Cycloartane triterpenoids from Cimicifuga yunnanensis induce apoptosis of breast cancer cells (MCF7) via p53-dependent mitochondrial signaling pathway

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PHYTOTHERAPY RESEARCH Phytother. Res. 25: 17–24 (2011) Published online 17 June 2010 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.3222

Cycloartane Triterpenoids from Cimicifuga yunnanensis induce Apoptosis of Breast Cancer Cells (MCF7) via p53-dependent Mitochondrial Signaling Pathway Zhong-Ze Fang,1,2 Yin Nian,2,3 Wei Li,1,2 Jing-Jing Wu,1 Guang-Bo Ge,1,2 Pei-Pei Dong,1,2 Yan-Yan Zhang,1,2 Ming-Hua Qiu,3 Lei Liu4 and Ling Yang1* 1

Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China 2 Graduate University of Chinese Academy of Sciences, Beijing, 100049, China 3 State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650204, China 4 Shenzhen Children’s Hospital, 7019 Hongli West Road, Futian District, Shenzhen, 518100, China

The present study was carried out to investigate the antitumor activity of five cycloartane triterpenoids isolated from Cimicifuga yunnanensis on the breast cancer cell line MCF7 and its corresponding drug resistant subline R-MCF7, including cimigenol-3-O-β-D-xylopyranoside (compound 1), 25-O-acetylcimigenol-3-O-β-D-xylopyranoside (compound 2), 25-chlorodeoxycimigenol-3-O-β-D-xylopyranoside (compound 3), 25-O-acetylcimigenol3-O-α-L-arabinopyranoside (compound 4) and 23-O-acetylcimigenol-3-O-β-D-xylopyranoside (compound 5). The results showed that compounds 2–5 have relatively high antitumor activity on both MCF7 and R-MCF7 cells. The involvement of apoptosis as a major cause of cycloartane triterpenoids-induced cell death was further confirmed. The results of RT-PCR showed that compounds 2–5 increased the expression of p53 and bax, which led to the loss of mitochondrial potential and then resulted in the activation of caspase-7. These findings collectively demonstrated that compounds 2–5 induced apoptosis of MCF7 via p53-dependent mitochondrial pathway. Copyright © 2010 John Wiley & Sons, Ltd. Keywords: cycloartane triterpenoids; MCF7; p53; apoptosis; mitochondrial membrane potential; caspase-7.

INTRODUCTION Breast cancer is regarded as the most common female malignant tumor in Western countries and is becoming more and more prevalent in Asia (Simstein et al., 2003; Lee et al., 2009). In terms of the therapy of breast cancer, chemotherapy drugs have been the mainstay for the past four decades (Early Breast Cancer Trialists Collab G, 1992). However, chemoresistance is a serious issue in the therapy of patients with breast cancer and limits the effectiveness of a large number of first-line chemotherapeutic agents including taxanes and anthracyclines (Coley, 2009). Therefore, the discovery of new agents that could overcome multi-drug resistance is highly desirable. Recently, the antitumor activities of the extracts of Cimicifugae plants and their major constituents cycloartane triterpenoids have been discovered both in vitro and in vivo and are drawing more and more attention (Hostanska et al., 2004; Tian et al., 2007, 2005; Einbond et al., 2004). The rhizome extracts of Cimicifuga racemosa (C. racemosa) and several isolated cycloartane triterpenoids (actein, 23-epi-26-deoxyactein, cimifugo-

side and cimiracemoside A) were demonstrated to exhibit marked cytotoxicity to estrogen receptorpositive (MCF-7) and estrogen receptor negative (MDA-MB-231 and MDA-MB-453) human breast carcinoma. The mechanisms were referred to as induction of cell cycle arrest and apoptosis (Hostanska et al., 2004; Einbond et al., 2004). Thus, cycloartane triterpenoids isolated from Cimicifugae plants might be a class of novel antineoplastic agents for the care of patients with breast cancer. In the present study, the breast cancer cell line MCF7 and its corresponding drug resistant cell subline R-MCF7 were utilized to investigate the antitumor activity of five cycloartane triterpenoids isolated from Cimicifuga yunnanensis (C. yunnanensis) which is a rare species indigenous to the north-west of Yunnan province of China and utilized by local people such as Tibetan and Naxi people to relieve fever, headache and some menopause symptoms. Further mechanisms were investigated and the results showed that compounds 2–5 induced apoptosis of MCF7 via p53-dependent mitochondrial signaling pathway.

MATERIALS AND METHODS * Correspondence to: Ling Yang, Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China. E-mail: [email protected]

Copyright © 2010 John Wiley & Sons, Ltd.

