Characterization and cytotoxic activity of apoptosis-inducing pierisin-5 protein from white cabbage butterfly

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In this study, caspase-dependent apoptosis-inducing pierisin-5 gene was identified and characterized from cabbage white butterfly, Pieris canidia. A thousand-fold increase in expression of pierisin-5 gene was observed from second to third instar larvae, gradually decreasing before pupation. Pierisin-5 was purified from the fifth-instar larvae and was found to exhibit cytotoxicity against HeLa and HepG2 human cancer cell lines. Pierisin-5 showed growth inhibition and several morphological changes such as cell shrinkage, chromatin condensation and apoptotic body formation with programmed cell death in HeLa and HepG2 cells. Moreover, DNA fragmentation was observed after gel electrophoresis analysis. Caspase substrate assay showed further cleavage of Ac-DEVD-pNA, suggesting the activation of Caspase-3. Flow cytometry analysis revealed the cell cycle arrest at G1 phase and increased the percentage of apoptotic cells in cancer cell lines treated with pierisin-5. These findings suggest that pierisin-5 could significantly induce apoptosis in cancer cell lines and is mediated by activation of caspase-3 in the mitochondrial pathway. Phylogenetic analysis using pierisin proteins from Pierid butterflies, ADP-ribosylating toxins from bacteria, human, rat, and mouse indicated the possibility of horizontal transfer of pierisin genes from bacteria to butterflies. The single copy of pierisin gene unlike other insect toxin genes also supports lateral transfer.