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Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells.


Background: The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor [alpha] positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet.

Material and methods: We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis). Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis.

Results: CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 [micro]M. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways.

Conclusion: CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis.



Breast cancer

Cell cycle





Breast cancer is one of the most common types of tumor (DeSantis et al. 2014; Donepudi et al. 2014; Siegel et al. 2014; Zagouri et al. 2014) and the American Cancer Society estimated around 230,000 new cases of invasive breast cancer diagnosed in women and about 40,000 breast cancer deaths in USA in 2013 (DeSantis et al. 2011). Some hormone receptors, i.e. estrogen and progesterone receptors (ER, PR) and growth factor receptors such as HER2 are referred as important prognostic factors for breast cancer (Donepudi et al., 2014; Kawano et al. 2013; Kwast et al. 2014; Pourzand et al. 2011). The majority of breast cancer cases are hormone-dependent and ER-positive. Anti-estrogenic therapies may be effective to improve the prognosis of breast cancer patients, however, breast tumors can develop resistance toward anti-hormonal drugs (Josefsson and Leinster 2010; Musgrove and Sutherland 2009; Osborne and Schiff 2011) and targeting one receptor is frequently inadequate due to multiple alternative routes to resist the detrimental effects of anticancer drugs (Osborne and Schiff 2011; Zhang et al. 2014b). Thus, the search for active compounds against breast cancer represents still a critical issue. Various studies have evaluated natural compounds in terms of their inhibitory effects on cancer-related signaling pathways (Cerella et al. 2015; Chinembiri et al. 2014; Jafari et al. 2014; Marrelli et al. 2014; Millimouno et al. 2014; Pourahmad et al. 2014; Sztiller-Sikorska et al. 2014; Turrini et al. 2014). Indeed, many anticancer drugs are derived from natural compounds (Al-Tweigeri et al. 2010; Driscoll and Marquez 1994; Fotia et al. 2012; Icli et al. 2011; Lee et al. 2014). Targeting breast cancer-related receptors (ER, PR, EGFR, etc.) and influencing cancer-related pathways related to apoptosis, ceil cycle and DNA damage possess a high potential against breast cancer.

BRCA-1 and -2 are important proteins for breast cancer progression by regulating cell cycle and DNA damage. Mutations in the BRCA-1 and BRCA-2 genes play a role for breast carcinogenesis (Gangi et al. 2014; Xu et al. 2012). Functional loss by mutation leads to deficient DNA damage repair and cell cycle control (Narod and Foulkes 2004; Shahid et al. 2014; Venkitaraman 2002; Wiltshire et al. 2007; Zhou and Elledge 2000). Down-regulation of BRCA-1 and/or -2 may cause increased DNA damage and apoptosis. In tumor cells, targeting those proteins may be an effective strategy to direct the fate of cancer cells through apoptosis.

Another critical protein in breast cancer is p21. This tumor suppressor is either mutated or downregulated to favor excessive cell proliferation and eventually metastasis. p21 also plays a role in DNA damage-related pathways. The mutation status of p21 may determine breast cancer susceptibility (Akhter et al. 2014), whereas upregulation of p21 is linked with anticancer activity by apoptosis induction (Aziz et al. 2014). Thus, natural compounds causing up-regulation of p21 and inducing apoptosis may be effective against breast cancer.

A novel compound from Pigeon Pea (Cajanus cajan (L.) Millsp.) is cajanin stilbene acid (CSA) (Wu et al. 2009). CSA's abundance is quite low and it is difficult to isolate. Thus, the bioactivity of CSA may have been overseen in the past. However, the profound cytotoxicity of this compound indicates that this compound may be valuable for cancer treatment The cellular and molecular mechanisms of action for CSA are still not well understood. In the present investigation, we analyzed the activity of CSA in ER-positive MCF-7 cells in terms of cell cycle and apoptosis regulation. For this purpose, we applied two experimental approaches:

(1) We investigated well-known parameters such as cell cycle distribution, mitochondrial membrane potential, DNA fragmentation, expression of Bax and Bcl-2 proteins, and activation of caspases.

(2) By using gene expression profiling, we found that the BRCA-1related-DNA damage response pathway was directly affected by CSA treatment and various genes playing role in this pathway were down-regulated. Moreover, p21 was up-regulated, implying the ability of CSA to affect both the DNA damage response and cell proliferation in MCF-7 breast cancer cells.

