Chemical Constituents from Cimicifuga dahurica and Their Anti-Proliferative Effects on MCF-7 Breast Cancer Cells

This study was designed to search for novel anti-cancer compounds from natural plants. The 70% ethanolic extract from the rizhomes of Cimicifuga dahurica (Turcz.) Maxim. (Ranunculaceae) was found to possess significant in vitro anti-proliferative effects on MCF-7 breast cancer cells. A phytochemical investigation using assay-guided fractionation of the ethanolic extract of C. dahurica resulted in the isolation of one new phenolic amide glycoside 3, two new lignan glycosides 4 and 7, one new 9,19-cycloartane triterpenoid glycoside 6, and thirteen known constituents 1, 2, 5, and 8–17. The structures of 3, 4, 6, and 7 were established using contemporary NMR methods and from their HRESIMS data. The anti-proliferative effects of isolated compounds were evaluated using the BrdU-proliferation kit. Five among the 17 isolated compounds showed significant anti-proliferative effects (p ≤ 0.05), wherein compound 7 showed the most significant anti-proliferative and cell cycle arresting effect (p ≤ 0.05) which followed a dose dependent manner. Western blot protein expression analysis showed a down expression of c-Myc and cyclin D1 which further elucidated the anti-proliferation mechanism of compound 7 while apoptotic effects were found in association with Bcl-2 family protein expression variations. Conclusively this study reports the isolation and identification of 17 compounds from C. dahurica, including four novel molecules, in addition to the fact that compound 7 possesses significant anti-proliferative and apoptotic effects in vitro that may require further exploration.


Introduction
The genus Cimicifuga (now known as Actaea) has been widely used all over the world and since ancient times has been a traditional Chinese herbal medicine. This genus consists of 28 species worldwide [1], among which nine species are indigenous to China. Three Cimicifuga species, namely, C. dahurica, C. foetida, and C. heracleifolia, commonly known as "Shengma" in Chinese, are officially listed in the Chinese Pharmacopoeia [2]. In traditional Chinese medicine, Shengma is widely used for treating aphtha, sore throat, toothache, and wind-heat headache. It also has been used in archoptosis, non-erupting measles, spot poison, uterine prolapse, and other diseases [1][2][3][4]. Complete assignments of all carbons and protons were based on HSQC, COSY, and HMBC experiments. After acid hydrolysis of 3, the absolute configuration of the sugar unit was confirmed using TLC, based on a sugar standard and analysis of the coupling constant of H-1 (J = 8.0 Hz). Thus, the sugar moiety was identified as β-D-allopyranoside. The position of an allose sugar and two methoxy groups were determined by HMBC experiments. The HMBC correlations between H-1 (δ H 5.32) and C-4 (δ C 149.7), H-2 (δ H 7.17) and H-6 (δ H 7.12) with C-4 suggested the location of the allose at C-4. Two methoxy groups attached at C-3 and C-3 were determined based on the HMBC correlations of the methoxy protons (δ H 3.88) with C-3 (δ C 150.9) and H-5 (δ H 7.17) with C-3, the methoxy protons (δ H 3.84) with C-3 (δ C 148.9) and H-5 (δ H 6.75) with C-3 , respectively. Using the COSY and HMBC spectra, the structure of 3 could be divided two parts, where part A was a (2E)-3-[4-(β-D-allopyranosyl)-3-methoxyphenyl unit and part B was a (4-hydroxy-3-methoxyphenyl)ethyl moiety ( Figure 1). These parts were connected through an amide bridge based on the HMBC correlation of H-8 (δ H 3.51) with C-9 (δ C 168.8) and the downfield-shift of the nitrogen-bearing methylene group at C-8 (δ C 42.4). From the evidence above, compound 3 was determined as a new compound with the (2E)-3-[4-(β-D-allopyranosyl)-3-methoxy-phenyl]-N-[2-(4-hydroxy-3-methoxyphenyl) ethyl]-2-propenamide structure.     Table 2). The 13 C-NMR spectrum of 4 exhibited the resonance signals of 16 carbons including the aglycone and sugar unit. The carbon chemical shifts of the aglycone at δ C 55.7 (C-1 and C-5), 87.2 (C-2 and C-6), 73.0 (C-4 and C-8), and aromatic carbons ranging from 104.8 to 154.4 suggested that 4 is a lignan skeleton with a tetrasubstituted aromatic ring. Comparing the 1 H-and 13 C-NMR spectroscopic data of 4 with those of a previously reported lignan named liriodendrin [16] revealed the similarity of their chemical shifts. However, the downfield-shift of the proton signal at H-1 , H-3 to 5.17 (d, J = 7.5 Hz), 4.15 (t, J = 2.5 Hz), respectively, and upfield-shift of C-2 ", 3 , 4 in the sugar unit (δ C 73.1, 72.2, and 68.7) indicated the presence of an allose moiety in the structure of 4 instead of glucose as in liriodendrin. These findings, together with the correlations of H-2 , H-3 , and H-4 and the absence of correlation between H-1 and H-3 in the ROESY spectrum indicated that the protons at C-1 were in a β-configuration and C-2 ", C-3 ", C-4 were the α-configuration. In addition, acid hydrolysis of 4 and comparison of the TLC, specific rotation, and properties of sugar unit with a sugar standard, in combination with the analysis of the coupling constant of H-1 (J = 7.5 Hz) confirmed the absolute the sugar to be a β-D-allopyranoside (Table 2). In the HMBC spectrum, the correlations between H-1 (δ H 5.17) and C-4 , C-4 (δ C 136.0), as well as H-2 , H-2 , H-6 , H-6" (δ H 6.74), with C-4 , C-4 determined the allose moiety to be linked at C-4 and C-4 . These evidences indicated that compound 4 was a new compound which was named syringaresinol di-O-β-D-allopyranoside.

