Background: Ferroptosis is an iron-dependent form of regulated cell death, which offers therapeutic potential in non-small cell lung cancer (NSCLC). This study aimed to evaluate whether Micheliolide (MCL) induces ferroptosis in NSCLC cells via the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway.

Methods: H1299 cells were treated with different concentrations of MCL. Cellular assays, including the Cell Counting Kit-8 (CCK-8), colony formation, scratch test, and Transwell, were used to evaluate the viability, proliferation, migration, and invasion of the cells. Ferroptosis-related indicators (reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), Fe²⁺) and protein expression (Nrf2, glutathione peroxidase 4 (GPX4), Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4), Transferrin Receptor 1 (TFR1) were assessed via flow cytometry, biochemical assays, and Western blot. Additionally, Nrf2-specific activator (tert-butylhydroquinone (tBHQ)) and ferroptosis inhibitors (Fer-1) were used to verify the involvement of this pathway.

Results: MCL inhibited proliferation, migration, and invasion of H1299 cells in a dose-dependent manner and induced ferroptosis as evidenced by elevated ROS, MDA, and Fe²⁺ levels, and reduced GSH concentration. Western blot analysis revealed significantly increased ACSL4 and TFR1 and decreased Nrf2 and GPX4 expression. These MCL-induced effects were reversed by tBHQ or Fer-1, confirming the involvement of Nrf2.

Conclusions: MCL induces ferroptosis in NSCLC cells by suppressing the Nrf2 pathway. Targeting Nrf2 may enhance the anticancer efficacy of Micheliolide, offering new therapeutic insights for NSCLC.