Ginkgolic acid targets HSPA8 to trigger ferroptosis in hepatocellular carcinoma via chaperone-mediated autophagy-dependent GPX4 degradation

Ginkgolic acid (GAC), one of the major active constituents of Ginkgo biloba L. (Ginkgoaceae) extract, has been reported as a potential anticancer agent. To investigate the effect of GAC on the viability of human hepatocellular carcinoma (HCC) cells and to identify its primary target and underlying mechanism. Human HCC cell lines and an orthotopic HCC mouse model were employed. The mechanism of GAC was elucidated through integrated analyses of cell viability, RNA sequencing, and biomarkers. The primary target of GAC was identified by drug affinity responsive target stability (DARTS) coupled with LC-MS/MS and further validated using molecular docking (MD), cellular thermal shift assay (CETSA), and microscale thermophoresis (MST) assay. Mechanistic roles were confirmed using genetic approaches, including gene knockdown and the construction of mutant plasmids. A KFERQ reporter system was used to detect the activity of chaperone-mediated autophagy (CMA). GAC effectively inhibits the viability of HCC by triggering ferroptosis. HSPA8 was identified as the direct target of GAC. The binding of GAC with HSPA8 enhances 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. Our results revealed a novel mechanism by which GAC induces ferroptosis in HCC cells through direct targeting of HSPA8 and promoting CMA-dependent GPX4 degradation. These findings suggest GAC-mediated ferroptosis as a potential therapeutic strategy against HCC and expand the pharmacological application of CMA-targeted cancer therapies.