Gene expression profiling of the anti-glioma effect of Cilengitide

Abstract

Cilengitide (EMD121974), an inhibitor of the adhesive function of integrins, demonstrated preclinical efficacy against malignant glioma. It is speculated that cilengitide can inhibit tumor growth, invasion, and angiogenesis. However, the effects of cilengitide on these processes have not been sufficiently examined. In this study, we investigated the anti-glioma effect of cilengitide using DNA microarray analysis. U87ΔEGFR cells (human malignant glioma cell line) were used for this experiment. The cells were harvested after 16 h of cilengitide treatment, and mRNA was extracted. Gene expression and pathway analyses were performed using a DNA microarray (CodeLink™Human Whole Genome Bioarray). The expression of 265 genes was changed with cilengitide treatment. The expression of 214 genes was up-regulated by more than 4-fold and the expression of 51 genes was down-regulated by more than 4-fold compared to the controls. In pathway analysis, "apoptotic cleavage of cellular proteins" and "TNF receptor signaling pathway" were over-represented. Apoptotic-associated genes such as caspase 8 were up-regulated. Gene expression profiling revealed more detailed mechanism of the anti-glioma effect of cilengitide. Genes associated with apoptosis were over-represented following cilengitide treatment.

Keywords: Apoptosis; Cilengitide; Gene expression profiling; Glioma; Integrin.

Figure 1

Immunohistochemical analysis of the αvβ3 and αvβ5 integrins, and WST-1 proliferation/viability assay. Cultured U87ΔEGFR cells were immunopositive for αvβ3 (a) and αvβ5 (b) integrins (Scale bar = 100 μm). Cilengitide reduced the number of viable cells in a dose and time -dependent manner (*P < 0.05) (mean ± SE) (c, d).

Figure 2

Microarray and QRT-PCR analyses of cilengitide-treated cells. There were 265 differentially expressed genes between cilengitide-treated U87ΔEGFR cells and untreated control U87ΔEGFR cells with 214 upregulated (a) and 51 downregulated (b) genes. Caspase-8 (c) and protein kinase C, zeta (d) were significantly increased by cilengitide treatment (*P < 0.05) (mean ± SE, n = 3).

Figure 3

QRT-PCR analysis and activation of caspase-3/7 and -8 in cilengitide-treated cells. U87ΔEGFR cells were treated with 0.5 μM cilengitide for 16 h with the colorimetric protease assay kit. For the activity of caspase-8, the relative absorbance (RA) of U87ΔEGFR cell clusters were higher than control (0.27 ± 0.01 RA vs. 0.36 ± 0.01 RA, respectively; P < 0.05) (a). Cilengitide-induced apoptosis was also detected with the CellEvent™ Caspase-3/7 Green Detection Reagent. The RFU of U87ΔEGFR cell clusters were higher than control (35.4 ± 0.78 RFU vs. 16.5 ± 0.5 RFU, respectively; P < 0.05) (b). Immunoblot analysis revealed that caspases 3 and 8 were processed in all examined cell lines following treatment with cilengitide in a concentration- (c) and time-dependent manner (d).

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 U87ΔEGFR control xenografts (g) and U87ΔEGFR 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).