1. National Institute of Genetic Engineering and Biotechnology 2. National Institute of Genetic Engineering and Biotechnology 3. National Institute of Genetic Engineering and Biotechnology
Abstract
Introduction
Bladder cancer is the most common urinary malignancy all over the world. . the pathologic angiogenesis plays an important role in bladder cancer tumor growth and progression .vegf is one of the primary required angiogenic factor for bladder cancer and is able to induce tumor angiogenesis and accelerate tumor growth. a dose-dependent increase in the expression of flt1 (fms-related tyrosine kinase 1) encoding vegfr-1 suggests that flt1 is an androgen target gene, linking ar.sflt01,which consist of the second immunoglobulin (igg)-likedomain of flt-1 fused to a human igg1 fc through a polyglycine linker 9gly has been previously generated with the inhibitory effect on vegf and placental growth factor(plgf).aim of this study is to evaluate the effect of sflt01 over expression on angiogenesis,proliferation, migration ,invasion ,and expression timp2,timp1, mmp2 and mmp9 of 5637 bladder cancer cell line
Methods
Sflt01-his tag-gfp sequence was designed synthesized and cloned in aav-mcs-gfp vactor. paav–sflt01-his tag-gfp vector was transfected to 5637cell line through lipofection 2000 transfection. then extracted mrna was analyzed by real time pcr. protein secretion into the conditioned medium of transfected 5637cells and hek293t cells proved by western blotting. the effect of the condition medium 5637 cells and hek293t cells on in vitro angiogenesis in huvec cells was investigated by the invitro angiogenesis assay.
5637 cell migration was evaluated by scratch assay. 5637 cell invation was evaluated by invation assay,mmp-2 and mmp-9 activities were assessed by gelatin zymography as well. the cytotoxic effect of the constructson 5637 cells was determined and cell proliferation assay was performed through the mtt assay.
Results
Real-time pcr results showed significant over expression of sflt01 in treated 5637 cells .western blot proved sflt01 protein secretion in conditioned media. in vitro angiogenesis assay results showed decreased potential of tube formation in conditioned medium of treated 5637 cells and hek293t cultures. scratch assay showed significant difference in treated 5637 cells when compared to the control untreated cultures. invasion assay demonstrated that over expression of sflt01 reduced the invasion and migration of the cells mtt assay showed that sflt01 had no cytotoxic effect on 5637 cells. analysis of gelatin zymography showed that overexpresstion of sflt01reduced the activity of mmp-2 and mmp-9 in 5637cells’ conditioned media.
Conclusion
Functional assessment of paav-sflt01 transfected cells, revealed that the secreted protein could be promising for inhibition of angiogenesis in animal model of cancer disease and tumorigenesis
