Inhibition On The B16 Mouse Melanoma Cells Of RLj-RGD4,A Novel Recombinant Peptide With Artificially Synthesized Gene

rLj-RGD4 is a recombinant peptide which gene was artificially synthesized.The gene of Lj-RGD4 was designed focus on the gene of Lj-RGD3 which was studied in our preliminary work.Lj-RGD4 is a deletion mutant without the purification tag.rLj-RGD3 is a 14.5 kDa recombinant RGD toxin protein from Lampetra japonicas which is a one of the marine species.rLj-RGD3 has strong anti-tumor functions and there are 3 RGD motifs in its' sequence,but which has a potential immunogenicity risk in future bio-medical research due to its higher molecular weight.We modified the the the gene of Lj-RGD3 in order to obtained a lower molecular RGD toxin peptide.The artificially synthesized gene of Lj-RGD4 was constructed to the plasmid of pET23 b,and the recombinant E.coli BL21 with pET23b-RGD4 was induced by 1 mM(final concentration)IPTG.Through optimization,the induced temperature was confirmed as 30 ? and the induced time was confirmed as 12 h.Because there 5 histidine in the sequence of Lj-RGD4,the nickel ion affinity chromatograph method was used in the purification.We obtained the purified rLj-RGD4 which is 6.27 kDa with 4 RGD motifs,and the sequence was confirmed by the method of N-terminal sequencing.In order to study the anti-tumor biological functions of rLj-RGD4,we selected the B16 mouse melanoma cells as the research model.We determined the effects of rLj-RGD4 to the critical processes of tumor growth and metastasis.The results of cell proliferation assay(MTT)showed that,rLj-RGD4 inhibited the bFGF induced proliferation of B16 cells in a dose-dependent manner,and the half maximal inhibitory concentration(IC50)value was 9.6?M.The result of cell adhension assay showed that,rLj-RGD4 inhibited the B16 cells adhesion to fibronectin in a dose-dependent manner.The Transwell and the cell wound scratch assays revealed that,rLj-RGD4 inhibited the B16 cells migration toward bFGF and the inhibition rate in the high-dose group was 100%.In the invasion assay,we utilized the Transwell and Matrigel to establish a simulation inner enviroment of the body.The results showed that,the inhibition rate of low-dose group was 31.7%,the inhibition rate of middle-rose group was 72.3%,and the inhibiton rate of high-rose group was 100%.Then,we tested whether rLj-RGD4 could induce the apoptosis of B16 cells.The results of Hochest33258 and TUNEL-FITC staining experiments showed that,rLj-RGD4 induced the apoptosis in a dose-dependent manner.This conclusion revealed that rLj-RGD4 inhibited B16 cells proliferation depended on the cells' apoptosis rather than the cells' necrosis.In order to study the functions of rLj-RGD4 on the cytoskeleton,the phalloidine was used.Theresults of the phalloidine-FITC staining assay showed that,rLj-RGD4 destroyed the cytoskeleton and inhibited the formation of pseudopod of B16 cells.Western blotting results showed that,the expression of Caspase-3 zymogen was up-regulated and the activation of which was increased.On the other hand,the expression of anti apoptosis-related genes Bcl-2was down-regulated.In conclusion,rLj-RGD4 inhibited the critical process of tumor growth and metastasis,include proliferation,adhension,migration,invasion and the cells' pseudopod formation.rLj-RGD4 also induced the apoptosis of B16 cells.Because rLj-RGD4 is a low molecular weight peptide,which is more promising in application of antineoplastic agents.Following the further studies,rLj-RGD4 will be hopefully to become the new drug candidates.