PHYTOTHERAPY RESEARCH Phytother. Res. 28: 1–10 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5241
Isoflavones Extracted from Chickpea Cicer arietinum L. Sprouts Induce MitochondriaDependent Apoptosis in Human Breast Cancer Cells Hua Chen,1,2,3 Hai-Rong Ma,1,2* Yan-Hua Gao,1,2 Xue Zhang,4 Madina Habasi,1,2 Rui Hu1,2,3 and Haji Akber Aisa1,2* 1
State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China 2 A Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China 3 University of Chinese Academy of Sciences, Beijing 100049, China 4 The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, China
Isoflavones are important chemical components of the seeds and sprouts of chickpeas. We systematically investigated the effects of isoflavones extracted from chickpea sprouts (ICS) on the human breast cancer cell lines SKBr3 and Michigan Cancer Foundation-7 (MCF-7). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that ICS (10–60 μg/mL) significantly inhibited the proliferation of both cell lines in a time-dependent and dose-dependent fashion. Wright-Giemsa staining as well as annexin V-fluorescein isothiocyanate and propidium iodide (Annexin V/PI) staining showed that ICS significantly increased cytoclasis and apoptotic body formation. Quantitative Annexin V/PI assays further showed that the number of apoptotic cells increased in a dose-dependent manner following ICS treatment. Semiquantitative reverse transcription PCR showed that ICS increased the expression of the apoptosis-promoting gene Bcl-2-associated X protein and decreased the expression of the antiapoptotic gene Bcl-2. Western blot analysis showed that treatment of SKBr3 and MCF-7 cells with ICS increased the expression of caspase 7, caspase 9, P53, and P21 in a dose-dependent manner. Flow cytometry assays using the fluorescent probe 3,3′-dihexyloxacarbocyanine iodide showed a dose-dependent decrease in mitochondrial membrane potential following ICS treatment. Treatment using ICS also induced a dose-dependent increase in reactive oxygen species production. This is the first study to demonstrate that ICS may be a chemopreventive or therapeutic agent against breast cancer. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Cicer arietinum L. sprouts; isoflavones; mitochondria-dependent apoptosis; breast cancer cells.
INTRODUCTION Breast cancer is the most common malignant tumor in women. The disease has the highest mortality rate of all cancers among women worldwide, and the incidence rate increases yearly (Siegel et al, 2013). It has been reported that Western women have a higher breast cancer incidence than women from Asian countries (Magee and Rowland, 2004). The lower incidence of breast cancer in Asian women has been attributed to their intake of soybeans, which are rich in phytoestrogen (Limer and Speirs, 2004). The preventative effects of phytoestrogens on breast cancer have attracted much attention and have become a major focus of breast cancer research. Isoflavones are a class of phytoestrogens that are structurally similar to mammalian estrogens. Clinical studies
* Correspondence to: Haji Akber Aisa and Hai-Rong Ma, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China. E-mail: [email protected] (Haji Akber Aisa); [email protected] (Hai-Rong Ma)
Copyright © 2014 John Wiley & Sons, Ltd.
and epidemiologic investigations show that isoflavones possess many bioactive functions, including bone preservation and improved learning ability and memory in menopausal women (File et al., 2001; Wei et al., 2012) and the prevention and treatment of cancer, heart disease, diabetes, and atherosclerosis (Farina et al., 2006; Tikkanen and Adlercreutz, 2000; Behloul and Wu, 2013; Chan et al., 2008). The antitumor properties of isoflavones have attracted wide attention. Possible mechanisms of their anticancer activity include inhibiting the activity of protein tyrosine kinase and type II topoisomerase, regulating the cell cycle, and inducing apoptosis (Spinozzi et al., 1994; Salti et al., 2000; Su et al., 2003; Mazzio et al., 2014; Yun et al., 2014). Cicer arietinum L. (chickpea) is an annual leguminous plant. The chickpea has been used as a traditional Uighur herb for over 2500 years in Xinjiang, China. In traditional Uighur medicine, chickpeas were used to treat bronchitis, dyspepsia, constipation, itchy skin, diabetes, hyperlipidemia, osteoporosis, and cancer (Liu and Yikemu, 1986). Chickpeas are high in protein, fiber, mineral, and unsaturated fatty acid content (White and Broadley, 2009). Eight isoflavones, including biochanin A, formononetin, genistein, calycosin, biochanin A-7Ob-D-glucoside, trifolirhizin, ononin, and sissotrin, are Received 24 February 2014 Revised 05 September 2014 Accepted 10 September 2014
H. CHEN ET AL.
found in chickpea seeds and sprouts (Zhao et al., 2009). The isoflavone content of chickpea sprouts is substantially higher than that of chickpea seeds (Cheng et al., 2008). Isoflavones extracted from chickpea sprouts (ICS) have dual functions. At low concentrations (10 3–1 μg/mL), they promote Michigan Cancer Foundation-7 (MCF-7) cell growth, while at high concentrations (>1 μg/mL), they inhibit cellular proliferation (Ma et al., 2013). There have been no reports evaluating the mechanism by which ICS inhibits breast cancer. We investigated the mechanism underlying ICS-related apoptosis in two human breast cancer cell lines, estrogen receptor(ER)positive MCF-7 cells and ER-negative SKBr3 cells.
MATERIALS AND METHODS Seed materials and ICS preparation. Fresh seeds of desi C. arietinum L. were provided by Dalong Food Company, Ltd., Mu Lei district, Xinjiang autonomous region, China. The sprouting procedure of the seeds of C. arietinum L. and the preparation of sprout extract were carried out in our lab (Lv et al., 2009). ICS was purified from sprout extract as previously described (Ma et al., 2013). The yield of dried sprout extract as a percentage of the weight of the dried sprouts was 5.06%. ICS was standardized to 96.26% isoflavones by weight (96.26 g of total isoflavones per 100 g of total extract) using a spectrophotometric method. Analysis using HPLC of ICS showed 13.74% ononin isoflavone, 19.17% biochanin A-7-O-β-D-glucoside isoflavone, 25.47% formononetin isoflavone, and 37.88% biochanin A isoflavone.
Cell culture. Michigan Cancer Foundation-7 (MCF-7) cells and SKBr3 cells were obtained from the Chinese Type Culture Collection, CAS (Shanghai, China). Cells of MCF-7 were maintained in Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/l glucose and 0.37% sodium bicarbonate (Gibco, Rockville, MD, USA). SKBr3 cells were cultured in DMEM/F12 medium (Gibco). Cell media were supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were grown in a fully humidified incubator (Binder, Germany) at 37°C with 95% air and 5% CO2.
Antiproliferation assay. The antiproliferative effects of ICS on human breast cancer cells were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, MCF-7 cells and SKBr3 cells were separately seeded on 96-well plates at a density of 5 × 103 cells per well. The cells were allowed to attach for 24 h. Thereafter, the cells were treated with 10, 20, 30, 40, 50, or 60 μg/mL of ICS for 24, 48, or 72 h. Next, 20 μL of 5 mg/mL MTT (Sigma, St. Louis, MO, USA) was added to each well, and the plates were incubated at 37°C. Four hours later, 200 μL dimethyl sulfoxide (DMSO) was added to each well to dissolve the resulting formazan crystals, and the multiwell plates were shaken for 30 min. The absorbance in each well was read at a wavelength of 540 nm using an enzyme-linked Copyright © 2014 John Wiley & Sons, Ltd.
immunosorbent assay reader (SpectraMax M5, Molecular Devices, USA). The percentage of ICS-induced cell growth inhibition was determined by comparison with DMSO-treated control cells.
Wright-Giemsa staining. The morphologic changes associated with ICS-induced apoptosis were evaluated using a Wright-Giemsa staining kit (Baso Diagnostics Inc., Zhuhai, China). Briefly, MCF-7 and SKBr3 cells were seeded onto glass cover slips placed in the wells of six-well plates. The cells were seeded at a density of 5 × 105 cells per cover slip and allowed to attach for 24 h. Next, the cells were exposed to half maximal inhibitory concentration (IC50) concentrations of ICS (41.6 μg/mL for SKBr3 cells, 32.8 μg/mL for MCF-7 cells) for 48 h. The glass cover slips were then carefully removed, fixed by air drying, and rinsed twice with PBS. The cover slips were first stained with 500 μL of Wright-Giemsa dye solution A for 1 min, and then, 1 mL of Wright-Giemsa dye solution B was added to each cover slip and mixed well with the dye solution A. The dye mixture was incubated on the cells for 5 min at room temperature (RT). The cover slips were then washed with water and dried completely. All of the cells were visualized using optical microscopy and photographed.
Annexin V-FITC and propidium iodide staining. The apoptotic effects of ICS on SKBr3 and MCF-7 breast cancer cells were evaluated using annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (Annexin V/PI) staining. Cells were seeded onto glass cover slips placed in six-well plates. The cells were seeded to approximately 5 × 105 cells per well and allowed to attach. After 24 h of attachment, log phase cultures were treated with 20, 40, or 60 μg/mL ICS for 24 h. Cells were then rinsed twice with 4°C PBS. Next, 100 μL of 1× binding buffer containing 5 μL of Annexin V-FITC (Signalway Antibody, Maryland, USA) and 10 μL of 20-μg/mL PI solution were added to each well. The cells were incubated at RT for 15 min in the dark. Stained cells were observed using fluorescence microscopy (Leica Microsystems Inc., Wetzlar, Germany) and photographed.
