Megumi Baba

Urokinase-type plasminogen activator expression correlates with tumor angiogenesis and poor outcome in gastric cancer

Urokinase plasminogen activating system (PA system) and vascular endothelial growth factor (VEGF) were recently suggested to contribute synergistically to tumor progression. To evaluate the roles of the PA system and VEGF in gastric cancer, the effects of the PA system and VEGF on tumor angiogenesis and the survival of patients with gastric cancer were investigated. Cancer tissues from 101 gastric cancer patients were assayed immunohistochemically for expression of urokinase‐type plasminogen activator (uPA), uPA receptor (uPAR), PA inhibitor‐1 (PAI‐1) and VEGF protein. The positive rates of uPA, uPAR, PAI‐1, VEGF expression were 22.8%, 32.7%, 36.6% and 26.7%, respectively. Positive staining was observed in tumor cells (uPA, uPAR, VEGF), or in both tumor cells and stromal cells (PAI‐1). The expressions of uPA, uPAR, PAI‐1 and VEGF were significantly correlated with the clinicopathological factors: uPA, depth of tumor invasion, differentiation, lymphatic and vascular invasion; uPAR, tumor size, depth, lymph node involvement, differentiation, vascular invasion; PAI‐1, tumor size, depth, lymph node involvement, differentiation, vascular invasion; VEGF, differentiation, vascular invasion. The microvessel density (MVD) assessed immunohistochemically was significantly higher in the patients with expression of uPA, uPAR or VEGF, and stepwise analysis identified uPA as an independent correlated factor with MVD. Furthermore, multivariate analysis demonstrated that depth of tumor invasion, lymph node involvement and uPA expression were independent prognostic factors. uPA is a key factor in the PA system, being associated with a poor outcome of gastric cancer, and contributing not only to invasive activity, but also to angiogenesis. (Cancer Sci 2003; 94: 43–49)

Anti-tumor and anti-metastatic effects of human-vascular-endothelial-growth-factor-neutralizing antibody on human colon and gastric carcinoma xenotransplanted orthotopically into nude mice

In order to determine whether the inhibition of vascular‐endothelial‐growth‐factor (VEGF) activity by administration of an immunoneutralizing antibody could suppress tumor growth and metastasis in spontaneous metastatic models of human colon and gastric carcinoma, 4 human carcinoma xenografts, 2 human colon carcinomas (TK4 and TK13) and 2 gastric carcinomas (MT2 and MT5) were transplanted orthotopically into nude mice. The anti‐VEGF antibody (MV833, 100 μg/mouse) or the same volume of saline was administered i.p. on alternative days from day 10 after transplantation. With each of the 4 xenografts, administration of MV833 significantly inhibited not only primary tumor growth but also macroscopic liver metastasis, although the growth rate varied. The inhibitory effect of MV833 on primary tumor growth appeared to have no correlation with the level of VEGF in tumor. Body‐weight gain in each treated group was comparable with that in the control group. No toxicity of the antibody was observed. These results suggest that an anti‐VEGF antibody can be effective against a wide variety of cancers, and that VEGF may be a possible target for cancer therapy. Int. J. Cancer 77:933–936, 1998.© 1998 Wiley‐Liss, Inc.

TSU68 Prevents Liver Metastasis of Colon Cancer Xenografts by Modulating the Premetastatic Niche

The aim of this study was to investigate the inhibitory effect of TSU68 [(Z)-5-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic acid; SU6668], an inhibitor of vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor beta, and fibroblast growth factor receptor 1 (FGFR1), on colon cancer liver metastasis, and to test the hypothesis that TSU68 modulates the microenvironment in the liver before the formation of metastasis. First, we implanted the highly metastatic human colon cancer TK-4 orthotopically into the cecal walls of nude mice, followed by twice-daily administration of TSU68 (400 mg/kg/d) or vehicle. Five weeks of treatment with TSU68 significantly inhibited liver metastasis compared with the control group (P<0.001). Next, we analyzed the gene expression profile in premetastatic liver using microarrays. Microarray and quantitative reverse transcription-PCR analysis showed that mRNA levels for the chemokine CXCL1 were significantly increased in tumor-bearing mice compared with non-tumor-bearing mice. Moreover, CXCL1 expression was significantly decreased by TSU68 treatment. CXCR2 expression was detected predominantly on tumor cells in orthotopic tumors compared with ectopic tumors. The number of migrating neutrophils in premetastatic liver was significantly decreased in the TSU68-treated group (P<0.001). The amount of interleukin-12 (IL-12) p40 in the portal vein was significantly decreased by TSU68 (P=0.02). Blockade of both CXCR2 and IL-12 p40 with a neutralizing antibody significantly inhibited liver metastasis. These results suggest that the CXCL1/CXCR2 axis is important in cancer metastasis and that TSU68 may modulate the premetastatic niche in the target organ through suppression of the inflammatory response, which might be an alternative mechanism used by antiangiogenic agents.

