The Comparison Of Tumor Cell Activities Among The Lj-RGD2Toxin Protein And Its’ RGD Delete Mutations

Lj-RGD2is a histidine-rich peptide with two RGD （Arg-Gly-Asp） motifs from the buccal gland of Lampetra japonica, which molecular weight is8kDa. Because RGD motifs and histidine-rich structure are all helpful to anti-tumor and anti-angiogenesis function, we studied the relationship between the function and the structure of Lj-RGD2. We synthesized the gene of the RGD delete mutations:the mutation which kept the second RGD motif was named Lj-738, the mutation which kept the first RGD motif was named Lj-739, the mutation which was deleted all two RGD motifs of Lj-RGD2was named Lj-740, and the mutation which the all2RGD motifs were replaced by2KGD was named Lj-741. After constructed above genes to pET23b, we transformed the recombination plasmids into E.coil BL21. Furthermore, the recombinant proteins expression was induced by IPTG and purified by His-affinity chromatography. The result of cell proliferation assay demonstrated that rLj-RGD2and the four forms of mutation protein rLj-738, rLj-739, rLj-740and rLj-741inhibited bFGF-induced proliferation of Lewis cells in a dose-dependent manner with IC₅0at1.12μmol/L,1.23μmol/L, l.llμmol/L,1.18μmol/L and1.03μmol/L, respectively. In order to determine the effect of rLj-RGD2and the four mutation protein on Lewis cells migration toward bFGF, we used the transwell containing insert filter （8.0μm pore size, polycarbonate filter）. The results showed that, all of rLj-RGD2, rLj-738, rLj-739, rLj-740and rLj-741showed significant inhibition on Lewis cells migration,the inhibition rates were30%,31%,31%,34%and25%, respectively. In the invasion assay, the Matrigel and Transwell were used to imitate environment in vivo. The results of invasion assay revealed that, rLj-RGD2and the mutation proteins significantly inhibited bFGF induced invasion of Lewis cells, but the inhibition effect without too much difference. rLj-RGD2and the mutation proteins significantly break down the cytoskeleton of the Lewis cells. All of rLj-RGD2and the mutation proteins could induce the apoptosis of Lewis cells. The partial signal pathway of above wild type Lj-RGD2and mutations were detected, the results showed that all of above proteins decreased the expression levels of FAK, ILK and FAK phosphorylation. Taken together, these results suggested that, the functions of Lj-RGD2are relative with RGD motifs or histidine-rich structure, and the mechanism of Lj-RGD2and the mutaions on the Lewis cells maybe different.