The Study Of Novel Oncolytic Adenoviruses On Leukemic Cells And Their Mechanisms

Leukemia is a series of clonal hematopoietic malignancies caused by mutations of stem cells or early progenitor cells(myeloid or lymphoid).Malignant proliferation of leukemic cells makes themselves into the circulation,infiltrating organs and tissues. Varying degrees of anemia,bleeding,fever can be observed as well as hepatomegaly, splenomegaly,bone pain and so on.So far,leukemia has become one of the most common malignancies in China with the incidence accounting sixth place,and leukemia has become the most common malignant disease among young people. Despite the integrated adoption of traditional combined chemotherapy,retinoic acid and arsenic trioxide-induced therapy,hematopoietic stem cell transplantation and supporting therapy has resulted in a complete remission rate of 70-90%and a 3-year disease-free survival rate of 40-50%,there are still chemotherapy-related toxicity, non-selectivity as well as drug resistance of relapsed or refractory leukemia.These questions prompt researchers to study for a variety of novel target therapies,while the gene-viral therapy is a important one among them.Oncolytic adenoviruses replicate in tumor cells and cause cell lysis,and then they release themselves to further infect surrounding tumor cells,until the total eradication of the tumor.In this study,we constructed novel oncolytic adenoviruses of ElB-55kDa deletion with chimeric Ad5/35 fibers or chimeric Ad5/ll fibers.These adenoviruses.can also carry TRAIL gene or BECN gene,so as to achieve the combination of gene therapy and viral therapy.In addition,we further designed chemo-gene-viral therapy in order to provide novel strategy for leukemia clinical practice.This study is divided into three parts:Partâ… :in vitro and in vivo study of oncolytic adenovirus SG235-TRAIL on leukemic cells;Partâ…¡:study of combination of SG235-TRAIL and homoharringtonine on leukemic cells;Partâ…¢:in vitro and in vivo study of oncolytic adenovirus SG511-BECN on leukemic cells.Partâ… :in vitro and in vivo study of oncolytic adenovirus SG235-TRAIL on leukemic cellsObjective:We constructed novel oncolytic adenovirus SG235 of E1B-55kDa deletion with a chimeric Ad5/35 fibers,and the adenovirus can also carry TRAIL cassette.We further confirm SG235 and SG235-TRAIL anti-leukemic cell effect by in vitro and in vivo experiments,and explore the mechanism.By this study,we try to provide target therapy for leukemia with the strategy of combined gene-viral therapy.Methods:We first constructed the adenovirus SG235,SG235-EGFP and SG235-TRAIL,and study the infection and replication ability of SG235 on leukemia cell lines,lymphoma cell lines,MM cell lines,primary leukemia cells as well as CFU-L via inverted fluorescence microscope,flow cytometry,viral titer testing and western blot for E1A;confirm the ElB-55kDa deletion of SG235 via western blot; confirm cytotoxicity of different adenoviruses on leukemia cell lines via MTT;prove SG235 and SG235-TRAIL inducing apoptosis on leukemia cell lines and primary leukemia cells via TUNEL and PS translocation;detect the mitochondrial membrane potential change of leukemia cell lines after SG235-TRAIL infection via Rh123 staining;use western blot to detect Caspase family proteins,Bcl-2 family proteins after various types of adenoviruses infection in leukemia cell lines and primary leukemia cells;add Caspase-3 inhibitor(z-DEVD-FMK) and Caspase-8 inhibitor (Z-IETD-FMK) to observe the apoptosis levels induced by SG235-TRAIL;observe SG235 and SG235-TRAIL anti-tumor effect on Hela cell line via crystal violet staining and western blot for Caspase family;collect bone marrow cells from clinically diagnosed leukemia patients to detect the expression of CD46;to determine the selective cytotoxicity of adenoviruses on leukemia cells via CFU-L and GM-CFU forming test;detect the supernatant TRAIL levels after adenoviruses infection via ELISA;observe the various types of adenoviruses on tumor growth of Kasumi-1 xenograft-bearing SCID mice and observe the in vivo anti-leukemic effect of adenoviruses by TUNEL staining.Results:(1) 122 cases of leukemia cells is collected,the overall CD46-positive rate is 78.7%,indicating that SG235 can effectively infect leukemia cells;(2) SG235 can effectively infect leukemia,lymphoma and MM cell lines in vitro with time-dependent and dose-dependent manners;(3) SG235 can induce apoptosis in leukemia cell lines and activate Caspase-9,Caspase-3 as well as PARP with a dose-dependent manner;(4) both SG235-TRAIL and SG235 have obvious cytotoxicity on leukemia cell lines with a dose-dependent manner,while SG235-TRAIL is superior to SG235;(5) SG235-TRAIL can induce apoptosis in leukemia cell lines and is superior to SG235;(6) the leukemia cell line infected by SG235-TRAIL has Caspase-8 and its substrate BID activated,BAX expression increased,mitochondrial membrane potential lost as well as Caspase-9 and Caspase-3 activated;(7) SG235-TRAIL-induced apoptosis in leukemia cell lines can be inhibited by Caspase-3 and Caspase-8 inhibitors,while Caspase-3 inhibitor