Paradoxical discrepancy between the serum level and the placental intensity of PP5/TFPI-2 in preeclampsia and/or intrauterine growth restriction: possible interaction and correlation with glypican-3 hold the key.

There have been controversies whether maternal serum placental protein 5 (PP5)/tissue factor pathway inhibitor (TFPI)-2 is increased in the patients with preeclampsia and/or intrauterine growth restriction (IUGR). Here, we have estimated the serum PP5/TFPI-2 in these patients by a sandwich enzyme-linked immunosorbent assay with a newly developed monoclonal antibody, coupled with placental immunohistochemical studies of their placentae with semiquantitative scoring. Serum PP5/TFPI-2 level was significantly elevated only in the patients with preeclampsia alone (p=0.033), while PP5/TFPI-2 was detected significantly less intensely in the placentae of the same patients (p=0.035) in immunohistochemistry, as compared to Controls. A proteoglycan present on the placental villous surface, glypican-3, showed the same pattern of staining as PP5/TFPI-2, and there was a positive correlation (C.I.=0.506, p=0.004) between the immunohistochemical scores for these. Further experiments using HepG2 cells transfected with PP5/TFPI-2 suggested that glypican-3 could anchor PP5/TFPI-2 on the placental villi. A possibility that a decrease in glypican-3 in the placenta increases the outflow of PP5/TFPI-2, which in turn increases its serum level, was proposed. Preeclampsia and IUGR, often regarded to have the same pathological basis in common, showed distinct distributions of PP5/TFPI-2, which could be a clue to elucidate the pathogenesis of preeclampsia and IUGR.

Plasma concentration of tissue inhibitor of matrix metalloproteinase 1 in patients with colorectal carcinoma.

BACKGROUND: The expression of tissue inhibitor of matrix metalloproteinase (TIMP) 1 in tumour tissue from patients with colorectal carcinoma has been reported to be related to disease progression. However, the clinical significance of plasma TIMP-1 has not been fully elucidated. METHODS: The plasma level of TIMP-1 protein was determined by enzyme-linked immunosorbent assay in samples from 54 patients who underwent resection of the primary tumour. RESULTS: Plasma TIMP-1 levels were associated significantly with depth of invasion and metastasis to lymph nodes and liver. Circulating TIMP-1 levels were significantly higher in patients with serosal invasion, liver metastases and Dukes' stage C tumours. Using a cut-off value of 160 ng/ml, serosal invasion and Dukes' C stage could be predicted with an accuracy of 68.5 per cent. With a cut-off value of 170 ng/ml, metastasis to the lymph node and liver could be predicted with an accuracy of 66.7 and 70.4 per cent respectively. These values were greater than those for carcinoembryonic antigen and CA19-9. CONCLUSION: These data suggest that the plasma concentration of TIMP-1 correlates with both invasion and metastasis in patients with colorectal carcinoma.

The inhibitory effects of TNP470 on tumour growth of head and neck carcinoma cell producing interleukin-8.

The anti-tumour effect of the angiogenic inhibitor TNP470, sigma-(chloro-acetyl-carbamoyl) fumagillol, a synthetic analogue of fumagillin, was studied in vitro and in vivo using KB cells, one of the human head and neck carcinoma cell lines that produce interleukin(IL)-8. In the in vitro study, the combination treatment of TNP470 and anti-IL-8 antibody significantly reduced the proliferation of KB cells. In the in vivo studies, TNP470 administration by any route (intratumoral: i.t., intraperitoneal: i.p., intravenous: i.v.) reduced the tumour volume significantly, compared to the control group. Among the groups administered TNP470, the anti-tumour effect was strongest in the it group. Furthermore, the concurrent treatment of anti-IL-8 antibody and TNP470 also maximally reduced the tumour volume. The combination therapy of TNP470 and anti-IL-8 antibody was very effective. These results suggest that combination therapy of TNP470 and anti-IL-8 antibody could be beneficial for solid tumours, such as head and neck cancer.

Intratumoral concentrations of tissue inhibitor of matrix metalloproteinase 1 in patients with gastric carcinoma a new biomartker for invasion and its impact on survival.

