In vitro and in vivo antitumour activities of puerarin 6″-O-xyloside on human lung carcinoma A549 cell line via the induction of the mitochondria-mediated apoptosis pathway

Abstract

Context Pueraria lobata (Leguminoseae) shows cytotoxic effects against cancer cells; however, its active components remain unclear.

Objective This study investigated the antitumour activity of puerarin 6″-O-xyloside (POS) on the human lung carcinoma A549 cell line.

Materials and methods The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxicity of POS (at 10, 20 and 40 μM) in vitro, and xenograft nude mice were established to evaluate the antitumour effect of POS (at 40 mg/kg/d) in vivo by 15 days intraperitoneal injection (ip). To explore its mechanism of action, flow cytometry was performed to determine the pro-apoptotic effect of POS (at 10, 20 and 40 μM). Subsequently, the expression of caspase-3, caspase-7, caspase-9, Bcl-2 and Bax in A549 cells were determined.

Results POS showed significant cytotoxicity toward A549 cells (p < 0.05) by inducing apoptosis. Treatment with POS significantly upregulated the levels of caspase-3 (p < 0.01), caspase-7 (p < 0.01), caspase-9 (p < 0.01) and Bax (p < 0.01) in A549 cells, and Bcl-2 was downregulated (p < 0.01). Additionally, the in vivo animal study showed that POS significantly inhibited tumour growth in A549 cells (p < 0.01).

Conclusion Our study demonstrated the POS has significant antitumour activities. The mechanisms are related to increased levels of caspase-3, caspase-7, caspase-9 and Bax, and reduced levels Bcl-2.

Keywords:

• Bcl-2
• caspase
• Pueraria lobata
• xenograft model in nude mice

Introduction

In recent years, lung cancer has been the leading cause of cancer-related deaths worldwide. In addition, it is reported that non-small cell lung cancers (NSCLC) represent more than 85% of the total incidence of lung cancer (Ko et al. 2010; Zhao et al. 2011; Siegel et al. 2015). Although the diagnosis and the treatment of lung cancers have improved greatly, the currently 5-year survival rate of lung cancer patients is still poor (Liu et al. 2007; Yang et al. 2014). Consequently, identifying novel strategies to cure lung cancer is important and urgent.

Previous investigations have focused on the possible capacities of natural agents derived from plants to prevent and treat lung cancers. The study of the medical uses of plants or herbs may provide useful clues for new effective drug discovery (Li & Vederas 2009; Skermana et al. 2011; Peng et al. 2014; Yang et al. 2014). Furthermore, previous investigations demonstrated that the extracts of the radix of Pueraria lobata (Willd.) Ohwi (Leguminoseae), a famous traditional Chinese medicine, possessed notable cytotoxic effect on cancer cells (Du et al. 1994; Chen & Hu 1997; Du et al. 1997; Yuan et al. 2007). However, there is little information regarding the active constituents in P. lobata that are responsible for its cytotoxic activity. In the present study, we determined the antitumour activity of puerarin 6″-O-xyloside (POS) (Figure 1), which is one of the major isoflavones of P. lobata. In addition, the mechanisms of action of its antitumour activity were also investigated.

Figure 1. The chemical structure of puerarin 6″-O-xyloside (POS).

Materials and methods

Chemicals and regents

Puerarin 6″-O-xyloside was obtained from the Shanghai Tauto Biotech Ltd. Co. (Shanghai, China), and the purity was 98%, as determined by HPLC; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulphoxide were purchased from Sigma (St. Louis, MO). RPMI 1640 media and foetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA); dimethyl benzene was purchased from Sino Pharm. (Chengdu, China); Annexin V-FITC/PI kit was purchased from BD Bioscience (San Diego, CA); cytochrome c, cleaved-caspase-3 (c-caspase-3), cleaved-caspase-7 (c-caspase-7) and cleaved-caspase-9 (c-caspase-9) antibodies were purchased from Cell Signaling Technology (Beverly, MA); Bcl-2, Bcl-xl, Bax, COX IV and β-actin antibodies were purchased from Abcam Biotechnology (Cambridge, MA); and the BCA protein assay reagent was purchased from Beyotime (Haimen, China).

Animals and ethics statement

Experimental groups consisted of Institute for Cancer Research (ICR) mice (4–5 weeks old) or BALB/C nude mice (5–6 weeks old) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). All animal treatments were strictly in accordance with International Ethical Guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals. The experimental protocols were carried out with approval of the Animal Experimentation Ethics Committee of the Changshu No. 1 People’s Hospital (No. cKY20140201).

Cell culture and determination of cell viability

The A549 cell line was purchased from the American Type Culture Collection (Manassas, MD). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS. The cell lines were cultured in 5% CO₂/95% air at 37 °C.

