Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines

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.

Cyclooxygenase-2 Upregulated by Temozolomide in Glioblastoma Cells Is Shuttled In Extracellular Vesicles Modifying Recipient Cell Phenotype

“…In this work, we show evidence that low and clinically relevant TMZ concentrations, able to upmodulate COX-2 in T98G and U87MG cells, led to a dose-dependent increase of the COX-2 levels in secreted EV. It is fair to point out that the results obtained with repeated exposure to TMZ for 5 consecutive days at clinically relevant concentrations, showing the TMZ-induced COX-2 increase as well as the increased sensitivity to TMZ in the presence of COXIB, have the same trend as those obtained in our previous work (8) where high doses of TMZ for 72 h were used on TMZ-resistant cells. Hence, the similarity between the effects of metronomic low dose application and those of single high dose protocol could slightly mitigate the criticalities raised toward in vitro studies in which the use of un-physiological high TMZ concentrations is questioned since it does not faithfully reproduce the clinical situation.…”

“…After 5 days of continuous, scheduled treatment with COXIB, the T98G and U87MG cell number was decreased compared to CNTR cells (Figures 1G, 2G). As previously reported, COX-2 inhibitors reduced the growth rate of GBM cell lines (8,32). The reduced cell growth rate following CXB or NS398 treatment could be due to the inhibition of basal COX-2 activity and COX-2-dependent signaling pathways.…”

“…As the proinflammatory enzyme COX-2 is recognized as a crucial mediator in GBM biology, selective COXIB were defined as an extremely promising GBM therapy increasing sensitivity to chemotherapy without other side effects (7). In this regard, recently, our group reported that the selective COX-2 inhibitor, NS398, counteracted chemoresistance to TMZ, used at high concentrations (200 µM) for 72 h, in resistant GBM cell line (T98G) abrogating TMZ-induced COX-2 upregulation and COX-2-dependent pathways involved in TMZ-resistance (8). TMZ exposure of resistant T98G, but not sensitive and COX-2 null U251MG cells, led to a significant and dose-dependent upregulation of COX-2.…”

“…In this study, the first aim was to analyze the effect of TMZ, at clinically relevant concentrations (5-20 mM), on T98G ability to release EV when daily exposed to long-term treatment (5 days), a condition conceived to mimic the clinical and therapeutical setting, during which intratumoral TMZ concentrations between 1 and 35 µM are achieved (16). Thus, to improve the transferability of in vitro results to in vivo studies, we initially evaluated whether TMZ at clinically relevant concentrations could induce a cytotoxic effect and a significant increase of COX-2 level similar to what we previously registered with TMZ at higher concentrations in T98G cells, chosen as TMZresistant/COX-2 positive cell line (8). The U251MG cells were used as a negative control, being TMZ-sensitive andCOX-2 null.…”

Gallic Acid Enhances the Anti-Cancer Effect of Temozolomide in Human Glioma Cell Line via Inhibition of Akt and p38-MAPK Pathway

“…This compound can readily spread across the blood-brain barrier. TMZ is a well-tolerated chemotherapeutic drug in the therapy of glioblastoma multiforme [1].…”