| Summary: | 博士 === 國立陽明大學 === 傳統醫藥學研究所 === 94 === The consumption of over-the-counter (OTC) natural products in perimenopausal women and patients with breast diseases remains a challenging problem. Previous studies have shown that the proliferation of breast cancer cells could be enhanced by Angelica sinensis Radix, ginsenoside Rg1, and red clover extracts. Therefore, the safety of OTC natural products required caution. Si-Wu-Tang (SWT) consists of Angelica sinensis Radix, Rehmanniae preparata Radix, Ligusticum chuanxiong Hortorum, and Paeonia lactiflora Pallas. It is a well-known Chinese formula for the treatment of gynecologic diseases, but the efficacy and mechanisms of SWT on breast disease have never been studied. The aims of this study were to investigate the growth-stimulation effect of SWT and its constituents on MCF-7 breast cancer cells and the SWT-modulated cell signaling as well as the HER2 gene expression.
Using primary fibroadenoma culture and breast cancer cell lines, MCF-7 (estrogen receptor-positive; ER+, HER2-low), BT-474 (ER+, HER2-high), MDA-MB-231 (ER-, HER2-low) and SK-BR-3 (ER-, HER2-high), as in vitro models, the mitogenic effects of SWT, its constituents, and its active compound, ferulic acid (FA), were assessed by trypan blue dye exclusion assay and DNA flow cytometry. Besides, SWT-modulated cell signaling and HER2 gene expression were analysed by Western blot and real-time RT-PCR. In addition, by oophorectomized athymic BALB/c nude mice as in vivo model, MCF-7 cells were implanted in subcutaneous sites of mice. The effects of Si-Wu-Tang on tumor growth and uterus weight were assessed after 21 days of treatment.
The results showed that SWT enhanced cell proliferation on primary fibroadenoma culture and breast cancer cells in a dose-dependent manner. Water extract of Angelica sinensis and ethanol extract of Ligusticum chuanxiong played key roles on SWT-induced proliferative effect on MCF-7 cells. Meanwhile, HER2 downstream signaling molecules, AKT and ERK1/2, were involved in SWT-enhanced cell proliferation. The ERα-modulated pS2, ESR1, and HER2 gene expression were also upregulated by SWT. Besides, addition of SWT and water extract of Angelica sinensis increased HER2 and ERα proteins on MCF-7 cells. Pretreatment of HER2-signaling blocker concentration-dependently inhibited SWT-upregulated HER2 overexpression, indicating the involvement of transmembrane HER2 for the synthesis of HER2 in this setting. In addition, ER antagonist, ICI182780, abrogated SWT-enhanced proliferative effect, indicating the involvement of ER in these phenomena.
Furthermore, FA (10-8~10-6 M) were demonstrated to promote cell proliferation of primary culture and breast cancer cells in a concentration-dependent manner. FA also activated the phosphorylation of HER2 on MCF-7 cells. HER2 downstream signaling molecules, AKT and ERK1/2, but not p38, were involved in FA-modulated ERα and cyclin D1 expression. The pS2, ESR1, and HER2 gene expression were also upregulated by FA treatment. The facts that ICI182780 blocked FA-increased HER2 protein synthesis and that trastuzumab decreased ERα protein synthesis suggested a crosstalk between ERα and HER2 signaling on breast cancer cells.
In oophorectomized BALB/c nude mice model, subcutaneous injection of E2 significantly increased uterus weight, but not body weight. Skin lesions were noticed on 75% of 1 x SWT group and 50% of E2 group, respectively. The thickness of dermal layer was increased in 1 x SWT group. In addition, hyalinization degenerations under dermal layer were also observed in 3 x SWT and E2 group. Furthermore, tumor cells were presented in E2 group.
In conclusion, herbal remedy SWT and its active compound FA enhance breast tumor cell proliferation by modulating the HER2 signaling and expression.
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