Kalantuboside B induced apoptosis and cytoprotective autophagy in human melanoma A2058 cells: An in vitro and in vivo study.

Kalantuboside B (KB), a natural bufadienolide derivative extracted from the succulent plant Kalanchoe tubiflora, is well-known for its cardiotonic, immunomodulatory, and anti-inflammatory properties. In this study, we tested in vitro and in vivo anti-cancer efficacy with low concentrations of KB (5-30 ng/mL; 8.7-52.2 nM) on A2058 melanoma cells; and for the molecular mechanisms that underlie them. KB significantly inhibited the cell viability and colony formation via arresting the cell cycle at G2/M phase. There was an association with a decrease in Cyclin A/B1, Cdc25C, and Cdc2 expressions. Further, this treatment indicated the induction of apoptosis, DNA fragmentation, cytochrome c release, and caspase-3, -8, -9, and -12 activation, and PARP cleavage, which shows that mitochondrial, death-receptor, and ER-stress signaling pathways are involved. KB-induced autophagy was apparent from enhanced LC3-II accumulation, GFP-LC3 puncta, and AVO formation. Surprisingly, KB-mediated cell death was potentiated by 3-MA and CQ to suggest the role of autophagy as a cytoprotective mechanism. Moreover, KB-treated A2058 cells enhanced intracellular ROS generation and antioxidant NAC prevented apoptosis and reversed cytoprotective autophagy. Interestingly, KB-induced apoptosis (PARP cleavage) and cytoprotective autophagy (LC3-II accumulation) were mediated by the up-regulation of the ERK signaling pathway. It was also shown that KB promoted cytoprotective autophagy by a calcium dependent-p53 downregulation pathway. In vivo data showed that KB suppressed tumor growth significantly in A2058-xenografted nude mice. A Western blot indicated cell-cycle inhibition (cyclin A reduction), apoptosis induction (PARP cleavage and Bcl-2 inhibition), and cytoprotective autophagy (LC3-II upregulation and p53 downregulation) in KB-treated A2058-xenografted mice. Our findings suggested that KB-induced ROS pathway plays a role in mediating the apoptosis and cytoprotective autophagy in human melanoma cells. Thus, KB is considered to be a putative anti-tumor agent.