Anti-tumor Effects Of Oncolytic Vaccinia Virus Harboring Therapeutic Genes For Hematological Malignancies

Despite significant progresses made in the treatment of hematopoietic malignancy,it is still incurable,especially for leukemia and multiple myeloma(MM).Oncolytic vaccinia virus(OVV)therapy has represented a potential antitumoral therapeutics for their promising phase ? clinical results.One reason for this has focused on their critical therapeutic importance of the immune response raised by these viruses.The other is.for their potent advantages as exogenous expressing vectors for therapeutic genes.This study is composed of two parts:Part ?:Combined expression of miR-34a and Smac mediated by oncolytic vaccinia virus synergistically promote anti-tumor effects in Multiple Myeloma;Part ?:Autophagic cell death induced by enforced expression of Beclinl via oncolytic vaccinia virus exerts potent antitumor activity in leukemia associated with SIRT1-LC3 pathway.Part ?:Combined expression of miR-34a and Smac mediated by oncolytic vaccinia virus synergistically promote anti-tumor effects in Multiple MyelomaAims:To construct the novel oncolytic vaccinia viruses OVV-miR-34a and explore its synergistic antitumor effects with OVV-Smac in MM and the underling mechanisms.Methods:OVV-miR-34a was constructed by genetic engineering technology.OVV-GFP was used to detect the infectious efficiency of OVV in MM cell lines with dose and time manner.MTT assay was applied to detect the cell proliferation of MM cell lines after being infected by OVV,OVV-miR-34a and OVV-Smac alone or combination and the normal human liver cells QSG-7701 and PBMCs were used as healthy cell controls to verify the safety of OVVs.Real-time PCR and Western blot were carried out to analysis the enforced expression of miR-34a and Smac as well as their downstream regulated genes.Activation of caspase pathway and induction of apoptosis were evidenced using immunoblot analysis and Annexin V/PI double staining.Confocal microscopy was administrated to monitor the release of Cytochrome c from the mitochondrial endomembrane to cytosol.To examine the therapeutic effectsof OVV-miR-34a combined with OVV-Smac in vivo,RPMI-8226 xenograft model was established and intratumorally injected with indicated OVVs.Tumor volumes were monitored and the survival rates were recorded regularly as well as histopathology changes in tumor tissues were evaluated by H&E,IHC,TUNEL and TEM assays.Results:(1)Results from sequencing and restriction enzyme digestion confirmed that we have constructed the OVV-miR-34a successfully.(2)Infectious efficiency of OVV-GFP in RPMI-8226 and U266 were showed at 49.28%and 59.1%respectively after 48h at MOI of 8.(3)MM cell viability and proliferation after infection of OVV,OVV-miR-34a and OVV-Smac alone or combination detected by MTT assay showed that co-infection caused significant decrease compared the individual treatment.(4)Real-time PCR and Western blot results showed that infection of OVV-miR-34a and OVV,Smac enforced the expression of miR-34a and Smac remarkably in MM cells.Moreover the target genes of miR-34a,such as Bcl-2 and SIRT1 were correspondently down regulated by OVV-miR-34a alone or combination in dose-dependent profile.The pro-survival genes regulated by Smac,such as XIAP,c-IAP1/2 were also changed after infection of OVV-Smac alone or combination.(5)Immunoblot and Annexin V/PI double staining results verified OVV-miR-34a combined with OVV-Smac caused intensive apoptosis induction.The apoptosis rate in the combined treatment was up to 58.1%,nearly two times more than that of OVV-Samc(32.4%)and OVV-miR-34a(25.9%)treatment alone,which were accompanied with the cleaved caspase-3,caspase-9 and PARP-1.