In Vitro Anti-Tumor Activity By Newcastle Disease Virus FMW Strain

In Recent years, oncolytic virus becomes to hot-point of tumor biotherapy, as so far more than ten kinds of oncolytic viruses was utilized in clinical trial, including Adenovirus(ONYX-015), Herpes simplex virus(G207), Reovirus(Dearins), Newcastle disease virus(PV701) etc. NDV as an oncolytic virus can't infect normal cells, only proliferate in tumor cells. Study on NDV suggests that NDV has highly oncolytic effect on many kinds of tumor cells. And now several strains of NDV (NDV-HUJ, 73T, MTH68, PV701, etc) have come into phaseâ…¡,â…¢clinic trial. The results show that they are promising in future use. A NDV strain from nearly 50 strains tested in this study showed obviously oncolytic effect on A549 and SMMC7721 cells and other human tumor cells. The NDV strain can activate apoptosis signal pathways which lead to oncolysis.Methods1 To isolate Newcastle disease virus strains with highly oncolytic effect on A549 and SMMC7721 cells by MTT assay.2 To determine Newcastle disease virus titer and multiplicity of infection (MOI) by plaque formation test.3 To observe morphological changes of tumor cells after infected with Newcastle disease virus by light microscopy.4 To detect impact of Newcastle disease virus towards proliferation of tumor cells by MTT and FACS.5 To distinguish apoptotic cells and detect genome change by Hochest33258 fluorescence staining.6 To measure cell apoptosis by propidium iodide(PI) staining and Annexinâ…¤/PI staining, to analyze cell cycle distribution by flow cytometry.7 To detect expression of apoptosis-related protein: PARP, Caspase-3, p-JNK, p-AKT, p-P38, p-ERK by Western Blot. ResultsWe obtained a NDV strain with highly oncolytic effect by MTT assay. The NDV strain was named NDV-FMW, which showed obvious growth inhibitory effect on A549 and SMMC7721 cells. After cells infected with NDV-FMW strain at 24 hours, cells turned round, at 48 hours infected cells blebed and condensed, detached from the bottom, at 72 hours cells died and produced fragments. Results of MTT assay showed that proliferation of SMMC7721 and A549 cells were significantly decreased after treated with NDV-FMW strain, when cells were treated with 20 MOI for 48 hours, growth inhibitory ratio was up to 60%. More than it we detected this impact with FACS, Trials demonstrated NDV-FMW strain can inhibit cell growth in a dose- and time- dependent manner. Hochest33258 fluorescence staining assay showed that NDV-FMW strain can induce apoptosis in SMMC7721 and A549 cells. Infected cells shrinked and chromatin condensed at 48 hours. PI staining showed apoptosis peak, cell cycle analysis suggested cell cycle was arrested in G0/G1 phase. Annexin-â…¤/PI staining detection showed that apoptotic rates of SMMC7721 cells were 2.1%, 18.5%, 23.8%, 30.4% after treated with 0MOI, 0.2MOI, 2MOI, 20MOI NDV-FMW strain for 48 hours.To further elucidate the apoptosis pathways induced by NDV-FMW strain, expression of Caspase-3, p-JNK, p-AKT, p-P38, p-ERK was detected by Western blot, results indicated that NDV-FMW strain could activate Caspase-3, JNK, decrease p-AKT expression, to further detect PARP, the substrate of Caspase-3, we observed 85KD fragment. All the results mentioned above showed that Caspase pathway is important mechanism in NDV-FMW strain inducing tumor cell apoptosis.Conclusions1 A Newcastle disease virus strain with effective oncolysis was named NDV-FMW strain which can infect many tumor cell lines.2 NDV-FMW strain significantly inhibited SMMC7721 and A549 cells growth in a dose-time-dependent manner.3 NDV-FMW strain blocked cell cycle which was arrested in G0/G1 phase.4 NDV-FMW strain could activate Caspase-3, JNK, PARP, and decrease p-AKT expression, inhibit the growth of tumor cells by apoptosis pathway.5 NDV-FMW strain can be used to further study, including clinical trial, also for the use of constructing recombinant Newcastle disease virus to incresae oncolytic capability.