[Show abstract][Hide abstract] ABSTRACT: Hepatocyte growth factor (HGF), acting through the c‑Met receptor, plays an important role in solid tumors. Various malignant cells utilize the biological actions of the HGF/c‑Met pathway for their dissociative, invasive and metastatic behaviors. HGF also binds to the receptor expressed on endothelial cells that stimulates angiogenesis, a process critical to continued growth of solid tumors. It is known that HGF induces in vitro expression of vascular endothelial growth factor (VEGF), a key agonist of tumor angiogenesis. In the present study, we showed using in vitro co-culture system with fibroblasts that VEGF expression of CT26 cells was amplified through tumor-stromal interaction, i.e., the HGF paracrine loop. This action was inhibited by interruption of the HGF paracrine loop by gene transfer of NK4, an HGF antagonist. In in vivo experiments, CT26 tumor growth and angiogenesis were markedly enhanced by fibroblast co-inoculation, while the effect of fibroblasts was not observed in NK4‑expressing CT26 cells. These findings suggest that NK4 exerted potent anti‑angiogenic action via indirectly inhibiting VEGF expression of tumor cells in addition to direct effects on endothelial cells. Thus, the HGF/c‑Met pathway may be a considerable candidate for molecular targeting strategy against tumor angiogenesis.
[Show abstract][Hide abstract] ABSTRACT: In the present study, we assessed the involvement of hepatocyte growth factor (HGF)/c-Met signalling with vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor (HIF)-1α expression in the downstream pathways phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) in CT26 cells, to determine the mechanisms of the potent anti-angiogenic effect of NK4. We established genetically modified CT26 cells to produce NK4 (CT26-NK4). VEGF expression in subcutaneous CT26 tumours in vivo and in culture supernatants in vitro was determined by ELISA. HIF-1α expression in nuclear extracts was evaluated by western blot analysis. VEGF and HIF-1α mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The DNA binding activity of HIF-1α was evaluated using an HIF-1α transcription factor assay kit. Our results demonstrated that VEGF expression was reduced in homografts of CT26-NK4 cells, compared to those of the control cells. In vitro, VEGF expression, which was induced by HGF, was inhibited by anti-HGF antibody, NK4 and by kinase inhibitors (PI3K, LY294002; MAPK, PD98059; and STAT3, Stattic). HGF‑induced HIF‑1α transcriptional activity was also inhibited by the kinase inhibitors. Real-time RT-PCR demonstrated that HGF‑induced HIF‑1α mRNA expression was not inhibited by LY294002 and PD98059, but was inhibited by Stattic. These data suggest that the PI3K̸Akt, MAPK and STAT3 pathways, downstream of HGF̸c‑Met signalling, are involved in the regulation of VEGF expression in CT26 cells. HGF̸c‑Met signalling may be a promising target for anti-angiogenic strategies.
Preview · Article · Dec 2012 · International Journal of Oncology
[Show abstract][Hide abstract] ABSTRACT: The hepatocyte growth factor (HGF)-Met pathway plays a role in progression to malignant characteristics in tumors, including that of resistance to anticancer drugs. The purpose of this study was to elucidate the possibility of the combination therapy of NK4, a competitive antagonist for HGF.
We established a genetically modified murine colon cancer cell, CT26, to produce abundant NK4 (CT26/NK4). Cell proliferation, apoptosis, cell cycle, intracellular signaling, and 5-fluorouracil (5-FU) metabolism of this cell line were examined.
There was no difference in thymidylate synthase mRNA level between mock-transfected control CT26 cells and CT26/NK4 cells, suggesting that NK4 expression does not change 5-FU metabolism. NK4 gene expression enhanced 5-FU-induced caspase-3 and -7 activation of CT26. Cell cycle analysis showed that NK4 gene expression and 5-FU treatment caused an increase in the proportion of sub-G(1) cells. On 5-FU treatment, phosphorylation of Akt and Erk1/2 was suppressed in CT26/NK4 less than in mock-transfected cells. 5-FU showed a stronger cytotoxic activity towards CT26/NK4 cells than control CT26 cells.
5-FU exerts an additional effect on apoptosis of NK4-expressing CT26 cells by down-regulating intracellular signaling of the HGF/c-Met pathway.
No preview · Article · Jun 2011 · Anticancer research
[Show abstract][Hide abstract] ABSTRACT: NK4, a proteolytic cleavage product of hepatocyte growth factor (HGF), acts not only as a competitive antagonist for HGF, but also as a potent angiogenesis inhibitor independently. In this study, we examined the correlation of NK4 with induction of CD8+ T cell-dependent immune reaction. We previously established NK4-expressing mouse colon adenocarcinoma CT26 cells (CT26/nk4). The animal experiments showed that the potent inhibition of subcutaneous CT26/nk4 tumor growth and prolonged survival of CT26/nk4 tumor-bearing mice shown in immunocompetent mice was weakened in nude mice. Re-challenge test showed that the immunocompetent mice that had shown complete regression of CT26/nk4 tumors rejected subsequent challenge with the parental CT26 cells, but not mouse fibrosarcoma Meth A cells. In immunohistochemical analysis, the tumor microvessel density was decreased and the percentage of apoptotic tumor cells was increased in CT26/nk4 tumors, as previously reported. Additionally, CT26/nk4 tumors contained markedly more tumor infiltrating lymphocytes, especially CD8+ T cells, than control tumors. Cytotoxicity assay clearly revealed that CT26 tumor-specific cytotoxic T lymphocytes were produced in CT26/nkc4-bearing mice. When we depleted CD4+, CD8+ T cells and NK cells in CT26/nkc4-bearing mice, the depletion of CD8+ T cells greatly reduced the antitumor activity of NK4. These data suggest that a CD8+ T cell-dependent cellular immune response is involved in the antitumor mechanism of NK4 in addition to HGF antagonism and antiangiogenesis.
