Abstract
Angiogenesis promotes growth and metastasis of tumor cells. In this study, we have developed two peptide antagonists of human angiogenin by deducing the codes from the antisense RNA sequence corresponding to the receptor-binding site of angiogenin in either 5' --> 3' (chANG) or 3' --> 5' (chGNA) direction. chANG and chGNA peptides bind to angiogenin with specificity and high affinity (Kd approximately 44 nM) and inhibit the interaction of angiogenin with actin, which is regarded as the angiogenin-binding protein on the surface of endothelial cells. The peptides inhibit the neovascularization induced by angiogenin in the chick chorioallantoic membrane assay. The anti-angiogenic activity of the peptides is specific for angiogenin, and the peptides do not have any apparent effect on embryonic angiogenesis or the preexisting blood vessels. chANG and chGNA also inhibit the angiogenesis induced by the angiogenin-secreting PC 3 human prostate adenocarcinoma cells and have no direct effect on the proliferation as well as the adhesion of PC 3 cells to angiogenin. Therefore, the inhibition of the tumor-induced angiogenesis by the peptides is most likely caused by neutralization of the extracellular angiogenin secreted by PC 3 cells. Based on our results, chANG and chGNA peptides may be effective for treatment of various human tumors which secrete angiogenin.
Highlights
Angiogenin is the tumor-derived potent angiogenic factor (1)
Design of Anti-angiogenin Peptides, chANG and chGNA—We designed two peptides that are complementary to the receptorbinding site of human angiogenin in the 5Ј 3 3Ј and 3Ј 3 5Ј directions using hydropathic complementary approach and designated as chANG and chGNA, respectively. chANG peptide is composed of 11 amino acids due to the presence of a termination codon at 12th position and has the following sequence: NH2-Val-Phe-Ser-ValArg-Val-Ser-Ile-Leu-Val-Phe-COOH. chGNA peptide is composed of 13 amino acids with the following sequence: NH2-LeuLeu-Phe-Leu-Pro-Leu-Gly-Val-Ser-Leu-Leu-Asp-Ser-COOH
Our goal in this work was development of the peptide antagonists of angiogenin using hydropathic complementary approach (15, 16). chANG and chGNA peptides, which are antipathic to the receptor-binding site of angiogenin, interact directly with angiogenin with somewhat high affinity (Kd Ϸ 44 nM) and show anti-angiogenin activities without any effect on the ribonucleolytic activity of angiogenin
Summary
Peptide Synthesis—chANG and chGNA peptides with the sequence of NH2-Val-Phe-Ser-Val-Arg-Val-Ser-Ile-Leu-Val-Phe-COOH and NH2Leu-Leu-Phe-Leu-Pro-Leu-Gly-Val-Ser-Leu-Leu-Asp-Ser-COOH, respectively, were synthesized and purified by Biosynthesis, Inc. (Lewisville, TX). Binding of Biotinylated Actin to Angiogenin—An ELISA plate (96 well, Maxisorb, Nunc) was coated with 50 ng of human angiogenin and blocked with 0.1% gelatin in PBS. An ELISA plate (96 well, Maxisorb, Nunc) was coated with recombinant human angiogenin (carrier-free, R & D Systems) or bovine pancreatic RNase A (Sigma) in 0.1 ml of PBS by incubation overnight at 4 °C. After 2-h incubation at 37 °C, the plates were washed three times with 0.2 ml of PBS, and the adhered cells were fixed and stained with methylene blue (24). Following incubation for 3 additional days, the number of viable cells was determined using cell titer 96 non-radioactive proliferation assay kit (Promega) according to the manufacturer’s instructions. Each experiment was carried out in triplicate, and the average value was used to determine percent viability
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