Cell Proliferation Activity
MCF-7 breast cancer cells were exposed to epidermal growth factor (EGF) which was known as a stimulator of proliferation (positive control). The anti-proliferative effects of the extract of C. dahurica and five purified compounds (compounds 1, 2, 7, 8, 9) under co-incubation of MCF-7 cells with 50 ng/mL EGF had statistically significant differences compared to control vehicle ( Figure 2A). Among these five purified compounds, compound 7 had the most potent anti-proliferative effect at the same concentration. In addition, the similar anti-proliferative effect of 7 was demonstrable under co-incubation of MCF-7 cells with 50 ng/mL EGF ( Figure 2B).  To date, only two lignans have been found in the plants of Cimicifuga genus, actaealactone, a neolignan derived from C. racemosa [30] and (+)-isolariciresinol-3-O-β-D-glucopyranoside from C. dahurica [31]. Actaealactone exhibited a small stimulating effect on the growth of MCF-7 breast cancer cells about 1.24-fold (14 µM) compared to untreated cells, while compound 7 exhibited the significantly anti-proliferative effect on MCF-7 cells even at the lower concentration (3 µM), showing more potential anti-proliferative effect.

Cell Cycle Distribution and Western Blot Analyses
For exploration of the anti-proliferation mechanism of compound 7, western blotting was performed to determine the effect of compound 7 to the levels of cyclin D1 and c-Myc. The cyclin D1 proto-oncogene exercises powerful control over the mechanisms that regulate the mitotic cell cycle, and overexpression of cyclin D1 is common in human cancers [32]. C-Myc encodes for a transcription factor that is a key regulator of a wide variety of cellular processes. In most non-malignant cells, Myc levels are low, its constitutive expression is linked to the pathogenesis of many human cancers [33]. As shown in Figure 3A-C, the downregulation of cyclin D1 protein level and c-Myc level were observed in the cells treated with compound 7. After observing the expression variation of Cyclin D1 and c-Myc, we further assessed the cell cycle phases to explore the variations. Our results showed that compound 7 caused cell cycle transition arrest in the G0/G1 phase that was markedly increased in dose dependent manner ( Figure 3D,E) in breast cancer MCF-7 cells. The increased arrest of cell cycle at G0/G1 phase was contrastingly reduced in subsequent S and G2/M phase indicting that the cell cycle was arrested within G0/G1 to S phase while a very small proportion was arrested in G2/M phase. These results seconded our earlier results of Cyclin D1 and c-Myc ( Figure 3A-C).
We examined the effect of the caspase inhibitors and the nucleoside addition on compound 7-induced cell cycle arrest in MCF-7 cells by adding a caspase 3 inhibitor (50 µM, Z-DEVD-FMK), a pan caspase inhibitor (50 µM, Z-VAD-FMK) or a nucleoside mixed solution of dATP, dCTP, dGTP and dTTP (125 µM of each) (dNTP Mixture) to the cultured media with compound 7 (30 µM). As shown in Figure 4, the cell cycle arrest at G0/G1 phase was reversed by a pan caspase inhibitor or a caspase 3 inhibitor but not the nucleoside addition. These results demonstrated that pan caspase and caspase 3 maybe had major roles in the mechanism of cell cycle arrest effects of compound 7 on MCF-7 cells. Figure 2. Proliferation of MCF-7 cells exposed to the extraction of Cimicifuga dahurica Maxim. at the concentration of 30 µ g/mL or 10 µ M of purified compounds (1-17) (A) and the increasing doses of compound 7 under simultaneous application of 50 mg/mL EGF (B). Means ± SD, n = 5 of three independent experiments: **** p < 0.0001 for the EGF group versus the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 versus the EGF group.  caspase inhibitor (50 μM, Z-VAD-FMK) or a nucleoside mixed solution of dATP, dCTP, dGTP and dTTP (125 µ M of each) (dNTP Mixture) to the cultured media with compound 7 (30 μM). As shown in Figure 4, the cell cycle arrest at G0/G1 phase was reversed by a pan caspase inhibitor or a caspase 3 inhibitor but not the nucleoside addition. These results demonstrated that pan caspase and caspase 3 maybe had major roles in the mechanism of cell cycle arrest effects of compound 7 on MCF-7 cells. After MCF-7 cells were treated with compound 7 (0, 1, 3, 10, 30 μM) for 48 h, cells were stained with annexin V/PI and examined using a BD flow cytometer. Early and late apoptosis and necrotic cells were discriminated. The corresponding quantities of total cell apoptosis and early apoptosis were 4.8 and 2.1%; 8.2 and 2.3%; 13.8 and 4.0%; 17.8 and 3.3%; 27.0 and 6.9%; respectively. The data demonstrated that apoptotic cells were found to be increased in a dose-dependent manner ( Figure   Figure 4. The effects of a caspase 3 inhibitor, a pan caspase inhibitor and nucleoside on compound 7-induced cell cycle arrest in MCF-7 cells. *** p < 0.001 versus the control group.