is superior to Caspase-8 inhibitor;(8) Bcl-2 and Mcl-1 is down regulated while BIM,BAK and Bcl-XL has no obvious change in Kasumi-1 cell lines after SG235 or SG235-TRAIL infection;(9) SG235 and SG235-TRAIL retain their cytotoxic effect on tumor cell lines;(10) SG235 and SG235-TRAIL can effectively infect primary leukemia cells, induce apoptosis,activate Caspase-8,Caspase-3 and PARP as well as kill primary leukemia cells,while SG235-TRAIL is superior to SG235;(11) SG235 and SG235-TRAIL can efficiently infect CFU-L,and selectively inhibit the formation of CFU-L,while SG235-TRAIL almost completely inhibits the formation of CFU-L,in addition,SG235 and SG235-TRAIL can not or slightly inhibit normal formation of GM-CFU;(12) the level of TRAIL expression is significantly higher after SG235-TRAIL infection than Ad5/35-TRAIL,while SG235 do not express TRAIL obviously;(13) SG235 can significantly inhibit the tumor growth in Kasumi-1 xenograft-bearing SCID mice while ZD55 can't;(14) SG235-TRAIL is superior to SG235 in inhibiting tumor growth in Kasumi-1 xenograft-bearing SCID mice and inducing apoptosis in vivo,while Ad5/35-TRAIL shows no anti-tumor effect.Partâ…¡:study of combination of SG235-TRAIL and homoharringtonine on leukemic cellsObjective:According to the thinking of chemo-gene-viral therapy,we combine SG235-TRAIL and HHT on leukemia cells,and confirm the cytotoxicity in vitro as well as conduct synergy analysis,furthermore,we explore the mechanism,with a view to provide new methods and strategies for clinical practice.Methods:We confirm the cytotoxic effect of combination of SG235-TRAIL and HHT via MTT and LDH test,then analyze the combination effect via Chou-Talalay method;SG235-TRAIL and HHT-induced apoptosis in leukemia cell lines is confirmed by PS translocation;E1A,TRAIL,Caspase family proteins and Bcl-2 family proteins is examined via western blot;SG235-TRAIL and HHT combination on normal bone marrow cells is tested via MTT;MSCs is cultured and identified,then HHT and SG235-TRAIL combination on MSCs is tested via crystal violet staining.Results:(1) SG235-TRAIL and HHT show anti-leukemia cell lines effect in vitro with different levels of synergy;(2) SG235-TRAIL and HHT synergistically induce apoptosis in leukemia cell lines;(3) the infection and and replication ability of SG235-TRAIL are not affected by HHT;(4) TRAIL expression level of SG235-TRAIL and HHT combination is more signigicant than SG235-TRAIL alone; (5) SG235-TRAIL mainly activates Caspase-8 and HHT mainly activates Caspase-9, Caspase-8 and its substrate BID,Caspase-9,Caspase-3 and PARP are significantly activated by SG235-TRAIL and HHT combination in leukemia cell lines;(6) Bcl-2 and Mcl-1 are down regulated by SG235-TRAIL and HHT combination in leukemia cell lines;(7) SG235-TRAIL and HHT combination has little effect on normal bone marrow cells.Partâ…¢:in vitro and in vivo study of oncolytic adenovirus SG511-BECN on Ieukemic cellsObjective:We constructed novel oncolytic adenovirus SG511 of ElB-55kDa deletion with chimeric Ad5/11 fibers,and the adenovirus can also carry BECN cassette.We further confirm SG511-BECN anti-leukemic cell effect by inducing autophagic cell death via in vitro and in vivo experiments,and explore the mechanism. By this study,we try to provide target therapy for leukemia with the strategy of combined gene-viral therapy.Methods:We first constructed the adenovirus SG511,SG511-EGFP and SG511-BECN,and study the infection and replication ability of SG511 on leukemia cell lines,lymphoma cell lines,MM cell lines,primary leukemia cells as well as CFU-L via inverted fluorescence microscope,flow cytometry and western blot for beclin-1;observe SG511 -BECN anti-tumor effect on tumor cell lines via crystal violet staining,and SG511-BECN anti-tumor effect on hematological malignant cell lines via MTT;detect autophagy induced by SG511-BECN via acridine orange staining, transmission electron microscopy and LC3-GFP-K562 cell fluorescence change; further observe the expression of LC3 and p62 via western blot in SG511-BECN-infected K562 cell line;in vivo study of SG511-BECN on the tumor growth of K562 xenograft-bearing SCID mice.Results:(1) SG511 can effectively infect leukemia,lymphoma and MM cell lines in vitro with a dose-dependent manner;(2) SG511 can efficiently infect CFU-L; (3) SG511-BECN can effectively infect K562 cell line,and express beclin-1;(4) SG511-BECN is superior to SG511 in killing tumor cell lines and malignant hematological cell lines;(5) SG511-BECN can efficiently induce autophagy in leukemia cell lines with LC3-â…¡expression and p62 degradation;(6) SG511-BECN is superior to SG511 in inhibiting tumor growth in K562 xenograft-bearing SCID mice.Conclusion:SG235 and SG511,as two viruses with novel chimeric fiber,can efficiently infect leukemia cell lines and primary leukemia cells.With TRAIL cassette, SG235-TRAIL can cause tumor cell lysis as well as induce apoptosis;with beclin-1 cassette,SG511-BECN can cause tumor cell lysis as well as induce autophagic cell death.In addition,SG235-TRAIL and HHT can exert their anti-leukemia effect synergistically.