BackgroundTransforming growth factor β (TGFβ) receptor signaling is closely associated with the invasion ability of gastric cancer cells. Although Smad signal is a critical integrator of TGFβ receptor signaling transduction systems, not much is known about the role of Smad2 expression in gastric carcinoma. The aim of the current study is to clarify the role of phosphorylated Smad2 (p-Smad2) in gastric adenocarcinomas at advanced stages.MethodsImmunohistochemical staining with anti-p-Smad2 was performed on paraffin-embedded specimens from 135 patients with advanced gastric adenocarcinomas. We also evaluated the relationship between the expression levels of p-Smad2 and clinicopathologic characteristics of patients with gastric adenocarcinomas.ResultsThe p-Smad2 expression level was high in 63 (47%) of 135 gastric carcinomas. The p-Smad2 expression level was significantly higher in diffuse type carcinoma (p = 0.007), tumours with peritoneal metastasis (p = 0.017), and tumours with lymph node metastasis (p = 0.047). The prognosis for p-Smad2-high patients was significantly (p = 0.035, log-rank) poorer than that of p-Smad2-low patients, while a multivariate analysis revealed that p-Smad2 expression was not an independence prognostic factor.ConclusionThe expression of p-Smad2 is associated with malignant phenotype and poor prognosis in patients with advanced gastric carcinoma.

Scirrhous gastric carcinoma (SGC) carries the highest mortality because of a frequent metastasis to lymph node (LN). S1, a 5-fluorouracil (5-FU) analog, is clinically available for gastric cancer at an advanced stage. Fibroblast growth factor receptor 2 (FGFR2) is required for the proliferation of SGC. The objective of this study is to clarify the benefit of a combination of S1 and kinase inhibitors including FGFR2 inhibitor Ki23057 in gastric cancer. OCUM-2MLN and KATO-III were derived from SGC. MKN-7 and MKN-74 were derived from non-SGC. MTT assay was used to examine the growth-inhibitory activity of 5 small-synthetic molecules including Ki23057, Sunitinib, Glivec, Lapatinib or SU11274, in cells cultured with 5-FU. Combination effects of 5-FU with Ki23057 on proliferation, apoptosis and mRNA expression were examined. S1 and/or Ki23057 were administered to murine models of SGC created by the orthotopic inoculation of OCUM-2MLN cells. Ki23057 at 100 nM significantly (p < 0.01) inhibited the proliferation and decreased the phosphorylation of FGFR2 in SGC cells, but not in non-SGC. Ki23057 showed synergistic antitumor effects for SGC cells in combination with 5-FU using CalcuSyn analysis, but Sunitinib, Glivec, Lapatinib and SU11274 did not. The combination of Ki23057 and 5-FU decreased DPD expression and increased apoptosis rates and p21 expression level of SGC cells. The combined administration of S1 and Ki23057 significantly (p < 0.05) decreased orthotopic tumors as well as LN metastasis more effectively than S1 alone. These findings suggested that the combined treatment with 5-FU and Ki23057 produced synergistic antitumor effects and is therapeutically promising for SGC treatment.The characteristic clinical features of scirrhous gastric carcinoma (SGC), a diffusely infiltrating type of gastric carcinomas also known as linitis plastica-type gastric carcinoma, include rapidly progressive growth and a high frequency of metastasis to the lymph node (LN).1-3 SGC carries a worse prognosis among other types of gastric carcinomas, with 5-year survival rates in the range of 10-15%. 4,5 Chemotherapy is frequently recommended treatment for LN metastasis of gastric cancer in Japan. 6-9 A novel chemotherapeutic agent S1, a 5-fluorouracil (5-FU) analog, has recently become first-line chemotherapy for gastric cancer patients with LN metastasis. 9 Even so, however, the median survival duration in patients with advanced SGC continues to be less than a year. 6,7 Fibroblast growth factor receptor 2 (FGFR2) overexpression has been reported in about 40% of advanced stomach cancer cases and is especially frequent in SGC.10,11 FGFR2 is a member of the FGFR receptor tyrosine kinase (RTK) family, which consists of 4 receptors and 23 ligands. 12 The signal through FGFR2 and FGF7 is important for the growth of SGC with an amplification of the activated FGFR2 gene (KsamII). 1,10,11,13 Ki23057, a newly developed FGFR2 inhibitor, competes with ATP for the binding site in the kinase and therefore blocks the autophosphorylation of FGFR2 as p...

