Abstract

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality worldwide. Bufalin, a bioactive bufadienolide derived from Venenum Bufonis, has shown antitumor activity in multiple cancer models, but its metabolic effects in NSCLC remain incompletely defined. In this study, we integrated untargeted metabolomics, U-13C6-glucose tracing, Seahorse extracellular flux analysis, and target-engagement assays to characterize bufalin-induced metabolic perturbations in A549 NSCLC cells. Bufalin reduced NSCLC proliferation in vitro and inhibited tumor growth in a xenograft model. Metabolic profiling indicated that bufalin altered glycolytic carbon flow and impaired metabolic flexibility, with concomitant changes in glycolysis-related intermediates and mitochondrial stress. Computational docking, peptide-centric local stability assay (PELSA), cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) supported a direct interaction between bufalin and pyruvate kinase M2 (PKM2), suggesting that PKM2 contributes to bufalin-associated metabolic reprogramming. Functional analysis using PKM2-modulated A549 cells further indicated that PKM2 expression influences cellular tolerance to bufalin-induced oxidative and mitochondrial stress. Because bufalin is a pleiotropic cardiotonic steroid with established targets such as Na+/K + -ATPase, the present findings are interpreted as evidence for a PKM2-involved mechanism rather than an exclusive PKM2-dependent pathway. These results provide a mechanistic basis for further investigation of bufalin as a metabolic modulator in NSCLC.