Plant material. Cimicifuga yunnanensis (C. yunnanensis) Hsiao rhizomes were collected from Napa lake, Received 21 January 2010 Revised 11 April 2010 Accepted 20 April 2010

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Xianggelila county, Yunnan Province, China, in August 2005. The plant was identified by Professor Bao-Gui Li, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Science. A voucher specimen (KUN No. 200508025) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Kunming, China. Tested cycloartane triterpenoids. Cimigenol-3-Oβ-d-xylopyranoside (compound 1), 25-O-acetylcimigenol-3-O-β-d-xylopyranoside (compound 2), 25-chlorodeoxycimigenol-3-O-β-d-xylopyranoside (compound 3), 25-O-acetylcimigenol-3-O-α-l-arabinoside (compound 4) and 23-O- acetylcimigenol-3-O-β-dxylopyranoside (compound 5) (Fig. 1) (Chen et al., 2007; Kadota et al., 1995; Liu et al., 1999; Kusano et al., 1999) were isolated from the rhizome of C. yunnanensis. The purity of the five compounds was more than 95%. All the tested compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/mL, then diluted in tissue culture medium and filtered before use. Cell culture. The breast-adenocarcinoma cell line MCF7 and its corresponding drug resistant cell subline R-MCF7 were obtained from the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and were available in our laboratory. The R-MCF-7 cell line was established by stepwise selection of original MCF-7 cells exposed to increasing concentrations of doxorubicin in the medium. Cells were grown in RPMI 1640 (Gibco, USA) supplemented

with 10% (v/v) fetal bovine serum (FBS, Hangzhou Sijiqing Biological Engineering Materials Co., Ltd, China) at 37°C and a 5% CO2 air atmosphere. Cell viability assay. The cells were seeded at a density of 6000 cells/well in 96-well plates and incubated for 24 h. And then, old media was replaced with fresh media containing various concentrations of cycloartane triterpenoids or 0.5% DMSO control. After the plates were incubated for 48 h, the SRB (sulforhodamine B) method was performed to assess the cytotoxicity of these compounds (Papazisis et al., 1997). In brief, 10% ice-cold trichloroacetic acid (TCA, 100 μL) was added. The plates were kept at 4°C for 1 h and washed five times with cold water, and then stained with sulforhodamine B (SRB, Sigma St Louis, MO, USA). After being washed with 1% acetic acid, the bound dye was solubilized with 100 μL Tris base (10 mm, pH 10.5) (Sigma St Louis, MO, USA) and the absorbance was read at 570 nm with a multi-detection microplate reader (BIO-RAD, USA). Hoechst 33342 staining assay. The Hoechst 33342 staining assay was employed to observe morphology alterations of the MCF7 cells and performed as reported previously (Yamakawa et al., 2008). In brief, the MCF7 cells were seeded at 2.4 × 104 cells per well in 24-well plates and after treatment of MCF7 cells with compound 2 (4 μg/mL), compound 3 (5.5 μg/mL), compound 4 (4.5 μg/mL) and compound 5 (50 μg/mL) for 48 h, all the cells were harvested and washed with PBS twice, and then incubated with Hoechst 33342 (5 μg/mL,

Figure 1. Chemical structure of compounds 1–5. Compound 1: cimigenol-3-O-β-D-xylopyranoside. Compound 2: 25-O-acetylcimigenol3-O-β-D-xylopyranoside. Compound 3: 25-chlorodeoxycimigenol-3-O-β-D-xylopyranoside. Compound 4: 25-O-acetylcimigenol-3-O-α-Larabinopyranoside. Compound 5: 23-O-acetylcimigenol-3-O-β-D-xylopyranoside. Copyright © 2010 John Wiley & Sons, Ltd.

