Cell culture and reagents
The U251 cell line was purchased from Shanghai Cell Bank (Shanghai, China), cultured in high-glucose DMEM (Thermo Fisher, USA) supplemented with 10% foetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin G and 0.1 mg/mL streptomycin), and then maintained in the exponential growth phase in an atmosphere of 5% CO2 at 37 °C. Bufalin (≥ 98% in purity) was purchased from Baoji Chenguang Technology Development Co., Ltd. (Baoji, Shaanxi, China).
Cell viability assay
Cell counting kit-8 (Dojindo Laboratories, Tokyo, Japan) was used to detect cell viability. U251 cells (5 × 103 cells/well) were seeded in 96-well plates and cultured overnight. The culture medium was replaced with medium containing different concentrations of bufalin, and 10% FBS culture medium (0.1% DMSO) was added to the control well. After incubation for 12 h, 24 h, and 48 h, the culture medium was discarded, and 100 µL of CCK-8 working solution was added.
Clone formation assay
U251 cells treated with 25 nM, 50 nM, and 100 nM bufalin for 24 h were seeded on new 6-well plates at a density of 100 cells/well and then incubated at 37 °C in 5% CO2 for 1 week. After staining with Giemsa (Solarbio Science & Technology, Beijing, China), the clone formation rate was calculated as follows: clone formation rate (%) = numbers of clones/numbers of inoculated cells × 100%.
Cellular ATP content detection
The ATP assay (Merck, Darmstadt, Germany) was used to detect cellular ATP content. Cells treated with different concentrations of bufalin were digested with trypsin, collected, and centrifuged for 5 min at 1500 rpm. A 10 µL cell suspension consisting of 104 cells was transferred onto a luminometer plate, and 100 µL of nucleotide lysate was added. After 5 min, 10 µL of ATP detection working solution was added to the cell lysate, and the luminescence level of each well was measured within 1 to 2 min with a fluorescence microplate reader. The steps in the reader manufacturer’s instructions were followed for drawing a standard curve and calculating the ATP content of each well.
Na+/K+ -ATPase activity assay
Cells treated with 25 nM, 50 nM, and 100 nM bufalin were collected after 24 h in culture, and 1 mL was extracted and added to each tube (adjusting cells at a density of 1 × 106 cells/mL in each group). The cells were sonicated at 20% power, with ultrasound applied for 3 s with 10 s intervals, and this cycle was repeated 30 times. Then, the cells were centrifuged for 10 min at 8000 g at 4 °C. The supernatant was transferred to 2-mL centrifuge tubes, and the enzymatic reaction and phosphorus levels were measured according to the instructions of an Na + /K + -ATPase activity kit (Solarbio Science & Technology, Beijing, China). Samples (200 µL) were placed in 96-well plates, and the absorbance was measured at 660 nm.
Intracellular Ca2+ level assay
Intracellular calcium ion levels were measured according to the Fluo-3AM manufacturer’s instructions. The Fluo 3-AM storage solution (1 mM) was prepared by dissolving 50 mg Fluo 3-AM powder (Dojindo Laboratories, Japan) with 44.2 μL of DMSO and stored at -20 °C in the dark. Furthermore, a Fluo 3-AM working solution (5 μM) was prepared by dissolving 44.2 μL of Fluo 3-AM storage solution and 16.2 μL Pluronic® F-127 (20% solution in DMSO, Invitrogen®, Thermo Fisher Scientific) with 8.80 mL of Hanks' Balanced Salt Solution (Gibco® HBSS, Thermo Fisher Scientific). Briefly, the cells were seeded in confocal dishes, incubated overnight and then exposed to bufalin in fresh culture medium at concentrations of 25 nM, 50 nM, and 100 nM. After 24 h of bufalin treatment, the cells were washed three times with PBS and inoculated with Fluo 3-AM working solution at 1.5 mL/dish for 45 min in a dark cell incubator and then washed with PBS. The cells were continuously incubated with HBSS at 37 °C with 5% CO2 for 20 min to ensure the complete de-esterification of Fluo 3-AM. The fluorescence intensities were measured at λex/λem = 490/520 nm by an Olympus FV1000 confocal microscope (Olympus; Center Valley, PA, USA).
Reactive oxygen species (ROS) assay
The reactive oxygen species (ROS) detection storage solution was prepared by dissolving ROS detection reagent (Sigma-Aldrich, St. Louis, MO, USA) with 40 μL of DMSO and stored at 4 °C in the dark. Five thousand cells were inoculated in a confocal culture dish. After they adhered to the dish walls, the cells were exposed to 25, 50 and 100 nM bufalin for 24 h. Two millilitres of ROS detection reagent in working solution was added to each dish and incubated at 37 °C for 40 min in the dark. The cells were washed 3 times with PBS solution, and 2 mL of PBS solution was added to each dish to cover the cells, and the fluorescence intensities were measured at λex/λem = 490/520 nm with an Olympus FV1000 confocal microscope (Olympus; Center Valley, PA, USA).
