Quercetagitrin targets EIF3D to activate NCOA4-mediated ferritinophagy-dependent ferroptosis for the treatment of non-small cell lung cancer.

[BACKGROUND] Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer. Although traditional treatment methods such as surgery, chemotherapy, and radiotherapy can extend patient survival to some extent, they still present significant challenges due to their limited efficacy and substantial side effects. Ferroptosis, a form of iron-dependent programmed cell death, has been shown to exhibit considerable potential in the treatment of NSCLC. However, the molecular mechanisms underlying ferroptosis and how to safely and effectively induce it remain to be fully explored. This study aims to investigate the mechanism by which the natural flavonoid compound Quercetagitrin (Que) regulates the ferritinophagy-ferroptosis pathway to suppress NSCLC and to identify its molecular targets.

[METHODS] This study evaluated the selective toxicity of Que against NSCLC cells (A549, PC9) and normal lung epithelial cells (BEAS-2B) using in vitro assays, including CCK-8, colony formation, and flow cytometry. Key pathways related to ferritinophagy and ferroptosis were identified through transcriptomic analysis and KEGG pathway analysis. Fluorescence imaging, Western blot (WB) and BODIPY C11 staining were used to assess the levels of ferritinophagy and ferroptosis in cells. Limited proteolysis-mass spectrometry (LiP-MS) and molecular dynamics simulations were employed to identify the direct targets of Que. Gene knockdown and overexpression experiments were conducted to verify that Que targets eukaryotic translation initiation factor 3 subunit D (EIF3D) and regulates the activation of nuclear receptor coactivator 4 (NCOA4) -mediated ferritinophagy. In vivo experiments using xenograft mouse models assessed the antitumor effect and safety of Que.

[RESULTS] Que selectively inhibited the proliferation and colony formation of NSCLC cells while showing minimal toxicity to normal lung epithelial cells. It promoted the release of Fe²⁺ and lipid peroxidation by activating the NCOA4-dependent ferritinophagy pathway, while simultaneously inhibiting the expression of ferroptosis markers such as glutathione peroxidase 4 (GPX4). LiP-MS and molecular dynamics simulations confirmed EIF3D as a direct target of Que. Knockdown of EIF3D mimicked the antitumor effect of Que, whereas overexpression of EIF3D diminished its antitumor effect. In vivo, Que significantly inhibited tumor growth without observable toxicity, accompanied by upregulation of NCOA4 and LC3 II, and downregulation of EIF3D and GPX4.

[CONCLUSION] Que exerts significant antitumor effects in NSCLC by targeting EIF3D to activate NCOA4-mediated ferritinophagy and ferroptosis. This study reveals a novel mechanism involving the EIF3D-NCOA4 axis in the regulation of ferritinophagy-dependent ferroptosis, providing a potential therapeutic strategy for the treatment of NSCLC.