Pivotal role of cyclooxygenase 2(COX-2) in temozolomide-resistance of glioblastoma cell lines

ABSTRACT Glioblastoma multiforme (GBM) is one of the most lethal brain cancers worldwide since it is a highly infiltrating and aggressive central nervous system tumor representing about 50% of all adult malignant primary brain tumors. Nowadays, treating malignant GBM is multidisciplinary since it is characterized by a high degree of genetic and cellular heterogeneity. Surgical resection is usually followed by radiotherapy and chemotherapy with alkylating agent temozolomide (TMZ). However, TMZ resistance remains a main limitation in GBM treatment. The cytotoxicity of TMZ is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. Despite continuous efforts to ameliorate the GBM therapeutic approaches, GBM often relapses due to the presence of GBM stem cells (GSCs), a small population of tumor-initiating cells which remain after surgical exeresis and show stem cell-like properties such as self-renewal and differentiation ability and can promote tumor progression, therapy resistance, and metastasis. GBM is characterized by the presence of an inflammatory milieu, and inflammation, a key component of the tumor microenvironment, is emerging as a target for new treatment modalities. Cyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in GBM tumorigenesis, progression, and stemness. The poor survival of GBM was mainly associated with the presence of GSCs and the markedly inflammatory microenvironment. To deeply explore the involvement of COX-2 in GBM biology, our first aim was to study the effects of NS398, a selective COX-2 inhibitor, on GSCs derived from COX-2- expressing GBM cell lines showing intrinsic diversity and individual genetic features, i.e., U87MG and T98G, assessing the neurospheres’ growth, autophagy induction, and extracellular vesicle (EVs) release. The previous results demonstrated that neurospheres derived from both GBM cell lines resulted highly influenced by NS398 exposure, showing remarkable morphological changes, a reduced growth rate, and a relevant level of autophagy. In addition, it has been verified that COX-2 inhibition induced a functional modification of the EVs released by neurospheres. In particular, EVs derived from neurospheres pre-treated with NS398 strongly reduced the migration ability and significantly triggered the autophagic process in the adherent U87MG and T98G cells, thus causing effects similar to those observed following NS398 direct addition. Recently, COX-2 inhibitors are increasingly considered an add-on treatment to improve GBM sensitiveness to traditional chemo- and radiotherapy, increasing apoptosis and reducing tumor migration and stemness potential. In this scenario, the effect of TMZ, as a single agent and in combination with NS398, on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG) was here investigated. Surprisingly, COX-2 expression resulted in dose-dependently upregulated by TMZ in T98G, COX-2 positive cells, while no influence was recorded in U251MG, COX-2 negative cell line. TMZ was also able to upregulate Wnt/β-catenin signaling, MGMT expression, and stemness potential, all crucial components implicated in the GBM-chemoresistance. Moreover, the combined effects of NS398 and TMZ (NS398+TMZ) were studied in two GBM cell lines. COX-2 inhibition by NS398 was able to overcome TMZ-induced overexpression of COX-2, β-catenin, MGMT, and SOX-2 in T98G, to significantly enhance the dead cell percentage, was able to induce apoptosis and to inhibit the clonogenic potential in T98G cells dramatically. These findings strongly support the key and the hierarchically superior role played by the COX-2/PGE2 system in the cascade of pathways activated by TMZ and implicated in chemoresistance.