吳茱萸鹼抑制穀胱甘肽過氧化物酶4以促進高惡性度膀胱癌細胞之鐵死亡和抑制上皮細胞間質化

胡哲源¹，歐建慧¹，謝嘉興²

¹成大醫院泌尿部

²衛生福利部台南醫院

Ferroptosis induction and glutathione peroxidase 4 suppression as mechanisms of Evodiamine's inhibition to high grade bladder cancer cell

Che-Yuan Hu¹, Chien‑Hui Ou¹, Gia-Shing Shieh²

¹Department of Urology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan

²Department of Urology, Tainan Hospital, Ministry of Health and Welfare, Executive Yuan, Tainan, Taiwan

Purpose: Evodiamine (EVO) has been shown to possess anti-cancer properties by inhibiting cell proliferation, but its underlying mechanism remains unclear. To investigate the role of ferroptosis in EVO's therapeutic effects, we examined critical factors, including lipid peroxidation levels and glutathione peroxidase 4 (GPX4) expression, in response to EVO treatment.

Materials and Methods: We employed multiple techniques to investigate the effects of EVO on poorly differentiated, high-grade bladder cancer TCCSUP cells. We employed the MTS assay to assess cell proliferation, cell cycle analysis to evaluate cell cycle arrest, DCFDA staining to detect reactive oxygen species, and C11-BODIPY to measure lipid peroxide levels. We also used the iron chelator deferoxamine (DFO) to interfere with the process of ferroptosis. Additionally, we utilized the Transwell migration assay and Western blotting to estimate migratory ability and epithelial-mesenchymal transition (EMT) markers, respectively. Finally, we confirmed our findings in vivo by using the TCCSUP bladder tumor xenograft tumor model.

Results: EVO induced cytotoxicity in a dose- and time-dependent manner and promotes cell cycle arrest at the G2/M phase in TCCSUP cells. Fluorescence microscopy revealed increased lipid peroxides in cancer cells after exposure to EVO. The treatment with EVO resulted in a dose-dependent reduction in GPX4, an enzyme that prevents cells from undergoing ferroptosis by converting lipid peroxides. Due to the characteristics of ferroptosis, which involve dependency on iron and death driven by lipid peroxidation, DFO was utilized to inhibit ferroptosis induced by EVO. When cells were pre-treated with DFO before exposure to EVO, the level of lipid peroxides significantly decreased. Additionally, DFO reduced EVO-induced cell death. The concomitant treatment of cancer cells with EVO and either a pan-caspase inhibitor or a necroptosis inhibitor did not mitigate cell death. It appears that EVO triggers ferroptosis as opposed to apoptosis or necroptosis. Moreover, EVO inhibited cancer cell migration, downregulated the expression of mesenchymal markers, and upregulated the expression of epithelial markers. Animal model corroborated the impact of EVO on suppressing tumor growth and inhibiting EMT.

Conclusion: EVO represents a new inducer capable of triggering ferroptosis in bladder cancer cells and could serve as a promising therapeutic option for bladder cancer.