ABSTRACT
We constructed a recombinant adenoviral vector expressing human tissue inhibitors of metalloproteinase-1(TIMP-1), and evaluated the inhibition of TIMP-1 secreted by primary fibroblasts after infection with adenovirus-mediated TIMP-1 gene (Ad-TIMP-1) on tumor cell invasion and metastasis in mouse melanoma. It was found that TIMP-1 was detected in the supernatants of cultured mouse primary fibroblasts after infection with Ad-TIMP-1 by indirect enzyme-linked immunosorbent assay (ELISA). The TIMP-1 secreted by Ad-TIMP-1 infected primary fibroblast significantly inhibited B16BL6 cell invasion and metastasis both in vitro and in vivo. We also demonstrated that the primary fibroblasts transfected by Ad-TIMP-1, after being subcutaneously injected into mouse, can secreted TIMP-1 into the blood of mouse and maintained at the therapeutic in vivo levels of TIMP-1. These results suggest that the preparation of Ad-TIMP-1 infected primary fibroblast be an effective method to deliver TIMP-1 gene in vivo, which provides a new strategy of gene therapy and has the potential for clinical applications in the treatment of tumor cell metastasis.
Subject(s)
Animals , Female , Humans , Mice , Adenoviridae , Genetics , Metabolism , Fibroblasts , Metabolism , Genetic Therapy , Melanoma, Experimental , Pathology , Therapeutics , Mice, Inbred C57BL , Neoplasm Invasiveness , Neoplasm Metastasis , Recombinant Proteins , Genetics , Pharmacology , Tissue Inhibitor of Metalloproteinase-1 , Genetics , PharmacologyABSTRACT
Objective To evaluate the effect of adenovirus-mediated siRNA targeting Survivin on tumor growth and metastasis of mouse melanoma.Methods A replication-defective recombinant adenovirus AdH1-siRNA-Survivin targeting mouse Survivin was constructed using the homologous recombination technique.The expression of Survivin protein in B16BL6 cells after infection was detected by Western blotting.Mouse melanoma models were established by subcutaneous injection of B16BL6 cells.AdH1-siRNA-Survivin of 2?109 pfu every week by multi-spot intratumoral or peritumoral injection was performed to every mouse(totally three times).The tumor volume was measured every five days after tumor cell implantation,and the microvessel density(MVD) of tumor was counted by immunohistochemistry under the microscope.For the experimental metastasis assay,B16BL6 cells infected with AdH1-siRNA-Survivin were injected into the tail veins of mice,then the visible black nodules on the lung surface were counted macroscopically at 21th day after tumor cell injection.Results AdH1-siRNA-Survivin could obviously decrease the expression of Survivin protein in B16BL6 cells.When compared with the blank control and AdH1-empty control,the tumor growth was inhibited and MVD was less in the mice injected with AdH1-siRNA-Survivin(P