Dihydroartemisinin Engages NQO1 and Promotes Ferroptosis‐Related Oxidative Stress in Non‐Small Cell Lung Cancer

ABSTRACT

Dihydroartemisinin (DHA), a bioactive derivative of Artemisia annua L. (AAL), has shown antitumor activity in several malignancies, including non‐small cell lung cancer (NSCLC). However, the molecular determinants linking DHA exposure to ferroptosis in NSCLC remain incompletely understood. This study aimed to identify functionally relevant targets of DHA‐associated ferroptotic stress and to investigate the role of NAD(P)H quinone dehydrogenase 1 (NQO1) in this process. An integrated network pharmacology and transcriptomic approach was used to identify ferroptosis‐related candidate targets of A. annua . Molecular docking and molecular dynamics simulations were used for target prioritization, followed by surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA) to evaluate the interaction between DHA and NQO1. The effects of DHA on ferroptosis‐related phenotypes and redox homeostasis were examined in A549 and PC9 cells. NQO1 overexpression, NQO1 knockdown, and GPX4 rescue experiments were performed to assess pathway involvement. Antitumor activity and NQO1‐associated effects were further evaluated in mouse tumor models. Network pharmacology identified 15 active components of AAL and 61 NSCLC‐associated therapeutic targets enriched in ROS‐related and ferroptosis‐associated pathways. Among 10 ferroptosis‐related core targets, NQO1 was prioritized based on disease relevance, predicted DHA interaction, selective protein suppression, and complementary computational and experimental evidence. DHA was selected for mechanistic validation based on its pharmacological relevance and anti‐NSCLC activity. SPR supported direct DHA‐NQO1 binding (KD = 7.61 × 10 ⁻⁶  M). Mechanistically, DHA suppressed NQO1 expression through transcriptional inhibition and enhanced protein degradation. DHA induced ferroptosis hallmarks, including ROS burst, LPO and Fe ²⁺ accumulation, GSH/GSSG depletion, MDA elevation, and GPX4 inactivation, in A549 and PC9 cells. NQO1 overexpression partially reversed these effects, whereas NQO1 knockdown phenocopied DHA‐induced ferroptotic responses. GPX4 overexpression partially rescued the ferroptotic phenotype induced by NQO1 depletion, supporting a functional link between NQO1 and the GPX4 antioxidant axis. In vivo, DHA suppressed tumor growth, an effect partially reversed by NQO1 overexpression. DHA triggers ferroptosis in the NSCLC models examined, particularly in LUAD‐derived cell models, in part through NQO1‐mediated ROS burst and impairment of the GSH/GPX4 antioxidant defense. These findings identify NQO1 as a critical mediator of DHA‐induced ferroptosis in these models and provide a mechanistic rationale for further investigation of DHA and related artemisinin derivatives in NSCLC.