Targeting P2X4 receptor triggers ER stress– mediated necroptosis in glioblastoma

Abstract

Glioblastoma (GBM) is a malignant brain cancer that remains extremely difficult to treat, in part because it readily evades apoptosis and relies on alternative survival programs. In this work, we examined the role of the purinergic receptor P2X4, which is elevated in GBM cells, and assessed whether its suppression influences cell fate. Selective pharmacological inhibition of P2X4 using PSB-12062 and 5-BDBD markedly reduced the viability of GBM cells and initiated necroptotic cell death rather than apoptosis. Using Fluo-3 AM and the ER-targeted Ca²⁺ indicator G-CEPIA1er, we found that loss of P2X4 activity caused a gradual decline in both cytosolic and ER-stored Ca²⁺. Immunocytochemistry showed that a subset of P2X4 molecules localize near ER membranes, supporting a potential contribution of ER-associated P2X4 to Ca²⁺ handling. Inhibitors targeting IP₃Rs, RyRs, SOCE did not rescue cell death, indicating that P2X4-dependent Ca²⁺ control operates independently of classical Ca²⁺ pathways. ER Ca²⁺ depletion triggered strong ER stress responses, including increased GRP78, ATF4 and CHOP. The chemical chaperone 4-phenylbutyric acid partially reversed this stress and diminished necroptotic signaling, indicating that ER dysfunction acts upstream of P2X4-inhibition–induced necroptosis. These results identify P2X4 as an important regulator of ER Ca²⁺ stability and stress tolerance in GBM cells, and suggest that targeting P2X4 could provide a therapeutic strategy for GBM that is refractory to apoptosis-based therapies.