Berbamine dihydrochloride (BBM, purity ≥ 98%) was purchased from Macklin (Shanghai China). PRMI 1640 Medium and Fetal Bovine Serum (FBS) were provided by Gibco (America). Matrigel was obtained from BD Biosciences (America). Cell Death Detection Elisa and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were supplied by Sigma (America). The antibodies of PI3K, MDM2, Akt, p-Akt, p53 and GAPDH were purchased from Abcam (England), Bcl-2, Bax and c-Maf were purchased from Santa Cruz Biotechnology (America), caspase-3 and β-actin were purchased from Proteintech (America). All the other reagents were got from Beyotime Biotechnology (Shanghai, China).
Cell line and cell culture
The human NSCLC cell line A549 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured with RPMI 1640 medium containing 10% FBS in a humidified atmosphere with 5 % CO2 at 37℃. The final DMSO concentration was less than 0.1% throughout all the study.
Cell viability was detected by MTT assay. Briefly, A549 cells were cultured in 96-well plates (1×104 cells/well), treated with vehicle or BBM (0, 1.25, 2.5, 5, 10, 20, 40 and 80 µM) for 24 h, 48 h and 72 h. Then the MTT solution (5 mg/ml) was added to each well and cultured for 4 h. Finally, the supernatant was gently removed and 200 µl DMSO was added to each well, the absorbance was detected by a Multiwell Microplate Reader (Bio-Rad Laboratories) at 560 nm.
Colony formation assay
A549 cells were inoculated into 6-well plates and treated with vehicle or different concentrations of BBM (10, 20 and 40 µM) for 24 h. Then the medium was gently replaced with complete medium (with 10% FBS) and the medium was replaced every 3 days. The cells were stained with crystal violet after incubating for 8 days. The colonies were counted.
EdU cell proliferation assay
A549 cell proliferation was evaluated by Edu Apollo-567 In Vitro Kit (Ribo Bio, Guangzhou, China). EdU is a thymine nucleoside analogue, which can replace thymine and insert into the DNA molecule that is replicating during the cell cycle. The activity of DNA replication could be quickly detected by the Apollo fluorescent dye. In Brief, A549 cells (8×103 cells/well) were planted into 96-well plates with vehicle and BBM (10, 20 and 40 µM) and cultured for 24 h. EdU was added and incubated for 2 h. Cell nuclei were stained with Hoechst 33342 for 30 min. Finally, the cells were observed via an inverted fluorescence microscope (Leica, Germany). Five views with at least 500 cells were randomly selected for EdU ratio assay (EdU/Hoechst 33342).
Trypan Blue Dye exclusion assay
A549 cells (8×104 cells/well) were inoculated into the 6-well plate and treated with vehicle or different concentrations (10, 20 and 40 µM) of BBM respectively. The cells were treated for 48 h. Then trypan blue staining was performed and the cells were photographed within 5 min. At least 500 cells were seen in each view.
Apoptosis was detected by Cell Death Detection Elisa Kit. This assay is based on the quantitative sandwich immunoassay using antibodies against histones and DNA. Thus the detection of mono- and oligo-nucleosomes in the cell lysates can indirectly represent the apoptosis of cells. A549 cells were seeded into the six well plates and cultured with or without different concentrations of BBM for 24 h. Then the cells were washed with ice-cold PBS. RIPA lysis buffer (with 1 mM PMSF) was added to the well and the supernatant was used for testing. The mono- and oligo-nucleosomal fragmented DNA was measured by a Multiwell Microplate Reader.
Wound scratch assay
A549 cells were seeded into the 6-well plate 24 h before scratching. A 200 µl plastic tip was used to generate a straight line. Fresh serum-free medium with vehicle or different concentrations (10, 20 and 40 µM) of BBM were added to each well. The cells were imaged at 0 h and 24 h in the same position of the wound. The migration distance was measured by NIH Imaje J software. Mitomycin (2 µg/ml) was always added to exclude the proliferation of the cells.
Transwell assay was performed to evaluate the effect of BBM on the migration (without Matrigel) and invasion (with Matrigel) ability of A549 cells. Briefly, cells (4×104 cells/well) were seeded into the upper chamber of the 8 µm pore (Corning, America) in serum-free medium, the lower chambers were added with full medium (with 10% FBS). The cells were allowed to invasion and metastasis through the chambers at 37℃ for 24 h. Then the cells invaded to the surface of the lower chambers were stained. The number of cells passing through the chambers represents the metastasis ability of the cells. Mitomycin (2 µg/ml) was always added to exclude the proliferation of the cells.
Male BALB/c nude mice (6 weeks old, weighing 18-20 g) were obtained from GemPharmatech Co., Ltd (Jiangsu, China). The mice were housed in polystyrene, well aerated cages with 12-h light/dark cycle. The animals were maintained on a standard pelleted diet and were provided with free access to food and water adlibitum. All studies were performed with the approval of ARRIVE Guidelines (Animal Research: Reporting of in Vivo Experiments) and approved by the Animal Care and Use Committee of Soochow University.
In vivo assay
Nude mice were transplanted to the right axillary of the nude mice. When the tumor volume (W2*L/2, W = wide, L = long) reached to 150 mm3, the mice were randomly divided into 3 groups, the control group (Ctrl) and the experimental group (20 mg/kg and 40 mg/kg). Each group had 6 mice. The mice were treated intraperitoneally with saline and different concentrations of BBM for 10 days. The body weight and tumor volume of the mice were recorded every 6 days. At the end of the experiment, the mice were sacrificed and the tumors were prepared for western blotting assay. The weight and the metastatic nodules of lungs were recorded. And the visceral tissues were sliced for histopathological examination.
To study the histopathological change of various organs, Hematoxylin/Eosin Staining kit (Beyotime, China) was employed. The tissues were fixed and sectioned at 3-4 µm. Then the slices were stained with the Hematoxylin/Eosin Staining kit according to the reagent instruction. Histopathological scrutiny was observed by the microscope (Leica, Germany).
Western blotting assay
For protein analysis of PI3K/Akt and MDM2-p53 signal pathways, PI3K, p-Akt, Akt, MDM2, p53, c-Maf, caspase-3, Bcl-2 and BAX were detected. The harvested cells or tumors were lysed with RIPA lysis buffer (containing 1mM PMSF). The protein samples were separated on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% skim milk for 2 h, then probed with primary antibodies of PI3K, Akt, p-Akt, MDM2, p53, c-Maf, Bcl-2, BAX, caspase-3,β-actin, and GAPDH at 4 ℃, followed by secondary antibodies at room temperature. The immunoreactive complexes were detected by Enhanced ECL Chemiluminescence Detection Kit (Beyotime, China). Relative optical density (ratio to GAPDH) was quantified by NIH Image J software.
The data were presented as mean ± standard deviation (SD). The statistics were analyzed using SPSS software (SPSS 21.0, CA). Statistical differences were evaluated by Student's t-test or one-way ANOVA method, the accepted level of significance was P＜0.05.