Figure 4

Nuclear DNA fragmentation in cilengitide-treated cells and in U87ΔEGFR-derived xenografts. To confirm the apoptosis of deformed glioma cells treated with cilengitide, the cells were stained with the In Situ Cell Death Detection Kit using TMR red. U87ΔEGFR cells in culture were composed of bipolar cells (a). They become spherical and agglutinated when cilengitide (1.0 μM) was added to their culture medium. Some of these deformed cells detached from the plate (b). Untreated U87ΔEGFR cells were negative (c), whereas U87ΔEGFR cell clusters treated with cilengitide were positive (d) (scale bar: 100 μm). The number of apoptotic cells in cilengitide-treated cells was significantly larger than in control. (*P < 0.05) (e). QRT-PCR analysis of cilengitide-treated caspase 8 gene expression in tumors treated with PBS or cilengitide. * P = .007 (f) (mean ± SE, n = 4) A subpopulation of apoptotic cells was visualized by TUNEL treatment (apoptotic cells: TMR red; nuclei: DAPI, blue) of U87Δ EGFR control xenografts (g) and U87Δ EGFR cilengitide-treated xenografts (h) (Scale bar = 100 μm). The control sections exhibited scattered red fluorescence, whereas more punctate red fluorescence was observed in the cilengitide-treated xenografts. To quantify the cytotoxic effect of cilengitide, the number or percentage of apoptotic cells per high-power field (HPF) in U87ΔEGFR control xenografts and U87ΔEGFR cilengitide-treated xenografts was assessed. The number (i) or percentage (j) of apoptotic cells in cilengitide-treated xenografts was significantly larger than in control xenografts (*P < 0.05) (mean ± SE, n = 5).