Molecular Biology Laboratory, Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar-243 122.
Better understanding of molecular oncology and virology has paved the path for development of novel virus-based therapeutics for the treatment of cancer. Oncolytic viruses are therapeutics that have either been naturally selected or engineered to specifically grow in and kill tumor cells. In general oncolytic viruses derive their specificity by exploiting cell surface or intracellular aberrations in gene expression that arise in malignancies during tumor evolution such as down regulation of p53 or over-expression of anti apoptotic genes. Chicken Infectious Anaemia Virus (CAV) protein apoptin is one such protein which has inherent ability to induce a p53-independent and bcl-2-stimulated apoptosis specifically in cancer cells leaving normal cells unharmed. The present study was designed to determine the apoptotic potential of apoptin and delineate the role of various signaling pathways and proteins involved in apoptin induced apoptosis of HeLa cells. In this study, the VP3 gene of CAV was amplified by PCR and cloned in eukaryotic expression vector pcDNA3.1(+) at BamHI and XbaI restriction sites and in vitro expression analysis of recombinant pcDNA.cav.vp3 was done by RT-PCR using total RNA extracted from cells transfected with recombinant pcDNA.cav.vp3.
To study the involvement of various apoptotic pathways induced by apoptin, HeLa cells grown to 80–90% confluency were transfected with recombinant pcDNA.cav.vp3 using lipofectamine. The cells were harvested at 24 and 48 h post transfection and cytochemical assays specific to various pathways were performed. The involvement of death receptor pathway was determined by assessing the activation of initiator caspase-8 by flow cytometry. The results of which shown no significance difference in activation of caspase-8 between VP3 transfected cells and vector control both at 24 and 48 h p.t. The results of western blot to detect the expression of caspase-12 also did not showed any significant difference in band intensity among VP3 transfected, vector and mock control. However the results of change in mitochondrial membrane potential, efflux of cytochrome c, activation of caspase-9 (the initiator caspase of intrinsic pathway) and flow cytometry and western blot to detect activation of caspase-3 revealed that the apoptosis of HeLa cells induced by apoptin is mediated by the intrinsic/mitochondrial pathway. Thus the findings of this study indicated that apoptin induced apoptosis is mediated by intrinsic pathway and is independent of extrinsic pathway. Further studies are being undertaken to determine the role of other important mediators of apoptosis induced by apoptin in order to develop a novel therapeutic agent against cancer.
