Bleomycin Inhibits Proliferation and Promotes Apoptosis of Brain Glioma Cells via TGF-β/Smad Signaling Pathway

J BUON. Mar-Apr 2020;25(2):1076-1083.

Abstract

Purpose: To investigate the influence of bleomycin (BLM) on the proliferation and apoptosis of brain glioma cells through transforming growth factor-β (TGF-β)/Smads signaling pathway.

Methods: The U87 brain glioma cells were cultured in vitro and reacted with different concentrations of BLM (5 and 10 mU/mL), and the cell growth status of each group was observed under a microscope. The cell proliferation activity was detected using Cell Counting Kit-8 (CCK-8) assay, the percentage of 5-Ethynyl-2'-deoxyuridine (EdU)-positive cells in each group was determined via EdU staining, and the apoptosis of U87 cells was tested by means of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to measure the messenger ribonucleic acid (mRNA) levels of genes related to proliferation, apoptosis and the TGF-β/Smads signaling pathway. Finally, western blotting assay was performed to analyze the expression of the TGF-β/Smads signaling pathway.

Results: In the 5 mU/mL BLM group, the glioma cells were in a poor growth status, with a low density, while the 10 mU/mL BLM group exhibited the poorest growth status and the lowest density, and the morphological structure trended toward normal. It was discovered via CCK-8 assay and EdU staining that the number of cells and proliferation activity were decreased markedly in the 10 mU/mL BLM group. According to TUNEL staining, 10 mU/mL BLM group had remarkably increased apoptotic cells, while negative control (NC) group had fewer apoptotic cells. The gene assay results revealed that the gene expressions of Bcl-2 and TGF-β1 declined notably in the 10 mU/mL BLM group but rose in the NC group, and the gene expression trends of Caspase-3 and Smad4 were the opposite. The protein assay results manifested that the expressions of TGF-β1 was obviously reduced, while that of Smad4 was evidently raised in the 10 mU/mL BLM group.

Conclusion: BLM at an appropriate concentration can inhibit the proliferation and promote apoptosis of brain glioma cells by repressing the TGF-β/Smads signaling pathway, thus ameliorating and treating brain glioma and other related diseases.