Ferroptosis mediates HMGB1-regulated autophagy in non-small cell lung cancer

This study aimed to determine whether ferroptosis regulates autophagy through high-mobility group box 1 (HMGB1) in non-small cell lung cancer (NSCLC) cells. A549 and H1299 NSCLC cells were assigned to the following groups: (i) control, treated with an equal volume of dimethyl sulfoxide (DMSO) vehicle; (ii) ferroptosis induction with 5 µM erastin; and (iii) ferroptosis rescue with 10 µM ferrostatin-1 (Fer-1) pretreatment followed by 5 µM erastin. To determine whether HMGB1 mediates ferroptosis-associated regulation of the autophagy-related protein Beclin-1, cells were additionally treated with ROCK/HDAC-IN-1 (1 µM), an inducer of DAMP/HMGB1 release, or the HMGB1 inhibitor HMGB1-IN-2 (1 µM). Colony formation assays, intracellular glutathione (GSH) and malondialdehyde (MDA) quantification, western blotting, reactive oxygen species (ROS) and ferrous iron (Fe²⁺) imaging, and flow cytometric analysis of lipid peroxidation (BODIPY 581/591 C11 and Liperfluo) were performed. Erastin reduced colony formation in a concentration-dependent manner and induced ferroptosis, characterized by a significant decrease in intracellular GSH levels ( P < 0.001) and elevated MDA, ROS, and Fe²⁺ levels ( P < 0.05), indicating oxidative stress and lipid peroxidation. Western blotting revealed downregulation of SLC7A11 and GPX4 with concomitant upregulation of HMGB1 and Beclin-1 ( P < 0.05); these alterations were reversed by Fer-1. Induction of HMGB1 release further upregulated Beclin-1 expression, whereas HMGB1 inhibition or ferroptosis suppression attenuated Beclin-1 upregulation ( P < 0.05). Erastin-induced ferroptosis is accompanied by HMGB1-dependent regulation of Beclin-1 in NSCLC cells, suggesting that HMGB1 may couple ferroptotic stress to autophagy-related responses. These preliminary in vitro findings warrant further mechanistic and in vivo validation.