Chamaejasmine Arrests Cell Cycle, Induces Apoptosis and Inhibits Nuclear NF-κB Translocation in the Human Breast Cancer Cell Line MDA-MB-231

In this study, the anticancer activity of chamaejasmine was characterized in the human breast cancer cell line, MDA-MB-231. Cell viability and cell cycle distribution were determined by MTT assay and flow cytometry, respectively. Western blotting was performed to determine changes in levels of various proteins. Results showed that treatment with chamaejasmine (4–16 μM) inhibited cell proliferation, which correlated with G2/M phase arrest and apoptosis in MDA-MB-231 cells. Chamaejasmine treatment of MDA-MB-231 cells resulted in induction of WAF1/p21 and KIP1/p27, decrease in cyclins A and cyclins B1. Cyclin-dependent kinase (cdk) 2 and cdc2 was also decreased after chamaejasmine treatment. Moreover, inhibition of nuclear translocation, phosphorylation of NF-κB, activation of IKKα and IKKβ, inhibition of phosphorylation and degradation of IκBα were also detected in this work. Our findings suggested that chamaejasmine could be explored as a preventive and perhaps as a chemotherapeutic agent in the management of breast cancer.

. Chemical structure of chamaejasmine and apigenin.
In the present study, the antiproliferation activity of chamaejasmine against three human breast cancer cell lines (HCC1937, MDA-MB-453 and MDA-MB-231) was evaluated by MTT assay first. The cell cycle arrest and apoptosis was further studied by flow cytometry. The expression of p21, p27, cdk2, cdc2, cyclin A and cyclin B1 was further detected by western blotting in MDA-MB-231 cells. Measurements of Bcl-2, Bax, caspase-3 and capspase-8 were used to assess apoptosis. Finally, we determined the chemotherapeutic potential of chamaejasmine on phosphorylation and activation of NF-κB in MDA-MB-231 cells.

Heading Cytotoxicity Assays
The cytotoxicity of chamaejasmine was evaluated on three human breast cancer cell lines (HCC1937, MDA-MB-453 and MDA-MB-231) using MTT assays. Apigenin was used as positive control. The results were listed in Table 1. Chamaejasmine exhibited stronger inhibition against all three cancer cell lines than apigenin. Among all of them, chamaejasmine showed more notable cytotoxicity against MDA-MB-231 than HCC1937 and MDA-MB-453, with IC 50 values of 4.72, 13.44 and 5.66 μM, respectively.

G2-M Phase Cell Cycle Arrest and Apoptosis by Chamaejasmine in MDA-MB-231 Cells
To determine whether chamaejasmine-induced apoptosis was related to arrest of cell cycle progression, flow cytometry was used to quantitate the cell cycle distribution in MDA-MB-231 cells under treatment with different chamaejasmine concentrations (4-16 μM). As shown in the concentration kinetic measurements (Figure 2), exposure to 4-16 μM chamaejasmine caused an increase of the G2/M phase population from 19.86% to 66.55%, as compared to 16.61% of G2/M phase cells in untreated control samples (p < 0.05). Hence, chamaejasmine exerted growth-inhibitory effects via G2/M phase arrest in a concentration-dependent manner.
The annexin V-FITC apoptosis detection kit was then employed to examine the influence of chamaejasmine on MDA-MB-231 cells apoptosis by flow cytometry. As shown in Figure 3, only a few untreated MDA-MB-231 (1.64%) cells bounded annexin V-FITC. Whereas, MDA-MB-231 cells binded annexin V-FITC highly increased in a concentration-dependent manner after treatment with 4-16 μM chamaejasmine (13.06% to 78.05%, p < 0.05). To sum up, dots were dispersed and shifted to the Q2 side in a dose-dependent manner when MDA-MB-231 cells were treated with chamaejasmine, indicating that the cells moved to the late apoptotic stage. These experimental results demonstrate that chamaejasmine induced apoptosis of MDA-MB-231 cells.

