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.
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.
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.
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.
Gliomas include neoplasm that originate from glial cells of the central nervous system (CNS). According to the World Health Organization Classification of Tumors of the CNS (CNS WHO) [
Notch signaling pathway plays an important role in cell differentiation and proliferation [
Propranolol is nonselective beta blocker that acts on both β1 and β2 receptors. Beta-adrenergic antagonists have tumor-suppressive effects on various cancers [
We investigated the effect of different propranolol concentration on the proliferation of glioblastoma cell lines and on the expression of Notch1 and Hes1.
This study was approved by the Hallym University Institutional Review Board (2019-03-007-001).
Two commercially available human glioblastoma cell lines (U87-MG and LN229) and one neuroblastoma cell line (SH-SY5Y) were de-identified and used in this study. Surgically resected, fixed a paraffin-embedded human glioblastoma tissues were obtained from our hospital.
Paraffin-embedded tissue sections of 31 primary human glioblastomas were used, and each tissue section was resected into six pieces (a total of 186 fields were evaluated). Immunostaining with rabbit polyclonal anti-Notch1 antibodies (ab27526; Abcam, Cambridge, UK) was performed at 1 : 50 dilution. The antibody was diluted in phosphate-buffered saline with 5% normal blocking serum. Biotinylated rabbit immunoglobulin G antibody (PK-6101; Vector Laboratories, Burlingame, CA, USA) was selected as the secondary antibody. Streptavidin-biotin-peroxidase complex was used to detect antibody-antigen reactions. Color development was performed with 3,3’-diaminobenzidine (SK-4100; Vector Laboratories) for one minute. Slides were counterstained with hematoxylin (H-3401; Vector Laboratories) and observed under the microscope. Normal brain tissues were used as negative controls. Notch1 signal was quantified by scoring 10 different tumor fields to determine the mean percentage of tumor cells with positive staining. The staining was divided as positive and negative for the qualitative verification of immunohistochemical staining.
For quantitative assessment, the staining was scored as follows : 1) negative, less than 10% area of positive cells; 2) weak positive, 10% to 20% area of positive cells; 3) moderate positive, 20% to 50% area of positive cells; and 4) strong positive, more than 50% area of positive cells. We did not calculate the percentage of positive cells but estimated the ratio in areas of the cancer cells.
The glioblastoma (U87-MG and LN229) and neuroblastoma (SH-SY5Y) cell lines were cultured in minimum essential medium containing 10% fetal bovine serum, an antibiotic combination (100 unit/mL penicillin and 0.1 mg/mL streptomycin; Gibco, Grand Island, NY, USA), and L-glutamine (2 mM). The cells were incubated at 37°C in an incubator containing 5% CO2. Cells were placed in a 96-well culture plate at a density of 1×104 cells/well in 200 µL culture media. After 24 hours incubation at 37°C, cells were treated for 48 hours with propranolol, isoproterenol and Notch1 small interfering RNA (siRNA) (HSS107248; Invitrogen, Carlsbad, CA, USA). The culture medium was thereafter replaced with 200 µL culture medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (M5655; Sigma-Aldrich, St. Louis, MO, USA). After incubation for 2 hours, the supernatant was removed and 200 µL dimethyl sulfoxide was added and the plates were incubated at 37°C for 30 minutes to dissolve the formazan precipitate, following which absorbance was measured at 570 nm using an automated microplate reader (Multiskan GO; Thermo Fisher Scientific, Vantaa, Finland). All experiments were repeated at least seven times.
The cell lines were dissolved in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium desoxycholate, 0.1% sodium dodecyl sulfate, 0.5 mM Tris, pH 8.0) on ice for 30 minutes and lysed for 30 minutes, followed by centrifugation for 20 minutes at 4°C. Protein quantification was performed using the Bradford assay (Bio-Rad, Glattbrugg, Switzerland). For all WBs, 50 µg total cellular protein was resolved per lane on a 7% Tris-acetate gel (Invitrogen) for Notch1 detection and transferred to nitrocellulose membranes (Schleicher and Schuell, Kassel, Germany). The transfer efficiency and loading accuracy was visually checked by Ponceau-S staining. Membranes were blocked overnight at 4°C with 5% weight/volume (w/v) nonfat dry milk/Tris-buffered saline and Tween-20 (0.05% w/v) and were treated thereafter with rabbit polyclonal anti-Notch1 antibodies (H-131; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for the detection of the extracellular domain of Notch1. All procedures were repeated more than three times. The densities of the bands were quantified using the ImageJ (1.47v; NIH, Bethesda, MD, USA), and the values were statistically analyzed.
