Impacts and Mechanisms of ADGRD1 on Ferroptosis and Oxidative Stress in Non-Small Cell Lung Cancer via the AKT/mTOR Pathway

Abstract

Background: Non-small cell lung cancer (NSCLC) is a common subtype of lung cancer that is usually associated with an increase in iron metabolism disorders and oxidative stress. Adhesion G protein-coupled receptor D1 (ADGRD1) is a receptor protein that plays important roles in a variety of biological processes. Therefore, we investigated the impacts and molecular mechanisms of ADGRD1 on ferroptosis and oxidative stress during the development of NSCLC. Methods: In this study, we used cells (BEAS-2B and A549) and mouse (specific pathogen-free (SPF) Balb/c nude male mice, n = 24) models. The tumor Gene Atlas (The Cancer Genome Atlas, TCGA) database was utilized to analyze the expression of ADGRD1 in NSCLC. We investigated the expression levels of ADGRD1, protein kinase B (AKT), mechanistic target of rapamycin (mTOR), glutathione peroxidase 4 (GPX4), cyclooxygenase-2 (Cox-2), cell cycle proteins, inflammatory factors and oxidative stress in NSCLC using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). ADGRD1 overexpression plasmid was constructed and transfected into the cells. The cells were treated with an AKT activator or AKT inhibitor. Additionally, subcutaneous implants of the transfected cells were made on the backs of nude mice to generate tumor tissues for further analysis. Results: In cell experiments, the model group had lower ADGRD1 expression and higher AKT and mTOR levels (p < 0.05). However, in the oe-ADGRD1 (cells with ADGRD1 overexpression) group, cell proliferation, migration, and invasion decreased, cell-cycle arrest in the G0-G1 phase, while apoptosis increased compared to the oe-NC (p < 0.05). Moreover, the oe-ADGRD1 group had lower AKT and mTOR expression (p < 0.05). Regarding inflammatory response and oxidative stress, the oe-ADGRD1 group exhibited significant reductions in interleukins-1β (IL-1β), interleukins-6 (IL-6), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA) levels (p < 0.05). Additionally, decreased Cox-2 and increased GPX4 expressions were observed in this group (p < 0.05). In animal experiments, the oe-ADGRD1 group demonstrated a considerable reduction in tumor volume and an improvement in pathological conditions based on hematoxylin-eosin (HE) staining. Moreover, it was revealed that AKT activation can reverse the therapeutic effect of oe-ADGRD1 on non-small cell lung cancer, while AKT inhibition can enhance its effect on NSCLC. Conclusions: ADGRD1 is downregulated, while AKT and mTOR are upregulated in NSCLC. Therefore, overexpression of ADGRD1 can synergistically inhibit AKT, reduce iron death and oxidative stress, and ultimately improve NSCLC.