Annexin V-FITC and propidium iodide assay. MCF-7 cells and SKBr3 cells were seeded onto six-well plates at a density of approximately 5 × 105 cells per well and allowed to attach for 24 h. The cells were then incubated with 20, 40, or 60 μg/mL ICS for 12 h. Treated cells were collected, washed twice with ice-cold PBS, and re-suspended in 100 μL of 1× binding buffer before being incubated with 5 μL Annexin V-FITC (Signalway Antibody) according to the manufacturer’s protocol. After 15 min of incubation at RT in the dark, 10 μL of 20 μg/mL PI was added to each tube, and the cells were gently re-suspended. Next, 400 μL PBS was added to each tube. The number of stained cells was quantified using a flow cytometer (Becton Dickinson, New Jersey, USA).
Semiquantitative RT-PCR analysis. MCF-7 cells and SKBr3 cells were seeded onto six-well plates to approximately Phytother. Res. 28: 1–10 (2014)
ISOFLAVONES EXTRACTED FROM CHICKPEA SPROUTS INDUCE APOPTOSIS
5 × 105 cells per well and allowed to attach for 24 h. The media were removed and replaced with fresh media containing ICS (20, 40, or 60 μg/mL) in DMSO. The cells were cultured at 37°C for 12 h. Total RNA was isolated using Trizol reagent (ComWin Biotech, Beijing, China) according to the manufacturer’s protocol. Reverse transcription was performed using the HiFiMMLV cDNA Kit (ComWin Biotech, Beijing, China). PCR was then conducted for 30 cycles using 1 μg RNA in a PCR machine (MJ, USA). The primers for detecting Bcl-2 expression were 5′-ACTTGTGGCCCAGATAG GCACCCA G-3′ (forward) and 5′-CGACTTCGCCGAGATGTCCAGCCAG-3′ (reverse). The primers for detecting Bcl-2-associated X protein (Bax) expression were 5′-TGCTTCAGGGTTTCATCCAG-3′ (forward) and 5′-GGCGGCAATCATCCTCTG-3′ (reverse). The primers for detecting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression were 5′-ACCACAGTCCATGCCTACAC-3′ (forward) and 5′-TTC ACCACCCTGTTGCTGTA-3′ (reverse). Primers were synthesized by Sangon Biotech (Shanghai, China). The PCR products of Bcl-2 (385 bp), Bax (170 bp), and GAPDH (452 bp) were separated using 1% agarose gel electrophoresis and visualized by staining with ethidium bromide. GAPDH was used as an internal control for both Bcl-2 and Bax expression. The net intensity of the bands was analyzed using ImageJ software (National Institutes of Health, Maryland, USA).
Western blot analysis. MCF-7 cells and SKBr3 cells were seeded onto 10-cm culture dishes at a density of approximately 1 × 106 per dish. After 24 h, the cells were treated with ICS (20, 40, or 60 μg/mL) or DMSO at 37°C for 12 h. The cells were then lysed with 200 μL radioimmunoprecipitation assay buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) and separated by 10% sodium dodecyl sulfate–polyacrylamide gel. The expression of the proteins caspase 7, caspase 9, P21, and P53 was detected using protein-selective antibodies (Boster Inc., Wuhan, China). Antibodies were incubated overnight at 4°C and then incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 h at RT. The immunolabeled proteins were detected using the enhanced chemiluminescence system (Thermo Fisher Scientific Inc., USA) and X-ray film. Band intensities were quantified using ImageJ software. β-actin was used as an internal control.
Mitochondrial membrane potential measurement. Mitochondrial membrane potential (MMP) was measured using the fluorescent probe 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)). Cells of MCF-7 and SKBr3 cells were seeded onto six-well plates at a density of approximately 5 × 105 cells per well and allowed to attach for 24 h. The cells were then treated with 20, 40, or 60 μg/mL ICS for 12 h. The treated cells were washed twice with PBS, re-suspended to a concentration of 1 × 106 cells/mL, and then incubated with 20 nM DiOC6(3) (Enzo Life Sciences International Inc., New York, USA) for 30 min at 37°C in the dark. The stained cells were washed twice with PBS and then Copyright © 2014 John Wiley & Sons, Ltd.
immediately analyzed using a flow cytometer (Epics Altra, Beckman Coulter, USA).
Intracellular reactive oxygen species evaluation. Intracellular reactive oxygen species (ROS) were evaluated based on intracellular peroxide-dependent oxidation of dichlorodihydrofluorescin diacetate (DCHF-DA), which produces the fluorescent compound 2′,7′-dichlorofluorescein (DCF). Cells of MCF-7 and SKBr3 cells were seeded onto six-well plates at a density of approximately 5 × 105 cells per well and allowed to attach for 24 h. The cells were then treated with 20, 40, or 60 μg/mL ICS for 12 h. The treated cells were washed twice with PBS and incubated with 20 μM DCHF-DA (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min in the dark. The cells were then washed with PBS, and ROS production was qualitatively evaluated using fluorescence microscopy. Quantitative evaluation was performed using cells treated as described previously, plated onto a 96-well black plate (1.5 × 105 cells/well) after digestion with trypsin, and assayed using a fluorescence microplate reader (SpectraMax M5, Molecular Devices, USA) with excitation at 488 nm and emission at 525 nm.
STATISTICAL ANALYSIS The results of each experiment are presented as the mean ± standard deviation (SD) of three experiments. The Student’s t-test was used to assess differences between ICS-treated and control groups. The data were analyzed using SPSS software. p < 0.05 was considered statistically significant.
RESULTS Antiproliferative effects of ICS on breast cancer cells The antiproliferative effects of various concentrations (10 to 60 μg/mL) of ICS on the human breast cancer cell lines SKBr3 and MCF-7 were assessed using the MTT assay. As shown in Fig. 1, ICS dramatically inhibited the proliferation of both SKBr3 and MCF-7 cells in a time-dependent and dose-dependent manner. The highest inhibition rates for SKBr3 cells were 36.1 ± 5.2% at 24 h (p < 0.01), 65.3 ± 2.6% at 48 h (p < 0.01), and 88.5 ± 3.2% at 72 h (p < 0.01; Fig. 1A). The highest inhibition rates for MCF-7 cells were 36.5 ± 5.2% at 24 h (p < 0.01), 76.8 ± 5.2% at 48 h (p < 0.01), and 86.9 ± 5.6% at 72 h (p < 0.01; Fig. 1B).
Morphologic observation of ICS-induced apoptosis A series of morphological features typically associated with apoptosis, including nucleus shrinkage, chromatin condensation, and DNA fragmentation, were observed in SKBr3 and MCF-7 cells treated with the IC50 of ICS (Fig. 2A). These features were not observed in control cells. Similar morphologic characteristics were Phytother. Res. 28: 1–10 (2014)
H. CHEN ET AL.
Figure 1. Antiproliferative activity of ICS. (A) SKBr3 cells and (B) MCF-7 cells were treated with different concentrations of ICS for 24, 48, or 72 h. Inhibition was determined by MTT assay. % inhibition rate was determined relative to control cells. Data are shown as the mean ± SD of three experiments.
also found with Annexin V/PI fluorescence staining (Fig. 2B and C).
concentration, ICS induced the highest apoptosis rate in both SKBr3 and MCF-7 cells (p < 0.01; Fig. 3C and D).
Quantitative assay of ICS-induced apoptosis
Apoptosis-related protein expression after ICS treatment
The Annexin V/PI assays showed significant changes in SKBr3 and MCF-7 cells treated with ICS (Fig. 3). ICStreated cells showed a dose-dependent increase in apoptosis compared with controls. The apoptotic rates ranged from 0.67 ± 0.15% to 34.30 ± 0.85% in SKBr3 cells (Fig. 3A). The apoptotic rates ranged from 0.20 ± 0.10% to 31.63 ± 0.75% in MCF-7 cells (Fig. 3B). At the 60-μg/mL
Levels of mRNA of the antiapoptotic family member Bcl-2 and the apoptosis-promoting Bax were determined using semiquantitative reverse transcription PCR. The constitutively expressed gene GAPDH was used as an internal control. In SKBr3 cells, Bcl-2 mRNA expression showed a dose-dependent decrease with increasing concentrations of ICS, while Bax
Figure 2. Micrographs of ICS-induced apoptosis in SKBr3 cells and MCF-7 cells. (A) Micrographs with Wright-Giemsa staining (×400) of SKBr3 cells or MCF-7 cells treated with IC50 concentrations of ICS for 48 h. Fluorescent micrographs with Annexin V-FITC and propidium iodide staining (×400) of SKBr3 cells (B) or MCF-7 cells (C) treated with ICS (20, 40, or 60 μg/mL) for 24 h. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. 28: 1–10 (2014)
ISOFLAVONES EXTRACTED FROM CHICKPEA SPROUTS INDUCE APOPTOSIS
Figure 3. ICS-induced apoptosis measured by Annexin V-FITC and propidium iodide (PI) assay. Characteristic dual-parameter fluorescence histograms were obtained with SKBr3 cells (A, C) and MCF-7 cells (B, D) stained with Annexin V-FITC and PI. Fluorescence was evaluated using flow cytometry after exposure to ICS (20, 40, and 60 μg/mL) for 12 h. Q1 [FITC( )/PI(+)], necrotic cells; Q2 [FITC(+)/PI(+)], late apoptotic cells; Q3 [Annexin V(+)/PI( )], living cells; Q4 [FITC(+)/PI( )], early apoptotic cells. The number in each quadrant represents the percentage of cells present. (C, D) Each point is the mean ± SD of three independent experiments. *, ** significantly different from control at p < 0.05 and p < 0.01, respectively.