BACKGROUND: Previously, the authors clarified that the plasma concentration of tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) in patients with gastric carcinoma was a significant predictor of tumor invasiveness and metastasis. METHODS: To further clarify the clinical significance of TIMP-1, the authors used an enzyme-linked immunoassay to assess TIMP-1 protein concentrations in samples of tumor tissue from 86 patients who underwent primary resection for gastric carcinoma. Concentrations in samples of normal gastric mucosa from 73 of these patients also were assessed. RESULTS: Tissue TIMP-1 concentrations were significantly greater in gastric tumors than in normal gastric mucosa and were associated significantly with a variety of pathologic factors, including macroscopic type, depth of tumor invasion in the gastric wall, presence of lymphatic vessel invasion, pattern of tumor infiltration into the surrounding tissue, and disease stage. Significantly greater TIMP-1 concentrations were found in tumors that were exposed to the serosal surface compared with tumors that were limited to the submucosal layer. TIMP-1 protein was significantly greater in tumors with lymphatic vessel invasion, an infiltrative pattern into the surrounding tissue (INF-gamma), and in tumors from patients with Stage III disease. Survival was significantly poorer in patients with TIMP-1 concentrations > or = 10.0 ng/mg total protein. When patients were stratified by disease stage, survival was significantly different in patients with Stage III disease. Multivariate analysis demonstrated that intratumoral concentrations of TIMP-1 were the most significant independent factor for survival. CONCLUSIONS: These findings suggest that the intratumoral concentration of TIMP-1 protein may be a good indicator of tumor aggressiveness and can serve as a significant independent predictor of survival in patients with gastric carcinoma.

[Induction of apoptosis in breast cancer cells by preoperative oral administration of 5-fluorouracil].

We investigated whether 5-fluorouracil (5-FU) can induce apoptosis in breast cancer cells. Preoperatively, 23 breast cancer patients were divided into a group treated with 5-FU at 200 mg/day for 2 weeks (Group A) and a non-treatment group (Group B), and breast cancer tissues were taken postoperatively. DNA fragmentation by agarose electrophoresis and the TUNEL method were used to investigate the induction of apoptosis. The labeling rate with Ki-67 was measured to study the reproductive activity of tumor cells. The involvement of p53 in the apoptosis decision mechanism was also studied. DNA was more fragmented in Group A than in Group B. The apoptosis index by the TUNEL method was 1.88 +/- 1.03 in Group A, which was significantly higher than 0.36 +/- 0.86 in Group B. The labeling rate with Ki-67 was significantly higher in Group B(62.3 +/- 21.7) than in Group A (29.8 +/- 16.0). There was no difference in the protein expression of p53 regardless of the presence or absence of DNA fragmentation. These results indicate that 5-FU administration induces apoptosis in breast cancer cells and significantly inhibits their reproductive activity. Involvement of p53 in the apoptosis decision mechanism was not demonstrated.

An experimental model of tumor dormancy therapy for advanced head and neck carcinoma.

An experimental model of tumor dormancy therapy for advanced head and neck carcinoma was developed. After transplantation of KB cells into nude mice, the mice were given tiracoxib, a selective cyclooxygenase (COX)-2 inhibitor, probucol, an antioxidant, and S-1, an oral pro-drug of 5-fluorouracil (5-FU), or combinations of two of them. The combined administration of tiracoxib with probucol significantly inhibited the tumor growth. The angiogenesis in this group was markedly reduced. Tiracoxib and probucol did not affect the intratumoral concentration of 5-FU when coadministered with S-1. The combined use of tiracoxib and probucol is thus a candidate for use in maintenance therapy after the primary therapy for patients with advanced head and neck carcinoma.

Therapeutic effect of 1 M tegafur-0.4 M 5-chloro-2, 4-dihydroxypridine-1 M potassium oxonate (S-1) on head and neck squamous carcinoma cells.

1 M Tegafur (FT)-0.4 M 5-chloro-2,4-dihydroxypridine (CHDP)-1 M potassium oxonate (Oxo) (S-1), was developed as a new oral antineoplastic agent based on biochemical modulation of fluorouracil (5-FU) by CHDP and Oxo. The antitumor effect of S-1 on human head and neck squamous carcinoma cells was evaluated in xenografts and a metastasis model, in comparison with combined drug of 1 M FT and 4 M uracil (UFT). Mice treatment with S-1 showed a significant higher concentration of 5-FU in the tumor and the serum than UFT treated mice. S-1 showed higher tumor growth inhibition and metastasis inhibition than UFT. The mice in which metastasis was inhibited lived more than twice as long as the control mice. These results suggest that S-1 will have a higher clinical therapeutic effect against advanced squamous cell carcinoma of the head and neck in humans.

Six-year disease-free survival of a patient with metastatic eyelid squamous cell carcinoma and colon adenocarcinoma after repeated postoperative adoptive immunotherapy.

