As breast cancer cells acquire a motile phenotype, this is translated into changes

in highly dynamic structures like actin learn more filaments and cytoplasmic microtubular complex [34]. We decided to investigate the effects on motility of over-expression or knockdown of Claudin-5. To achieve this, an in vitro motility assay and a traditional wound healing assay was carried out, both revealing that MDACL5rib2 showed a reduction in motility. Moreover, ECIS was used in order to measure in real time how fast cells migrate after wounding. Similar results were obtained; MDACL5rib2 was indeed slower when compared to the control. However, MDACl5exp cells were the fastest in each of the assays mentioned above. Until now, we have shown that knockdown of Claudin-5 expression in a breast cancer cell line see more resulted in a less adhesive and less motile cell phenotype when compared to controls. The opposite was seen when Claudin-5 expression was forced, resulting in a more adhesive and more motile phenotype but with no differences in invasiveness in vivo and in vitro. We might tentatively conclude from this that Claudin-5 might be a motility regulator, or at least XAV-939 cost have a role in the motility of these human breast cancer cells. Previously, we have carried out a significant body of work on the role and effect of HGF in epithelial

cancer cells. HGF is a powerful motogen able to promote proliferation, invasion, and migration of epithelial cells by binding to its tyrosine kinase receptor c-met [35] as well as modulating expression and function of TJ molecules in human breast cancer cell lines and decreasing trans-epithelial resistance

[21]. Cells displaying enhanced or suppressed expression of Claudin-5 respond in keeping with the well established effect after treatment with HGF, showing reduced epithelial resistance and increased motility. ECIS experiments corroborated these results. It is interesting that claudin-7 expressing human lung cancer cells have been shown to have a reduced response to HGF, are less motile, and form fewer foot processes than untreated cells. In addition, cells transfected with claudin-7 dramatically decreased their invasive PLEKHM2 ability after HGF treatment. It has been shown that this is mediated through the MAPK signalling pathway since the phosphorylation level of ERK1/2 was significantly lower in claudin-7 transfected cells than in control cells [36]. To address the possibility that Claudin-5 might play a role in regulating cell motility, different motility-regulators were studied in order to search for any possible links between Claudin-5 and a range of motility-related proteins. Cell motility was analysed using ECIS after being treated with different motility inhibitors. In particular the N-WASP inhibitor (Wiskostatin) and the ROCK inhibitor (Y-27632) responded in an unexpected way in our transfected cells.