Cell culture and treatment
MG63, U2OS, MNNG and 143B human osteosarcoma cell lines were obtained from Stem Cell Bank of Chinese Academy of Sciences. The cells were tested for Mycoplasma before experiments. Cells were cultured in DMEM (Gibco, Grand Island, NY) medium containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY) and were incubated in a CO2 incubator with 5% CO2 at 37 °C. Alternatively, Cells were treated with inhibitors OTX015 (MCE, MedChem Express, HY-15743) and WT-161 (MCE, MedChem Express, HY-100871) for 48 h before analyzing by qRT-PCR, western blotting, proliferation assay or transwell assay.
Cell viability assay
OS cells were seeded at the density (3 × 103 cells/well) in a 96-well culture plate for 18–24 h, and then each cell line was treated independently or cooperatively for 24, 48, or 72 h with OTX015 and WT-161 at different concentrations. Meanwhile, an equal volume of DMSO was added as a negative control. After that, 10 µL of CCK-8 (Sigma-Aldrich, m4839) solution was added to each well and incubated for 1 h away from light in a cell incubator. A microplate reader was used to calculate the absorbance value (OD) of each well at 450 nm. The cellular viability was calculated using the following formula: (OD of control − OD of treatment)/(OD of control − OD of blank) × 100%. Each sample was tested in triplicate.
Calculation of combination index
As for the synergistic assay, the concentrations of MG63, U2OS, 143B and MNNG cells treated by OTX015/WT-161 for 48 h were listed in Additional file 1: Fig. S1A. The Chou-Talalay method and CompuSyn software were used to analyse the synergistic effect of the two drug combinations. The combination index (CI) of Chou-Talalay was calculated from the data derived from monotherapy and combination treatment by CompuSyn, and the CI value quantitatively defines additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations. Finally, appropriate concentration was selected for the following experiment and listed in Additional file 1: Fig. S1D.
Apoptosis and cell cycle assay
Cell apoptosis was evaluated using an apoptosis assay kit (KeyGEN BioTECH, KGA108-1) according to the manufacturer’s instructions. In brief, cells exposed to appropriate concentrations of OTX015 and/or WT-161 or control DMSO for 48 h were resuspended in 500 µL of binding buffer after being washed with PBS three times. Then, 1% annexin V-FITC and 1% propidium iodide (PI) were added to the cells and incubated for 30 min at room temperature in the dark before measurement by flow cytometry (Becton-Dickinson, USA). For cell cycle analysis, cells treated with OTX015 and/or WT-161 and DMSO for 48 h were washed with PBS three times and fixed with 75% ethanol at 4 °C overnight. Then, the cells were centrifuged, washed three times, resuspended in precooled PBS, and incubated in the dark for 30 min with PI and RNase. Finally, flow cytometry was used to evaluate the cell cycle profile.
Transwell analysis was performed to assess the effects of OTX015 and/or WT-161 on the migration and invasion ability of osteosarcoma cells. The cells were inoculated into Transwell chambers with polycarbonate membranes. In the invasion experiment, 2 × 104 cells in 200 µL of serum-free medium were inoculated into Matrigel (BD Biosciences, Franklin Lakes, NJ)-coated upper chambers after being treated with a particular concentration of OTX015, WT-161, or their mixture for 48 h. In the lower chamber, 750 µL of medium supplemented with 10% FBS was added. The steps were the same for the migration experiment, except there was no Matrigel coating in the Transwell chambers. The cells stained with 0.1% crystal violet in the upper compartment were washed clean with cotton swabs after 24 h, and the number of invading cells in the lower chamber was observed under a microscope. Finally, 5 fields/chamber were selected for cell counting at 20× magnification. The number of every field cell was counted by ImageJ software.
Colony forming assay
Cells treated with OTX-015, WT-161, or their combination were seeded into 6-well plates at a density of 500 cells/well for 14 days. Then, the cells were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet and photographed. The number of stained colonies (≥ 10 mm2) was also counted by ImageJ software.
Plasmid construction and cell transfection
Full-length β-catenin was amplified from cDNAs reverse transcribed from mRNA extracted from 293T cells and cloned into the pcDNA3.1 vector. The primers for cloning β-catenin were as follows: 5′-GCTACTCAAGCTGATTTGATGGA-3′ and reverse: 5′-TTACAGGTCAGTATCAAACCAGGC-3′. The transfection of plasmids was performed using Lipofectamine® 2000 (Invitrogen) according to the manufacturer’s instructions.
