Metformin promotes apoptosis of A549 cells via regulation of p-AMPK protein expression, bax/bcl-2 ratio and ROS levels

Ziying Yu,Fengtao Liu,Xiaoli Zhang

doi:10.4314/tjpr.v20i9.7

Abstract

Purpose: To investigate the influence of metformin on apoptosis of pulmonary carcinoma cells (A549), and the associated mode of action.Methods: Pulmonary carcinoma cells in logarithmic growth phase were treated with graded concentrations of metformin, and the anti-proliferative and apoptotic effects of the drug were measured using MTT assay and flow cytometry, respectively. The levels of reactive oxygen species (ROS) in A549 cell suspension were determined with 2, 7- dihydrodichlorofluorescein diacetate (DCFH-DA) assay. The expression levels of phosphorylated AMP-activated protein kinase (p-AMPK), mammalian target of rapamycin (mTOR), and bax/bcl-2 ratio were measured using Western blotting and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR).Results: Metformin significantly promoted A549 cell apoptosis, but suppressed its proliferative potential in a dose- and time-based fashion. The levels of ROS, superoxide anion and MDA in A549 cells were significantly and dose-dependently increased by metformin (p < 0.05). Moreover, metformin markedly upregulated the mRNA and protein expressions of p-AMPK as well as bax/bcl-2 ratio, but had no impact on the expression level of mTOR (p < 0.05).Conclusion: Metformin promotes apoptosis in A549 cells via regulation of p-AMPK protein expression, bax/bcl-2 ratio, and ROS levels, and hence may play a role in lung cancer therapy.

Highlights

Pulmonary carcinoma, a frequently diagnosed malignancy in clinical practice, is associated with high morbidity and mortality
Bax antibody was bought from Shanghai Hengyuan Biotechnology Company; bcl-2 antibody was obtained from EmMETt Technologies, while phosphorylated AMP-activated protein kinase (p-AMPK) antibody was supplied by Xiamen Huijia Biotech
A549 cells treated with varied concentrations of metformin were PBS-rinsed after they were incubated for 72 h, followed by addition of DCFH-DA (10 μM) to each well

Summary

Introduction

A frequently diagnosed malignancy in clinical practice, is associated with high morbidity and mortality. It has been speculated that metformin inhibits the growth of tumor cells via activation of adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, as well as promotion of protein synthesis and cell apoptosis [5]. This research was carried out to determine the influence of metformin on A549 cell apoptosis, as well as the associated mode of action. Bax antibody was bought from Shanghai Hengyuan Biotechnology Company; bcl-2 antibody was obtained from EmMETt Technologies, while p-AMPK antibody was supplied by Xiamen Huijia Biotech.

Results

Conclusion