Fig 2: NRF2 expression is downregulated by procyanidin B1 through ubiquitin‐dependent protein degradation via PSMC3 in U251 cells. (A) Venn diagram illustrating interactions between proteins and NRF2 as detected by IP and LC–MS analysis in U251 cells treated as indicated. (B) LC–MS analysis showing NRF2 protein modification sites. (C) Gene ontology analysis depicting the enrichment of BP pathways using the specific proteins associated with procyanidin B1 treatment. (D) STRING analysis showing proteins interacting with NRF2 and proteins associated with ubiquitin‐dependent protein degradation. (E) U251 cells were treated with procyanidin B1 or vehicle, followed by IP using an anti‐NRF2 antibody and probed with an anti‐PSMC3 antibody. (F) Scatterplots of correlations between PSMC3 expression and NRF2 expression in glioblastoma (GBM) sample of The Cancer Genome Atlas data (Agilent‐4502A) (R = −0.24, p = 0). (G) Quantitative analysis of PSMC3 mRNA levels in U251 cells treated as indicated. (H) Representative western blotting and quantitative analysis of NRF2 protein levels in U251 cells treated as indicated. (I) U251 cells were treated as indicated, then treated further with 10 μM of MG132 for 6 h. NRF2 was immunoprecipitated using an anti‐NRF2 antibody and probed with an anti‐ubiquitin (UB) antibody; siPSMC3 represents PSMC3 knockdown in U251 cells. BP, biological process; GO, Gene Ontology; IP, immunoprecipitation; LC–MS, liquid chromatography–mass spectrometry; STRING, Search Tool for the Retrieval of Interacting Genes/proteins; WB, western blot. Data are presented as the mean ± SEM. All experiments were conducted at least in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, ns: p ≥ 0.05.

Fig 3: Downregulation of NRF2 by procyanidin B1 promotes ferroptosis in U251 cells. (A) Fe2+ levels in U251 cells with the treatment indicated as determined by iron ion detection. (B) Representative western blotting and quantitative analysis of TFR1, SLC7A11, and COX‐2 protein levels in U251 cells with the treatment indicated. (C and D) Immunostaining quantification of TFR1, SLC7A11, and COX‐2 protein levels in U251 cells with the treatment indicated. (E) Representative images and quantitative analysis of LOOH levels in U251 cells with the treatment indicated. (F) Fe2+ levels in U251 cells with procyanidin B1 treatment and NRF2 overexpression as determined by iron ion detection. (G) Representative western blotting and quantitative analysis of TFR1, SLC7A11, and COX‐2 protein levels in U251 cells after procyanidin B1 treatment and NRF2 overexpression. (H and I) Immunostaining quantification of TFR1, SLC7A11, and COX‐2 protein levels in U251 cells with procyanidin B1 treatment and NRF2 overexpression. (J and K) Representative images and quantitative analysis of LOOH levels in U251 cells with procyanidin B1 treatment and NRF2 overexpression. (L) Iron ion detection showed the Fe2+ levels with procyanidin B1 treatment and NRF2 knockdown in U251 cells. (M) Representative western blotting and quantitative analysis of TFR1, SLC7A11, and COX‐2 protein levels in U251 cells with procyanidin B1 treatment and NRF2 knockdown. (N and O) Immunostaining quantification of TFR1, SLC7A11, and COX‐2 protein levels in U251 cells after procyanidin B1 treatment and NRF2 knockdown. (P) Representative images and quantitative analysis of LOOH levels in U251 cells after procyanidin B1 treatment and NRF2 knockdown. OE‐NRF2 represents NRF2 overexpression achieved by transfection with the pCMV‐NRF2‐3×Myc‐Neo plasmid. siNRF2 represents NRF2 knockdown achieved by siRNA transfection. Scale bar = 25 μm. IF, immunofluorescence; LOOH, lipid hydroperoxide; WB, Western blotting. Data are presented as the mean ± SEM. All experiments were conducted in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, ns: p ≥ 0.05.

Fig 4: Procyanidin B1 binds to NRF2 in U251 cells. (A) Molecular docking analysis shows the binding configurations and interactions of procyanidin B1 with NRF2 (binding energy = −7.677 kcal/mol). (B) 3D tomography with z‐stack imaging (0° rotation) in confocal microscopy after treating with procyanidin B1 (linked with CY5) for 48 h at 394 μM in U251 cells with transfected pCDNA3‐EGFP‐C4‐NRF2 plasmid (scale bar = 25 μm). (C) Images depicting maximum intensity projection in 2D‐XY (scale bar = 25 μm). (D) Kinetics fit curve of the affinity between procyanidin B1 and NRF2 protein by surface plasmon resonance (SPR). All experiments were conducted at least in triplicate.

Fig 5: Efficacy of procyanidin B1 on glioblastoma (GBM). (A) Traditional Chinese medicines targeting NRF2 were obtained from the SymMap database. Line thickness represents the possibility value of targeting p value <0.05. (B) Blood–brain barrier (BBB) permeability of temozolomide (TMZ) and procyanidin B1 was determined using an in vitro BBB model. (C and D) Quantification of half maximal inhibitory concentration (IC50) in the U251, GL‐261, and bEnd.3 cells with procyanidin B1 and TMZ treatment. (E) Quantitative analysis of NRF2 mRNA levels in U251 cells with procyanidin B1 or vehicle treatment. (F) Representative western blotting and quantitative analysis of NRF2 protein levels in U251 cells with procyanidin B1 or vehicle. (G) Schematic illustration of procyanidin B1 therapeutic schedule in an orthotopic GBM model. (H) Bioluminescence images in GBM‐bearing mice model using an IVIS (n = 6 mice/group). (I) Survival curve of tumor‐bearing mice with procyanidin B1 or PBS treatment (n = 6 mice/group). (J) Quantification of bioluminescence images in the GBM‐bearing mice treated with procyanidin B1 at day 0 and day 44. (K) HE staining in the brain of GBM‐bearing mice (n = 3 samples/group). Scale bar = 0.50 cm. (L and M) Representative immunostaining and quantitative analysis of KI67+ cells in GBM‐bearing mice treated with PBS or procyanidin B1 (n = 3 samples/group). Left scale bar = 0.20 cm. Right scale bar = 50 μm. Data are presented as the mean ± SEM. All experiments were conducted at least in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, ns: p ≥ 0.05.