Inhibition of e-cadherin-mediated homotypic adhesion of Caco-2 cells: a novel evaluation assay for peptide activities in modulating cell-cell adhesion.
Transient modulation of E-cadherin-mediated cell-cell adhesion may improve paracellular drug delivery through biological barriers. Therefore, there is a need to develop an efficient method to evaluate cadherin peptides that can modulate the intercellular junctions. The objective of this study was to establish a novel assay to evaluate peptide activity in modulating E-cadherin-mediated homophilic interactions, based on the homotypic adhesion of Caco-2 cells. Fluorescence-labeled Caco-2 single cells were incubated with Caco-2 monolayers that were treated beforehand with Ca(2+)-free medium. The homotypic adhesion in the presence or absence of peptide and antibody was determined fluorometrically. The Ca(2+)-deficient pretreatment dramatically increased the number of single cells bound to the monolayers. Immunofluorescence staining showed that some of E-cadherins became accessible without surfactant-induced permeabilization of Caco-2 cell monolayers after the Ca(2+)-deficient pretreatment. The homotypic adhesion was largely dependent on extracellular Ca(2+) concentrations and significantly inhibited by the presence of anti-E-cadherin monoclonal antibody DECMA-1. In contrast, DECMA-1 did not inhibit E-cadherin-independent adhesion, such as the homotypic adhesion of Caco-2 cells in the absence of Ca(2+) or the heterotypic adhesion of Molt-3 T cells to Caco-2 monolayers. These results indicate the predominant involvement of E-cadherin-mediated cell-cell adhesion in this assay. E-cadherin-derived peptides, which had been shown in our previous studies to inhibit E-cadherin-mediated cell-cell adhesion, significantly inhibited homotypic adhesion in a dose-dependent manner. These results, taken together, suggest that the present assay can be used for evaluation of peptide, protein, or antibody activity in modulating the E-cadherin-mediated homophilic interactions in the context of whole live cells.
Kobayashi
Naoki
N
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, 66049-3729, USA.
Ikesue
Atsutoshi
A
Majumdar
Sumit
S
Siahaan
Teruna J
TJ
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018938:Caco-2 Cells; D015820:Cadherins / Q000276:immunology / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects; D003470:Culture Media; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D015820:Cadherins; D003470:Culture Media; D010455:Peptides