Characterization of Antiestrogenic Activity of the Chinese Herb, Prunella vulgaris (PV), Using In Vitro and In Vivo (Mouse Xenograft) Models

Collins NH, Lessey EC, DuShell CD, McDonnell DP, et al. Biol Reprod. Feb 2009; 80(2): 375–383. Nov 5, 2008. doi: 10.1095/biolreprod.107.065375

Prunella vulgaris (Xia Ku Cao), a commonly used Chinese herb, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182 780 and PV extract.
Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner—responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women. DISCUSSION Herbal medicines are widely used in the United States, with up to one quarter of adults reporting use of such remedies within the past few years [23]. Not surprisingly, the primary physician is often unaware that their patients are using these treatments. The availability and perception that herbal and complementary remedies are safer than prescription medications may have increased the use of these remedies, often leading to self-treatment for a variety of ailments. Failure to recognize side effects of herbal remedies could lead to interactions or unanticipated health consequences. On the other hand, these medicinal agents may have beneficial effects that could be advantageously applied if the mechanisms of action were better understood in the context of Western medicine. There are several conditions affecting women in which herbal therapies have found a niche. These include menopausal symptoms, menstrual disorders, and treatment for benign conditions, such as endometriosis or uterine fibroids [44, 45]. Many traditional US Food and Drug Administration-approved medications for these conditions have significant side effects or expense [45, 46]. Public concerns about hormone replacement using commercially available estrogens or progestins have also increased after the Women's Health Initiative studies were published [47, 48]. Pre- and postmenopausal women are thus actively seeking natural or nontraditional medical approaches for a wide variety of complaints. In the present study, we screened 21 herbs using the alkaline phosphatase assay of Littlefield and coworkers [40] to detect either estrogenic or antiestrogenic characteristics. PV was the only herbal extract that had strong biological activity in this assay. In review of the literature, there is one report that supports this finding. Huang and colleagues [49] studied the Chinese herb Prunella stica based on its use for the treatment of dysmenorrhea. They showed that this herb blocked the proliferation of Ishikawa cells, but did not interfere with prostaglandin production [49]. Similar to that study, we found that PV extract specifically blocked cell proliferation without affecting cell viability in ECC-1 cells. The in vivo studies suggest that PV is effective when taken orally, and functions in vivo to block proliferation of ectopic endometrium in response to estrogen. The apparent reduction in CYR61 and MKI67 expression in the endometriotic implants provides evidence that PV interferes with estrogen receptor (ESR1) actions. The ability of PV to block estrogen action without affecting fecundity in mice suggests that this herb may have advantages for the treatment of women with gynecologic disease. We [12, 13] and others [15, 50–52] have pointed out the relationship between elevated estrogen action and benign endometrial disorders. ESR1 normally declines at the time of implantation, around Cycle Day 19–20 in normal, fertile women [53]. The failure to down-regulate this receptor may be a primary defect leading to altered gene expression patterns seen in endometriosis [1], or the infertility and pregnancy loss in women with polycystic ovary syndrome [12, 14]. Lack of ESR1 down-regulation is a feature of underlying P resistance seen in some women with benign gynecologic disease [1, 2]. Methods to block the inappropriate actions of estrogen during the putative window of implantation could theoretically be helpful and promote improved uterine receptivity toward embryo attachment and implantation. Studies to investigate this possibility are underway. Understanding the mechanism of the antiestrogenic activity of PV would likely be informative for the design of improved therapies for endometriosis. There is increasing interest in the role of the AHR pathways regulated by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and other environmental toxins in endometriosis. Antagonists of AHR found in food are increasingly being studied [54]. Red clover, thought to have SERM-like activities, is also an AHR ligand [55]. The trihydroxystilbene resveratrol, an active component in red wine, appears to have antagonistic actions via AHR. While many natural flavinoids appear to be AHR antagonists and, therefore, inhibit CYP1A1 expression [56, 57], PV appears to be an AHR agonist with antiestrogenic properties. Like other herbs noted to inhibit estrogen [58, 59], PV uses novel mechanisms to achieve this action. PV appears to activate the AHRE and stimulate AHR target genes in ECC-1 cells. We found little indication that PV had direct effects on ER in endometrial cells. The mechanism of action of PV to inhibit estrogen responses, potentially through regulation of AHR, remains elusive. AHR, besides regulating target genes, also interacts with both the androgen and estrogen receptors [60], and promotes degradation of these receptors through ubiquitin-mediated mechanisms. Direct action of the AHR to compete with ER activity is another possible explanation for the noted effects of PV on estrogen activity in vivo and in vitro. In addition, evidence points to regulatory elements of AHR as targets for estrogen, further complicating this puzzle [61]. Like resveratrol, PV may also act as a competitive antagonist for TCDD, providing yet another beneficial effect of PV on endometriosis in women who consume this herb. Such a mechanism could account for reported improvements in dysmenorrhea seen in women with endometriosis that take PV. There remains much to learn about herbal and complementary treatments, especially given the potential for cross-reactivity and unintended consequences when taken with other pharmaceuticals. Unrecognized properties of nontraditional therapies, including their actions as AHR ligands, could provide new opportunities for safe and effective treatment of disorders that have not been adequately addressed by conventional Western medical treatments. The discovery of the antiestrogenic properties of PV provides such an opportunity. This herbal treatment might now be understood at the molecular level as a treatment that blocks estrogen action in endometriosis, and may protect individuals from environmental factors that favor its development. Unlike resveratrol, found in red wine, which cannot as yet be conveniently consumed by women seeking pregnancy, PV may provide a potent alternative, with potential benefits for both pain and infertility. Further investigation and evaluation of the active component(s) of this herb are currently underway. 1. Burney RO, Talbi S, Hamilton AE, Vo KC, Nyegaard M, Nezhat CR, Lessey BA, Giudice LC. 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