Glutathione (GSH) assay
The intracellular GSH level was measured by a GSH/GSSG-Glo assay kit (Solarbio Science & Technology, Beijing, China). U251 cells treated with different concentrations of bufalin for 24 h were collected, and the contents of GSH and GSSG in each group of cells were determined according to the instructions of the GSH and GSSG activity assay kit. Then, the GSH/GSSG ratio was calculated.
Flow cytometry (FCM)
Cells were collected in aseptic tubes after drug treatment, 490 μL of assay buffer, 5.0 μL of Annexin V labelled with fluorescein (FITC-Annexin V) and 5.0 μL of propidium iodide (PI) were added, and the cells were incubated for 20 min and detected by FCM. According to the staining results, the proportion of living cells (Annexin V-/PI-), early apoptotic cells (Annexin V + /PI-), late apoptotic cells and necrotic cells (Annexin V + /PI +) were distinguished in each group.
Cell cycle analysis was carried out by FCM. Through the combination of PI and DNA, FCM was used to categorize cell stage directly on the basis of the fluorescence intensity indicating DNA content. G1/G0 phase cells have DNA content of diploid cells, G2/M phase cells have DNA content of tetraploid cells, and S phase cells have DNA content of both diploid and tetraploid cells. After bufalin treatment, the cells were collected, fixed with 70% ethanol and incubated overnight at 4 °C. After PBS washes, PI staining solution containing RNA enzyme was added to the tubes containing the cells. The cells were incubated at 37 °C for 30 min and subsequently subjected to FCM.
Cell apoptosis analysis
A MitoTracker red CMX Ros kit (Beyotime Biotechnology, Shanghai, China) was used to identify apoptotic U251 cells treated with 25, 50 or 100 nM bufalin for 24 h. An Olympus FV1000 confocal microscope was used to detect red fluorescence at λex/λem = 579/599 nm, green fluorescence at λex/λem λex/λem = 492/520 nm, and blue fluorescence at λex/λem = 350/461 nm.
Transmission electron microscopy (TEM)
The cells treated with different concentrations of bufalin for 24 h were digested and collected in a centrifuge tube. After the cells were washed twice with PBS, 2.5% glutaraldehyde solution was added along the tube wall to cover the cell clumps, and the cells were incubated in these tubes overnight at 4 °C. The cells were then washed 2 times with PBS, fixed with 1% osmium acid, dehydrated in a gradient, and embedded in resin that was then cut into ultrathin sections and stained with 2% uranyl acetate for 5 min. The ultrastructural changes of the cells were observed by TEM.
Mitochondrial membrane potential (MMP) assay
JC-10 is a cationic lipophilic dye that forms reversibly emitting red fluorescence when concentrated in cells with polarized mitochondrial membranes. When the mitochondrial membrane potential decreases and JC-10 cannot accumulate in the matrix of mitochondria, JC-10 is a monomer that emits green fluorescence. By calculating the ratio of red/green fluorescence, the MMP level in U251 cells was determined to evaluate the effect of bufalin on cell mitochondrial function. The MMP was determined using a mitochondria membrane potential kit (Sigma-Aldrich, St. Louis, MO, USA), which contains JC-10 dye. U251 cells were added to confocal Petri dishes (1 × 105 cells/dish) and treated with 25, 50 or 100 nM bufalin for 24 h. Then, the medium was discarded, and the cells were washed with PBS, 2 mL of JC-10 working solution was added to each dish to cover the cells, and the cells were incubated at 37 °C in the dark for 40 min. Then, the cells were washed with PBS, the fluorescence intensity was observed under a confocal microscope, and the ratio of red/green fluorescence was calculated.
RT-QPCR
Total RNA was extracted using TRIzol reagent. cDNA was synthesized using TaqMan™ reverse transcription reagents (Applied Biosystems, Foster City, CA). Quantitative reverse transcriptase-polymerase chain reaction (RT-QPCR) analyses were carried out to determine the mRNA levels (Chk1, Chk2, ATM, ATR, CDK2 and CDC25A mRNA) using SYBR Green Real-Time PCR Master Mixes (Applied Biosystems). β-Actin was the internal control. The primers used for RT-QPCR are shown in Additional file 1: Table S1; they were designed by Shanghai Novelbio Medical Technology Co., Ltd.
