Evaluation of Estrogenic Activity of Extract from the Herbal Mixture Cynanchum wilfordii Hemsley, Phlomis umbrosa Turczaninow, and Angelica gigas Nakai원문보기
Hormone replacement therapy (HRT) consists of highly effective prescription medications for treating menopausal symptoms; however, these agents have exhibited side effects including the risk of estrogen-induced carcinogenesis. Therefore, interest in phytotherapy-based materials as a natural source o...
Hormone replacement therapy (HRT) consists of highly effective prescription medications for treating menopausal symptoms; however, these agents have exhibited side effects including the risk of estrogen-induced carcinogenesis. Therefore, interest in phytotherapy-based materials as a natural source of alternatives to estrogen therapy has increased. However, some of these herbal medicines have been reported to increase the risk of estrogen-induced cancer. Herbal formulations composed of a combination of Cynanchum wilfordii Hemsley (CW), Phlomis umbrosa Turczaninow (PU), and Angelica gigas Nakai (AG) extracts (CPAE) have been used for treating menopausal symptoms. Therefore, in this study, we aimed to examine the safety of CPAE by determining its potential adverse estrogenic activity using the Organization for Economic Cooperation and Development (OECD) test guideline 455 (TG455) in a stably transfected transcriptionally activated human estrogen receptor ${\alpha}$ ($hER{\alpha}$)-HeLa9903 cell model. We found that CPAE did not how any estrogenic activity or stimulate promoters containing estrogen response elements in MCF-7 cells. In addition, CPAE showed no significant selective activity against $hER{\alpha}$ and $hER{\beta}$, non-selective activity against the ER, or effects on ER target gene expression. Furthermore, CPAE did not significantly induce MCF-7 cell proliferation and uterine weight increase in ovariectomized rats. These results demonstrate that CPAE can be used as beneficial herbal drug for prevention and therapeutic intervention of estrogen carcinogenesis in menopausal women.
Hormone replacement therapy (HRT) consists of highly effective prescription medications for treating menopausal symptoms; however, these agents have exhibited side effects including the risk of estrogen-induced carcinogenesis. Therefore, interest in phytotherapy-based materials as a natural source of alternatives to estrogen therapy has increased. However, some of these herbal medicines have been reported to increase the risk of estrogen-induced cancer. Herbal formulations composed of a combination of Cynanchum wilfordii Hemsley (CW), Phlomis umbrosa Turczaninow (PU), and Angelica gigas Nakai (AG) extracts (CPAE) have been used for treating menopausal symptoms. Therefore, in this study, we aimed to examine the safety of CPAE by determining its potential adverse estrogenic activity using the Organization for Economic Cooperation and Development (OECD) test guideline 455 (TG455) in a stably transfected transcriptionally activated human estrogen receptor ${\alpha}$ ($hER{\alpha}$)-HeLa9903 cell model. We found that CPAE did not how any estrogenic activity or stimulate promoters containing estrogen response elements in MCF-7 cells. In addition, CPAE showed no significant selective activity against $hER{\alpha}$ and $hER{\beta}$, non-selective activity against the ER, or effects on ER target gene expression. Furthermore, CPAE did not significantly induce MCF-7 cell proliferation and uterine weight increase in ovariectomized rats. These results demonstrate that CPAE can be used as beneficial herbal drug for prevention and therapeutic intervention of estrogen carcinogenesis in menopausal women.
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제안 방법
One week after surgery, the OVX rats were divided into the following four groups of seven rats each and treated as indicated: OVX control group, E2 (10 μg/kg), and low- and high-dose CPAE (125 and 250 mg/kg, respectively) groups.
The quantity of each transcript was calculated as described in the instrument manual and normalized to the amount of β-ACTIN, which was the housekeeping gene.
대상 데이터
Cells were harvested and assayed for luciferase activity. All experiments were performed in triplicate. Bars represent mean ± SD.
The effects of CPAE on the uterus wet weight were determined by normalizing the uterine absolute and wet weights to the respective body weights of each rat. The animal experiments were conducted after approval (WJIACUC20160120-1-01) by WOOJUNG Life Science Research Center (WOO-JUNGBSC, Suwon, Korea).
