Expression of interleukin-6 and its effect on the cell growth of gastric carcinoma cell lines

The expression and the effect of IL-6 were examined in human gastric carcinoma cell lines to determine whether IL-6 serves as a growth stimulator. The expression of IL-6 mRNA was detected in three (TMK-1, MKN-1, MKN-7) of 8 gastric carcinoma cell lines. All three cell lines secreted IL-6 into the culture fluid, in large amounts in the cases of MKN-1 and MKN-7 cells. Scatchard plot analysis of IL-6 binding revealed that MKN-1 and MKN-7 cells had both high- and low-affinity receptors. Cell growth of MKN-1 and MKN-7 cells was stimulated by IL-6, while anti-IL-6 antibody inhibited growth. The expression of IL-1 alpha mRNA by these three cell lines was induced by IL-6. IL-1 alpha increased the expression of mRNA for IL-6 by TMK-1 cells. These findings indicate that IL-6 induced by IL-1 alpha is an autocrine growth factor for some gastric carcinomas.     

Expression of sialosyl-Tn antigen (monoclonal antibody MLS102 reactive) in normal tissues and malignant tumors of the digestive tract

Oncogenic transformation is often associated with changes in the glycosylation state of malignant cells. We investigated the immunohistochemical localization of sialosyl-Tn antigen [O-linked NeuAc(alpha 2-->6)GAINAc] using a novel monoclonal antibody MLS102 in normal and malignant digestive-tract tissues. In normal tissues, weak MLS102 immunoreactivity was observed in the epithelium of the esophagus, stomach and colon. However, MLS102 immunoreactivity was strong in the goblet cells of the duodenum, but not in the Brunner glands. In carcinomas of the esophagus, stomach, colon, pancreas and biliary tract, positive staining was detected with a high frequency (80%-100%). In mucinous carcinomas and signet-ring cell carcinomas, malignant cells themselves and the mucins they secreted were strongly positive for sialosyl-Tn antigen. There was no significant correlation between the frequency of expression of sialosyl-Tn antigen and the degree of differentiation (grade). However, in the case of well-differentiated adenocarcinomas, sialosyl-Tn antigen was found mainly in the supranuclear areas (Golgi area), on the apical surface and in the adjacent cytoplasm. In poorly differentiated adenocarcinomas, the antigen was often detected in the whole plasma membrane and cytoplasm. Therefore, monoclonal antibody MLS102 may be useful in further elucidating the characteristics of digestive-tract cancers, and possibly in their treatment.     

Expression of amphiregulin, a novel gene of the epidermal growth factor family, in human gastric carcinomas

The expression of mRNA for amphiregulin (AR), a novel gene of the epidermal growth factor family, was examined in 8 human gastric carcinoma cell lines and 32 gastric carcinoma tissues as well as corresponding normal mucosa. Of the 8 gastric carcinoma cell lines, 7 expressed 1.4 kb AR mRNA at various levels. The expression of AR mRNA by TMK-1 and MKN-28 cells was increased by treatment with epidermal growth factor or transforming growth factor a. In surgical cases, all the gastric carcinoma tissues and their adjacent normal mucosa expressed AR mRNA. Interestingly, 20 (62.5%) out of 32 tumors expressed AR mRNA at higher levels than their corresponding normal mucosas (tumor/normal > or = 1.2). No obvious correlation was observed between the AR mRNA levels and the histological types or tumor staging of gastric carcinoma. Immunohistochemically, AR protein was localized to the cytoplasm and/or nucleus in tumor cells. These results suggest that AR produced by tumor cells may be involved in the pathogenesis and/or progression of human gastric carcinoma.     