mRNA expression showed a dose-dependent increase with increasing concentrations of ICS in SKBr3 cells (Fig. 4A). Similar findings were observed in MCF-7 cells (Fig. 4B). The ratio of Bcl-2/Bax mRNA expression in SKBr3 and MCF-7 cells was significantly lower than that of control cells (5.5 ± 0.2 vs 0.6 ± 0.03 in SKBr3 cells and 7.8 ± 0.1 vs 0.6 ± 0.02 in MCF-7 cells) (Fig. 4A and B). The expression of the caspase family members caspase 7 and caspase 9 was detected using Western blot analysis. As Fig. 4C and D show, treatment of SKBr3 and MCF-7 cells with increasing doses of ICS induced a dose-dependent increase in the protein expression of caspase 7 and caspase 9. The maximum relative protein expression level of caspase 7 was approximately 1.6 times higher in SKBr3 cells than in control cells and approximately 3.4 times higher in MCF-7 cells than in control cells. Caspase 9 expression was approximately 4.4 times higher in SKBr3 cells than in the control group and approximately 6.0 times higher in MCF-7 cells than in the control group. Copyright © 2014 John Wiley & Sons, Ltd.
Mitochondrial membrane potential disruption after ICS treatment Treatment using ICS led to a dose-dependent decrease in MMP in both SKBr3 and MCF-7 cells (Fig. 5A and B). The 40-μg/mL ICS treatment was associated with a 30.95 ± 1.62% decrease in the MMP of SKBr3 cells (p < 0.05) and an 18.21 ± 1.51% decrease in the MMP of MCF-7 cells (p < 0.05). The 60-μg/mL ICS treatment was associated with a 41.70 ± 2.78% decrease in the MMP of SKBr3 cells (p < 0.01) and a 35.49 ± 2.98% decrease in the MMP of MCF-7 cells (p < 0.01; Fig. 5C and D). ROS generation in ICS-treated breast cancer cells The fluorescent intensity of DCF was used to evaluate ROS generation after cells were treated with 20, 40, or 60 μg/mL ICS. As shown in Fig. 6, the incubation of both Phytother. Res. 28: 1–10 (2014)
H. CHEN ET AL.
Figure 4. ICS-induced apoptosis-related protein expression. (A, B) Bcl-2 and Bax mRNA expression. (A) SKBr3 cells and (B) MCF-7 cells were treated with ICS (20, 40, or 60 μg/mL) for 12 h. Total RNA was extracted, and Bcl-2 and Bax mRNA levels were determined using semiquantitative reverse transcription PCR. Representative bands are shown in the left panels. Bcl-2/Bax mRNA expression in SKBr3 cells or MCF-7 cells with different doses of ICS is shown in the right panels. (C, D) Caspase 7 and caspase 9 protein expression. (C) SKBr3 and (D) MCF-7 cells were treated with ICS (20, 40, or 60 μg/mL) for 12 h. Total protein was extracted, and caspase 7 and caspase 9 protein expression was determined by Western blot. Representative bands are shown in the left panels. The average caspase 7 and caspase 9 levels of SKBr3 cells or MCF-7 cells treated with different doses of ICS are shown in the right panels. β-actin was used to normalize protein loading. All results are the mean ± SD of three independent experiments. *, ** significantly different from control at p < 0.05 or p < 0.01, respectively. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. 28: 1–10 (2014)
ISOFLAVONES EXTRACTED FROM CHICKPEA SPROUTS INDUCE APOPTOSIS
Figure 5. ICS-induced mitochondrial membrane potential depolarization. Representative fluorescence histograms of SKBr3 cells (A) and MCF-7 cells (B) stained with DiOC6. Fluorescence was evaluated using flow cytometry after exposure to ICS (20, 40, and 60 μg/mL) for 12 h. M1 represents the percentage of cells with a low mitochondrial membrane potential. Data are expressed in the percentage of cells with mitochondrial depolarization in SKBr3 cells (C) and in MCF-7 cells (D). The data are presented as the mean ± SD. Experiments were performed in triplicate. *, ** significantly different from control at p < 0.05 or p < 0.01, respectively.
SKBr3 and MCF-7 cells with ICS (20, 40, or 60 μg/mL) was associated with a dose-dependent increase in ROS production, compared with controls (Fig. 6A and B). Production of ROS in SKBr3 cells was highest when a concentration of 60 μg/mL ICS was used. This was a 2.8-fold and 2.0-fold increase compared with untreated SKBr3 cells (p < 0.01; Fig. 6C) and untreated MCF-7 cells (p < 0.01; Fig. 6D), respectively. P21 and P53 have key roles in oxidative-damageinduced apoptosis (Forman, et al., 2008). P21 and P53 protein expression increased in a dose-dependent fashion with ICS treatment. The maximum protein expression of P21 in SKBr3 cells was about 2.8 times higher than that of controls. The maximum protein expression of P21 in MCF-7 cells was about 3.6 times higher than that of controls. SKBr3 cell P53 expression was about 2.8 times higher than that of controls, and MCF-7 cell P21 expression was 6.8 times higher than that of controls (Fig. 7A and B). Copyright © 2014 John Wiley & Sons, Ltd.
DISCUSSION We evaluated the antiproliferative and apoptotic effects of ICS on two human breast cancer cell lines, SKBr3 and MCF-7. ICS, at concentrations from 10 to 60 μg/mL, significantly inhibited the growth of both cell lines in a dose-dependent and time-dependent manner. Moreover, the growth inhibition occurred via a mitochondria-dependent apoptotic mechanism. The ICS-induced apoptosis suggests that ICS may be useful for the chemoprevention or chemotherapy of breast cancer. In vitro studies have confirmed that isoflavonoids act as ER agonists and promote the growth of estrogenresponsive human breast cancer cells at low concentrations. Isoflavonoids have ER antagonist effects and inhibit the growth of estrogen-responsive and ER-negative Phytother. Res. 28: 1–10 (2014)
H. CHEN ET AL.
Figure 6. ICS-induced reactive oxygen species (ROS) generation. (A, B) Representative micrographs of DCHF-DA staining of breast cancer cells. (A) SKBr3 cells and (B) MCF-7 cells were treated with ICS (20, 40, or 60 μg/mL) for 12 h and then incubated with DCHF-DA for 30 min. The upper panels of panels A and B show representative fluorescent staining. The lower panels of panels A and B are light micrographs of the same field (×100). (C, D) Quantitative ROS generation assay with a fluorescence microplate reader. (C) SKBr3 cells and (D) MCF-7 cells were incubated with ICS (20, 40, or 60 μg/mL) for 12 h. Generation of ROS was measured using a fluorescence microplate reader. The data are presented as the mean ± SD. Experiments were performed in triplicate. *, ** significantly different from control at p < 0.05 or p < 0.01, respectively. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. 28: 1–10 (2014)
ISOFLAVONES EXTRACTED FROM CHICKPEA SPROUTS INDUCE APOPTOSIS
Figure 7. ICS induced the expression of the oxygen stress-reaction-related proteins P53 and P21. (A) SKBr3 and (B) MCF-7 cells were treated as described previously, and protein expression was determined by Western blot. Representative bands are shown in the left panels. The average P53 and P21 levels in SKBr3 and MCF-7 cells treated with different doses of ICS are shown in the right panels. β-actin was used to normalize protein loading. All results are the mean ± SD of three independent experiments. *, ** significantly different from control at p < 0.05 or p < 0.01, respectively.
human breast cancer cells at higher concentrations (Chinni et al., 2003; Robb and Stuart, 2014). We found that ICS at 10 to 60 μg/mL inhibited the growth of ER-positive MCF-7 cells and ER-negative SKBr3 cells (Fig. 1). This result suggests that ICS-mediated inhibition of the two breast cancer cell lines was ER independent. These findings are consistent with previous reports that isoflavonoids inhibited the growth of human breast carcinoma cells (Choi and Kim, 2008; Lin et al., 2009). The induction of apoptosis is an important mechanism in inhibiting cell proliferation. Isoflavonoids like genistein and formononetin inhibit cancer cell growth via apoptosis (Chinni et al., 2003). In this study, Wright-Giemsa staining and Annexin V/PI staining revealed morphological features typically associated with apoptosis in ICS-treated SKBr3 and MCF-7 cells (Fig. 2). In addition, the Annexin V/PI assay demonstrated that exposure of SKBr3 and MCF-7 cells to ICS (20, 40, and 60 μg/mL) for 48 h caused a remarkable accumulation of apoptotic cells in a concentrationdependent manner (Fig. 3). These three methods of determining apoptosis demonstrated similar results. To further understand the molecular mechanism associated with the development of apoptosis, we measured alterations in the expression of the apoptosis regulators Bcl-2 and Bax. The regulators Bax and Bcl-2 are two important members of the apoptosis-regulating Bcl-2 family. These proteins play key roles in the integration and regulation of apoptotic signals (Gross et al., 1999). The protein Bax belongs to the proapoptotic subfamily and binds to Bcl-2, which belongs to the antiapoptotic subfamily. The gene Bax accelerates the progression of Copyright © 2014 John Wiley & Sons, Ltd.
