The Experiment Of Antitumor Therapy With GALV Membrane Fusion Glycoprotein In Lung Adenocarcinoma

Objective: Package of constructed recombinant tumor-selective type I Herpes simplex virus vector plasmid containing UL38 and CMV promoter and GALV.fus gene(HSV-UL38P- GALV.fus,HSV-CMVP- GALV.fus), GALV.fus gene was replaced by EGFP in control plasmid(HSV-CMVP-EGFP). The three recombinant viruses were named Synco-2,Synco-1 and Baco-1 respectively。Methods: At first the three constructed plasmids were amplificated in coliform bacterium and transfected into Vero cells by using lipofectamine respectively. Then, these three recombinant type I Herpes simplex viruses were propagated in Vero cells and purified by cwsium chloride density purification, titrated by TCID50 method. In the end, Total RNA was extracted from the HepG2 cells which were transfected by Synco-1 and Synco-2 and GALV.fus mRNA was detected by RT-PCR.Results: The three recombinant HSV-1 were packaged successfully,which were propagated in Vero cells and purified by cesium chloride density purification, titrated by TCID50 method. The titre of Baco-1,Synco-1 and Synco-2 were 3×1010pfu/ml,1×1011pfu/ml and 4×1010pfu/ml respectively. GALV.fus gene were identificated in the infected HepG2 cells.PARTⅡKILLING EFFICIENCY OF HUMAN LUNG CANCER CELLS BY GALV.FUS SYSTEM MEDIATED BY TYPE I HERPES SIMPLEX VIRUS UNDER THE CONTROL OF UL38 AND CMV PROMOTER IN VITROObjective: To observe the selective killing effect of recombinant HSV-I mediated GALV.fus gene system controlled by UL38 promoter and CMV promoter on lung cancer cells in vitro.Methods: Lung adenocarcinoma cell line A549 was cultured in RPMI1640 medium containing 10% FBS, and human fibroblast cell line HFL-I GNHu5 was cultured in DMEM medium containing 10% FBS.To phenotypically characterize the recombinant HSV-I virus, we seeded A549 cells into six-well plates, and then infected them the following day with Baco-1,Synco-1and Synco-2 at a dose of 0.01 pfu/cell. Cells were cultured in a maintenance medium(containing 1% fetal bovine serum) and were incubated for up to 2 days to allow the fusion pattern and plaques to develop. The Phenotypic characterization were observed and photographed under the inverted microscopy.To measure the in vitro killing effect of the viruses, we seeded A549 tumor cells into 24-well plates and infected them with the recombinant HSV-I of different multiplicities of infection(MOI) at 1, 0.1, or 0.01 pfu/cell, or left them uninfected. Cells were harvested 24 or 48 hours later by trypsinization, and the number of viable cells determined with a hemocytometer after trypan blue staining under the inverted microscopy. The percentage of viable cells was calculated by dividing the number of cells excluding trypan blue in the infected well by the number excluding the stain in the well that was left uninfected. The experiments were done in triplicate, with mean cell numbers used for the final calculation.To observe the selective killing effect of recombinant HSV-I mediated GALV.fus gene system controlled by UL38 promoter and CMV promoter in vitro, the embryonic fibroblasts were either kept in the cycling phase by growth in a medium containing 10%FBS, or were arrested with 20μM lovastatin for 30 h in serum-free medium,before the cells were infected with virus. Lovastatin is a chemical that induces cell-cycle arrest but does not directly interfere with HSV replication.The cells were then infected with either Synco-1 or Synco-2 at 0.01 pfu /cell, The photos were taken 48 h after infection. Total protein was extracted from the transfected A549 cells and HFL-I GNHu5 cells ,the expression of GALV.fus gene was detected by Western Blot.Results: We monitored the cultures for a cytopathic effect and syncytia formation. syncytia formation was clearly present in cultures infected with Synco-1and Synco-2, but not in cells infected with the Baco-1. Instead, infection with Baco-1 induced a typical cytopathic effect, characterized by cell rounding and swelling. These results indicate that Synco-1and Synco-2 are fusogenic in human lung cancer cells.A549 cells were harvested at either 24 or 48 hours postinfection in 24-well plates, and cell viability was determined by trypan blue staining. As early as 1 day postinfection , Synco-1and Synco-2 killed a substantial number of tumor cells even in the wells infected with virus at a multiplicity of infection as low as 0.01 pfu/cell. By 48 hours, Synco-1and Synco-2 had almost completely eradicated the tumor cells exposed to virus doses of 1 or 0.1 pfu/cell. Even at the lowest virus dose, 0.01 pfu/cell,more than half of the tumor cells were killed. Overall, the oncolytic activity of HSV-GALV.fus was superior to that of HSV-CMVP-EGFP(p<0.01).We tested directly whether Synco-2 loses its ability to cause syncytial formation in normal nondividing human cells, by infecting primary human fibroblasts in either a quiescent or a cycling state.infection with either Synco-1 or Synco-2 caused syncytial formation in these normal human cells when they were in cycle; the syncytia formed