Construction Of Adenovirus Vector Carrying HSulf-1Gene And Investigation Of Its Antitumor Mechanism

human sulfatase-1（hSulf-1） is a negative modulating factor in cell growth, it iswidely expressed in normal tissue, but inactivated in majority of various human cancercells and tissues, e.g., the ovarian, breast, pancreatic, renal, hepatic cancers. Previousstudies demonstrated that re-expression of hSulf-1gene in cancer cells resulted inreduced proliferation, migration and invasion, as well as sensitivity to induction ofapoptosis by the chemotherapeutic agent, inhibition tumorigenesis and angiogenesis.hSulf-1is a heparin-degrading endosulfatase that desulfates cell surface heparan sulfateproteoglycans （HSPGs） in extracellular matrix and negatively modulatesheparin-binding growth factor and cytokine signaling. Therefore, the aim of study is totransport hSulf-1gene to endothelial cell and cancer cells by the recombinantadenovirus vector, and further investigate the effect of hSulf-1protein on cellproliferation and migration in vivo and in vitro. This study provides a new moleculartarget for clinical gene therapy.PartⅠ Construction of adenovirus vector carrying hSulf-1gene【Objective】To clone hSulf-1gene and construct adenovirus vector Ad5-hSulf1which could express hSulf-1gene stably.【Methods】Plasmid pcDNA3.1-hSulf1was used to amplify the hSulf-1cDNA, thePCR product was digested with BamHI and SalI, then inserted into the adenovirusplasmid pDC315to generate pDC315-hSulf1. The plasmids pDC315-hSulf1and theadenovirus packaging plasmid pBHGloxdelE13cre were transfected into HEK293cellsusing the PolyFect Transfection Reagent. After a homologous recombination inHEK293cells, we obtained adenoviruses named Ad5-hSulf1. The control adenoviruscarrying enhanced green fluorescent protein （EGFP）, Ad5-EGFP, was also recombinedin this way. All adenoviruses were amplified and purified. The viral titers were detectedwith the Tissue Culture Infectious Dose50（TCID50） method. 【Results】The results of PCR and DNAsequencing demonstrated the recombinantadenovirus Ad5-hSulf1was successfully constructed. TCID50method detect viral titers,the titer of Ad5-hSulf1was1.15×1010pfu mL, the titer of control virus Ad5-EGFP was1.80×108pfu mL.【Conclusion】 The recombinant adenovirus Ad5-hSulf1was constructedsuccessfully, which lays a foundation for further studies in vivo and in vitro.PartⅡ Over-expression of hSulf-1gene and its effect onendothelial cell proliferation and migration【Objective】To investigation the effect of hSulf-1gene over-expression on theproliferation and migration of human umbilical vein endothelial cells, ECV-304.【Methods】The expression of hSulf-1gene and phospho-Akt、ERK were detectedby Western blotting; the cell viability and migration were evaluated by MTT-assay andwound-healing assay in ECV-304cell line.【Results】Western blotting analysis showed that over-expression of hSulf-1down-regulated the phosphorylation levels of Akt and ERK in ECV-304cells. MTTassay showed that over-expression of hSulf-1inhibited cell proliferation of ECV-304cells, with the cell survival rate decreased to （68.49±0.05）%at an MOI of50pfu mLand （67.78±0.06）%at an MOI of100pfu mL（P<0.05）. Wound-healing assay showedthat over-expression of hSulf-1significantly inhibited the migration of ECV-304cellscompared with the control group （P<0.01）.【Conclusion】 The recombinant adenovirus Ad5-hSulf1mediated hSulf-1over-expression can remarkably inhibit the cell proliferation and migration of ECV-304cells, laying a foundation for gene therapy of tumor and angiogenesis related diseases.Part Ⅲ hSulf-1gene exhibits anticancer efficacy throughnegatively regulating VEGFR-2signaling in the in vitroexperiment【Objective】To study how hSulf-1gene regulated VEGFR-2signaling to exhibitantitumor effect in vitro.【Methods】To demonstrate its inactivation in majority of various human cancers, we examined hSulf-1expression in many types of human cancer specimens byimmunohistochemistry; The level of p-VEGFR2^Tyr1175after Ad5-hSulf1infection andhSulf-1shRNA transfection were detected by Western blotting; BEL-7404and SKOV3cancer cells were infected by Ad5-hSulf1or transfected by VEGFR-2shRNA or both,cell viability were evaluated by MTT assay.【Results】The positive rates of hSulf-1were23.1%（6/26）,16.7%（2/12）,31.8%（7/22）,11.1%（1/9）,44.4%（8/18） in hepatocellular, breast, gastric, renal and coloncancers, respectively. Among26cases of hepatocellular carcinoma, there is an obviousdecrease of p-VEGFR2^Tyr1175level in the hSulf-1-positive hepatocellular carcinoma thanthat in the hSulf-1-negative hepatocellular carcinoma （P <0.05）, but no difference oft-VEGFR2expression between them （P>0.05）. Western blotting analysis showed thatthe level of p-VEGFR2^Tyr1175had an obvious decrease after infection of Ad5-hSulf1.When the hSulf-1shRNA was transfected into the Ad5-hSulf1infected cancer cells, thecontent of p-VEGFR2^Tyr1175recovered nearly to the normal levels. MTT assay showedthat Ad5-hSulf1infection or VEGFR-2shRNA transduction both decreased cellviability to some extent, BEL-7404cancer cells were infected with Ad5-hSulf1and thentransfected with VEGFR-2shRNA vector, the results showed cell viability was furtherdecreased.【Conclusion】Inactivation of hSulf-1is a common molecular event in majority ofhuman cancers. In BEL-7404and SKOV3cancer cells, re-expression of hSulf-1genecould reduce the level of p-VEGFR2^Tyr1175. Hereby, adenovirus-mediated hSulf-1reactivation inhibits cancer cell proliferation.Part Ⅳ Establishment of human cancer xenografts models innude mice and study of hSulf-1gene effect on tumor microvesseldensity and tumor growth【Objective】To establish the human cancer xenograft models and study the effectof hSulf-1re-expression on tumor growth and microvessel density（MVD） in vivo.【Methods】The ovarian and hepatocellular cancer xenograft models in nude micewere established, and treated with Ad5-hSulf-1. The effect of hSulf-1on tumor growthand the effect of re-expression on tumor microvessel density were investigated. 【Results】In the ovarian and hepatocellular cancer xenograft models in nude mice,suppression of tumor growth in the Ad5-hSulf1treated group was more effective, withthe tumor inhibition rates of46.19%and49.56%in SKOV3and BEL-7404models,respectively, compared with the blank control group （P <0.01）. There was no differencebetween the negative virus control group and the blank control group （P>0.05）.SKOV3xenografts were removed and weighed. The tumor weights of the Ad5-hSulf1group were lower than that of the other two groups （P <0.01）. Immunohistochemistryshowed that the microvessel density in tumor tissues were24.67±6.51and52.33±12.34in the Ad5-hSulf1and control groups, there was a significant difference betweenthem （P <0.05）. The expression of p-VEGFR2^Tyr1175and p-AKTThr308wasdown-regulated in the Ad5-hSulf1group examined by Western blotting andimmunohistochemistry.【Conclusion】The ovarian and hepatocellular cancer xenografts in nude mice wereestablished and treated with adenovirus expressing hSulf-1. The results found that thetumor growth was inhibited after treatment. Re-expression of hSulf-1resulted indown-regulation of phosphorylated VEGFR-2and phosphorylated AKT, thensignificantly reduced tumor microvessel density, indicating that hSulf-1expression wasassociated with antiangiogenesis.