Materials and methods


Cajanin stilbene acid (CSA, purity [greater than or equal to] 98%) was isolated from Pigeon Pea (Cajanus cajan (L.) Millsp.) roots (Fig. 1). A 10 mg/ml stock solution of CSA was prepared in dimethyl sulfoxide (DMSO) and stored at -80[degrees]C. Rhodamine 123 (R123) and propidium iodide (PI) were obtained from Sigma-Aldrich Inc. (St. Louis, MO). Deionized water was used in all experiments. Hoechst 33258 was purchased Sigma-Aldrich (Taufkirchen, Germany).

Cell culture

Human MCF-7 breast cancer cells were obtained from the Institute of Molecular Biology (University of Mainz, Germany). They were maintained under standard conditions (37[degrees]C, 5% C[O.sub.2]) in DMEM medium (Gibco BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (100 U/ml penicillin, 100 [micro]g/ml streptomycin). Cells were passaged twice weekly. All experiments were performed with cells in the logarithmic growth phase.

Cell cycle analysis

MCF-7 cells (1 x [10.sup.6] cells/well) were seeded into 6-well plates. After 24 h, cells were treated with CSA at serial dilutions (0, 8.88, 11.83 or 14.79 [micro]M) for 48 h. Then, cells were centrifuged, washed with PBS, stained with 50 pig/ml DAPI (Partee, Munster, Germany) and analyzed by flow cytometer (Partee, Munster, Germany). The cell cycle phases were analyzed with FACScan and CellQuest software (Becton Dickinson, Mountain View, CA).

Measurement of mitochondrial membrane potential

The mitochondrial membrane potential has been measured by R123 (Cao et al. 2007). MCF-7 cells (1 x [10.sup.6] cells/well) were seeded into 6-well plates. After 24 h, cells were treated with CSA (8.88, 11.83 or 14.79 [micro]M) for 48 h or left untreated. Then, cells were harvested and washed twice with PBS. Cell pellets were resuspended in 2 ml fresh medium containing 1.0 [micro]M R123 and incubated at 37[degrees]C in a thermostatic bath for 30 min with gentle shaking. MCF-7 cells were separated by centrifugation, washed twice with PBS, stained with 2 [micro]g/ml PI and analyzed by flow cytometry.

For microscopic analysis, cell monolayers were treated with the same protocol as described above and subjected to a confocal laser scanning microscope (C1-LU3EX, Nikon, Sendai, Japan).

DNA fragmentation assay

Apoptotic DNA fragments appear as DNA ladder consisting of multimers of 180-200 bp (Tilly and Hsueh 1993). MCF-7 cells (1 x [10.sup.6] cells/ml) were seeded in 6-well plates and exposed to CSA (8.88, 11.83 or 14.79 [micro]M) for 48 h or left untreated. Then, cells were collected by centrifugation. DNA was isolated with a commercial isolation kit (Watson Biotechnologies Inc, Shanghai, China) according to the manufacturer's instructions. The DNA was separated in 1 % agarose gel and visualized by ultraviolet illumination (Image Master VDS-CL, Tokyo, Japan) after staining with ethidium bromide.

Morphological observation of nuclear change

MCF-7 cells (1 x [10.sup.6] cells/ml) were seeded in 6-well plates and treated with CSA (8.88, 11.83 or 14.79 [micro]M) for 48 h at 37[degrees]C or left untreated. Cells were collected, washed, fixed in 4% paraformaldehyde for 30 min and stained with 5 (xg/ml Hoechst 33258 for 5 min at room temperature. The apoptotic cells were visualized using inverted fluorescence microscope (Nikon TE2000, Tokyo, Japan).

Western blotting

MCF-7 cells were treated with CSA (8.88,11.83 or 14.79 [micro]M) for 48 h or left untreated. For protein isolation, medium was removed, cells were washed twice with ice-cold PBS, then lysed using cell lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% [Na.sub.3]C[O.sub.4], 0.5 [micro]g/ml leupeptin, 1 mM phenylmethanesulfonyl fluoride (PMSF)]. The lysates were collected by scraping from the plates and then centrifuged at 10,000 rpm at 4[degrees]C for 5 min.

