Ammonium Ferric Citrate induced Ferroptosis in Non-Small-Cell Lung Carcinoma through the inhibition of GPX4-GSS/GSR-GGT axis activity

Abstract

The morbidity and mortality rates associated with non-small-cell lung carcinoma (NSCLC) are increasing every year, placing new demands on existing therapies and drugs. Ammonium ferric citrate (AFC) is often used as a food additive for iron supplementation; however, to our knowledge, no studies have investigated whether AFC can induce ferroptosis in NSCLC. In this study, we demonstrated that specific concentrations of AFC effectively inhibit the proliferation and invasion of lung cancer cell lines in vitro using a cell proliferation inhibition test, a transwell assay, and flow cytometry analysis of cell cycle and apoptosis. In addition, AFC significantly induced oxidative stress injury in lung cancer cell lines. A quantitative polymerase chain reaction assay showed that AFC markedly reduced the expression levels of cell growth factors, negative regulators of ferroptosis, and autophagy regulators. Lastly, a protein-protein interaction analysis revealed that glutathione peroxidase 4 (GPX4) exerted its biological role through the regulation of the GSS/GSR complex and downstream GGT family proteins. When the expression of GPX4 changes, its biological activities, such as the glutathione metabolic process, cellular biosynthetic process, cellular response to chemical stimulus, and antioxidant activity, change accordingly, thereby affecting the survival quality and physiological and biochemical activities of cells. Overall, this study verifies that AFC has the biological activity of activating oxidative stress injury in NSCLC cell lines, leading to a decrease in their autophagy and inducing ferroptosis. We also confirmed that the GPX4-GSS/GSR-GGT axis is a crucial target of AFC-induced ferroptosis.

Keywords: ammonium ferric citrate; autophagy; ferroptosis; non-small-cell lung carcinoma.

Figure 1

AFC significantly inhibited the proliferation and invasion of the NSCLC cell line in vitro. (A) The molecular structure of AFC. (B) The MTT assay results showing that AFC significantly inhibited the proliferation of the NSCLC cell line in vitro. **p < 0. 01 vs. PBS control group, t-test, n=3. (C) The transwell assay results showing that AFC significantly inhibited the migration of the NSCLC cell lines in vitro. **p < 0. 01 vs. PBS control group, t-test, n=3.

Figure 2

AFC significantly inhibited cell cycle progression in NSCLC cell lines. (A) PI staining/FCM assay results showing that AFC significantly inhibited cell cycle progression in the NSCLC cell lines. **p < 0.01 vs. PBS control group, *p < 0.05 vs. PBS control group, t-test, n=3. (B) Annexin V-FITC staining/FCM assay results showing that AFC significantly promoted apoptosis in NSCLC cell lines. **p < 0. 01 vs. PBS control group, t-test, n=3.

Figure 3

AFC caused elevated Fe²⁺ content in NSCLC cell lines and induced oxidative stress injury. (A) The FCM assay results showing that AFC significantly elevated the Fe²⁺ content in NSCLC cell lines. (B) The FCM assay results showing that AFC significantly increased the ROS levels in NSCLC cell lines. (C) The biochemical assay results showing that AFC significantly increased the MDA content in NSCLC cell lines but significantly decreased SOD, ATP, and GPX levels in the NSCLC cell lines.

Figure 4

AFC promoted differential gene expression profiles of proliferation, autophagy, and ferroptosis in NSCLC cell lines. (A) Heat map of qPCR assay results. (B) The qPCR assay results showing that the mRNA expression levels of some cell proliferation inhibitors and ferroptosis promoting factors were significantly increased in the AFC cell group. **p < 0.01vs. PBS control group, *p < 0.05 vs. PBS control group, t-test, n=3. (C) The qPCR assay results showing that the mRNA expression levels of some cell cycle proliferation factors, autophagy promoting factors, and negative regulators of ferroptosis were significantly decreased in the AFC cell group. ** p < 0.01vs. PBS control group, *p < 0.05 vs. PBS control group, t-test, n=3. (D) A comprehensive analysis showing that the mRNA expression levels of two genes were significantly upregulated, and those of eight genes were significantly downregulated in AFC-treated NSCLC cell lines.

Figure 5

AFC promoted differential gene expression profiles of proliferation, autophagy, and ferroptosis in NSCLC cell lines. (A) A protein-protein interaction network prediction software result showing that GPX4 catalysed the expression of downstream GSS/GSR-GGT axis. (B) GO analysis results showing that the expression level of GPX4 significantly affected biological processes, such as small molecule metabolic processes, as well as molecular functions, such as catalytic activity. (C) KEGG pathway analysis results showing that GPX4 was highly correlated with the regulation of glutathione metabolism and ferroptosis signalling pathways. (D) Western blotting results showing that AFC significantly affected the expression of the GPX4-GSS/GSR-GGT axis, autophagy-associated proteins, and ferroptosis regulatory proteins in NSCLC cell lines.

Figure 6

Ammonium ferric citrate induced ferroptosis in non-small-cell lung carcinoma through the inhibition of gpx4-gss/gsr-ggt axis activity.