Drugs and antibodies
AKT inhibitor SC66 was purchased form MedChem Express ((HY-19832, Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO), which was purchased form Servicebio (G5051, Wuhan, China). The GS3K-β inhibitor IM-12was obtained from Selleck (S7566, USA). The antibodies included the following: anti-Phospho-AKT (#4060, Cell Signaling Technology, USA), anti-AKT (#4691, Cell Signaling Technology), anti-Phospho-GSK-3β (#9323,Cell Signaling Technology),anti-GSK-3β (#12456,Cell Signaling Technology)，anti-Phospho-β-catenin (#9561,Cell Signaling Technology), anti-cleaved-caspase3(ab32042, Abcam, UK), anti-β-catenin (ab32572, Abcam）,anti-GAPDH (60004-1-Ig,Proteintech,Wuhan, China),anti-Snai1（13099-1-AP,Proteintech）,anti -BAX (50599-2-Ig,Proteintech),anti-Bcl-2 ( 12789-1-AP, Proteintech),anti-Cyclin D1(60186-1-Ig, Proteintech),anti-caspase3 (19677-1-AP,Proteintech),anti-MMP2(10373-2-AP,Proteintech),anti-Vimentin ( sc-6260 Santa Cruz Biotechnology, USA)
Humans glioblastoma cell lines (U87 and U251) were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in high-glucose DMEM (Genom, Hangzhou, China) supplemented with 10% foetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, USA) and 1% penicillin/streptomycin (Genom, Hangzhou, China). These cells were incubated at 37̊C in a humidified atmosphere containing 5% carbon dioxide.
Cell viability assay
The ability of SC66 exerts antiproliferative activity was measured with Cell Counting Kit-8 (CCK-8) (Dojindo, Shanghai, China) according to the manufacturer's instructions. U87 and U251 cells were planted into 96-well plate (5000 cells/well) and treated with 0,5,10,15,20,25,30umol/L of SC66 for 24 hours. Then 10u per well of CCK8 was added and then incubated at 37̊C for 1 hour. The absorbance value (OD) was measured with a spectrophotometric plate reader at 450 nm. Three independent assays were carried out.
Colony formation assay
An approximate number of 500 cells per well were planted into 6-well plate and cultured with 2ml DMEM containing 10%FBS.Then, the cells were treated with 0,6,10 and 15umol/L of SC66 for two to three weeks until there was significant single-cell colony formation. The cells were fixed in 4% paraformaldehyde for 30 minutes and then stained with 0.5% crystal violet for 15 minutes. The counts of colonies were counted using Image J software (National Institutes of Health, Bethesda, MD, USA).
5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay.
Approximately 5x103cells were seeded into 96-well plates containing 100ul DMEM and treated with 0,6,10,15umol/L SC66 for 24 hours the next day. The cell growth was measured using the Cell -Light Edu imaging detecting kit (RiboBio, Guangzhou, China) according to the manufacturer's protocol. Cells were cultured in medium supplement with 50uM EdU for 2 hours, and fixed in 4% paraformaldehyde for 30 minutes. Subsequently, 100ul of 1X Apollo® reaction cocktail was added to each well and incubated at room temperature for 30 minutes, and then the DNA was stained with 1X Hoechst 33342 in dark for 30 minutes. Fluorescence images of the Hoechst 33342 and EdU were visualized using a fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan). The number of DAPI and EdU-positive cells was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Wound -healing assay
Cells were seeded into 6-well plates and growth until 70% confluency. A wound was created by scratching the monolayer cell gently and slowly with a yellow 200-ul pipette tip in each well. Whereafter, the cells were gently washed twice with PBS and cultured with DMEM supplement with serum-free medium. Meanwhile ,0 or 10 uM of SC66 was added into DMEM at 0 and 24 hours respectively. The images were captured under a microscope (Olympus BX51; Olympus, Tokyo, Japan).
Invasion assay was performed using a Transwell chamber with an 8.0-μm pore polycarbonate
membrane. The polycarbonate Transwell filters coated with Matrigel, a number of 8x103cells treated with 0,6,10 or 15uM of SC66 for 24 hours were seeded into the top chambers. Simultaneously, 200 ul serum-free DMEM was added into the top chambers, and 600 ul DMEM supplement with 10%FBS was added into the bottom as a chemoattractant. The cells was incubated at 37℃ for 36 hours and fixed in 4% paraformaldehyde for 30 minutes. The non-invasive cells in the top chambers were moved with cotton swabs, and the cells on the lower side of membrane surface were stained with 0.5% crystal violet for 15 minutes. Air dried and the results were obtained under a microscope (Olympus BX51; Olympus, Tokyo, Japan).
Flow cytometry analysis of the cell cycle distribution
Cells were harvested with 0.25 trypsin after treated with 0.6,10 or 15 of SC66 for 24 hours. Next, cells were fixed in 70% cold ethanol overnight at −20 °C. Then the cells were washed twice with PBS and incubated with PBS containing RNAase for 30 minutes. Eventually, the cells stained with
propidium iodide (PI) and incubated in dark for 15 minutes. The cells were analysed by BD FACSAria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the data were quantified using ModFit LT 5.0 software (http://www.vsh.com/products/mflt/mfFeatures.asp; Verity Software House, Topsham, ME, USA).