Total protein samples (20 [micro]g) were loaded on 12% of SDS-polyacrylamide gels for electrophoresis, and then transferred onto PVDF transfer membranes (Millipore, Billerica, USA) at 0.8mA/[cm.sup.2] for 2 h. Membranes were blocked at room temperature for 2 h with blocking solution (1% BSA in PBS plus 0.05% Tween-20). Membranes were then incubated overnight at 4[degrees]C with primary antibodies (anticaspase-3, anti-Bax, anti-Bcl-2, anti-[beta]-actin) at a dilution of 1:250 (Biosynthesis Biotechnology Company, Beijing, China) in blocking solution. After thrice washings in TBST for each 5 s, membranes were incubated for 1 h at room temperature with alkaline phosphatase peroxidase-conjugated anti-mouse secondary antibody (1:500 dilution) in blocking solution. Detection was performed by the BCIP/NBT Alkaline Phosphatase Color Development kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Bands were then recorded by a digital camera (Canon, EOS 350D, Tokyo, Japan).

mRNA microarray analysis

Total RNA from MCF-7 cells after 72 h of treatment with CSA at [IC.sub.50] concentration (54.77 [micro]M) or DMSO solvent control was extracted using Rneasy[R] mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. RNA quality was verified by electrophoresis using the Nano Chip assay on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany). Only samples with RNA index values greater than 8.5 were selected for expression profiling. RNA concentrations were determined using the NanoDrop spectrophotometer (Nano-Drop Technologies, Wilmington, DE). Total RNA was labeled and converted to cDNA (Eberwine et al. 1992). Then, fluorescent cRNA (Cyanine 3-CTP) was synthesized and purified using QIAgen Rneasy[R] kit. After fragmentation of the cRNA, samples were hybridized on Whole Human Genome RNA chips (8 x 60 K Agilent) by following the One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technologies GmbH) for 17 h at 65[degrees]C. Microarray slides were washed and scanned with Agilent Microarray Scanning system. Images were analyzed and data were extracted, background subtracted and normalized using the standard procedures of Agilent Feature Extraction Software. The expression data obtained was filtered with Chipster data analysis platform. These steps include filtering of genes by two times standard deviation of deregulated genes and subsequent assessment of significance using empirical Bayes t-test (p < 0.05). Pathway analysis was done by using the Ingenuity Pathways Analysis software (version 5.5) from Ingenuity Systems (Redwood City, CA, USA) and -log (p-value) was used to estimate the significances of pathways and biological functions.

Real time RT-PCR

In order to validate the microarray data, BRCA-1, BRCA-2 and p21 genes were selected and the total RNA isolated for the microarray experiment was used for real time RT-PCR. RPS-13 was used as reference gene for standardization. All measurements were done in duplicates and the average fold change values were provided. Table 1 depicts the primer nucleotide sequences, primer concentrations and annealing temperatures. Real time RT-PCR reactions and the fold change calculations were conducted as described previously (Panossian et al. 2013).


Cell cycle analysis

Exposure of MCF-7 cells to CSA (8.88-14.79 piM) for 48 h in vitro caused a dose-dependent [G.sub.2]M arrest (19.64-27.48% compared to 12.88% in untreated cells) and S arrest (17.46-32.16% compared to 13.40% in untreated cells) (Fig. 2A). CSA(5 [micro]g/ml) arrested MCF-7 cells at the S and [G.sub.2]M phase also in a time-dependent manner (Fig. 2B). Then, we used single-cell suspensions obtained from MCF-7 xenograft tumors after excision from nude mice. Again, a dose-dependent increase in S and [G.sub.2]M phase cells was observed after treatment with 15 or 30 mg/kg CSA (Fig. 2C, histograms 3 and 4) compared to untreated controls (Fig. 2C, histogram 1). Cyclophosphamide is a standard drug for breast cancer therapy and was used as control compound. Cyclophosphamide did not affect the [G.sub.2]M phase, but the S phase (Fig. 2C, histogram 2).

Assessment of apoptosis

Hoechst 33258 staining showed considerable morphological changes in nuclear chromatin of CSA-treated MCF-7 cells. Untreated control cells did not show chromatin condensation and their nuclei were stained in less bright and homogeneous blue color (Fig. 3A, photograph 1). In contrast, CSA (14.79 [micro]M for 48 h), caused very intense staining of condensed and fragmented chromatin and the formation of typical apoptotic bodies. Only a few nuclei displayed normal morphology (Fig. 3A, photograph 2).

