Analysis and Identification of Active Compounds from Gami-Soyosan Toxic to MCF-7 Human Breast Adenocarcinoma Cells

Gami-soyosan is a medicinal herbal formulation prescribed for the treatment of menopausal symptoms, including hot flashes and osteoporosis. Gami-soyosan is also used to treat similar symptoms experienced by patients with breast cancer. The incidence of breast cancer in women receiving hormone replacement therapy is a big burden. However, little is known about the components and their mechanism of action that exhibit these beneficial effects of Gami-soyosan. The aim of this study was to simultaneously analyze compounds of Gami-soyosan, and determine their cytotoxic effects on estrogen receptor (ER)-positive MCF-7 human breast adenocarcinoma cells. We established a simultaneous analysis method of 18 compounds contained in Gami-soyosan and found that, among the various compounds in Gami-soyosan, gallic acid (1), decursin (17), and decursinol angelate (18) suppressed the viability of MCF-7 cells. Gallic acid (1), decursin (17), and decursinol angelate (18) induced apoptotic cell death and significantly increased poly (ADP-ribose) polymerase (PARP) cleavage and the Bcl-2-associated X protein/ B-cell lymphoma 2 (Bax/Bcl-2) ratio. Decursin (17) increased the expression of cleaved caspases-8, -9, -7, and -3. Decursinol angelate (18) increased the expression of cleaved caspase-8 and -7. These three components altered the different apoptosis signal pathways. Collectively, gallic acid (1), decursin (17), and decursinol angelate (18) may be used to inhibit cell proliferation synergistically in patients with ER-positive breast cancer.


Introduction
Breast cancer originates in breast ducts and lobes, and is a very common cancer that accounts for 25.2% of all gynecological cancers worldwide. It is generally known that factors, such as older age, history of contralateral breast cancer, late menopause, family history, and hormone replacement therapy (HRT), increase the risk of breast cancer. Before menopause, the risk of breast cancer incidence increases two-fold every decade [1][2][3].
Among the types of breast cancer, estrogen receptor (ER)-positive breast cancer accounts for more than a 70% incidence rate. Moreover, estrogen plays a significant role in the growth and development of cancer cells [4]. Tamoxifen, which inhibits the action of estrogen, and aromatase inhibitors, which block estrogen production, are usually used to treat breast cancer via endocrine therapies [5].

Gami-Soyosan Sample
The Gami-soyosan decoction was prepared in the Korea Institute of Oriental Medicine (KIOM), as described in our previous study [20]. Briefly, the 12 raw herbs of Gami-soyosan (Table 1) were obtained from Kwangmyungdang Medicinal Herbs (Ulsan, Korea) and identified by Professor Jung-Hoon Kim of the School of Korean Medicine, Pusan National University (Yangsan, Korea). A voucher specimen of each raw material (from 2012-KE45-1 to  and the Gami-soyosan decoction (2012-KE45) were stored at the KIOM. To obtain the Gami-soyosan water decoction, the mixture of 12 herbs was extracted in distilled water at 100 • C for 2 h under pressure (98 kPa) using an electric extractor (COSMOS-660; Kyungseo Machine Co., Incheon, Korea). The extract solution was freeze-dried by using a LP100R freeze dryer (IlShinBioBase, Yangju, Korea) to afford 970.4 g of the Gami-soyosan extract.

HPLC Analysis of Gami-Soyosan
High-performance liquid chromatography (HPLC) analysis for 18 marker components for quality assessment of the Gami-soyosan sample are described in detail in our previous study [20]. In brief, simultaneous determination was carried out using an LC-20A Prominence HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a photo diode array (PDA) detector and the analytical parameters for simultaneous analysis are shown in Table 2.

Cells and Cell Culture
MCF-7 cells are from a human breast adenocarcinoma cell line obtained from the American Type Culture Collection (ATCC, Bethesda, MD, USA). They were cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Corning, Manassas, VA, USA) containing 10% fetal bovine serum (Gibco BRL, Carlsbad, MD, USA) and 1% penicillin/streptomycin solution (1000 IU/mL penicillin and 10,000 µg/mL streptomycin; Life Technologies, Waltham, MA, USA) in a CO 2 incubator at 37°C. In all cell experiments, dimethyl sulfoxide (DMSO) was used as a vehicle to dissolve samples. The final concentration of DMSO was kept under 0.1%, which exerts no effect of the vehicle (DMSO) compared to the naive cell.

Determination of Cell Viability
The Ez-Cytox assay kit, which is a WST-based Cell Viability/Cytotoxicity Assay Kit (Daeil Lab Service Co., Seoul, Korea), was used for quantifying cell viability. MCF-7 cells (1 × 10 4 cells/well) were seeded into a 96-well plate and cultured until confluent. Vehicle (dimethyl sulfoxide, DMSO) or indicated concentrations of each compound were added into the wells and incubated for 24 h. Subsequently, Ez-Cytox solution was added into each well and the cells were incubated for 1 h. The absorbance at 450 nm compared with absorbance of a reference at 600 nm was determined through a SPARK 10 M microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Cell viability was calculated based on a ratio to 100% of the vehicle control (DMSO).

