Rg3 suppresses gastric cancer progression via TFEB-mediated lysosomal autophagy pathway.

BACKGROUND: Gastric cancer (GC) ranks among the most prevalent malignant tumors globally, characterized by elevated incidence and mortality rates. As a natural active component isolated from Panax ginseng, Rg3 has demonstrated anti-tumor activity across multiple malignancies. Nevertheless, its specific functions and underlying molecular mechanisms in GC remain incompletely elucidated. METHODS: Rg3 exerted effects in viability, proliferative capacity, apoptotic progression, and autophagy-associated protein expression in AGS cells, evaluated via CCK-8 assay, colony formation, EdU staining, flow cytometry, and Western blot. To investigate the direct interaction between Rg3 and TFEB, as well as the mechanism by which Rg3 modulated apoptosis via lysosomal autophagy, the molecular docking, CETSA, immunofluorescence, mRFP-GFP-LC3 dual-labeling system, LysoTracker/Magic Red staining, and gene knockdown/overexpression approaches were employed. The expression of AKT/mTOR pathway-related proteins was analyzed by Western blot. A nude mouse xenograft tumor model was established to monitor tumor development, while H&E staining and IHC were employed to dissect histopathological alterations and the expression of pivotal proteins. RESULTS: Rg3 attenuated the proliferation of AGS cells in a concentration-dependent manner, induced apoptosis, and inhibited the AKT/mTOR pathway. Mechanistic investigations unveiled that Rg3, on one hand, reduced TFEB phosphorylation by inhibiting the AKT/mTOR pathway, thereby driving TFEB nuclear translocation; on the other hand, it bound to TFEB to enhance its protein stability, maintained the activated state of TFEB, and upregulated TFEB protein expression, consequently activating the lysosomal autophagy pathway. TFEB knockdown reversed Rg3-mediated autophagy activation, apoptosis induction, and development inhibition, whereas TFEB overexpression recapitulated these effects, which were abrogated by the autophagy inhibitor chloroquine. In vivo experiments further verified that Rg3 prominently suppressed xenograft tumor development, concurrent with upregulated TFEB and LC3B expressions, downregulated Ki67, and elevated Bax levels in tumor tissues. CONCLUSION: Rg3 upregulates and activates TFEB, thereby triggering the lysosomal autophagy pathway, which ultimately induces GC cell apoptosis and suppresses tumor progression.