Investigatıon of Apoptotic Effects of Thymoquinone on Glioblastoma Cells

Background: Glioblastoma multiforme (GBM) is the most aggressive and fatal primary brain tumor, characterized by rapid proliferation, resistance to therapy, and poor prognosis. Current treatment strategies, including surgery, radiotherapy, and chemotherapy, have limited efficacy and significant side effects. Phytotherapeutic agents such as thymoquinone (TQ), the major bioactive compound of Nigella sativa, have gained attention for their potential anticancer properties. TQ has been reported to exhibit pro-apoptotic, anti-inflammatory, and anti-proliferative effects in various cancer models. Objective: This study aimed to investigate the apoptotic effects of TQ on U87 human glioblastoma cells by evaluating its impact on cell viability, migration, colony formation, and the expression of key genes and proteins involved in apoptosis and inflammation. Methods: U87 glioblastoma cells were treated with increasing concentrations of TQ (10–100 µM) for 24, 48, and 72 h. Cell viability was assessed using the Sulforhodamine B (SRB) assay. A scratch assay was performed to evaluate cell migration, and a colony formation assay was used to assess clonogenic potential. Quantitative real-time PCR (qRT-PCR) and Western blotting were conducted to analyze the expression levels of genes and proteins associated with apoptosis, inflammation, and cell cycle regulation. Results: TQ treatment significantly reduced U87 cell viability in a time- and dose-dependent manner, with IC50 values of 75 µM at 24 h, 45 µM at 48 h, and 36 µM at 72 h. The scratch assay demonstrated that TQ had no significant effect on U87 cell migration. Colony formation was inhibited at 10 µM after 24 h. qRT-PCR analysis revealed that at 50 µM TQ, caspase-3 and Bax mRNA levels were significantly upregulated. At 75 µM, caspase-9, caspase-3, Bax, and p21 mRNA levels increased, while caspase-8, Bcl-2, and Akt1 expression decreased. Western blot analysis showed increased expression of cleaved caspase-3, p21, and ATG5 proteins, along with decreased expression of caspase-8 and TNF-α at 24 h. Conclusion: TQ exhibits potent pro-apoptotic effects in U87 glioblastoma cells by modulating key apoptotic and inflammatory pathways, including the PI3K/AKT signaling axis. These findings suggest that TQ may serve as a promising adjuvant therapeutic agent for GBM. Further studies are required to elucidate its full molecular mechanisms and potential clinical applications.