Antibacterial activity of extracts and constituents of Pelargonium sidoides and Pelargonium reniforme

Tumor angiogenesis is essential for solid tumor growth. The object of this study was to investigate the presence of newly identified angiogenic factor, placenta growth factor (P1GF) in human cervical cancers. The expression of P1GF mRNA was assessed by RT-PCR in 29 patients with cervical cancer. Fifteen out of 29 cervical cancers expressed a certain level of P1GF mRNA. In addition, the expression levels of P1GF mRNA in squamous cell carcinomas were significantly higher than those in adenocarcinomas. There was no correlation between the expression of P1GF mRNA and FIGO stage. These results indicate that the expression of P1GF may be implicated in the promotion of angiogenesis in human cervical squamous cell carcinomas, but not in human cervical adenocarcinomas.     

Vascular endothelial growth factor expression is higher in differentiated thyroid cancer than in normal or benign thyroid

Vascular endothelial growth factor (VEGF) is an angiogenic factor, and its expression has been rarely demonstrated in thyroid tumors. We, therefore, investigated the expression of VEGF messenger RNA (mRNA) and production of VEGF protein in cell lines from human primary and metastatic follicular (FTC-133, FTC-236, and FTC-238), papillary (TPC-1), Hürthle cell (XTC-1), and medullary thyroid cancers (MTC-1.1 and MTC-2.2), and in human thyroid tissues (papillary, follicular, medullary, and Hürthle cell cancers, follicular adenomas, and Graves' thyroid tissue) by Northern blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) studies. All thyroid cell lines expressed a 4.2-kilobase VEGF mRNA. The VEGF mRNA levels were higher in the thyroid cancer cell lines than in primary cultures of normal thyroid cells, and higher in thyroid cancers of follicular than those of parafollicular cell origin. The VEGF mRNA levels were similar in primary and metastatic thyroid tumors. Immunohistochemical staining and Northern blot analysis of the cell lines correlated positively, thus thyroid cancer cell lines stained more intensely than normal thyroid cells and follicular tumor cells more intensely than parafollicular tumor cells. Again, no difference was noted in VEGF staining between primary and metastatic thyroid tumors. Deparafinized sections of papillary, follicular, and Hürthle cell cancers also stained much stronger than those of medullary thyroid cancers, benign, or hyperplastic (Graves' disease) thyroid tissue. Thyroid cancer cell lines (XTC-1 > TPC-1 > FTC-133 > MTC-1.1) also secreted more VEGF protein as measured by ELISA than did normal thyroid cells. VEGF secretion of cell lines derived from primary and metastatic thyroid tumors were similar. VEGF mRNA is therefore expressed, and VEGF protein is secreted by normal, hyperplastic, and neoplastic thyroid tissues. The higher levels of VEGF expression in differentiated thyroid cancers of follicular cell origin suggests a role in oncogenesis.     

Significance of vessel count and vascular endothelial growth factor and its receptor (KDR) in intestinal-type gastric cancer

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors by host and/or tumor cells. The role of angiogenesis and angiogenic factor expression in intestinal- and diffuse-type gastric cancer are undefined. Archival specimens of 51 intestinal-type and 38 diffuse-type human gastric carcinomas were examined for tumor vessel counts, angiogenic factor expression, and the presence or absence of angiogenic factor receptors on tumor endothelium using antibodies against vascular endothelial growth factor (VEGF) and its receptors (KDR and flt-1), basic fibroblast growth factor (bFGF) and its receptors (bek and flg), and factor VIII (endothelial cells). Vessel count and VEGF and bFGF expression were higher in intestinal-type than in diffuse-type gastric cancers (P = 0.01, P < 0.001, and P < 0.001, respectively). Similarly, vessel count and VEGF expression were higher in patients with liver metastasis than in patients with peritoneal dissemination (P = 0.003 and P = 0.01, respectively). Vessel count correlated with VEGF expression and the presence of endothelial KDR in intestinal-type gastric cancer (P = 0.003 and P = 0.02, respectively) but not diffuse-type gastric cancer. Vessel count, VEGF expression, and presence of endothelial KDR increased with increasing stage of disease in intestinal-type gastric cancer but not diffuse-type gastric cancer. The expression of bFGF and its receptors did not correlate with vessel count in either cancer type. These findings suggest that the pattern of metastasis in intestinal-type gastric cancer is angiogenesis dependent. The correlation of VEGF expression and its endothelial receptor with vessel count and stage of disease suggests that VEGF is at least one of the factors responsible for the induction of angiogenesis in intestinal-type gastric cancer.     

