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The invasion of malignant cells through the basement membrane is a critical step in local infiltration and metastasis. Adhesion and invasion of malignant cells may be modulated by their receptor mediated binding to the basement membrane glycoprotein-laminin. Laminin consists of the sequences with anticancer and antimetastatic activity. Its peptide fragment YIGSR is known to inhibit the experimental metastases of several tumors. This sequence and a tripeptide RGD of laminin inhibits both angiogenesis and tumor growth. In contrary, the sequence SIKVAV initiates angiogenesis and tumor progression. Moreover, laminin plays also other functions in human organism. One of them is the influence on platelet aggregation and thrombogenesis.

Both the laminin composition of the basement membrane and the keratin intermediate filament composition of the epithelial cell differs between cornea and conjunctiva, suggesting that at least some aspects of ocular surface epithelial cell differentiation may be regulated by extracellular matrix. The purpose of this study was to analyse the role of beta1 integrin in intracellular signaling pathways in human conjunctival epithelial cells adherent to laminin. In addition, the purpose was to compare the phosphorylation kinetics of signaling intermediates in cells adherent to different laminin isoforms. Cell adhesion assays, integrin clustering experiments, and integrin function blocking experiments demonstrated that beta1 but not beta4 integrin mediated human conjunctival epithelial cell adhesion to placental laminin isoforms (laminin-10/11) and induced focal adhesion kinase (FAK) tyrosine phosphorylation. Western blot analysis of cell lysates adherent to placental laminin showed that the tyrosine phosphorylation of p130Cas and FAK was maximally above constitutive levels after 60 min. In cells adherent to EHS laminin (laminin-1), the tyrosine phosphorylation kinetics of tensin, p130Cas, FAK and unknown proteins of 138 kDa and 110 kDa were similar, and peaked above constitutive levels after 30 min. Tyrosine phosphorylation of a 70 kDa protein was induced by cell adhesion to EHS laminin after 5 min, and phosphorylation peaked at 15 min. In contrast, the tyrosine phosphorylation of the 70 kDa protein was undetected in cells adherent to placental laminin. Erk-1 phosphorylation and activation was not differentially modulated by conjunctival epithelial cell adhesion to laminins. However, phosphorylation and activation kinetics of Erk-2 in cells adherent to placental laminin was similar to that observed for FAK and p130Cas. Erk-2 phosphorylation and activation was essentially undetectable in cells adherent to EHS laminin. These observations suggest that human conjunctival epithelial cell adhesion to different laminin isoforms activates different intracellular signaling pathways, and provides support for the hypothesis that extracellular matrix molecules can modulate ocular surface epithelial cell differentiation via alternate signaling pathways.

Laminin, a cell adhesion protein, consists of three peptide chains (alpha-1, beta-1 and gamma-1). The beta-1 chain contains a Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence that has been found to inhibit experimental metastasis in mice. We have prepared a hybrid of a water-soluble chitosan and a laminin-related peptide, and have examined its inhibitory effect on experimental metastasis in mice. A laminin-related peptide, acetyl-Tyr-Ile-Gly-Ser-Arg-betaAla-OH (Ac-YIGSRbetaA-OH), was prepared by a solid-phase method. Ac-YIGSRbetaA-OH was then reacted with a water-soluble chitosan. BetaAla is a spacer and was placed to avoid racemization of the Arg residue when the peptide was coupled with chitosan. Although chitosan has amino groups, they did not react with the peptide. Four methods were tried to achieve a coupling reaction, the diphenylphosphoryl azide method, the diisopropylcarbodiimide/1-hydroxybenzotriazole method, the water-soluble carbodiimide (WSC), and the 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) method, but all four methods were unsuccessful. Therefore, a small spacer, tert-butyloxycarbonyl-Gly, was intercalated in chitosan, by the TBTU method, to facilitate its coupling with the peptide. After removal of the protecting group, the Gly-chitosan was coupled with Ac-YIGSRbetaA-OH by the water-soluble carbodiimide method to give Ac-YIGSRbetaAG-chitosan. Conjugation of the peptide with the larger chitosan molecule did not reduce the inhibitory effect of the peptide on experimental metastasis in mice, it actually potentiated the antimetastatic effect, demonstrating that chitosan may be effective as a drug carrier for peptides.

