Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion - PubMed (original) (raw)

Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion

Apaporn Menakongka et al. World J Gastroenterol. 2010.

Abstract

Aim: To investigate the role of hepatocyte growth factor (HGF) in cholangiocarcinoma (CCA) cell invasiveness and the mechanisms underlying such cellular responses.

Methods: Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines, HuCCA-1 and KKU-M213, using Transwell in vitro assay. Levels of proteins of interest and their phosphorylated forms were determined by Western blotting. Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography.

Results: Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line. HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization, but did not affect the levels of secreted matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator, key matrix degrading enzymes involved in cell invasion. Concomitantly, HGF stimulated Akt and extracellular signal-regulated kinase (ERK)1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines. Inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway by the PI3K inhibitor, LY294002, markedly suppressed HGF-stimulated invasion of both CCA cell lines, and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells.

Conclusion: These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type.

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Figures

Figure 1

Figure 1

Steady state level of Met expression in cholangiocarcinoma cell lines and activation by hepatocyte growth factor (HGF). Cell lysates from 80% confluent cells cultured in 10% fetal bovine serum (FBS) medium were examined for Met expression by Western blotting analysis (A). Lysates from HuCCA-1 (B) and KKU-M213 (C) cells treated with or without 50 ng/mL HGF for various times were analyzed by Western blotting for levels of Met and phospho-Met (pY1234/1235). The graphs show band densities of phospho-Met relative to those at zero time points. Data are presented as mean ± SE of results obtained from three independent experiments. a_P_ < 0.05 vs untreated control.

Figure 2

Figure 2

HGF induction of cholangiocarcinoma motility and invasiveness. In vitro invasion and motility assays of HuCCA-1 (A) and KKU-M213 (B) cells were conducted in a Transwell unit coated with and without Matrigel. Cells (105) in serum-free medium were plated in the upper chamber of a Transwell unit and 0-100 ng/mL HGF added to the lower chamber. After 6 h of incubation, cells invading to the lower compartment of the Transwell unit were stained and counted. The numbers of invaded/motile cells are presented as mean ± SE of results obtained from three independent experiments. a_P_ < 0.05, b_P_ < 0.01, vs untreated control.

Figure 3

Figure 3

Effects of HGF on E-cadherin expression and localization. A: cholangiocarcinoma (CCA) cells were treated with HGF for 6 h, then cell lysate was analyzed by Western blotting with anti-E-cadherin and -β-actin monoclonal antibodies; B: After treatment with 0 and 100 ng/mL HGF for 6 h, cells were analyzed by immunofluorescence using anti-E-cadherin antibody and visualized under confocal laser scanning microscopy (60 × objective magnification plus 2 × digital magnification).

Figure 4

Figure 4

Effect of HGF on levels of secreted matrix degrading enzymes from cholangiocarcinoma HuCCA-1 and KKU-M213 cell lines. Cells were treated with various concentrations of HGF (0-100 ng/mL) in serum-free medium for 6 h. Conditioned media were then analyzed for MMP-2 (approximate 65 kDa) and MMP-9 (approximate 85 kDa) gelatinolytic activity by gelatin zymography (A) and for uPA by plasminogen-gelatin zymography (B).

Figure 5

Figure 5

HGF induction of ERK1/2 and Akt phosphorylation in cholangiocarcinoma HuCCA-1 and KKU-M213 cell lines. About 80% confluent cells were treated with 50 ng/mL HGF in serum-free medium for 15, 60, 360 min. Lysates from HuCCA-1 (A) and KKU-M213 (B) cells were assessed for total and phosphorylated forms of ERK1/2 and Akt by Western blotting assay. The graphs showed band densities of phospho-ERK1/2 and phospho-Akt relative to those at zero time points. Data are presented as mean ± SE of results obtained from three independent experiments. a_P_ < 0.05 vs untreated control.

Figure 6

Figure 6

Suppression of HGF-induced cholangiocarcinoma cell invasiveness by PI3-kinase inhibitor, LY294002. HuCCA-1 and KKU-M213 cells were treated with 50 ng/mL HGF in the absence (control) or presence of 10 and 50 μmol/L LY294002 for 6 h, and subsequently Akt phosphorylation was determined by Western blotting (A). In vitro invasion of HuCCA-1 (B) and KKU-M213 (C) cells was evaluated in the absence or presence of HGF with or without 50 μmol/L LY294002. Numbers of invaded cells are presented as mean ± SE of results obtained from three independent experiments. b_P_ < 0.01 vs control.

Figure 7

Figure 7

Suppression of HGF-induced cholangiocarcinoma cell invasiveness by MEK1 inhibitor, U0126. HuCCA-1 and KKU-M213 cells were treated with 50 ng/mL HGF in the absence (control) or presence of 0.1, 1 and 5 μmol/L U0126 for 6 h, and subsequently ERK1/2 phosphorylation was determined by Western blotting (A). In vitro invasion of HuCCA-1 (B) and KKU-M213 (C) cells was evaluated in the absence or presence of HGF with or without 1 and 5 μmol/L U0126. Numbers of invaded cells are presented as mean ± SE of results obtained from three independent experiments. a_P_ < 0.05 and b_P_ < 0.01 vs control.

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