Astragalus Polysaccharides and Metformin May Have Synergistic Effects on the Apoptosis and Ferroptosis of Lung Adenocarcinoma A549 Cells

Abstract

Astragalus polysaccharides (APSs), the compounds extracted from the common herb Astragalus membranaceus, have been extensively studied for their antitumor properties. In this study, we investigated the effect of APS on lung adenocarcinoma A549 cells. The effects of APS and the anti-diabetic drug metformin on apoptosis and ferroptosis were compared. Furthermore, the combination treatment of APS and metformin was also investigated. We found that APS not only reduced the growth of lung cancer cells but also had a synergistic effect with metformin on A549 cells. The study results showed that it may be promising to use APS and metformin as a combination therapy for the treatment of lung adenocarcinoma.

Keywords: A549 cells; Astragalus polysaccharides (APS); apoptosis; ferroptosis; lung cancer; metformin.

Figure 1

Synergistic effect of APS and metformin on A549 cell viability. A549 cell viability under the treatment of (a,b) APS/metformin alone for (a) 24 h and (b) for 48 h. (c,d) APS in combination with different concentrations of metformin after treatment of (c) 24 h and (d) 48 h. The combination group significantly decreased compared to using either APS or metformin alone. All the results are representative of at least three independent experiments. (Error bars = mean ± S.E.M. Asterisks mark samples significantly different from control group with (*) p < 0.05; (**) with p < 0.01; (***) with p < 0.001).

Figure 2

The apoptotic effect on A549 cells of APS, metformin detected by flow cytometry with annexin V-FITC/PI dual staining: (a,c) A549 cells treated for 24 h and (b,d) for 48 h. (Error bars = mean ± S.E.M. Asterisks mark samples significantly different from control group with (**) with p < 0.01; (***) with p < 0.001).

Figure 3

The mitochondrial depolarization stained with Mitoscreen JC-1 assay detected by flow cytometry. Dot plots showing depolarisation of mitochondria in treated A549 cells. The percentage of events in the upper gate (P2) and the percentage of events in the (P3) represent the population of treated A549 cells with normal and depolarised mitochondria, respectively. (a,c) A549 cells treated for 24 h and (b,d) for 48 h. (Error bars = mean ± S.E.M. Asterisks mark samples significantly different from control group with (*) p < 0.05; (**) with p < 0.01; (***) with p < 0.001).

Figure 4

A549 were treated with indicated treatments for (a) 24 h and (b) 48 h and then harvested. The prepared cell protein was immunoblotted with polyclonal antibodies specific for PARP and cleaved PARP. Vinculin was used as an internal loading control. (Error bars = mean ± S.E.M. Asterisks mark samples significantly different from control group with (*) p < 0.05; (**) with p < 0.01).

Figure 5

Effect of APS, metformin, and their combination on ferroptosis of A549 cells in vitro. The A549 cells were treated with either APS, metformin, or their combination under different concentrations for 24 h and then collected for the following test: (a) ROS assay; (b) MAD assay; (c) GSH levels; (d) Western blot for GPx4 protein. B-actin was used as the internal control. (Error bars = mean ± S.E.M. Asterisks mark samples significantly different from control group with (*) p < 0.05; (**) with p < 0.01; (***) with p < 0.001).