Induction of Apoptosis by Compound 7 in MCF-7 Cells
After MCF-7 cells were treated with compound 7 (0, 1, 3, 10, 30 µM) for 48 h, cells were stained with annexin V/PI and examined using a BD flow cytometer. Early and late apoptosis and necrotic cells were discriminated. The corresponding quantities of total cell apoptosis and early apoptosis were 4.8% and 2.1%; 8.2% and 2.3%; 13.8% and 4.0%; 17.8% and 3.3%; 27.0% and 6.9%; respectively. The data demonstrated that apoptotic cells were found to be increased in a dose-dependent manner ( Figure 5A). We further assessed the expression variations of Bcl-2 family proteins including Bcl-2, Bcl-XL (anti-apoptotic proteins) and Bax (pro-apoptotic protein). Our results showed that after 48 h treatment of compound 7, the expression of anti-apoptotic proteins (Bcl-2, Bcl-XL) were decreased in dose dependent manner alternatively the expression of pro-apoptotic protein (Bax) was increased in breast cancer MCF-7 cells ( Figure 5B). 5A). We further assessed the expression variations of Bcl-2 family proteins including Bcl-2, Bcl-XL (anti-apoptotic proteins) and Bax (pro-apoptotic protein). Our results showed that after 48 h treatment of compound 7, the expression of anti-apoptotic proteins (Bcl-2, Bcl-XL) were decreased in dose dependent manner alternatively the expression of pro-apoptotic protein (Bax) was increased in breast cancer MCF-7 cells ( Figure 5B). To determine whether cell apoptosis was dependent upon caspase activation and the influence of nucleoside in the cultured media on the cells apoptosis produced by compound 7, we examined the effects of the caspase inhibitors and the nucleoside addition on compound 7-induced apoptosis in MCF-7 cells using Annexin V-FITC/PI kit. As shown in Figure 6, apoptosis was reduced by a pan caspase inhibitor or a caspase 3 inhibitor but not the nucleoside addition. These results demonstrate that compound 7 induces apoptosis in MCF-7 cells by activating a pan caspase and caspase 3dependent apoptotic pathway. To determine whether cell apoptosis was dependent upon caspase activation and the influence of nucleoside in the cultured media on the cells apoptosis produced by compound 7, we examined the effects of the caspase inhibitors and the nucleoside addition on compound 7-induced apoptosis in MCF-7 cells using Annexin V-FITC/PI kit. As shown in Figure 6, apoptosis was reduced by a pan caspase inhibitor or a caspase 3 inhibitor but not the nucleoside addition. These results demonstrate that compound 7 induces apoptosis in MCF-7 cells by activating a pan caspase and caspase 3-dependent apoptotic pathway. Collectively in our present study we conclusively found four new compounds including one new phenolic amide glycoside 3, two new lignan glycosides 4 and 7, and one new 9,19-cycloartane triterpenoid glycoside 6. In addition to these four novel compounds we also isolated and purified thirteen other previously known compounds 1, 2, 5, and 8-17 from the ethanol extract of C. dahurica. In continuation of our studies searching for novel anti-cancer agents from natural products, we found that the ethanol extract from the rhizomes of C. dahurica exhibited considerable inhibitory effects on the proliferation of MCF-7 cells through the BrdU-proliferation test. Moreover we found that compound 7 had significant anti-proliferatory and apoptotic effects against breast cancer MCF-7 cells. In additions, we also found that compound 7 had significant cell cycle arresting (at G0/G1-S) effects following graded dose response effectiveness. The apoptotic effects and cell cycle arresting (at G0/G1-S) effects of compound 7 maybe through caspase 3 and pan caspase-dependent pathways. Although compounds 7 and 4 were two chemically similar lignans with the same sugar moieties, the higher anti-proliferative activity in MCF-7 cells of 7 compared to 4 suggested that two methoxy units in the benzene ring (rather than four methoxy units) may be associated with increased biological potency.