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CYCLOARTANE TRITERPENOIDS INDUCING APOPTOSIS OF MCF7

Sigma) for 30 min at 37°C. The stained cells were visualized under a fluorescence microscope (Nikon). Annexin V-FITC/PI analysis. Flow cytometric analysis was performed to identify and quantify the apoptotic cells using an Annexin V-FITC/PI (propidium iodide) apoptosis detection kit (BD Bioscience). In brief, the MCF7 cells were seeded in 6-well plates at a density of 1 × 105 cells/well. After treatment with compound 2 (4 μg/mL and 6 μg/mL), compound 3 (6 μg/mL and 8 μg/mL), compound 4 (4 μg/mL and 6 μg/mL) and compound 5 (30 μg/mL and 75 μg/mL) for 24 h, both adherent and floating cells were harvested and stained with Annexin V-FITC/PI according to the manufacturer’s procedure. The samples were analysed with a Becton Dickinson FACSVantage SE instrument. Mitochondrial membrane potential (MMP, ΔΨm) analysis. The uptake of the cationic fluorescent dye rhodamine 123 (rho 123) has been used for the estimation of mitochondrial membrane potential (Yang et al., 2006). After treatment with 6 μg/mL of compound 2, 8 μg/mL of compound 3, 6 μg/mL of compound 4 and 60 μg/mL of compound 5 for 24 h, control and treated cells were harvested and washed twice with PBS buffer (pH 7.4), and then incubated with 0.5 μm rhodamine 123 (ICN Biomedicals, Inc) for 30 min at 37°C. The fluorescence intensity was measured using a FACSVantage SE flow

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cytometer (BD) at an excitation and an emission wavelength of 490 nm and 520 nm. The results were expressed as the fluorescence retained within the cells. Caspase-7 activity measurement. The activity of caspase7 was measured with a fluorometric substrate of caspase3/7, Asp-Glu-Val-Asp-p-nitroanaline (DEVD-pNA) (R&D Systems, Inc., USA) according to the manufacturer’s procedure. In brief, the MCF7 cells were treated with 6 μg/mL of compound 2, 8 μg/mL of compound 3, 6 μg/mL of compound 4 and 75 μg/mL of compound 5, respectively. After 24 h of treatment, the cells were collected and washed with PBS, and then treated with 50 μL of cold lysis buffer (R&D Systems, Inc) for 10 min. Then 50 μL reaction buffer and 5 μL DEVDpNA were added and incubated at 37oC for 2 h in the dark. These samples were analysed using a multi-detection microplate reader (BIO-RAD, USA) at 405 nm. Data were calculated as fluorescence units/mg protein and presented as the fold increase relative to the control. RT-PCR assay. Following treatment of cells with 6 μg/ mL of compound 2, 8 μg/mL of compound 3, 6 μg/mL of compound 4 and 60 μg/mL of compound 5 for 24 h, total RNA was extracted using TRIzol reagent (invitrogen, USA) according to the manufacturer’s instructions. The RNA yield and purity were assessed by spectrophotometric analysis. Total RNA (500 ng) from each sample

Figure 2. Growth inhibition of compounds 2–5 on MCF7 and R-MCF7 cells. MCF7 and R-MCF7 cells were treated with various concentrations of compounds 2–5 for 48 h. Cell viability was measured by SRB assay. The experiment was carried out in three independent experiments in triplicate. Data are given as mean ± SD. Copyright © 2010 John Wiley & Sons, Ltd.

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was subjected to reverse transcription with random nonamer, dNTP and AMV reverse transcriptase in a 10 μL reaction mixture. The PCR of cDNA was carried out using Takara Ex Taq Hotstart polymerase, dNTPs and the following related primers: 5′-CAA GAG ATG GCC ACG GCT GCT-3′ (sense) and 5′-TCC TTC TGC ATC CTG TCG GCA-3′ (antisense) for β-actin; 5′-CAG CTC TGA GCA GAT CAT GAA GAC-3′ (sense) and 5′-GCC CAT CTT CTT CCA GAT GGT GAG-3′ (antisense) for bax; 5′-CAG CTC CTA CAC CGG CGG CCC CTG CAC CAG-3′ (sense) and 5′-GAG CCA ACC TCA GGC GGC TCA TAG GGC ACC-3′ (antisense) for p53. After denaturation for 3 min at 94°C, the total amount of reaction products was amplified for 30 cycles for p53 (94°C, 30 s; 58°C, 30 s; 72°C, 120 s) and bax (94°C, 30 s; 58°C, 30 s; 72°C, 60 s) and 25 cycles for β-actin (94°C, 30 s; 58°C, 30 s; 72°C, 120 s) on the TAKARA PCR Thermal Cycler.

Data analysis and statistics. The results were expressed as mean ± standard deviation (SD). Statistical differences were evaluated using the two-tailed Student’s t-test and considered significant at the *p < 0.05, **p < 0.01 level.

RESULTS Cytotoxic activity After 48 h of incubation, compounds 2–5 displayed a dose-dependent growth inhibitory activity on MCF7 and R-MCF7 cells among all studied cycloartane triterpenoids (Fig. 2). The IC50 values are listed in Table 1. However, compound 1 exhibited weak activity even at a high concentration of 100 μg/mL.

