High-throughput screening identifies stevioside as a potent agent to induce apoptosis in bladder cancer cells

Background: Bladder cancer (BC) is a global health issue that lacks effective treatment strategies. Growing evidence suggests that various natural products possess anti-tumour effects. This study aims to identify a novel agent that can be used in the treatment of BC. Methods: High-throughput screening was conducted to search for potential anti-BC natural agents. Cell viabilities were measured by the CCK-8 assay. Cell death, cellular reactive oxygen species (ROS), and mitochondrial outer membrane potential (MOMP) were measured by flow cytometry. RNA sequencing was conducted to identify the affected signalling pathways. Western blots were used to measure the change of proteins. Xenografts models were used to assess the anti-tumour effects in vivo. Results: Through high-throughput screening, we identified stevioside, a diterpenoid glycoside isolated from Stevia rebaudiana, which selectively inhibited the viability of BC cells and induced their intrinsic apoptosis sparing normal cells. Stevioside also induced mitochondrial stress in BC cells, and activated Bax by downregulating Mcl-1 and upregulating Noxa. RNA sequencing revealed that stevioside treatment caused activation of GSK-3 beta and endoplasmic reticulum (ER) stress signalling pathways. Activation of GSK-3 beta induced upregulation of FBXW7, which effectuated the downregulation of Mcl-1. In addition, activation of GSK-3 beta triggered ER stress, leading to the upregulation of Noxa. Further investigations revealed that the accumulation of ROS was responsible for the activation of the GSK-3 beta signalling pathway in BC cells. Moreover, we also found that stevioside inhibited the growth of BC cells in vivo. Conclusions: Collectively, our data suggest that stevioside can be a potential agent for the treatment of BC.