Alkalization of the intracellular pH reprograms TNF-α signaling from inflammatory NF-κB activation to tumoricidal RIP kinase-dependent necroptosis in cancer cells.

Tumor necrosis factor-alpha (TNF-α) plays paradoxical roles in cancer, promoting tumor survival via NF-κB activation while also inducing necroptotic cell death. Here, we identify the intracellular pH (pHᵢ) as a decisive switch that reprograms TNF-α signaling from a prosurvival inflammatory response toward tumoricidal necroptosis. In both hepatocellular and cervical carcinoma models, alkalizing the pHᵢ with bicarbonate inhibits TNF-α-mediated IκB phosphorylation and NF-κB nuclear translocation while simultaneously activating the TNF-α-driven necroptotic cascade, which is characterized by the sequential phosphorylation of RIPK1, RIPK3, and MLKL. Mechanistically, bicarbonate induces the depolarization of the mitochondrial membrane potential, reactive oxygen species (ROS) generation, and a cyclophilin D-dependent permeability transition-effects that are synergistically amplified by TNF-α. Genetic knockdown of RIPK1 or RIPK3 abrogates necroptosis, whereas the pharmacological inhibition of ROS suppresses necroptosis and concurrently restores NF-κB nuclear translocation. ROS are identified as the key mediators determining the switch between these opposing TNF-α signaling pathways. In vivo, bicarbonate and TNF-α treatment suppresses tumor growth, enhances necroptotic signaling, and inhibits NF-κB activation. Thus, our work indicates that intracellular alkalization acts as a modifiable regulator to redirect TNF-α from a toxic, proinflammatory cytokine into a potent and selective tumoricidal agent. Graphical Abstract: Alkalization of the pHi by bicarbonate reprograms TNF-α signaling from a prosurvival NF-κB axis to a cytotoxic necroptotic pathway. a TNF-α alone activates NF-κB signaling, promoting inflammation and tumor growth. b Bicarbonate alkalizes the pHi and induces mitochondrial dysfunction and reactive oxygen species (ROS) production, which is further amplified by TNF-α. Elevated ROS levels inhibit NF-κB signaling and activate RIPK1-dependent necroptosis. This ROS-mediated switch redirects TNF-α signaling from a proinflammatory, tumor-promoting response to a cytotoxic, necroptotic pathway, resulting in the suppression of both tumor growth and inflammation. This diagram highlights the translational potential of combining bicarbonate with TNF-α for cancer therapy.