Flow cytometric analysis of apoptosis
Cells in each group was plated in 6-well plates, and harvested the cells after treated with 0,6,10 or 15 uM of SC66 for 24 hours. The cells were suspended in 1ml 1X binding buffer, and stained with 5 μl of PE Annexin V and 5 μl of 7-amino-actinomycin (7-ADD) for 15 minutes at room temperature away from light. For each experiment ,2x105 cells were analyzed using BD FACSAria flow cytomer (BD Biosciences, Franklin Lakes, NJ, USA).
Western blot analysis
Cells treated with 0,6,10 or 15 uM of SC66 for 24 hours, and then lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Roche, Indianapolis, IN) and phosphatase inhibitors (Applygen, Beijing, China) at 4 °C for 30 min. The cell lysate was centrifuged at 1.2x105 rpm for 15 minutes at 4℃ and protein concentrations were measured by BCA method (Beyotime, shanghai, China).The protein was loaded onto a 10% or 12% SDS-PAGE and transferred to a PVDF membrane (Millipore, Germeny). After block with 5% skim milk powder for 1 hour, membranes were immunoblotted with primary antibodies to Phospho-AKT,Phospho-GSK-3β，Phospho-β-catenin，AKT，GSK-3β，β-catenin，GAPDH，BAX，Bcl-2,cleaved-caspase3,casapse3,snai1,MMP2,vimtenin and Cyclin D1 with an appropriate dilution concentration overnight at 4℃. Subsequently, the membranes were incubated with Alex Fluor 680/790-labelled secondary antibodies (LI-COR Bioscience, USA) for 1hour. The bands were captured using a LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences).
Immunofluorescence staining of cells
The sterilized slides were placed in a 6-well plate, and approximately 3x104 cells were planted. After 12 hours of sc66 treatment, they were fixed in 4% paraformaldehyde for half an hour. Permeabilized with 0.5% Triton X-100 (Amresco, USA) for 20 min, and blocked with 5% bull serum albumin (Amresco, USA) for 30 min at room temperature. Cells were incubated with primary antibodies overnight at 4℃ according to the manufacturer's recommended concentration. The next day, cells were washed and then incubated with FITC-labeled goat anti-rabbit IgG (1:50 dilution; Servicebio, China) in dark for 1 hour at room temperature. Subsequently, cells nuclei were counterstained with diamidino-phenyl-indole (DAPI) (ANT046, Antgene,China) in dark for 5minutes.The images were visualized under a fully automatic Microscope (Olympus BX63; Olympus, Tokyo, Japan).
The tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were deparaffinized, hydrated and antigen repaired with 10mM sodium citrate (pH, 6.0). After removing endogenous peroxidase by using 3% H2O2. Then, the samples were blocked with 5% bovine serum albumin (Amresco, USA) for 30 minutes. The samples were incubated with primary antibody overnight at 4 °C according to the manufacturer's recommended concentration. The reactions were visualized using a 3,3′-diaminobenzidine visualization kit (Servicebio,China) and counterstained with hematoxylin to visualize nuclei for 1minute.The images were visualized under a microscope (Olympus BX51; Olympus, Tokyo, Japan)
T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase reporter assay
Cells were seeded in 6-well plates and treated with 0 or 6uM of SC66 for 24hours. The cells were harvested after co-transfection with TCF/LEF1 luciferase reporter plasmid and pGMLR-TK luciferase reporter plasmid (Yeasen Biotech Co, Ltd. Shanghai, China) for 48 hours. The Renilla and firefly luciferase activities was measured by the Dual Luciferase Reporter Gene Assay Kit (Yeasen Biotech Co., Ltd.Shanghai, China) according to manufacturer’s protocol.
All animal experiments were approved by the Institutional Animal Care and Use Committee at Renmin Hospital of Wuhan University and complied with the internationally recognized Animal Research: Reporting of In vivo Experiments guideline. U87 cells were resuspended in PBS at a concentration of 2x107/ml and subcutaneously injected a 100 μL cell suspension into the left armpit of 5-week-old male Balb/c nude mice. After 15 days, when the tumor volume reached about 100 mm3, the mice were randomly divided into two groups (n = 8) and treated with SC66 at 25 mg/kg by i.p. injection every other 3 days for 18 days. Meanwhile, the control group was intraperitoneally injected with DMSO of the same volume. In addition, we measured tumor volume every 3 days. Tumor volume was calculated using the formula V = L ×W2 × 1/2 (V, volume; L, length of tumor;
W, width of tumor). Two days after the last drug treatment, the mice were killed and the tumor was stripped. Half of each tumor was fixed with 4% paraformaldehyde and paraffin-embedded (FFPE) sections, the other half was extracted for western blot.
Statistical analyses were performed using GraphPad Prism 6.0 software. All experimental results were expressed as the mean ± SD. One-way ANOVA was used to measure differences between various groups. P<0.05 was considered statistically significant. Statistical significance was indicated in the figures as follows: *P<0.05, **P<0.01 and ***P<0.001.