Compounds
To synthesize (Z)-2-((2-((1-Ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-di-hydrobenzofuran-6-yl)oxy) acetonitrile (A14), 6 mmol anhydrous potassium carbonate was added to 2 mmol of (2Z)-2- [(1-ethyl-5-methoxy-1 h-indole-3-yl)meth-ylene]-6-hydroxy-1-benzofuran-3(2H)-one in 10 mL n,n-dimethylformamide (DMF) solution. The mixture was heated to 60 °C, mixed with 2.4 mmol/L chloroacetonitrile and stirred at 60 °C for another 8 h before being cooled. The resulting mixture was then added to 100 mL of 0.05 mol/L sulfuric acid aqueous solution. The precipitate was collected by filtration, washed with water, dried and recrystallized in DMF methanol to obtain A14 yellow crystal: mp 230–232 °C.
A14 was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and prepared into corresponding concentrations for cell experiments. In the animal experiments, A14 was dissolved in solvent (50% PEG-400 + 35% PBS + 10% DMSO + 5% Tween-80) to prepare a 0.2% A14 solution. Briefly, A14 was added to 10% DMSO, mixed well, added to 50% PEG-400 and 5% Tween-80, mixed ultrasonically for 2 h, supplemented with 35% PBS, and stirred using a vortex mixer before use.
Cell culture
Human leukemia cell lines Jurkat and THP-1, purchased from China Fenghui Biotechnology Co., Ltd. Cells were cultured in RPMI-1640 and supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin and 100 U/mL penicillin (Gibco Life Technologies, Carlsbad, CA, USA). The cells were kept in a humid incubator at 37 °C with 5% CO2.
Cell proliferation assay
We detected the proliferation of Jurkat and THP-1 cells according to the protocol. Cells were treated with different concentrations of A14 (10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM), and each concentration was repeated three times. Equal volumes of DMSO were added to the blank wells. After 5 days of treatment, Vi-cell (Beckman Coulter, Indianapolis, IN, USA) counter was used to calculate the inhibition rate.
Colony formation assay
1.2% and 0.7% agar were prepared and maintained at 42 °C without solidification after high-pressure sterilization.1.2% agar mixed with RPMI 1640 medium (RPMI 1640 + 20% FBS + 2% PS), was laid on a 6-well plate as the lower glue and incubated at 37 °C for 30 min to fully solidify. The prepared Jurkat or THP-1 cell suspension were mixed with 0.7% agar. The mixed medium was incubated in a 6-well plate as the upper glue. After solidification, the complete medium was supplemented with corresponding A14, and the culture medium was changed every 3 days. After 2 weeks, the clone size was stained with 0.1% crystal violet (Sigma-Aldrich) and observed by Nikon ECLIPSE Ts2 light microscope (Nikon Corporation, Tokyo, Japan).
Cell cycle assay
We measured the cell cycle of Jurkat and THP-1 cells according to the instructions of cell cycle kit. Cells with a density of 1 × 106/well were inoculated into 6-well plates, and different concentrations of A14 were added to each well for treatment. The blank control well was treated with DMSO. After 24 h of treatment, cells were collected and transferred to a centrifuge tube, supernatant was discarded, and washed with PBS. To detect the cell cycle by flow cytometry (Beckman Coulter, Indianapolis, IN, USA), DNA Staining solution and permeability solution were added to each tube, mixed well with a vortex and incubated at room temperature in darkness for 30 min.
Cell apoptosis analysis
We also measured the apoptosis of Jurkat and THP-1 cells via the apoptosis kit. Cells with a density of 1 × 106 were incubated into 6-well plates and challenged with different concentrations of A14. After 24 h of treatment, the cells were collected by centrifugation. After washing the cells with precooled PBS, we added AnnexinV-FITC and propidium iodide (PI) (BioLegend Inc., San Diego, CA, USA Biolegend, San Diego, CA) into each tube, and incubated them at room temperature for 5 min. We were then able to detect AnnexinV-FITC by flow cytometry (Beckman Coulter).
Cell mitochondrial membrane potential analysis
As above, Jurkat cells and THP-1 cells were stimulated with corresponding concentrations of A14. Carbonyl cyanide 3-chlorophenylhy drazone (CCCP) was added to the cell culture medium and treated for 20 min as a positive control. Cells were stained with 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) and incubated at 37 °C for 20 min. We then washed the cells with JC-1 staining buffer five times, and analyzed them using cell flow cytometry (Beckman Coulter).
Cell derived xenograft model
First, we constructed a cell line with stably expressing luciferase gene. Specifically, Jurkat cells were cultured in 96 well plates with 5% virus per well. After incubation at 37 °C for 6 h, the medium was changed to 2 μg/mL puromycin for continuous screening over several days. Then the monoclonal antibodies were selected and isolated.
Female NOD/SCID mice aged 6–8 weeks were fed in a SPF room with free access to water and food. Mice were divided into five groups randomly and injected with 1 × 106 Jurkat-Luc (luciferase reporter gene) cells per mouse through the tail vein. Three days later, the fluorescence was detected by IVIS Lumina Series III (PerkinElmer, Waltham, MA, USA) to judge whether the CDX model was established successfully. The mice that successfully expressed fluorescence were randomly divided into A14 (20 mg/kg) group and control group (n = 15). The weights of the mice were recorded every day, and the fluorescence was detected every five days. All the animal procedures were performed in strict conformance to the Guidelines for Care and Use of Laboratory Animals of Hebei Normal University and approved by the Animal Ethics Committee of Hebei Normal University.
Immunoblot analysis
Total proteins were extracted from cells using RIPA buffer with cocktail and phenylmethanesulfonyl fluoride (Cell Signaling Technology, Inc, Boston, MA, USA). The protein content was determined by the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Aliquots were separated on 10% sodium dodecyl sulphate–polyacrylamide (SDS-PAGE) gel, and then transferred to the polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After being sealed with 5% milk, the membrane was washed with TBST and incubated overnight with primary antibodies at 4 °C. The primary antibodies include CDK1 (1:1000, Abcam, Cambridge, MA, USA), cyclin B1 (1:1000, Abcam), CDK2(1:1000, Abcam), cyclin D1 (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Caspase 3 (1:1000, Cell Signaling), Cleaved Caspase 3 (1:1000, Cell Signaling), Caspase 9 (1:1000, Cell Signaling), Cleaved Caspase 9 (1:1000, Cell Signaling), poly [ADP-ribose] polymerase (PARP) (1:1000, Cell Signaling), Bcl-2 (1:1000, Abcam) and GAPDH (1:1000, Cell Signaling). The membrane was washed three times and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000, Abcam) at room temperature for one hour. ChemiDocMP Imaging Systems (BioRad Laboratories, Inc., Hercules, CA, USA) used an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology) to detect the protein band. The optical density of protein bands were quantified by the Quantity One software (Bio-Rad), and the values were expressed as the ratio of protein to GAPDH with the values of the DMSO group set to one.
Statistical analysis
Statistical analyses were performed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. All results were presented as the mean ± standard deviation (SD), p < 0.05 was thought to be statistically significant.