Cell. CRC tissues compared with their normal counterpart tissues and was significantly correlated with lymph node metastasis and poor survival. The overexpression of S100A4 protein was also positively correlated with S100P or Trx\1 protein overexpression in our cohort of CRC tissues. In addition, overexpression of S100P reversed the Trx\1 knockdown\induced inhibition of S100A4 expression, EMT and migration and invasion in SW620 cells. The Rabbit polyclonal to Anillin data suggest that interplay between Trx\1 and S100P promoted CRC EMT as well as migration and invasion by up\regulating S100A4 through AKT activation, thus providing further potential therapeutic targets for suppressing the EMT in metastatic CRC. value of less than .05 was considered statistically significant. 3.?RESULTS 3.1. The expression levels of Trx\1 and S100P influence the EMT phenotype of CRC cells In this study, the CRC cell lines SW480 and SW620 that are derived from main (SW480) and metastatic lesions (SW620) of the same individual were chosen as model systems for studying EMT.23 Protein expression levels were determined by Western\blot assays, and protein levels relative to \actin protein levels were assessed by densitometric analysis. Physique ?Physique1A1A shows that protein levels of S100P, Trx\1, S100A4, vimentin and fibronectin in the SW620 are higher than that seen in SW480 cells, while the level of epithelial marker E\cadherin is lower in SW620 than in SW480 cells. As SW480 cells exhibited lower expressions of Trx\1 and S100P than SW620 cells do, we overexpressed Trx\1 or S100P in SW480 cells by lentiviral\mediated gene transfer. Overexpression of S100P or Trx\1 showed an elongated, mesenchymal morphology as compared to the parental SW480 cells (Physique ?(Figure1B).1B). In contrast, SW620 cells with S100P or Trx\1 knockdown showed a reversed EMT morphology: Myelin Basic Protein (68-82), guinea pig the cells were more epithelial\like as compared to the control cells (Physique ?(Figure1B).1B). In addition, ectopic overexpression of Trx\1 or S100P in SW480 cells resulted in down\regulation of E\cadherin, whereas the expressions of the 2 2 mesenchymal markers vimentin and fibronectin were up\regulated (Figures ?(Figures2A2A and B). On the other hand, knockdown of Trx\1 or S100P in SW620 by shRNA resulted in an increased expression of E\cadherin and decreased expressions of vimentin and fibronectin. In addition, overexpression of Trx\1 or S100P up\regulated the levels of S100A4 and P\AKT in SW480 cells, whereas knockdown of Trx\1 or S100P down\regulated the levels of S100A4 and P\AKT in SW620 cells (Physique ?(Physique2A,B).2A,B). Moreover, the expression of the mesenchymal marker, vimentin, and the epithelial marker, E\cadherin, were examined by immunofluorescence. Immunofluorescent staining showed that E\cadherin expression decreased while vimentin expression increased after the overexpression of Trx\1 or S100P in SW480 cells (Physique ?(Physique2C,D).2C,D). Conversely, knockdown of Trx\1 or S100P in SW620 cells caused an increase in E\cadherin expression and a decrease in vimentin expression (Physique ?(Physique2E,F).2E,F). These results suggested that S100P or Trx\1 could induce EMT in CRC cells. Open in a separate window Physique 1 The expression levels of S100P, Trx\1, S100A4 and EMT\associated proteins in SW480 and SW620 cells. A, S100P, Trx\1, S100A4 and EMT\associated proteins (E\cadherin, vimentin and fibronectin) were examined by Western blotting. \actin was used as the loading control. B, EMT morphological changes induced by S100P or Trx\1. Representative microscopic views of SW480 and SW620 cells were shown. Scale bar, 50 m Open in a separate window Physique 2 Effects of Trx\1 and S100P on epithelialCmesenchymal transition of colorectal carcinoma cells. (A) Western blotting revealed that overexpression of Trx\1 resulted in a decreased expression of epithelial marker E\cadherin and increased expressions of mesenchymal markers (vimentin and fibronectin), S100A4 and phosphorylated AKT (P\AKT) in SW480 cells, whereas knockdown of Trx\1 by shRNA resulted in an increased expression of E\cadherin and decreased expressions of vimentin, fibronectin, S100A4 and P\AKT in SW620 cells. (B) Western blotting showed that overexpression of S100P resulted in a decreased expression of E\cadherin and increased expressions of vimentin, fibronectin, S100A4 and P\AKT in SW480 cells, whereas knockdown of S100P by shRNA resulted in an increased expression of E\cadherin Myelin Basic Protein (68-82), guinea pig and decreased expressions of vimentin, fibronectin, S100A4 and P\AKT in SW620 cells. \Actin Myelin Basic Protein (68-82), guinea pig was used as the loading control. (C) Immunofluorescence staining of Trx\1 overexpression down\regulated E\cadherin expression while up\regulating vimentin expression in SW480 cells. (D) Knockdown of Trx\1 by shRNA up\regulated E\cadherin expression and down\regulated vimentin expression in SW620 cells. (E) S100P.