Vitexin induces apoptosis through mitochondrial pathway and PI3K/Akt/mTOR signaling in human non-small cell lung cancer A549 cells

Background Currently, the prognosis of patients with non-small cell lung cancer (NSCLC) remains dismal; hence, it is critical to identify effective anti-NSCLC agents with limited side effects. This study aimed to evaluate the therapeutic potential of flavonoid compound vitexin in human NSCLC cells and the underlying mechanisms. Results The experimental results indicated that vitexin reduced the viability of A549 cells in a dose-dependent manner with nearly no toxicity against normal human bronchial epithelial 16HBE cells. Vitexin also dose-dependently increased A549 cell apoptosis, accompanied by the decreased Bcl-2/Bax ratio and the increased expression of cleaved caspase-3. Moreover, the in vivo anticancer activity of vitexin was further determined in nude mice bearing A549 cells. In addition, vitexin induced the release of cytochrome c from the mitochondria to the cytosol and the loss of mitochondrial membrane potential. Vitexin also significantly reduced the levels of p-PI3K, p-Akt and p-mTOR, and the pro-apoptotic effect of vitexin on A549 cells was partly blocked by SC79, an Akt activator. Conclusions Accordingly, we believed that vitexin could be used as a potential therapeutic agent for the treatment of NSCLC in the future.


Introduction
Lung cancer is the leading cause of cancer-related mortality in China [1]. There are two major types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of all lung cancer cases [2]. The mechanisms underlying the pathogenesis of NSCLC are complicated. Conventional therapeutic options for NSCLC in clinics include chemotherapy and surgery, but these methods exert limited effects for patients with advanced NSCLC [3]. Undoubtedly, it is of critical importance to identify novel therapeutic agents for the treatment of this fatal malignancy.
Recently, natural products, especially plant-derived compounds, have attracted the attention of many researchers for their potential antitumor properties. Among them, vitexin (apigenin-8-C-D-glucopyranoside; Fig. 1a), a naturally-derived flavonoid compound found in the traditional Chinese herb Crataegus pinnatifida (hawthorn) [4], has shown anti-tumor efficacy against a wide variety of human cancers, including leukemia [5], hepatocellular carcinoma [6] and glioblastoma [7]. Therefore, the objectives of the present study were to characterize the anti-NSCLC role of vitexin both in vitro and in vivo, and to clarify the underlying molecular mechanisms.

Cell culture and treatment
NSCLC cell line (A549) and normal human bronchial epithelial cell line (16HBE) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). These cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO 2 in air.

MTT assay
Cell viability was monitored by 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, cells were seeded into 96-well plates at a density of 5 × 10 3 cells per well. Following treatment with different doses of vitexin for 48 h, 20 μl MTT (5 mg/ml; Sigma-Aldrich) was added to each well, and the cells were incubated for additional 4 h at 37 °C. Formazan cyrstals that formed in living cells was dissolved in 150 μl of DMSO, and the absorbance of the plate was then read with a microplate reader (Dynex, Chantilly, VA, USA) at 490 nm.

LDH release assay
Cell injury was determined based on lactate dehydrogenase (LDH) leakage into the culture medium from cells using an LDH assay kit (Jiancheng, Nanjing, China) [8]. Following vitexin treatment for 48 h, 100 μl of working solution was added to each well and the plate was incubated for additional 30 min. Then 50 μl stop solution was added to each well, and the absorbance of all samples was detected at 490 nm with a microplate reader.

Cell apoptosis analysis
Cell apoptosis was determined using an Annexin V-FITC/PI apoptosis detection kit (BestBio, Shanghai, China). In brief, cells were harvested after 48 h of the aforementioned treatment by trypsinization and then double stained with Annexin V-FITC and propidium iodide (PI) for 30 min in the dark. The samples were then analyzed using a FACSCaliber flow cytometer (BD Biosciences).

Measurement of mitochondrial membrane potential
The mitochondrial membrane potential (MMP) of cells was determined by the classical JC-1 staining method [9]. Briefly, following vitexin treatment, cells were harvested, washed with PBS twice and then incubated with 500 μl JC-1 staining solution (5 μg/ml) for 20 min at 37 °C in darkness. Next, the cells were suspended with trypsin, and analyzed using a flow cytometer.

Tumor formation assay
Fifteen male athymic BALB/c nude mice aged 5-6 weeks were purchased from Shanghai Laboratory Animals Center (Shanghai, China) and maintained under SPF conditions. 2 × 10 6 A549 cells were subcutaneously injected into a single side of the posterior flank of nude mice. Tumor volume was measured using a caliper every 3 days and calculated as follows: Tumor volume (mm 3 ) = length × width 2 /2. When the tumor size reached approximately 100 mm 3 , the mice were randomized into three groups (five mice/group). The mice in low dose group and high dose group were treated daily for 4 weeks by intraperitoneal injection with 1 mg/kg and 2 mg/kg vitexin, respectively, and the mice in control group received 0.1% DMSO. At the end of the study (Day 19), the tumors were excised and weighed. All animal handling and procedures were approved by the Ethics Committee of Affiliated Cancer Hospital of Zhengzhou University (Zhengzhou, China). All necessary steps were taken to minimize suffering of the mice.

