Proteomic Analysis Reveals That P97 Inhibitors Induce Ferroptosis in Gastric Cancer AGS Cells

Background: P97 regulates diverse cellular processes, including proteostasis, metabolism and pluripotency. Pharmacological inhibitors of P97 protein have shown antitumor activity in various cancers. However, the downstream pathways and effectors remain poorly characterized, limiting clinical translation. Methods: We performed a quantitative proteomic analysis using liquid chromatography–mass spectrometry (LC–MS) to identify differentially expressed proteins (DEPs) (fold change > 1.5, p < 0.05) and pathway alterations in gastric cancer AGS cells following treatment with two distinct P97 inhibitors, Eeyarestatin I (EerI) and NMS873. Results: Proteomic analysis revealed that both inhibitors significantly perturbed multiple signaling pathways. The endoplasmic reticulum (ER) proteostasis network was markedly disrupted, evidenced by activation of the unfolded protein response (UPR) and upregulation of ER-associated degradation (ERAD) components. Pathway enrichment further indicated that cell death processes, particularly ferroptosis, were prominently affected (p < 0.05). Key ferroptosis regulators showed altered expression, which was validated by Western blotting. Functional assays demonstrated that P97 inhibition suppressed cell proliferation and increased the accumulation of ferroptosis-associated lipid hydroperoxides in vitro, suggesting execution of ferroptotic cell death. In vivo, using a xenograft mouse model established by subcutaneous injection of AGS cells into nude mice, P97 inhibition significantly reduced tumor growth, confirming its antitumor efficacy. Conclusions: This integrated proteomic and functional study delineates that pharmacological P97 inhibitors disrupt ER proteostasis and simultaneously trigger ferroptosis in gastric cancer cells. These findings identify ferroptosis as a key downstream mechanism of P97 inhibition and provide molecular mechanisms for developing P97-targeted therapies in gastric cancer.