RGD-Modified Endostatin Peptide 30 Derived from Endostatin Suppresses Invasion and Migration of HepG2 Cells Through the αvβ3 Pathway

Abstract

The nucleotide sequence encoding amino acids 1-30 of endostatin (peptide 30, with amino acids 25-31 mutated from RGIRGAD to RGDRGD) was artificially synthesized and cloned into the plasmid pTYB2 and expressed in Escherichia coli (DE3). Peptide 30 was purified by chitin affinity chromatography followed by dithiothreitol removal by gel filtration and protein identification using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similarly, peptide 27, corresponding to amino acids 1-27 of endostatin, was produced as control. The effects of peptide 30 on the metastatic potential of HepG2 cells were then investigated. Peptide 30 was found to effectively suppress the adhesion, invasion, and migration of HepG2 cells. Flow cytometry demonstrated that peptide 30 did not alter the expression of membrane integrin αvβ3, although immunofluorescent staining revealed that these integrins formed clusters on the cell surface. Therefore, the effects of peptide 30 on cancer cell invasion may involve the αvβ3 pathway. Moreover, peptide 30 inactivated metalloproteinase-2 (MMP-2) and MMP-9 and downregulated the expression of COX-2 (cyclooxygenase 2), MMP-2, and MMP-9 at both mRNA and protein levels. Peptide 30 also upregulated the expression of tissue inhibitor of metalloproteinase-1 and tissue inhibitor of metalloproteinase-2 at mRNA and protein levels. Lastly, an antibody against αvβ3 enhanced the biological effects of peptide 30.