General Experimental Proceduces
The 1 H and 13 C, DEPT, HSQC, HMBC, ROESY, and COSY NMR spectra were recorded on a Avance 500 MHz instrument (Bruker, Switzerland). The high-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained from an Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies Co., Ltd., Waldbronn, Germany). Semi-preparative HPLC was performed on a Agilent 1100 series system,)using an ODS column (Zorbax SB-C18, 9.4 × 250 mm, 5 µm, Agilent Technologies Inc., Santa Clara, CA, USA). Column chromatography was performed on silica gel (200-300 mesh and 300-400 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex TM LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and octadecyl silica (ODS, 50 μm, YMC Co., Ltd., Tokyo, Japan). Thin-layer chromatography was performed on a precoated GF254 plate (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), RP-18 F254S plates (Merck, Kenilworth, NJ, USA) and compounds were visualized by spraying with aqueous 10% H2SO4 and then heating for 3-5 min. Collectively in our present study we conclusively found four new compounds including one new phenolic amide glycoside 3, two new lignan glycosides 4 and 7, and one new 9,19-cycloartane triterpenoid glycoside 6. In addition to these four novel compounds we also isolated and purified thirteen other previously known compounds 1, 2, 5, and 8-17 from the ethanol extract of C. dahurica. In continuation of our studies searching for novel anti-cancer agents from natural products, we found that the ethanol extract from the rhizomes of C. dahurica exhibited considerable inhibitory effects on the proliferation of MCF-7 cells through the BrdU-proliferation test. Moreover we found that compound 7 had significant anti-proliferatory and apoptotic effects against breast cancer MCF-7 cells. In additions, we also found that compound 7 had significant cell cycle arresting (at G0/G1-S) effects following graded dose response effectiveness. The apoptotic effects and cell cycle arresting (at G0/G1-S) effects of compound 7 maybe through caspase 3 and pan caspase-dependent pathways. Although compounds 7 and 4 were two chemically similar lignans with the same sugar moieties, the higher anti-proliferative activity in MCF-7 cells of 7 compared to 4 suggested that two methoxy units in the benzene ring (rather than four methoxy units) may be associated with increased biological potency.

Plant Material
The rizhomes of Cimicifuga dahurica were collected from Panshi City, Jilin Province, China in November 2015. The plant materials were authenticated by Prof. Li Hui-Jun of the Department of Pharmacognosy, China Pharmaceutical University. A voucher specimen (No. XASM201511) was deposited in the State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China.

Acid hydrolysis and Sugar Identification
The new compounds 3, 4, 6 and 7 (2 mg of each) were dissolved in MeOH (2 mL) and refluxed with 1.0 N HCl (2 mL) for 3 h and following by extracting with ethyl acetate. The water layer was then neutralized by Ag 2 CO 3 , and filtered to give a monosaccharide. Each monosaccharide of those compounds had an Rf

Anti-Proliferative Activity
After treatment with C. dahurica extract at the concentration of 30 mg/mL and 10 µM of purified compounds (1-17) under co-incubation with 50 ng/mL EGF, MCF-7 cells were incubated with BrdU labelling solution (10 µM) for 2 h and fixed with fixation solution for 30 min at room temperature. 100 µL anti-BrdU peroxidase-labelled antibody was added for 90 min and washed three times. 100 µL/well of substrate solution for colorimetric quantification was added at room temperature for 5-30 min until colour development was sufficient for photometric detection. The absorbance was measured at 405 nm using a Synergy 2 multi-detection microplate reader (Bioteck, Winooski, VT, USA).

