TKKK, TGBC24TKB, and HuCCT1 were established from IHCC, and OZ was from EHCC. MKN45 was a gastric cancer cell line that was used as a positive control, because of its high expression of c-Met and phospho-Met (Smolen et al, 2006). selleck All of the cell lines had been derived from Japanese patients. The originally established six CC cell lines, HuCCT1 and MKN45 were maintained in RPMI with 10% bovine serum. TGBC24TKB, TKKK, and OZ were maintained in Dulbecco’s modified Eagle medium with 10% bovine serum. Western blotting Subconfluent cells were lysed at 4��C for 30min using lysis buffer containing 10m Tris-HCl (pH 7.5), 1% Triton X-100, and 150m NaCl with a complete protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphate inhibitor cocktail (Nacalai Tesque, Kyoto, Japan).
The protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Lysates (5��g protein per well) were separated by SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in PBS for 30min and then probed with the following primary antibodies: anti-c-Met (rabbit polyclonal; IBL; 1:1000), anti-phospho-Met (pY1234/1235, rabbit monoclonal, clone D26; Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti-EGFR (mouse monoclonal, clone 31G7; Zymed, South San Francisco, CA, USA; 1:1000), and anti-phospho EGFR (pY1173, rabbit monoclonal, clone 53A5; Cell Signaling Technology) at 4��C overnight. After washing with PBS-Tween 20 (0.
5%), the membranes were re-blocked and then incubated at room temperature for 1h with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibody at a dilution of 1:1000. Following three washes, bands were visualised using the ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd, Buckinghamshire, England). Anti-��-actin (mouse monoclonal; clone AC-15, Sigma, St Louis, MO, USA) was used as a loading control. Statistics Correlations between the results of IHC and clinicopathological factors were determined by Fisher’s exact probability test, except for histopathological classification, which was analysed by ��2-test. Cumulative survival rates and survival curves were calculated by the Kaplan�CMeier method, and log-rank test was performed for the comparison of survival curves between low and high groups defied by c-Met expression level.
The Cox proportional hazards model was used to estimate the hazard ratio and 95% confidence interval of each outcome (tumour death and recurrence). Multivariate analysis was performed for factors selected as risk factors by univariate analysis, except for UICC pT and UICC stage, which are composed of other factors. Cilengitide Correlations between the intensity of c-Met and that of EGFR in IHC or Western blotting were determined by Spearman’s rank correlation. Statistical analysis was done using the Statview 5.