Chrysin sensitizes glioblastoma cells and spheroids to temozolomide treatment by reducing EMT and stemness phenotypes, as well as targeting multidrug resistance proteins

Yunus Aksüt^*Aslıhan ŞengelenDudu Melek Gürsoyİrem ÖğütcüÖzge KuvetMurat Pekmez^*

• Istanbul University, Istanbul, Türkiye

Glioblastoma (GB, grade-IV astrocytoma) represents a highly aggressive and treatment-resistant brain tumor characterized by a poor prognosis, notwithstanding advancements in conventional therapies. Temozolomide (TMZ) serves as the standard chemotherapeutic agent for GB; however, its clinical efficacy is significantly constrained by multidrug resistance (MDR). Given this limitation, there is increasing interest in identifying natural compounds that can enhance TMZ sensitivity. Chrysin (CHR), a natural flavonoid, has been shown to possess anti-inflammatory, antioxidant, and anticancer properties. Nonetheless, its potential role in mitigating TMZ resistance in glioblastoma remains unclear. This study aimed to evaluate the possible synergistic effects of CHR and TMZ on the U-87MG glioblastoma cell line in both 2D and 3D culture models. The combined treatment significantly reduced cell viability and proliferation in a dose-and time-dependent manner, surpassing the effects observed with individual treatments. CHR enhanced TMZ-induced cytotoxicity by promoting mitochondrial membrane potential collapse, endoplasmic reticulum stress, autophagy, and apoptosis, while also reducing cell motility, clonogenic potential, epithelial-mesenchymal transition (EMT) status, and stemlike characteristics. CHR co-treatment mitigated TMZ-induced elevations in the expression levels and nuclear translocation of P-glycoprotein (P-gp, a MDR protein) and the transcription factor nuclear factor-kappaB p65 subunit (NF-κB-p65). This was accompanied by a decrease in levels of stress/heat shock proteins (Hsp60, Hsp70, and Hsp90) and MDR-associated protein 1 (MRP1). Network pharmacology identified 20 common target genes for CHR, TMZ, and glioma. P-gp, a crucial glioma-related target, was confirmed as a key target of CHR through immunoblotting and immunofluorescence labeling. In 3D-spheroid cultures (mimicking in vivo tumor conditions), co-treatment with CHR and TMZ significantly impaired spheroid growth and viability. These findings suggest that CHR may be a promising adjuvant to TMZ therapy, providing novel insights into overcoming chemoresistance in GB treatment.