BRIP1 Induced Ferroptosis to Inhibit Glioma Cells and was Associated with Increased Oxidative Stress

Background: Glioma, a malignant brain tumour, poses a significant threat to human life and well-being. Identifying new treatment targets is crucial. This study aimed to explore the impact of BRIP1 (BRCA1 interacting helicase 1) on glioma cell ferroptosis and its underlying mechanisms.

Methods: We utilized GEPIA (Gene Expression Profiling Interactive Analysis) to predict the expression of BRIP1 in glioma. The expression of BRIP1 was evaluated in normal brain glial cell lines (HEB) as well as two glioblastoma (GBM) cell lines (U87 and U251) using qRT-PCR (quantitative RT-PCR) and Western blot analyses. U251 cells were specifically chosen to investigate the impact of BRIP1 down-regulation and treatment with erastin (a ferroptosis activator) on cell viability and proliferation. In U251 cells, si-BRIP1 was administered in combination with the necroptosis inhibitor Necrostain-1 (Nec-1), apoptosis inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl- [O-methyl]-fluoromethylketone), autophagy inhibitor CQ (Chloroquine), pyroptosis inhibitor VX765 (Belnacasan), or ferroptosis inhibitor Fer-1 (ferrostain-1), as well as erastin+Fer-1, to determine the mode of programmed cell death using the CCK-8 (Cell counting kit-8) assay. Malondialdehyde (MDA) and glutathione (GSH) levels were measured using ELISA (Enzyme linked immunosorbent assay). Intracellular Fe²⁺ content was detected using a commercial reagent kit. Gpx4 (Glutathione peroxidase 4) levels were measured using Western blot analysis. The relationship between BRIP1 and SLC7A11 (Solute Carrier Family 7 Member 11) was verified by co-IP (co-immunoprecipitation) experiments. The level of SLC7A11 and SLC3A2 (Solute Carrier Family 3 Member 2) was analyzed through qRT-PCR and Western blot analyses. A rescue experiment was conducted to observe the effects of SLC7A11 overexpression on si-BRIP1-treated U251 cells.

Results: The GEPIA database predicted that the expression level of BRIP1 was increased in glioma. The expression level of BRIP1 was higher in U251 cells compared to HEB and U87 cells (p < 0.05). Both down-regulation of BRIP1 and treatment with erastin resulted in inhibited cell viability and proliferation in U251 cells (p < 0.05). The mode of programmed cell death in si-BRIP1-treated U251 cells was ferroptosis. Following si-BRIP1 transfection or erastin treatment, there was an increase in the levels of MDA and intracellular Fe²⁺ content, as well as a decrease in the levels of GSH, Gpx4, and SLC7A11 (p < 0.05). However, these alterations observed in the si-BRIP1 group were reversed by Fer-1 treatment (p < 0.05). The co-IP results demonstrated that BRIP1 and SLC7A11 were able to bind to each other. Up-regulation of SLC7A11 reversed the reduction in cell viability, the increase in MDA, the reduction in GSH, the increase in Fe²⁺ content, and the down-regulation of Gpx4 in si-BRIP1-treated U251 cells (p < 0.05).

Conclusion: In this study, we found that down-regulation of BRIP1 could inhibit cell viability and proliferation in glioma cells through the induction of ferroptosis. This process was associated with increased oxidative stress, which was mediated by the down-regulation of SLC7A11 (xCT (Cysteine/glutamate transporter)) expression.