Masashige Tendo

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.

Novel models for human scirrhous gastric carcinoma in vivo

Human scirrhous gastric carcinoma, a diffusely infiltrating type of poorly differentiated gastric carcinoma also known as linitis plastica type carcinoma, is characterized by cancer cell infiltration and proliferation accompanied with extensive stromal fibrosis. We established two new gastric cancer cell lines, designated OUCM‐8 and OCUM‐11, which developed the characteristic biology of scirrhous gastric carcinoma upon orthotopic implantation in mice. Involvement of lymph nodes and liver metastasis was also found in both orthotopic models. Histologically, these orthotopic models showed proliferation with extensive fibrosis, resembling human scirrhous gastric cancer. Both cell lines were derived from ascites of patients with scirrhous gastric cancer. The growth of OCUM‐8 and OCUM‐11 cells following the addition of KGF, FGF, and EGF was increased significantly relative to untreated cells. An increase in the number of attached and spreading cells occurred following the addition of TGF‐β1 in both cell lines. OCUM‐11 cells showed microsatellite instability. Although subcutaneous scirrhous gastric cancer cells show medullary growth, most in vivo studies of scirrhous gastric cancer have used xenografted tumors implanted subcutaneously. Only in a few cases was it confirmed that these scirrhous gastric cancer cell lines retained the original histologic characteristics. Our orthotopic models should contribute to the elucidation of disease progression in situ and to the development of therapy for scirrhous gastric cancer.

Effect of organ-specific fibroblasts on proliferation and differentiation of breast cancer cells.

SummaryBreast carcinomas contain both tumor cells and stromal cells, including fibroblasts, endothelial cells, and lymphocytes. Proliferation of breast cancer cells may be controlled partly by mesenchymal cells, especially fibroblasts. We studied effects of fibroblasts on tumorigenicity and histologic features of breast cancer cells vivo, and analyzed fibroblast-produced growth-promoting factors in vitro. Breast carcinoma cells from four lines, and fibroblasts from lines obtained from skin and breast tissue of four patients with breast cancer were used. A suitable number of breast tumor cells and fibroblasts were inoculated subcutaneously into nude mice; resulting tumors were examined. Then conditioned medium from fibroblasts was added to cultures of breast cancer cells to study growth effects, and growth-promoting factors from breast fibroblasts were analyzed. Co-inoculation of breast cancer cells with breast fibroblasts into mice significantly increased tumorigenicity and tumor size beyond those obtained with breast cancer cells alone. Histologically, tumors resulting from co-inoculation with breast fibroblasts showed a scirrhous pattern with extensive fibrosis, while those formed by breast cancer cells injected alone or co-inoculation with skin fibroblasts showed a solid pattern. Medium from breast fibroblasts significantly increased breast cancer cell growth in vitro, while the various skin fibroblasts did not all show this effect. Structural and functional interactions between organ-specific fibroblasts and breast cancer cells may importantly regulate breast cancer growth and progression.

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.

Selective cyclooxygenase-2 inhibitor downregulates the paracrine epithelial–mesenchymal interactions of growth in scirrhous gastric carcinoma

The importance of cancer‐mesenchymal interactions in the aggressive behavior of scirrhous gastric cancer is supported by experimental and clinical evidences. We have previously reported that gastric fibroblasts secretion of keratinocyte growth factor (KGF) underline the remarkable proliferation of scirrhous gastric cancer cells. Cyclooxygenase‐2 (COX‐2) is not only expressed in cancer cells, but also in interstitial fibroblasts in gastric carcinoma. To clarify the mechanisms responsible for the antiproliferation effect of COX‐2 inhibitors, effect of COX‐2 inhibitor on the paracrine epithelial–mesenchymal interactions of growth was examined. Scirrhous gastric cancer cell line, OCUM‐2M, gastric fibroblasts, NF‐21, and COX‐2 inhibitor, JTE‐522, were used. Growth‐interaction was examined by calculating the number of cancer cells or by measuring [3H] thymidine incorporation of cancer cells. Effect of JTE‐522 on KGF expression from NF‐21 cells and OCUM‐2M cells was analyzed by ELISA and RT‐PCR. The conditioned medium from gastric fibroblasts significantly stimulated the growth of scirrhous gastric cancer cells. JTE‐522 at the concentrations of 10−5 and 10−6 M significantly decreased the growth‐stimulating activity of gastric fibroblasts. JTE‐522 reduced the expression of KGF mRNA and the production of KGF from gastric fibroblasts. Oral administration of JTE‐522 significantly decreased the size of xenografted tumor coinoculated with OCUM‐2M cells and NF‐21 cells in nude mice. JTE‐522 decreased COX‐2 expression and Ki67 labeling index within the coinoculated tumor. These findings suggested that a selective COX‐2 inhibitor, JTE‐522, downregulates KGF production from gastric fibroblasts, resulting in the inhibition of paracrine epithelial–mesenchymal interactions of proliferation between scirrhous gastric cancer cells and gastric fibroblasts.