apoptosis by inhibiting the function of Bcl-2 (Oltval et al., 1993). The ratio of Bcl-2/Bax expression determines the cell’s susceptibility to apoptosis (De Angelis et al., 1998; Findley et al., 1997). We found that ICS induced an increase in the mRNA expression of Bax, as well as a decrease in the mRNA expression of Bcl-2, in both MCF-7 and SKBr3 cell lines. Furthermore, the ratio of Bcl-2/Bax mRNA significantly decreased in a concentration-dependent manner following exposure to ICS (Fig. 4A and B). A typical characteristic of apoptosis is the activation of caspases, a class of cysteine proteases that selectively cleave proteins after aspartic acid residues. Caspases are activated by sequential cleavage of their inactive procaspase forms (Hu et al., 2001). Caspase 7 and caspase 9 are two key members of the caspase family. Activation of caspase 9 triggers a cascade of downstream signals, including caspase 3 and caspase 7, members of the intrinsic mitochondria-dependent apoptotic pathway (Chen, et al., 2000). Because there is a lack of caspase 3 expression in MCF-7 cells (Mc Gee, et al., 2002), we evaluated the expression of the activated forms of caspase 7 and caspase 9 using Western blots. Exposure of SKBr3 and MCF-7 cell lines to ICS increased the expression of active caspase 9 and caspase 7 in a dose-dependent manner (Fig. 4A and D). We presumed that mitochondria were involved in the ICS-induced apoptosis because caspase 9 and caspase 7 expression was upregulated by ICS. The disruption of MMP is a crucial step in intrinsic mitochondriamediated apoptosis induction (Gupta, 2003). Mitochondria are a prerequisite for ROS generation. The ROS Phytother. Res. 28: 1–10 (2014)
H. CHEN ET AL.
generated in and around mitochondria act as second messengers in the apoptosis-mediated signaling pathway (Forman et al., 2008). We found that ICS treatment led to a dose-dependent decrease in MMP in both SKBr3 and MCF-7 cells (Fig. 5). ICS induced a high level of ROS production by SKBr3 cells and MCF-7 cells (Fig. 6). ICS also induced an increase in the expression of two tumor suppressor proteins, P53 and P21, which play a vital role in oxidative stress responses (Fig. 7; Forman, et al., 2008). ICS-induced MMP damage and ROS generation, leading to breast cancer apoptosis. In conclusion, we found that ICS induced mitochondriamediated apoptosis in ER-positive and ER-negative human breast cancer cell lines. This finding has greatly extended the potential use of ICS for chemoprevention
and chemotherapy. In future research, we will elucidate the detailed mechanism underlying ICS-induced apoptosis and chemoprevention in animal models.
Acknowledgements This work was supported, in part, by grants from the National Natural Science Foundation of China (Grant No 81102890) and the Joint Funds of the National Natural Science Foundation of China (Grant No. U1203203).
Conflict of Interest The authors have declared that there is no conflict of interest.
REFERENCES Behloul N, Wu GZ. 2013. Genistein: a promising therapeutic agent for obesity and diabetes treatment. Eur J Pharmacol 698: 31–38. Chan YH, Lau KK, Yiu KH, et al. 2008. Reduction of C-reactive protein with isoflavone supplement reverses endothelial dysfunction in patients with ischaemic stroke. Eur Heart J 29: 2800–2807. Chen Q, Gong B, Almasan A. 2000. Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death Differ 7: 227–233. Cheng Z, Ayiguli A, Ma QL, et al. 2008. Determination of isoflavones in extracts of Cicer afiefinum L. and bean sprout by ultrabiolet spectrophotometry. Lishizhen Med Mater Med Res 19: 2612–2613. Chinni SR, Alhasan SA, Multani AS, et al. 2003. Pleotropic effects of genistein on MCF-7 breast cancer cells. Int J Mol Med 12: 29–34. Choi EJ, Kim GH. 2008. Daidzein causes cell cycle arrest at the G1 and G2/M phases in human breast cancer MCF-7 and MDAMB-453 cells. Phytomedicine 15: 683–690. De Angelis PM, Stokke T, Thorstensen L, et al. 1998. Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype. Mol Pathol 51: 254–261. Farina HG, Pomies M, Alonso DF, et al. 2006. Antitumor and antiangiogenic activity of soy isoflavone genistein in mouse models of melanoma and breast cancer. Oncol Rep 16: 885–891. File SE, Jarrett N, Fluck E, et al. 2001. Eating soya improves human memory. Psychopharmacology (Berl) 157: 430–436. Findley HW, Gu L, Yeager AM, et al. 1997. Expression and regulation of Bcl-2, Bcl-xl, and Bax correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia. Blood 89: 2986–2993. Forman HJ, Fukuto JM, Miller T, et al. 2008. The chemistryof cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal. Arch Biochem Biophys 477: 183–195. Gupta S. 2003. Molecular signaling in death receptor and mitochondrial pathways of apoptosis. Int J Oncol 22: 15–20. Gross A, Mcdonnell JM, Korsmeyer SJ. 1999. BCL-2 family members and the mitochondria in apoptosis. Gene Dev 13: 1899–1911. Hu CCA, Tang CHA, Wang JJ. 2001. Caspase activation in response to cytotoxic Rana catesbeiana ribonuclease in MCF-7 cells. FEBS Lett 503: 65–68. Limer JL, Speirs V. 2004. Phyto-oestrogens and breast cancer chemoprevention. Breast Cancer Res 6: 119–127. Lin YJ, Hou YC, Lin CH, et al. 2009. Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells. Biochem Biophys Res Comm 378: 683–688. Liu YM, Yikemu S. 1986. Wei Wu Er Yao Zhi, 1st edn. Xinjiang People’s Medical Publishing House: Urumqi, Xinjiang. Copyright © 2014 John Wiley & Sons, Ltd.
Lv Q, Yang Y, Zhao Y, et al. 2009. Comparative study on separation and purification of isoflavones from the seeds and sprouts of chickpea by high-speed countercurrent chromatography. J Liq Chrom Relat Tech 32: 2879–2892. Magee PJ, Rowland IR. 2004. Phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer. Br J Nutr 91: 513–531. Ma HR, Wei HB, Chen Z, et al. 2013. The estrogenic activity of isoflavones extracted from chickpea Cicer arietinum L sprouts in vitro. Phytother Res 27: 1237–1242. Mazzio E, Badisa R, Mack N, et al. 2014. High throughput screening of natural products for anti-mitotic effects in MDA-MB-231 human breast carcinoma cells. Phytother Res 28: 856–867. Mc Gee MM, Hyland E, Campiani G, et al. 2002. Caspase-3 is not essential for DNA fragmentation in MCF-7 cells during apoptosis induced by the pyrrolo-1,5- benzoxazepine, PBOX-6. FEBS Lett 515: 66–70. Oltval ZN, Milliman CL, Korsmeyer SJ. 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 74: 609–619. Robb EL, Stuart JA. 2014. Multiple phytoestrogens inhibit cell growth and confer cytoprotection by inducing manganese superoxide dismutase expression. Phytother Res 28: 120–131. Salti GI, Grewal S, Mehta RR, et al. 2000. Genistein induces apoptosis and topoisomerase II-mediated DNA breakage in colon cancer cells. Eur J Cancer 36: 796–802. Siegel R, Naishadham D, Jemal A. 2013. Cancer statistics, 2013. CA Cancer J Clin 63: 11–30. Spinozzi F, Pagliacci MC, Migliorati G, et al. 1994. The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells. Leukemia Res 18: 431–439. Su SJ, Chow NH, Kung ML, et al. 2003. Effects of soy isoflavones on apoptosis induction and G2-M arrest in human hepatoma cells involvement of caspase-3 activation, Bcl-2 and Bcl-XL downregulation, and Cdc2 kinase activity. Nutr Cancer 45: 113–123. Tikkanen MJ, Adlercreutz H. 2000. Dietary soy-derived isoflavone phytoestrogens: could they have a role in coronary heart disease prevention? Biochem Pharmacol 60: 1–5. Wei P, Liu M, Chen Y, et al. 2012. Systematic review of soy isoflavone supplements on osteoporosis in women. Asian Pac J Trop Med 5: 243–248. White PJ, Broadley MR. 2009. Biofortification of crops with seven mineral elements often lacking in human diets–iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytol 182: 49–84. Yun SM, Jung JH, Jeong SJ, et al. 2014. Tanshinone IIA induces autophagic cell death via activation of AMPK and ERK and inhibition of mTOR and p70 S6K in KBM-5 leukemia cells. Phytother Res 28: 458–464. Zhao SH, Zhang LP, Gao P, et al. 2009. Isolation and characterisation of the isoflavones from sprouted chickpea seeds. Food Chem 114: 869–873. Phytother. Res. 28: 1–10 (2014)
Isoflavones Extracted from Chickpea Cicer arietinum L. Sprouts Induce MitochondriaDependent Apoptosis in Human Breast Cancer Cells Hua Chen,1,2,3 Hai-Rong Ma,1,2* Yan-Hua Gao,1,2 Xue Zhang,4 Madina Habasi,1,2 Rui Hu1,2,3 and Haji Akber Aisa1,2* 1
State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China 2 A Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China 3 University of Chinese Academy of Sciences, Beijing 100049, China 4 The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, China
Isoflavones are important chemical components of the seeds and sprouts of chickpeas. We systematically investigated the effects of isoflavones extracted from chickpea sprouts (ICS) on the human breast cancer cell lines SKBr3 and Michigan Cancer Foundation-7 (MCF-7). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that ICS (10–60 μg/mL) significantly inhibited the proliferation of both cell lines in a time-dependent and dose-dependent fashion. Wright-Giemsa staining as well as annexin V-fluorescein isothiocyanate and propidium iodide (Annexin V/PI) staining showed that ICS significantly increased cytoclasis and apoptotic body formation. Quantitative Annexin V/PI assays further showed that the number of apoptotic cells increased in a dose-dependent manner following ICS treatment. Semiquantitative reverse transcription PCR showed that ICS increased the expression of the apoptosis-promoting gene Bcl-2-associated X protein and decreased the expression of the antiapoptotic gene Bcl-2. Western blot analysis showed that treatment of SKBr3 and MCF-7 cells with ICS increased the expression of caspase 7, caspase 9, P53, and P21 in a dose-dependent manner. Flow cytometry assays using the fluorescent probe 3,3′-dihexyloxacarbocyanine iodide showed a dose-dependent decrease in mitochondrial membrane potential following ICS treatment. Treatment using ICS also induced a dose-dependent increase in reactive oxygen species production. This is the first study to demonstrate that ICS may be a chemopreventive or therapeutic agent against breast cancer. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Cicer arietinum L. sprouts; isoflavones; mitochondria-dependent apoptosis; breast cancer cells.