Pathological characteristics of gastric carcinomas in the very old

A total of 69 gastric carcinomas of very old people (aged > or = 85) were collected and pathologically analyzed in comparison with those of young to middle-aged (30-39) and elderly (65-69) people, with special attention to their phase. In the very old, almost all (34/35) carcinomas in the early phase belonged to well-differentiated categories. In the advanced phase, half of them (17/34) were classified into poorly differentiated categories when determined from the predominant pattern, but a well-differentiated pattern almost always coexisted in the superficial site. Thus, the gastric carcinomas in the very old may principally develop as well-differentiated carcinomas which then progress to poorly differentiated carcinomas with time, in contrast to those of the young to middle-aged, most of which emerged from the very early phase as poorly differentiated lesions. The gross features of the carcinomas were also in line with these histological observations. The carcinomas of the elderly showed distinct similarity to those of the very old. The results suggest that poorly differentiated carcinomas of the young to middle-aged and the old may be better classified and analyzed separately in view of the generally recognized etiological (e.g., specifically close causal relationship with environmental factors of the intestinal-type carcinoma of the old) and biological (e.g., practically no tendency for hematogenous metastasis of the diffuse-type carcinoma of the young to middle-aged) differences, although in the General Rules for Gastric Cancer Study of Japan, both are placed in the same category, por (por2).     

Immunohistochemical study of growth factors and oncogenes in gastric carcinomas

Immunohistochemical staining for EGF, EGFR, c-erbB-2, p53, K-ras and PCNA was performed on the formalin-fixed, paraffin embedded sections of resected gastric carcinomas. A relatively high positive rate was observed for EGFR and c-erbB-2 in the well-differentiated adenocarcinomas and p53 in the poorly-differentiated adenocarcinomas. The positive rate of these factor was higher in the advanced cases than in the early cases, and also in the deep invasive area than the superficial area. According to the PCNA staining, a relatively high positive rate was observed in the well-differentiated adenocarcinomas compared with the early cases of poorly-differentiated adenocarcinomas, but the positive rate was markedly higher in the advanced cases of the latter. Typical signet-ring cell carcinomas showed the lowest positivity rate compared with the other histological types of gastric carcinomas.     

Vascular endothelial growth factor, wild-type p53, and angiogenesis in early operable non-small cell lung cancer

Vascular endothelial growth factor (VEGF) is a cytokine that is involved in tumor angiogenesis. Wild-type p53 (wt-p53) protein has been shown in cell lines to suppress angiogenesis through thrombospondin regulation. In this study, we immunohistochemically examined the expression of VEGF, nuclear and wild-type cytoplasmic p53, bcl-2, epidermal growth factor receptor, and c-erbB-2 oncoprotein; vascular grade; proliferation index; and extent of necrosis in non-small cell lung cancer (NSCLC). We analyzed 120 cases of early-stage NSCLCs (81 squamous cell carcinomas and 39 adenocarcinomas) treated with surgery alone (median follow-up, 63 months; range, 45-74 months). VEGF expression showed a positive association with high vascular grade (microvessel score of >75 per x250 field; P = 0.008), although about half of the LVG cases also expressed VEGF. None of the p53 antibodies examined correlated with angiogenesis. However, wt-p53 expression was inversely associated with VEGF expression, suggesting that wt-p53 is involved in the suppression of the VEGF gene. Combined analysis of VEGF, wt-p53, and microvessel counting showed that, although wt-p53 loss associates with VEGF switch-on, p53 protein may not be involved in the regulation of the angiogenic events downstream of VEGF expression. Moreover, no significant association of bcl-2 and c-erbB-2 oncoprotein expression with VEGF expression was observed. T/N stage, grade, Ki67 proliferation index, and extent of necrosis were not correlated with VEGF expression. Survival analysis showed that VEGF correlated with poor survival (P = 0.04) and was significant in node-negative cases (P = 0.03). We conclude that VEGF is an important angiogenic factor in NSCLC, its expression being dependent on wt-p53 loss.     

