The Flavonoid Rutin Enhances Temozolomide Sensitivity in Glioblastoma Spheroids by Modulating Chemoresistance via PI3K/AKT, STAT3, Redox and Kynurenine Pathways, and Altering ECM Remodeling Associated with Reduced Migration

INTRODUCTION: Glioblastoma (GBM) is the most aggressive primary tumor of the central nervous system and is highly resistant to temozolomide (TMZ). Rutin is a potent antioxidant with immunomodulatory and anti-glioma effects in vitro, although its mechanisms of action remain incompletely understood. This study investigated the effects of rutin on morphology, viability, redox balance, and pro-tumoral signaling in GBM 2D cultures and 3D spheroids, as well as its association with TMZ sensitivity. METHODS: GL15 and U343 human GBM cell lines and primary astrocytes were treated with rutin (5-30 μM) and/or TMZ (125-4000 μM). Cell metabolic activity and viability were assessed by MTT, PI/DiOC18(3) or PI/Hoechst. Cell migration was assessed from spheroid-derived cells, and extracellular matrix (ECM) components (fibronectin and laminin) were evaluated by immunofluorescence. Intracellular reactive oxygen species (ROS) were measured by DCFH-DA fluorescence. IL-6, STAT3, NOS2, and IDO1 gene expression were determined by RT-qPCR, and protein expression of MMP2, fibronectin, STAT3, PI3K, and AKT by Western blotting. Nitric oxide (NO) and L-kynurenine levels were quantified in the supernatant by colorimetric assays. RESULTS: Rutin reduced cell viability and enhanced TMZ cytotoxicity in both 2D and 3D cultures, while exerting selective effects by increasing metabolic activity and attenuating TMZ-induced effects in non-tumoral primary astrocytes. In 3D spheroids, rutin affected structural organization and reduced spheroid-derived cell migration, accompanied by changes in ECM components, including MMP2, fibronectin, and laminin. Rutin decreased intracellular ROS levels and suppressed the TMZ-induced increase in ROS and NOS signaling. These effects were accompanied by modulation of IL-6/STAT3 signaling, along with reduced STAT3, PI3K, and AKT protein levels. Rutin also modulated immunometabolic parameters, including extracellular L-kynurenine and nitric oxide levels, and enhanced TMZ responsiveness following pre-sensitization. CONCLUSIONS: Rutin enhances TMZ responsiveness by modulating interconnected pro-tumoral mechanisms, including redox balance, pro-survival signaling, ECM remodeling and migratory behavior, and immunometabolic pathways linked to chemoresistance, supporting its potential as an adjuvant therapeutic strategy.