Cell lines and reagents
Human GBM cells lines (U87, U251, A172, T98G, LN229 and LN18) used in this study were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). These cell lines were grown in a humidified incubator containing 5% CO2 at 37 °C. MELK (cat.no.2274s), AKT (cat.no.4691s), p-AKT(Ser473, cat.no.4058s), p-mTOR (Ser2448, cat.no.5536s), p-S6 (Thr389, cat.no.9206s), p21 (cat.no.2947s), Cyclin B1 (cat.no.12231s), Cdc2 (cat.no.77055s), FOXM1 (cat.no.20459s), p-FOXM1(Ser35, cat.no.14170s) and β-actin (cat.no.8457S) primary antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). Antibody for Ki-67 (Cat.PA5-16446) was purchased from Thermo Fisher (Waltham, MA, USA). MELK antibody for immunohistochemical experiments was obtained from Proteintech (Cat.11403-1-AP, Chicago, IL, USA). MELK inhibitor OTSSP167 and AKT inhibitor MK-2206 were purchased from Sellect Chemicals (Houston, TX, USA). OTSSP167 and MK-2206 were dissolved in DMSO to create a 10 mmol/L solution, which was diluted to different concentrations of DMEM medium before use.
Culture of GSCs
Two GSC lines, GSC1 and GSC2, were cultured in neurobasal medium containing basic fibroblast growth factor, epidermal growth factor, B27 supplement, harpin, L-glutamine, and N2 supplement to form a neurosphere culture that is enriched with GSCs. A third volume of fresh medium was added every three days, and neurospheres were dissociated using a NeuroCult Chemical Dissociation Kit (StemCells Technologies) for cell passage according to manufacturer’s protocol.
Cell counting kit (CCK)-8 assay
Cell viability was examined using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) as previously described . The GBM cells were seeded into 96-well plates with 3,000 cells per well and cultured overnight, followed by the addition of different concentrations of OTSSP167. After 72 h of treatment, 10 μL of CCK-8 solution were added to each well, followed by incubation for 2 h and measuring the absorbance (optical density, OD) at a wavelength of 450 nm. Three independent experiments were conducted with each experiment having three replicate wells, and the background reading of media was subtracted from each well for result standardization.
EdU incorporation assays
The Cell-Light EdU Cell Proliferation Detection Kit (Ruibo Biotech, Guangzhou, China) was used for the detection of cell proliferation. The human glioblastoma cell lines, U87 and LN229, were seeded into 96-well plates. After overnight culture, the adhered cells were treated with 0–200 nM OTSSP167. After 24 h, the cells were added and continuously incubated with 50 μΜ 5-ethynyl-2'-deoxyuridine (EdU) for 4 h. Subsequently, the cells were fixed with 4% paraformaldehyde solution for 15 min and treated with 0.5% Triton X-100 for 20 min, followed by incubating with 1× Apollo® reaction cocktail in the dark for 30 min before DPAI staining for 20 min. After washing thrice with phosphate-buffered saline (PBS), the cell images were taken under a fluorescent inverted microscope. This experiment was conducted three times.
Colony formation assay
The U87 and LN229 cells were seeded into a 6-well plate (400 cells/well), with 3 replicate wells per group. After cell adhesion, the cells in the experimental group were treated with 0–200 nM OTSSP167, and the cells in the control group were treated with DMSO. After 12 h, cell lines were further incubated with fresh DMEM medium containing 10% FBS for approximately 14 days, until colony formation stopped (judged by naked eye). The cells were washed with PBS and fixed in 4% paraformaldehyde solution. After staining with crystal violet working solution and rinsing the staining solution, the colonies were observed, photographed, and counted.
Cell cycle analysis
We examined the effect of OTSSP167 treatment on cell cycle distribution by flow cytometry as previously described . The U87 and LN229 cell lines were treated with OTSSP167, followed by cycle analysis 24 h later. Propidium iodide (PI) staining was used to analyze cell cycle distribution. The cells were collected after staining and centrifuged at 1,000 rpm for 5 min at 4°C. Subsequently, the cells were fixed in 3 mL of 70% ice-cold methanol overnight. The fixed cells were washed twice with PBS and stained with PI solution containing RNase A for 30 min. Finally, the stained cells were detected by flow cytometry, and the cell cycle distribution was analyzed using a flow cytometry software (Becton-Dickinson).
In vitro cell migration and invasion assays
Cell invasion and migration assays were performed using a Transwell system. The U87 and LN229 cells were re-suspended in serum-free DMEM medium containing various concentrations of OTSSP167 or DMSO. The cell suspensions were seeded into the upper layer of the Transwell chamber at a density of 10,000 cells per well, and the lower layer of the Transwell chamber contained DMEM medium supplemented with 10% FBS. For the cell invasion assay, diluted Matrigel in cold distilled water was applied to polycarbonate membrane filters with an 8-μm pore size. However, the migration assay did not require the addition of Matrigel. After 24 h of incubation, the non-invasive or non-migrating cells were wiped off with a cotton swab, and the cells that invaded or migrated to the lower layer of the Transwell system were fixed with 4% methanol for 20 min, followed by staining with 0.3% crystal violet solution for 30 min and observing the cells under a light microscope before imaging and counting.
