Isoxazole based nucleosides induce autophagy through the production of ROS and the suppression of the β-catenin pathway in human colorectal carcinoma cells

Abstract

β-catenin is frequently implicated in signaling pathways that regulate autophagy, and the production of reactive oxygen species (ROS) has been linked to autophagy activation. Isoxazole-based nucleoside compounds have demonstrated anti-cancer properties. In this study, we report the identification of novel isoxazole-nucleosides as anti-tumor agents and their impact on autophagy in human colorectal carcinoma (CRC) cells. Among the ITP series, ITP-7 and ITP-9 (ITP-7/9) exhibited significant cytotoxicity compared to other compounds. Treatment with ITP-7/9 upregulated the expression of key autophagy-related proteins, including LC3 II, Atg7, and phosphorylated Beclin-1. Additionally, ITP-7/9 promoted the formation of LC3 II puncta and increased the number of AO-stained and MDC-stained cells, indicating enhanced autophagy. ROS levels were elevated following ITP-7/9 exposure, and treatment with N-acetyl l-cysteine (NAC), a ROS inhibitor, reduced the ITP-7/9-induced expression of LC3 II. Furthermore, ITP-7/9 inhibited β-catenin's role as a transcription factor, as observed in ICC assays. Moreover, cells with β-catenin gene deletion exhibited stronger autophagy when treated with ITP-7/9 compared to those treated with ITP-7/9 alone. These findings suggest that ITP-7/9 induces autophagy and promotes CRC cell death by downregulating β-catenin.