Osamu Shinto

Cancer stem cell-like SP cells have a high adhesion ability to the peritoneum in gastric carcinoma.

Cancer stem cells (CSCs) are considered to be responsible for cancer metastasis, but the evidence to conclusively prove this hypothesis remains uncertain. The side population (SP), as evaluated by a flow cytometric analysis using Hoechst 33342, has been known as CSC‐rich population. The aim of this study was to clarify the characterization of the SP cells in peritoneal metastasis of gastric carcinoma. Gastric cancer cell lines OCUM‐2M, OCUM‐2D, and OCUM‐2MD3 (a daughter cell line with high potential for peritoneal metastasis) were used. We isolated SP cells from OCUM‐2M and OCUM‐2D using flow cytometry. Serial sorting was performed three times to enrich SP cells, and they were designated as OCUM‐2M/SP and OCUM‐2D/SP cells. Flow cytometric analysis showed 0.46%, 0.29%, 5.24%, 6.49%, and 11.3% of the SP cells to be found in OCUM‐2M, OCUM‐2D, OCUM‐2MD3, OCUM‐2M/SP, and OCUM‐2D/SP cells, respectively. The intraperitoneal inoculation of SP cells and OCUM‐2MD3 cells produced peritoneal metastasis, but parent cells did not. The adhesion ability of SP and OCUM‐2MD3 cells was significantly high in comparison to that of parent cells. The expression level of adhesion molecules α2‐, α5‐, β3‐, and β5‐integrin, and CD44, was high in SP cells compared to parent cells. The expression of stemness markers, Oct3/4 and Sox2, increased in the SP‐cell‐injected tumors. These findings suggested that CSC‐like SP cells expressing α2‐, α5‐, β3‐, and β5‐integrin, and CD44, may play an important role for peritoneal metastasis in gastric carcinoma. Oct3/4 and Sox2 may be associated with CSC in gastric cancer. (Cancer Sci 2009)

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

Background:Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-β (TGF-β) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-β receptor (TβR) 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 TβR type I (TβR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TβR-I. We investigated the expression levels of TβR and phospho-Smad2, and the effects of TGF-β 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:TβR-I, TβR-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-β1 in scirrhous gastric cancer cells. Transforming growth factor-β1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-β1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-β1 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-β1 or Ki26894 treatment.Conclusion:A TβR-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.

Hypoxia Stimulates the EMT of Gastric Cancer Cells through Autocrine TGFβ Signaling

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.

DNA methyltransferase inhibitor 5-aza-CdR enhances the radiosensitivity of gastric cancer cells

The National Comprehensive Cancer Network guidelines recommend radiotherapy as a standard treatment for patients with a high risk of recurrence in gastric cancer. Because radiation is harmful to the surrounding organs, a radiation sensitizer might therefore be useful to decrease the side effects of patients with advanced gastric carcinoma. The aim of the current study was to clarify the effect of a DNA methyltransferase inhibitor, 5‐aza‐2′‐deoxycytidine (CdR), on radiation sensitivity in gastric cancer cells. Five gastric cancer cell lines, OCUM‐2M, OCUM‐12, KATO‐III, MKN‐45, and MKN‐74, were used. The effects of 5‐aza‐CdR with irradiation on the growth activity, cell‐cycle distribution, apoptosis, and apoptosis‐associated gene expression were examined. 5‐aza‐CdR sensitized three of five gastric cancer cell lines to radiation. A combination of irradiation and 5‐aza‐CdR significantly (P < 0.05) decreased the growth activity compared with irradiation alone in OCUM‐2M, OCUM‐12, and MKN‐45 cells, but not in KATO‐III and MKN‐74 cells. The percentage of cells in G2–M phase and the apoptotic rate with irradiation in combination with 5‐aza‐CdR were increased in OCUM‐2M, OCUM‐12, and MKN‐45 cells compared with irradiation alone, but not in KATO‐III and MKN‐74 cells. 5‐aza‐CdR increased the expression of p53, RASSF1, and death‐associated protein kinases (DAPK) genes compared with the control or irradiation alone. These findings suggest that 5‐aza‐CdR might therefore be useful as a radiation sensitizer to treat some types of gastric carcinoma. The arrest at G2–M phase and increased apoptotic rate might be partly mediated by enhanced expression of the p53, RASSF1, or DAPK gene families by 5‐aza‐CdR. (Cancer Sci 2009; 100: 181–188)

Establishment and characterization of a new hypoxia-resistant cancer cell line, OCUM-12/Hypo, derived from a scirrhous gastric carcinoma

