase activity by using human ovarian granulosa KGN cells. Two natural products, egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae, were identified to significantly promote estrogen biosynthesis, and their effects on aromatase activity in vitro and in vivo were examined.

Section snippets

Materials

Testosterone, formestane, forskolin, dibenzylfluorescein, and nilestriol were purchased from Sigma (Shanghai, China), dissolved in DMSO to a concentration of 100 mM, and stored at −20 °C. The NADPH regenerating system was purchased from Promega (Madison, WI, USA). The recombinant expressed human aromatase plus reductase was purchased from BD Biosciences (San Jose, CA, USA). The magnetic particle-based 17β-estradiol enzyme-linked immunosorbent assay (ELISA) kit was purchased from Bio-Ekon

Effect of S. perkinsiae extract on estrogen biosynthesis in human ovarian granulosa-like cells

To obviate the limitations caused by the use of isotope-labeled androgen substrates and cancer cell lines expressing weak aromatase activity, we first established a convenient and sensitive assay to examine estrogen biosynthesis in the human granulosa-like tumor cell line (KGN), using a magnetic particle-based ELISA. To minimize the effects of steroids present in the fetal bovine serum, experiments were performed using culture medium without serum, and there was no significant cytotoxicity when

Discussion

Currently, aromatase activity is determined by measuring the amount of 3H-water released upon the enzymatic conversion of radiolabeled androgens in a cell-free assay using human placental microsomes or recombinant human aromatase protein (Njar et al., 1995, Vinggaard et al., 2000) or in a cell-based assay using mammalian cell lines such as the human JEG-3 and JAr cell lines (Drenth et al., 1998, Brueggemeier et al., 1997). However, these assays require the use of radioactive materials and the

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grants nos. 20932007, 30572221, and 30900769), the West Light Foundation of Chinese Academy of Sciences, and the National New Drug Innovation Major Project of China (2011ZX09307-002-02).

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