Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture medium was purchased from Life Technologies (Gaithersburg, MD, USA). Fetal bovine serum (FBS) and B27 supplement were purchased from Gibco-BRL (Grand Island, NY, USA). Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were obtained from PeproTech (Rocky Hill, NJ, USA). Normal goat serum was provided by Wuhan Boshide Biotechnology Co., Ltd. (Wuhan, China). Poly-L-lysine, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody and Cy3-conjugated goat anti-rabbit antibody were provided by Sigma (St. Louis, MO, USA). Rabbit anti-mouse CD133 monoclonal antibody and glial fibrillary acidic protein (GFAP) polyclonal antibody were purchased from Abcam (Cambridge, MA, USA) and Zhongshan Jinqiao Biotechnology, Ltd., (Beijing, China), respectively. Anti-caspase-3 antibody (rabbit polyclonal against mouse, rat or human) and anti-β-actin antibody were obtained from Abcam Inc. (Cambridge, MA, USA). Cell Counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Tokyo, Japan).

GL261 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in DMEM/F12 culture medium containing 10% FBS. Cells were maintained at 37°C in a humidified environment containing 5% CO₂. The medium was changed every 4 days. To induce the formation of neurospheres (NS), FBS was gradually withdrawn from culture medium in a gradient reduction pattern (10, 5, 2 and 0%). Cells were then maintained in serum free DMEM/F12 medium supplemented with 20 ng/ml bFGF, 20 ng/ml EGF, B27 (1X), 2 mM L-glutamine and 4 U/l insulin. The floating cells formed NS-like clones and secondary spheres derived from single cells of these clones were used to induce cell differentiation. To induce differentiation, the GL261-NS cells were seeded onto coverslips and cultured in DMEM/F12 culture medium containing 10% FBS. These cells were defined as GL261 adherent cells (AC).

The spheres were placed onto coverslips precoated with poly-L-lysine (Sigma) and then fixed with 4% paraformaldehyde for 20 min at room temperature. Cell samples were blocked with normal goat serum and then incubated with the following respective primary antibodies: Rabbit anti-mouse CD133/1 monoclonal antibody (1:50 dilution) and GFAP polyclonal antibody (1:100 dilution) overnight. The cells were then washed three times in phosphate-buffered saline (PBS) and then stained with Cy3-conjugated goat anti-rabbit (1:100 dilution) or FITC-conjugated goat anti-rabbit secondary antibody (1:50 dilution) for 30 min. Cell samples were then counterstained with 100 mg/ml DAPI for 10 min to visualize nuclei and then analyzed with a confocal laser scanning microscope (Leica, Mannheim, Germany).

The effect of drug treatment on cell viability was determined using the CCK-8 kit. Briefly, cells were seeded onto a 96-well-plate and then treated 24 h after seeding with different concentrations (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 μM) of drug. Dimethyl sulfoxide (DMSO) was used as a negative control. Four days after drug incubation, 10 μl of thawed CCK-8 solution was added to each well. Plates were incubated for 4 h at 37°C and the absorbance was read at 450 nm with a reference wavelength of 600 nm using the Thermo Scientific™ Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA).

In order to evaluate the effects of agents on the colony formation of GL261-NS, cells were seeded onto a 96-well plate at a density of 1×10³ cells/ml. The cells were then incubated with 0.1 μM salinomycin, 30 μM 1-(4-amino-2-methyl-5-pyrimidyl)-methyl-3-(2-chloro ethyl)-3-nitrosourea hydrochloride (ACNU) or 10 μM vincristine (VCR) dissolved in DMSO. DMSO was used as a negative control. A total of 12 wells were assessed for each treatment group. Six days after incubation, the colony forming ability of cells were examined and the number of colonies was counted under an Olympus CX22 microscope (Olympus Corp., Inc., Tokyo, Japan).

Apoptotic cell death was measured using the Annexin V-FITC/propidium iodide Apoptosis Detection kit (Dojindo Molecular Technologies, Inc., Kunamoto, Japan) according to the manufacturer’s instructions and assessed on a fluorescence activated cell sorting Calibur instrument (Beckman Coulter Inc., Miami, FL, USA). Data were analyzed using CellQuest software (Becton-Dickinson, San Jose, CA, USA).

Cells were washed with ice-cold PBS and then lysed in ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM sodium orthovandate and 1 mM NaF) for 30 min. Following this, samples were centrifuged at 16,000 × g at 4°C for 30 min. The supernatant was collected and the protein concentration was determined using the Bradford protein assay. Total cell protein was separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk then incubated with anti-caspase-3 primary antibody at 4°C overnight. On the following day, the membrane was washed with 0.1% Tween 20 in PBS and probed with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The bound antibody complexes were detected using an electrochemiluminesence reagent (GE Healthcare, Amersham, UK). β-actin was used as an internal control. Band images were analyzed with a gel imaging analysis system (Kodak ID, Kodak, Rochester, NY, USA). The intensities of the immunoreactive bands were quantified by densitometric analysis.

