4-Methylumbelliferone triggers early oxidative stress, bioenergetic rewiring, and remodeling of cell-surface N-glycosylation in glioblastoma cells

Abstract Glioblastoma (GBM) is an aggressive brain tumor characterized by high metabolic plasticity and resistance to standard therapies. Targeting metabolic vulnerabilities has emerged as a promising therapeutic strategy. 4-Methylumbelliferone (4MU), a known inhibitor of hyaluronan synthesis, has demonstrated antitumor effects beyond its canonical mechanism; however, its impact on tumor metabolism and associated pathways in GBM remains poorly understood. Human glioblastoma cell lines (U251 and LN229) were treated with 4MU (1000 µM) or vehicle. Metabolic alterations were assessed using $${}_{ }{}^{1}H$$ -NMR-based untargeted metabolomics, followed by exploratory metabolite set quantitative enrichment analysis (QEA) to evaluate pathway-level associations. Oxidative stress was evaluated using a dihydrorhodamine (DHR) assay. N-glycosylation patterns were analyzed by lectin-binding assays and flow cytometry. Glycolytic activity was determined using a Seahorse Glycolysis Stress Test. Statistical analyses included Student’s t-test, one-way ANOVA, and multiple comparison tests, considering p < 0.05 as significant. 4MU rapidly induced oxidative stress in both glioblastoma cell lines, detectable as early as 1.5 h. This early redox imbalance preceded broader metabolic alterations observed at 24 h. Metabolomic profiling identified significant depletion of NAD + and shifts in nucleotide-sugar precursors. Exploratory QEA highlighted coordinated associations with glycan and amino-sugar pathways, which were functionally corroborated by altered cell-surface N-glycan profiles (lectin-binding assays). Additionally, 4MU treatment increased glycolytic activity and capacity in U251 cells, pointing to a compensatory bioenergetic response in this model. These findings indicate that 4MU elicits a rapid redox imbalance alongside pronounced metabolic rewiring (characterized by NAD⁺ depletion, altered glycolytic activity, and disrupted N-glycosylation) in glioblastoma cells. Rather than a purely linear causal chain, our data suggest a multi-tiered stress response where direct UDP-sugar consumption and early oxidative stress converge to impair tumor cell bioenergetics and glycosylation patterns. Our results provide new mechanistic insights into the antitumor activity of 4MU and support its potential repositioning as a strategy targeting metabolic vulnerabilities in GBM.