Characterization of the tissue-selectivity of traditional Chinese medicine (TCM)-derived phytoestrogen and the possible mechanisms involved

Abstract

Icariin is the most abundant flavonoid in Herba Epemedii (HEP), a commonly used Chinese herb for treatment of bone disease, that has been reported to exert estrogenic effects. Danggui Buxue Tang (DBT), consisting of Radix Astragali and Radix Angelicae Sinensis, is the most popular traditional Chinese Medicine (TCM) decoction prescribed for management of menopausal symptoms in China and its chemical composition has been well characterized. Recent studies found that DBT may contain phytoestrogens and majority of the activities of DBT are mediated by these phytoestrogens. This study aimed to investigate the estrogenic effects of icariin and DBT in different estrogen-sensitive tissues and the possible mechanisms involved. In the first part of my study, the tissue-selectivity of icariin was evaluated by using mature ovariectomized (OVX) Sprague Dawley rats and four estrogen-responsive cell lines (human breast cancer MCF-7 cell, human endometrial Ishikawa cell, human neuroblastoma SH-SY5Y cell and human osteosarcoma MG-63 cell). Upon treatment for 12 weeks, icariin dose-dependently increased the bone mineral density (BMD) and improved the trabecular bone properties at distal femur, proximal tibia and lumbar spine in OVX rats. Moreover, icariin reversed the changes in mRNA expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in striatum of OVX rats but did not induce estrogenic responses in uterus and breast. In addition, icariin significantly stimulated cell proliferation or differentiation but failed to induce the expression of estrogen-responsive genes and estrogen response element-dependent luciferase activities in MCF-7, Ishikawa cell, SH-SY5Y cell and MG-63 cell. These results indicate that icariin did not induce estrogen-dependent transcriptional events in these cell lines. In the second part of my study, the tissue-selectivity of DBT was investigated using both mature OVX rats and estrogen receptor (ER)-positive cell lines. The results showed that DBT reversed the decrease in estradiol level as well as the accompanying increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) level in rats induced by ovariectomy. In addition, DBT increased BMD and improved micro-architecture of both long bone and vertebra in OVX rats. DBT also up-regulated mRNA expression of TH and suppressed mRNA expression of DAT in striatum of OVX rats without inducing estrogenic responses in uterus and breast tissue of OVX rats. Our in vitro studies showed that DBT directly stimulated cell proliferation and differentiation as well as ERE luciferase activities in MCF-7, Ishikawa cell, SH-SY5Y cell and MG-63 cell, suggesting the direct estrogenic effects of DBT. Co-treatment of DBT-treated MG-63 cells with ICI182,780 (ER antagonist, 10-6M), U0126 (mitogen-activated protein kinase MAPK inhibitor, 10-6M) and LY294002 (phosphoinositide 3-kinase PI3K inhibitor, 10-6M) completely or partially blocked the stimulating effects of DBT on cell proliferation and cell differentiation. These results indicate that ER, MAPK and PI3K pathways might be involved in mediating the actions of DBT in bone cells. It is also of concern to determine if the use of herbal medicine will be safe for postmenopausal women who are simultaneously prescribed with selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene for treatment of breast cancer and osteoporosis as many of their activities are mediated by the same receptors. In the third part of my study, the potential interactions between DBT and SERMs (tamoxifen and raloxifene) were thereby investigated. Upon co-treatment for three months, DBT did not alter the estrogenic effects of tamoxifen and raloxifene in the uterus, breast tissues, brain and bone of OVX rats. In addition, DBT at 0.1mg/ml did not interact with the estrogenic effects of SERMs in MCF-7 cells. However, DBT at 0.5mg/ml antagonized the effects of tamoxifen on cell proliferation in MCF-7 cell and enhanced the stimulating effects of tamoxifen and raloxifene on cell proliferation of SH-SY5Y cell ras well as the alkaline phosphatase (ALP) activity in Ishikawa and MG-63 cells. These results suggest that DBT does not interact with western drugs (tamoxifen and raloxifene) in estrogen-sensitive tissues in vivo while the in vitro interactive effects appear to be dose-dependent and tissue-specific. This discrepancy between in vivo and in vitro studies might be due to the fact that DBT applied in the cells was without biological activation and such concentrations are too high to be achieved in vivo. In conclusion, both icariin and DBT selectively exerted protective effects in bone and brain without causing side effects in uterus and breast in vivo. Our study provide evidence to support our hypothesis that phytoestrogens derived from TCM selectively exert estrogenic effects in estrogen-sensitive tissues and might be useful for the management of postmenopausal syndromes.