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 Ca2+-free medium. The homotypic adhesion in the presence or absence of peptide and antibody was determined fluorometrically. The Ca2+-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 Ca2+-deficient pretreatment. The homotypic adhesion was largely dependent on extracellular Ca2+ 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 Ca2+ or the heterotypic adhesion of Molt-3 T cells to Caco-2 monolayers. These results indicate the predominant involvement of E-cadherinmediated cell-cell adhesion in this assay. E-cadherinderived 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.