A 74-year-old male was affected concurrently with squamous cell carcinoma of the left eyelid and adenocarcinoma of the colon, both with lymph node metastasis. He underwent exenteration of the left orbit with left modified radical neck dissection and subsequently resection of the transverse colon with regional lymph node dissection. The patient has been treated by an adoptive immunotherapy as a sole postoperative modality without receiving any chemotherapeutic agents causing immunosuppression. For the adoptive immunotherapy, autologous peripheral blood lymphocytes were activated with an immobilized anti-CD3 antibody and IL-2 for 14 days (the CD3-AT cells). The infusion with 1.38 x 10(10) CD3-AT cells has been repeated 150 times in total at the time of writing. Neither recurrence nor additional metastasis has been detected for 6 years after surgery.

Prognostic value of tissue inhibitor of matrix metalloproteinase-1 in plasma of patients with gastric cancer.

Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in plasma has been reported to be related to disease progression in patients with gastric cancer. However, the prognostic significance of plasma TIMP-1 concentrations has not been clarified. Concentrations of TIMP-1 protein were measured by enzyme-linked immuno-sorbent assay in plasma samples of 147 preoperative patients who subsequently underwent gastric resection, and prognosis was compared. The cut-off value of plasma TIMP-1 concentrations was defined as 112.5 ng/ml, referring to the TIMP-1 levels in patients with intramucosal gastric cancer. Twenty-nine out of 147 patients had higher plasma TIMP-1 levels than the cut off value. When the patients were divided into those with elevated values and those with normal TIMP-1, such parameters as age, serosal invasion, metastases to lymph nodes, peritoneum, and liver, lymphatic invasion, curability, and stage were significantly different between the two. By univariate analysis of the factors affecting survival, macroscopic type, histology, serosal invasion, metastasis to lymph node, peritoneum, and liver, vessel invasions, curability, and plasma TIMP-1 were significant. However, multivariate analysis revealed that TIMP-1 was the only significant factor. In patients with gastric cancer, plasma TIMP-1 seem to be an independent and most powerful prognosticator for the survival.

Plasma concentrations of VEGF and bFGF in patients with gastric carcinoma.

We examined plasma levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in 54 patients with gastric carcinoma. Postoperative survival was significantly poorer in patients with plasma VEGF levels more than 10.0 pg/ml at the time of surgery. By an univariate analysis of the factors affecting survival, serosal invasion, lymph node metastasis, peritoneal dissemination, lymphatic vessel invasion, curability, and VEGF proteins were significant. By a multivariate analysis only VEGF levels and curability remained significant. Patients with recurrent disease, including liver metastasis, had significantly higher plasma VEGF concentrations than those with resectable primary tumors. VEGF, not bFGF, may serve as an independent prognosticator and a sensitive indicator for liver recurrence in patients with gastric carcinoma.

Marked increase of trypsin(ogen) in serum of linitis plastica (gastric cancer, borrmann 4) patients.

Linitis plastica, or Borrmann 4 gastric cancer, shows very poor prognosis, and the reason has not been understood. In the present study, we examined serum levels of trypsin(ogen) in 44 gastric cancer patients, including 17 early gastric cancer, 18 non-Borrmann 4 advanced gastric cancer, and 9 Borrmann 4 gastric cancer, by using the RIA gnost Trypsin kit (Hoechst Japan, Tokyo, Japan), which was expected to detect trypsin-1, trypsin-2, trypsinogen-1, and trypsinogen-2 in sera. The trypsin(ogen) concentration was much higher in the patients with linitis plastica than in the other gross types of gastric cancer. Hypertrypsinemia was identified in approximately 60% of advanced gastric cancer cases. Lymph node involvement, liver metastasis, or poorly differentiated adenocarcinoma is an important factor of hypertrypsinemia. The serum trypsin(ogen) level in linitis plastica patients was 3484.4 +/- 2319.7 ng/ml (mean +/- SD), which was significantly higher not only than that of the early gastric cancer (384.1 +/- 92.1) but also the stage IV gastric cancer patients (578 +/- 440.4), excluding those with linitis plastica. The elevated serum trypsinogen level in linitis plastica patients may be related to the malignant behavior of this type of cancer cell. Serum trypsin(ogen) of linitis plastica shows significantly higher concentrations than do the other types of advanced gastric cancer. Therefore, serum concentration of trypsin(ogen) might be a good marker of gastric cancer of linitis plastica.

Therapeutic efficiency of IL-2 gene transduced tumor vaccine for head and neck carcinoma.