Figure 3. Apoptotic morphological changes in MCF7 cells. MCF7 cells treated with vehicle; (2) MCF7 cells treated with compound 2 at 4 μg/mL for 48 h; (3) MCF7 cells treated with compound 3 at 5.5 μg/mL for 48 h; (4) MCF7 cells treated with compound 4 at 4.5 μg/ mL for 48 h; (5) MCF7 cells treated with compound 5 at 50 μg/mL for 48 h. Cells were stained by Hoechst 33342 and observed under Nikon fluorescence 40 × 10 microscopy. Copyright © 2010 John Wiley & Sons, Ltd.

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Table 1. Cytotoxicity of compounds 1–5 on MCF7 and R-MCF7 cells IC50 (μg/mL) Cell line

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Taxol

MCF7 R-MCF7

>100 >100

4.0 ± 0.13 5.29 ± 0.03

5.74 ± 0.06 6.18 ± 0.17

4.32 ± 0.10 4.81 ± 0.47

39.13 ± 1.52 59.75 ± 1.13

0.033 ± 0.004 4.81 ± 0.47

Taxol, positive control.

Figure 4. Representative figure of flow cytometric analysis of apoptosis. MCF7 cells were treated with compounds 2–5 for 24 h, and then stained with Annexin V/PI. This experiment was repeated three times. Control; (2–3) MCF7 cells treated with 4 μg/mL and 6 μg/ mL of compound 2 for 24 h; (4–5) MCF7 cells treated with 6 μg/mL and 8 μg/mL of compound 3 for 24 h; (6–7) MCF7 cells treated with 4 μg/mL and 6 μg/mL of compound 4 for 24 h; (8–9) MCF7 cells treated with 30 μg/mL and 75 μg/mL of compound 5 for 24 h.

Induction of apoptosis in MCF7 cells by compounds 2–5 In order to explore whether compounds 2–5 showed cytotoxicity to MCF7 cells through inducing apoptosis, the Hoechst 33342 staining assay was employed to Copyright © 2010 John Wiley & Sons, Ltd.

observe morphology alterations of the MCF7 cells after treatment with compound 2 (4 μg/mL), compound 3 (5.5 μg/mL), compound 4 (4.5 μg/mL) and compound 5 (50 μg/mL) for 48 h. Compared with the control, chromatin aggregation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into Phytother. Res. 25: 17–24 (2011)

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membrane-bound vesicles (apoptotic bodies) were observed in MCF7 cells after treatment with compounds 2–5 (Fig. 3). To further confirm and quantify apoptosis in MCF7 cells induced by compounds 2–5, the cells were stained with Annexin V-FITC/PI and analysed by flow cytometry. In the dual parameter fluorescent dot plots, the cells in early apoptosis (annexin V+/PI−, in the lower right quadrant) and in late apoptosis (annexin V+/PI+, in the upper right quadrant) were counted. As shown in Fig. 4, 3.81% of cells were Annexin V+/PI− and 6.82% of cells were Annexin V+/ PI+ in untreated cells. After treatment with compound 2 at 4 μg/mL and 6 μg/mL, compound 3 at 6 μg/mL and 8 μg/mL, compound 4 at 4 μg/mL and 6 μg/mL, or compound 5 at 30 μg/mL and 75 μg/mL for 24 h, the number of cells which were Annexin V+/PI− and Annexin V+/PI+ were increased significantly. These results collectively demonstrated that compounds 2–5 showed antitumor activities through inducing apoptotic cell death. Depolarization of mitochondrial membrane potential (MMP) Considering the significant role of mitochondria in the transduction of apoptotic signals, the involvement of mitochondria in cycloartane triterpenoids-induced apoptosis was investigated. As shown in Fig. 5, when the MCF7 cells were treated with 6 μg/mL of compound 2, 8 μg/mL of compound 3, 6 μg/mL of compound 4 and 60 μg/mL of compound 5 for 24 h, the percentage of cells having low fluorescence was 27.1 ± 7.3%, 22 ± 0.1%, 23.4 ± 0.9% and 9.3 ± 0.2% respectively, compared with 2.9 ± 0.3% in the control. These results suggested that depolarization of MMP was involved in the mechanism of apoptosis induced by compounds 2–5.

Figure 5. Analysis of mitochondrial membrane potential in the process of apoptosis. MCF7 cells were treated with the indicated concentration of compounds 2–5 for 24 h. Cells were stained with rhodamine 123 and the percentage of depolarized cells was evaluated by flow cytometry based on mean values obtained from three independent experiments. *p < 0.05, **p < 0.01 (vs control cells). Copyright © 2010 John Wiley & Sons, Ltd.

