Ginkgolic Acid Targets HSPA8 to Trigger Ferroptosis in Hepatocellular Carcinoma via Chaperone-Mediated Autophagy-Dependent GPX4 Degradation

Context: Ginkgolic acid (GAC), one of the major active constituents of Ginkgo biloba L. (Ginkgoaceae) extract, has been reported as a potential anticancer drugs. Objective: To investigate the effect of GAC on the viability of Human hepatocellular carcinoma (HCC) cells and its underlying mechanisms. Materials and methods: Human HCC cell lines and an orthotopic HCC mouse model were employed. RNA sequencing, drug affinity responsive target stability (DARTS) with LC-MS/MS, protein–protein interaction analysis, molecular docking, and cellular thermal shift assay (CETSA) were used to identify and validate GAC targets. Ferroptosis was evaluated by BODIPY probes, Annexin-V/PI staining, Western blotting, malondialdehyde (MDA), GSH/GSSG assays, and transmission electron microscopy (TEM). Cell viability was assessed by CCK-8 and colony formation assays. HSPA8 knockdown, mutant, and KFERQ-reporter cells were generated to investigate the role of HSPA8 and chaperone-mediated autophagy (CMA). Results: GAC effectively inhibits the viability of HCC by triggering ferroptosis. HSPA8 was identified as the directly target of GAC. The binding of GAC with HSPA8 enhancing its interaction with glutathione peroxidase 4 (GPX4), which leads to the degradation of GPX4 via CMA. This process depleted glutathione (GSH) and caused lipid peroxidation, finally inducing ferroptosis in HCC cells. Furthermore, GAC suppressed tumor growth in an orthotopic HCC model, increased lipid peroxidation and GPX4 degradation in tumor tissues. Discussion and conclusions: Our results revealed a novel mechanism by which GAC induces ferroptosis in HCC cells through direct targeting HSPA8 and promoting CMA-dependent GPX4 degradation. These findings demonstrate the potential of GAC as therapeutic approaches for HCC.