Inhibitory effect of a TGFbeta receptor type-I inhibitor, Ki26894, on invasiveness of scirrhous gastric cancer cells

Abstract

Background: Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-beta (TGF-beta) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-beta receptor (TbetaR) phosphorylation inhibitor on the invasiveness of gastric cancer cells.

Methods: Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TbetaR type I (TbetaR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TbetaR-I. We investigated the expression levels of TbetaR and phospho-Smad2, and the effects of TGF-beta in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells.

Results: TbetaR-I, TbetaR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-beta1 in scirrhous gastric cancer cells. Transforming growth factor-beta1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-beta1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-beta1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-beta1 or Ki26894 treatment.

Conclusion: A TbetaR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.

Figure 1

Expression of TGF-β signalling and inhibitory effect of Ki26894. (A) Expression level of TGF-β receptor and phospho-Smad2. The overexpression of TGF-β receptor type I (TβR-I), type II (TβR-II), and phospho-Smad2 (P-Smad2) was observed in scirrhous gastric cancer cell lines, OCUM-2MLN and OCUM-12, but not in non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74. (B, C) Effects of Ki26894 on TGF-β-induced Smad2 phosphorylation of gastric cancer cells. PANC-1 was used as positive control of Smad2 phosphorylation. Transforming growth factor-β stimulated Smad2 phosphorylation in PANC-1, OCUM-2MLN, and OCUM-12 cells. Total Smad2/3 expression was recognised in all four gastric cancer cell lines and no difference in expression level was found on addition of TGF-β1 or Ki26894. Transforming growth factor-β1 (10 ng ml⁻¹) stimulated Smad2 phosphorylation in both scirrhous gastric cancer cell lines, but not in non-scirrhous cancer cell lines, MKN-45 and MKN-74. Smad2 phosphorylation was decreased in a dose-dependent manner by Ki26894 in scirrhous gastric cancer cell lines. PANC-1, a pancreas cancer cell line, was used as positive control of Smad2.

Figure 2

Effect of Ki26894 on the wound healing ability of cancer cells. (A) Representative pictures from three experiments are shown. The number of OCUM-2MLN cells that migrated over the wound line (dotted line) was increased by TGF-β1 treatment when compared with that of control. The migrating cells displayed spindle shapes characteristic of epithelial-to-mesenchymal transition (EMT). Ki26894 (10 μM) downregulated the ability of OCUM-2MLN cells to migrate over a wound line. (B) TGF-β1 significantly stimulated the migration of scirrhous gastric cancer cells, but not of non-scirrhous gastric cancer cells. Ki26894 (10 μM) significantly inhibited the migration of scirrhous gastric cancer cells.

Figure 3

Ki26894 inhibits invasion by scirrhous gastric cancer cells. (A) Representative pictures of invading gastric cancer cells. OCUM-12 cells invaded into a 12 μm-pore membrane filter in the presence of TGF-β1 when compared with control. Most of the invaded cells show epithelial-to-mesenchymal transition (arrow heads). Ki26894 (10 μM) inhibited the invasion induced by TGF-β1. (B) Transforming growth factor-β1 significantly stimulated the invasive behaviour of OCUM-2MLN and OCUM-12 cells. Ki26894 (10 μM) significantly inhibited the invasion seen in these cells. In contrast, invasion by non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74, was not affected by TGF-β1 or by Ki26894.

Figure 4

Effects of TGF-β1 and Ki26894 on morphological characteristics of gastric cancer cells. Epithelial-to-mesenchymal transition (arrow heads) by TGF-β1 (10 ng ml⁻¹) was shown in scirrhous gastric cancer cell lines, OCUM-2MLN and OCUM-12, but not in non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74. Pseudopod formation was remarkable in OCUM-12 cells in the presence of TGF-β1. Epithelial-to-mesenchymal transition by TGF-β1 in scirrhous gastric cancer cells was inhibited by Ki26894 (10 μM).

Figure 5

Effects of Ki26894 on cellular migration signals in gastric cancer cells. (A) ELISA of RhoA. Transforming growth factor-β1 (10 ng ml⁻¹) significantly upregulated the active form of RhoA in scirrhous gastric cancer cell lines, OCUM-2MLN and OCUM-12, and Ki26894 (10 μM) significantly inhibited this effect. In contrast, the active form of RhoA was not increased by TGF-β1 or by Ki26894 in non-scirrhous cancer cell lines, MKN-45 and MKN-74. (B) Transforming growth factor-β1 (10 ng ml⁻¹) increased myosin light chain-2 phosphorylation (p-myosin) and decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cell lines, but not in non-scirrhous gastric cancer cell lines. Ki26894 (10 μM) decreased p-myosin, and increased ZO-2 and E-cadherin expression in scirrhous gastric cancer cell lines.