A total of 40 six-week-old specific pathogen free level C57BL/6 male mice weighing 22±2 g were obtained from the Laboratory Animal Center, Third Military Medical University (Chongqing, China). Animals were randomly divided into the following four groups each including 10 animals: Sham-surgery, sham-surgery plus salinomycin, tumor-bearing plus placebo and tumor-bearing plus salinomycin. In tumor-bearing groups, animals received orthotopic transplantation of GL261-NS cells. Briefly, mice were anesthetized with 400 mg/kg chloral hydrate via intraperitoneal injection. Animal experiment procedures were approved by the Ethics Committee of Southwest Hospital, Third Military Medical University (Chongqing, China). The cell density was adjusted to 2×10⁶ cells/ml in serum free DMEM/F12 culture medium and then 5 μl of cell suspension was injected into the right caudate nucleus. Mice were placed on a stereotaxic instrument. Following sterilization and skin incision, a hole with a diameter of 3 mm was made with a micro-electrical drill (RWD Life Science, Inc., Shenzhen, China) on the skull at the position of 1.4 mm anterior to the anterior fontanel and 2.0 mm lateral to the sagittal suture. Stereotaxic coordinates were obtained from the bregma and the dura mater according to a mouse brain atlas (11). In the sham surgery group, mice received 1 ml of normal saline by intraperitoneal injection 24 h after cell injection and for salinomycin treatment, mice were subjected to intraperitoneal administration of salinomycin. The two groups of animals received daily injections for 10 days. The animal survival time was assessed for up to 60 days post-surgery. Animal experiments were conducted according to the guidelines of the Institutional Committee for Laboratory Animal Usage. The study was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (Chongqing, China).

Statistical analyses were performed using the SPSS 13.0 statistical software package (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean. The results were analyzed using a one-way analysis of variance with Fisher’s Least Significant Difference test. LogRank (Mantel-Cox) analysis was used to compare the significance of median survival time of animals. P<0.05 was considered to indicate a statistically significant difference.

Neurosphere-like clones formed when the cells were cultured in serum-free culture medium supplemented with bFGF, EGF, L-glutamine and insulin (Fig. 1A). In addition, abundant expression of the stem cell marker, CD133, was observed in these neurosphere-like GL261-NS cells (Fig. 1B). In addition, the level of GFAP expression, which is a biomarker for differentiated glial cells, was relatively low. In order to induce cell differentiation, the culture medium was changed and cells were maintained in DMEM/F12 culture medium containing 10% FBS. The majority of the cells became adherent and began to differentiate, which were defined as GL261-AC cells.

The effects of salinomycin on the cell viability of GL261-NS and GL261-AC cells were then determined. Two commonly used anti-cancer agents, ACNU and VCR, were also used as controls. As shown in Fig. 2, salinomycin (0.01 μM-30 mM) significantly decreased the cell viability of GL261-NS and GL261-AC cells in a dose-dependent manner (P<0.05, compared with the DMSO control). Salinomycin appeared to preferentially inhibit the cell growth and viability of GL261-NS cells compared with GL261-AC cells with IC50 values of 0.06 and 0.58 μM, respectively. In addition, a salinomycin concentration of 0.1 μM or higher was more effective in decreasing cell viability than ACNU or VCR (P<0.05).

The effect of drug treatment on GL261-NS colony formation was then assessed. Although 30 μM ACNU or 10 μM VCR significantly decreased the sphere-forming ability of GL261-NS cells after 6 days of treatment, the administration of only 0.1 μM salinomycin markedly inhibited GL261-NS colony formation (SAL, 4.31 ± 1.53%; ACNU, 17.31 ± 8.53%; VCR, 10.41 ± 2.53%; DMSO, 52.31 ± 8.53%; P<0.05, SAL compared with the other three groups; Fig. 3). These results indicate that salinomycin is significantly more effective at reducing colony formation than standard chemotherapy.

Following this, the effect of salinomycin on GL261-NS apoptosis was assessed. The administration of SAL markedly increased apoptotic cell death (P<0.05; Fig. 4A). The expression of the apoptotic cascade protein caspase-3 was further examined in GL261-NS cells following 12 or 24 h of salinomycin treatment. Significantly elevated expression of activated caspase-3 was observed in cells treated with 10 μM compared with 0.1 μM salinomycin (P<0.05; Fig. 4B). The level of activated caspase-3 was also significantly reduced after 24 h of treatment compared with 12 h (P<0.05). Collectively, these data indicate that salinomycin promotes apoptosis of GL261-NS cells.

In order to determine the in vivo effects of salinomycin on the survival time of tumor-bearing mice, xenografts were treated with salinomycin and the overall survival rate was monitored. Treatment with salinomycin significantly prolonged the median overall survival of mice injected with GL261-NS cells compared with untreated controls (23 vs. 21 days, respectively; P<0.05; Fig. 5). All animals in the sham-surgery and sham-surgery plus salinomycin groups survived for the entire study period (60 days). These findings suggest that salinomycin may be an effective therapy for targeting GICs in glioma, reducing tumor burden and extending survival.