DNA laddering

Apoptosis-related DNA laddering was visible after treatment of MCF-7 cells with increasing CSA concentrations for 48 h (Fig. 3B, lanes 2-4). Untreated control cells did not induce apoptosis (lane 1). DNA-laddering was also observed in MCF-7 xenograft tumors treated with CSA or cyclophosphamide (data not shown).

Markers of the mitochondrial apoptosis

Western blot analysis revealed that CSA-treated MCF-7 cells down-regulated Bd-2 expression, but up-regulated Bax expression (Fig. 3C). CSA-induced apoptosis was associated with mitochondrial depolarization. In MCF-7 cells, CSA at doses of 8.88-14.79 [micro]M led to dose-dependently increased percentages of mitochondrial depolarization ([DELTA][[PSI].sub.m]) from 97.03 to 61.26% (Fig. 3D). Mitochondrial membrane potentials were measured by laser scanning microscopy and comparable results were obtained (Fig. 3E). Furthermore, CSA led to a dose-dependent increase of caspase-3 activity as observed by Western blotting (Fig. 3F).

Differential gene regulation by CSA

Upon CSA treatment at the [IC.sub.50] concentration, 363 genes were differentially regulated after 24 h and 659 genes after 72 h, as analyzed by microarray-based mRNA hybridizations. We subjected these genes to Ingenuity Pathway Analysis. Many cell cycle and apoptosis related pathways were observed to be affected upon CSA treatment as shown in Fig. 4A. BRCA1 in DNA damage response (Fig. 4B) and cell cycle control of chromosomal replication (Fig. 4C) were the most affected pathways with -log (p-value) of 12.5 and 11.3 respectively. BRCA-1 and BRCA-2 were down-regulated by 2.990- and 3.364-fold, respectively, whereas p21 was up-regulated by 5.223-fold upon CSA treatment. Microarray data and the deregulation of those genes were validated by real time RT-PCR as can be seen in Table 2. The correlation coefficient between mRNA expression values determined by microarray hybridization and real-time RT-PCR was 0.99 (Pearson Correlation Test). These results clearly indicate that CSA influences DNA damage and cell cycle related pathways and the expression of three important genes playing role in DNA damage response pathway and cell cycle control.


In the present study, we investigated the anti-cancer activity of CSA, a compound isolated by us from Pigeon Pea (Cajanus cajan) (Wu et al. 2009) in terms of apoptosis and cell cycle regulation. The cellular and molecular mechanisms of CSA's mode of action are still not well understood and we hypothesized that CSA may act on cell cycle and apoptosis related pathways. CSA induced arrest in the [G.sub.2]M phase of the cell cycle in a time- and concentration-dependent manner. If unreleased, [G.sub.2]M arrest can ultimately lead to apoptosis. By DNA laddering assay and fluorescence microscopy, we found that CSA indeed induced apoptosis. Apoptosis induction was associated with Bd-2 down-regulation, Bax up-regulation, caspase-3 activation and depolarization of the mitochondrial membrane potential, suggesting that CSA activated the mitochondrial pathway of apoptosis in MCF-7 cells. Various studies have shown that targeting those pathways and inducing cell cycle arrest and apoptosis serve as a valuable strategy for cancer drug discovery process (Evan and Vousden 2001; Kim et al. 2014; Wang et al. 2014; You and Park 2014; Zhang et al. 2015; Zhang et al. 2014a; Zheng et al., 2014).

Gene expression profiling studies yield valuable information to understand molecular mechanisms of different cancer types (Drukker et al. 2014; Fina et al. 2015; Fu et al. 2014; Yuan et al. 2014; Zubor et al. 2015). The mode of action of a compound and its potential as an anticancer agent can be evaluated via gene expression profiling studies (lorio et al. 2009; Nunez et al. 2008; Righeschi et al. 2012; Schmeits et al. 2014; Zhou et al. 2005). Therefore, we applied mRNA microarray analyses to unravel modes of action of CSA. The deregulated genes after CSA treatment were subjected to Ingenuity Pathway analyses to identify affected signaling routes. Intriguingly, among the top signaling pathways were [G.sub.2]M arrest pathways. This is a strong hint that cell cycle arrest in [G.sub.2]M and induction of apoptosis are important modes of action of CSA toward cancer cells. BRCA-1 and BRCA-2 were down-regulated upon CSA treatment, indicating that DNA damage and repair pathways were affected. Proteins (p21, BRCA-1 and BRCA-2) playing role in DNA damage response pathway (Pawlik and Keyomarsi 2004) were deregulated upon CSA treatment. Up-regulation of p21, down-regulation of BRCA-1 and BRCA-2 imply that uncontrolled proliferation was to some extent normalized and DNA damage was accumulated leading to apoptosis. As tumor suppressor p21 plays a critical role in cell cycle regulation, excessive cell proliferation and metastasis can be halted via p21 up-regulation (Garcia-Tunon et al. 2006; Tanaka and lino 2014). Our results on CSA can be reconciled with more general findings in cancer biology that tumors activate DNA damage response pathways such as BRCA-1/2 upon exposure to DNA-damaging agents (Cheung-Ong et al. 2014). It is worth speculating that CSA may be even more cytotoxic, if combined with other DNA-damaging drugs such doxorubicin and cisplatin.