Transduction of the human interleukin-2 (IL-2) gene into tumor cells was carried out in order to develop a new immunotherapy for advanced head and neck carcinomas with a poor outcome. We transduced the IL-2 gene into KB cells, a head and neck squamous cell carcinoma cell line, using a defective herpes simplex viral (HSV) amplicon vector as a gene transfer vehicle. A high level of IL-2 was secreted by IL-2 gene-transduced KB cells (KB/IL-2). The IL-2 producibility of irradiated KB/IL-2 cells was almost the same as that of non-irradiated cells. In the tumor establishment model in nude mice, IL-2 and interferon-gamma (IFN-gamma) at high concentrations were detected in the sera of mice transplanted with KB/IL-2 cells. The spleen cells of nude mice transplanted with KB/IL-2 cells exhibited high cytotoxic activity compared to those from mice transplanted with KB cells and from untreated mice. Three of five mice transplanted with KB/IL-2 cells rejected tumors. In the treatment of established tumors, therapeutic effects due to irradiated KB/IL-2 were dose-dependent. The suppressive effects on tumor growth were blocked by anti-asialo GM1, anti-human IL-2 and anti-IFN-gamma antibodies. Immunohistochemical observation revealed the presence of asialo GM1(+) cells among the KB/IL-2 cells in tumors transplanted into nude mice.

Angiogenesis inhibitor, TNP-470, suppresses growth of peritoneal disseminating foci.

BACKGROUND/AIMS: Angiogenesis is critical not only for growth of primary tumors but also for cells established at distant organs. We investigated the effects of angiogenesis inhibitor, TNP-470, on the establishment and growth of intraperitoneally inoculated human gastric cancer cell line, MKN-45, and survival of nude mice with this tumor. METHODOLOGY: Human gastric cancer cell line, MKN-45, were injected into the peritoneal cavity of an ICR nude mouse and a model of peritoneal dissemination was developed. TNP-470 was injected subcutaneously every other day from day 1 until sacrifice or death. The effects of TNP-470 on MKN-45 cells were also examined in vitro. RESULTS: Although the number of disseminated foci was not significantly different, the maximum size was significantly smaller in a TNP-treated group than those of a control. Survival time was significantly longer in a TNP-treated group. TNP-470 demonstrated no growth inhibition of MKN45 cells in vitro. CONCLUSIONS: Those results suggested that anti-angiogenic agent, TNP-470, might be effective in treating peritoneal dissemination of gastric cancer by inhibiting growth of the seeded tumor cells on the peritoneum.

Overexpression of laminin gamma2 chain monomer in invading gastric carcinoma cells.

Laminin (LN)-5, a heterotrimer of alpha3, beta3, and gamma2 chains, has been suggested to be involved in tumor cell invasion. The present immunohistochemical study investigated the distribution of the LN gamma2 chain in 48 different human gastric adenocarcinomas. The immunohistochemical analysis showed two distinct patterns of LN gamma2 chain expression: (a) extracellular deposition; and (b) cytoplasmic accumulation. The extracellular deposition of the LN gamma2 chain was typically observed at neoplastic basement membranes of well-differentiated adenocarcinomas. The immunoreactivity was continuous along tumor basement membranes in these tumors but was irregular and diffuse in poorly differentiated carcinomas. These tumor cells coexpressed the LN alpha3 and beta3 chains, suggesting that the LN gamma2 chain was deposited as the LN-5 complex. In contrast, tumor cells at the invading fronts showed strong cytoplasmic staining for the LN gamma2 chain without any detectable signal for the LN alpha3 or beta3 chain in both well- and poorly differentiated carcinomas. On the other hand, in vitro analysis by two-dimensional SDS-PAGE demonstrated that human gastric carcinoma cells secrete a high level of LN gamma2 chain monomer in addition to the LN-5 complex into culture medium. These results indicate that the LN gamma2 chain can be secreted as a single subunit and might be involved in tumor cell invasion.

A selective cyclooxygenase-2 inhibitor suppresses tumor growth in nude mouse xenografted with human head and neck squamous carcinoma cells.

The anti-tumor effect of a selective cyclooxygenase (COX)-2 inhibitor, JTE-522, was examined with the human head and neck squamous cell carcinoma cell line KB. KB cells do not produce prostaglandin (PG)-E2. In vitro, JTE-522 induced an increase of G1 phase-arrested cells, suppression of platelet-derived growth factor (PDGF) production and inhibition of telomerase activity. No cytotoxic effect was detected. In vivo, the growth of the tumor xenografted into nude mice was significantly suppressed by JTE-522. Suppression of angiogenesis at the periphery of the tumor, increase of G1-arrested cells and suppression of telomerase activity were observed, together with an increase of apoptotic cell death in the tumor. Immunological enhancement did not play a role. We concluded that the anti-tumor effect of JTE-522 was caused by anti-angiogenesis action, cell cycle arrest and inhibition of telomerase activity of the tumor cells. These combined effects might induce apoptosis.

