Live-attenuated Mumps Virus Causes Oncolytic Effect Against Nephroblastoma

Part Ⅰ Oncolytic effect against nephroblastoma of Live-attenuated Mumps virus in vitroObjectiveTo investigate the oncolytic effect of MuVS79 against nephroblastoma cells in vitro and provide new perspectives for therapeutic strategies against nephroblastoma.Methods1.Virus amplification was on Vero cells.Virus titers were determined by a plaque forming unit(PFU)assay.2.Following infection with MuVS79 at a MOI of 0.5 for 4 h,the virus was removed.MuVS79 N protein was determined by indirect immunofluorescence(IFA)at 72 h.3.To assess the oncolytic effect of MuVS79,5×103 G401,SK-NEP-1 and 293 cells were plated into 96-well plates,then treated with MuVS79 at a series of MOIs(0.1,0.2,0.5,1,and 2).Cell viability was monitored with the CCK8 assay.4.Quantitative assessment of apoptotic G401 and SK-NEP-1 cells was performed by flow cytometry via annexin V/propidium iodide(PI)staining.5.The whole genome DNA was extracted after infection with MuVS79 for 72h.DNA Ladder was detected by agarose gel electrophoresis.6.The whole protein was extracted after infection with MuVS79 or without MuVS79,Western blot was performed to measure the expression of apoptosis and autophagy-related proteins(P62、MAPILCB3、Caspase-3、Bcl-2).7.Autophagy-related gene(ATG7)was knocked-down by lentivirus expressing SiRNA-ATG7 in G401 and SK-NEP-1.Cell viability was detected in autophagy-impaired and autophagy-normal groups after MuVS79 infection by CCK8 assay.8.Cell viability was detected after MuVS79 infection with rapamycin(autophagy inducer)treated or without rapamycin treated groups by CCK8 assay.Results1.We successfully harvested numerous of MuVS79.The titer of MuVS79 was measured by PFU.2.MuVS79 was able to effectively infect nephroblastoma cell lines.MuVS79 infection induced the formation of typical syncytia,resulting from the fusion of infected cells with neighboring cells3.MuVS79 infection effectively induced cell death and reduced cell viability in G401 and SK-NEP-1 cells.Cell viability was 37%,32%respectively in MOI=2 at 72 h.4.The expression of cleaved Caspase-3 and Bcl-2 did not increase or decrease.We did not find multiples of 200-bp DNA fragments.The apoptotic rates of G401 and SK-NEP-1 cells were 9.29%and 4.47%,respectively.The oncolytic effects of MuVS79 are not due to increased apoptosis.5.Infection with MuVS79 induced the expression of MAPILC3B-II in nephroblastoma cells lysates compared with uninfected cells.The expression of MAPILC3B-II was further increased in the presence of CQ compared with the MuVS79-alone infected group.MuVS79 infection reduced the levels of P62 at 48 h and 72 h.These results demonstrate that MuVS79 stimulates the complete autophagic process through strengthening autophagic flux in G401 and SK-NEP-1 cells.6.A significant correlation between MuVS79-induced cell death and the level of an autophagy protein marker(P62)in G401 and SK-NEP-1 cells was found.7.When blocked ATG7,an essential protein for autophagy,cell viability was increased significantly in autophagy-impaired groups after MuVS79 infection.Cell viability was decreased after MuVS79 infection with rapamycin(autophagy inducer)treated compared to untreated group.These results show that autophagy contributes to MuVS79-induced oncolytic effects in nephroblastoma cell lines.Part Ⅱ Oncolytic effect against nephroblastoma of Live-attenuated Mumps virus in vivoObjectiveTo study the antitumor activity of MuVS79 in vivo,we implanted human nephroblastoma into BALB/C nude mice and explore new therapeutic strategies for nephroblastoma.MethodsG401 cells(106)in 100 μL PBS were implanted subcutaneously into the right flanks of nude mice.When tumor volumes reached 30-50 mm3,the mice were divided randomly into four groups,five per group,and injected intratumorally with a 0.1 mL total volume of 1×106,1×105,or 1×104 PFU/mL of MuVS79 or PBS,every other day,for a total of seven times.1.Tumor volumes were measured twice weekly and calculated as follows:tumor volume(mm3)＝length × width2 × 1/2.Tumor mass were measured when mice were sacrificed.2.To investigate the stimulation of autophagic tumor cell death in vivo,we detected the expression of MAP1L3B,P62,and Becinl by immunohistochemistry3.The tumor whole protein was extracted.Western blot was performed to measure the expression of autophagy-related proteins(P62、MAPILCB3、Bcl-2).4.The tumor whole protein was extracted.RT-PCR was performed to measure the expression of autophagy-related proteins(P62、MAPILCB3、Bcl-2).Results1.MuVS79-treated nude mice showed slower tumor development and lighter tumor mass compared with the control group.2.We found that levels of MAP1L3B and Beclinl were higher in the MuVS79-treated group than in the PBS-treated group.Degradation of P62 was found in MuVS79-treated nude mice.3.MuVS79 treatment enhanced tumor suppression and autophagy in a G401 xenograft tumor modelConclusion1.MuVS79 was able to effectively infect nephroblastoma cell lines and induce cytopathic effects(CPEs)and cell death2.MuVS79-induced cell death in G401 and SK-NEP-1 cells is not via apoptosis3.MuVS79 induced autophagy and preserved autophagic flux.Autophagy is related to the MuVS79-induced oncolytic effects in nephroblastoma cell lines4.MuVS79 treatment resulted in an inhibition of tumor growth.The secretion of TNF and activation of autophagy in tumor tissues promotes and enhances the antitumor effect.