Image-Based Cytometric Assay
MCF-7 cells (1 × 10 6 cells/well) were seeded into a 6-well plate and cultured for 24 h. Vehicle (DMSO) or indicated concentrations of each compound were added into the wells and incubated for 24 h. Following incubation, the cells were detached and then stained with an image-based cytometric assay kit (Invitrogen, Temecula, CA, USA) in the dark for 20 min at 20 ± 5 • C. The presence of apoptotic cells was determined using TaliImage-based cytometer (Invitrogen, Carlsbad, CA, USA) and analyzed with TaliPCApp (version 1.0, Invitrogen, Carlsbad, CA, USA). The ratio of apoptotic cells was calculated based on a ratio to 100% of the vehicle control (DMSO).

Western Blotting
MCF-7 cells (1 × 10 6 cells/well) were seeded into a 6-well plate and cultured for 24 h. Vehicle (DMSO) or indicated concentrations of each compound were added into the wells and incubated for 24 h. Following incubation, the cells were collected and lysed with RIPA buffer (Elpis Biotech, Daejeon, Korea). The protein concentrations of lysates were determined using a Pierce™ BCA protein assay kit (Thermo Scientific, Carlsbad, CA, USA). Equal amounts of protein and sample buffer (4× NuPAGE LDS, Thermo Scientific) were mixed and boiled for 5 min at 95 • C. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using acrylamide gels and transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). After blocking with 2.5% skim milk, membranes were blotted with specific primary antibodies antibody against poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cleaved caspase-3, -7, -8, -9, and β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA) for 6 h at 20 ± 5°C. After washing, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Inc.) at 20 ± 5°C for 1 h. The blotted membrane was developed with Pierce ECL Western Blotting Substrate (Rockford, IL, USA) and visualized with the chemiluminescence System (FUSION Solo; PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Statistical Analysis
All quantitative data are presented as the mean ± standard deviation (S.D.) (n = 3). Comparisons between the groups were performed using repeated measures analysis of variance (ANOVA). When a significant interaction was present, Dunnett's post-hoc test was performed. The test was considered statistically significant when p < 0.05 as compared to untreated cells.