Cell viability was assessed using the MTT assay, as previously described (Wu et al. 2014). Cells (5 × 10⁵ cells/mL) were plated to adhere in 96-well plates and cultured for 24 h. Then, POS (10, 20 and 40 μM) was added to the wells, and cells were cultured for 32 h. Subsequently, the absorbance (OD value) was read at 570 nm by a microplate reader (Bio-rad, Hercules, CA) to determine the percentage of cell proliferation inhibition. The inhibition rate was calculated accor-ding to the following formula: [(OD_control–OD_treatment)/OD_control] × 100%.

Apoptosis assay by flow cytometry

Apoptotic cells were identified by staining with fluorescein isothiocyanate (FITC)-conjugated Annexin V/PI by flow cytometry, according to the instructions of a commercial kit. The percentage of cells in early apoptosis was calculated by Annexin V-positivity and PI-negativity, while the percentage of cells in late apoptosis was calculated by Annexin V-positivity and PI-positivity.

Western blotting

Total proteins of the cells were extracted and the protein concentration was determined by using the BCA protein assay reagent. Equal amounts of proteins (35 μg) were separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE), blotted onto PVDF membranes and probed with various primary antibodies, followed by incubation with horseradish-peroxidase-conjugated secondary antibodies and chemiluminescence detection. To normalize the protein loading, antibodies directed against β-actin/COX IV were used, and the proteins expression levels were expressed as a relative value to that of β-actin/COX IV.

Xenograft model in nude mice

Nude mice were injected in the right flank subcutaneously with A549 cells (2 × 10⁶ cells per mouse). When the tumours grew to approximately 2–3 mm in diameter, the mice were treated with POS (40 mg/kg/d, intraperitoneal injection, ip) or an equal volume of solvent control (0.5% DMSO, ip) as a control, respectively. Mice of the two groups (n = 10) were observed for 15 d, and tumour sizes were measured every 5 d. Tumour diameters were determined by using a vernier caliper, and the tumour volumes were calculated according to the formula: volume = (width² × length)/2 (He et al. 2009; Zhou et al. 2013).

Statistical analysis

The significance of the differences between the different groups was determined using Student’s t-test. The results are presented as the mean ± SD, and the differences were considered significant at p < 0.05.

Results

Anti-proliferative effect of POS on A549 cells in vitro

The anti-proliferative activity of POS in vitro was using human lung cancer cell line A549, a classical NSCLC cell line. POS showed obvious anti-proliferative effect on A549 cancer cells (p < 0.05, p < 0.01 and p < 0.01 for 10, 20 and 40 μM POS, respectively), with a significant concentration dependence (Figure 2).

Figure 2. Anti-proliferative effect of puerarin 6″-O-xyloside (POS) on A549 cells in vitro. A549 cell lines were treated with POS (10, 20 and 40 μM) for 32 h, and cell proliferation was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The data are represented as the mean ± SD (n = 4). *p < 0.05 and **p < 0.01 versus control.

Pro-apoptotic effect of POS on A549 cells

Cytotoxicity assays demonstrated that POS had notable antitumour activity against human lung cancer A549 cells. To determine whether the antitumour activity of POS resulted from apoptosis, A549 cells were further analyzed by staining with Annexin V-FITC/PI followed by flow cytometry (Figure 3). The results indicated that apoptotic cells increased gradually when treated with increasing concentrations of POS (10, 20 and 40 μM). Thus, POS induced death of human A549 lung cancer cells by inducing apoptosis.

Figure 3. Flow cytometric analysis of A549 cells apoptosis following exposure to puerarin 6″-O-xyloside (POS). A549 cell lines were treated POS (10, 20 and 40 μM) for 32 h. The flow cytometry assay was performed to determine the apoptosis rate by Annexin-V/PI double-staining. (A–D) The control, 10, 20 and 40 μM groups.

Exposure of A549 cells to POS results in the increase of cytochrome c release in cytoplasm

As can be seen from Figure 4, after cells were exposed to the POS (10, 20 and 40 μM), the release of cytochrome c into the cytosol from the mitochondria was significantly increased in a concentration-dependent manner (p < 0.05, p < 0.01 and p < 0.01, for the 10, 20 and 40 μM, respectively); whereas the cytochrome c levels in the mitochondria decreased (p < 0.05, p < 0.01 and p < 0.01, for the 10, 20 and 40 μM, respectively).

Figure 4. Effects of puerarin 6″-O-xyloside (POS) on the expressions of cytochrome c (cytoplasm and mitochondria) in A549 cells. Cytochrome c (C) and cytochrome c (M) represent the expressions of cytochrome c in the cytoplasm and mitochondria, respectively. A549 cell lines were treated POS (10, 20 and 40 μM) for 32 h, and the levels of the different proteins were measured by western blotting. The data are represented as mean ± SD (n = 4). *p < 0.05 and **p < 0.01 versus control.

