STONIN2, A Novel Therapeutic Target of Bacopasaponin C Promotes Lysosome Mediated Cell Death in Oral Cancer

Abstract

BACKGROUND: Oral cancer is one of the most common malignancies with major clinical challenges worldwide. STONIN2 (STON2), an endocytic protein, has recently been reported to be vital for lysosomal quality control in oral cancer cells. AIM: For the present study, we hypothesize that a potent STON2 inhibitor, bacopasaponin C (BSC), bioactive component of the Indian traditional medicine Bacopa monnieri, efficiently activates autophagy while progressively promoting lysosomal damage, hence leading to cell death. METHODS: The effect of BSC on lysosome integrity in oral cancer cells was examined through LysoTracker staining, dextran internalization, GAL3 accumulation, and tandem-fluorescent-GAL3 (tf-GAL3) assay. Further, BSC treatment inhibited autophagy flux (tandem-fluorescent LC3) and accumulated LC3 puncta (confocal microscopy) along with induction of various autophagy proteins, including ULK1, ATG5, ATG7, and LC3 (western blotting). Additionally, cell viability and apoptosis were examined by anti-proliferative assays, caspase activation, and Annexin V/PI staining. RESULT: Upon BSC treatment, the mTOR pathway is inhibited, leading to autophagy induction while simultaneously disrupting the lysosomal membrane integrity, leading to a loss in functional lysosomes. However, preventing mTOR reactivation leads to the suppression of autophagic lysosome reformation (ALR), promoting further study to investigate the role of lysosomal biogenesis. Initially, BSC treatment triggered the nuclear translocation of TFEB, a master regulator for genes involved in lysosomal biogenesis, whose activity was subsequently inhibited. Enhanced lysosomal damage triggered calcium-mediated ROS production, which in turn activated the caspase cascade for apoptosis. Mechanistically, BSC promotes ubiquitin-mediated proteasomal degradation of STON2, leading to failure in recovery of damaged lysosomes. Notably, under STON2 overexpression, BSC-triggered lysosomal damage was rescued while promoting oral cancer cell survival. CONCLUSION: Taken together, our study reveals BSC as a novel modulator of STON2, capable of disrupting lysosomal function and triggering apoptosis in oral cancer cells. Thus, targeting STON2-mediated lysosome quality control using BSC holds a promising therapeutic option against oral cancer.