We conclude that CSA may act on breast cancer cells by targeting multiple tumorigenic pathways leading to cell cycle arrest and apoptosis. Our data indicate that CSA possesses therapeutic potential against breast cancer. Further predinical and clinical studies are warranted to clarify the therapeutic potential of CSA.


BCIP/NBT    5-bromo-4-chloro-3'-indolyphosphate
            p-toluidine salt/nitro-blue tetrazolium chloride
BRCA 1/2    breast cancer resistance genes 1/2
BSA         bovine serum albumine
CSA         cajanin stilbene acid
DAPI        4',6-diamidine-2-phenylindole
DMEM        dulbecco's minimal essential medium
DMSO        dimethyl sulfoxide
EGFR        epidermal growth factor receptor
ER          estrogen receptor
HER2        human epidermal growth factor receptor 2
MMP         mitochondrial membrane potential
PBS         phosphate buffered saline
PI          propidium iodide
PMSF        phenylmethanesulfonyl fluoride
PR          progesterone receptor
R123        rhodamine 123
SDS         sodium dodecyl sulfate
TBST        tris-buffered saline/Tween 20


Article history:

Received 19 February 2015

Accepted 26 February 2015

Conflict of interest

The authors declare that they have no conflict of interest.


We gratefully acknowledge the financial support from Special Fund of Forestry Industrial Research for Public Welfare of China (201004040), Importation of International Advanced Forestry Science and Technology, National Forestry Bureau (2012-4-06), Heilongjiang Province Science Foundation for Excellent Youths (JC200704) and Project for Distinguished Teacher Abroad, Chinese Ministry of Education (MS2010DBLY031).


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Yujie Fu (a,b,1), Onat Kadioglu (c,1), Benjamin Wiench (c), Zuofu Wei (a,b), Chang Gao (d), Meng Luo (a,b), Chengbo Gu (a,b), Yuangang Zu (a,b), Thomas Efferth (c), *

(a) Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China

(b) Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin, China

(c) Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry. Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany

(d) Peking University People's Hospital, Beijing 100044, China

* Corresponding author. Tel.: +49 6131 3925751; fax: +49 6131 23752.

E-mail address: (T. Efferth).

(1) Both authors contributed equally to this work.

Table 1
Primer nucleotide sequences, concentrations and annealing

Gene    Sequence                           Concentration  Annealing
                                           (nM)           temperature

BRCA-1  Fw: 5'-TCAATGGAAGAAACCACCAAGGT-3'  250            59
BRCA-2  Fw: 5'-GTTTGTGAACCGTCGTCACA-3'     250            59
P21     Fw: 5'-GCGATGGAACrrCGACTTTGT-3'    250            59
RPS13   Fw: 5'-GGTTGAAGTTGACATCTGACGA-3'   250            59

Table 2
Validation of microarray-based mRNA expression by
quantitative real-time RT-PCR.

Gene         Method         Fold change

BRCA-1       Microarray     -2.99
             RT-PCR         -4.93
BRCA-2       Microarray     -3.36
             RT-PCR         -4.91
P21          Microarray      5.22
             RT-PCR          1.66

mRNA expression values from microarray hybridization and
real-time RT-PCR were significantly correlated (R = 0.99;
p = 0.013; Pearson correlation test).
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Author:Fu, Yujie; Kadioglu, Onat; Wiench, Benjamin; Wei, Zuofu; Gao, Chang; Luo, Meng; Gu, Chengbo; Zu, Yua
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9JAPA
Date:Apr 15, 2015
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