Plumbagin activates NLRP3/caspase-1/GSDMD-mediated pyroptosis by decreasing DUSP4 expression to exhibit anticancer property in cholangiocarcinoma.

BACKGROUND: Plumbagin is effective in treating cholangiocarcinoma (CCA), however, the potential mechanism of plumbagin therapy is still unclear. This study aims to uncover the anti-tumor nature of plumbagin and the specific mechanism by which plumbagin alleviates NLRP3/caspase-1/GSDMD-induced pyroptosis.

METHODS: Human CCA cells lines HuCCT1 and RBE and the CCA mouse model injected with HuCCT1 cells were used for the mechanism exploration. Different concentrations of plumbagin were incubated with the cells or injected to the CCA mouse to investigate the anti-tumor role of pumbagin. CCK-8 and Transwell was used to detect the cell viability and migration, respectively. ELISA kits were used to detect the ROS, LDH, IL-1β, and IL-18 levels. SEM was used for pyroptosis condition observation. qRT-PCR and WB were used to detect the expression levels of DUSP4, NLRP3, GSDMD-N, and cle-caspase-1 levels. Immunohistochemical staining was used for detection of tumor tissue injury.

RESULTS: In CCA cells, plumbagin inhibited the cell viability and migration. In CCA mouse model, plumbagin inhibited the tumor growth. Along with the plumbagin treatment, the decreased DUSP4 expression, and increased ROS, LDH, NLRP3, clecaspase-1, GSDMD-N proteins, and pyroptosis degree, were investigated. After addition of NLRP3 inhibitor MCC950 and transfection of oe-DUSP4, the promoted pyroptosis and inhibited tumor growth induced by plumbagin were reversed, indicating that the plumbagin treatment inhibited the CCA progression through decreasing the DUSP4 expression and thus activating the pyroptosis.

CONCLUSION: DUSP4/NLRP3/caspase-1/GSDMD-mediated pyroptosis is demonstrated to be the therapeutic mechanism of plumbagin in CCA.