br The above data support Bruceine D
The above data support Bruceine D as being a potential candidate to treat cancer. In the present study, we investigate the cytotoxic eﬀects of BD on NSCLC cell lines in vitro and explore the underlying mechanism of BD in cytotoxicity and apoptosis.
2. Materials and methods
2.1. Chemicals and cell culture
Bruceine D (CAS No.: 21499-66-1, ≥98% pure) was purchased from the Shanghai Sheng Zhong Pharmaceutical Chemical Co., Ltd, China. The chemical was dissolved in DMSO (Sigma-Aldrich, St Louis, Missouri, USA) and stored at -20℃. The NSCLC cell lines H460, A549, H1975, PC9 were obtained from the Shanghai Institute of Biochemistry r> Fig. 1. BD inhibited the cell growth and colony formation in human NSCLC H460 and A549 cells. (A) The chemical structure of BD. (B) CCK-8 assay was used to examine the cell-pro-liferation inhibitory activities in human NSCLC H460, A549, H1975, PC9, and BEAS-2B (Human normal lung epithelial cell) cell lines. The concentrations used included 0, 0.78, 1.56, 3.12, 6.25 and 12.5 μmol/L. Cells were plated in 96-well plates and exposed to serial con-centrations treatment of BD for 48 h. Dose–response curves for BD are shown. Each condition was performed in triplicate. (C) H460 or A549 K 252a were plated in 60-mm dishes and then treated with 0.2, 0.4 μmol/L or 0.4, 0.8 μmol/L BD for 14 days. The colonies were stained with a 0.1% crystal violet solution and photographed. Representative images are pre-sented. (D) The bars represent the colony numbers for diﬀerent groups relative to the control group. The experiments were per-formed in triplicate, and the error bars re-present the standard deviation. Significance was determined by the Student's t-test (*P < 0.05, ***P < 0.001 vs. untreated controls).
and Cell Biology, routinely maintained in RPMI-1640 medium (HyClone, USA). All media were supplemented with 10% (v/v) fetal bovine serum (HyClone, USA) plus 1% penicillin-streptomycin. All cells were incubated at 37℃ in a 5% CO2 atmosphere.
2.2. Cell proliferation assay
Cell viability was measured by CCK-8 assay. Briefly, 3–5 × 103 cells were cultured in 96-well plates. After 24 h, the cells were treated with the indicated concentrations of BD. Cells were evaluated finally using the Cell Counting Kit-8 (MedChem Express, USA). The absorbance was measured at 450 nm using a Multiskan Spectrum spectrophotometer (Thermo Scientific, Rockford, IL, USA).
2.3. Colony formation assay
H460 and A549 cells were inoculated in 6-well plates at the density of 500/well and incubated for 48 h. The cells were then treated with continuous concentrations (0.2, 0.4, 0.8 μmol/L) of BD for another 48 h. The compound-containing medium was replaced every 2–3 days. After two weeks, the cells were fixed with a 0.1% crystal violet solution and photographed.
H460 and A549 cells were cultured in 6-well plates. After BD treatment, the cells were fixed with 4% paraformaldehyde for 30 min and then stained with DAPI for 20 min at 37℃. After washing with 1 × PBS, the cells were observed under a fluorescence microscope (Nikon, Japan).
2.5. Apoptosis assay
Apoptosis was carried out by Annexin V (Invitrogen, Carlsbad, CA) staining according to manufacturer’s protocol. Briefly, cells (5 × 105/ ml) were seeded into 6-well plates and incubated with BD for 24 h, then harvested and washed with PBS, resuspended in indicated binding buﬀer. Subsequently, Annexin V FITC and propidium iodide (PI) was
added to the cell suspension and incubated for 15 min in dark at room temperature. After incubation, the samples were analyzed by flow cy-tometric (BD Biosciences), and data were analyzed by BD CellQuest Pro software (version 2.0, BD Pharmingen, BD Biosciences).
2.6. Western blot analysis
After treatment with BD, cells were harvested, washed once with PBS, and resuspended in lysis buﬀer (50 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1% NP-40, 2.0 μg/ml aprotinin, 0.02% sodium azide, and 1 mM phenylmethylsulfonyl fluoride) on ice for 30 min. The lysates were centrifuged at 13,000 rpm for 30 min at 4 °C. Total lysate protein concentrations were measured with the Bradford kit (Beyotime).
2.7. Statistical analysis
The data was calculated as mean ± S.D. from at least three in-dependent experiments; Statistical analysis was performed with Student’s t-test. All analyses were realized by using two-tail student’s t-test or one way ANOVA, *p < 0.05 was considered a statistically significant diﬀerence. Graphs were prepared using SigmaPlot 12.5 software.
Fig. 2. BD triggered cell apoptosis in H460 and A549 cells. (A) H460 and A549 cells were exposed to BD as indicated concentrations for 24 h. After exposure, the cells were stained with DAPI, and the apoptotic nuclear changes were examined under a fluorescence microscopy. White arrows indicate the apoptotic cells. (B and D) H460 and A549 cells were treated with BD for 48 h. The cells were then double-stained with Annexin V-FITC and PI, and analysed by flow cytometry to determine the level of apoptosis. (C and F) Protein extracts of treated cells were immunoblotted with specific antibodies directed against caspase-3, cleaved caspase-3, PARP, Bcl-2 and BAX. GAPDH was used as a loading control. Data are expressed as the mean ± S.D. of three independent experiments. Significance was determined by the Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated controls). Scal bar, 10 μm.