Abstract
TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). β-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte® system, cell cycle/apoptosis, and clonogenic potential. COX-2, β-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, β-catenin, MGMT, and SOX-2 up-regulation.
Highlights
Glioblastoma (GBM) represents the most frequent and aggressive primary malignancy of the central nervous system in adults
Sci. 2022, 23, 1545 either U87MG or T98G cells, significantly inhibited cell migration and induced autophagy in recipient adherent U87MG and T98G cells, leading to effects quite comparable with those directly produced by the inhibitor in the same cells. These findings showed that NS398 influenced both GBM cell lines likewise, despite their different COX-2 expression levels, as well as the intrinsic genetic diversity, including TP53 gene status, methylguanine-DNA methyltransferase (MGMT)
Our results show that COX-2 inhibitor, NS398, down-regulated, even though not significantly, the β-catenin expression compared with CNTR, while TMZ alone significantly up-regulated its expression (Figure 11)
Summary
Glioblastoma (GBM) represents the most frequent and aggressive primary malignancy of the central nervous system in adults. Cyclooxygenase-2 (COX-2), the inducible isoform of prostaglandin H synthase, a proinflammatory enzyme, is often up-regulated in GBM cells and tissues, and higher COX-2 expression is associated with most malignant histological grade [9,10]. The glioma-promoting effects of COX-2 induction are mainly associated with its product, prostaglandin E2 (PGE2) [14], which, in turn, at high levels, favors tumor initiation and progression and is implicated in the development of therapy resistance [15,16,17]. In GBM cell lines and mouse models, COX-2-derived PGE2 promotes glioma stem cells (GSC) self-renewal and therapy resistance through the EP4 receptor that activates the MAPK signaling cascade, leading to the up-regulation of the inhibitor of differentiation 1 (Id1), a main mediator that abrogates differentiation signals in GSC and contributes to chemoresistance in GBM. Id1 could regulate stemness through Wnt signaling, and when Id1 was genetically or pharmacologically inhibited, the cytotoxic effect of TMZ was enhanced in vitro, and the survival in a GBM xenograft model was extended [18,19]
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