Exposure of A549 cells to POS results in the upregulation of C-caspase-3, C-caspase-7, C-caspase-9, bax and downregulation of Bcl-2

As shown in Figures 5 and 6, compared with the control group, POS (10, 20 and 40 μM) significantly increased the protein levels of caspase-3 (p < 0.05), caspase-7 (p < 0.05), caspase-9 (p < 0.05) and Bax (p < 0.05), in a concentration-dependent manner; whereas Bcl-2 was obviously decreased (p < 0.01), in a concentration-dependent manner.

Figure 5. Effects of puerarin 6″-O-xyloside (POS) on the expressions of cleaved-caspase-3 (C-capase-3), cleaved-caspase-7 (C-capase-7) and cleaved-caspase-9 (C-caspase-9) in A549 cells. A549 cell lines were treated POS (10, 20 and 40 μM) for 32 h, and the levels of the different proteins were measured by western blotting. The data are represented as the mean ± SD (n = 4). *p < 0.05 and **p < 0.01 versus control.

Figure 6. Effects of puerarin 6″-O-xyloside (POS) on the expressions of Bax and Bcl-2 in A549 cells. A549 cell lines were treated with POS (10, 20 and 40 μM) for 32 h, and the levels of the different proteins were measured by western blotting. The data are represented as the mean ± SD (n = 4). *p < 0.05 and **p < 0.01 versus control.

Antitumour activity of POS in vivo

These results from the experiments in vitro indicated that POS possessed cytotoxic effects on A549 cells via inducing mitochondria-mediated apoptosis. Therefore, we studied the antitumour effect of POS in vivo using a transplanted tumour nude mouse model. To achieve better bioavailability, POS was delivered by intraperitoneal injection (i.p.). The results demonstrated that exposure of mice to POS (40 mg/kg) significant suppressed tumour growth during a 15 d observation period (p < 0.05), compared with the control group (Figure 7A). No significant difference in body weight was observed between the POS and control mice (p > 0.05) (Figure 7B).

Figure 7. Antitumour activity of puerarin 6″-O-xyloside (POS) on A549 tumour-bearing nude mice. (A) Effect of POS on tumour volumes; (B) effect of POS on the weights of A549 tumour-bearing nude mice. The data are represented as the mean ± SD (n = 10). *p < 0.05 and **p < 0.01 versus control.

Discussion

In recent years, increasing evidence has demonstrated that uncontrolled cell proliferation and insufficient apoptosis would eventually lead to cancer (Wu et al. 2014). Apoptosis, known as a programmed cell death, is an active process of cell death, the promotion of which is considered as an ideal method of cancer therapy (Yang et al. 2007; Chipuk et al. 2012). Apoptosis is characterized by distinct morphological changes and biochemical hallmarks, including cell shrinkage, nuclear DNA fragmentation, apoptotic bodies and membrane blebbing, and is regulated by a series of biochemical events that eventually result in cell death (Gao et al. 2009). Mitochondria-mediated apoptosis is a major apoptotic pathway induced by the release of cytochrome c into the cytoplasm by mitochondrial outer membrane permeabilization, which activates caspases proteins (Chipuk et al. 2012; Xiong et al. 2014). Caspases (cysteine-aspartic acid proteases) are key mediators of apoptosis, and the activation of caspase-3 is a biomarker for cells undergoing apoptosis. Caspase-9 is the initiating caspase protein in the caspase cascade, and caspase-3, the crucial activated death proteases, is activated by caspase-9 (Cohen 1997; Li et al. 2004; Gao et al. 2009). The Bcl-2 family is another species of proteins that play a crucial role in the mitochondria-mediated intrinsic apoptosis pathway, and is considered to be the first regulatory step in mitochondrial apoptosis (Kuwana & Newmeyer 2003; Xiong et al. 2014). In addition, the Bcl-2 family proteins are commonly divided into two types with opposing effects on the regulation of mitochondrial apoptosis. Bax is the pro-apoptotic protein, whereas Bcl-2 is anti-apoptotic protein (Oltvai et al. 1993; Antonsson, 2001). Our results revealed that POS has significant cytotoxic activity toward human A549 lung cancer cells, which involves the induction of apoptosis. Our results showed that POS induces apoptosis in A549 cells by altering the levels of proteins related to mitochondria-mediated intrinsic apoptosis. Thus, exposure of A549 cells to POS led to a significant increase in the levels of C-caspase-3, C-caspase-7, C-caspase-9 and Bax, whereas the level of Bcl-2 significantly decreased. These findings indicated that POS induced mitochondria-mediated intrinsic apoptosis in the human lung cancer cell line A549. In addition, the in vivo experiments were consistent with the results obtained in vitro, which suggested that POS has antitumour activity toward the human lung cancer A549.

In conclusion, the present study demonstrated that POS has significant antitumour activities in vitro and in vivo. The mechanisms of its antitumour activities involve induction of mitochondria-mediated apoptosis via increased abundances of caspase-3, caspase-7, caspase-9, Bax and reduced levels of Bcl-2. Thus, the results suggested that POS could be regarded as a new candidate to treat lung cancer.

Declaration of interest

The authors report that they have no conflicts of interest.