The human estrogen receptor α (hERα)-expressing HeLa-9903 cell line (HeLa9903), which is stably transfected with a human ERα gene containing a luciferase reporter gene, was provided by the Chemicals Evaluation and Research Institute (CERI), Japan. The human breast cancer MCF-7 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in a humidified 5% CO2 incubator at 37°C with 10% dextran-coated charcoal-treated fetal bovine serum (FBS) and phenol-free Dulbecco’s modified Eagle’s medium (DMEM).
데이터처리
Statistical analysis. All experiments were repeated at least thrice, and a one-way analysis of variance (ANOVA) was used to determine the significance of differences between the treatment groups. The Newman-Keuls test was used for multi-group comparisons and a p-value < 0.
이론/모형
Stably transfected transcriptional activation (STTA) assay. The luciferase activities were determined in accordance with the OECD TG 455 method (11). HeLa9903 cells were exposed to reference chemicals and the serially diluted test extract, followed by incubation for 20~24 hr (at 37°C in an atmosphere of 5% CO2).
The accumulated PCR products were detected directly by monitoring the increase in SYBR® reporter dye fluorescence. The mRNA expression levels of E2-inducible genes (progesterone receptor [PGR] and trefoil factor 1 [TFF1]) in the MCF-7 cells treated with CPAE, E2, ICI, or a combination of these compounds were compared to those in the control cells at each time point using the comparative cycle threshold (Ct) method. The following primer sequences were used: hPGR, forward, 5'-GAC GTG GAG GGC GCA TAT-3' and reverse, 5'-GCA GTC CGC TGT CCT TTT CT3'; hTFF1, forward, 5'-CCC TCC CAG TGT GCA AAT A-3' and reverse, 5'-CTG GAG GGA CGT CGA TGG TA-3'; and hβ-ACTIN, forward, 5'-TGG CAC CCA GCA CAA TGA A-3' and reverse, 5'-CTA AGT CAT AGT CCG CCT AGA AGC A-3'.
성능/효과
In conclusion, our present study provided evidence that CPAE does not show any estrogenic effects in the STTA and ERE reporter assays on HeLa9903 and MCF-7 cells, respectively. CPAE showed no significant activity against hERα, hERβ, and non-selective ER, as well as the expression of ER target genes.
This assay is used to evaluate the ability of chemicals to function as ERα ligands and induce agonistic responses in HeLa9903 cells stably transfected with the hERα-Luc gene. In the STTA assay performed in this study, CPAE did not exhibit any estrogenic activity at any tested concentrations while E2 and genistein, well-known phytoestrogens used as reference chemicals, showed significant estrogenic activity in a concentration-dependent manner (Fig. 1).
In addition, a randomized, double-blinded, placebo-controlled clinical study was performed in non-Asian American women at Friends Medical Group and Shady Canyon Medical Group in CA, USA. In this study, the CPAE-treated group showed a more significant improvement in various menopausal symptoms such as hot flashes and night sweats, paresthesia, insomnia, nervousness, melancholia, vertigo, fatigue, rheumatic pain, and vaginal dryness than the placebo group did. Furthermore, no adverse effects or significant changes were observed in body weight, BMI, serum E2 and FSH levels, and liver enzymes that showed changes following treatment with HRT and other phytoestrogens (10).
CPAE was tested in vivo to determine if it induces uterine weight gain in rats and the results show that it did not increase uterine wet weight; however, E2 significantly increased both the absolute and relative wet weight compared to that of animals receiving saline only. The result demonstrated that CPAE was not estrogenic in adult female Sprague-Dawley rats in vivo (Fig. 5). A previous study reported that the mixture of CW and PU extracts decreased the uterus wall thickness and increased the body weight of OVX rats (26), which is consistent with our in vivo data.
We attempted to determine if CPAE exposure affects the proliferation of MCF-7 cells. The result showed that the CPAE did not affect the proliferation of MCF-7 cells compared with the effects of the positive control E2 (Fig. 4), indicating that CPAE did not affect the development of E2-mediated breast cancer. Wild yam extract, which is known as “natural progesterone” and popularly used to alleviate menopausal symptoms, was reported not to induce the growth of MCF-7 cells (22), which is similar to our present result.
후속연구
Unfortunately, the National Institutes of Health reported that genistein, an aglycone of isoflavone from soybean, shows carcinogenic potential through its binding with estrogen receptors (ER) α and β (3,4). Although the genistein contents in isoflavone-related ingredients may not be high enough to cause cancer, further investigations are needed to determine the possible side effects of phytoestrogens.
참고문헌 (26)
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