博来霉素通过 TGF-β/Smad 信号通路抑制脑胶质瘤细胞的增殖并促进其凋亡。

Bleomycin inhibits proliferation and promotes apoptosis of brain glioma cells via TGF-β/Smad signaling pathway.

机构信息

Department of Neurosurgery, the first People's Hospital of Huaihua City, Huaihua, Hunan 418000, China.

出版信息

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

PMID:32521909

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.

摘要

目的

通过转化生长因子-β（TGF-β）/Smads 信号通路研究博来霉素（BLM）对脑胶质瘤细胞增殖和凋亡的影响。

方法

体外培养 U87 脑胶质瘤细胞，分别用不同浓度 BLM（5 和 10 mU/mL）处理，显微镜下观察各组细胞生长状态。用细胞计数试剂盒-8（CCK-8）法检测细胞增殖活性，用 5-乙炔基-2'-脱氧尿苷（EdU）染色法检测各组 EdU 阳性细胞的百分比，用末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）染色法检测 U87 细胞的凋亡。此外，采用逆转录-聚合酶链反应（RT-PCR）法检测与增殖、凋亡及 TGF-β/Smads 信号通路相关的基因的信使核糖核酸（mRNA）水平。最后，采用蛋白质印迹法（western blotting）分析 TGF-β/Smads 信号通路的表达。

结果

在 5 mU/mL BLM 组，胶质瘤细胞生长状态差，密度低，而在 10 mU/mL BLM 组，生长状态最差，密度最低，形态结构趋于正常。CCK-8 法和 EdU 染色结果显示，10 mU/mL BLM 组细胞数量和增殖活性明显减少。TUNEL 染色结果显示，10 mU/mL BLM 组凋亡细胞明显增多，而阴性对照组（NC）组凋亡细胞较少。基因检测结果显示，10 mU/mL BLM 组 Bcl-2 和 TGF-β1 基因表达明显下降，但 NC 组表达升高，Caspase-3 和 Smad4 基因表达趋势相反。蛋白检测结果显示，10 mU/mL BLM 组 TGF-β1 表达明显减少，Smad4 表达明显升高。