Cordycepin Induces Ferroptosis in A549 Lung Cancer Cells via Nrf2/HO-1 Pathway Suppression: An In Vitro and Network Pharmacology Study

Background: Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide, and novel therapeutic strategies are urgently needed to overcome treatment resistance. Cordycepin (Cor), a bioactive nucleoside isolated from Cordyceps militaris, exhibits antitumor activity in multiple malignancies. However, whether Cor induces ferroptosis in NSCLC cells and the underlying mechanisms remain unclear. Therefore, this study aimed to investigate the modulatory effects of Cor on ferroptosis in NSCLC cells and to elucidate the underlying molecular and network pharmacology mechanisms. Methods: Human NSCLC A549 cells were treated with Cor (25, 50, and 100 μM). Cell viability was assessed using Cell Counting Kit-8 assays. Ferroptosis involvement was evaluated using ferroptosis inhibitors (ferrostatin-1, deferoxamine, and liproxstatin-1) and other programmed cell death inhibitors. Intracellular reactive oxygen species (ROS), Fe2+ accumulation, lipid peroxidation, glutathione (GSH) levels, and ferroptosis-related proteins were measured using fluorescence probes, spectrophotometric assays, and western blotting. Network pharmacology, protein-protein interaction analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, and molecular docking were performed to identify potential molecular targets. Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expression was further examined by western blotting. Results: Cor inhibited A549 cell viability in a time- and dose-dependent manner (n = 6). Among the tested death inhibitors, only ferroptosis inhibitors significantly reversed Cor-induced cytotoxicity (p < 0.05, n = 3). Cor treatment elevated intracellular ROS levels, Fe2+ accumulation, and lipid peroxidation while reducing GSH content (p < 0.05, n = 3). At the protein level, Cor downregulated glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression but upregulated transferrin and acyl-CoA synthetase long-chain family member 4 (ACSL4) expression (p < 0.05, n = 3). Database screening identified 28 mutual targets linked to Cor, NSCLC, and ferroptosis. Molecular docking confirmed that Cor spontaneously binds to the Nrf2 and HO-1 proteins encoded by the key pathway genes nuclear factor erythroid 2 like 2 (NFE2L2) and heme oxygenase 1 (HMOX1), which correlated with a significant, dose-dependent downregulation of Nrf2 and HO-1 protein expression (p < 0.05, n = 3). Conclusions: Cordycepin induces ferroptosis in A549 NSCLC cells, characterized by oxidative stress, iron accumulation, lipid peroxidation, and suppression of antioxidant defenses. The Nrf2/HO-1 pathway may contribute to this process and represents a potential therapeutic target for NSCLC.