Subbarayan Sarathbabu, Satheesh K. Marimuthu, Souvik Ghatak, Subramanian Vidyalakshmi, Guruswami Gurusubramanian , Sankar K. Ghosh, Selvi Subramanian*, Wenqing Zhang and Nachimuthu S. Kumar* Pages 337 - 346 ( 10 )
Background: 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, HepG2 human cancer cell lines and L-132 human lung epithelial cell line. Among the target cells, HeLa was the most sensitive to Pierisin-6. 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 the 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.
Pierisin-6, cytotoxicity, flow cytometry, cytochrome c, caspase-3, transmembrane potential.
Department of Biotechnology, Mizoram University, Aizawl -796004, Mizoram, Department of Biotechnology, PSG College of Technology, Coimbatore -641004, TN, Department of Biotechnology, Mizoram University, Aizawl -796004, Mizoram, Department of Biotechnology, PSG College of Technology, Coimbatore -641004, TN, Department of Zoology, Mizoram University, Aizawl -796004, Mizoram, Department of Biotechnology, Assam University, Silchar -788011, Assam, Department of Biotechnology, PSG College of Technology, Coimbatore -641004, TN, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Department of Biotechnology, Mizoram University, Aizawl -796004, Mizoram