3A), and the PMNs still caused dyshesion of the cell layer (Table 1). The PMN-mediated dyshesion was greatly reduced in the presence of the protease inhibitor α1-antitrypsin, or peptide substrates of the PMN elastase, or a selective elastase inhibitor, indicating a major contribution of elastase (data summarized in Table 2). As expected, purified PMN elastase also caused a dyshesion of the tumor cells (data summarized in Table 1), which again was reversible, and could be inhibited by high serum concentrations

(data not shown). Pancreas elastase when used in comparable concentrations did not cause a dyshesion of cells after 2 h; only concentrations of more than 5 μg/mL and prolonged Idasanutlin order incubation (up to 4 h) resulted in some dyshesion. A likely target for elastase is the adhesion molecule E-cadherin that is expressed by T3M4 cells as shown by indirect immunofluorescence of confluent cell layers and by flow cytometry of dispersed cells (Figs 3B and C and Fig. 4A and D). Following exposure of T3M4 to PMNs or to PFA-fixed PMMs,

surface expression of E-cadherin was reduced (Fig. 3B). The loss of E-cadherin amounted to 45.9 ± 17.7% (mean ± SD of n = 5). Alpha-1-antitrypsin prevented the PMN-induced loss of E-cadherin, as did the elastase inhibitor or the respective substrate (examples in Fig. 3C). Also isolated PMN elastase caused a reduction of E-cadherin surface expression (example in Fig. 4C). By using an Ab that binds to LDK378 nmr the N-terminus of E-cadherin, the surface expression

was reduced, on average by 33.55 ± 19.2% within 2 h (mean ± SD of n = 7) (example in Fig. 4F). A mAb to E-cadherin that binds to a domain near the membrane showed no differences in binding to T3M4 compared with that of elastase-treated T3M4 cells (data not shown). The flow cytometry forward-sideward scatter image revealed that the majority of T3M4 cells were viable after the elastase Staurosporine solubility dmso treatment. The data so far implied an elastase-mediated loss of E-cadherin from the surface. Indeed, when T3M4 were treated with elastase for 2 h, E-cadherin within the membrane fraction was greatly reduced, but was conserved in the cytoplasm, as shown by western blotting (Fig. 4G). Since the Ab is directed to N-terminal region of the molecule, the data indicate cleavage of E-cadherin. Furthermore, a cleavage product of E-cadherin was detected in cell culture supernatants by ELISA. In untreated cells, a cleavage product concentration of 18.7 pg/mL was detected compared with one of 198.3 pg/mL in the elastase-treated cells (mean of three experiments performed in duplicates; p = 0.017 calculated by ANOVA). E-cadherin was not detectable in supernatants of MiaPaCa-2. Transfection of T3M4 with specific siRNA reduced the E-cadherin surface expression by more than 90% when measured after 48 h (Fig. 5A and B).