Inhibition of Cyclins, Cdk2, cdc2 and Induction of WAF1/p21 and KIP1/p27 by Chamaejasmine in MDA-MB-231 Cells
Many reports have revealed that cell cycle regulators are frequently mutated in most common malignancies [21,22]. Thus, we examined the effects of chamaejasmine on cell cycle inhibitory proteins KIP1/p27 and WAF1/p21, which are involved in cell cycle progression. Western blotting analysis showed a significant induction of these proteins in a dose-dependent manner ( Figure 4A). The effects of chamaejasmine on the proteins levels of cyclins, cdk2 and cdc2 (which are known to be regulated by KIP1/p27 and WAF1/p21) were next evaluated. Chamaejasmine treatment of cells resulted in a significant dose-dependent decrease in the protein levels of cyclin A and B1 as well as cdk2 and cdc2 ( Figure 4B,C). These results suggested that chamaejasmine restored proper checkpoint control via modulation of the cyclins, cdk2, cdc2 and the expression of their inhibitors.

Induction of Bax and Inhibition of Bcl-2 and Procaspases in MDA-MB-231 Cells
In order to investigate the mechanism by which chamaejasmine induces apoptosis, the changes in the level of apoptosis-related proteins (Bax, Bcl-2, caspase-3 and caspase-8) were examined ( Figure 5A,B). As shown in Figure 5A, western blotting analysis revealed a significant increase in the expression of Bax in chamaejasmine treated MDA-MB-231 cells, while there was a significant decrease in Bcl-2 expression, indicating that the Bax/Bcl-2 ratio increased significantly. Pro-caspase-3 levels decreased upon treatment with chamaejasmine, while the levels of active caspase-3 increased. Similarly, pro-caspase-8 levels decreased upon treatment with chamaejasmine which induced increase active caspase-8 ( Figure 5B).

Inhibition of NF-κB Pathway by chamaejasmine in MDA-MB-231 Cells
Treatment of MDA-MB-231 cells with chamaejasmine (4-16 μM) resulted in a significant inhibition in the phosphorylation of IκBα protein ( Figure 5C). To evaluate the possible inhibitory mechanism of chamaejasmine on IκBα protein increase, IKKα and IKKβ protein level was then measured. Western blotting analysis showed that pre-treatment of MDA-MB-231 cells with chamaejasmine inhibited IKKα and IKKβ in a dose-dependent manner ( Figure 5C). Furthermore, chamaejasmine treatment cells resulted in decreased phospho-NF-κB/p65 at Ser536 and inhibition of translocation of NF-κB/p65 in the nuclear fraction ( Figures 5D and 6).

