Anti-tumor Effects Of Oncolytic Adenovirus Ad-VT On Human Lung Cancer Stably-expressing Luciferase

At present,lung cancer(LC)is the highest incidence and mortality malignant tumor in the worldwide scale,and the number of cases are continuing to rise.Traditional therapies such as surgical,radiotherapeutic,and chemotherapeutic and monoclonal antibody approaches,only for a few patients at the early stage.In spite of having certain efficacy,it is difficult to be completely cured,and usually has the characteristics of great minus effects and high-rate of relapse.With the rapid development of genetic engineering in the biological and medical community,it is possible to use a gene targeted therapy for cancer.Gene therapy shows more and more prominent advantages,especially the adenovirus vector targeting cancer cell therapy receives widespread attention,which will be the focus study,and is expected to replace the traditional therapy for the treatment of cancer.Using immunodeficient animals(such as BALB/c nude mice)to establish tumor models is an important tool to study the anti-tumor effects of oncolytic adenovirus,but traditional animal models cannot study tumor growth and metastasis,and the anti-tumor effect of oncolytic adenovirus continually and dynamically.Luciferase gene is a common reporter gene,with high sensitivity,low endogenous and good stability,which is widely used in tagging tumor cels;Tumor cels stably-expressing luciferase were used to establish tumor model,and then by in vivo bioluminescence imaging system to monitor tumor growth and metastasis in noninvasively,real-time,continually;Even if the subtle changes of tumor cell during treatment can be timely detected,providing an ideal animal model for further studying the anti-tumor effects of oncolytic adenovirus in integrity.Our research group had constructed recombinant adenovirus(Ad-VT,Ad-T,Ad-VP3,Ad-Mock)by RAPAd.I packaging system.Ad-VT(Ad-Apoptin-hTERTpE1A)is a recombinant adenovirus in which tumor specific promoter(hTERTp,human telomerase reverse transcriptase drives E1Agene(gene required for viral replication)and cytomegalovirus promoter drives apoptin gene,so Ad-VT has the effect of tumor specific cytotoxicity and replication competent;Ad-T(Ad-hTERTp-E1A),Ad-VP3(Ad-CMV-Apoptin)and Ad-Mock are control virus.Objective:The research established human lung cancer cells stably-expressing luciferase(A549-luc)and subcutaneous tumor model of BALB/c nude mice,and then investigated the inhibitory effect of oncolytic adenovirus Ad-VT on human lung cancer cells stably-expressing luciferase(A549-luc)in vitro and in vivo.Contents:1.Screen human lung cancer cells stably-expressing luciferase,and detect luciferase activity,luciferase stability,in vitro imaging and biological characteristics;2.Study the inhibitory effects of oncolytic adenovirus Ad-VT on human lung cancer cells stably-expressing luciferase in vitro;3.Study the inhibitory effects of oncolytic adenovirus Ad-VT on human lung cancer cells stably-expressing luciferase in vivo.Methods:1.Construction and identification of human lung cancer cells stably-expressingluciferaseScreen human lung cancer cells stably-expressing luciferase by plasmid transfection and G418 selection,luciferase activity,luciferase stability and in vitro imaging,and detect the biological characteristics of A549-luc cells and A549 cells,which includes growth characteristics,migration and invasion and cell cycle.2.Study the inhibitory effect of oncolytic adenovirus Ad-VT on human lung cancer cells stably-expressing luciferas in vitroStudy the cytotoxicity effect of oncolytic adenovirus Ad-VT on A549-luc cells and A549 cells through the crystal violet staining and WST-1,and verify the inhibition pathway of oncolytic adenovirus Ad-VT on A549-luc cells and A549 cells according to the morphological change of mitochondrial membrane potential(JC-1 staining),cell nuclear(Hoechst staining)and cell membrane(AnnexinV-FITC/PI staining),and then measure the migration and invasive ability of oncolytic adenovirus Ad-VT on A549-luc cells and A549 cells via the wound healing,Transwell chambers migration and BioCoat chambers invasion experiments.3.Study the inhibitory effect of oncolytic adenovirus Ad-VT on human lung cancer cells stably-expressing luciferas in vivoA549-luc cells were injected subcutaneously into the right hind limb near back area of the BALB/c nude mice,and subcutaneous tumor models was successfully established in nude mice,which were randomly divided into six groups;From 0 week to 7 week,take photos with in vivo imaging system,and measure the parameters of tumor growth by vernier caliper weekly;After two weeks begin to treat them with the recombinant adenovirus for four weeks,and study the inhibitory effect of oncolytic adenovirus virus Ad-VT on tumor and the survival changes of nuce mice.Results:1.The highest luciferase activity and most stably-expressing luciferase was clone 28 through plasmid transfection and G418 selection,luciferase activity and luciferase stability detection;Bioluminescence intensity and cell number of clone 28 were positively correlated,R2=0.9948,by in vitro imaging;Construction process had been proved to be no significant effects on cells whose growth characteristics,migration,invasion and cell cycle through the A549-luc cells and A549 cells biological characterization experiments.2.There was no significant difference in the killing effect of oncolytic adenovirus Ad-VT on A549-luc cells and A549 cells through crystal violet staining and WST-1 assay,with significant time effects and dose effects,and the suppression rate of recombinant adenovirus was as follows: Ad-VT > Ad-T > Ad-VP3 > Ad-Mock;JC-1 staining,Hoechst staining and Annexin V-FITC/PI staining experiments show no significant difference in the inhibitory pathways of oncolytic adenovirus Ad-VT on A549-luc cells and A549 cells,and Ad-VT exerted their suppression mainly through inducing cell apoptosis(such as the changes of mitochondrial membrane potential,nuclear fragmentation,hyperchromatic nuclei and membrane phosphatidylserine eversion),and the apoptosis rate of recombinant adenovirus was as follows: Ad-VT > Ad-T > Ad-VP3 > Ad-Mock;Wound healing assay,Transwell chambers migration and BioCoat chambers invasion Experiments show no significant difference in the migration and invasion ability of oncolytic adenovirus Ad-VT on A549-luc cells and A549 cells,with significant time effects and dose effects,and the inhibition of recombinant adenovirus on migration and invasion ability was as follows: Ad-VT > Ad-T > Ad-VP3 > Ad-Mock.3.A549-luc cells were injected subcutaneously into BALB/c nude mice,successfully establishing nude mice tumor model.0W ~ 3W,The mean bioluminescence intensity and mean tumor volume of Ad-VT treatment group and the other treatment groups had no significant difference;3W ~ 5W,The mean bioluminescence intensity and mean tumor growth rate of Ad-VT treatment group,compared with the control group,decreased significantly,while the mean tumor supression rate increased significantly;There was a positive correlation between tumor volume and bioluminescence intensity,and the bioluminescence intensity was enhanced with the increase of tumor volume;Ad-VT treatment group compared with other groups,can significantly prolong the average survival of the mice.Conclusion:1.Human lung cancer cells stably-expressing luciferase A549-luc were constructed successfully,and there was no significant difference in the biological characteristics of A549-luc cells and A549 cells.2.There was significant killing effect of oncolytic adenovirus Ad-VT on human lung cancer cells stably-expressing luciferas A549-luc,and Ad-VT exerted their suppression mainly through inducing cell apoptosis,and could effectively decrease the migration and invasion ability of A549-luc cells.3.The subcutaneous tumor model of BALB/c nude mice could monitor in real-time the change of tumor bioluminescence intensity and tumor volume in vivo;Oncolytic adenovirus Ad-VT could significantly inhibit tumor growth and prolong the average survival of mice.