Results and Discussion
Breast cancer can be categorized as ER-positive or ER-negative based on the presence or absence of ER expression, respectively; usually, breast cancer is ER-positive [21]. The development of ER-positive breast cancer is closely related to the action of estrogen. High estrogen levels in the blood or excessive estrogen exposure leads to an increased risk of developing breast cancer [22]. Estrogen directly or indirectly increases the risk of genetic mutations associated with cell proliferation and apoptosis in the breast tissue. In addition, estrogen induces cell proliferation through the stimulation of growth factors in breast cancer cells and the suppression of TNF-α, which induces apoptosis. Estrogen facilitates cancer cell survival by conferring insensitivity to cell death signaling [23].
In addition, estrogen is widely used as the most effective treatment for menopausal symptoms [9]; consequently, side effects have been reported. Estrogen-progestin therapy, which is a type of HRT, is known to increase the risk of invasive breast cancer [7,8]. Studies have also reported that HRT increases the risk of breast cancer [8,9].
Generally, therapeutic agents that suppress the action and production of estrogen are utilized to treat patients with ER-positive breast cancer [24]. Tamoxifen is a representative drug that works as an ER antagonist in the prevention and therapy of breast cancer. This therapy has contributed significantly to the reduction in breast cancer recurrence and mortality [25,26]. Aromatase inhibitors, which are agents that suppress the production of estrogen, are utilized as alternatives to tamoxifen in postmenopausal women. Aromatase converts other hormones to estrogen. In postmenopausal women, aromatase is the main regulator of estrogen production, whereas aromatase inhibitors suppress estrogen production [27,28]. However, when these drugs are used to treat breast cancer through estrogen inhibition, estrogen deficiency symptoms, such as vaginal dryness, unstable emotion, osteoporosis, and arthritis, are observed. Because these symptoms cannot be treated with HRT, as is usual in postmenopausal women, other treatments are required [29]. In traditional Korean medicine, Gami-soyosan is prescribed to ameliorate menopausal symptoms, especially for the treatment of hot flashes [30]. Gami-soyosan is used not only for menopausal women, but also for patients with breast cancer who experience symptoms similar to menopause symptoms [16][17][18].
Our preliminary experiments indicated that some Gami-soyosan compounds, such as gallic acid (1), decursin (17), and decursinol angelate (18), suppressed the viability of MCF-7 cells. In this study, the morphological changes induced by these compounds were examined using a phase-contrast inverted microscope. Compared with untreated cells, the treatment with gallic acid (1), decursin (17), and decursinol angelate (18) induced a large number of morphological changes, including membrane blebbing, cell shrinkage, cell condensation, and detachment from the plate ( Figure 3A). Cellular morphological changes, such as cell shrinkage, membrane bleb formation, nuclear condensation, and phagocytosis by neighboring cells, are characteristic of apoptotic cell death [35]. Gallic acid (1) significantly increased cytotoxicity in a concentration-dependent manner ( Figure 3B, IC 50 : 81.3 µM); similar results were obtained for decursin (17) and decursinol angelate (18)   Apoptosis occurs via extrinsic or intrinsic pathways [40,41]. The extrinsic pathway is regulated by signals from other cells and is initiated by FAS (Fas cell surface death receptor), TNF-α (tumor necrosis factor-alpha), and andligands that penetrate the cell membrane. Subsequently, caspase-8, -3, or -7 are activated and apoptosis occurs. The intrinsic pathway, also called the mitochondrial pathway, is predominantly controlled by the Bcl-2 (B-cell lymphoma 2) family, which comprises antiapoptotic and pro-apoptotic proteins [42][43][44][45]. Bcl-2 and Bcl-xl, which are representative antiapoptotic proteins, inhibit the penetration of the mitochondrial membrane and suppress apoptosis while disrupting the action of pro-apoptotic proteins, such as Bax (Bcl-2-associated X protein) and Bak (BCL2-antagonist/killer). However, if these proteins do not function correctly, Bax promotes apoptosis through the induction of caspase-9, -3, and-7 activation. Following the intrinsic and extrinsic pathways, the executioner caspases, caspase-3, -6, and-7, are activated. This in turn causes continuous morphological and biochemical changes in apoptotic cells through the activation of Gallic acid is an indicator component of Paeoniae Radix (the roots of Paeonia lactiflora Pallas) [19]. It promotes the expression of Bax, which induces cancer cell apoptosis and suppresses the expression of apoptosis inhibitory proteins [36]. Through these mechanisms, gallic acid exerts anticancer effects in esophageal, stomach, colon, and uterine cancer cells. In particular, in MCF-7 cells, gallic acid increases p27 kip1 , which inhibits cell cycle regulation and controls MCF-7 cell proliferation through its preventative action on cell division in the G 2 /M phase of the cell cycle [37].
Decursin and decursinol angelate are indicator components of Angelicae Gigantis Radix (roots of Angelica gigas Nakai) [19]. They are known to suppress cell division in the G1 phase of the MCF-7 cell cycle and act as anti-cancer agents by inhibiting the mRNA and protein expression of ERα [38]. They also suppress vascular endothelial growth factor (VEGF)-induced phosphorylation of VEGF-2 and prevent angiogenesis, which plays a key role in the growth of cancerous tumors [39].
Although the results of Bax were differently expressed, gallic acid (1), decursin (17), and decursinol angelate (18) increased the Bax/Bcl-2 ratio and PARP, respectively. It has been shown that these compounds cause apoptosis in MCF-7 cells via PARP cleavage and the Bax/Bcl-2 ratio increase.
In MDA-MB-231 cells, gallic acid (1) enhanced the activity of initiator caspase-8 and -9 MCF-7 cells [49]; however, our study showed that the expression of cleaved caspase-9 was significantly increased in MCF-7. The present study demonstrated that gallic acid (1) induced only extrinsic apoptotic pathways in MCF-7 cells.
Treatment with decursin (17) also increased the protein expression of cleaved caspase-9 (apoptotic initiator caspase) and -3 (apoptotic effector caspase) in a concentration-dependent manner. In addition, decursin (17) significantly increased the expression of cleaved caspase-8 (apoptotic initiator caspase) and -7 (apoptotic effector caspase). These results demonstrated that decursin induced both the intrinsic and extrinsic apoptotic pathways in MCF-7 cells.
In contrast with decursin (17), decursinol angelate (18) induced the protein expression of cleaved caspase-9 and -3 without changing caspase-8 and -7. Decursin (17) and decursinol angelate (18) are structural isomers on the side chain [38]. We found that the distinguished structures of the side chain affect the activation of apoptosis related caspases.
Although the results of Bax were differently expressed, gallic acid (1), decursin (17), and decursinol angelate (18) increased the Bax/Bcl-2 ratio and PARP, respectively. It has been shown that these compounds cause apoptosis in MCF-7 cells via PARP cleavage and the Bax/Bcl-2 ratio increase.

Conclusions
We established a method to simultaneously analyze 18 compounds of Gami-soyosan, a prescription used to improve menopausal symptoms. Among the various compounds of Gami-soyosan, gallic acid (1), decursin (17), and decursinol angelate (18) were found to promote apoptosis in MCF-7 cells by increasing pro-apoptotic and decreasing anti-apoptotic protein levels. In conclusion, gallic acid (1), decursin (17), and decursinol angelate (18) may be the Gami-soyosan compounds that inhibit cell proliferation in ER-positive breast cancer. These results suggest that gallic acid, decursin, and decursinol angelate, which are components of Gami-soyosan, may be used to inhibit cell proliferation synergistically in patients with positive breast cancer.