Purified Brazilin from Sappan wood extract has been reported with significant antitumor effect, especially on human T24 cells and bladder cancer mouse models. Brazilin can significantly induce expression of c-Fos and GADD45β and transfection expression of c-Fos and GADD45β in T24 cells can induce significant cell morphology changes, reduced viability and cell death, while transfection of siRNA-c-Fos and siRNA-GADD45β can reverse the induced cell death. Co-transfection of both c-Fos and GADD45β into T24 cells resulted in a significantly additive effect when compared to single transfection with only c-Fos or GADD45β. Meanwhile, transfection of interfering siRNA-c-Fos or siRNA-GADD45β can partially rescue the cell viability and siRNA co-transfection showed increased rescue rate. The transfection expression and interference with pcDNA3.1-c-Fos/siRNA-c-Fos or pcDNA3.1-GADD45β/siRNA-GADD45β did not affect each other's expression. Moreover, analysis of c-Fos and GADD45β regulated genes and signal pathways showed that no common regulated genes or pathways were present. All the results indicated that c-Fos and GADD45β mediate independent Brazilin-inducible genes and pathways. © 2018 IUBMB Life, 70(11):1101-1110, 2018.

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 Ca2+ 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 Ca2+ 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.