Cantharidin Induces Pyroptosis in triple-negative breast cancer cells via vitamin D receptor-targeted inhibition and activation of ROS/caspase/GSDME axis.

Cantharidin, a natural small-molecule compound with anticancer activity, has been reported to induce programmed cell death in various cancers. However, its molecular targets in triple-negative breast cancer (TNBC) remain unclear. This study explored the mechanism and potential target of Cantharidin in TNBC. Cantharidin significantly reduced TNBC cell viability and triggered pyroptosis, as indicated by membrane bubbling, lactate dehydrogenase (LDH) release, membrane damage, and increased annexin V/PI and annexin V/PI populations. Mechanistically, it activated the Caspase-9/Caspase-3/GSDME axis, elevated intracellular reactive oxygen species (ROS), and suppressed the JAK2/STAT3 pathway. Molecular docking predicted a strong binding affinity between Cantharidin and the vitamin D receptor (VDR) (binding energy: 7.1 kcal/mol), further supported by Molecular Dynamics (MD) simulations, MM/PBSA calculations, and confirmed by drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) assays. Western blotting showed that Cantharidin downregulated VDR protein expression. These findings identify VDR as a direct target of Cantharidin and highlight its potential as a potent pyroptosis inducer for TNBC therapy.