Temozolomide resistance mechanisms: unveiling the role of translesion DNA polymerase kappa in glioblastoma spheroids in vitro

Temozolomide (TMZ) is the leading therapeutic agent for combating Glioblastoma Multiforme (GBM). Nonetheless, the persistence of chemotherapy-resistant GBM cells remains an ongoing challenge, attributed to various factors, including the translesion synthesis (TLS) mechanism. TLS enables tumor cells to endure genomic damage by utilizing specialized DNA polymerases to bypass DNA lesions. Specifically, TLS polymerase Kappa (Pol'() has been implicated in facilitating DNA damage tolerance against TMZ-induced damage, contributing to a worse prognosis in GBM patients. To better understand the roles of Pol'( in TMZ resistance, we conducted a comprehensive assessment of the cytotoxic, antiproliferative, antimetastatic, and genotoxic effects of TMZ on GBM (U251MG) wild-type (WTE) and TLS Pol'( knockout (KO) cells, cultivated as three-dimensional (3D) tumor spheroids in vitro. Initial results revealed that TMZ: (i) induces reductions in GBM spheroid diameter (10-200 (<= 25 mu M) and promotes cell cycle arrest (G2/M phase) in Pol'( KO spheroids when compared with WTE counterparts. Furthermore, Pol'( KO spheroids exhibit elevated levels of cell death (Caspase 3/7) and display greater genotoxicity (53BP1) than WTE following TMZ exposure. Concerning antimetastatic effects, TMZ impedes invadopodia (3D invasion) more effectively in Pol'( KO than in WTE spheroids. Collectively, the results suggest that TLS Pol'( plays a vital role in the survival, cell death, genotoxicity, and metastatic potential of GBM spheroids in vitro when subjected to TMZ treatment. While the precise mechanisms underpinning this resistance remain elusive, TLS Pol'( emerges as a potential therapeutic target for GBM patients.