Targeting GSS-mediated glutathione synthesis: 6-Met induces oxidative stress dependent apoptosis via triggering mitochondrial bioenergetics metabolism crisis and mTOR suppression in glioblastoma.

BACKGROUND: Alkaloids from Macleaya cordata have demonstrated to possess anti-tumor effects against various cancer types, but their efficacy on GBM remains unexplored. In this study we investigated the anti-GBM activity of 6-Met and its underlying mechanisms. METHODS: GBM cell proliferation was assessed using CCK-8, RTCA, and colony formation assays in vitro. A xenograft mode used to investigate the anti-tumor efficacy of 6-Met. Apoptosis and ROS were quantified by Flow cytometry. GSH content was determined using a commercial GSH/GSSG assay kit. In parallel, the activities of GSS, GCLC, and GR, were quantified by corresponding commercial assay kits. Cellular bioenergetics was evaluated by Seahorse XFe96 analyzer. Ubiquitination analysis was conducted by immunoprecipitation (IP) using an anti-ubiquitin antibody. Finally, expression changes were analyzed by Western blotting and quantitative reverse transcription PCR. RESULTS: Our results revealed that 6-Met significantly suppressed GBM cell growth in vitro and transplanted tumor growth in vivo. Additionally, 6-Met induced caspase-dependent apoptosis in GBM cells. Mechanistically, 6-ME suppressed GSS expression by enhancing its ubiquitination, resulting in impaired GSH synthesis and subsequent ROS accumulation. The elevated ROS levels further disrupted mitochondrial function by damaging Fe-S clusters, as evidenced by reduced expression of Fe-S cluster-associated proteins, ultimately impairing mitochondrial respiration. Concurrently, ROS activation suppressed the mTOR signaling via modulation of its phosphorylation. Notably, exogenous GSH supplementation and ectopic GSS expression mitigates 6-Met cytotoxicity in GBM cells. Furthermore, exogenous expression of GSS abrogated 6-Met-induced transplanted tumor growth inhibition in vivo. Clinically, we observed that GSS expression was significantly elevated in GBM tissues compared to normal brain tissues, and lower GSS expression correlated with better patient prognosis, highlighting its potential as a prognostic marker. CONCLUSION: Collectively, these findings establish that 6-Met inhibits GBM tumor growth by disrupting GSS-dependent glutathione synthesis, thereby promoting oxidative stress, impairing mitochondrial respiration and mTOR signaling, and ultimately triggering apoptosis.