1. Amino acids and peptides. XXIX. Synthesis and antimetastatic effects of peptides and peptide-poly(ethylene glycol) hybrids related to the core sequence of the type III connecting segment domain of fibronectin
K Kawasaki, M Maeda, S Inoue, Y Yamashiro, Y Kaneda, Y Mu, Y Tsutsumi, S Nakagawa, T Mayumi Biol Pharm Bull. 1996 Dec;19(12):1574-9. doi: 10.1248/bpb.19.1574.
Peptides (H-Glu-Ile-Leu-Asp-Val-NH2, H-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr-NH2, H-Arg-Glu-Asp-Val-NH2) and their poly(ethylene glycol) (PEG) hybrids related to the core sequence of the type III connecting segment domain of fibronectin A chain were prepared by the solution method or the solid phase method. Their inhibitory effects on the adhesion and migration of B16-BL6 melanoma cells to fibronectin were assessed in vitro, and their therapeutic potency against tumor metastasis were also examined. Anti-adhesive and anti-migrative effects of the synthetic fibronectin-related peptids were superior to those of their PEG hybrids, so we found that the in vitro bioactivity of peptides decreased by PEGylation. In the in vivo assay, we found that the synthetic peptides containing Glu-Ile-Leu-Asp-Val and Arg-Glu-Asp-Val sequences exhibited an inhibitory effect on the experimental metastasis of B16-BL6 melanoma. Of the synthetic peptides, H-Glu-Ile-Leu-Asp-Val-NH2 exhibited the most potent inhibitory effect. Hybrid formation of Arg-Glu-Asp-Val with poly(ethylene glycol) resulted in potentiation of the inhibitory effect of the parent peptides. A mixture composed of PEG hybrids of Glu-Ile-Leu-Asp-Val, Arg-Glu-Asp-Val and Tyr-Ile-Gly-Ser-Arg dramatically inhibited tumor metastasis.
2. Two integrin-binding peptides abrogate T cell-mediated immune responses in vivo
T A Ferguson, H Mizutani, T S Kupper Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8072-6. doi: 10.1073/pnas.88.18.8072.
Two VLA proteins (or beta 1 integrins; originally called very late activation antigens) that bind to distinct determinants on fibronectin (FN) are increased on activated immune or memory T cells. VLA-4 binds to the peptide sequence Gly-Pro-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr (GPEILDVPST in single-letter code) on the alternatively spliced CS-1 form of FN, whereas VLA-5 binds to an Arg-Gly-Asp sequence found on all forms of FN. It has been proposed that the migration of immune T cells out of blood vessels and through connective tissue to a site of antigenic challenge is facilitated by the interaction of such integrins with matrix protein molecules. We have examined directly the role of T-cell integrins in vivo by using the well-characterized, T-cell-mediated contact hypersensitivity (CHS) response to the hapten trinitrochlorobenzene (TNCB). We demonstrate that the cells that transfer CHS to TNCB adhere to FN in the presence of Ca2+/Mg2+, and T-cell populations depleted of FN-adherent cells do not transfer immunity. We further show that TNCB-immune T cells treated with the synthetic peptides GPEILDVPST or Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP in single-letter code), ligands for VLA-4 and VLA-5, respectively, lose their ability to mediate this immune response in a murine model, whereas the control peptides Val-Ile-Pro-Asp-Leu-Thr-Glu-Ser-Pro-Gly and Gly-Arg-Gly-Glu-Ser-Pro have no effect. Neither GPEILDVPST nor GRGDSP significantly inhibited the proliferative response of TNCB-immune T cells in vitro. These data suggest that FN-binding integrins on T cells play a role in the localization of T cells to sites of antigenic challenge in tissue.
3. Antimetastatic effect of synthetic Glu-Ile-Leu-Asp-Val peptide derivatives containing D-amino acids
Y Kaneda, Y Yamamoto, N Okada, Y Tsutsuml, S Nakagawa, M Kakiuch, M Maeda, K Kawasaki, T Mayumi Anticancer Drugs. 1997 Aug;8(7):702-7. doi: 10.1097/00001813-199708000-00009.
The aim of this study was to increase the antimetastatic potency of the fibronectin-related peptide, Glu-Ile-Leu-Asp-Val (EILDV), and to determine the minimal core sequence of EILDV required to inhibit tumor metastasis in vivo. The EILDV subpeptide analog, ILDV, markedly inhibited the adhesion of B16-BL6 melanoma cells to fibronectin. EILD and ILD were only slightly inhibitory, and the smaller overlapping tripeptide, LDV, was inactive. The inhibitory activities of ILDV and LDV on the migration of B16-BL6 melanoma cells were as potent as those of EILDV, whereas ILD did not inhibit cell migration. These results suggested that the minimal sequences essential for cell adhesion and migration are ILD and LDV, respectively. However, the antimetastatic effects of all subpeptide analogs were lower than that of EILDV. In order to improve the stability in vivo, we synthesized various EILDV-related peptides substituted with a D-amino acid. EILDV containing D-Glu or D-Ile inhibited cell adhesion and migration as potent as EILDV, whereas replacing Leu, Asp or Val with the corresponding D-isomer reduced the antiadhesive activities. The inhibitory effect of EILDV-related peptides containing D-Leu, D-Asp or D-Val on migration was also lower than that of EILDV. All synthetic EILDV-related peptides containing D-amino acids inhibited metastasis by B16-BL6 melanoma cells to the same extent as EILDV, whereas the specific activity of EILDV was decreased by the D-amino acid substitution. These results indicated that the balance of stability in vivo and biological activity in vitro is important in inhibiting tumor metastasis.