by Synco-1 infection were noticeably larger than those produced by Synco-2 . Synco-1-mediated cell fusion was only marginally affected in cells whose cycling was either slowed by serum starvation or completely arrested with lovastatin. Synco-2-mediated cell fusion, on the other hand, was absent in cells whose cycling was decreased or arrested.By Western Blot, the expression of GALV.fus gene could be detected in both A549 cells and embryonic fibroblasts transfected by Synco-1 and Synco-2.PARTⅢKILLING EFFICIENCY OF HUMAN LUNG CANCER BY GALV.FUS SYSTEM MEDIATED BY TYPE I HERPES SIMPLEX VIRUS IN VIVOObjective: To explore the killing effect of recombinant HSV-I mediated GALV.fus gene system controlled on lung cancer cells in vivo,and the best schema on establishing tumor-bearing nude mice model with human lung adenocarcinoma.Methods: The tumour-bearing nude mouse model was established with scapular subcutaneous injection with cell suspension in advance. A549 cells were transplanted subcutaneously to the nude mice by injection with three different doses(1 X 106,1 X 107and 1X 109 cells per mouse)of cell suspension and tissue block implantation with trocar, the cells were washed once with serum-free medium before they were resuspended in PBS. Only single cell suspensions with >95% viability were used for in vivo injections.The tumor groth information were recorded and weiwere after transplantation. The mice were sacrificed 4 weeks after inoculation and the tumours were removed for histopathological analysis.All sacrificed mice were evaluated macroscopically for the presence of metastases in the thoracic and abdominal cavity.A suspension of 1 X 107 A549 cells in a volume of 200μl was inoculated subcutaneously into the right scapular of 6-week-old nude mice to establish tumors, mice were divided into 5 groups(Group A,B,C,D,E and F ). With the s.c. tumor model, treatment was initiated 2 weeks after tumor cell implantation, when the tumors reached 7 mm in diameter. Mice received a single intratumor injection of either 1 X 107 pfu of recombinant virus in a volume of 100μl or the same volume of PBS. Tumor size was measured weekly for 5 weeks, and there volume were calculated by the formula:tumor volume (mm3) = 0.5 x [length (mm)] x[width (mm)]2. Group E was sacrificed 15 days later after virus injection and tumors tissues were used to analyses the expression of GALV.fus gene by RT-PCR or Western Blot. For the examination of viral cytotoxicity test,1 X 107 pfu of Synco-2 in a volume of 100μl injected in Group F by caudalis intravenous injection .Mice were sacrificed 15 days after virus injection and internal organs were removed, fixed, and stained with hematoxylin and eosin for pathological examination.Results:The rate of tumor formation of both the subcutaneous injection with cell of 1X107cells,1X109 cells per mouse and the tissue block implantation were 100%,The rate of tumor formation of subcutaneous injection with cell suspension of 1X 106cells per mouse was 60% . The time of tumor formation was longer in the groups subjected to subcutaneous injection with cell suspension,approximately13一16days.Whereas that of the group subjected to tissue block implantation was7 -10days.we did not observe any metastases in Internal organs in nude mice.The heterotransplantative tumourigenecity of the tumour in nude mice was 100% after A549 cells were inoculated.The growth rate of the tumour in nude mice in Group C and D decreased significantly compared those of tumor in nude mice in Group A and B. There were no obviously difference in Group C and D. we detected the expression of GALV.fus gene by RT-PCR and Western Blot in infected tumour tissues.Conclusions1 The three recombinant tumor-selective type I Herpes simplex virus HSV-UL38P- GALV.fus,HSV-CMVP- GALV.fus and HSV-CMVP-EGFP were packed successfully. After they were propagated in Vero cells and purified by cesium chloride density purification, their titres were very high. The expression of GALV.fus gene were detected in infected liver cancer cell line HepG2 with RT-PCR method.2 Recombinant type I Herpes simplex virus HSV-UL38P- GALV.fus and HSV-CMVP- GALV.fus could infected and killed lung cancer cell line A549 cells selectively, with the infection rate associated with the MOI of recombinant type I Herpes simplex virus. type I Herpes simplex virus vector containing UL38 promoter for the GALV.fus gene treatment appears to be highly selective in killing lung cancer cell line and no killing effection in human fetal fibroblast HFL-I GNHu 5 in vitro. The targeting therapeutical effect existed.3 Cell suspension or tissue block implantation are all ideal methods to establish tumor-bearing nude mice model,The heterotransplantative tumourigenecity of the tumour in nude mice was 100% after A549 cells were inoculated. The growth of the tumour in nude mice could be inhibited obviously by recombinant HSV- GALV.fus oncolytic gene system.The use of HSV-UL38P- GALV.fus in vivo did not appear adverse reaction obviously which can be investigated sequencely in metastatic therapy of tumour in vivo.