Brazilin inhibits bladder cancer by promoting cell necroptosis

Abstract Brazilin possesses anticancer effects, but the mechanisms are poorly understood. This study investigated the mechanisms of brazilin‐induced cell death in the T24 human bladder cancer cell line. Low serum cell culture and the lactate dehydrogenase assay were used to confirm the antitumor effect of brazilin. Annexin V and propidium iodide double staining, transmission electron microscopy, fluo‐3‐AM assay for Ca 2+ mobilization and caspase activity assay were performed to identify the type of cell death after brazilin treatment. Mitochondria membrane potentials were measured using JC‐1. Quantitative real‐time polymerase chain reaction and western blot analyses were performed to verify the expression of the necroptosis‐related genes and proteins receptor interacting protein 1 (RIP1), RIP3 and mixed lineage kinase domain‐like (MLKL). The results showed that brazilin induced necrosis in T24 cells and upregulated the mRNA and protein levels of RIP1, RIP3 and MLKL and Ca 2+ influx. The necroptosis‐mediated cell death was rescued by the necroptosis inhibitor necrostatin‐1 (Nec‐1), but not by the apoptosis inhibitor z‐VAD‐fmk. Brazilin repressed caspase 8 expression and decreased the mitochondrial membrane potentials; both effects were partially reversed by Nec‐1. Brazilin induced physiological and morphological changes in T24 cells and RIP1/RIP3/MLKL‐mediated necroptosis might be involved. In conclusion, the results confirm the involvement of necroptosis in brazilin‐induced cell death and suggest that brazilin could be explored as an anticancer agent against bladder cancer.