The structure of laminin peptide 11 (CDPGYIGSR-NH2) contains valuable information for the design of mimetic compounds with anti-invasive and anti-metastatic properties. An alanine scan replacement experiment identified Tyr5, Ile6 and Arg9 residues as contributing significantly to anti-invasive activity. Circular dichroism spectra and NMR alphaH chemical shift values both supported the existence of populations of nonrandom coil solution structures for the analogs tested. A D-Ala4 for Gly4 substituted analog completely lost activity, while an L-Ala4 for Gly4 substituted analog retained half the activity of the parent peptide. These results complement our previous findings with D/L alanine substitutions at the Gly7 position, and together they suggest an 'S'-shaped backbone as likely for the active peptide conformation. NMR-constrained molecular modeling supported a direct involvement of the Tyr5 and Ile6 sidechains in conferring bioactivity, and indicated that the Tyr5 sidechain was buried in the Ala2 for Asp2 substitution. Based on the fact that the peptide 11 sequence derives from the disulfide bonded c-loop of an LE-repeat, we synthesized the cyclic CDPGYIGSRC-NH2 peptide. This analog exhibited good anti-invasive and anti-metastatic activity. NMR modeling experiments suggested that the trans-proline cyclic peptide, would favor an 'S'-shaped backbone conformation. Full retro-inverso analogs of peptide 11 were shown to have anti-invasive activity inferior to that of peptide 11. This weak bioactivity was probed using NMR-constrained molecular dynamics, and revealed potential conformations which limited the ability of the required sidechains to mimic the positions of those in the native peptide conformations.

This paper describes the synthesis and biological evaluation of six partial retro-inverso peptidomimetic analogs of YIGSR-NH2, a synthetic peptide from the beta 1 chain of laminin, which has antimetastatic activity. The intent was to improve the antimetastatic potency of YIGSR-NH2 by limiting the in vivo enzymatic degradation through the incorporation of fraudulent peptide bonds. We have prepared the following retro-inverso peptides, Tyr-Ile-Gly-Ser-gArg-CHO (1), Tyr-gIle-mGly-Ser-Arg-NH2 (2), Tyr-gIle-mGly-Ser-gArg-CHO (3), gTyr-D-rIle-mGly-Ser-Arg-NH2 (4), Tyr-Ile-Gly-gSer-D-rArg-CHO (5) and Tyr-gIle-rGly-D-rSer-D-rArg-CHO (6). In vitro assays for B16F10 melanoma cell adhesion showed no significant activity for these six peptides. Peptides 1-3, 5 and 6 were further tested, in vivo, for their ability to inhibit tumor metastases to the lung in mice injected in the tail vein with B16F10 melanoma cells. All five of the retro-inverso peptides tested showed statistically significant inhibition of metastasis, but the most active peptides were 5 and 6, which showed 57 and 69% inhibition of metastasis, respectively.

Laminin-related peptide poly(ethylene glycol) hybrid, Tyr-Ile-Gly-Ser-Arg-aminopoly(ethylene glycol) was prepared by the solution method and carboxylated poly(ethylene glycol)-Tyr-Ile-Gly-Ser-Arg was prepared by the solid phase method. Their inhibitory effects on experimental tumor metastasis were examined in mice. The inhibitory effect of Tyr-Ile-Gly-Ser-Arg was significantly potentiated by hybrid formation with poly(ethylene glycol) either at amino- or carboxyl terminals of the peptide. Of the hybrids, Tyr-Ile-Gly-Ser-Arg-amino(polyethylene glycol) #6000 hybrid exhibited about 10 times more potent anti-metastatic effect than the peptide itself. The inhibitory effect of a mixture of the carboxylated poly(ethylene glycol) hybrid and Arg-Gly-Asp-aminopoly(ethylene glycol) hybrid also exhibited an inhibitory effect.

This paper describes our efforts to study structure-activity relationships, improve the antimetastatic potency, and limit the in vivo enzymatic degradation of YIGSR-NH2, a synthetic peptide from the B1 chain of laminin, which reportedly has potential as an antimetastatic agent. To this end we have synthesized a series of psi (CH2NH) peptide analogs (5-9) of YIGSR-NH2 and a number of peptides in which the Tyr residue was replaced with D-Tyr (1), Phe (2), Phe (p-F) (3), and Phe(p-NH2) (4). All new peptides were assayed in vitro for their ability to promote cell attachment in both B16F10 mouse melanoma cells and HT-1080 human fibrosarcoma cells. On the basis of the in vitro assay results, peptides 3-5, 8, and 9 were tested in vivo for their ability to inhibit tumor metastasis to the lungs in mice that were coinjected in the tail vein with B16F10 melanoma cells and 1 mg of peptide. In summary, of the nine new peptides only the Phe(p-NH2) peptide 4 showed consistent in vitro cell attachment activity, but only low in vivo antimetastatic activity.

