Research On Effect And Signal Transduction Pathway Of Newcastle Disease Virus-Induced Apoptosis Of Human Esophageal Cancer Cells ECA109

Newcastle disease virus, which is also called Asian fowl plague virus, pseudo- fowl plague virus or Avian pneumo-encephalitis virus, can cause avian Newcastle disease. In the 1950s, people accidentally discovered that NDV had anti-tumor activity. In the past several decades, people had confirmed that NDV could selectively kill many human tumor cells such as KB825211, IMR32, G104, HT1080 and HCV29. Whereas NDV has no adverse effect to normal human fibroblasts. People have started to understand its anti-tumor mechanisms. The main mechanisms of NDV anti-tumor effects can be summarized as follows: Induce the production of cytokines, regulate the immune cells, selectively kill tumor cells, inhibit the growth of tumor cells by its HN protein. But its exact anti-tumor mechanisms are still not fully unknown. To make a further study of NDV anti-tumor mechanism and elucidate its signal transduction pathway, have been problems that urgently to be solved.Preparation and purification of NDV, subcultivation of ECA109 cells with RPMI1640 medium. ECA109 cells were treated with various concentrations of NDV for various times. Anti-proliferation activities were measured by CCK-8 assay. Inhibition rate and proliferation rate of ECA109 cells to NDV was calculated. Detection of NDV-induced apoptosis was performed cytofluorimetric analysis by FITC-PI double staining. The total cellular DNA was extracted from the tumor cells. DNA fragment was determined with agarose gel electrophoresis.Transmission nelectron microscopy was used to study the changes of the ultrastructure in ECA109 cells which were infected by NDV. Western blot, immunocytochemistry staining were used to detect the expressions and activations of caspase 8, caspase 9, Cytochrome C, Bcl-2, and Bax in ECA109 cells.CCK-8 assay showed that NDV could suppress cell growth and proliferation of ECA109 cells. NDV inhibited the viability of ECA109 cells in a dose- and time-dependent manner (p<0.05). Cytofluorimetric analysis by FITC-PI double staining showed the percentage of early apoptotic ECA109 cells treated with different concentrations of NDV. They were 8.96±0.31%,10.93±1.11%,18.30±1.21% and 22.13±1.53% respectively. Compared to 2.4±0.36% in the negative control, the percentages were significantly higher in the cells treated with NDV (p<0.05). These results showed NDV-induced apoptosis in ECA109 cells. The clear DNA ladders were observed in the DNA gel electrophoresis. The occurrence of DNA fragmentation provided an evidence for NDV efficiently stimulate apoptosis in ECA109 cells.Electron microscope was used to observe mitochondrion in ECA109 cells. We could see the mitochondrion in ECA109 cells swelling, the ridges were fragmented and decreased after the treatment of NDV. Results of western blot and immunocytochemistry staining showed that the pro-apoptotic factors caspase 9, caspase 8, Bax, and cytochrome C levels were remarkably higher, whereas the anti-apoptotic factor, the Bcl-2, was decreased than that of in the negative control.In this study, the growth inhibited of esophageal carcinoma cell line (ECA109) induced by NDV was assessed by using the CCK-8 assay, flow cytometry, immunocytochemistry, DNA fragment assay, western blot analysis, electron microscope technique, in order to investigate the apoptotic mechanisms and its signal transduction pathway. It will become an important theoretical foundation for the biotherapy of esophageal cancer.