Gambogic acid induces cell death via covalent binding with PRDX1 to regulate ER stress and autophagy.

Abstract

Background: Gambogic acid (GA) is a natural bioactive compound derived from Garcinia hanburyi Hook. F, has proven anticancer activity and is currently in Phase II clinical trials for the treatment of cancer. However, the molecular mechanisms and targets underlying GA's anti-renal cell carcinoma effects remain unclear.

Methods: CCK-8 assay evaluated kidney cancer cell viability via formazan formation. Flow cytometry identified apoptotic cells using characteristic fluorescence signals. Western blotting assessed autophagy- and apoptosis-related protein expression through extraction, SDS-PAGE, transfer, and antibody detection. JC-1 assay determined mitochondrial health by measuring red-to-green fluorescence ratio in kidney cancer cells. The production of reactive oxygen species (ROS) was quantified through CM-H2DCFDA staining. Additionally, a range of techniques, such as proteomics, activity-guided protein profiling (ABPP), and cellular thermal stability assays (CETSA), were employed to ascertain the molecular targets involved.

Results: GA induces cell death by inducing ER stress and modulating autophagy. GA-induced autophagy is involved in ER stress activation. Importantly, GA-induced ER stress and subsequently cell death is caused by increased ROS levels. Mechanistic studies show that peroxiredoxin-1 (PRDX1), a key antioxidant enzyme, is a directly covalent binding target of GA, and overexpression of PRDX1 mitigates GA-induced ROS production and subsequently cell death.

Conclusion: The study identified PRDX1 is a potential directly covalent binding protein of gambogic acid, and elucidated its mechanism for inducing cell death that involving in ROS-mediated ER stress and autophagy regulation. The results obtained offer fresh perspectives on the mechanisms underlying the cytotoxic effects induced by gambogic acid, further suggesting that PRDX1 may represent a promising candidate for targeted therapy in the development of novel anticancer agents, notably for the treatment of kidney cancer.