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Introduction

According to a previous study, ~3,120,000 novel cases of cancer are identified in China annually, which is equivalent to ~8,550 patients being diagnosed with cancer every day or 5 patients/min on average (1). Lung cancer is one of the most common types of malignant tumors of the respiratory system, and has the highest rates of incidence and mortality of malignant tumors (1,2). Non-small cell lung cancer (NSCLC) is a common type of cancer. Numerous previous studies have confirmed that the leading cause of mortality for patients with lung cancer is metastasis. The prevention of metastasis is therefore the focus of lung cancer treatment (3–5).

Angiogenesis serves an important role in tumor growth and metastasis (6). The induction of angiogenesis requires the angiogenesis phenotype of lung cancer tumor cells (7). Angiogenesis induction also requires the promotion of tumor angiogenesis factors using inhibiting factors; vascular endothelial growth factor (VEGF) is the primary regulating factor of angiogenesis, and serves an important role in the development, invasion and metastasis of lung cancer (8). Matrix metalloproteinases (MMPs) are a group of metal ion-dependent proteases, which are able to degrade the various proteins in the basement membrane and extracellular membrane (ECM), thus promoting the invasion and metastasis of lung cancer to adjacent and distant tissues (9). A previous study indicated that MMPs and VEGF are closely associated with the occurrence, development, invasion, metastasis and prognosis of lung cancer (10).

According to a previous study on the effect of berberine hydrochloride on tumor cells, it has been revealed that this compound exhibits cytotoxicity, and is able to inhibit the growth and proliferation of tumor cells (11). Berberine hydrochloride may inhibit the proliferation of colon, gastric, breast, esophageal and liver cancer cells (12,13). The inhibitory effect previously demonstrated was revealed to be dose and time-dependent in a number of studies (14–16). The effects of numerous treatment concentrations of berberine hydrochloride on various cellular locations and stages of the cell cycle in a human melanoma cell line were investigated. A previous study revealed that a low dose of berberine hydrochloride may be concentrated in mitochondria and inhibit the growth of melanoma cells in the G1 phase cell cycle (17). A high dose of berberine hydrochloride may promote the accumulation of melanin tumor cells in the G2 phase and inhibit the synthesis of DNA, whereas a low dose of berberine may not affect the synthesis of DNA (18). Furthermore, the present study investigated the inhibition of cell proliferation and the promotion of apoptosis induced by the berberine hydrochloride treatment of NSCLC cells, which was induced by the suppression of MMP-2 and the Bcl-2/Bax signaling pathway.

Materials and methods

Materials and the cell line

Human NSCLC A549 cells were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (Cellgro; Corning Incorporated, Corning, NY, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin, in a humidified atmosphere with 5% CO2 at 37°C. The chemical structure of berberine hydrochloride (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) is presented in Fig. 1.

Figure 1. The chemical structure of berberine hydrochloride.

Proliferation assay

A549 human NSCLC cells were seeded at a density of 5×103 cells/well in 96-well culture plates and treated with various concentrations (0, 30, 60, 90, 150 and 200 µM) of berberine hydrochloride at 37°C for 0, 1, 2 and 3 days. A total of 150 µl MTT (Invitrogen; Thermo Fisher Scientific, Inc.) was added to each well and incubated for 4 h at 37°C. A total of 150 µl dimethyl sulfoxide (Invitrogen; Thermo Fisher Scientific, Inc.) was then added to each well for 10 min at 37°C with agitation. The absorbance was evaluated using a Varioskan Flash microplate reader (Thermo Fisher Scientific, Inc.) at 450 and 630 nm.

Cell apoptosis detection

A549 human NSCLC cells were seeded at a density of 2×106 cells/well in 96-well culture plates and treated with various concentrations (30, 60 and 90 µM) berberine hydrochloride at 37°C for 2 days. The cells were subsequently harvested by careful trypsinization without EDTA, and washed twice with PBS. Subsequently, the cells were harvested using 10 µl Annexin V binding buffer and stained with Annexin V-fluorescein isothiocyanate/5 µl propidium iodide (PE Annexin V Apoptosis Detection kit I, BD Biosciences, Franklin Lakes, NJ, USA) at 4°C for 15 min at darkness. Flow cytometry was performed using a FACS-420 flow cytometer (BD Biosciences) in order to analyze the rate of cell apoptosis.

