Differential Apoptotic Mechanisms Exploited By Different Virulent NDV Strains

Oncolytic viruses can specificly replicate in tumor cells that lead to cell death. Although NDV(Newcastle disease virus) is an avian virus, studies in vitro and in vivo have proved that NDV can be safely used as an efficient oncolytic virus. Apoptosis was reported to play an important role in the oncolytic processs induced by NDV. Although the results of ongoing clinical trials are encouraging, the cellular basis of NDV cytotoxcity were not fully understood, especially, the reported apoptosis signaling pathways induced by NDV in tumor cells are inconsistent. In this study, NDV-FMW, NDV-BC and NDV-LaSota were used as representive of velogenic, mesogenic, lentogenic NDV strain, with the three different virulent NDV strains here we mainly demostrarte the time course induction of intrinsic and extrinsic apoptoic pathways following NDV infection using A549 cells as the model system.Methods1 MTT assay to validate oncolytic efficacy of different virulent NDV strains in A549 cells with a range of MOIs (0.1,1,10) at 24h, 48h, 72h post infection respectively.2 Western blot assay to detect the activity of apoptosis signalling related proteins PARP, Caspase-3, Caspase-8, Caspase-9, Caspase-12 and MAPK signaling proteins p-JNK, p-P38, p-Erk and p-AKT.ResultsOur data show that NDV strains of different viulence have disparities in oncolytic efficacy, virus replication and apoptotic mechanisms. A549 cells were infected with 0.1moi,1moi,10moi NDV virus respectively, MTT assay cell viability at 24h, 48h, 72h postinfection(p.i.). Results indicate oncolytic efficacy of NDV-FMW>NDV-BC>NDV-LaSota, cell toxicity of velogenic and mesogenic NDV increased while extending incubating time, but no evident trend was observed in lentogenic group; oncolytic efficacy of each strain used here is proportion to virus dose.Result of virus replication assay shows all strains but lentogenic strains replicate in A549, which is corresponding to the above MTT results.Research on apoptotic signaling indicates all strains activate caspase-8, caspase9, caspase-3 and cleave PARP at the same time, demonstrating both intrinsic and extrinsic apoptotic pathways were exploited simultaneously; as a primary signal of endocytoplasmic reticulum stress to initiate apoptosis, no evident activation of caspase-12 was detected comparing to control. we also detect elevation of main MAPK proteins activation, of which JNK activation reach peak at 12h p.i., p38 show a significant activation up regulation compare to control, all strains but velogenic group didn't show detectable change of Erk1/2 phosphorylation, while initiation of caspase and p38 activation in velogenic and mesogenic groups were earlier than those in lentogenic group. Decreased Serine phosphorylation of AKT was observed in A549cells treated with the three strains individually.Our research systematically investigate the oncolytic efficacy and apoptotic mechanisms induced by different virulent NDV strains, providing a basis for further invivo study and clinical trails.