MUC-1 expression as a predictor of the curative endoscopic treatment of submucosally invasive colorectal carcinoma

PURPOSE: This study was undertaken to clarify the clinical significance of MUC-1 expression in the endoscopic treatment of colorectal carcinoma with submucosal invasion. METHODS: One hundred eighty-four colorectal carcinomas with submucosal invasion were examined. The depth of submucosal invasion was classified as scanty or massive. The histologic subclassification at the deepest invasive portion was defined as well-differentiated, moderately well-differentiated, moderately to poorly differentiated, poorly differentiated, or mucinous adenocarcinoma. MUC-1 expression was examined immunohistochemically at the deepest invasive portion. In addition, the Ki67 labeling index was also examined immunohistochemically. RESULTS: Lymph node metastases were detected in 28 (15.2 percent) of 184 lesions. Lesions with both scanty submucosal invasion and well-differentiated or moderately well-differentiated adenocarcinomas had no lymph node metastases. MUC-1 expression was detected in 88 (47.8 percent) of 184 lesions and correlated significantly with the presence of lymph node metastases. The Ki67 labeling index also correlated significantly with lymph node metastases. Furthermore, lesions with both MUC-1-negative and low Ki67 labeling index showed no lymph node metastases, even in lesions with massive submucosal invasion. Multivariate analysis indicated that MUC-1 expression was one of the most important risk factors for lymph node metastases and histologic grade among the clinicopathologic factors usually examined. CONCLUSION: MUC-1 expression is one of the accurate predictors of the presence of lymph node metastases among the clinicopathologic factors commonly used. Combined analysis of MUC-1 expression and Ki67 labeling index may be a useful indicator of lymph node metastases and may broaden the indications for the curative endoscopic treatment of carcinoma with massive submucosal invasion.     

Angiogenesis correlates with vascular endothelial growth factor expression but not with Ki-ras oncogene activation in non-small cell lung carcinoma

A total of 195 non-small cell lung carcinoma (NSCLC) specimens were studied for the presence of mutations in their ras family genes, for tumor vascularity, and for their immunostaining pattern with an antibody to vascular endothelial growth factor (VEGF). ras mutation was found in 37 of 104 (34.6%) adenocarcinoma specimens, in 0 of 64 squamous cell carcinomas, and in 2 of 27 (7.4%) large cell undifferentiated carcinomas. All mutations were found on the Ki-ras gene, with 37 (95%) of them on codon 12 and the remaining 2 on codon 13. Thirty (77%) of the mutations were G to T transversions. There was a correlation between increasing tumor vascularity and VEGF immunostaining score, but there was no correlation between either of them with the activation of the ras oncogene. A study of VEGF mRNA expression in 14 NSCLC cell lines also demonstrated a lack of correlation between the constitutive expression levels of VEGF and the presence or absence of ras mutation in these cell lines. The results suggest that VEGF is a major angiogenesis factor in NSCLC but that other factors beside ras mutations may influence tumor vascularity in these tumors. The two parameters may potentially serve as independent prognostic factors in NSCLC.     

