Inhibition of Glutathione Pathway by Oleanolic Acid via PSAT1 Leads to Ferroptosis in Colorectal Cancer

ABSTRACT Phosphoserine aminotransferase 1 (PSAT1), a pivotal regulator of serine metabolism, is overexpressed in a variety of cancers, yet its role in colorectal cancer (CRC) remains to be elucidated. Oleanolic acid (OA), a naturally occurring pentacyclic triterpenoid, is suspected to have the potential to regulate ferroptosis, though this capability has not been confirmed. Utilizing bioinformatics tools, we conducted an analysis of PSAT1 expression in CRC, pathway enrichment, and its correlation with proteins pivotal to glutathione metabolism and the induction of ferroptosis. Expression levels of PSAT1 were validated by Quantitative Polymerase Chain Reaction (qPCR) and western blot analysis (WB). Cell Counting Kit‐8 (CCK‐8) was employed to measure the viability of CRC cells. Ferroptosis was assessed via flow cytometry, Fe 2+ detection, malondialdehyde (MDA) levels, and WB. The metabolic pathway of glutathione was examined by analyzing the levels of reduced glutathione (GSH) and glutathione disulfide (GSSG), glutamate, glutamine, and α‐ketoglutarate, and by assessing the expression of rate‐limiting enzymes in glutamine metabolism. The interaction between OA and PSAT1 was predicted by molecular docking and validated with the cellular thermal shift assay (CETSA) ‐WB assay. The results showed that PSAT1 was found to be highly expressed in CRC and enriched in pathways associated with ferroptosis and glutathione metabolism, instrumental in preserving the vitality of CRC cells. PSAT1 knockdown increased cellular lipid reactive oxygen species (ROS) and MDA, and ferrous ion accumulation, while also inhibiting the expression of ferroptosis markers Solute carrier family 7, membrane 11 (SLC7A11) and Glutathione peroxidase 4 (GPX4). Overexpression of PSAT1 raised the levels of glutathione metabolic intermediates and the GSH ratio, inhibiting ferroptosis, and these effects were reversed by methionine sulfoximine. OA was identified through molecular docking and CETSA‐WB to effectively dock with PSAT1 and target it to inhibit glutathione metabolism, enhancing lipid peroxidation and Fe 2+ accumulation in CRC cells. In conclusion, OA, upon binding to PSAT1, curbs its expression, which in turn downregulates glutathione metabolism and enhances ferroptosis in CRC cells.