(6)Animal experiments demonstrated that OVV-miR-34a in combination with OVV-Smac have severe antitumoral effects on tumor growth of MM xenograft and prolonged the survival rates of mice.Furthermore,the results from histopathology analysis showed the significant antiangiogensis,apoptosis induction and irreversible cell death appeared in the treatment of OVV-miR-34a in combination of OVV-Smac.Conclusion:In this study,we constructed OVV-miR-34a and OVV-Smac successfully and evaluated their anti-myeloma potential as monotherapies and in combination.We found that these OVVs were able to replicate in and kill human MM cells in vitro and in vivo.Furthermore,combination therapy with OVV-miR-34a and OVV-Smac synergistically improved anti-tumoral efficacy.Part ?:Autophagic cell death induced by enforced expression of Beclinl via oncolytic vaccinia virus exerts potent antitumor activity in leukemia associated with SIRT1-LC3 pathwayAims:To construct the novel oncolytic vaccinia virus OVV-Beclinl and explore its function in the aspect of autophagic cell death induction and the underling mechanism on the growth inhibition of hematologic malignancies.Methods:OVV-Beclinl was constructed by genetic engineering technology.OVV-GFP infecting the leukemia cell lines K562 and HL-60 was programmed to detect the infectious efficiency of OVV.IF assay was carried out and confocal microscope was used to observe the subcellular localization of the enforced Beclinl with the Calreticulin as reference substance.Real-time PCR and Western blot assays were used to detect the over expression of Beclinl by OVV-Belcinl on mRNA and protein levels.MTT assay was applied to examine the growth inhibition effect on leukemia or MM cell lines,at the same time PBMCs from healthy donors were applied to verify the safety of OVVs.Following experiments were carried out to test whether apoptosis can be induced by infection of OVV-Beclinl:(1)Western blots were used to test caspase-related proteins such as caspase-9,caspase-3,PARP-1 activation;(2)Annexin V/PI double staining was programmed to analysis the apoptotic rates induced by the indicated OVVs;(3)Combined with pan-caspase inhibitor Z-LEHD-FMK or constructed the caspase-3 shRNA K562 cell line to further examine whether OVV-Beclinl can activate the apoptosis cell death pathway.The manners of autophagy induced by OVV-Belcinl were detected as following experiments:(1)autophagy inhibitor(3-MA,BafAl)and autophagy inducer(Rapa)were used as co-effectors to determine the effect of OVV-Belcinl on leukemia cell lines;(2)Western blots were programmed to examine the regulation of p62 and conversion of LC3 ?/?;(3)AO and MDC staining assays were employed to test the appearance of the acidic vesicular organelles;(4)Endogenous LC3 detection with IF or exogenous Ad-mRFP-GFP-LC3 transduction was arranged to detect the aggregation of LC3 and the autophagosomes formation in cytosol;(5)TEM was further used to test the autophagic vacuole and autolysosome formation.Further Real-time PCR,Western blot and Co-IP were programmed to explored the relationship between autophagic cell death induced by OVV-Belcinl and SIRT1-LC3 pathway.A K562-luciferase mouse model was constructed to verify the systematic antitumoral effect of OVV-Beclinl in vivo.Histopathology changes in tumor tissues were evaluated by H&E,IHC and TEM assaysResults:(1)We constructed the OVV-Beclinl successfully.(2)Infectious efficiency of OVV-GFP in leukemia cell lines were up to 51.85%and 44.92%after 48 h at MOI of 20,respectively.(3)Enforced expression of Beclinl was subcellular localized in ER.(4)OVV-Beclinl exerted severe cytotoxicity to leukemia or MM cell lines,while spared the normal cells.(5)Real-time PCR and Western blot showed OVV-Belcinl enforced expression of Belcinl at mRNA and protein levels in time and dose-dependent profile.(6)Results from apoptosis determination experiments confirmed that OVV-Beclinl can not cause the apoptosis induction.(7)Autophagy determination experiments demonstrated that OVV-Beclinl can cause autophagic cell death,regulate the autophagic proteins p62 and LC3 ?/? changes,induce the formation of autophagosomes and autolysosome.(8)Further results demonstrated the relationship between OVV-Beclinl induced-autophagic cell death and the SIRT1-LC3 signal pathway.(9)Animal experiments demonstrated that OVV-Beclinl was superior to OVV in inhibiting the tumor growth of leukemia xenograft and prolonging the survival rates of mice.Histopathology analysis showed the apparent autophagosomes existing in the tumor lesions injected with OVV-Beclinl.Conclusion:We constructed the novel OVV-Beclinl successfully in this study.Large amount of results evidenced OVV-Beclinl has the ability to induce the autophagic cell death and the underlying mechanism is associated with SIRT1-LC3 signal pathway.Our study is the first to combine the OVV therapeutics and autophagic cell death,which provides a promising groundwork for future clinical therapy of hematologic malignancy.