J Korean Neurosurg Soc.  2021 Sep;64(5):716-725. 10.3340/jkns.2021.0068.

Propranolol Inhibits the Proliferation of Human Glioblastoma Cell Lines through Notch1 and Hes1 Signaling System

• Kim HS
• Park YH
• Lee HS
• Kwon MJ
• Song JH
• Chang IB

• KMID: 2519698
• DOI: http://doi.org/10.3340/jkns.2021.0068

Abstract

Objective
: The anti-tumor effect of the beta-adrenergic receptor antagonist propranolol in breast cancer is well known; however, its activity in glioblastoma is not well-evaluated. The Notch-Hes pathway is known to regulate cell differentiation, proliferation, and apoptosis. We investigated the effect of propranolol to human glioblastoma cell lines, and the role of Notch and Hes signaling in this process.
Methods
: We performed immunohistochemical staining on 31 surgically resected primary human glioblastoma tissues. We also used glioblastoma cell lines of U87-MG, LN229, and neuroblastoma cell line of SH-SY5Y in this study. The effect of propranolol and isoproterenol on cell proliferation was evaluated using the MTT assay (absorbance 570 nm). The impact of propranolol on gene expression (Notch and Hes) was evaluated using real-time polymerase chain reaction (RT-PCR, whereas protein levels of Notch1 and Hes1 were measured using Western blotting (WB), simultaneously. Small interfering RNA (siRNA) was used to suppress the Notch gene to investigate its role in the proliferation of glioblastoma.
Results
: Propranolol and isoproterenol caused a dose-dependent decrease in cell proliferation (MTT assay). RT-PCR showed an increase in Notch1 and Hes1 expression by propranolol, whereas WB demonstrated increase in Notch1 protein, but a decrease in Hes1 by propranolol. The proliferation of U87-MG and LN229 was not significantly suppressed after transfection with Notch siRNA.
Conclusion
: These results demonstrated that propranolol suppressed the proliferation of glioblastoma cell lines and neuroblastoma cell line, and Hes1 was more closely involved than Notch1 was in glioblastoma proliferation.

Keyword

Glioblastoma; Neuroblastoma; Isoproterenol; Propranolol

Figure

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  Fig. 1. Immunohistochemical staining of primary human glioblastoma tissues for Notch1 by the streptavidin-biotin-peroxidase complex technique. Glioblastoma tissue shows negative staining with less than 10% of staining area (A), weak positive staining with 10–20% of staining area (b), moderate positive staining with 20–50% of staining area (c), and strong positive staining with more than 50% of staining area (d). Scale bar, 200 μm.

• 

  Fig. 2. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay shows that propranolol suppresses U87-MG, SH-SY5Y, and LN229 cell lines in a dose-dependent manner (p=0.013).

• 

  Fig. 3. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay shows that isoproterenol suppresses U87-MG, SH-SY5Y, and LN229 cell lines in a dose-dependent manner (p=0.013).

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  Fig. 4. Relative expression fold of Notch1 and Hes1 genes of U87-MG (A) and LN229 (b) in real time polymerase chain reaction (PcR) shows propranolol increases copy number of Notch1 and Hes1 genes (p=0.035). Expression of Notch1 and Hes1 protein of U87-MG (c) and LN229 (d) in Western blot demonstrates that propranolol stimulates Notch1 expression but suppresses Hes1 expression (p=0.021).

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  Fig. 5. Proliferation of glioblastoma cell lines in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. There are no significant differences between lipofectamine with negative control small interfering RNA (siRNA) and lipofectamine with active siRNA groups in control of both cell lines (p=0.157).

Reference

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