Inhibitory effect of a selective cyclooxygenase inhibitor on the invasion-stimulating activity of orthotopic fibroblasts for scirrhous gastric cancer cells

The cyclooxygenase (COX)‐2 inhibitor has been reported to impede the progression of gastric cancer, but underlying mechanisms remain unclear. We therefore investigated the effect of a COX‐2 inhibitor, JTE‐522, on the ability of orthotopic fibroblasts to stimulate invasion of scirrhous gastric carcinoma cells. The human scirrhous gastric cancer cell lines OCUM‐2D or OCUM‐2M, and human gastric fibroblasts (NF‐21) were cultured in the absence or presence of JTE‐522 at various concentrations. Cancer cells were then assayed for invasiveness in vitro by invasion assay. The effect of prostaglandins (PG) on growth factor production in NF‐21 cells was examined by ELISA. Finally, the effects of orally administrated JTE‐522 on orthotopically transplanted tumors were examined in nude mice. NF‐21 cells stimulated invasion by OCUM‐2D cells, an effect suppressed by JTE‐522 at 5 × 10−6 M. Hepatocyte growth factor (HGF) and PGE2 production by NF‐21 cells were suppressed by JTE‐522 (P < 0.01). PGE2 stimulated HGF production by NF‐21 cells in a dose‐dependent manner. JTE‐522 significantly suppressed orthotopic tumor growth and lymph node metastasis, and also decreased HGF expression by fibroblasts within the gastric tumor. In conclusion, we found that gastric fibroblasts stimulated invasiveness in scirrhous gastric cancer cells, whereas a selective COX‐2 inhibitor inhibited this paracrine effect by decreasing fibroblast PGE2 production, resulting in downregulation of HGF production. (Cancer Sci 2005; 96: 451–455)

Salvage surgery after chemotherapy with S-1 plus cisplatin for α-fetoprotein-producing gastric cancer with a portal vein tumor thrombus: a case report

BackgroundPatient with α-Fetoprotein (AFP)-producing gastric cancer usually has a short survival time due to frequent hepatic and lymph node metastases. Gastric cancer with portal vein tumor thrombus (PVTT) is rare and has an extremely poor prognosis.Case presentationA 63-year-old man was found to have a huge Type 3 gastric cancer with a PVTT and a highly elevated serum AFP level. Chemotherapy with S-1 plus cisplatin was given to this patient with unresectable gastric cancer for 4 months. The serum AFP level decreased from 6,160 ng/mL to 60.7 ng/mL with chemotherapy. Since the PVTT disappeared after the chemotherapy, the patient underwent total gastrectomy. Histological findings of the primary tumor after chemotherapy showed poorly differentiated adenocarcinoma without hepatoid cells and viable tumor cells remaining in less than 1/3 of the neoplastic area of mucosa and one lymph node. The cancerous cells were immunohistochemically stained by anti-AFP antibody. The patient has survived for 48 month without recurrence.ConclusionsAFP-producing gastric cancer with a PVTT has an extremely poor prognosis, but long-term survival was achieved for this dismal condition by salvage surgery after chemotherapy.

Abstract 2552: Synergistic antitumor effects of FGFR2 inhibitor with 5-fluorouracil on diffuse-type gastric carcinoma

Purpose: Diffuse-type gastric carcinoma carries the highest mortality because of a frequent metastasis to lymph node. S1, a 5-fluorouracil (5-FU) analog, is clinically available for gastric cancer at advanced stage. Fibroblast growth factor receptor 2 (FGFR2) is required for the proliferation of diffuse-type gastric carcinoma. 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. Experimental Design: OCUM-2MLN and KATO-III were derived from diffuse-type gastric carcinoma. MKN-7 and MKN-74 were derived from intestinal-type gastric carcinoma. 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 diffuse-type gastric carcinoma created by the orthotopic inoculation of OCUM-2MLN cells. Results: Ki23057 at 100 nM significantly (p Conclusion: The combined treatment with 5-FU and Ki23057 produced synergistic anti-tumor effects, and is a therapeutically promising for diffuse-type gastric carcinoma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2552. doi:10.1158/1538-7445.AM2011-2552