Stigmasterol suppresses glioblastoma growth through mitochondrial dysfunction and oxidative stress

Glioblastoma (GBM) is an aggressive primary brain tumor characterized by rapid progression, metabolic reprogramming, and poor therapeutic outcomes. Increasing evidence suggests that mitochondrial dysfunction contributes to glioblastoma progression and may represent a potential therapeutic vulnerability. Stigmasterol has demonstrated anti-tumor activity in several malignancies; however, its effects on mitochondrial metabolism in GBM remain unclear. Tumor growth was evaluated using a U87-MG xenograft model. Transcriptomic analysis was performed to identify differentially expressed genes and enriched pathways associated with stigmasterol treatment. Mitochondrial function was assessed by measuring mitochondrial membrane potential, complex I activity and mitochondrial reactive oxygen species (ROS) levels. Colony formation assays were used to evaluate long-term proliferative capacity. The role of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2) was investigated using small interfering RNA (siRNA)-mediated knockdown. Key mechanistic findings, including complex I activity, mitochondrial ROS accumulation and NDUFA4L2 expression, were further validated in U251 glioblastoma cells. Stigmasterol significantly inhibited xenograft tumor growth and reduced clonogenic capacity in glioblastoma cells. Mechanistically, stigmasterol decreased mitochondrial membrane potential and complex I activity while increasing mitochondrial ROS accumulation, indicating impaired mitochondrial function and enhanced oxidative stress. Transcriptomic analysis revealed coordinated alterations in metabolism-related genes, including downregulation of Aconitase 2 (ACO2) and 3-hydroxyacyl-CoA dehydratase 2 (HACD2), together with upregulation of NDUFA4L2. Functional experiments showed that NDUFA4L2 knockdown partially restored complex I activity, further increased ROS accumulation, and enhanced the inhibitory effect of stigmasterol on colony formation. Stigmasterol suppresses glioblastoma growth by inducing mitochondrial dysfunction and oxidative stress. Upregulation of NDUFA4L2 appears to represent an adaptive response to mitochondrial stress and may contribute to the maintenance of redox homeostasis in glioblastoma cells. These findings suggest that targeting mitochondrial metabolic adaptation may represent a potential therapeutic strategy for glioblastoma.