Induction of Apoptosis by pierisin-6 in HPV positive HeLa and HepG2 Cancer Cells is mediated by the caspase-3 dependent mitochondrial pathway.

OBJECTIVE: To explore the cytotoxic and apoptotic activity of the pierisin-6 protein in HPV HeLa and HepG2 cell lines.

METHODS: In this study, isolation and purification of cytotoxic prierisin-6 from the larvae of Pieris napi by affinity column chromatography techniques.Characterization of full-length mRNA of pierisin-6 gene was performed using 3'/5' RACE PCR. The quantitative RT-PCR used to study the developmental stage-specific expression of pierisin-6 mRNA. The most effective concentration of pierisin-6 protein was determined by measuring cell proliferation. Apoptosis was assessed using AO/Et-Br, Propidium Iodide and Rhodamine 123 assays, whereas protein levels of caspase 3, cytochrome C were evaluated by ELISA method. Pierisin-6 induced cell cycle arrest was determined using Propidium iodide by FACS.

RESULTS: In this study, Pierisin-6, a novel apoptotic protein was found to have cytotoxicity against HeLa at IC50 of 2.02 ng/ml and HepG2 human cancer cell lines. Flow cytometry analysis confirms an increased percentage of apoptotic cells in sub G1 phase and cell cycle arrest at S phase. Alteration in the transmembrane potential of mitochondria, Cytochrome c released from mitochondrial membrane, and caspase substrate assay demonstrated the cleavage of Ac-DEVD-pNA signifying the activation of Caspase-3. These findings suggested that Pierisin-6 significantly induce apoptosis in HeLa and HepG2 cells and is attributed mainly through a mitochondrial pathway by activation of caspases. The developmental and stage-specific expression of pierisin-6 mRNA was one thousand-fold increased from second to third instar larvae and gradually declined before pupation.

CONCLUSION: Pierisin-6 represents a promising therapeutic approach for liver cancer patients.