Experimental Study On Inhibitory Effects Of The Defective Vesicular Stomatitis Virus On Malignant Melanoma

PurposeChoroidal melanoma is the most common primary intraocular malignant tumor in adults,it may distant spread by intravascular invasion in early stage.The mortality rate is very high after choroidal melanoma transfer.Traditionally,enucleation of an eye with a choroidal melanoma was the mainstay treatment.Recently,some new forms of theraphy have been used in CM,such as particularly local resection,radiotherapy with proton or radioactive plaque therapy,transpupillary thermotherapy and photodynamic therapy.All these new therapy have preserve some eyes and partial visual functions,however,the metastasis rate and mortality rate of CM were not changed.In recent years,tumor biotherapy has become the new model of tumor therapy with curative effect.As one of the biotherapy,oncolytic virus are perfectly capable of killing tumor cells in some researches.Vesicular Stomatitis Virus(VSV)was an excellent vector for tumor therapy,which has mature and efficient virus rescue system and rapid virus replication.For the characteristics of the eye anatomy,the VSV should be avirulent or more hypovirulent to the eye healthy tissue as an oncolytic virus used in CM.This study intends to rescue defective VSV over WT-VSV and to investigate the specific killing effect of the defective VSV on malignant melanoma cells,and to investigate the inhibitory effect and inhibition mode of the defective VSV on malignant melanoma cells and its mechanism.Methods1.Virus Rescue:By site directed mutagenesis PCR and reverse genetics system,recombinant Vesicular Stomatitis Virus VSV-Y1650A and VSV-F1691A with individual alanine substitution in conserved aromatic residues(Y1650 and F1691)of L protein,were successfully recovered entirely from an infectious cDNA clone.2.Comparative study on Neuronophagia:To compare the neuronophagia in WT-VSV and recombinant VSV(Y1650A and F1691 A)by observing the pathological changes in brain tissues inoculation groups.3.In vitro experiment:Three different VSV was proliferated in BHK-21 cells,and viral titers were determined by TCID50 analysis.The WT-VSV and recombinant Y1650A-VSV and VSV-F1691A were infected with murine melanoma B16 cells.To investigate the targeted effect of three different VSV on B16 cells by CCK-8 which observed the effect of the three VSV on proliferation of B16 cellls and flow cytometry which assayed the cell cycle and apoptosis of the B16 cells.4.In vivo experiment:BALB/c(nu/nu)mice bearing tumor model was constructed by subcutaneous injection of B16 cells.The WT-VSV and recombinant Y1650-VSV was injected into tumor respectively.To investigate the effect of WT-VSV and Y1650-VSV on tumor inhibition in vivo by observing the growth trend of tumor and the expression of apoptosis related proteins in tumor tissue by Western blot.Results1.The recombinant VSV-Y1650A and VSV-F1691A with individual alanine substitution in conserved aromatic residues(Y1650 and F1691)of L protein were successfully re covered entirely from an infectious cDNA clone by site directed mutagenesis PCR and reverse genetics system,2.By day 7 postinfection,brain sections from the WT-VSV group showed moderate histological changes characterized by multifocal meningeal mononuclear cell infiltrates with formation of perivascular cuffs.Brain tissues from the F1691A-VSVinfected mice had mild inflammatory changes,whereas those from Y1650A-VSV infected mice had no apparent histological change.3.In vitro experiment,the three VSV had obvious inhibition effect on B16 cells by CCK-8 anf the inhibition effect on B16 cells in vitro was dose-dependent.The FCM analysis showed that the apoptosis rate increased significantly after B16 cells were infected by three different VSV compared with control group.(44.44%/WT,28.36%/Y1650A,24.19%/F1691A vs 2.14%,P<0.01)。The result from the effect of three different VSV on B16 cells’ cycles showed that cells of G1 phase decreased significantly compared with control group.WT-VSV(67.96%vs 86.42%,P<0.01);Y1650A-VSV(76.42%vs 86.42%,P<0.05);F1691A-VSV(81.70%vs 86.42%,P>0.05);S phase from three VSV groups had significantly increased,Cell cycle on VSV-infected B16 cells were arrestted at S phase.4.In vivo experiment showed that VSV-WT and VSV-Y1650A by intra-tumor injection could significantly inhibited the proliferation of tumor in BALB/c(nu/nu)mice bearing tumor model,and no obvious toxic and side effects.The expression levels of caspase-3,Bax and CDK-2 were detected by Western blot assay.The results showed that WT-VSV or Y1650A-VSV intra-tumor injection induced up-regulation of caspase-3 and Bax expression and down-regulation of CDK-2 of the tumor tissue in vivo.Conclusions1.The infection ability to nervous tissue of recombinant VSV(Y1650A and F1691 A)were significantly weakened compared with the infection ability of WT-VSV.2.The three different VSV could kill B16 cells specifically and induce apoptosis and cell cycle arrest in S phase in vitro experiment.VSV-WT and VSV-Y1650A by intra-tumor injection could significantly inhibited the proliferation of tumor in vivo experiment and no obvious toxic and side effects.3.The result of apoptosis by FCM analysis in vitro and the expression levels of caspase-3,Bax and CDK-2 in vivo experiment suggested that the killing effect of VSV on B16 cells may be related to pathway of apoptosisIn summary,Y1650A-VSV shows potential for using in the treatment of malignant melanoma.This study provided the reference for the further research on oncolytic virus therapy of choroidal melanoma.