Nerve Growth Factor Stimulates Glioblastoma Proliferation through Notch1 Receptor Signaling

Abstract

Objective: Notch receptors are heterodimeric transmembrane proteins that regulate cell fate, such as differentiation, proliferation, and apoptosis. Dysregulated Notch pathway signaling has been observed in glioblastomas, as well as in other human malignancies. Nerve growth factor (NGF) is essential for cell growth and differentiation in the nervous system. Recent reports suggest that NGF stimulates glioblastoma proliferation. However, the relationship between NGF and Notch1 in glioblastomas remains unknown. Therefore, we investigated expression of Notch1 in a glioblastoma cell line (U87-MG), and examined the relationship between NGF and Notch1 signaling.

Methods: We evaluated expression of Notch1 in human glioblastomas and normal brain tissues by immunohistochemical staining. The effect of NGF on glioblastoma cell line (U87-MG) was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. To evaluate the relationship between NGF and Notch1 signaling, Notch1 and Hes1 expression were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To confirm the effects of NGF on Notch1 signaling, Notch1 and Hes1 small interfering RNAs (siRNAs) were used.

Results: In immunohistochemistry, Notch1 expression was higher in glioblastoma than in normal brain tissue. MTT assay showed that NGF stimulates U87-MG cells in a dose-dependent manner. RT-PCR and Western blot analysis demonstrated that Notch1 and Hes1 expression were increased by NGF in a dose-dependent manner. After transfection with Notch1 and Hes1 siRNAs, there was no significant difference between controls and 100 nM NGF-β, which means that U87-MG cell proliferation was suppressed by Notch1 and Hes1 siRNAs.

Conclusion: These results indicate that NGF stimulates glioblastoma cell proliferation via Notch1 signaling through Hes 1.

Keywords: Glioblastoma; Nerve growth factor; Notch1.

Fig. 1.

Normal brain tissue (A) and negative control (B) show no staining, but human glioblastoma tissue (C) shows brown colored changes in the nucleus and cytoplasm by the streptavidin-biotin-peroxidase complex technique. Scale bar=200 μm.

Fig. 2.

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay shows that nerve growth factor (NGF) stimulates U87-MG cell proliferation in a dose-dependent manner (p=0.005).

Fig. 3.

Expression of Notch1 in the reverse transcription polymerase chain reaction (RT-PCR) (A) and a dose-dependent effect of nerve growth factor (NGF) on Notch1 expression in the U87-MG cell line (p=0.044) (B); expression of Notch1 in the Western blot analysis (C) and a dose-dependent effect of nerve growth factor (NGF) on Notch1 expression in the U87-MG cell line (p=0.037) (D). GAPDH : glyceraldehyde-3-phosphate dehydrogenase.

Fig. 4.

Expression of Hes1 in the reverse transcription polymerase chain reaction (RT-PCR) (A) and a dose-dependent effect of nerve growth factor (NGF) on Hes1 expression in the U87-MG cell line (p=0.020) (B); Expression of Hes1 in the Western blot analysis (C) and a dose-dependent effect of NGF on Hes1 expression in the U87-MG cell line (p=0.037) (D). GAPDH : glyceraldehyde3-phosphate dehydrogenase.

Fig. 5.

Expression of Notch1 mall interfering RNA (siRNA) in the Western blot analysis (A). Notch1 siRNA suppresses the effects of nerve growth factor (NGF) on U87-MG cell proliferation. There was difference in without lipofectamin, in lipofectamin only, and in negative control siRNA (p<0.05), but no significant difference in Notch1 siRNA (p=0.887) (B). Expression of Hes1 siRNA in the Western blot analysis (C). Hes1 siRNA suppresses the effects of NGF on U87-MG cell proliferation. There was difference in without lipofectamin, in lipofectamin only, and in negative control siRNA (p<0.05), but no significant difference in Hes1 siRNA (p=0.075) (D).