Total RNA was isolated using TRIzol reagent (Invitrogen) and converted to cDNA using amfiRivert cDNA synthesis Platinum Master Mix (R5600; GenDEPOT, Barker, TX, USA) according to the manufacturer’s instructions. The primers (mentioned below) were used to amplify cDNA (PCR), and the products were separated on a 1% agarose gel containing ethidium bromide. All procedures were repeated more than five times. The band densities on the gel were quantified by the ImageJ. The density values were analyzed statistically. The following primers were used : 1) the Notch1 primer; sense : 5’-AGATCAACCTGGATGACTGTGCCA-3’, antisense : 5’-ACACGTAGCCACTGGTCATGTCTT-3’; 2) the Hes1 primer; sense : 5’-AGATCAACCTGGATGACTGTGCCA-3’, antisense : 5’-ACACGTAGCCACTGGTCATGTCTT-3’; 3) the β-actin primer; sense : 5’-GCACCACACCTTCTACAATA-3’, antisense : 5’-TGCTTGCTGATCCACATCTG-3’.
Statistical package for the Social Science software version 26 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Cell proliferation and Notch1 and Hes1 expression were analyzed by paired t-test and cross-tabulation.
Immunohistochemical staining was performed using the streptavidin-biotin-peroxidase complex technique to investigate Notch1 expression level in glioblastoma (
We investigated the effect of propranolol and isoproterenol on U87-MG, LN229, and SH-SY5Y cell lines. After treatment of cell lines with 50, 100, 150, 200, and 250 µM propranolol, cell proliferation was measured by MTT assay (
Similarly, mean OD values for propranolol-treated SH-SY5Y cells declined to 0.971±0.089, 0.892±0.077, 0.497±0.052, 0.401±0.033, and 0.253±0.023, respectively, when compared to untreated controls (0.994±0.071). The mean OD values showed a dose-dependent decrease after propranolol treatment of LN229 cells to 0.478±0.054, 0.452±0.042, 0.426±0.027, 0.350±0.031, and 0.298±0.019, respectively, compared to untreated controls (0.503±0.037).
In the case of isoproterenol treatment (0.1, 1, 5, 10, and 30 µM), the mean OD values of U87-MG cells declined to 0.639±0.057, 0.621±0.052, 0.583±0.048, 0.554±0.037, and 0.521±0.020, respectively, compared to untreated controls (0.663±0.054,
These results demonstrated that propranolol and isoproterenol suppressed U87-MG, SH-SY5Y, and LN229 cell lines in a dose-dependent manner (
After observing that propranolol suppressed proliferation of all the cell lines, Notch1 and Hes1 expression was evaluated in U87-MG and LN229 cell lines by RT-PCR and WB analysis to evaluate the effect of propranolol on Notch1 and Hes1 signaling (
The mean density of Notch1 in the WB analysis was 178±21 in U87-MG and 691±72 in LN229 cells, whereas the mean density of Hes1 was 48.35±6.73 in U87-MG and 31.33±5.54 in LN229 cells.
These results demonstrated that propranolol increased Notch1 and Hes1 gene expression (
To verify the effects of propranolol on cell line proliferation, siRNA targeting Notch1 was used. The efficacy of siRNA for reducing the target was quantified using quantitative PCR (
Rundle-Thiele et al. [
One study reported that propranolol downregulated primary tumor expression of mesenchymal genes [
As mentioned above, Notch signaling is involved in cell differentiation, proliferation, migration, self-renewal and apoptosis [
Protein and mRNA levels of Notch1 and Hes1 are higher in brain tumor cells than normal brain cells [
Several reports using the same type of cell lines as in this paper have been studied regarding glioblastoma proliferation. Kusaczuk et al. [
This study has some limitations. First, commercialized glioblastoma cell lines were used with
This study suggests that propranolol suppresses glioblastoma proliferation in a dose-dependent manner. Propranolol could be a new therapeutic option for glioblastoma patients. While various efforts and treatment modalities are being made to treat glioblastoma, survival is still very poor in the majority of patients. As discussed in this study, further exploration for understanding the molecular-level mechanisms should be made to design new effective strategies to cure glioblastoma in the future.
No potential conflict of interest relevant to this article was reported.
This type of study does not require informed consent.
Conceptualization : IBC, HSK
Data curation : IBC, HSK, YHP, MJK, JHS, HSL
Formal analysis : IBC, HSK
Methodology : HSK, YHP, MJK, JHS
Project administration : HSK, IBC
Visualization : HSK, HSL
Writing - original draft : HSK
Writing - review & editing : IBC
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.
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 (
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 (
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 (
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 (