The Time-Dependent Effects of Temozolomide on Autophagy Gene Expression in Glioblastoma Cells

Abstract

Background: Temozolomide (TMZ) resistance represents a major therapeutic challenge in glioblastoma treatment, where autophagy has emerged as a key adaptive survival mechanism. Although numerous studies have implicated autophagy in TMZ resistance, most have assessed this process at a single point, thereby overlooking its dynamic and time-dependent nature. Methods: In this study, we systematically investigated the temporal regulation of autophagy-related gene expression in two human glioblastoma cell lines with distinct MGMT methylation status and TMZ sensitivities (T98G and U87) following TMZ treatment. Cells were exposed to TMZ and harvested at defined time points (0 h, 6 h, 24 h, and 48 h). The expression levels of genes representing distinct stages of the autophagy pathway, including initiation, nucleation, elongation, selective autophagy, lysosomal function, and transcriptional regulation, were analyzed using RT-qPCR. Relative gene expression was calculated using the 2-Delta Delta CT method with GAPDH as the reference gene. Results: Our results reveal a time-dependent and phase-specific transcriptional reprogramming of the autophagy machinery in response to TMZ-induced stress. Early time points were characterized by modulation of autophagy initiation and nucleation genes, whereas intermediate and late phases showed prominent regulation of genes associated with autophagosome elongation, selective autophagy, autophagic flux, and transcriptional control. Conclusions: Collectively, these findings demonstrate that autophagy in TMZ-treated glioblastoma cells is not a static response but a dynamically regulated, multi-phase program. Specifically, in TMZ-resistant T98G cells, this process matures into a sustained adaptive program with robust late-phase lysosomal integration, while in TMZ-sensitive U87 cells, the early autophagic response is transient and fails to support long-term lysosomal coordination. This temporal perspective provides new insights into the role of autophagy in TMZ tolerance and underscores the importance of time-resolved analyses when targeting autophagy to overcome chemoresistance in glioblastoma.