Cyclooxygenase-2 expression correlates with uPAR levels and is responsible for poor prognosis of colorectal cancer

Considering recent findings that cyclooxygensase-2 (COX-2) is involved in the progression of colorectal carcinoma (CRC), the role of COX-2 in promoting invasion and angiogenesis was investigated by evaluating the relationship of COX-2 expression to various clinicopathological variables, including plasminogen activating system (PA system) and vascular endothelial growth factor (VEGF). Tumor tissues from 71 patients with CRC were assayed to determine the antigen levels of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor-1 and -2 (PAI-1 and PAI-2), as well as immunohistochemical expression of VEGF. COX-2 was assayed immunohistochemically in 56 patients. COX-2 expression was detected in cancer cells and it was also expressed by stromal cells in some patients. Fourteen patients (25%) were COX-2 positive, whereas 42 were negative. COX-2 expression was significantly related to lymphatic invasion (P=0.0317), but was not related to microvessel density or VEGF expression. In the PA system, uPAR antigen levels were significantly higher in tumors with COX-2 expression than in tumors without (P=0.0233). Univariate analysis showed that significant prognostic variables for survival were tumor size, lymph node involvement, lymphatic invasion, vascular invasion, liver metastasis, high uPAR level, and COX-2 expression, but only liver metastasis was an independent prognostic factor (P=0.0065) in multivariate analysis. COX-2 expression was a more important prognostic indicator than any other factor except liver metastasis (P=0.0526). The significant relationship between the presence of COX-2 protein and uPAR antigen levels contributed to the enhancement of tumor invasion and the poor outcome in patients with CRC.

Effect of angiogenesis inhibitor TNP-470 on the progression of human gastric cancer xenotransplanted into nude mice.

The effect of an angiogenesis inhibitor, TNP‐470, on primary tumor growth, liver metastasis and peritoneal dissemination of gastric cancer was investigated by means of an orthotopic xenotransplanted model of 2 human gastric cancers, MT‐2 and MT‐5. TNP‐470 showed a significant inhibitory effect on the growth of primary tumors after orthotopic transplantation of both xenografts when given at a dose of 30 mg/kg on alternate days from day 7 after transplantation (early treatment). However, growth of the MT‐2 primary tumor was not inhibited by administration from day 14 after transplantation (late treatment). Liver metastasis was prevented significantly by early treatment of TNP‐470. In particular, early treatment of MT‐2 completely inhibited the development of macroscopic foci in the liver and was significantly more effective than late treatment. Peritoneal dissemination also was inhibited. Thus, TNP‐470 was revealed to have strong inhibitory activity not only on primary tumors and liver metastases but also against peritoneal dissemination. These results suggest that this agent may provide a new approach to the treatment of gastric cancer. Int. J. Cancer 71: 838‐841, 1997.

Antitumor Effect of a Neutralizing Antibody to Vascular Endothelial Growth Factor on Liver Metastasis of Endocrine Neoplasm

Distant metastasis of gastrointestinal endocrine neoplasm is resistant to currently available treatments. Because hematogenic metastasis is dominant, anti‐angiogenic drugs are expected to be a novel therapy for this neoplasm. In the present study, the therapeutic effect of vascular endothelial growth factor neutralizing antibody (VEGFAb) on liver metastasis of an endocrine neoplasm was investigated experimentally. Cecal transplantation into nude mice of small pieces of EN‐1, a xenotransplanted human intestinal endocrine neoplasm, resulted in liver metastasis. A treated group (n=19) received 100 μg/mouse of VEGFAb intraperitoneally on alternate days from day 10 after tumor transplantation, and the control group (n=19) received saline. Five of the 19 control mice died of tumor progression, of which 2 could not be evaluated. The cecal tumor weighed 6316±2333 mg (n=17) in the control group and 1209±837 mg (n=19) in the treated group (P<0.01) 6 weeks after transplantation. Liver metastasis developed in 16 of 17 control mice and in 2 of 19 treated mice (P<0.01). The VEGF level of the whole cecal tumor in the control group was significantly higher than that in the treated group (305.1±174.1 vs. 54.7±41.2 mg; P<0.001). VEGFAb did not cause any body weight loss (28.52±1.63 in the control vs. 28.44±1.71 g in the treated group). These results indicate that VEGFAb may be a novel therapeutic agent for endocrine neoplasm with distant metastasis.

