Artemisitene targets GLIS3 to inhibit the NF-κB-TNF signaling pathway and suppress bladder cancer cell growth

BACKGROUND: Artemisia annua, a medicinal and edible herb best known for Artemisinin, exhibits broad pharmacological activities and considerable clinical value. However, its therapeutic potential in bladder cancer remains largely unexplored. PURPOSE: Here, we systematically investigated the anti-bladder cancer efficacy of Artemisitene (ATT) in vitro and in vivo and delineated its underlying mechanisms. METHODS: Using cell-based and animal models, we found that ATT robustly suppressed bladder cancer cell growth. Integrated transcriptomic and proteomic sequencing, combined with MST assays and transient transfection, revealed the mechanism of ATT inhibiting the progression of bladder cancer. In addition, we analysed single-cell RNA-sequencing datasets and performed virtual knockout experiments to investigate the expression pattern and potential functional role of the ATT target gene GLIS3 across different pathological stages of bladder cancer. RESULTS: GLIS3 expression was higher in tumour than in adjacent nontumor tissues in BLCA patients and was further elevated in intermediate/late stages (T3-T4) compared with early stages (T1-T2). Consistently, single-cell RNA-seq data showed that GLIS3 levels in epithelial cells were higher in T3-T4 than in T1-T2 BLCA. Moreover, in virtual GLIS3 knockout in BLCA epithelial cells negatively regulated TNF and NF-κB signalling pathways. ATT directly interacts with GLIS3 and destabilizes the protein, consequently suppressing the transcription of NF-κB and TNF. Mutation of GLIS3 at Arg-404 and Pro-411 markedly weakened its interaction with ATT and partly abrogated the pharmacological effects of ATT. Moreover, GLIS3 knockdown enhanced the inhibitory effect of Gem on bladder cancer cells, while ATT further potentiated Gem-mediated suppression of BLCA by downregulating GLIS3 expression. CONCLUSIONS: ATT binds to GLIS3 and decreases its protein stability, thereby suppressing the transcription of NF-κB and TNF, inducing apoptosis in bladder cancer cells, and inhibiting cell proliferation, migration, and invasion. These findings provide a mechanistic rationale for developing ATT as a potential therapeutic agent or dietary supplement for bladder cancer and expand the medicinal value of Artemisia annua derivatives.