See the Supporting Materials and Methods for details regarding DNA sequencing, HGF immunoassay, flow cytometry, and cell viability analysis. The Student t test was used to compare data between two groups. Analysis of variance was used to evaluate Selleckchem DAPT the difference among multiple groups. A prior report demonstrated that MHCC97-L and MHCC97-H cell lines have low and high metastatic

potential, respectively.25 Given the current hypothesis proposed by Thiery31 and Bernards and Weinberg32 that links a mesenchymal phenotype to metastasis, we investigated whether metastatic HCC cells have mesenchymal features in comparison with nonmetastatic Huh7 and Hep3B cells.33 In terms of morphology, MHCC97-L and MHCC97-H cells demonstrated a fibroblast-like appearance, whereas Huh7 and Hep3B cells displayed a cobblestone appearance (Fig. 1A). In terms of gene expression, MHCC97-L and MHCC97-H cells demonstrate low E-cadherin expression, consistent with a mesenchymal phenotype, and high expression of

E-cadherin repressor Zeb2 compared with Huh7 and Hep3B cells (Fig. 1B). There was no significant difference in expression of Snail, Twist, and Zeb1 between the four cell lines (data not shown). Protein expression confirmed a mesenchymal phenotype in MHCC97-L and MHCC97-H cells, with decreased E-cadherin expression and increased fibronectin expression (Fig. 1C). The mesenchymal phenotype of MHCC97-L and MHCC97-H cells correlates with strong expression and constitutive phosphorylation of c-Met (Fig. 1B,C). Several published reports have indicated that PLX3397 cost mutations in the c-Met gene correlate with activation in multiple different cancers.20-22 Therefore, we investigated whether the activation of c-Met in MHCC97-L and MHCC97-H cells was due to a mutation. Sequencing of the c-Met gene demonstrated none of the reported

mutations, as identified through alignment analysis (Supporting Materials and Methods, data not shown). Because sequencing demonstrated no previously reported mutation, we hypothesized that the activation was due to autocrine secretion of HGF. MCE公司 An analysis of secreted proteins in conditioned media failed to demonstrate any HGF secreted by MHCC97-L and MHCC97-H cells (Supporting Materials and Methods, data not shown). Thus, the precise mechanism of c-Met activation in MHCC97-L and MHCC97-H cells remains unknown. Constitutive activation or HGF-stimulated tyrosine phosphorylation of c-Met is blocked by PHA665752, a small molecular compound that functions as a selective inhibitor of c-Met phosphorylation26 in gastric, lung, and pancreatic cancer cells.27 Using PHA665752, we investigated the effect of tyrosine kinase inhibition on the activation of c-Met and downstream signaling pathways in human HCC. As shown in Fig. 2, PHA665752 treatment eliminated c-Met phosphorylation at multiple tyrosine residues (Y1234/Y1234 and Y1349) and reduced downstream phosphorylation of Akt and Erk (P44/42) in c-Met–positive MHCC97-L and MHCC97-H cells.