The U87 and LN229 cells were treated with different concentrations of OTSSP167 separately. After 24 h, the cells were collected for total protein extraction, which was quantified by the Bradford protein assay before western blotting. The specific methods were as described previously [28,. Briefly, 50 μg of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein gels were transferred onto polyvinylidene fluoride (PVDF) membranes, which were subsequently blocked with 5% skim milk at room temperature for an hour before incubating with specific primary antibodies at 4°C overnight. The next day, the membranes were incubated with the corresponding secondary antibodies before developing using an enhanced chemiluminescence (ECL) reagent. The relative protein expression of Cyclin B1 (dilution 1:1000), Cdc2 (dilution 1:1000), MELK (dilution 1:500), p21 (dilution 1:1000), AKT (dilution 1:1000), p-AKT(473) (dilution 1:1000), phosphorylated-mammalian target of rapamycin (p-mTOR) (dilution 1:800), and p-S6 (dilution 1:750) were analyzed using β-actin (dilution 1:2000) as loading control.
In vitro cell viability and neurosphere formation assays
The GSC1 and GSC2 cells were seeded into 96-well plates at a density of 1,000 cells per well and treated with the indicated concentrations of OTSSP167 or DMSO. The CellTiter-Glo luminescent cell viability kit (Promega) was used to assess cell viability on days 0, 3, 6, 9, and 12. For the neurophere formation assay, the GSC1 and GSC2 cells were seeded into 96-well plates at a density of 1,000 cells per well. The cells were cultured in neurobasal medium containing various concentrations of OTSSP167 or DMSO. After 10–14 days, the neurophere formation were observed under a light microscope. Neurospheres containing more than 50 cells were scored. The numbers of neurospheres in each well were calculated.
Luciferase reporter assay
Transcriptional activity of FOXM1 was detected as our previous report . The LipofectamineTM 2000 was used to transfect 6× FOXM1-luc and Renilla luciferase reporter vector into the GSC2 cells. After 24 h, 0.1% DMSO or different concentrations of OTSSP167 were added to the transfected cells and further incubated for 24 h. The luciferase reporter assay was performed using the Dual Luciferase Assay kit (Promega). The activity of sea cucumber luciferin was used as control for the transfection and expression efficiency of the experiment. All samples were repeated in triplicate.
In vivo studies
All animal protocols were approved by the Ethics Committee of the Xuzhou Medical University (Jiangsu Province, China). Fifty-seven male athymic BALB/c nude mice aged 5–6 weeks were purchased from Beijing Vital River Experimental Animal Technology Co. Ltd., China. This study used a subcutaneous animal model and orthotopic transplantation tumor model to evaluate the therapeutic effect of OTSSP167 on GBM treatment. For the subcutaneous tumor model, the U87 cells (5 × 105) were inoculated on the right lateral flank of nude mice. Once the tumor grew to a volume of approximately 50 to 100 mm3, the nude mice were randomly divided into 3 groups: the control group, a 5 mg/kg OTSSP167-treatment group, and a 10 mg/kg OTSSP167-treatment group. The animals in the OTSSP167-treatment groups were intraperitoneally injected with the corresponding concentrations of OTSSP167 twice a week, and their tumor size was measured with calipers every 3 days. The volume of the subcutaneous tumors was calculated as follows: Tumor volume = (Length × Width2)/2 (assuming a prolate shape).
For the intracranial tumor model, the U87 (5 × 105 cells per mouse) were injected in situ into the right striatum of nude mice using a small animal stereotaxic instrument as previously described . Five days after the tumor cells were inoculated, the nude mice bearing tumor cells were randomly divided into 3 groups (n = 15 mice per group). The mice were treated with OTSSP167 (5 μL of 1 μM or 2 μM OTSSP167 in 1% DMSO in PBS per mouse) or vehicle control by intratumoral injection once a week for 4 weeks. After 30 days, 5 mice were randomly selected from each group and euthanized, followed by brain perfusion to assess brain tumor size. The remaining 10 mice in each group were used for statistically survival analysis.
Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC)
The whole brains of mice in the control and treatment groups were fixed in 4% paraformaldehyde solution overnight, followed by paraffin-embedding and tissue sectioning at a thickness of 5 μm. The brain sections were placed on glass slides and dried in an oven. For HE staining, the brain sections were deparaffinized in xylene solution, hydrated across an ethanol gradient, and then rinsed with tap water. The brain sections were then stained with HE staining solutions in sequence for 5 min, followed by dehydration and mounting with neutral gum. Brain morphology was then assessed by light microscopy and imaged.
For IHC, the brain sections were heat retrieved in a microwave oven at 60°C for 30 min, followed by deparaffinizing in xylene solution for 15 min thrice and hydrating across a decreasing ethanol gradient (100%, 85%, and 75%) for 5 min each. The brain sections were incubated with citric acid antigen repair buffer, further washed with PBS thrice, and quenched in 3% hydrogen peroxide solution in the dark for 25 min. The brain sections were then blocked with bovine serum albumin (BSA) for 30 min followed by incubation with the corresponding primary antibodies individually (anti-MELK (dilution 1:150), anti-p-Akt473 (dilution 1:200), and anti-ki67 (dilution 1:150) primary antibodies) at 4°C overnight. Subsequently, the brain sections were incubated with the corresponding secondary antibodies (dilution 1:200) at room temperature for 50 min before developing with 3,3′-diaminobenzidine (DAB) solution. The brain sections were subjected to nuclear counterstaining with a hematoxylin solution for 3 min, followed by routine dehydration, mounting, and observation and imaging under a light microscope.
Each experiment was independently repeated for at least three times. The figures show representative images of our results of repeated experiments. The experimental results were statistically analyzed using GraphPad Prism 6.0 (San Diego, CA). The data were presented as the mean ± standard deviation of the mean. Comparison between two samples was performed using an independent sample t-test. The Kaplan-Meier method was used for survival analysis of the mice. The log-rank test was used to compare survival time between the two study groups. P <0.05 was considered statistically significant.