Quantitative real-time PCR
Total RNA was extracted by TRIzol reagent (Invitrogen), and then the RNA concentration was measured using a spectrophotometer (Thermo Fisher Scientific). The single-peak concentration curve showed high RNA purity. cDNA was synthesized by a PrimeScript™ RT reagent kit (TaKaRa, RR047A). PCR was carried out with a SYBR Premix Ex Taq II kit (TaKaRa, RR820A) according to the manufacturer’s instructions. GAPDH, a housekeeping gene, was used as the internal control. The result was analysed via the 2−ΔΔCt method. The primer sequence was as follows:
MG63 and U2OS cells (1000 cells/well) suspended in DMEM/F12 (Invitrogen, Carlsbad, CA) medium supplemented with B27 (Invitrogen, 17504-044), human EGF (10 ng/mL, PeproTech) and human BFGF (10 ng/mL, PeproTech) were inoculated in six-well ultralow attachment plates (Corning Inc., NY, 3471) and incubated in a CO2 incubator with 5% CO2 at 37 °C. The spheres over 50 μm diameter were photographed and counted under a microscope. The limiting dilution assay was performed according to Xiang [22, 23]. Briefly, the OSCs were implanted into 96-well plates at a gradient of 5, 10, 20, 50, 100, or 200 cells/well, with 10 replicates for each gradient. And formation of tumor spheres in each well was examined after 9 days. The wells without spheres in each group were counted and the proportions of wells without spheres in each gradient were calculated. Then the sphere formation efficiency was calculated using the Extreme Limiting Dilution Analysis (http://bioinf.wehi.edu.au/software/elda) .
Western blot analysis
Cells were washed three times with PBS, lysed with RIPA buffer on ice for half an hour and centrifuged at 12,000 g/s for 20 min to collect the supernatant. We used a BCA kit (Boster Biological Technology) to detect the protein concentration. Then, the proteins (10 µL/lane) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk, probed with primary antibodies (1:1000) and stained with horseradish peroxidase (HRP)-linked secondary antibodies (1:15,000, Promega). Then, the blots were visualized using an ECL kit (Tiangen). The antibodies used in this study were as follows: anti-PTEN (Proteintech, 22034-1-AP), anti-β-actin (Proteintech, 20536-1-AP), and anti-β-Catenin (Proteintech, 66379-1-lg). Anti-p-mTOR (5536), anti-mTOR (2983), anti-P65 (8242), anti-IKB (4814), anti-p-ERK1/2 (4370), anti-ERK1/2 (5013), anti-p-STAT3 (9131), anti-STAT3 (9132), anti-CDK2 (18048), anti-CyclinB1 (12231), anti-P21 (2947), anti-cleaved caspase-3 (9664), anti-cleaved caspase-9 (20750), anti-p-GSK-3β (5558) and anti-GSK-3β (12456) were purchased from Cell Signaling Technology [CST, Danvers, MA (20750)]. Anti-p-JAK2 (ab32101), anti-JAK2 (ab108596), anti-vimentin (ab92547), anti-N-cadherin (ab18203) and anti-E-cadherin (ab40772) were purchased from Abcam (MA, United States).
The tumour tissues were stored with 4% paraformaldehyde, embedded in paraffin, sliced and mounted on a slide. The tumour tissue slices were dewaxed, hydrated, and sealed before being incubated overnight at 4 °C with a 1% primary antibody (β-catenin, PTEN, pGSK-3β). After 30 min of incubation with the secondary antibody (MXB Biotechnologies, 201126S407r), the slides were stained with DAB (Solarbio, DA1010) and haematoxylin (Servicebio, G1004) before being sealed with gelatine. HE staining was performed using the HE staining kit (Solarbio, G1120).
All nude mice (age of 4–6 weeks) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). A total of 1 × 106 U2OS cells in 200 µL of PBS were injected subcutaneously into the left armpits of each mouse. After 5 to 7 days of inoculation when the xenograft grew to 200 cm3, the mice were randomly divided into four groups (6 mice per group), and each group was injected intraperitoneally with drugs every 3 days: vehicle as a negative control; OTX015 (50 mg/kg); WT-161 (50 mg/kg); and OTX015 combined with WT-161 (same concentrations as for a single agent). Approximately 2 weeks later, the nude mice were sacrificed by cervical dislocation, and the tumours were removed and weighed. All animal experiments were approved by the institutional ethical review boards of the Third Affiliated Hospital of Nanchang University.
All of the experimental data were repeated at least three times and expressed as the mean ± SD. Student’s t-test was used to compare differences between two groups. P values < 0.05 were considered to be statistically significant.