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

Abstract

Temozolomide (TMZ) resistance is frequent in patients with glioblastoma (GBM), a tumor characterized by a marked inflammatory microenvironment. Recently, we reported that cyclooxygenase-2 (COX-2) is upregulated in TMZ-resistant GBM cells treated with high TMZ concentrations. Moreover, COX-2 activity inhibition significantly counteracted TMZ-resistance of GBM cells. Extracellular vesicles (EV) are considered crucial mediators in orchestrating GBM drug resistance by modulating the tumor microenvironment (TME) and affecting the surrounding recipient cell phenotype and behavior. This work aimed to verify whether TMZ, at low and clinically relevant doses (5-20 µM), could induce COX-2 overexpression in GBM cells (T98G and U87MG) and explore if secreted EV shuttled COX-2 to recipient cells. The effect of COX-2 inhibitors (COXIB), Celecoxib (CXB), or NS398, alone or TMZ-combined, was also investigated. Our results indicated that TMZ at clinically relevant doses upregulated COX-2 in GBM cells. COXIB treatment significantly counteracted TMZ-induced COX-2 expression, confirming the crucial role of the COX-2/PGE2 system in TMZ-resistance. The COXIB specificity was verified on U251MG, COX-2 null GBM cells. Western blotting of GBM-EV cells showed the COX-2 presence, with the same intracellular trend, increasing in EV derived from TMZ-treated cells and decreasing in those derived from COXIB+TMZ-treated cells. We then evaluated the effect of EV secreted by TMZ-treated cells on U937 and U251MG, used as recipient cells. In human macrophage cell line U937, the internalization of EV derived by TMZ-T98G cells led to a shift versus a pro-tumor M2-like phenotype. On the other hand, EV from TMZ-T98G induced a significant decrease in TMZ sensitivity in U251MG cells. Overall, our results, in confirming the crucial role played by COX-2 in TMZ-resistance, provide the first evidence of the presence and effective functional transfer of this enzyme through EV derived from GBM cells, with multiple potential consequences at the level of TME.

Involvement of Cyclooxygenase-2 in Establishing an Immunosuppressive Microenvironment in Tumorspheres Derived from TMZ-Resistant Glioblastoma Cell Lines and Primary Cultures

“…Interestingly, the treatment with the selective COX-2 inhibitor, NS398, concurrent with TMZ, overcame the TMZ-induced overexpression of β-catenin, O-6-methylguanine-DNA methyltransferase (MGMT), and SOX-2 in T98G and lowered the levels of COX-2 shuttled in extracellular vesicles. These data confirmed the crucial role of the COX-2/PGE2 system in the cascade of events activated by TMZ and implicated in GBM chemoresistance [27].…”

Repurposed Drugs Celecoxib and Fmoc-L-Leucine Alone and in Combination as Temozolomide-Resistant Antiglioma Agents—Comparative Studies on Normal and Immortalized Cell Lines, and on C. elegans

“…Higher COX-2 production may be stimulated via a negative feedback loop that was described previously [ 53 , 54 ]. Lombardi et al observed that temozolomide, one of the three chemotherapy agents available for the treatment of glioma, also causes increase of COX-2 level in glioma cells [ 55 ]. This may indicate that the anti-glioma action of CXB possesses a similar mechanism of action.…”

Monitoring of single extracellular vesicle heterogeneity in cancer progression and therapy

“…Temozolomide is a promising chemotherapeutic drug to alkylate the DNA and is used in treating malignant glioma ( 102 ). Likewise, temozolomide could elicit the cyclooxygenase-2 expression in glioma cells, resulting in the upregulation of cyclooxygenase-2-carrying EVs, which have the activity to shift the M2-like pro-tumor phenotype of macrophage ( 103 ). Also, temozolomide-treated glioma cells release a different repertoire of small and large EVs, which could stimulate the macrophage to transit the M2-like phenotype with increased cellular expression of IL-6 and IL-10 ( 104 ).…”