Mechanistic insights into Nrf2- and PARP1-mediated radioresistance of glioblastoma stem cells under photon, proton, and carbon ion irradiation: An in vitro study

Glioblastoma multiforme (GBM) exhibits strong resistance to radiotherapy, partly driven by glioblastoma stem-like cells (GSCs) with enhanced redox homeostasis and DNA repair capacity. This study evaluated whether targeting Nrf2-mediated antioxidant signaling and PARP1-dependent DNA repair enhances GSC radiosensitivity to different radiation modalities. Pharmacological inhibition of Nrf2 (ML385, 6 µmol/L) or PARP1 (olaparib, 5 µmol/L) reduced tumorsphere formation to 74.5 ± 10% and 58.56 ± 14.5% of control levels, respectively, while combined treatment further reduced formation to 51 ± 11% and sphere size to 29% of control. Western blotting confirmed effective pathway inhibition, with complete suppression of PARP activity and approximately 30% reduction in Nrf2 downstream proteins (SOD1, PRDX2, and NQO1). Dose-response analysis showed D₅₀ values of 5.03 ± 0.09 Gy (photons), 2.96 ± 0.91 Gy (protons), and 2.04 ± 0.47 Gy (carbon ions), corresponding to RBE₅₀ values of 1, 1.70 ± 0.55, and 2.46 ± 0.57, respectively. ML385 enhanced radiosensitivity to photons and protons and showed a similar radiosensitizing trend following carbon-ion irradiation, whereas olaparib showed its strongest effect with photons and limited effects with protons and carbon ions. Combined treatment produced a greater reduction in radiation survival than either inhibitor alone under selected conditions, particularly following photon irradiation. Nrf2 inhibition reduced downstream antioxidant proteins and increased late apoptotic/necrotic fraction, while PARP1 inhibition was associated with altered DNA damage persistence. Combined inhibition further increased γ-H2AX foci at selected time points following proton irradiation, consistent with delayed or incomplete repair of radiation-induced DNA damage. These findings support Nrf2 and PARP1 as potential regulators of GSC radioresistance and provide a rationale for further investigation of their therapeutic targeting in combination with radiotherapy.