The role of circulating IL‐8 and VEGF protein in the progression of gastric cancer

Both vascular endothelial growth factor (VEGF) and interleukin 8 (IL‐8) play an important role in the progression of gastric cancer (GC). In this study, we investigated whether circulating levels of VEGF or IL‐8 in drainage veins of GC patients were correlated with any clinicopathological factors. Thirty‐seven patients with primary GC who underwent gastrectomy at our department between 1999 and 2002 were analyzed. Blood samples were drawn from a peripheral vein just before surgery and from a drainage vein immediately after laparotomy. Plasma VEGF levels were significantly higher than those in 10 healthy controls. There was no correlation between VEGF levels in drainage veins and any clinicopathological variable, whereas there was a significant relationship in the case of VEGF levels in peripheral veins; the levels were higher in patients with venous invasion. We found a significant relationship between IL‐8 levels in drainage veins and both tumor size and lymph node metastasis, whereas no significant relationship between IL‐8 levels in peripheral veins and any variable was found. There was no correlation between VEGF and IL‐8 levels in drainage veins. Large tumors, deeply invasive tumors, lymph node involvement, venous invasion and high IL‐8 levels in drainage veins were all significantly associated with shorter disease‐free survival, although multivariate analysis revealed that lymph node involvement was the only independent prognostic factor. In conclusion, the measurement of IL‐8 levels in drainage veins of GC patients may reflect production mainly by the primary lesion and is valuable as an indicator of risk for recurrent disease.

Prevention of liver metastasis of human colon cancer by selective matrix metalloproteinase inhibitor MMI-166.

MMI-166 is a selective inhibitor of matrix metalloproteinase (MMP)-2 and MMP-9. Mice implanted a human colon cancer orthotopically received 200 mg/kg of MMI-166 orally for 5 weeks. Gelatin zymography demonstrated that the administration of MMI-166 remarkably decreased the active MMP-2 expression. Histological examination revealed that MMI-166 showed prominent effect on reduction of the invasive feature of the cancer cells and showed inhibitory effect on tumor vasculature, resulting in the significant decrease of microvessel density of the implanted tumor and liver metastasis compared with the control group. Conclusively, MMI-166 is a potent antiangiogenic oral agent for a human colon cancer.

Optical diagnosis of gastric cancer using near-infrared multichannel Raman spectroscopy with a 1064-nm excitation wavelength

BackgroundGastric cancer is one of the most common cancers in Japan. The use of endoscopy is increasing, along with the number of histological examinations of specimens obtained by endoscopy. However, it takes several days to reach a diagnosis, which increases the medical expense. Raman spectroscopy is one of the available optical techniques, and the Raman spectrum for each molecule and tissue is characteristic and specific. The present study investigated whether Raman spectroscopy can be used to diagnose gastric cancer.MethodsA total of 251 fresh biopsy specimens of gastric carcinoma and non-neoplastic mucosa were obtained from 49 gastric cancer patients at endoscopy. Without any pretreatment, the fresh specimens were measured with a near-infrared multichannel Raman spectroscopic system with an excitation wavelength of 1064 nm, and Raman spectra specific for the specimens were obtained. A principal component analysis (PCA) was performed to distinguish gastric cancer and non-neoplastic tissue, and a discriminant analysis was used to evaluate the accuracy of the gastric cancer diagnosis.ResultsThe Raman spectra for cancer specimens differed from those for non-neoplastic specimens, especially at around 1644 cm−1. Sensitivity was 66%, specificity was 73%, and accuracy was 70%. The accuracy of diagnosis using the single Raman scattering intensity at 1644 cm−1 was 70%, consistent with the PCA result.ConclusionsThe present results indicate that near-infrared multichannel Raman spectroscopy with a 1064-nm excitation wavelength is useful for gastric cancer diagnosis. Establishment of a Raman diagnostic system for gastric cancer may improve the clinical diagnosis of gastric cancer and be beneficial for patients.

The significance of circulating vascular endothelial growth factor (VEGF) protein in gastric cancer.

We examined the vascular endothelial growth factor (VEGF) levels in peripheral blood and drainage vein (plasma and serum), and then these were compared with local VEGF expression from gastric cancer. Peripheral blood plasma VEGF levels was increased in the patients with venous invasion, and moderately correlated with the number and ratio of lymph nodes with metastasis. Local VEGF expression was correlated significantly with tumor size, advanced stage and lymph node metastasis, but not correlated with peripheral VEGF levels. The level of plasma VEGF in peripheral veins is one of the sensitive markers of the status of gastric cancer.