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte growth factor (HGF) has been shown to be involved in malignant behaviors, such as invasion and metastasis, in different tumors. Hence, HGF could be a target molecule for control of the malignant potential of cancer. NK4 is a competitive antagonist for HGF and exerts an antitumor activity, not only by HGF antagonism but also by antiangiogenesis. Here, we studied the participation of cellular immunity in CT26 tumor regression by NK4 gene transfer. In vivo experiments showed that NK4-induced inhibition of subcutaneous tumor growth (as demonstrated in immunocompetent BALB/c mice) was weakened in T lymphocyte-deficient nude mice. In addition, the immunocompetent BALB/c mice that had shown complete regression of CT26-NK4 tumors generated an immune memory against repeated challenge with the same tumor antigen. Immunohistochemistry of tumor-infiltrating lymphocytes showed that the ratio of CD8/CD4 in CT26-NK4 tumors was significantly higher than that in control tumors. Also, the presence of tumor-specific cytotoxic T lymphocytes (CTL) was demonstrated by cytotoxicity assays. Depletion of CD8+ T lymphocytes markedly abrogated the antitumor activity of NK4. However, NK4 had no direct effect on the in vitro cellular immune system. Taken together, these data indicate that NK4 expression by gene transfer, at the tumor site, triggers tumor-specific CTL activation, resulting in complete CT26 tumor regression in vivo. This action was considered to be due to apoptosis induced by NK4's potent antiangiogenic and HGF antagonistic effects.
Full-text · Article · Dec 2009 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte growth factor (HGF) plays a definitive role in invasive, angiogenic, and metastatic activities of tumor cells by binding to the c-Met receptor. NK4, a competitive antagonist for HGF and the c-Met receptor, prevents tumor cell growth and metastasis via its bifunctional properties to act as an HGF antagonist and angiogenesis inhibitor. In the present study, we investigated the inhibitory effectiveness of NK4 on hematogenous pulmonary metastasis of the CT26 murine colon cancer cell line, focusing on tumor cell adhesion to endothelial cells. In an in vitro adhesion assay, HGF facilitated adhesion of CT26 cells to a murine endothelial cell line (F-2) in a dose-dependent manner. Furthermore, the enhancing effect of HGF on CT26-F-2 cell interaction was blocked by NK4 as well as by anti-HGF antibody. Similarly, HGF-induced phosphorylation of focal adhesion kinase (FAK), downstream of integrin signaling, was reduced by NK4 and by anti-HGF antibody. However, distinct integrin expression on the surface of CT26 cells was not altered by HGF. In an in vivo experimental pulmonary metastasis assay, stable NK4 expression potently decreased the number of pulmonary metastatic foci. The NK4-induced suppression of pulmonary metastasis was partially reversed when HGF was intraperitoneally administered in an adhesive phase. These results suggest that NK4 could act on tumor cells to inhibit CT26 adhesion to endothelial cells by reducing FAK phosphorylation, which is regulated by inside-out HGF/c-Met signaling, and thereby suppress hematogenous pulmonary metastasis.
No preview · Article · Mar 2009 · Clinical and Experimental Metastasis
[Show abstract][Hide abstract] ABSTRACT: A 77-year-old woman visited our hospital with the chief complaint of left supraclavicular lymph node redness and swelling. Needle biopsy revealed metastatic, epithelial, undifferentiated carcinoma. However, the primary tumor remained unknown despite further thorough examinations, FDG-PET showed abnormal FDG accumulation at the lymph nodes of para-aortic and left external iliac artery area in addition to left supraclavicular lymph node. However, CT and MRI showed no lymph node swelling in the peritoneal cavity. Nedaplatin (CDGP) combined with S-1 therapy was carried out for this primary unknown cancer with lymph node metastases. Three months after CDGP/S-1 therapy was begun, the swollen left supraclavicular lymph node was obviously reduced by 42.5%. Moreover, abnormal FDG accumulation at left supraclavicular and para-aortic lymph nodes dramatically decreased and that at the left external iliac artery area disappeared. The anti-tumor effect was evaluated as a partial response by use of Response Evaluation Criteria in Solid Tumors (RECIST). Standard treatment for primary unknown cancer was not established, because it includes various carcinomas. Here we report a case of primary unknown cancer successfully treated with CDGP/S-1. This combined therapy was considered to be one of the promising strategies for a primary unknown cancer.
No preview · Article · Dec 2008 · Gan to kagaku ryoho. Cancer & chemotherapy