Cilengitide induces cell detachment and cytotoxic effects in glioma cells. U251 and U87 cells were plated onto vitronectin-coated dishes and treated with cilengitide (25 μg/mL) for the indicated times. The detachment of the cells was evaluated using phase-contrast microscopy (A). Cell viability was measured in U251 and U87 cells treated with cilengitide 25 μg/mL for different time points (B) or with different concentrations of cilengitide for 24 h (C) using the WST assay. Cell apoptosis of the U251 and U87 cells was determined using propidium iodide staining and FACS analysis (D) or measurement of active caspase-3 expression (E) in cells treated with cilengitide (25 μg/mL) after 24 and 48 h of treatment. The results represent 1 of 3 experiments with similar results (A and E) or show the mean ± SE of 3 independent experiments (B–D). *P < .01, **P < .001.

Cilengitide induces autophagy in glioma cells. U251 cells were transfected with LC3-GFP, and after 24 h the cells were treated with cilengitide for 8 h (25 μg/mL). The U251 cells were visualized using a confocal microscope (C1, Nikon), and cells with punctated LC3-GFP appearance were observed (A). U87 and U251 cells were transfected with LC3-GFP and treated with either different concentrations of cilengitide for 12 h or with temozolomide (TMZ, 100 μM) for 48 h. The percentage of cells with puncta LC3-GFP of the total cells was determined for control and treated cells (B). The expression of LC3I and LC3II was measured in U251 and U87 cells treated with cilengitide (25 μg/mL) for different time points (C). U251 cells were treated with cilengitide (25 mg/mL) for 24 h and with TMZ (100 ng/mL) for 48 h. The development of AVOs in control and treated cells was determined using acridine orange staining and FACS analysis (D). The results are representative of 3 different experiments (A, C. and D) or are the mean ± SE of 3 different experiments (B). *P< .01, **P< .001.

Inhibition of autophagy decreases the cytotoxic effect of cilengitide in glioma cells. U251 and U87 cells were treated with 3-methyladenine (3-MA) (1 mM) or bafilomycin (10 nM) for 30 min followed by cilengitide (25 μg/mL). The cells were then incubated for 24 h and examined for cell viability using the WST-1 assay (A) or for the presence of AVOs using acridine orange staining and FACS analysis (B). U251 and U87 cells were transfected with control or beclin-1 shRNA plasmids using electroporation. The expression of beclin-1 was examined in the cells after 72 h (C). Cells in which the expression of beclin-1 was silenced were treated with cilengitide (25 μg/mL), and the presence of AVOs (D) or the viability of the cells using the WST-1 assay (E) was examined after 24 h. The results are the mean ± SE of 3 different experiments (A, B, D and E) or are representative of 3 different experiments (C). *P < .001.

Combined treatment of glioma cells with cilengitide and γ-radiation. U251 and U87 cells were treated with cilengitide for 4 h followed by γ-irradiation of the cells (1 and 2 Gy). Following 48 h of incubation, the cells were assessed for the expression of LC3I and LC3II (A) and for cell viability using the WST assay (B). The clonogenic assay was performed as described in Materials and Methods, and clonogenic survival was determined following 14 days of treatment (C). The results are representative of 3 different experiments (A) or are the mean ± SE of 3 different experiments (B and C). *P < .01; **P < .001.

Cilengitide induces autophagy and inhibits the self-renewal of GSCs. The GSCs HF2414 (A) and HF2354 (B) were treated with cilengitide (10 and 25 μg/mL), and the neurosphere formation assay was performed. The number of neurospheres per well was quantified after 14 days for 8 different wells. Spheres that contained more than 20 cells were scored, and the results are presented as percentages of maximal neurospheres formed in control untreated cells (A and B). The morphology of HF2354 treated with cilengitide (25 mg/mL) for 24 h was followed using a phase-contrast microscope (C). The expression of LC3I and LC3II was measured in the HF2414 and HF2354 cells treated with cilengitide (25 mg/mL) for 24 h (D). HF2414 and HF2354 cells were treated with cilengitide (25 mg/mL) for 4 h followed by γ-irradiation of the cells (1 and 2 Gy). The expression of LC3I and LC3II was measured after 24 h (E), and the neurosphere formation assay was performed after 14 days of treatment (F). Results are representative of 3 different experiments (C–E) or are the mean ± SE of 3 experiments (A, B, and F). *P < .001 compared with cilengitide-treated cells.

Induction of autophagy by cilengitide in U251-derived xenografts. U251 cells expressing LC3-GFP (400 000 in 5 µL) were intracranially implanted into nude rat brains and allowed to grow for 21 days. Cilengitide was then administered intraperitoneally, and the animals were killed 48 h later. Brains were harvested and immersed in cold saline, and 2-mm sections were then embedded in optimal cutting temperature embedding medium and snap-frozen in an isopentane bath. Each block was mounted on a cryostat tissue holder, and 8-µL frozen sections were cut. Tumor sections were examined using fluorescent microscopy (A, magnification ×60) or were immersed in a solution of propidium iodide and viewed using confocal microscopy (B, magnification ×60). The images are representative of 6 rats per group.