Antiangiogenic Activity Of Arresten And Genetic-modification By Its CDNA In The Therapy Of Three Human Carcinoma Xenografts

As for the newly identified angiogenesis inhibitor, arresten, there exits two dramatic slant upon it. One considers it being a potent inhibiting substance on the proliferation of vascular endothelial cell and even stronger, the inhititory activity being concerned, than endostatin, which is thought as having kept ahead of others among the categories, while the other holds that its activity is weak, even if it has some. In fact, the activity change resulting from such production method of arresten by prokaryotic engeneering, as above two research groups having adapted, which lacks the post translation modification for protein, added the complexity of the subject. Would arresten inhibit the proliferation of vascular endothelial cell? We insist that the questen will not be a question if the real human derived arresten is obtained, for it is a human body endogenesis fragment of protein. In another word, the eukaryotic expressed arresten is needed firstly whenever its activitis is put into further disscusion. That is the backgrouds of the research.Objective: To study the influence of eukaryotic-expressed arresten on proliferation of vascular endothelial cell in vitro, and to investigate its effect upon the growth of human gastric cancer(SGC-7901, MGC-803) and human breast cancer(MCF-7) in vivo, following the exploration of the biological influence of the genetic-modification with arresten cDNA on above cell lines in vitro.Methods: arresten cDNA was ligated downstream and in frame with the sequence of the signal peptide from Kappa chain of murine Ig by PCR strategy, before it being cloned into pcDNA3.1(+), a eukaryotic expression vector. Then the pcDNA3.1(+) vector encoding arresten was transfected using liposome into the cell SGC-7901, MGC-803, and MCF-7 respectively, followed by G418 method to select definedly the positive-transformed clones, whose biological behaviors were further studied with cellcounting, MTT, and FCAS(flow cytometry analysis system). And then Western blot method was adapted to detect the protein expressed in cell medium, accompanied with the vascular endothelial cell-inhibitory experiment to find whether arresten could or not inhibit its proliferation. At last, the positive-transformed cell clones were respectively implanted subcutaneously into nude mice and the data of tumors size were recorded 25 days after.Results: The secretable eukaryotic expression vector of arresten cDNA was constructed successfully. The SGC-7901, MGC-803, and MCF-7 cell lines with the character of arresten expression were obtained. And the most importantly, arresten was found did having the activity in vitro of inhibitory effect on the proliferation of vascular endothelial cell. In mice mide xenograft models, the data implicated that the tumors from above genetic-modified cancer cells expanded remarkedly slowlier as compared with the control did.Conclusion: arresten is a potent inhibitor of proliferation of endothelial cell, and probably through the mechanism the substance contributes to the good results in in vivo experiments of two human cancers cell lines(SGC-7901, MGC-803). At the same time, the successful construct of pcDNA3.1(+)-arresten plasmid brings possibility of arresten-gene therapy against human carcinoma.