INTRODUCTION Breast cancer is the most common malignant tumor in women. The disease has the highest mortality rate of all cancers among women worldwide, and the incidence rate increases yearly (Siegel et al, 2013). It has been reported that Western women have a higher breast cancer incidence than women from Asian countries (Magee and Rowland, 2004). The lower incidence of breast cancer in Asian women has been attributed to their intake of soybeans, which are rich in phytoestrogen (Limer and Speirs, 2004). The preventative effects of phytoestrogens on breast cancer have attracted much attention and have become a major focus of breast cancer research. Isoflavones are a class of phytoestrogens that are structurally similar to mammalian estrogens. Clinical studies
* Correspondence to: Haji Akber Aisa and Hai-Rong Ma, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China. E-mail: [email protected] (Haji Akber Aisa); [email protected] (Hai-Rong Ma)
Copyright © 2014 John Wiley & Sons, Ltd.
and epidemiologic investigations show that isoflavones possess many bioactive functions, including bone preservation and improved learning ability and memory in menopausal women (File et al., 2001; Wei et al., 2012) and the prevention and treatment of cancer, heart disease, diabetes, and atherosclerosis (Farina et al., 2006; Tikkanen and Adlercreutz, 2000; Behloul and Wu, 2013; Chan et al., 2008). The antitumor properties of isoflavones have attracted wide attention. Possible mechanisms of their anticancer activity include inhibiting the activity of protein tyrosine kinase and type II topoisomerase, regulating the cell cycle, and inducing apoptosis (Spinozzi et al., 1994; Salti et al., 2000; Su et al., 2003; Mazzio et al., 2014; Yun et al., 2014). Cicer arietinum L. (chickpea) is an annual leguminous plant. The chickpea has been used as a traditional Uighur herb for over 2500 years in Xinjiang, China. In traditional Uighur medicine, chickpeas were used to treat bronchitis, dyspepsia, constipation, itchy skin, diabetes, hyperlipidemia, osteoporosis, and cancer (Liu and Yikemu, 1986). Chickpeas are high in protein, fiber, mineral, and unsaturated fatty acid content (White and Broadley, 2009). Eight isoflavones, including biochanin A, formononetin, genistein, calycosin, biochanin A-7Ob-D-glucoside, trifolirhizin, ononin, and sissotrin, are Received 24 February 2014 Revised 05 September 2014 Accepted 10 September 2014
H. CHEN ET AL.
found in chickpea seeds and sprouts (Zhao et al., 2009). The isoflavone content of chickpea sprouts is substantially higher than that of chickpea seeds (Cheng et al., 2008). Isoflavones extracted from chickpea sprouts (ICS) have dual functions. At low concentrations (10 3–1 μg/mL), they promote Michigan Cancer Foundation-7 (MCF-7) cell growth, while at high concentrations (>1 μg/mL), they inhibit cellular proliferation (Ma et al., 2013). There have been no reports evaluating the mechanism by which ICS inhibits breast cancer. We investigated the mechanism underlying ICS-related apoptosis in two human breast cancer cell lines, estrogen receptor(ER)positive MCF-7 cells and ER-negative SKBr3 cells.
MATERIALS AND METHODS Seed materials and ICS preparation. Fresh seeds of desi C. arietinum L. were provided by Dalong Food Company, Ltd., Mu Lei district, Xinjiang autonomous region, China. The sprouting procedure of the seeds of C. arietinum L. and the preparation of sprout extract were carried out in our lab (Lv et al., 2009). ICS was purified from sprout extract as previously described (Ma et al., 2013). The yield of dried sprout extract as a percentage of the weight of the dried sprouts was 5.06%. ICS was standardized to 96.26% isoflavones by weight (96.26 g of total isoflavones per 100 g of total extract) using a spectrophotometric method. Analysis using HPLC of ICS showed 13.74% ononin isoflavone, 19.17% biochanin A-7-O-β-D-glucoside isoflavone, 25.47% formononetin isoflavone, and 37.88% biochanin A isoflavone.
Cell culture. Michigan Cancer Foundation-7 (MCF-7) cells and SKBr3 cells were obtained from the Chinese Type Culture Collection, CAS (Shanghai, China). Cells of MCF-7 were maintained in Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/l glucose and 0.37% sodium bicarbonate (Gibco, Rockville, MD, USA). SKBr3 cells were cultured in DMEM/F12 medium (Gibco). Cell media were supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were grown in a fully humidified incubator (Binder, Germany) at 37°C with 95% air and 5% CO2.
Antiproliferation assay. The antiproliferative effects of ICS on human breast cancer cells were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, MCF-7 cells and SKBr3 cells were separately seeded on 96-well plates at a density of 5 × 103 cells per well. The cells were allowed to attach for 24 h. Thereafter, the cells were treated with 10, 20, 30, 40, 50, or 60 μg/mL of ICS for 24, 48, or 72 h. Next, 20 μL of 5 mg/mL MTT (Sigma, St. Louis, MO, USA) was added to each well, and the plates were incubated at 37°C. Four hours later, 200 μL dimethyl sulfoxide (DMSO) was added to each well to dissolve the resulting formazan crystals, and the multiwell plates were shaken for 30 min. The absorbance in each well was read at a wavelength of 540 nm using an enzyme-linked Copyright © 2014 John Wiley & Sons, Ltd.
immunosorbent assay reader (SpectraMax M5, Molecular Devices, USA). The percentage of ICS-induced cell growth inhibition was determined by comparison with DMSO-treated control cells.
Wright-Giemsa staining. The morphologic changes associated with ICS-induced apoptosis were evaluated using a Wright-Giemsa staining kit (Baso Diagnostics Inc., Zhuhai, China). Briefly, MCF-7 and SKBr3 cells were seeded onto glass cover slips placed in the wells of six-well plates. The cells were seeded at a density of 5 × 105 cells per cover slip and allowed to attach for 24 h. Next, the cells were exposed to half maximal inhibitory concentration (IC50) concentrations of ICS (41.6 μg/mL for SKBr3 cells, 32.8 μg/mL for MCF-7 cells) for 48 h. The glass cover slips were then carefully removed, fixed by air drying, and rinsed twice with PBS. The cover slips were first stained with 500 μL of Wright-Giemsa dye solution A for 1 min, and then, 1 mL of Wright-Giemsa dye solution B was added to each cover slip and mixed well with the dye solution A. The dye mixture was incubated on the cells for 5 min at room temperature (RT). The cover slips were then washed with water and dried completely. All of the cells were visualized using optical microscopy and photographed.
Annexin V-FITC and propidium iodide staining. The apoptotic effects of ICS on SKBr3 and MCF-7 breast cancer cells were evaluated using annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (Annexin V/PI) staining. Cells were seeded onto glass cover slips placed in six-well plates. The cells were seeded to approximately 5 × 105 cells per well and allowed to attach. After 24 h of attachment, log phase cultures were treated with 20, 40, or 60 μg/mL ICS for 24 h. Cells were then rinsed twice with 4°C PBS. Next, 100 μL of 1× binding buffer containing 5 μL of Annexin V-FITC (Signalway Antibody, Maryland, USA) and 10 μL of 20-μg/mL PI solution were added to each well. The cells were incubated at RT for 15 min in the dark. Stained cells were observed using fluorescence microscopy (Leica Microsystems Inc., Wetzlar, Germany) and photographed.