Western blotting

A549 human NSCLC cells were seeded at a density of 2×106 cells/well in 96-well culture plates and treated with various concentrations (30, 60 and 90 µM) of berberine hydrochloride at 37°C for 2 days. Subsequently, the cells were harvested and lysed with lysis buffer (RIPA assay, Beyotime Institute of Biotechnology, Haimen, China) at 4°C for 15 min. The supernatant was centrifuged at 12,000 × g for 15 min at 4°C. The soluble protein concentration was determined using a Bradford assay (Beyotime Institute of Biotechnology, Haimen, China). Protein samples were separated by SDS-PAGE (8–12%) and transferred onto a polyvinylidene fluoride membrane by wet transfer. The membranes were blocked with 1% Tween-20 PBS (TBST) at 4°C overnight prior to incubation with anti-MMP-2 (cat. no. sc-13594; dilution, 1:2,000), anti-Bax (cat. no. sc-20067; dilution, 1:2,000), anti-Bcl-2 (cat. no. sc-7382; dilution, 1:2,000, anti-Janus kinase 2 (Jak2) (cat. no. sc-390539; dilution, 1:2,000), anti-VEGF (cat. no. sc-390741; dilution, 1:2,000), anti-NF-κB/p65 (cat. no. sc-372; dilution, 1:2,000) and anti-transcription factor AP-1 (AP-1; cat. no. sc-12629; dilution, 1:2,000; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and β-actin (cat. no. D110007; dilution, 1:2,000, Shanghai Sangon Pharmaceutical Co., Ltd., Shanghai, China). Following washing with TBST, the membranes were incubated with anti-rabbit IgG-HRP (cat. no. sc-2357, dilution, 1:3,000, Santa Cruz Biotechnology, Inc.) at 37°C for 1 h and visualized using an ECL Prime kit (Merck Millipore).

Statistical analysis

The data are presented as the mean ± standard error of the mean, which was determined using SPSS 19.0 software (IBM SPSS, Armonk, NY, USA). The paired t-test and one-way analysis of variance were performed in order to determine the statistical significance between various groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Berberine hydrochloride inhibits the proliferation of A549 cells

In order to determine the anticancer effect of berberine hydrochloride on the proliferation of A549 cells, the cells were treated with a variety of concentrations of berberine hydrochloride. As presented in Fig. 2, berberine hydrochloride suppressed the growth of cells in a time- and dose-dependent manner. Following a 48-h treatment, 60–200 µM of berberine hydrochloride effectively suppressed the proliferation of A549 cells, compared with the control group (Fig. 2). However, 90–200 µM berberine hydrochloride treatments effectively suppressed the proliferation of A549 cells following 24 h (Fig. 2).

Figure 2. Berberine hydrochloride treatment inhibits the proliferation of A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group (control group).

Berberine hydrochloride promotes the apoptosis of A549 cells

Further analysis demonstrated that berberine hydrochloride dose-dependently promote the apoptosis of A549 cells (Fig. 3). Berberine hydrochloride treatment at concentrations of 60 and 90 µM for 24 h effectively induced the apoptosis of A549 cells (Fig. 3; P=0.0056 and P=0.0031, respectively).

Figure 3. Berberine hydrochloride treatment promotes the apoptosis of A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group.

Berberine hydrochloride inhibits MMP-2 protein expression in A549 cells

In order to evaluate the association between the anticancer effect of berberine hydrochloride and MMP-2 expression level in A549 cells, western blotting was performed. As indicated in Fig. 4, 90 µM berberine hydrochloride treatments significantly inhibited the MMP-2 protein expression level in A549 cells following 24 h, compared with control group (P=0.0074).

Figure 4. Berberine hydrochloride treatment inhibits the expression level of MMP-2 in A549 cells. Western blot analysis and statistical analysis of MMP-2 expression levels in berberine hydrochloride-treated A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group. MMP-2, matrix metalloproteinase 2.