Activation of caspase-7 activity In the present study, caspase-3-deficient MCF-7 cell line was used. Therefore, caspase-7 activity was investigated to demonstrate whether caspase-7 activation is involved in the apoptotic process triggered by compounds 2–5. As expected, by comparison of the control, after 24 h of treatment with compounds 2–5 at concentrations of 6 μg/mL, 8 μg/mL, 6 μg/mL and 75 μg/mL respectively, the activity of caspase-7 was increased significantly (Fig. 6). These results showed that apoptosis induced by compounds 2–5 was related to the activation of caspase-7. Analysis of the up-regulation of p53 and bax using RT-PCR assay After treatment of MCF7 cells with compounds 2–5 as described in Materials and Methods, the mRNA levels of p53 and bax were increased (Fig. 7).

DISCUSSION Multi-drug resistance is a serious problem in terms of breast cancer treatment, which makes chemotherapy more complicated and influences the effectiveness of therapy (Coley, 2009). In this study, the drug resistant cell subline R-MCF7 and the parental cell line MCF7 were used to investigate the antitumor activity of cycloartane triterpenoids. The similar IC50 values for MCF7 and R-MCF7 cells showed compounds 2–5 were all effective on drug resistant cells as well as the parental cancer cells. To our best knowledge, this is the first report on the antitumor activity of compound 3 and compound 4. Compound 2 and compound 5 have been reported to have anti-liver cancer activity (Tian et al., 2005), and this study displayed that they also have anti-

Figure 6. Changes in activities of caspase-7 in MCF7 cells. MCF7 cells were treated with vehicles or with compounds 2–5 at indicated concentrations. The experiment was carried out in three independent experiments in triplicate. Data are given as mean ± SD. p < 0.05, ** p < 0.01 (vs control cells). Phytother. Res. 25: 17–24 (2011)

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Figure 7. Effects of compounds 2–5 on expression of p53 and bax genes. MCF7 cells were treated with vehicle, compound 2 at 6 μg/ mL compound 3 at 8 μg/mL, compound 4 at 6 μg/mL or compound 5 at 60 μg/mL for 24 h. The data for p53 and bax were adjusted to β-actin (OD of p53 and bax mRNA versus OD of β-actein) and the fold increase was calculated.

breast cancer activity. On the basis of the structures of studied cycloartane triterpenoids, it was found that the antitumor activity will increase apparently when some hydrophobic groups such as acetyl and halogen were introduced to the C23 and the C25 carbon sites, especially for the C25 carbon site, which is in agreement with previous reports (Shao et al., 1998). Apoptosis, a program of cellular suicide, is critical for both proper embryonic development and homeostasis of adult tissues (Schafer and Kornbluth, 2006). Induction of apoptosis, which can destroy the cancer cells innocuously, is a significant strategy in anticancer drug research and development (Tian et al., 2006). The results obtained from the Hoechst 33342 staining assay and Annexin V-FITC/PI analysis demonstrated that compounds 2–5 induced apoptosis of MCF7. Furthermore, the RNA level of p53 and pro-apoptotic gene bax were demonstrated to increase after treatment of MCF7 with compounds 2–5, which led to the disruption of the mitoCopyright © 2010 John Wiley & Sons, Ltd.

chondrial membrane. Caspases are a family of cysteine proteases, often activated during the execution phase of the apoptosis process. When activated, caspases can activate downstream caspases, leading to apoptosis (Ho and Hawkins, 2005). Activated caspase-3/7 is a key executioner of apoptosis, which can lead to the cleavage and inactivation of key cellular substrates, such as PARP (Sun et al., 1999). Our data showed that compounds 2–5 induced the up-regulation of caspase-7 activity. Taken together, compounds 2–5 are potential antitumor agents on both parental and drug resistant breast tumors and the antitumor activity of these analogues will apparently increase when some hydrophobic groups are introduced to the C23 and the C25 carbon sites, such as acetyl and halogen. Compounds 2–5 exert their antitumor activity by inducing apoptosis. Furthermore, it was found that the p53-dependent mitochondrial signaling pathway contributed to the action mechanism of apoptosis induced by compounds 2–5. Phytother. Res. 25: 17–24 (2011)

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Acknowledgement

Conflict of interest

This work was supported by the 973 program (2009CB522808) of the Ministry of Science and Technology of China, Innovation Methodology Program of the Ministry of Science and Technology of China (2008IM020900) and the National Natural Science Foundation of China (No. 30772636).

The authors have declared that there is no conflict of interest.

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