Immunoprecipitation of Claudin-5 followed by immunoblotting with N-WASP and ROCK 1 was used in order to investigate a possible interaction between Claudin-5 and N-WASP as well as with ROCK 1. Results showed a protein-protein interaction between Claudin-5 and these motility-related proteins in MDA-MB-231pEF6 and MDA-MB-231Cl5exp (Figure 7b, negative controls shown below). In keeping with this, immunoprecipitation with either N-WASP (Figure 7c) or ROCK1 (Figure 7d) followed by immunoblotting with

Claudin-5 produced consistent results. Discussion In this present study, we used cells transfected with Claudin-5 expression sequence and ribozyme transgenes to assess the impact of reducing the expression of our protein of interest as well as enhancing it in order to evaluate changes in the aggressive nature of MDA-MB-231 breast Selisistat order cancer cells. We also demonstrated for the first time that there is a link between Claudin-5 and cell motility. DMXAA in vivo The disruption of the Tight Junction (TJ) structure is a common feature of many human cancer cells. Downregulation of different TJ proteins has been linked with staging and metastatic potential in various cancers including selleck chemicals llc breast [28]. Indeed, in human breast cancer, tumour tissues show truncated and/or variant

signals for occludin. Knockdown of occludin resulted in increased invasion, reduced adhesion and significantly reduced TJ functions, whilst Q-RT-PCR showed occludin to be significantly decreased in patients with metastatic disease [29]. This loss of or aberrant expression has clear repercussions as to the importance of Thalidomide occludin in maintaining TJ integrity in breast tissues and could play a part in breast cancer development. In addition, in vivo and in vitro data has revealed that over-expression of TJ proteins

in cancer cells, such as Claudin-4, leads to a decrease in invasiveness and metastases in animal models [29]. Similar conclusions were found when cells breast cancer cells overexpressing Claudin-16, showed a decrease in invasiveness and motility [26]. Since claudin-18 is overexpressed in precursor lesion PanIN and pancreatic duct carcinoma, it serves as a diagnostic marker and a target of immunotherapy [30]. The upregulation of claudin-18 by TPA in human pancreatic cancer cell lines can be prevented by inhibitors of PKCδ, PKCϵ, and PKCα, whereas the upregulation of claudin-18 by TPA in hTERT-HPDE cells is prevented by inhibitors of PKCδ, PKCθ, and PKCα. This suggests that in human pancreatic cancer cells claudin-18 is primarily regulated at the transcriptional level via specific PKC signaling pathways and modified by DNA methylation [30]. These studies have provided promising evidence that TJ proteins might serve as useful molecular targets in the prognosis of cancer. In prostate, claudin-4 was down-regulated and claudins-2, -3, and -5 were overexpressed in prostate adenocarcinomas compared with benign prostatic hyperplasia samples.