Rat ascites hepatoma AH7974 cells strongly expressed antilaminin antibody-reactive substances (laminin-like substances) and Griffonia simplicifolia isolectin B4 (GS)-reactive carbohydrate (alpha-D-galactose; alpha-Gal) on their cell surface. The alpha-Gal expression was not apparently influenced by the pretreatment of cells with methanol. The cell membrane laminin-like substances has approximate molecular weights of 150, 62 and 56 kDa in denaturating reducing conditions, of which the 62 and 56 kDa bands were stained with GS. The cell membrane molecules bearing alpha-Gal were 62 and 56 kDa and were stained with antilaminin antibody. Therefore, the major molecules bearing alpha-Gal residues of AH7974 cell membrane are considered to be laminin-like substances. To determine the role of the substances in metastasis, we selected four cell lines (74AD, 74AD-f, 74FL, 74FL-a) from AH7974 in culture. 74AD and 74FL-a are adherent lines and 74AD-f and 74FL are floating lines. All of these cell lines strongly expressed laminin-like substances, but a marked difference was found in expression of alpha-Gal, which was most strongly expressed by 74FL, followed by 74AD, and rarely by 74AD-f and 74FL-a; the staining intensity was positively correlated with their experimental lung-colonizing potential. Cell membrane laminin-like substances were 200, 97, 62, 56 and 46 kDa and among them 62 and 56 kDa molecules were glycosylated with alpha-Gal. The pretreatment of 74FL cells with antilaminin antibody or with human type A serum (containing natural antibody to alpha-Gal epitope) depressed remarkably the lung-colonizing potential of the cells. These results suggest that the expression of 62 and 56 kDa laminin-like substances with alpha-Gal residues on tumor cell surfaces is one of the determinants associated with lung-colonizing potential of these cells.

Laminin-related peptides, Tyr-Ile-Gly-Ser-Arg analogs, were prepared and their inhibitory effects on experimental metastasis were examined. Of the amino acids in the Tyr-Ile-Gly-Ser-Arg sequence, L-Arg was very important and Ile was not essential for the inhibitory effect. To obtain a potent inhibitor of metastasis, hybrids of Tyr-Ile-Gly-Ser-Arg-Gly and 2 types of poly(ethylene glycol) were prepared. The inhibitory effects of the hybrids were more potent than that of Tyr-Ile-Gly-Ser-Arg-Gly.

The locomotion of murine myoblasts over the extracellular matrix components laminin and fibronectin was analyzed using quantitative videomicroscopy, and the organization of the cytoskeleton was observed in parallel immunofluorescence studies. Cells plated on the laminin-nidogen complex locomoted twice as fast as on laminin alone. The main form of translocation on laminin was a jerky cycle of prolonged lamellipod extension followed by rapid (approximately 200- less than 500 microh h-1) movement of the cell body into the extended lamellipod. The locomotion-stimulating activity of laminin resides in the elastase digestion fragment E8, part of the laminin long arm, while the E1-4 fragment containing the three short arms is inactive. Myoblasts moved poorly over fibronectin irrespective of whether high, intermediate, or low coating concentrations were used (approximately 5,000- approximately 10 fmol cm-2). In contrast, the locomotory responses both to laminin and to E8 peaked sharply at coating concentrations approximately 20-50 fmol cm-2 and decreased at higher concentrations. This response corresponds to that expected for a haptotactic stimulant. When cells locomoted over a mixed substrate of laminin and fibronectin, the fibronectin effects appeared to predominate. The cytoskeleton has been implicated in many cellular motile processes. Within 6 h on fibronectin many cells expressed vinculin-containing focal contacts, elaborated stress fibers and had periodically organized alpha actinin, whereas on laminin, most cells showed diffuse vinculin and alpha actinin and a fine meshlike actin cytoskeleton. We conclude that the poor locomotion of cells over fibronectin is because of the cytoskeletal stabilization it induces.