Berberine hydrochloride promotes the Bcl-2/Bax signaling pathway in A549 cells

In order to determine the association between the anticancer effect of berberine hydrochloride on lung cancer cells and the Bcl-2/Bax signaling pathway, Bax and Bcl-2 protein expression levels were evaluated using western blot analysis. Treatment with 60 and 90 µM berberine hydrochloride significantly increased the activity of the Bcl-2/Bax signaling pathway in A549 cells, compared with control group (Fig. 5; P=0.0047 and P=0.0025, respectively).

Figure 5. Berberine hydrochloride treatment promotes Bcl-2 and Bax protein expression levels in A549 cells. Western blotting and statistical analysis revealed that berberine hydrochloride treatment increased Bax and Bcl-2 protein expression levels in berberine hydrochloride treated A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Berberine hydrochloride inhibits Jak2 protein expression in A549 cells

In order to determine the anticancer effect of berberine hydrochloride treatment on the protein expression levels of the apoptosis signaling molecules, A549 cells were treated with berberine hydrochloride for 24 h and analyzed using western blotting. It was revealed that berberine hydrochloride treatment at 60 and 90 µM significantly inhibited Jak2 protein expression level in A549 cells, compared with control group (Fig. 6; P=0.0082 and P=0.0068, respectively).

Figure 6. Berberine hydrochloride treatment inhibits the expression of Jak2 in A549 cells. Western blotting and statistical analysis were performed in order to evaluate the expression level of Jak2 in berberine hydrochloride treated A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group. Jak2, Janus kinase 2.

Berberine hydrochloride inhibits VEGF protein expression level in A549 cells

In order to determine the association between the VEGF signaling pathway and the anticancer effect of berberine hydrochloride on lung cancer, changes in expression of VEGF protein were analyzed following treatment of A549 cells with various concentrations (0–90 µM) of berberine hydrochloride. As presented in Fig. 7, VEGF expression was significantly inhibited following treatment with 60 and 90 µM berberine hydrochloride (P=0.0061 and P=0.0028, respectively).

Figure 7. Berberine hydrochloride treatment inhibits the expression of VEGF protein in A549 cells. Western blotting and statistical analysis evaluated the VEGF protein expression levels in berberine hydrochloride treated A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group. VEGF; vascular endothelial growth factor.

Berberine hydrochloride inhibits NF-κB protein expression in A549 cells

In order to confirm the anticancer effect of berberine hydrochloride on NF-κB protein expression levels in A549 cells, NF-κB/p65 protein expression levels were determined by western blot analysis. As indicated in Fig. 8, NF-κB/p65 protein expression level was significantly inhibited by 60 and 90 µM berberine hydrochloride treatment for 24 h, compared with the control group (P=0.0045 and P=0.0017, respectively).

Figure 8. Berberine hydrochloride treatment inhibits NF-κB protein expression levels in A549 cells. Western blotting and statistical analysis were performed in order to evaluate the expression level of NF-κB protein in berberine hydrochloride treated A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group. NF-κB, nuclear factor κB.

Berberine hydrochloride inhibitsAP-1 protein expression in A549 cells

In order to determine the association between the anticancer effect of berberine hydrochloride and the AP-1 signaling pathway in A549 cells, the effect of berberine hydrochloride on AP-1 protein expression was evaluated. As indicated in Fig. 9, pre-incubation with berberine hydrochloride significantly increased the rate of apoptosis in A549 cells via suppression of the AP-1 signaling pathway, compared with the control group (P=0.0089 and P=0.0055).

Figure 9. Berberine hydrochloride treatment inhibits AP-1 protein expression in A549 cells. Western blotting and statistical analysis were performed in order to evaluate the expression level of AP-1 protein in berberine hydrochloride treated A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group. AP-1, transcription factor AP-1.

Discussion

NSCLC is a threat to human health and life (10). The morbidity and mortality rates of NSCLC have significantly increased worldwide over the past 50 years, and the disease is a major type of cancer (19). Previously, studies have revealed that the occurrence of NSCLC is closely associated with the inactivation of certain oncogene and tumor-suppressor genes (20). The overexpression of oncogenes, the downregulation of tumor-suppressor genes and the decreased rate of apoptosis may induce the malignant transformation of cells (21). The results of the present study have demonstrated that berberine hydrochloride inhibits cell proliferation and promotes the apoptosis of A549 cells. Certain studies have revealed that berberine suppresses the growth of MCF-7 breast cancer cells, colon cancer and ovarian cancer cells (22–24).