Discussion
Recently, the antitumor activity of constituents obtained from Stellerachamaejasme L. has become a research hotspot. For example, chamaejasmine showed notable anticancer activity against HEp-2, NCI-H1975, HT-29 and SKOV-3. MCF-7, A549, SGC-7901, HCT [25]. Besides, chamaejasmine exhibited strong inhibition against all three human breast cancer cell lines (HCC1937, MDA-MB-453 and MDA-MB-231). Based on all above, it seemed that the presence of hydroxyl group may contribute to the obvious anticancer activity of chamaejasmine. The antitumor activity of biflavanones or their structural derivatives need to be further evaluated in cancer cell lines.
Several studies have shown that the induction of apoptosis might be due to cell cycle arrest [26,27]. Cell cycle control is a major regulatory mechanism of cell growth [28]. Blockade of the cell cycle is considered as an effective strategy for the development of novel cancer therapies [29,30]. Cell cycle analysis of the treated culture revealed that chamaejasmine induced a concentration-dependent G2/M phase cell cycle arrest with an accompaniment decrease in G1 and S phase.
It is known that cell cycle is primarily regulated by complexes containing cdks and cyclins, which are critical for the progression of cell cycle and whose inactivation leads to cell cycle arrest [31,32]. Cdk activity is additionally regulated by cdk inhibitors such as the WAF1/p21 and KIP1/p27 proteins families. Among Cdks that regulate cell cycle progression, Cdk2 and Cdc2 kinases are primarily activated in association with cyclin A and cyclin B1 during the progression of the G2/M phase [33,34]. Thus, our data suggest that cell cycle arrest at the G2/M phase is mediated by reduction of cyclin A and cdc2/cyclin B complex formation, which is an essential step in regulating the cells passage into mitosis. It could be conclude the cell cycle arrest may partly explain apoptosis and anti-proliferative effects induced by chamaejasmine.
Bcl-2, an upstream effect or molecule in the apoptotic pathway, has been identified as a potent suppressor of apoptosis [35]. It has been proven that most cancers, including lung cancer, generally overexpress Bcl-2, thereby escaping apoptosis and undermining therapy. Bcl-2 forms a heterodimer with the apoptotic protein Bax and thereby neutralizes its apoptotic effect. Therefore, alteration in the ratio of Bax/Bcl2 is a decisive factor that plays an important role to determine whether cells will undergo apoptosis [36,37]. Based on the above results, chamaejasmine significantly down-regulated Bcl-2 protein and up-regulated levels of Bax protein in MDA-MB-231 cells, suggesting the involvement of an intrinsic apoptotic pathway by which chamaejasmine induces apoptosis in MDA-MB-231 cells.
Caspases are part of a growing family of cysteine proteases which have been involved in many forms of apoptosis [38][39][40]. Activation of caspase proteases was required for the induction of apoptosis in different cell types [41,42]. Caspases include initiator caspases and effector caspases. Once the initiator caspases (caspases 8, 9, and 10, etc) are activated through intrinsic or extrinsic pathway, they are proteolytically cleaved and thus activate the effector caspases (caspases 3, 6, and 7, etc.) whose functions are known to be responsible for the cleavage of the intracellular substrates that leads to cell death. Caspase 3 is one of the key executioners of apoptosis. Upon activation, caspase 3 can cleave 5 substrates, including other effector caspases and fodrin, which form a cytoskeletal network [43]. The activated caspase-3 and caspase-8 detected in the results further explained the signaling pathway of chamaejasmine-induced apoptosis in MDA-MB-231 cells.
Activation of NF-κB has been confirmed to block apoptosis and promote cell proliferation, and its increased activity is positively associated with many cancer types, including breast cancer [44]. Inhibition of tumorigenesis often involves modulation of signal transduction pathways, leading to cell cycle arrest and apoptosis. NF-κB is sequestered in the cytoplasm in an inactive form through interaction with IκB. Phosphorylation of IκB by IκB kinase (IKK) causes ubiquitination and degradation of IκB, thus releasing NF-κB, which then translocates to the nucleus. Then it binds to specific B binding sites in the promoter regions of several genes [45]. Our results showed that treatment of chamaejasmine in MDA-MB-231 cells significantly inhibited IKKα and IKKβ as well as phosphorylation and degradation of IκBα protein. This suggested that the effects of chamaejasmine on NF-κB/p65 are through inhibition of phosphorylation and subsequent proteolysis of IκBα. NF-κB is a ubiquitous transcription factor that controls the expression of genes involved in immune responses, apoptosis, and cell cycle. It has been shown to modulate the expression of cyclin B1 [46,47]. Moreover, NF-κB plays important role in p53-mediated apoptosis. P53 can induce activation of NF-kB, and loss of NF-kB activity specially abrogated the p53-mediated apoptotic response, without impinging on the ability to activate expression of target genes or induce cell-cycle arrest [48].

