Toosendanin induces ferroptosis in gastrointestinal stromal tumor cells through the regulation of the NCOA4 ferritinophagy pathway: implications for tumor proliferation, migration, and invasion

Background: Ferroptosis, a regulated form of cell death marked by iron-dependent lipid peroxidation, has gained recognition as a potential therapeutic target in diverse cancers, including gastrointestinal stromal tumors (GIST). The NCOA4-mediated ferritinophagy pathway is integral to the regulation of cellular iron homeostasis and the process of ferroptosis. Nevertheless, the effects of modulating this pathway on the viability of GIST cells and the induction of ferroptosis are yet to be elucidated. This study sought to examine the impact of toosendanin (TSN), a natural compound with prospective anticancer attributes, on ferroptosis in GIST cells, specifically emphasizing its regulatory influence on the NCOA4-mediated ferritinophagy pathway. Methods: GIST-T1 cells were exposed to different concentrations of TSN. Cell viability, apoptosis, and ferroptosis were evaluated through flow cytometry (Annexin V/7-AAD), transmission electron microscopy (TEM), and biochemical detection. The proliferation, migration, and invasion capacities were assessed utilizing the Cell Counting Kit-8 (CCK-8) assay, clone formation assay, and Transwell assay, respectively. The impact of NCOA4 silencing and ferrostatin-1, an inhibitor of ferroptosis, was analyzed in conjunction with TSN treatment. Results: The results showed that TSN treatment markedly decreased cell viability and induced ferroptosis in GIST-T1 cells, as demonstrated by elevated levels of lipid reactive oxygen species (ROS), increased ferrous ion content, and membrane damage. Mechanistically, western blot analysis demonstrated that TSN downregulated the expression of key ferroptosis inhibitors glutathione peroxidase 4 (GPX4) and SLC7A11, while simultaneously upregulating NCOA4 and LC3II/I. The application of small interfering RNA (siRNA)-NCOA4 and ferrostatin-1, which inhibit NCOA4-mediated autophagy and ferroptosis, resulted in a significant restoration of GPX4 and SLC7A11 expression, thereby mitigating TSN-induced ferroptosis. Furthermore, TSN was found to effectively suppress the proliferation, migration, and invasion of GIST cells; these effects were reversed upon NCOA4 silencing and inhibition of ferroptosis. Conclusions: This study underscores the potential of TSN as a therapeutic agent for GIST, particularly through the exploitation of NCOA4-mediated ferritinophagy to induce ferroptosis.