Nrf2 and PGC-1α signaling in temozolomide resistance in glioblastoma under hypoxia

Hypoxia is a key contributor to chemoresistance in glioblastoma (GBM), partly through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent cellular stress responses and mitochondrial adaptive programs. The present study investigated Nrf2 signaling in hypoxia-associated resistance to temozolomide (TMZ) in U87MG cells, a glioblastoma cell line, and evaluated whether pharmacological suppression of Nrf2 pathway output with brusatol enhances TMZ sensitivity under hypoxic conditions. U87MG cells were cultured under normoxic or hypoxic conditions (1% O₂) and treated with TMZ, brusatol, or their combination. DAPI-based cell-counting assays were used to determine half-maximal inhibitory concentrations (IC₅₀ values) and assess drug interactions by combination index analysis. Hypoxia-inducible factor 1α (HIF-1α) expression, epidermal growth factor receptor (EGFR) phosphorylation and Nrf2 nuclear accumulation were analyzed by western blotting. The expression of antioxidant and growth factor-related signaling genes and downstream targets of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a key regulator of mitochondrial bioenergetics, was quantified by real-time PCR (qPCR). PGC-1α subcellular localization and oxidative DNA damage, assessed using 8-hydroxy-2'-deoxyguanosine, were evaluated by immunofluorescence. Hypoxia markedly increased the TMZ IC₅₀ and was accompanied by elevated HIF-1α expression and enhanced Nrf2 nuclear accumulation. Combination index analysis identified synergistic interactions between TMZ and brusatol under normoxia, whereas no synergistic interaction was detected under hypoxia; nonetheless, brusatol still enhanced TMZ sensitivity under hypoxia based on direct cell-count analysis. Hypoxia markedly upregulated antioxidant genes (GCLM, GPX1, GSTP1, HO-1, NQO1 and SOD1) and growth- and survival-related genes (NF-κB, TGF-β, EGF and EGFR) and induced a PGC-1α-associated mitochondrial transcriptional program, as evidenced by increased expression of sirtuin 3, cytochrome c and ATP synthase F1 subunit β. Co-treatment with TMZ and brusatol suppressed antioxidant gene expression, reduced EGFR phosphorylation, attenuated PGC-1α-linked mitochondrial gene expression, and increased oxidative DNA damage. These findings indicated that pharmacological suppression of Nrf2 pathway output by brusatol was associated with enhanced TMZ sensitivity under hypoxic conditions in U87MG cells, together with reduced antioxidant gene expression, lower EGFR phosphorylation, attenuation of PGC-1α-associated mitochondrial responses, and increased oxidative DNA damage in GBM, potentially reflecting suppression of HIF-1α-dependent as well as Nrf2-dependent signaling.