Timosaponin AIII inhibits gastric cancer by causing oxidative stress and blocking autophagic flux.

BACKGROUND: Gastric cancer (GC) is a prevalent malignant tumor worldwide, with limited treatment targets. Timosaponin AIII (Tim AIII) is the naturally steroid saponin isolated from Anemarrhena, while this study initially confirms the anti-GC effect of Tim AIII. METHODS: MTT assay, cell cycle analysis, and wound healing assay were used to evaluate the inhibitory effects of Tim AIII on GC cells (AGS and HGC27). Evaluate the oxidative stress (OS) by measuring reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the Kelch-like ECH-associated protein 1 (Keap1) - Nuclear factor erythroid-derived 2-like 2 (Nrf2) pathway. RNA sequencing and proteomics analysis were utilized to investigate deeper molecular mechanisms. To track the autophagic flux using transmission electron microscope, detecting changes in autophagy-related pathway proteins, staining with LC3B and lysosome. Experiments related to cell viability, OS, and autophagy levels were performed on normal gastric mucosal epithelial cells (GES-1) as parallel controls. Finally, Nude mouse subcutaneous tumor model to evaluate the anti-GC ability in vivo. RESULTS: Tim AIII inhibits the viability, proliferation, and migration of GC cells. Tim AIII causes OS in GC cells by the increasing intracellular ROS and MDA levels and inhibiting the Keap1-Nrf2 pathway. RNA sequencing and proteomics analysis mainly focused on the autophagy-associated pathways and lysosome in GC cells. Tim AIII activates autophagy, as indicated by an increase in the number of autophagosomes, inhibition of the PI3K-AKT pathway, and activation of the AMPK pathway in GC cells. However, Tim AIII inhibits autophagy-lysosome fusion and impairs lysosomal function, which results in autophagic flux blockage in GC cells. The Tim AIII concentration that significantly inhibited GC cells in this study was applied to GES-1 cells. The results showed that at this concentration, Tim AIII exhibited no significant cytotoxic effects on GES-1 cells, did not induce OS, and had no impact on autophagy. Finally, Tim AIII also has the ability to inhibit tumor growth in vivo. SIGNIFICANCE: In summary, the results of our study indicate Tim AIII as a novel late-stage autophagy inhibitor, which may provide novel medical possibilities for GC.