The following primary antibodies were used: anti-thrombin (clone 12, BD Biosciences, Franklin Lakes, NJ), anti-OPN (R&D Systems, Minneapolis, MN), anti-FAK, Phospho-FAK (Tyr397), or anti-GAPDH (Cell Signal

Tech, Danvers, MA). Values are expressed as the mean ± standard deviation. P < 0.05 was considered statistically significant. Both the mRNA and protein expression levels of thrombin in six established HCC cell lines were found to be significantly increased in comparison to nontransformed hepatic cell lines (L-02 and Chang liver cell U0126 price lines) detected by real-time PCR (Fig. 1A) and western blot (Fig. 1B). Among these HCC cell lines, HepG2, PLC, and MHCC97-L cells had relatively lower invasive and metastatic capabilities (low-metastatic group), whereas MHCC97-H, HCCLM3, and HCCLM6 had higher invasive and metastatic potential (high-metastatic group). Both the mRNA and protein

Belnacasan order expression levels of thrombin in these three high-metastatic HCC cell lines were much higher than those of the three low-metastatic HCC cell lines (P < 0.01, Fig. 1A,B). These data indicate that expression of thrombin is up-regulated in HCC cell lines, and its increased expression is positively correlated with the malignant phenotype of HCC cells. The mRNA expression levels of thrombin were evaluated in 72 human HCC tumors, in their adjacent nontumor liver tissues, and 20 normal liver tissues. The amount of thrombin mRNA was significantly increased in HCC tissues (2-&Dgr;Ct, 46.4 ± 23.5) compared with their adjacent nontumor liver tissues (19.6 ± 8.1, P = 0.005; Fig. 2A, Supporting Information PDK4 Table S2). Moreover, the higher amount of thrombin mRNA in HCC tissue was significantly positively correlated with tumor recurrence (P = 0.037; Fig. 2A). These findings were confirmed by elevated thrombin protein levels in eight out of the above 72 HCC tissue samples using western blot analyses (Fig. 2B). The mRNA level of OPN in HCC tissues (2-&Dgr;Ct,

137.4 ± 69.1) were also significantly higher than that in nontumor liver tissues (2-ΔCt, 22.2 ± 7.6, P = 0.043) and normal liver tissues (19.9 ± 8.7) (Supporting Information Table S2). A scatterplot of thrombin and OPN expression revealed no correlation between thrombin and OPN mRNA levels in HCC tissues (r = −0.17, P = 0.15) (Supporting Information Fig. S1). To determine whether thrombin influences OPN-related prognosis, we divided the HCC patients into low-OPN and high-OPN groups using the median OPN expression level as the cutoff. As shown in Fig. 2C, among the HCC tissues in the high-OPN group the expression level of thrombin was significantly increased in HCCs from the patients with tumor recurrence compared with those without recurrence (P = 0.027). Importantly, this difference was not statistically significant in the low-OPN group (P = 0.457).