The Construction Of An Armed Oncolytic Adenovirus Vector Carrying Apoptin And IL-24Gene And Its Anti-tumor Effect

Cancer is a kind of disease that seriously depresses our life quality. The statistic resultsshow that, for lung cancer, mortality has increased rapidly since1970s. Non-small-cell lungcancer, including squamous carcinoma, adenocarcinoma, and large cell carcinoma, merisisslowly, metastasis later, accounts for about80%-85%of lung cancer. As a kind of popularvector in Cancer Target Gene-Viro Therapy, oncolytic adenovirus has been used in clinicaltrial. And the modification of it has always been concentrated on its effect and safety. Apoptinand IL-24both can specifically induce the tumor cells apoptosis in a p53independentpathway. Based on the high activity of hTERT promoter in human tumor cell lines, we haveutilized it to promote the expression of virus E1A. We have also deleted the24bp in E1A thatassociated with the interaction with Rb and inserted the Apoptin and IL-24expression cassetteinto this E1B55K deleted adenovirus vector.As is shown in our experimental results, recombinant adenoviruses have killing effect tovarious tumor cells, and they can specifically target lung cancer cell with low influence onnormal cells. Cytotoxicity of our viruses on tumor cells is time and dose dependent. Viruseswith Apoptin and IL-24heterogenous expression show no significant cytotoxicity than mockvirus when low MOIs virus affect tumor cells, however the effect is significant different whenhigh MOIs virus affected (p<0.001). Recombinant adenoviruses show much higher replicationcapacity in tumor cells than in normal cells (p<0.005), and viruses with Apoptin and IL-24heterogenous expression show lower ability in replication than mock. The recombinantadenovirus with IL-24expression cassette shows better cytotoxicity and replication ability,while the viruses with Apoptin expression cassette can not achieve hypothetical killing effect,possibly because of their low replication ability result from gene effect or insertion site. Thestudy of mechanisms of our recombinant adenovirus on A549cell shows that our viruses caninduce A549cell apoptosis mainly through the mitochondrial pathway. In addition, ourrecombinant adenoviruses are likely to induce tumor cells G2/M arrest and autophagy to killtumor cells. Although recombinant adenovirus with dual genes inserted shows low cytotoxicity,combination of viruses with Apoptin and IL-24inserted achieves much better effect, and theresult suggests that there is no antagonistic action between Apoptin and IL-24, and it also lightthe possibility of linking Apoptin and IL-24with a appropriate linker and then insert thisfusion gene into our oncolytic adenovirus vector. However, the option of linker, the order ofgenes and the insert site are all problems that should be considered. As a member of2Apeptide, P2A has the advantage of high cutting efficiency and no adverse effects from thepresence of residual sequence.