Glioblastoma (GB) is the most aggressive and common primary malignant tumor of the brain and central nervous system. Without treatment, the average patient survival time is about six months, which can be extended to fifteen months with multimodal therapies. The chemoresistance observed in GB is, in part, attributed to the presence of a subpopulation of glioblastoma-like stem cells (GSCs) that are characterized by heightened tumorigenic capacity and chemoresistance. GSCs are situated in hypoxic tumor niches, where they sustain and promote the stem-like phenotype and have also been correlated with high chemoresistance. GSCs have the particularity of generating high levels of extracellular adenosine (ADO), which causes the activation of the A₃ adenosine receptor (A3AR) with a consequent increase in the expression and activity of genes related to chemoresistance. Therefore, targeting its components is a promising alternative for treating GB. This analysis determined genes that were up- and downregulated due to A3AR blockades under both normoxic and hypoxic conditions. In addition, possible candidates associated with chemoresistance that were positively regulated by hypoxia and negatively regulated by A3AR blockades in the same condition were analyzed. We detected three potential candidate genes that were regulated by the A3AR antagonist MRS1220 under hypoxic conditions: LIMD1, TRIB2, and TGFB1. Finally, the selected markers were correlated with hypoxia-inducible genes and with the expression of adenosine-producing ectonucleotidases. In conclusion, we detected that hypoxic conditions generate extensive differential gene expression in GSCs, increasing the expression of genes associated with chemoresistance. Furthermore, we observed that MRS1220 could regulate the expression of LIMD1, TRIB2, and TGFB1, which are involved in chemoresistance and correlate with a poor prognosis, hypoxia, and purinergic signaling.

Slc4a genes encode various types of transporters, including Na⁺-HCO₃⁻ cotransporters, Cl⁻/HCO₃⁻ exchangers, or Na⁺-driven Cl⁻/HCO₃⁻ exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl⁻/HCO₃⁻ exchanger, which can be driven by either Na⁺ or K⁺, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na⁺ or K⁺ drive Cl⁻/HCO₃⁻ exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl⁻/HCO₃⁻ exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl⁻ and HCO₃⁻-dependent K⁺ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K⁺ at the Na⁺ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K⁺ in the same direction as HCO₃⁻ ions in a similar fashion to that described for Na⁺ transport in the rat Slc4a8 structure.