Modulation of Wnt/β‐Catenin Pathway by Aesculus hippocastanum Extract Enhances Temozolomide Sensitivity in Glioblastoma Cells

Glioblastoma (GB) is a highly aggressive brain tumour with a poor prognosis and limited responsiveness to standard chemotherapy, particularly temozolomide (TMZ), due to intrinsic resistance mechanisms. This study investigates the potential of Aesculus hippocastanum, known as horse chestnut extract (HCE), to enhance the therapeutic efficacy of TMZ in GB cells through modulation of the Wnt/β-catenin signalling pathway. Combined treatment of HCE (500 μg/mL) and TMZ (100 μM) significantly reduced cell viability and inhibited wound healing and colony formation compared to either agent alone at 48 h. Notably, the expression of β-catenin and Wnt-1 was significantly reduced in the combination group, followed by a significant downregulation of Nestin and β3-tubulin, markers of glioma stem-like cells and aggressiveness, respectively. Furthermore, apoptotic activity was significantly increased following the combined treatment. In a 3D U87-spheroid model, the combination therapy resulted in a substantial reduction in spheroid area, suggesting impaired tumour growth. Propidium iodide (PI) staining revealed increased membrane permeability in cells treated with the combination, which was accompanied by an increase in p53 expression, supporting the induction of apoptosis. Collectively, these findings demonstrate that HCE increases the cytotoxic effects of TMZ by inhibiting Wnt/β-catenin signalling, reducing tumour stemness, and promoting apoptotic pathways in GB cells.