Annexin V-FITC and propidium iodide assay. MCF-7 cells and SKBr3 cells were seeded onto six-well plates at a density of approximately 5 × 105 cells per well and allowed to attach for 24 h. The cells were then incubated with 20, 40, or 60 μg/mL ICS for 12 h. Treated cells were collected, washed twice with ice-cold PBS, and re-suspended in 100 μL of 1× binding buffer before being incubated with 5 μL Annexin V-FITC (Signalway Antibody) according to the manufacturer’s protocol. After 15 min of incubation at RT in the dark, 10 μL of 20 μg/mL PI was added to each tube, and the cells were gently re-suspended. Next, 400 μL PBS was added to each tube. The number of stained cells was quantified using a flow cytometer (Becton Dickinson, New Jersey, USA).
Semiquantitative RT-PCR analysis. MCF-7 cells and SKBr3 cells were seeded onto six-well plates to approximately Phytother. Res. 28: 1–10 (2014)
ISOFLAVONES EXTRACTED FROM CHICKPEA SPROUTS INDUCE APOPTOSIS
5 × 105 cells per well and allowed to attach for 24 h. The media were removed and replaced with fresh media containing ICS (20, 40, or 60 μg/mL) in DMSO. The cells were cultured at 37°C for 12 h. Total RNA was isolated using Trizol reagent (ComWin Biotech, Beijing, China) according to the manufacturer’s protocol. Reverse transcription was performed using the HiFiMMLV cDNA Kit (ComWin Biotech, Beijing, China). PCR was then conducted for 30 cycles using 1 μg RNA in a PCR machine (MJ, USA). The primers for detecting Bcl-2 expression were 5′-ACTTGTGGCCCAGATAG GCACCCA G-3′ (forward) and 5′-CGACTTCGCCGAGATGTCCAGCCAG-3′ (reverse). The primers for detecting Bcl-2-associated X protein (Bax) expression were 5′-TGCTTCAGGGTTTCATCCAG-3′ (forward) and 5′-GGCGGCAATCATCCTCTG-3′ (reverse). The primers for detecting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression were 5′-ACCACAGTCCATGCCTACAC-3′ (forward) and 5′-TTC ACCACCCTGTTGCTGTA-3′ (reverse). Primers were synthesized by Sangon Biotech (Shanghai, China). The PCR products of Bcl-2 (385 bp), Bax (170 bp), and GAPDH (452 bp) were separated using 1% agarose gel electrophoresis and visualized by staining with ethidium bromide. GAPDH was used as an internal control for both Bcl-2 and Bax expression. The net intensity of the bands was analyzed using ImageJ software (National Institutes of Health, Maryland, USA).
Western blot analysis. MCF-7 cells and SKBr3 cells were seeded onto 10-cm culture dishes at a density of approximately 1 × 106 per dish. After 24 h, the cells were treated with ICS (20, 40, or 60 μg/mL) or DMSO at 37°C for 12 h. The cells were then lysed with 200 μL radioimmunoprecipitation assay buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) and separated by 10% sodium dodecyl sulfate–polyacrylamide gel. The expression of the proteins caspase 7, caspase 9, P21, and P53 was detected using protein-selective antibodies (Boster Inc., Wuhan, China). Antibodies were incubated overnight at 4°C and then incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 h at RT. The immunolabeled proteins were detected using the enhanced chemiluminescence system (Thermo Fisher Scientific Inc., USA) and X-ray film. Band intensities were quantified using ImageJ software. β-actin was used as an internal control.
Mitochondrial membrane potential measurement. Mitochondrial membrane potential (MMP) was measured using the fluorescent probe 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)). Cells of MCF-7 and SKBr3 cells were seeded onto six-well plates at a density of approximately 5 × 105 cells per well and allowed to attach for 24 h. The cells were then treated with 20, 40, or 60 μg/mL ICS for 12 h. The treated cells were washed twice with PBS, re-suspended to a concentration of 1 × 106 cells/mL, and then incubated with 20 nM DiOC6(3) (Enzo Life Sciences International Inc., New York, USA) for 30 min at 37°C in the dark. The stained cells were washed twice with PBS and then Copyright © 2014 John Wiley & Sons, Ltd.
immediately analyzed using a flow cytometer (Epics Altra, Beckman Coulter, USA).
Intracellular reactive oxygen species evaluation. Intracellular reactive oxygen species (ROS) were evaluated based on intracellular peroxide-dependent oxidation of dichlorodihydrofluorescin diacetate (DCHF-DA), which produces the fluorescent compound 2′,7′-dichlorofluorescein (DCF). Cells of MCF-7 and SKBr3 cells were seeded onto six-well plates at a density of approximately 5 × 105 cells per well and allowed to attach for 24 h. The cells were then treated with 20, 40, or 60 μg/mL ICS for 12 h. The treated cells were washed twice with PBS and incubated with 20 μM DCHF-DA (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min in the dark. The cells were then washed with PBS, and ROS production was qualitatively evaluated using fluorescence microscopy. Quantitative evaluation was performed using cells treated as described previously, plated onto a 96-well black plate (1.5 × 105 cells/well) after digestion with trypsin, and assayed using a fluorescence microplate reader (SpectraMax M5, Molecular Devices, USA) with excitation at 488 nm and emission at 525 nm.
STATISTICAL ANALYSIS The results of each experiment are presented as the mean ± standard deviation (SD) of three experiments. The Student’s t-test was used to assess differences between ICS-treated and control groups. The data were analyzed using SPSS software. p < 0.05 was considered statistically significant.
RESULTS Antiproliferative effects of ICS on breast cancer cells The antiproliferative effects of various concentrations (10 to 60 μg/mL) of ICS on the human breast cancer cell lines SKBr3 and MCF-7 were assessed using the MTT assay. As shown in Fig. 1, ICS dramatically inhibited the proliferation of both SKBr3 and MCF-7 cells in a time-dependent and dose-dependent manner. The highest inhibition rates for SKBr3 cells were 36.1 ± 5.2% at 24 h (p < 0.01), 65.3 ± 2.6% at 48 h (p < 0.01), and 88.5 ± 3.2% at 72 h (p < 0.01; Fig. 1A). The highest inhibition rates for MCF-7 cells were 36.5 ± 5.2% at 24 h (p < 0.01), 76.8 ± 5.2% at 48 h (p < 0.01), and 86.9 ± 5.6% at 72 h (p < 0.01; Fig. 1B).
Morphologic observation of ICS-induced apoptosis A series of morphological features typically associated with apoptosis, including nucleus shrinkage, chromatin condensation, and DNA fragmentation, were observed in SKBr3 and MCF-7 cells treated with the IC50 of ICS (Fig. 2A). These features were not observed in control cells. Similar morphologic characteristics were Phytother. Res. 28: 1–10 (2014)
H. CHEN ET AL.
Figure 1. Antiproliferative activity of ICS. (A) SKBr3 cells and (B) MCF-7 cells were treated with different concentrations of ICS for 24, 48, or 72 h. Inhibition was determined by MTT assay. % inhibition rate was determined relative to control cells. Data are shown as the mean ± SD of three experiments.
also found with Annexin V/PI fluorescence staining (Fig. 2B and C).
concentration, ICS induced the highest apoptosis rate in both SKBr3 and MCF-7 cells (p < 0.01; Fig. 3C and D).
Quantitative assay of ICS-induced apoptosis
Apoptosis-related protein expression after ICS treatment
The Annexin V/PI assays showed significant changes in SKBr3 and MCF-7 cells treated with ICS (Fig. 3). ICStreated cells showed a dose-dependent increase in apoptosis compared with controls. The apoptotic rates ranged from 0.67 ± 0.15% to 34.30 ± 0.85% in SKBr3 cells (Fig. 3A). The apoptotic rates ranged from 0.20 ± 0.10% to 31.63 ± 0.75% in MCF-7 cells (Fig. 3B). At the 60-μg/mL
Levels of mRNA of the antiapoptotic family member Bcl-2 and the apoptosis-promoting Bax were determined using semiquantitative reverse transcription PCR. The constitutively expressed gene GAPDH was used as an internal control. In SKBr3 cells, Bcl-2 mRNA expression showed a dose-dependent decrease with increasing concentrations of ICS, while Bax
Figure 2. Micrographs of ICS-induced apoptosis in SKBr3 cells and MCF-7 cells. (A) Micrographs with Wright-Giemsa staining (×400) of SKBr3 cells or MCF-7 cells treated with IC50 concentrations of ICS for 48 h. Fluorescent micrographs with Annexin V-FITC and propidium iodide staining (×400) of SKBr3 cells (B) or MCF-7 cells (C) treated with ICS (20, 40, or 60 μg/mL) for 24 h. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. 28: 1–10 (2014)
ISOFLAVONES EXTRACTED FROM CHICKPEA SPROUTS INDUCE APOPTOSIS
Figure 3. ICS-induced apoptosis measured by Annexin V-FITC and propidium iodide (PI) assay. Characteristic dual-parameter fluorescence histograms were obtained with SKBr3 cells (A, C) and MCF-7 cells (B, D) stained with Annexin V-FITC and PI. Fluorescence was evaluated using flow cytometry after exposure to ICS (20, 40, and 60 μg/mL) for 12 h. Q1 [FITC( )/PI(+)], necrotic cells; Q2 [FITC(+)/PI(+)], late apoptotic cells; Q3 [Annexin V(+)/PI( )], living cells; Q4 [FITC(+)/PI( )], early apoptotic cells. The number in each quadrant represents the percentage of cells present. (C, D) Each point is the mean ± SD of three independent experiments. *, ** significantly different from control at p < 0.05 and p < 0.01, respectively.