Western blot analysis
Proteins from cells treated with bufalin for 24 h or protein samples taken after bufalin incubation with U251 cell were separated by SDS electrophoresis. The following primary antibodies were used for western blotting: anti-p53 (#9282), anti-p-p53 (#9284), anti-Chk1 (#2360), anti-p- anti-Chk1 (#2344), anti-c-myc (#13,987), anti-cytochrome C (#11,940), anti-caspase 3 (#9662) and anti-cleaved caspase 3 (#9661) (Cell Signaling Technology, Danvers, MA, USA); anti-DRP1 (WL03028), anti-TUBb (WL01931), anti-Annexin A2 (WL0033a) and anti-COX IV (WL02203) (Wanleibio Technology, Shenyang, China); anti-HSPA8 (E-AB-22118), anti-HSPA9 (E-AB-11284), anti-mitochondrial fusion protein-2 (E-AB-32025) (Elabscience, Wuhan, China) and anti-GAPDH (G9545) (Sigma-Aldrich, St. Louis, USA), which was used as the internal control. After treatment with primary antibodies, the membrane was treated with the appropriate secondary antibody conjugated to horseradish peroxidase (HRP; Santa Cruz). Western blotting was performed three times. The intensity of each band was quantified with ImageJ software.
Drug affinity responsive target stability (DARTS) assay
Total protein in U251 cells was extracted, 66 μL of 10 × TNC solution was added to 600 μL of total protein after BCA quantification. The protein in TNC solution was divide into three aliquots, and 2.0 μL of 100 μM DMSO and 1000 μM bufalin were added to each aliquot, which was mixed gently and incubated overnight in a refrigerator at 4 °C. Fifty microlitres of the cell protein extract combined with bufalin was added to 2.0 μL of 1.25 mg/mL 1 × TNC solution and 0.25 mg/mL pronase working solution, enzymatically digested at room temperature for 15 min, and then added to 5 × loading buffer for denaturation. The proteins were separated by SDS-PAGE electrophoresis, and LC–MS/MS analysis was performed after silver staining in accordance with the protocol of a Pierce silver stain kit (Thermo Fisher, USA).
LC–MS/MS and proteomics analysis
A SCIEX triple TOF 5600 LC–MS/MS system was used to perform mass spectrometry analysis of the different bands, and the peptide samples bound to the C18 capture column were gradient eluted for analysis. Ultrapure water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) constituted the mobile phase, and the gradient elution programmes were as follows: 0 min-15 min, 5%–35% B; 15 min-16 min, 35%–80% B; 16 min-21 min, 80% B; 21 min-21.1 min, 80%–5% B; and 21.1 min-29 min, 5% B. The flow rate was 0.3 µL/min. Mass spectrometry IDA mode analysis included one full MS scan (at m/z 350–1500, 250 ms) in each scan cycle, followed by 40 MS/MS scans (at m/z 100–1500, 50 ms). MS/MS collected any precursor ion signal greater than 120 cps, the charge number was + 2– + 5, and the exclusion time of repeated ion collection was set to 18 s.
The mass spectrum data were retrieved by ProteinPilot (V4.5), the Paragon database retrieval algorithm was used, and the human proteome reference database in UniProt was referenced. The search results were screened with unused ≥ 1.3 as the standard, the entries and contaminating proteins retrieved with an anti-database were deleted, and the remaining identification information was assessed in a followed-up analysis.
Molecular docking
The MOE-DOCK module was used to dock and predict the affinity of the ligand and the receptor. In this procedure small-molecule drugs are defined as ligands, and proteins are defined as receptors. The 3D structures of the Annexin A2, TUBb, DRP1, HSPA9 and HSPA8 proteins were downloaded from the RCSB Protein Data Bank (http://www.rcsb.org/). With LigX, the protonation state and hydrogen orientation of the protein were optimized at pH 7 and 300 K. The docking process adopts a flexible induced fit mode, the side chain in the amino acid binding pocket can be optimized and adjusted according to the ligand conformation, and the weight of restrained side chain rotation was set to 10. For each ligand, a total of 1000 conformations were obtained, and all docking poses were ranked by London dG scoring, and the top 30 poses were then rescored by the GBVI/WSA dG method. Finally, the representative conformation was selected based on the binding score. The interaction mode of ligand and receptor was mapped by PyMOL software (http://www.pymol.org).
Small interfering RNA transfection
U251 cells were seeded in a 6-well plate at a density of 1 × 106 cells/well. LipofectamineTM 2000 reagent was used to transfect Annexin A2 siRNA (GenePharma, Shanghai, China) with the sequence 5'-TGTGTGGTGGAGATGACTGA-3' into U251 cells to represent transfection into humans. A genomic sequence without a matching negative siRNA was used as the negative control, and U251 cells treated only with LipofectamineTM 2000 reagent were used for the mock control group. Seventy-two hours after Annexin A2 siRNA was transfected into U251 cells, western blotting was performed to detect the expression level of Annexin A2 protein in cells.
Statistical analysis
Each experiment was performed at least three times and analysed by GraphPad Prism 7 software. The data are expressed as the means ± SD. P values were calculated using one-way ANOVA when the variances were uniform, and when the variances were not uniform, nonparametric tests were performed for statistical analysis. P < 0.05 was considered statistically significant.