Background:Many kinds of solid tumour have heterogeneously a hypoxic environment. Tumour hypoxia reported to be associated with more aggressive tumour phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear.Methods:We established a scirrhous gastric carcinoma cell line (OCUM-12) from ascites associated with scirrhous gastric carcinoma, and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) was cloned from OCUM-12 cells by continuous exposure to 1% oxygen.Results:Histologic findings from orthotopic tumours derived from parent OCUM-12 cells and daughter OCUM-12/Hypo cells revealed poorly differentiated adenocarcinoma with extensive fibrosis that resembled human scirrhous gastric cancer. Necrotic lesions were frequently detected in the OCUM-12 tumours but were rarely found in the OCUM-12/Hypo tumours, although both types had multiple hypoxic loci. Apoptosis rate of OCUM-12 cells was increased to 24.7% at 1% O2, whereas that of OCUM-12/Hypo was 5.6%. The OCUM-12/Hypo orthotopic models developed multiple metastases to the peritoneum and lymph nodes, but the OCUM-12 models did not. OCUM-12/Hypo cells showed epithelial-to-mesenchymal transition and high migratory and invasive activities in comparison with OCUM-12 cells. The mRNA expression levels of both E-cadherin and zonula occludens ZO-1 and ZO-2 decreased in OCUM-12/Hypo cells, and that of vimentin, Snail-1, Slug/Snail-2, Twist, ZEB-1, ZEB-2, matrix metalloproteinase-1 (MMP-1), and MMP-2 were increased in OCUM-12/Hypo cells.Conclusion:OCUM-12 and OCUM-12/Hypo may be useful for the elucidation of disease progression associated with scirrhous gastric cancer in the setting of chronic hypoxia.

Synergistic antitumor effects of FGFR2 inhibitor with 5-fluorouracil on scirrhous 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.

A Novel Transforming Growth Factor β Receptor Kinase Inhibitor, A-77, Prevents the Peritoneal Dissemination of Scirrhous Gastric Carcinoma

Purpose: Transforming growth factor β receptor (TGFβ-R) is reported to correlate with the malignant potential of scirrhous gastric carcinoma. The aim of the current study is to clarify the possibility of molecular target therapy with a TGFβ-R inhibitor, A-77, for the treatment of peritoneal dissemination of scirrhous gastric cancer. Experimental Design: Three scirrhous gastric cancer cell lines and two fibroblasts were used. For in vivo experiments, the A-77 was administered i.p. to mouse models of peritoneal dissemination. The influences of A-77 on the adhesion ability, invasion ability, and the expression of adhesion molecules were examined in vitro. Results: The A-77 administration resulted in a significantly (P < 0.01) better prognosis for the mice with peritoneal dissemination (median survival time, 51 days), compared with the control (median survival time, 25 days). A-77 therefore significantly (P < 0.01) decreased the weight and number of metastatic nodes. The adhesive ability and invasion ability of cancer cells were significantly decreased by A-77. A-77 decreased the expression of α2, α3, and α5 integrins in gastric cancer cells. The histologic findings showed the degree of fibrosis to be less in the tumors treated by A-77. A-77 decreased the growth of fibroblast and invasion-stimulating activity of fibroblasts on cancer cells. Conclusion: The TGFβ-R inhibitor, A-77, decreased the expression of integrins in cancer cells and the proliferation of fibroblasts, which resulted in the decreased adhesive and invasive abilities of scirrhous gastric cancer cells to peritoneum. A-77 is thus considered to be useful for the inhibition of peritoneal dissemination of scirrhous gastric carcinoma.

Phosphorylated Smad2 in Advanced Stage Gastric Carcinoma

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.

Effects of VEGFR-3 phosphorylation inhibitor on lymph node metastasis in an orthotopic diffuse-type gastric carcinoma model

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 IC50 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 (P<0.01) 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 (P<0.05) 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.

RhoA/ROCK signaling mediates plasticity of scirrhous gastric carcinoma motility.

The small guanosine triphosphatase (GTPase) Rho and its downstream effector Rho-associated kinase (ROCK) is one of a key mediator involved in controlling focal adhesions and the dynamics of actin stress fibers. The molecular mechanisms for the function of Rho/ROCK pathway leading to the progression in scirrhous gastric carcinoma cells have not been defined. The activation of RhoA in several gastric carcinoma cells was examined. The role of RhoA/ROCK pathway in the metastatic processes of gastric carcinoma cells, using a human scirrhous gastric cancer cell line, OCUM-2MD3 was investigated by in vitro adhesion and invasion assay. The effect of ROCK inhibitor, Y-27632 on the mRNA expression of the integrin family and MMP in gastric carcinoma cells was subsequently examined by Reverse transcriptional (RT)-PCR analysis. Finally, Random OCUM-2MD3 cell motility was evaluated using Time-lapse microscopy. ROCK inhibitor significantly increased the adhesion of OCUM-2MD3 cells to the extracellular matrix (ECM) protein matrigel. Further examination using ECM components showed enhanced binding ability was obtained only in laminin and Integrin subunits α3-integrin was clearly up-regulated by treatment with Y-27632 in OCUM-2MD3 cells. ROCK inhibitor also enhanced the invasion of OCUM-2MD3 cells through matrigel and the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP). Time-lapse microscopy showed conversion of OCUM-2MD3 cells from round to more elongated morphology in the presence of Y-27632, suggesting that inhibition of RhoA/ROCK pathway undergo a so-called ‘amoeboid to mesenchymal’ transition. The fact that Rac1 inhibitor decreased the facilitated invasion by ROCK inhibitor suggested the possibility that increased invasion ability of OCUM-2MD3 cells was related to Rac activity. These data may suggest that RhoA/ROCK regulate plasticity of metastatic gastric carcinoma via mesenchymal-amoeboid transition, leading to provide new insights for designing a new and effective treatment for this type of refractory carcinoma.