Antileukemic activity and mechanism of ardisianone-a crucial role of mitochondrial stress in apoptosis and necroptosis

Abstract Background Ardisianone was reported to induce apoptosis in prostate cancer cells, but necroptosis in leukemia cell lines. It is needed to clarify whether ardisianone could cause apoptosis and necroptosis in leukemia cells and investigate its underlying mechanisms. Methods Cytotoxicity was examined using the MTT assay. Cytoflowmetric analysis of JC-1 and PI staining was used to examine mitochondrial membrane potential and cell cycle progression, respectively. Apoptosis was examined by annexin V-PI staining assay. Protein expression was detected by Western blot and apoptosis antibody array chip. Immunofluorescence staining detected necroptosis effect. Results Ardisianone inhibited cell viability in HL-60, a promyelocytic leukemia cell line, with IC50 value of 1.87 µ M in a 24h exposure. Further detection showed that ardisianone induced time- and concentration-dependent apoptosis. JC-1 staining demonstrated that ardisianone caused a profound loss of mitochondrial membrane potential. Western blot also confirmed the decrease of pro-survival Bcl-2 family protein and the downregulation of necroptosis relatives (e.g., RIPK1 and RIPK3). The caspase cascade was profoundly activated by ardisianone. Notably, the specific pan-caspase inhibitor Q-VD-OPh significantly blunted ardisianone induced loss of membrane potential and apoptosis, suggesting that caspase cascade activation may further amplify mitochondrial damage and apoptotic signaling cascade. The expression of RIPK1/RIPK3/MIKL as significantly reduced by pre-treatment of necroptosis inhibitor (Necrostatin-1, Nec-1), suggesting that Nec-1 attenuates ardisianone-induced necroptosis. Conclusion The data suggest that ardisianone induces mitochondrial dysfunction and apoptosis in leukemia cells through modification of Bcl-2 family members and causes necrotic cell death.