Abstract
Objectives Glioblastoma is the most common and aggressive form of primary brain cancer with a median survival of 15 months from diagnosis. The purinergic receptor P2X7 (P2X7R) is a regulator of several cell signalling pathways, and its expression is upregulated in glioblastoma. This study examined the expression and function of P2X7R in a human glioblastoma cell line, U251 and utilised a pharmacological antagonist of P2X7R, AZ10606120, to inhibit receptor function and delineate downstream consequences of receptor inhibition.
Methods P2X7R expression and function in the U251 cell line was determined using immunocytochemistry, live cell Ca2+ imaging and live cell dye uptake assays. Effect of AZ10606120 on cell viability was investigated via cell counts and LDH assay. The mode of tumour cell death was subsequently investigated via annexin V and cleaved caspase-3 staining, and multiplex RNA analysis.
Results P2X7R was expressed and functional in U251 cells. AZ10606120 treatment72 significantly decreased tumour cell number (p<0.0001), and significantly increased tumour cell death, as evidenced by increased LDH release (p<0.001). This was concentration-dependent, modelled by a least squares linear regression (R2 = 0.8221). No difference was observed in annexin V or cleaved caspase 3 staining, indicating minimal apoptosis occurring upon AZ10606120 treatment. Multiplex mRNA analysis demonstrated downregulation of genes associated with apoptosis, pyroptosis and necroptosis. Inhibition of P2X7R using small interfering RNA significantly decreased cell number and increased extracellular LDH.
Conclusions This study describes findings of significant translational potential in neurooncology. It highlights AZ10606120 related P2X7R inhibition as a novel therapeutic target in glioblastoma.
