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Berberine, also known as berberine hydrochloride and isoquinoline alkaloid, is a major alkaloid from
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 (
Angiogenesis serves an important role in tumor growth and metastasis (
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 (
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
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.
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.
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 ×
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.
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
Further analysis demonstrated that berberine hydrochloride dose-dependently promote the apoptosis of 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
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 (
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 (
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
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
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
NSCLC is a threat to human health and life (
MMP-2 and MMP-9 are gelatinases belonging to the MMP family (
VEGF belongs to a group of endothelial growth factors mainly produced by tumor cells and intercellular substances (
Numerous genes are involved in the apoptosis of lung cancer and Bcl-2 is closely associated with Bax and NSCLC (
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.
The chemical structure of berberine hydrochloride.
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 treatment promotes the apoptosis of A549 cells. **P<0.01, compared with the 0 µM berberine hydrochloride treatment group.
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 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 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 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 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 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.