MMP-2 and MMP-9 are gelatinases belonging to the MMP family (25). They are able to degrade the constitutive proteins in the ECM and basement membrane, and gather on the surface of collagen IV (25). MMPs contribute to the loss of basement membrane by degrading the ECM and basement membrane, which then allows tumor cells to spread and infiltrate into the adjacent tissues through the loose ECM and the absent basement membrane, eventually contributing to the invasion and metastasis of lung tumors (26,27). Previous studies also indicated that MMP-2 or MMP-9 may promote the growth of blood vessels and lymphatic vessels by enhancing the binding of VEGF to VEGF receptors, thus promoting tumor metastasis into lymph glands or other tissues and the formation of metastatic tumors (28,29). The expression levels of MMP-2 and MMP-9 were significantly increased in tumor cells, compared with normal tissues, and positively correlated with tumor invasion ability (27,30,31). In the present study, a 24 h treatment with berberine hydrochloride significantly inhibited MMP-2 protein expression levels in A549 cells. Liu et al (27) suggested that berberine suppressed human aortic smooth muscle cells by decreasing the expression levels of MMP-2 and inhibiting the NF-κB/AP-1 signaling pathways. Therefore, berberine hydrochloride inhibition of lung cancer may occur primarily via the MMP-2 signaling pathway.

VEGF belongs to a group of endothelial growth factors mainly produced by tumor cells and intercellular substances (32). VEGF belongs to a group of endothelial growth factors mainly produced by tumor cells and intercellular substances. VEGF may promote the formation and development of novel blood vessels (29). Angiogenesis is essential to tumor growth, infiltration and metastasis, and it is regulated by a series of promotion and inhibition factors; one of the most important factors is VEGF, which is secreted by tumor cells in the growth stage (33). The expression level of VEGF in the sera of patients with lung cancer is significantly increased compared with in healthy patients, and is significantly higher in cancerous tissues and benign lesions, compared within healthy lung tissues (8). In the present study, berberine hydrochloride induced a significant upregulation of the VEGF signaling pathway in A549 cells. Zhang et al (30) demonstrated that berberine inhibits the expression of hypoxia-inducible factor 1α and increases the expression level of VEGF in prostate cancer. Therefore, the anticancer effect of berberine hydrochloride on A549 cells may occur via the activation of the VEGF signaling pathway.

Numerous genes are involved in the apoptosis of lung cancer and Bcl-2 is closely associated with Bax and NSCLC (34). The overexpression of the anti-apoptotic gene, Bcl-2, and the low expression level of the apoptosis-promoting gene, Bax, inhibits cell apoptosis and extends the cell survival time, which is one of the underlying mechanisms of lung cancer (35). The abnormal expression level of Bax in NSCLC may be associated with to the occurrence, development and prognosis of NSCLC (36). In the present study, berberine hydrochloride significantly increased the activity of the Bcl-2/Bax signaling pathway in A549 cells. Xie et al (34) revealed that berberine induced apoptosis via the downregulation of Bcl-2 in human breast cancer cells. Li et al (11) suggested that berberine hydrochloride inhibited the invasion of and promoted cell apoptosis in MDA-MB-231 cells through inactivation of the Jak2/NF-κB/AP-1 signaling pathways. Thus, the suppression of Bcl-2/Jak2/NF-κB/AP-1 expression levels were involved in the cell apoptosis induced by berberine hydrochloride.

In conclusion, berberine hydrochloride may be a possible anticancer therapeutic strategy, as it inhibits cell proliferation and promotes apoptosis of NSCLC cells via suppression of the MMP-2, Bcl-2/Bax and Jak2/VEGF/NF-κB/AP-1 signaling pathways. Therefore, the results of the present study suggest that berberine hydrochloride may be an effective and safe therapeutic candidate drug for treating NSCLC in humans.

References