mRNA expression showed a dose-dependent increase with increasing concentrations of ICS in SKBr3 cells (Fig. 4A). Similar findings were observed in MCF-7 cells (Fig. 4B). The ratio of Bcl-2/Bax mRNA expression in SKBr3 and MCF-7 cells was significantly lower than that of control cells (5.5 ± 0.2 vs 0.6 ± 0.03 in SKBr3 cells and 7.8 ± 0.1 vs 0.6 ± 0.02 in MCF-7 cells) (Fig. 4A and B). The expression of the caspase family members caspase 7 and caspase 9 was detected using Western blot analysis. As Fig. 4C and D show, treatment of SKBr3 and MCF-7 cells with increasing doses of ICS induced a dose-dependent increase in the protein expression of caspase 7 and caspase 9. The maximum relative protein expression level of caspase 7 was approximately 1.6 times higher in SKBr3 cells than in control cells and approximately 3.4 times higher in MCF-7 cells than in control cells. Caspase 9 expression was approximately 4.4 times higher in SKBr3 cells than in the control group and approximately 6.0 times higher in MCF-7 cells than in the control group. Copyright © 2014 John Wiley & Sons, Ltd.
Mitochondrial membrane potential disruption after ICS treatment Treatment using ICS led to a dose-dependent decrease in MMP in both SKBr3 and MCF-7 cells (Fig. 5A and B). The 40-μg/mL ICS treatment was associated with a 30.95 ± 1.62% decrease in the MMP of SKBr3 cells (p < 0.05) and an 18.21 ± 1.51% decrease in the MMP of MCF-7 cells (p < 0.05). The 60-μg/mL ICS treatment was associated with a 41.70 ± 2.78% decrease in the MMP of SKBr3 cells (p < 0.01) and a 35.49 ± 2.98% decrease in the MMP of MCF-7 cells (p < 0.01; Fig. 5C and D). ROS generation in ICS-treated breast cancer cells The fluorescent intensity of DCF was used to evaluate ROS generation after cells were treated with 20, 40, or 60 μg/mL ICS. As shown in Fig. 6, the incubation of both Phytother. Res. 28: 1–10 (2014)
H. CHEN ET AL.
Figure 4. ICS-induced apoptosis-related protein expression. (A, B) Bcl-2 and Bax mRNA expression. (A) SKBr3 cells and (B) MCF-7 cells were treated with ICS (20, 40, or 60 μg/mL) for 12 h. Total RNA was extracted, and Bcl-2 and Bax mRNA levels were determined using semiquantitative reverse transcription PCR. Representative bands are shown in the left panels. Bcl-2/Bax mRNA expression in SKBr3 cells or MCF-7 cells with different doses of ICS is shown in the right panels. (C, D) Caspase 7 and caspase 9 protein expression. (C) SKBr3 and (D) MCF-7 cells were treated with ICS (20, 40, or 60 μg/mL) for 12 h. Total protein was extracted, and caspase 7 and caspase 9 protein expression was determined by Western blot. Representative bands are shown in the left panels. The average caspase 7 and caspase 9 levels of SKBr3 cells or MCF-7 cells treated with different doses of ICS are shown in the right panels. β-actin was used to normalize protein loading. All results are the mean ± SD of three independent experiments. *, ** significantly different from control at p < 0.05 or p < 0.01, respectively. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. 28: 1–10 (2014)
ISOFLAVONES EXTRACTED FROM CHICKPEA SPROUTS INDUCE APOPTOSIS
Figure 5. ICS-induced mitochondrial membrane potential depolarization. Representative fluorescence histograms of SKBr3 cells (A) and MCF-7 cells (B) stained with DiOC6. Fluorescence was evaluated using flow cytometry after exposure to ICS (20, 40, and 60 μg/mL) for 12 h. M1 represents the percentage of cells with a low mitochondrial membrane potential. Data are expressed in the percentage of cells with mitochondrial depolarization in SKBr3 cells (C) and in MCF-7 cells (D). The data are presented as the mean ± SD. Experiments were performed in triplicate. *, ** significantly different from control at p < 0.05 or p < 0.01, respectively.
SKBr3 and MCF-7 cells with ICS (20, 40, or 60 μg/mL) was associated with a dose-dependent increase in ROS production, compared with controls (Fig. 6A and B). Production of ROS in SKBr3 cells was highest when a concentration of 60 μg/mL ICS was used. This was a 2.8-fold and 2.0-fold increase compared with untreated SKBr3 cells (p < 0.01; Fig. 6C) and untreated MCF-7 cells (p < 0.01; Fig. 6D), respectively. P21 and P53 have key roles in oxidative-damageinduced apoptosis (Forman, et al., 2008). P21 and P53 protein expression increased in a dose-dependent fashion with ICS treatment. The maximum protein expression of P21 in SKBr3 cells was about 2.8 times higher than that of controls. The maximum protein expression of P21 in MCF-7 cells was about 3.6 times higher than that of controls. SKBr3 cell P53 expression was about 2.8 times higher than that of controls, and MCF-7 cell P21 expression was 6.8 times higher than that of controls (Fig. 7A and B). Copyright © 2014 John Wiley & Sons, Ltd.
DISCUSSION We evaluated the antiproliferative and apoptotic effects of ICS on two human breast cancer cell lines, SKBr3 and MCF-7. ICS, at concentrations from 10 to 60 μg/mL, significantly inhibited the growth of both cell lines in a dose-dependent and time-dependent manner. Moreover, the growth inhibition occurred via a mitochondria-dependent apoptotic mechanism. The ICS-induced apoptosis suggests that ICS may be useful for the chemoprevention or chemotherapy of breast cancer. In vitro studies have confirmed that isoflavonoids act as ER agonists and promote the growth of estrogenresponsive human breast cancer cells at low concentrations. Isoflavonoids have ER antagonist effects and inhibit the growth of estrogen-responsive and ER-negative Phytother. Res. 28: 1–10 (2014)
H. CHEN ET AL.
Figure 6. ICS-induced reactive oxygen species (ROS) generation. (A, B) Representative micrographs of DCHF-DA staining of breast cancer cells. (A) SKBr3 cells and (B) MCF-7 cells were treated with ICS (20, 40, or 60 μg/mL) for 12 h and then incubated with DCHF-DA for 30 min. The upper panels of panels A and B show representative fluorescent staining. The lower panels of panels A and B are light micrographs of the same field (×100). (C, D) Quantitative ROS generation assay with a fluorescence microplate reader. (C) SKBr3 cells and (D) MCF-7 cells were incubated with ICS (20, 40, or 60 μg/mL) for 12 h. Generation of ROS was measured using a fluorescence microplate reader. The data are presented as the mean ± SD. Experiments were performed in triplicate. *, ** significantly different from control at p < 0.05 or p < 0.01, respectively. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. 28: 1–10 (2014)
ISOFLAVONES EXTRACTED FROM CHICKPEA SPROUTS INDUCE APOPTOSIS
Figure 7. ICS induced the expression of the oxygen stress-reaction-related proteins P53 and P21. (A) SKBr3 and (B) MCF-7 cells were treated as described previously, and protein expression was determined by Western blot. Representative bands are shown in the left panels. The average P53 and P21 levels in SKBr3 and MCF-7 cells treated with different doses of ICS are shown in the right panels. β-actin was used to normalize protein loading. All results are the mean ± SD of three independent experiments. *, ** significantly different from control at p < 0.05 or p < 0.01, respectively.
human breast cancer cells at higher concentrations (Chinni et al., 2003; Robb and Stuart, 2014). We found that ICS at 10 to 60 μg/mL inhibited the growth of ER-positive MCF-7 cells and ER-negative SKBr3 cells (Fig. 1). This result suggests that ICS-mediated inhibition of the two breast cancer cell lines was ER independent. These findings are consistent with previous reports that isoflavonoids inhibited the growth of human breast carcinoma cells (Choi and Kim, 2008; Lin et al., 2009). The induction of apoptosis is an important mechanism in inhibiting cell proliferation. Isoflavonoids like genistein and formononetin inhibit cancer cell growth via apoptosis (Chinni et al., 2003). In this study, Wright-Giemsa staining and Annexin V/PI staining revealed morphological features typically associated with apoptosis in ICS-treated SKBr3 and MCF-7 cells (Fig. 2). In addition, the Annexin V/PI assay demonstrated that exposure of SKBr3 and MCF-7 cells to ICS (20, 40, and 60 μg/mL) for 48 h caused a remarkable accumulation of apoptotic cells in a concentrationdependent manner (Fig. 3). These three methods of determining apoptosis demonstrated similar results. To further understand the molecular mechanism associated with the development of apoptosis, we measured alterations in the expression of the apoptosis regulators Bcl-2 and Bax. The regulators Bax and Bcl-2 are two important members of the apoptosis-regulating Bcl-2 family. These proteins play key roles in the integration and regulation of apoptotic signals (Gross et al., 1999). The protein Bax belongs to the proapoptotic subfamily and binds to Bcl-2, which belongs to the antiapoptotic subfamily. The gene Bax accelerates the progression of Copyright © 2014 John Wiley & Sons, Ltd.
