The Tumoricidal Effect Of Adenovirus Mediated IL2-KDRscFv-sTRAIL Fusion Gene

Objective:To explore the lethal effect of adenovirus mediated IL2-sTRAILgene,IL2-KDRscFv gene and IL2-KDRscFv-sTRAIL gene (Ad-IL2-sTRAIL , Ad-IL2-KDRscFv and Ad-IL2-KDRscFv-sTRAIL) on gastric cancer cell line SGC-7901, hepatocellular carcinoma cell line HepGII , colon cancer cell line SW480 and normal human hepatocyte line LO2. Meanwhile, their effects on the growth and angiogenesis of established hepatocellular carcinoma nodules in nude mice were observed.Methods : Both TRAIL and IL-2 cDNA were cloned f rom PBMC by RT-PCR, and KDRscFv gene was synthesized by Shanghai Sangon Biological Engineering Technology & Services Co, Ltd.The signal peptide of IL-2 was cloned to the N tip of KDRscFv and sTRAIL respectively with SOE,and the fusion gene of IL2-KDRscFv and sTRAIL was obtained by using a polypeptide linker ( Gly4Ser).After sequencing, the IL2-KDRscFv gene,IL2-sTRAILgene and the fusion gene of IL2-KDRscFv-sTRAIL fragment was cloned respectively into the shuttle plasmid pAdTrack - CMV to form the transfer vector and then transformed to competent BJ5183 cells previously transformed with pAdEasy-1 to carrying out homogenous recombination in bacteria. Screening recombinant plasmid pAdEasy-sTRA IL,pAdEasy-IL2-KDRscFv and pAdEasy -IL2-KDRscFv-sTRAIL and transfected to 293 cells in mediation of Lipofectamine to preparing replication-deficient adenovirus Ad-IL2- sTRAIL,Ad-IL2-KDRscFv and Ad-IL2-KDRscFv-sTRAIL. The recombinant adenovirus vector was identified by restriction enzyme digestion and polymerase chain reaction (PCR). The prepared Ad-IL2-sTRAIL,Ad-IL2-KDRscFv and Ad-IL2-KDRscFv-sTRAIL virus particles were purified by density gradient centrifugation with cesium chloride and determining the titer by OD260. The transcription of interest gene in 293 cells was detected by PCR.After the virus particles were constructed successfully,they were transfected into gastric cancer line SGC7901, hepatocellular carcinoma cell line HepGII , colon cancer cell line SW480 and normal human hepatocyte line LO2 respectively and the apoptosis rate was determinded by MTT assasy and flow cytometry.In vivo,the adenovirus were injected into tumor in nude mice and the expressions of interest proteins were detected by western blot and immunohistochemistry assay.Results:(1) Recombinant adenovirus Ad-IL2-sTRAIL , Ad-IL2-KDRscFv and Ad-IL2 -KDRscFv-sTRAIL were successfully constructed, and the titer of purified adenovirus particles was 1012 VP /ml .(2) All the three adenovirus have killing effect on cancer cells and the one bearing the fusion gene of IL2-KDRscFv-sTRAIL had the most powerful effect. When the tumor cells were treated with adenovirus in concentration of 20 MOI for 48 hours ,Ad- IL2-KDRscFv-sTRAIL could induce the apoptosis of 39.16 % in HepGII cells ,which was significantly higher than that in the control group of PBS and the vector control group of GFP(p<0.05).(3) Among the three cancer cell lines, hepatocellular carcinoma cell line HepGII had the most distinguished sensitivity to adenovirus.In concentration of 20 MOI for 48 hours, Ad-IL2-sTRAIL could kill 19.88% while Ad-IL2-KDRsvFv killed 9.09% HepGII cells.At the same condition, the apotosis rate of gastric cancer cell line SGC-7901was 8.09% and 5.75% respectively.(4) All the three adenovirus had no killing effect on normal human hepatocyte line LO2 and there had no statistic distinction compared with the control group of PBS and the vector control group of Ad-GFP.(p>0.05).(5) The interest proteins could be detected both in the culture supermatant and cell precips after the tumor cells were incubated with adenovirus.(6) In vivo,injection of adenovirus into hepatocellular carcinoma nodules in nude mice could shrink its volume significantly. Ad-IL2-KDRscFv-sTRAIL had the most powerful lethal effect, and the tumor volume suppression rate was 75.60±4.8891%, while that of Ad-IL2-sTRAIL and Ad-IL2-KDRscFv was 63.69±6.6706% and 56.17±7.3983% respectively.(7) The microvessel density( MVD) was significantly lower in the experiment groups than those in the control group ( P < 0. 01 ) and Ad-IL2-KDRscFv-sTRAIL had the most powerful inhibited effct. (8) The interest proteins could only be detected in hepatic tissues and tumor tissues when the nude mice were treated with adenovirus.(9) There had no obvious pathological change in the hepatic tissues and other tissues after treated with adenovirus.Conclusions:(1) Recombinant adenovirus Ad-IL2-sTRAIL , Ad-IL2-KDRscFv and Ad-IL2- KDRscFv-sTRAIL all have killing effect on cancer cells and among them IL2- KDRscFv- sTRAIL had the most powerful tumoricidal effect.(2) In vitro, all the three adenovirus are safe to normal human hepatocyte line.(3) In vivo studies, injection of adenovirus into tumor significantly inhibited the growth of the hepatocellular carcinoma nodules in nude mice, and the MVD were significantly lower too in the experiment groups than those in the control group and Ad-IL2-KDRscFv-sTRAIL had the most powerful role.(4) The adenovirus mediated KDRscFv-sTRAIL fusion gene have double lethal effect on tumor cells and tumor angiogenesis and presented synergia effect.(5) In vivo and vitro studies, all the three adenovirus are safe to normal cells and tissues.