Glioma-initiating cells are a small population of cells that have the

Glioma-initiating cells are a small population of cells that have the ability to undergo self-renewal and initiate tumorigenesis. and induced cell apoptosis. In addition salinomycin long term the median survival time of glioma-bearing mice (P<0.05). Therefore the present study indicated that salinomycin may preferentially inhibit glioma-initiated cell growth by inducing apoptosis suggesting that salinomycin may provide a valuable restorative strategy for the treatment of malignant glioma. which functions as an ionophore with a high affinity for potassium (6 7 In addition to its well-established antimicrobial activities it has previously been demonstrated to act as a specific inhibitor of breast tumor stem cells (8). Even though mechanism of BILN 2061 action of salinomycin remains to be elucidated it has been reported that salinomycin may serve as a permeability-glycoprotein inhibitor therefore impairing the viability of malignancy stem cells (9). By contrast salinomycin has also been demonstrated to induce apoptosis in malignancy cells and overcome apoptotic resistance in human breast tumor cells (10). Nevertheless the activity of salinomycin in growth suppression and tumorsphere formation in glioma particularly GICs remains to be elucidated. In the present study the and effects of salinomycin on GL261 glioma cells were investigated. The present study may provide important insights into understanding the pathogenesis of GICs and may offer a novel therapeutic approach for the treatment of human being malignant glioma. Materials and methods Reagents Dulbecco’s revised Eagle’s medium (DMEM)/F12 culture medium was purchased from Life Systems (Gaithersburg MD USA). Fetal bovine serum (FBS) and B27 product were purchased from Gibco-BRL (Grand Island NY USA). Fundamental fibroblast CIT growth element (bFGF) and epidermal growth factor (EGF) were from PeproTech BILN 2061 (Rocky Hill NJ USA). Normal goat serum was provided by Wuhan Boshide Biotechnology Co. Ltd. (Wuhan China). Poly-L-lysine 4 6 (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 being) and anti-β-actin antibody were from Abcam Inc. (Cambridge MA USA). Cell Counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Tokyo Japan). Cell tradition GL261 cells were from the American Type Tradition Collection (ATCC Manassas VA USA) and were cultured in DMEM/F12 tradition medium comprising 10% FBS. Cells BILN 2061 were managed at 37°C inside a humidified environment comprising 5% CO2. The medium was changed every 4 days. To induce the formation of neurospheres (NS) FBS was gradually withdrawn from tradition medium inside a gradient reduction pattern (10 5 2 and 0%). Cells were then managed 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 created NS-like clones and secondary spheres derived from solitary 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 tradition medium comprising 10% FBS. These cells BILN 2061 were defined as GL261 adherent cells (AC). Immunocytochemistry analysis The spheres were placed onto coverslips precoated with poly-L-lysine (Sigma) and then fixed with 4% paraformaldehyde for 20 min at space temperature. Cell samples were blocked with normal goat serum and then incubated with the following respective main antibodies: BILN 2061 Rabbit anti-mouse CD133/1 monoclonal antibody (1:50 dilution) and GFAP polyclonal antibody (1:100 dilution) over night. 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 having a confocal laser scanning microscope (Leica Mannheim Germany). Dedication of cell viability The effect of drug treatment on cell viability was identified using the CCK-8 kit. Briefly cells were seeded onto a 96-well-plate and then treated 24 h after seeding with different.