Cytotoxicity Assay
Inhibition of cell proliferation of chamaejasmine was measured by the MTT assay [49]. Briefly, cells were plated in 96-well culture plates (1 × 10 5 cells/well) separately. After 24 h incubation, cells were treated with chamaejasmine or apigenin (1,2,4,8,16,32 and 64 μM, eight wells per concentration) for 72 h, MTT solution (5 mg/mL) was then added to each well. After 4 h incubation, the formazan precipitate was dissolved in dimethyl sulfoxide (100 μL) and then the absorbance was measured in an ELISA reader (Thermo Molecular Devices Co., Union City, CA, USA) at 570 nm. The cell viability ratio was calculated by the following formula: Inhibitory ratio (%) = [(OD control − OD treated )/(OD control )] × 100%. Cytotoxicity was expressed as the concentration of chamaejasmine inhibiting cell growth by 50% (IC 50 value).

Flow Cytometric Analysis of Cell Cycle and Apoptosis
Cell cycle was studied with CyStain (Partec GmbH, Görlitz, Germany) [49]. Briefly, 1 × 10 6 cells/well MDA-MB-231 cells were seeded in six-well plate and left for 24 h in incubator to resume exponential growth. Cells were exposed to chamaejasmine (0, 4, 8 and 16 μM) and incubated for 48 h. Then, the cells were harvested and washed with PBS. After suspension in PBS (800 μL) and CyStain (200 μL) the cell cycle distribution of 10,000 cells was recorded by flow cytometry (Partec), and the percentage of cells at G0/G1, S, and G2/M phases was analyzed using the FloMax software (Partec).
The extent of apoptosis was measured through annexinV-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, Jiangsu, China) as described by the manufacture's instruction [49].

Western Blotting Assay
To evaluate the expression levels of various intracellular proteins related to apoptosis, MDA-MB-231 cells were treated with chamaejasmine (0, 4, 8 and 16 μM) for 48 h, respectively. For isolation of total protein fractions, cells were collected, washed twice with ice-cold PBS, and lysed using cell lysis buffer [20 mMTris pH 7.5, 150 mMNaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% Na 2 CO 3 , 0.5 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The lysates were collected by scraping from the plates and then centrifuged at 10,000 rpm at 4 °C for 5 min. Total protein samples (20 μg) were loaded on a 12% of SDS polyacrylamide gel for electrophoresis, and transferred onto PVDF transfer membranes (Millipore, Billerica, MA, USA) at 0.8 mA/cm 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 incubated overnight at 4 °C with primary antibodies at a 1:1,000 dilution in blocking solution. After thrice washings in TBST for each 5 min, membranes were incubated for 1 h at room temperature with an alkaline phosphatase peroxidase conjugated anti-mouse secondary antibody at a dilution of 1:500 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 recorded by a digital camera (Nikon, Tokyo, Japan).

Immunofluorescence Assay
The cells were grown at a density of 2 × 10 4 /well on 6-well culture plates (Nunc Inc., Naperville, IL, USA). After the treatments, the cells were washed with PBS and fixed with cold methanol for 5 min at −20 °C. The cells were incubated with a 1:100 dilution of anti-p65 antibody, followed by probing with a 1:800 dilution of Alexa Fluor 488-conjugated goat anti-rabbit IgG. The cells were examined with a Zeiss Axiovert 200M microscope, and data were analyzed using Carl Zeiss Axiovision software (Carl Zeiss Instruments, Jena, Germany).

Statistical Analysis
The data were expressed as mean ± S.D. All statistics were calculated using the STATISTICA program (StatSoft, Tulsa, OK, USA). A p-value of <0.05 was considered as significant.

Conclusions
In summary, the present study showed that chamaejasmine inhibited the growth of MDA-MB-231 cells resulted from cell cycle arrest at G2/M phase, accompanied by cell apoptosis. Chamaejasmine induced cell cycle arrest through cyclinA, cyclinB1, cdk2 and cdc2 inhibition. These events were found to be associated with alterations in the levels of Bax, Bcl-2, caspase-8 and caspase-3. Our results further showed that the effects of chamaejasmine on NF-κB/p65 are through inhibition of phosphorylation and subsequent proteolysis of IκBα. All these evidences provide a rationale to explore chamaejasmine as a preventive and perhaps as a chemotherapeutic agent in the management of breast cancer.