Statistical analysis
All statistical analyses were performed using Graph-Pad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA). All experimental data are shown as the mean ± standard deviation (SD) and analyzed using oneway analysis of variance (ANOVA) and Dunnett's post hoc test. P < 0.05 was considered to indicate a statistically significant difference.

Vitexin reduces viability in A549 cells
First, A549 cells were treated with different doses of vitexin, and the inhibitory effect of vitexin on cell viability was estimated by MTT assay. As demonstrated in Fig. 1b, exposure of A549 cells to vitexin for 48 h led to a dosedependent reduction in cell viability. However, vitexin exerts nearly no toxicity against normal human bronchial epithelial 16HBE cells (Fig. 1c). In addition, we also found that vitexin treatment remarkably increased the LDH leakage of A549 cells (Fig. 1d).

Vitexin induces apoptosis in A549 cells
To determine whether vitexin exert a pro-apoptotic effect on NSCLC cells, flow cytometry analysis via Annexin V/ PI staining was performed. As shown in Fig. 2a, vitexin treatment dose-dependently increased the number of Annexin V-positive A549 cells. Next, we investigated the expression levels of several apoptosis-associated proteins by western blot analysis, and the results indicated that vitexin treatment led to the downregultion of Bcl-2/Bax ratio and upregulation of cleaved caspase-3 in A549 cells (Fig. 2b).

Vitexin inhibits NSCLC tumor growth in vivo
We also analyzed the anti-NSCLC potential of vitexin in vivo. We confirmed that all mice developed xenograft tumors at the injection sites, and as shown in   Fig. 3a, vitexin treatment led to significant inhibition of NSCLC tumor growth. The average weight of tumors was also significantly reduced following vitexin treatment (Fig. 3b). Moreover, we found that Bcl-2 expression was decreased, whereas the expression levels of Bax and cleaved caspase-3 were increased in the tumor tissues of vitexin-treated mice (Fig. 3c).

Vitexin induces mitochondrial dysfunction in A549 cells
It is well known that mitochondria play important roles the regulation of cell apoptosis. The results of JC-1 staining indicated that vitexin exposure enhanced the loss of MMP in A549 cells (Fig. 4a). We also observed that vitexin exposure significantly reduced the levels of mitochondrial cytochrome c and increased the levels of cytoplasmic cytochrome c in A549 cells (Fig. 4b).

Vitexin inactivates PI3K/Akt/mTOR signaling
Targeting PI3K/Akt/mTOR signaling is a promising approach for the treatment of NSCLC [10]. We further investigated the effect of vitexin on PI3K/Akt/mTOR signaling in NSCLC cells. The results of western blot analysis demonstrated that vitexin treatment dosedependently reduced the levels of p-PI3K, p-Akt and p-mTOR in A549 cells (Fig. 5a). Additionally, as demonstrated in Fig. 5b, the apoptosis-inducing role of vitexin in A549 cells was also significantly blocked by pretreatment with 5 μM of Akt activator, SC79.

Discussion
Currently, chemotherapy remains the major therapeutic option for NSCLC patients [11]; however, anticancer agents often have harmful side effects. Major progress has been made in identifying novel anti-NSCLC agents with low toxicity. Vitexin possesses potential antitumor activities against many human cancers. For example, vitexin could inhibit esophageal cancer cell growth and induce apoptosis [12]. In the present study, we found that vitexin treatment reduced the viability of A549 cells in vitro, accompanied by an increase in LDH release due to cell membrane damage. In addition, administration of vitexin also inhibited the NSCLC tumor growth in vivo. Hence, the anti-NSCLC potential of vitexin was clearly indicated.
Apoptosis is an evolutionary conserved program of cell death, and activation of apoptotic pathways is an important anti-cancer strategy [13]. The Bcl-2 family proteins, including pro-apoptotic Bax and anti-apoptotic Bcl-2, play important roles in the regulation of apoptosis and tumorigenesis [14]. Mitochondrion is an important organelle involved in cell death [15], and loss of MMP can induce the release of pro-apoptotic molecules. Our findings demonstrated that vitexin reduced the Bcl-2/Bax ratio and caused the release of cytochrome c from mitochondria to cytosol, which further led to the cleavage of caspase-3, an executor caspase, in A549 cells. Therefore, we considered that vitexin induces A549 cell apoptosis, in part, through mitochondria-dependent pathway.
The PI3K/AKT/mTOR signaling is one of the most important intracellular pathways, which serve a critical regulatory role in a number of key cancerous behaviors [16,17]. Over-activation of this signaling is observed in NSCLC and many others. Drugs that target PI3K/Akt/ mTOR signaling have the potential to induce apoptosis in NSCLC cells [18]. In this study, we observed that treatment of A549 cells with vitexin reduced the levels of p-PI3K, p-Akt and p-mTOR, and more importantly, pretreatment with Akt activator, SC79, effectively blocked vitexin-induced A549 cell apoptosis. These results suggested that vitexin induces apoptosis partly through suppressing PI3K/Akt/mTOR signaling in A549 cells.

Conclusion
Taken together, by performing in vitro and in vivo experiments, our study might be the first to show that vitexin treatment impairs the viability and induces the apoptosis of A549 cells partly through mitochondrial pathway and PI3K/Akt/mTOR signaling. Although Authors' contributions XL, QJ and HL designed and performed the experiments. XL and QJ analyzed the data and wrote the manuscript. SL supervised the study and reviewed the manuscript. All authors read and approved the final manuscript.