Fulvestrant Enhances Temozolomide Effectiveness Against Glioblastoma: Insights from a Preclinical Study

Introduction: Glioblastomas (GBM) are among the most aggressive primary brain tumors, as they are characterized by a median survival of less than 15 months. The standard treatment for GBM includes surgical resection, radiotherapy, and temozolomide chemotherapy. However, these tumors exhibit high levels of resistance, posing a significant clinical challenge. One factor contributing to resistance is the overexpression of the DNA repair enzyme O6-methylguanine- DNA-methyl transferase (MGMT). This study evaluated whether fulvestrant, an antagonist of the estrogen receptor, could function as an MGMT inhibitor to improve temozolomide effectiveness against glioblastoma cell lines, spheroids, and in vivo xenograft models. Methods: Cell viability was assessed following temozolomide treatment, either individually or combined with fulvestrant. Spheroid size was monitored by confocal microscopy along with the density of viable and dead cells as detected by the Cell Titer-Glo assay (in vitro experiments were performed in triplicate). Additionally, in vivo glioblastoma xenografts were established in male nude mice ((n=8), and tumor growth was monitored by near-infrared fluorescence imaging of the αvβ3 integrin (RGD). Their metabolic activity was characterized by micro-PET/CT with 18FFluorodeoxyglucose (18F-FDG). MGMT protein levels were detected by Western blot (n=3) Results: The temozolomide/fulvestrant combination significantly enhanced cytotoxicity by 80% in cells (p<0.001), and by 40% (p<0.01) in spheroids. Furthermore, the combination reduced tumor growth when compared to temozolomide alone (p<0.01). A statistically significant three-fold decrease in MGMT protein levels was detected after the temozolomide/fulvestrant treatment (p<0.01). Discussion: The enhancement of temozolomide’s effect may be partly explained by the reduction of MGMT levels induced by fulvestrant, underscoring the need to further explore this pathway in glioblastoma resistance. This includes investigating the inhibition of MGMT-regulating transcription factors such as Sp1, AP-1, NF-κB, and HIF-1, as well as the disruption of ER signaling. By promoting ER degradation and reducing ER-dependent gene transcription, fulvestrant could suppress MGMT expression, thereby contributing to increased temozolomide sensitivity. Conclusion: These findings suggest that fulvestrant may be a promising chemo-sensitizing agent for glioblastoma treated with temozolomide.