Tasuku Matsuoka

Hepatocyte growth factor (HGF) produced by peritoneal fibroblasts may affect mesothelial cell morphology and promote peritoneal dissemination.

Mesothelial cell monolayers have been reported to prevent infiltration of cancer cells into the peritoneum. We have previously reported that peritoneal fibrosis induced by gastric cancer cells prior to metastatization may provide a congenial environment for peritoneal metastases. In this study, we investigated the effects of peritoneal fibroblasts on peritoneal mesothelial cell morphology. Human gastric cancer (OCUM‐2MD3), peritoneal fibroblast (NF‐2P) and mesothelial (MS‐1) cell lines were established in our laboratory. Histology of the peritoneum was investigated following intraperitoneal inoculation of serum‐free conditioned media (SF‐CM) from OCUM‐2MD3 cells into nude mice. SF‐CM from peritoneal fibroblasts was added to monolayer‐cultured mesothelial cells, and their morphology was examined by phase‐contrast microscopy. This experiment was conducted in the presence and absence of neutralizing antibodies against various factors. Mesothelial cells exposed to fibroblasts proliferation became hemispherical and separated from each other, while unexposed mesothelium remained as a flat monolayer. Cultured‐mesothelial cells rounded up or exhibited a fibroblast‐like shape following the addition of peritoneal fibroblast SF‐CM. Anti‐hepatocyte growth factor (HGF) neutralizing antibody partly inhibited this effect. We suggest that soluble factors, such as HGF, produced by peritoneal fibroblasts affect the morphology of mesothelial cells in monolayers so that the resulting environment may become prone to the peritoneal dissemination of cancer cells.

The Role of PI3K/Akt/mTOR Signaling in Gastric Carcinoma

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is one of the key signaling pathways induced by various receptor-tyrosine kinases. Accumulating evidence shows that this pathway is an important promoter of cell growth, metabolism, survival, metastasis, and resistance to chemotherapy. Genetic alterations in the PI3K/Akt/mTOR pathway in gastric carcinoma have often been demonstrated. Many kinds of molecular targeting therapies are currently undergoing clinical testing in patients with solid tumors. However, with the exception of the ErbB2-targeting antibody, targeting agents, including PI3K/Akt/mTOR inhibitors, have not been approved for treatment of patients with gastric carcinoma. This review summarizes the current knowledge on PI3K/Akt/mTOR signaling in the pathogenesis of gastric carcinoma and the possible therapeutic targets for gastric carcinoma. Improved knowledge of the PI3K/Akt/mTOR pathway in gastric carcinoma will be useful in understanding the mechanisms of tumor development and for identifying ideal targets of anticancer therapy for gastric carcinoma.

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.

Predictive value of Bcl-2 and Bax protein expression for chemotherapeutic effect in gastric cancer : A pilot study

We investigated tissue staining for Bcl-2 and Bax proteins, which regulate apoptosis, as indicators of chemotherapeutic effect in patients with gastric cancer. In 23 patients with gastric carcinoma biopsy specimens were obtained endoscopically prior to chemotherapy and stained immunohistochemically with anti-Bcl-2 and anti-Bax antibodies. Patients then were treated with continuous infusion of 5-FU and cisplatin. No correlation was seen between chemotherapeutic effect and Bcl-2 or Bax alone. However, among the Bax-positive cases, the patients with Bcl-2-positive tumors were significantly more chemoresistant (p = 0.036) and had worse prognoses (p = 0.008) than Bcl-2-negative cases. Therefore, immunohistochemical staining for Bcl-2 protein may predict chemotherapeutic efficacy or guide specific therapeutic choices in treating Bax-positive tumors.

Rho/ROCK signaling in motility and metastasis of gastric cancer

Gastric cancer is one of the most frequent and lethal malignancies worldwide because of high frequency of metastasis. Tumor cell motility and invasion play fundamental roles in cancer metastasis. Recent studies have revealed that the Rho/Rho-associated protein kinases (ROCK) pathway plays a critical role in the regulation of cancer cell motility and invasion. In addition, the Rho/ROCK pathway plays important roles in invasion and metastasis on the basis of its predominant function of cell cytoskeletal regulation in gastric cancer. According to the current understanding of tumor motility, there are two modes of tumor cell movement: mesenchymal and amoeboid. In addition, cancer cell movement can be interchangeable between the mesenchymal and amoeboid movements under certain conditions. Control of cell motility through the actin cytoskeleton creates the potential for regulating tumor cell metastasis. In this review we discuss Rho GTPases and ROCK signaling and describe the mechanisms of Rho/ROCK activity with regard to motility and metastasis in gastric cancer. In addition, we provide an insight of the therapeutic potential of targeting the Rho/ROCK pathway.

Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer.

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both 2β1 and α3β1 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequenc e intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhe-sion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients.

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.

Elevated dietary linoleic acid increases gastric carcinoma cell invasion and metastasis in mice

Background:Dietary (n-6)-polyunsaturated fatty acids influence cancer development, but the mechanisms have not been well characterised in gastric carcinoma.Methods:We used two in vivo models to investigate the effects of these common dietary components on tumour metastasis. In a model of experimental metastasis, immunocompromised mice were fed diets containing linoleic acid (LA) at 2% (LLA), 8% (HLA) or 12% (VHLA) by weight and inoculated intraperitoneally (i.p.) with human gastric carcinoma cells (OCUM-2MD3). To model spontaneous metastasis, OCUM-2MD3 tumours were grafted onto the stomach walls of mice fed with the different diets. In in vitro assays, we investigated invasion and ERK phosphorylation of OCUM-2MD3 cells in the presence or absence of LA. Finally, we tested whether a cyclooxygenase (COX) inhibitor, indomethacin, could block peritoneal metastasis in vivo.Results:Both the HLA and VHLA groups showed increased incidence of tumour nodules (LA: 53%; HLA: 89%; VHLA: 100%; P<0.03); the VHLA group also displayed increased numbers of tumour nodules and higher total volume relative to LLA group in experimental metastasis model. Both liver invasion (78%) and metastasis to the peritoneal cavity (67%) were more frequent in VHLA group compared with the LLA group (22% and 11%, respectively; P<0.03) in spontaneous metastasis model. We also found that the invasive ability of these cells is greatly enhanced when exposed to LA in vitro. Linoleic acid also increased invasion of other scirrhous gastric carcinoma cells, OCUM-12, NUGC3 and MKN-45. Linoleic acid effect on OCUM-2MD3 cells seems to be dependent on phosphorylation of ERK. The data suggest that invasion and phosphorylation of ERK were dependent on COX. Indomethacin decreased the number of tumours and total tumour volume in both LLA and VHLA groups. Finally, COX-1, which is known to be an important enzyme in the generation of bioactive metabolites from dietary fatty acids, appears to be responsible for the increased metastatic behaviour of OCUM-2MD3 cells in the mouse model.Conclusion:Dietary LA stimulates invasion and peritoneal metastasis of gastric carcinoma cells through COX-catalysed metabolism and activation of ERK, steps that compose pathway potentially amenable to therapeutic intervention.

Plasminogen activator inhibitor 1 RNAi suppresses gastric cancer metastasis in vivo

Cancer metastasis remains the primary cause of pain, suffering, and death in cancer patients, and even the most current therapeutic strategies have not been highly successful in preventing or inhibiting metastasis. In most patients with scirrhous gastric cancer (one of the most aggressive of diffuse‐type gastric cancer), recurrence occurs even after potentially curative resection, most frequently in the form of peritoneal metastasis. Given that the occurrence of diffuse‐type gastric cancers has been increasing, the development of new strategies to combat metastasis of this disease is critically important. Plasminogen activator inhibitor‐1 (PAI‐1) is a critical factor in cancer progression; thus, PAI‐1 RNAi may be an effective therapy against cancer metastasis. In the present study, we used an RNAi technique to reduce PAI‐1 expression in an in vivo model system for gastric cancer metastasis. Ex vivo plasmid transfection and adenovirus infection were tested as mechanisms to incorporate specific PAI‐1 RNAi vectors into human gastric carcinoma cells. Both approaches significantly decreased peritoneal tumor growth and the formation of bloody ascites in the mouse model, suggesting that this approach may provide a new, effective strategy for inhibiting cancer metastasis. (Cancer Sci 2012; 103: 228–232)