Cell Cycle Distribution Analyses
We cultured breast cancer MCF-7 cells in small dishes and after 24 h of incubation the cells were treated with varying concentrations (1, 3, 10 and 30 µM) of compound 7. The cells were then further incubated for 48 h and then collected. The harvested cells were centrifuged at 1000 rpm for 5 min, washed thoroughly with ice cold PBS and were then subjected to fixation in 75% ethanol over night at 4 • C. After fixation the ethanol was removed and cells (about 1 × 10 6 ) were re-suspended in PBS having RNase (25 µg/mL), then the cells were further incubated for another half hour in propidium iodide (50 µg/mL) at 37 • C and subjected to flow cytometer analyses. Cell cycle distribution was analyzed by ModFitLT (Verity, Software House, Topsham, ME, USA).
To determine whether cell cycle distribution is dependent upon caspase activation and the influence of the nucleoside addition to apoptotic activity of compound 7, a caspase 3 inhibitor (50 µM, Z-DEVD-FMK, Medchem Express, Monmouth Junction, NJ, USA), a pan caspase inhibitor (50 µM, Z-VAD-FMK, Beyotime Biotechnology, Shanghai, China) or a nucleoside mixed solution of dATP, dCTP, dGTP and dTTP (125 µM of each) (dNTP Mixture, Beyotime Biotechnology) were added to the media with compound 7 (30 µM) to incubation for 48 h and followed by flow cytometer analyses as described above.

Annexin V/PI Analyses
The Annexin V-FITC/PI apoptosis detection kit (Keygen Biotechnology, Nanjing, China) was used to detect the effects of compound 7. MCF-7 cells (3 × 10 5 cells/well) were seeded in 6-well plates and cultured for 24 h. After treatment of cells with compound 7 (0, 3, 10, 30 µM) for 48 h, adherent and floating cells were collected and washed with ice cold PBS, then cells were centrifuged. The pellet was suspended with binding buffer (500 µL), stained with Annexin V-FITC (5 µL) and PI (5 µL), and then incubated for 15 min at room temperature in the dark. After incubation, cells were analyzed with a BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA) to determine the percentage of apoptotic cells.
To determine whether cell apoptosis is dependent upon caspase activation and the influence of the nucleoside addition on apoptotic activity of compound 7, a caspase 3 inhibitor (50 µM Z-DEVD-FMK, Medchem Express, Monmouth Junction, NJ, USA), a pan caspase inhibitor (50 µM Z-VAD-FMK, Beyotime Biotechnology, Shanghai, China) or a nucleoside mixed solution of dATP, dCTP, dGTP and dTTP (125 µM of each) (dNTP Mixture, Beyotime Biotechnology) were added to the media with compound 7 (30 µM) and incubated for 48 h.
3.5.5. Western Blotting MCF-7 cells were seeded in 6-well plates and treated with compound 7. Cells were collected and washed with cold phosphate-buffered saline (PBS). The collected cells were lysed on ice for 30 min in 100 µL lysis buffer (120 mM NaCl, 40 mM Tris (pH 8), 1% Nonidet P-40 and protease inhibitors) and centrifuged at 12,000 rpm for 30 min. BCA protein assay kit (Beyotime, Shanghai, China) was used to measure protein concentrations as described in a previous report [34]. Whole-cell extracts were subjected to electrophoresis on polyacrylamide gels and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST). Then, anti-cyclin D1, anti-Bcl-2, anti-Bcl-XL, anti-Bax antibodies (Cell Signaling Technology, Danvers, MA, USA), anti c-Myc antibody (Abcam, Cambridge, MA, USA) or anti-actin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was bound overnight at 4 • C. After washing with TBST, HRP conjugated secondary antibody (Keygen, Nanjing, China) was bound for 1 h at room temperature. Finally, protein bands were detected using a Tanon 5200 Multi Chemiluminescent Imaging System (Tanon, Shanghai, China). The ratio of cyclin D1/β-actin and c-Myc/β-actin were analyze using NIH Image J.