Reagents and chemicals
Brucine was purchased from APExBIO (Boston, MA, USA). Antibodies were purchased from Abcam (Cambridge, UK). All other chemicals were purchased from MedChemExpress (Monmouth Junction, NJ, USA), unless indicated otherwise. The U87 human glioblastoma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA).
Human U87 glioblastoma cells were cultured in Gibco Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 100 U/mL of penicillin and streptomycin. Cells were maintained in a humidified incubator with 95% air atmosphere and 5% CO2 at a temperature of 37°C. Before the experiments, the cells were passaged for four generations.
U87 cells were harvested in the log phase and then adjusted to a density of 25,000 cells/mL. The cells were seeded into 96-well plates at 200 μL per well and incubated for 6 h. The medium of every three wells was replaced with 200 μL of DMEM containing 1, 0.5, 0.25, or 0 mM brucine. After 24-h incubation, 20 μL of MTT (5 mg/mL) was added to each well and the cells were incubated for an additional 4 h. After this, the supernatant was removed, 150 μL of dimethyl sulfoxide was added, and the 96-well plate was rocked on a shaker at an appropriate speed for 10 min. The optical density (15) of each well was measured at 492 nm using an enzyme-immunoassay instrument.
Flow cytometry assay
U87 cells were incubated with or without brucine (1, 0.5, 0.25, or 0 mM) for 24 h, harvested, and counted for use in subsequent assays. For the cell cycle assay, the cells were washed with cold (4°C) PBS and centrifuged at 1,000 rpm for 5 min. Then, 70% alcohol was added, and the cells were washed again with PBS and stained with propidium iodide (4) in the presence of 100 µL of RNase A for 30 min prior to analysis by flow cytometry. For the Annexin V/PI assay, the cells were washed with cold PBS. The supernatant was removed and then 1× binding buffer (100 μL) was added to each sample. After adding Annexin V (5 μL) and PI (5 μL), the volume of each sample was adjusted to 500 μL with 1× binding buffer. The cells were kept in the dark and analyzed through flow cytometry within 1 h.
Reverse transcription-polymerase chain reaction (RT-PCR)
U87 cells were incubated with or without brucine (1, 0.5, 0.25, or 0 mM) for 24 h. Total cellular RNA was extracted using TRIzol reagent according to the manufacture’s protocol. After measuring the amount of total RNA with an enzyme-immunoassay instrument, RNase-free water was added to adjust the samples to the same density. Next, cDNA was acquired by reverse transcription using Superscript II Reverse Transcriptase according to the manufacturer’s protocol. The SYBR Green method was used for performing RT-PCR on an iCycler (Bio-Rad, Hercules, CA, USA). cDNA (2 μL) from each sample was added as template to 18-μL reaction mixture containing 10 μL of Hieff qPCR SYBR Green Master Mix (YEASEN, Shanghai, China), 0.2 μM of each specific forward and reverse primer (Sangon Biotech, Shanghai, China), and 7.2 μL of RNase-free water. RT-PCR was performed according to the following protocol: 95°C for 5 min, 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s. At the end of the first round of RT-PCR, 80 cycles of 55°C + 0.5°C per cycle were used for melting curve analysis. GADPH was used as a control. The comparative CT method was used to determine relative expression levels.
Western blot assay
U87 cells were incubated with or without brucine (1, 0.5, 0.25, or 0 mM) for 24 h. The cells were harvested, washed with cold PBS, and homogenized on ice in RIPA lysis buffer with 1% 100× protease inhibitor cocktail. After centrifugation at 14,000 rpm for 15 min, the supernatants were collected, and protein levels were measured using the standard bicinchoninic acid assay. Thirty micrograms of protein from each sample were separated on a 10% sodium dodecyl sulfide-polyacrylamide gel and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free milk containing Tris-buffered saline with Tween 20 for 2 h at 20°C. The membranes were separately incubated with primary antibodies against c-Myb, β-tubulin, BAX, cyclin-D1, caspase-9, and caspase-3 overnight at 4°C. After incubation, the membranes were washed with Tris-buffered saline with Tween 20 and incubated for 2 h at 20°C with anti-mice or anti-rabbit secondary antibodies according to the primary antibody. Finally, the protein bands were detected through Amersham electrochemiluminescence.
Dual luciferase reporter assay
Transfected cells were assessed using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The guanine-rich sequence, 5′-GGGCTGGGCTGGGCGGGG-3′, located in the c-Myb promoter region was synthesized to replace the sequence from site KpnI to NheI of pGL3-basic (Promega)(20). Next, 293 cells were seeded in 12-well plates and co-transfected with 1.0 μg of moderated pGL3 or pGL3-basic and 0.2 μg of pRL-SV40 (Promega) according to the manufacture’s protocol. After 48-h incubation following transfection, the cells were incubated with or without brucine (1, 0.5, 0.25, or 0 mM) for 24 h.
Electrospray ionization mass spectrometry (ESI-MS)
The sequence, 5′-GGGCTGGGCTGGGCGGGG-3′, set as S1, and the complementary strand, set as S2, were synthesized (Sangon Biotech) with high-performance liquid chromatography. Annealing buffer was configured with Tris (10 mM), EDTA (1 mM), NaCl (50 mM), and pure water. DNA samples were divided into 4 groups: groups A and B with 0.1 mM S1 and groups C and D with 0.1 mM of both S1 and S2 in annealing buffer. Groups B and D were heated to 94°C, maintained for 2 min, and gradually cooled to room temperature. The four groups were separately divided into 4 subgroups with different concentrations of brucine (A1, B1, C1, and D1: 0 mM, A2, B2, C2, and D2: 5 mM, A3, B3, C3, and D3: 10 mM, and A4, B4, C4, and D4: 20 mM). Finally, the samples were detected with SYNAPT G2-Si (Waters, Milford, MA, USA).
Tumor xenograft model in nude mice
Female nude mice (25–30 g, 6 weeks old) were purchased from the Animal Center of Chinese Academy of Sciences (Shanghai, China). All mice were allowed free access to food and water under controlled conditions (12/12 h light/dark cycle with humidity of 60% ± 5% and temperature of 22 ± 3°C). All experimental protocols were approved by the Ethics Committee of the First Hospital of Jilin University and its institutional animal-care committee. Approximately 5 × 106 U87 cells were suspended in 200 μL ECM-gel (Sigma-Aldrich, St. Louis, MO, USA) with 50% 1× PBS and injected into the subcutaneous right forelimb axilla of 6-week-old female nude mice. After two weeks, mice with palpable tumors were randomly placed into two groups for treatment with either brucine or an equivalent volume of normal saline (NS). Mice in the experimental group were intraperitoneally injected with brucine (10 mg/kg) every 24 h and for 10 days. During treatment, body weights of mice were monitored and tumor sizes were measured every two days. After treatment, all mice were sacrificed and their tumors were resected and stored at –80°C. All studies were performed under the China Association for the Accreditation of Laboratory Animal Care guidelines for humane treatment of animals and adhered to national and international standards.
Data analyses were conducted using GraphPad Prism 7 software (GraphPad, Inc., La Jolla, CA, USA). The results were presented as the mean ± standard deviation. Student’s t test or one-way analysis of variance was used to compare two or more than two groups, respectively. P < 0.05 was considered as statistically significant.