[Distribution of lymphocytes to tumor tissue and regional lymph nodes in patients with gastric cancer--the effects of oral administration of OK-432].

We attempted to clarify the accumulation of radiolabeled lymphocytes to tumors and regional lymph nodes in patients with gastric carcinoma. The effects of oral administration of OK-432 were also studied. Five patients with gastric cancer and one who underwent endoscopic mucosal resection, were entered in the study. Prior to the tracer study in 3 patients with gastric cancer, 5 KE of OK-432 was administered for 2 days. Peripheral mononuclear cells were separated and labeled with [111] In-tropolone. After the resection of stomach, tumor tissue, normal gastric mucosa, regional lymph nodes, and non-regional lymph nodes were dissected and radioactivity was measured by a gamma-counter. Accumulation of lymphocytes to the tumor tissue and n1 lymph node station was more than two times that in the normal gastric mucosa and ten times that in non-regional lymph nodes. Accumulation of lymphocytes to the n2 station was strongly enhanced by oral administration of OK-432.

Tissue inhibitor of matrix metalloproteinase-1 in the plasma of patients with gastric carcinoma. A possible marker for serosal invasion and metastasis.

BACKGROUND: Expression of the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in tumor tissue from patients with gastric carcinoma has been reported to be related to disease progression. However, to the authors' knowledge the clinical significance of plasma TIMP-1 concentrations in these patients has not been clarified. METHODS: Concentrations of TIMP-1 protein were examined by enzyme-linked immunoadsorbent assay in plasma samples from 149 patients who underwent resection of their primary tumors and from 18 patients with nonresected or recurrent disease. RESULTS: In the 149 patients whose primary tumors were resected, plasma TIMP-1 concentration was associated significantly with a variety of pathologic factors including macroscopic type, depth of invasion, lymph node and peritoneal metastases, vessel invasion, pattern of tumor infiltration into surround ing tissue, and disease stage. Plasma TIMP-1 concentration was significantly higher in patients with serosal invasion, lymph node metastasis, peritoneal dissemination, or liver metastasis than in those without these factors. Neither carcinoembryonic antigen (CEA) nor CA 19-9 concentrations appeared to be related to these measures of disease progression. In the 18 patients with nonresected or recurrent disease, TIMP-1, CEA, and CA 19-9 were similarly sensitive in predicting peritoneal, liver, and lymph node metastases. The combination of these three factors was able to detect 73.3% of patients with peritoneal metastasis, 83.3% of patients with liver metastasis, and 88.9% of patients with disease recurrence. CONCLUSIONS: In patients with gastric carcinoma, plasma concentration of TIMP-1 appears to correlate with both serosal invasion and metastasis.

Expression of Epstein-Barr virus in mesopharyngeal and hypopharyngeal carcinomas.

Epstein-Barr virus (EBV) causes various tumors, including nasopharyngeal carcinoma (NPC). There have been no reports as to whether the carcinogenicity of EBV is restricted to the nasopharynx or extends into the mesopharynx and hypopharynx. We attempted to ascertain the relation between EBV and mesopharyngeal (MPC) and hypopharyngeal carcinomas (HPC). Messenger RNA in situ hybridization showed that all 29 cases of MPC and 5 of 12 HPC expressed EBV mRNA. For further analysis, we established 7 cell lines from 5 MPC and 2 HPC. All cell lines and 5 tumors formed by these cultured cells in nude mice expressed EBV transcripts. Moreover, immunofluorescence staining showed expression of EBV-related nuclear antigen-2 and latent membrane protein-1 (LMP1) in the original tumors and the cell lines, as well as in nude mouse tumors. Study by reverse transcription polymerase chain reaction (RT-PCR) also showed EBER1 and LMP1 expression. Furthermore, lytic-cycle antigens of EBV were detectable in most cell lines. Nested PCR showed the EBV genome in 3 cases of MPC and 4 cases of HPC. These results suggest that EBV plays an important role in the development of MPC and HPC as well as in NPC.

Expression of trypsin in human cancer cell lines and cancer tissues and its tight binding to soluble form of Alzheimer amyloid precursor protein in culture.

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.