The aim of this study is to clarify the benefit of combination chemotherapy in gastric cancer based on a cell-signal inhibitor and an anticancer drug. Two scirrhous gastric cancer cell lines and two non-scirrhous gastric cancer cell lines were used. Five anticancer drugs (5-fluorouracil [5FU], paclitaxel, oxaliplatin, irinotecan, and gemcitabine) and four cell-signal inhibitors, mammalian target of rapamycin (mTOR) inhibitor, glycogen synthase kinase 3b, p38abMAPK, and cyclin-dependent kinase, were used. The proliferation of cancer cells was examined by MTT assay and in vivo study. The apoptosis of cancer cells and the expression of apoptosisrelated molecules were examined by flow cytometry, real-time PCR, and immunostaining. mTOR inhibitors with 5FU showed a synergistic antiproliferative effect in scirrhous gastric cancer, whereas the other signal inhibitors showed no synergistic effect with any anticancer drugs. mTOR inhibitor decreased the IC 50 of 5FU and increased the apoptosis rate in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. The pan-caspase inhibitor, zVAD-fmk, inhibits apoptosis induced in combination with 5FU and mTOR inhibitor. mTOR inhibitor decreased dihydropyrimidine dehydrogenase, thymidylatesynthase, and bcl-2 expression, and increased caspase-3 and p21 expression of scirrhous gastric cancer cells, but did not affect those of non-scirrhous gastric cancer cells. In an in vivo study, mTOR inhibitor significantly enhanced the therapeutic efficacy of S1, an analog of 5FU. These findings suggest that mTOR inhibitor interacts with 5FU in a synergistic manner in scirrhous gastric cancer cells by the activation of the apoptosis signal. Therefore, mTOR inhibitor is a promising therapeutic agent in combination with 5FU in scirrhous gastric cancer.

Abstract. ERas is a recently identified oncogene that supports the tumorigenic growth of embryonic stem cells, it is constitutively active in the absence of mutation. ERas oncogene is expressed only in viviparity phase cells, but not in somatic cells because of epigenetic transcriptional silencing in the somatic phase. The aim of this study was to clarify the ERas expression and its epigenetic regulation in gastric cancer of somatic phase. Fifteen gastric cancer cell lines were used. ERas mRNA expression and its epigenetic regulation were examined by reverse transcription-polymerase chain reaction and bisulfite sequencing analysis. To identify a subset of cancer stem cells, termed 'side population' (SP) cells, flow cytometry analysis was performed. ERas is expressed in 8 of the 15 gastric cancer cell lines, but is silenced in the remaining 7 cancer cell lines and normal cell lines. Six of 7 cancer cell lines without ERas expression had promoter methylation, which correlated with silencing of ERas expression. ERas expression is re-activated following treatment with the DNA methyltransferase inhibitor 5-azaCdR. The percentage of SP fraction of ERas-positive gastric cancer cells was significantly (p=0.024) higher (3.4±1.8%), in comparison to that of ERas-negative cells (1.6±0.4%). These findings suggested that the activating ERas oncogene might be associated with tumorigenic growth of somatic cells, and might be a putative molecule responsible for cancer stem celllike characteristics in gastric cancer. Loss of methylation in the promoter of ERas might be one of mechanisms responsible for the re-expression of an embryonic oncogene in gastric cancer.

BACKGROUND: Vascular endothelial growth factor receptor-3 (VEGFR-3) signalling mediates lymphangiogenesis and lymphatic invasion; however, the effect of VEGFR-3 inhibition on the lymph node (LN) metastasis remains unclear. The aim of this study is to clarify the benefit of a VEGFR-3 inhibitor Ki23057 for LN metastasis. METHODS: Ki23057 was administered orally to gastric cancer models created by orthotopic inoculation of diffuse-type gastric cancer cells, OCUM-2MLN. The effects of Ki23057 on lymphatic vessel invasion, lymphatic vessel density, and VEGFR-3 phosphorylation were examined by immunostaining or immunoblotting. RESULTS: Ki23057 inhibited the autophosphorylation of VEGFR-3, with IC 50 values of 4.3 nM in the cell-free kinase assay. Murine gastric cancer models created by the orthotopic inoculation of OCUM-2MLN cells showed the diffusely infiltrating growth and frequently developed LN metastasis. The oral administration of Ki23057 significantly (Po0.01) reduced the size of orthotopic tumours and the number of the metastatic LN in gastric cancer models. The degree of lymphatic invasion and lymphangiogenesis was significantly (Po0.05) lower in the gastric tumours treated by Ki23057. Ki23057 inhibited the phosphorylation of VEGFR-3 of lymphatic endothelial cells in gastric tumours. CONCLUSION: The inhibition of lymphangiogenesis targeting VEGFR-3 phosphorylation is a therapeutic strategy for inhibiting LN metastasis of diffuse-type gastric cancer.