apoptosis by inhibiting the function of Bcl-2 (Oltval et al., 1993). The ratio of Bcl-2/Bax expression determines the cell’s susceptibility to apoptosis (De Angelis et al., 1998; Findley et al., 1997). We found that ICS induced an increase in the mRNA expression of Bax, as well as a decrease in the mRNA expression of Bcl-2, in both MCF-7 and SKBr3 cell lines. Furthermore, the ratio of Bcl-2/Bax mRNA significantly decreased in a concentration-dependent manner following exposure to ICS (Fig. 4A and B). A typical characteristic of apoptosis is the activation of caspases, a class of cysteine proteases that selectively cleave proteins after aspartic acid residues. Caspases are activated by sequential cleavage of their inactive procaspase forms (Hu et al., 2001). Caspase 7 and caspase 9 are two key members of the caspase family. Activation of caspase 9 triggers a cascade of downstream signals, including caspase 3 and caspase 7, members of the intrinsic mitochondria-dependent apoptotic pathway (Chen, et al., 2000). Because there is a lack of caspase 3 expression in MCF-7 cells (Mc Gee, et al., 2002), we evaluated the expression of the activated forms of caspase 7 and caspase 9 using Western blots. Exposure of SKBr3 and MCF-7 cell lines to ICS increased the expression of active caspase 9 and caspase 7 in a dose-dependent manner (Fig. 4A and D). We presumed that mitochondria were involved in the ICS-induced apoptosis because caspase 9 and caspase 7 expression was upregulated by ICS. The disruption of MMP is a crucial step in intrinsic mitochondriamediated apoptosis induction (Gupta, 2003). Mitochondria are a prerequisite for ROS generation. The ROS Phytother. Res. 28: 1–10 (2014)
H. CHEN ET AL.
generated in and around mitochondria act as second messengers in the apoptosis-mediated signaling pathway (Forman et al., 2008). We found that ICS treatment led to a dose-dependent decrease in MMP in both SKBr3 and MCF-7 cells (Fig. 5). ICS induced a high level of ROS production by SKBr3 cells and MCF-7 cells (Fig. 6). ICS also induced an increase in the expression of two tumor suppressor proteins, P53 and P21, which play a vital role in oxidative stress responses (Fig. 7; Forman, et al., 2008). ICS-induced MMP damage and ROS generation, leading to breast cancer apoptosis. In conclusion, we found that ICS induced mitochondriamediated apoptosis in ER-positive and ER-negative human breast cancer cell lines. This finding has greatly extended the potential use of ICS for chemoprevention
and chemotherapy. In future research, we will elucidate the detailed mechanism underlying ICS-induced apoptosis and chemoprevention in animal models.
Acknowledgements This work was supported, in part, by grants from the National Natural Science Foundation of China (Grant No 81102890) and the Joint Funds of the National Natural Science Foundation of China (Grant No. U1203203).
Conflict of Interest The authors have declared that there is no conflict of interest.
REFERENCES Behloul N, Wu GZ. 2013. Genistein: a promising therapeutic agent for obesity and diabetes treatment. Eur J Pharmacol 698: 31–38. Chan YH, Lau KK, Yiu KH, et al. 2008. Reduction of C-reactive protein with isoflavone supplement reverses endothelial dysfunction in patients with ischaemic stroke. Eur Heart J 29: 2800–2807. Chen Q, Gong B, Almasan A. 2000. Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death Differ 7: 227–233. Cheng Z, Ayiguli A, Ma QL, et al. 2008. Determination of isoflavones in extracts of Cicer afiefinum L. and bean sprout by ultrabiolet spectrophotometry. Lishizhen Med Mater Med Res 19: 2612–2613. Chinni SR, Alhasan SA, Multani AS, et al. 2003. Pleotropic effects of genistein on MCF-7 breast cancer cells. Int J Mol Med 12: 29–34. Choi EJ, Kim GH. 2008. Daidzein causes cell cycle arrest at the G1 and G2/M phases in human breast cancer MCF-7 and MDAMB-453 cells. Phytomedicine 15: 683–690. De Angelis PM, Stokke T, Thorstensen L, et al. 1998. Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype. Mol Pathol 51: 254–261. Farina HG, Pomies M, Alonso DF, et al. 2006. Antitumor and antiangiogenic activity of soy isoflavone genistein in mouse models of melanoma and breast cancer. Oncol Rep 16: 885–891. File SE, Jarrett N, Fluck E, et al. 2001. Eating soya improves human memory. Psychopharmacology (Berl) 157: 430–436. Findley HW, Gu L, Yeager AM, et al. 1997. Expression and regulation of Bcl-2, Bcl-xl, and Bax correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia. Blood 89: 2986–2993. Forman HJ, Fukuto JM, Miller T, et al. 2008. The chemistryof cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal. Arch Biochem Biophys 477: 183–195. Gupta S. 2003. Molecular signaling in death receptor and mitochondrial pathways of apoptosis. Int J Oncol 22: 15–20. Gross A, Mcdonnell JM, Korsmeyer SJ. 1999. BCL-2 family members and the mitochondria in apoptosis. Gene Dev 13: 1899–1911. Hu CCA, Tang CHA, Wang JJ. 2001. Caspase activation in response to cytotoxic Rana catesbeiana ribonuclease in MCF-7 cells. FEBS Lett 503: 65–68. Limer JL, Speirs V. 2004. Phyto-oestrogens and breast cancer chemoprevention. Breast Cancer Res 6: 119–127. Lin YJ, Hou YC, Lin CH, et al. 2009. Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells. Biochem Biophys Res Comm 378: 683–688. Liu YM, Yikemu S. 1986. Wei Wu Er Yao Zhi, 1st edn. Xinjiang People’s Medical Publishing House: Urumqi, Xinjiang. Copyright © 2014 John Wiley & Sons, Ltd.
Lv Q, Yang Y, Zhao Y, et al. 2009. Comparative study on separation and purification of isoflavones from the seeds and sprouts of chickpea by high-speed countercurrent chromatography. J Liq Chrom Relat Tech 32: 2879–2892. Magee PJ, Rowland IR. 2004. Phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer. Br J Nutr 91: 513–531. Ma HR, Wei HB, Chen Z, et al. 2013. The estrogenic activity of isoflavones extracted from chickpea Cicer arietinum L sprouts in vitro. Phytother Res 27: 1237–1242. Mazzio E, Badisa R, Mack N, et al. 2014. High throughput screening of natural products for anti-mitotic effects in MDA-MB-231 human breast carcinoma cells. Phytother Res 28: 856–867. Mc Gee MM, Hyland E, Campiani G, et al. 2002. Caspase-3 is not essential for DNA fragmentation in MCF-7 cells during apoptosis induced by the pyrrolo-1,5- benzoxazepine, PBOX-6. FEBS Lett 515: 66–70. Oltval ZN, Milliman CL, Korsmeyer SJ. 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 74: 609–619. Robb EL, Stuart JA. 2014. Multiple phytoestrogens inhibit cell growth and confer cytoprotection by inducing manganese superoxide dismutase expression. Phytother Res 28: 120–131. Salti GI, Grewal S, Mehta RR, et al. 2000. Genistein induces apoptosis and topoisomerase II-mediated DNA breakage in colon cancer cells. Eur J Cancer 36: 796–802. Siegel R, Naishadham D, Jemal A. 2013. Cancer statistics, 2013. CA Cancer J Clin 63: 11–30. Spinozzi F, Pagliacci MC, Migliorati G, et al. 1994. The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells. Leukemia Res 18: 431–439. Su SJ, Chow NH, Kung ML, et al. 2003. Effects of soy isoflavones on apoptosis induction and G2-M arrest in human hepatoma cells involvement of caspase-3 activation, Bcl-2 and Bcl-XL downregulation, and Cdc2 kinase activity. Nutr Cancer 45: 113–123. Tikkanen MJ, Adlercreutz H. 2000. Dietary soy-derived isoflavone phytoestrogens: could they have a role in coronary heart disease prevention? Biochem Pharmacol 60: 1–5. Wei P, Liu M, Chen Y, et al. 2012. Systematic review of soy isoflavone supplements on osteoporosis in women. Asian Pac J Trop Med 5: 243–248. White PJ, Broadley MR. 2009. Biofortification of crops with seven mineral elements often lacking in human diets–iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytol 182: 49–84. Yun SM, Jung JH, Jeong SJ, et al. 2014. Tanshinone IIA induces autophagic cell death via activation of AMPK and ERK and inhibition of mTOR and p70 S6K in KBM-5 leukemia cells. Phytother Res 28: 458–464. Zhao SH, Zhang LP, Gao P, et al. 2009. Isolation and characterisation of the isoflavones from sprouted chickpea seeds. Food Chem 114: 869–873. Phytother. Res. 28: 1–10 (2014)
