The Role of Claudin- 5 and the Paracellular Barrier Function in Endothelial Cells during Invasion of Breast Cancer Cells.

Abstract

Abstract Material and Methods: Human endothelial (HECV) and breast cancer cells (MDA-MB-231) were used to assess the effect of over-expression (HECVCL-5, MDACL-5) or knockdown (HECVDCL-5, MDADCL-5) of Claudin-5. Cell adhesion and growth were evaluated using in vitro assays. An in vivo tumour model using athymic nude mice was used to assess growth of tumours. Transendothelial and transepithelial resistance (TER) was employed to measure changes in TJ function. Cellular motility was analyzed using Cytodex-2 bead motility assay. ECIS (electrical cell impedance sensing) was used to assess the attachment and migration of the cells.Results: There were no significant differences in in vitro growth of cells that over-expressed or had knockdown of Claudin-5 (both HECV and MDA-MB-231 cells). Expression of CL5 in both HECV and MDA-MB-231 did not have a significant impact on in vivo growth. There were however, significant differences in adhesion between control cells (HEVCpEF6) and HECVCL-5, HECVDCL-5 (changes in adhesion: HEVC pEF6 vs. HECVCL-, P>0.05; HECVDCL-5, P=0.046). There was a distinct modulation of TJ function as assessed using TER. HECVDCL-5 cells exhibited increased resistance compared to HEVC pEF6 and HECVCL-5 (change in TER: HEVC pEF6: -151.33±6.11 vs. HECVCL-5 -187±1.73, HECVDCL-5-89.33±2.08, P <0.01). There was no difference in TER in MDA CL-5 and MDADCL-5cells. HGF, a powerful motogen and modulator of TJ function, caused a significant increase in TER in HECVCL-5 and HECVDCL-5, but resulted in a significant decrease in TER in MDA-MB-231 cells. Motility studies revealed that HECVCL-5 and MDA CL-5 after treatment with HGF cells were significantly more motile than cells without treatment (HECVCL-5 vs. HECVCL-5+HGF, P=0.015; MDACL-5 vs. MDACL-5+HGF, P=0.033). There was no difference in motility in HECVDCL-5 cells, however, MDADCL-5 cells were significantly less motile after treatment with HGF (P=0.017). Interestingly, ECIS results showed that the ROCK inhibitor Y27632 inhibited the attachment and migration of HECVCL-5 cells but was no longer able to inhibit attachment and migration of HECVDCL-5 cells.Discussion: These results demonstrate for the first time that Claudin-5 can be modulated to effect changes in cell motility, attachment and TJ function in human endothelial and breast cancer cells. Moreover, there appears to be a link between Claudin-5 expression and cell motility, particularly with respect to the ROCK signalling cascade. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6170.