Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1

Figure 1 Immunofluorescent study in HuCCA-1 cells shows positive staining to human CK-19 mAb (A) and negative staining to SMA mAb (B) (40×magnification).

Figure 2 mRNA expression level of HGF (A) and c-Met (B) was observed by RT-PCR technique. HuCCA-1 (CCA) expressed low HGF but a high level of c-Met mRNA. Liver myofibroblasts (MF) and hepatocellular carcinoma cell line (HepG2) were used as positive controls for HGF and c-Met, respectively. Equal amount of total RNA from each cell was confirmed by GAPDH (C).

Figure 3 Cell proliferation assay showed significantly increased proliferating cell number after 10 ng/mL rhHGF treatment in HuCCA-1 cells for 24 and 48 h (A). Invasion assay also showed significantly increased invading cells after 24 and 48 h treatment of 20 ng/mL rhHGF (B). Data are expressed as mean±SE, ^aP < 0.05 and ^bP < 0.01, compared to the HGF-untreated cells at the corresponding time.

Figure 4 Treatment of HuCCA-1 cells with 20 ng/mL rhHGF for 0, 5, 15, 30, 60, and 120 min showed increased degree of FAK phosphorylation in a time-dependent manner. The maximal level of FAK phosphorylation was reached at 60 min and then sharply decreased at 120 min (A). Equal amount of FAK protein used in each time point was confirmed by reprobing the membrane with anti-FAK antibody (B). The density of bands was measured and pFAK/FAK value was plotted in a graph (C).

Figure 5 Treatment of HuCCA-1 cells with 20 ng/mL rhHGF for 0, 5, 15, 30, 60, and 120 min showed an increased degree of Src phosphorylation in a time-dependent manner. The maximal level of Src phosphorylation was reached at 30 min (A). Equal amount of Src protein (upper bands) used in each time point was confirmed by reprobing the membrane with anti-Src antibody (B). The density of bands was measured and pSrc/Src value was plotted in a graph (C).

Figure 6 The interaction between Src and FAK was demonstrated in HGF-treated HuCCA-1 cells by co-immunoprecipitation at varied time points. The resulting bands showed a positive correlation of FAK with Src phosphorylation, which reached a maximum at 30 min and then declined (A). The amount of Src protein (upper bands) used in each time point was confirmed (B). The density of bands was measured and the ratio between FAK and Src was plotted in the graph (C).

Figure 7 Treatment of cells with 0. 1, 0.5, and 1.0 µmol/L of AZM555130 for 48 h showed no inhibition of Src phosphorylation both in HGF-treated and untreated cells (A). The HGF-induced FAK phosphorylation was not inhibited after 48 h treatment of0.1 µmol/L AZM555130 but showed 34% and 63% inhibition by 0.5 and 1.0 µmol/L treatment, respectively. The 37% inhibition of FAK phosphorylation was observed by treatment of 1.0 µmol/L AZM555130 in non-induced cells (B). These values were compared with the levels of Src and FAK phosphorylation in AZM555130-untreated cells which is equal to 100%.

Figure 8 The HGF-mediated cell proliferation was not inhibited by 0. 1 µmol/L AZM555130 but was significantly inhibited by 0.5 and 1.0 µmol/L of AZM555130 for 25% and 34%, respectively. Without HGF, cell proliferation was inhibited by 0.1, 0.5, and 1.0 µmol/L of AZM555130 for 9%, 33%, and 41%, respectively. All values are mean±SE, ^aP < 0.05 and ^bP < 0.01, compared to the control in HGF-untreated cells; ^dP < 0.01, compared to the control in HGF-treated cells.

Figure 9 The HGF-mediated invasion of HuCCA-1 cells was significantly decreased by 0. 1 and 1.0 µmol/L of AZM555130 for 32% and 85%, respectively. Without HGF, cell invasion was also significantly decreased by 0.1 and 1.0 µmol/L of AZM555130 for 23% and 98%, respectively. All values are mean±SE, ^bP < 0.01, compared to the control in HGF-untreated cells; ^dP < 0.01, compared to the control in HGF-treated cells.