Expression of costimulatory molecules, B7-1 and B7-2 on human gastric carcinoma

Costimulation of T cells via B7-1 and B7-2 molecules on a tumor has been shown to be important for eliciting cell-mediated antitumor immunity. We studied the surface expression of B7-1 and B7-2 in 24 cases of gastric carcinoma from the primary locus, 20 cases of metastatic carcinoma from malignant ascites, 20 cases of benign gastric mucosa and 7 gastric carcinoma cell lines by two-color flow cytometry with mAb CD80 and CD86. The B7-1 and B7-2 molecules were expressed by 6 cell lines, and 1 cell line showed the predominant expression of B7-2 but not B7-1. Almost all patients with primary gastric carcinoma and benign gastric mucosa showed high levels of expression of the B7-1 and B7-2, revealing approximately 40%-60% positive cells. However, the percentage of B7-1-positive cells of poorly differentiated primary carcinomas was significantly lower than that of well-differentiated carcinoma and normal mucosa (P < 0.01). Furthermore, all of the metastatic carcinoma cells revealed consistently very low or undetectable levels of expression of the B7-1 molecule, only 8% (mean) of cells being positive, despite showing higher levels of B7-2 expression. Thus, it seems likely that decreased or deleted expression of B7-1 correlates with the grade of tumor differentiation, tumor progression and metastasis. These results suggest that the B7-1 molecule on the gastric carcinoma bearing CD80+CD86+ is abrogated during tumor invasion and/or metastasis, and the tumor finally acquires the CD80-CD86+ phenotype. Consequently, inadequate B7-1 costimulation may contribute to the escape of tumors from destruction by the host's immune system.     

Vascular endothelial growth factor expression in head and neck squamous cell carcinoma

BACKGROUND: Angiogenesis is an essential process required for growth and metastasis in cancer. In breast, gastric, and prostate cancer, vascular endothelial growth factor (VEGF) has been implicated in angiogenesis; however, little is known about VEGF in HNSCC. In this study, we hypothesize that VEGF is present in elevated levels in HNSCC and may therefore play a role in promoting angiogenesis. METHODS: We obtained tumor tissue from 63 HNSCC patients undergoing primary resection. All tissue samples were analyzed by immunohistochemistry (IHC) techniques for the presence and localization of VEGF; however, only 36 had sufficient amounts of tissue for quantitative analysis of VEGF by ELISA. Nine control specimens taken from patients undergoing uvulopalatopharyngoplasty were also analyzed. RESULTS: In all 63 of our patient samples we found VEGF to be present and localized to the cancer cells and endothelial cells. The poorly differentiated cancer cells stained more intensely in comparison with the well-differentiated ones. There was a 20-fold increase in the patient levels when compared with controls levels (P > or =0.05). Analysis by enzyme-linked immunosorbent assay revealed elevated mean levels of VEGF (241 +/- 326 pg/mg total protein [TP]) with a range of 2 to 1484 pg/mg TP. The control specimens had mean levels of 13 +/- 11 pg/mg TP and a range of 1 to 78 pg/mg TP. Patients who exhibited higher levels of VEGF tended to have a higher rate of disease recurrence (P < or =0.048) and shorter disease-free interval (P < or =0.05). CONCLUSIONS: The expression of VEGF in elevated levels in the HNSCC tumor microenvironment appears to be associated with more aggressive disease. Based on our results, VEGF may be an important angiogenic factor associated with cancer cells and endothelial cells in HNSCC. Further studies are needed to better define the role of VEGF in HNSCC and its role as a potential target for therapeutic intervention.     

Sociosomatics and illness in chronic fatigue syndrome

Angiogenesis is essential for tumour growth and important in tumour metastasis and prognosis. Vascular endothelial growth factor (VEGF) stimulates endothelial proliferation in vitro and angiogenesis in vivo. VEGF expression has been correlated with high vascularity in tumours, including carcinoma of the breast. This study investigated VEGF expression and vascularity of invasive lobular (n = 10) and invasive ductal carcinoma (n = 28), and pure ductal carcinoma in situ of the breast (n = 33). VEGF protein expression was studied with immunohistochemistry and VEGF mRNA with in situ hybridization. Vascular density was assessed on sections stained for von Willebrand factor. There was more expression of both VEGF protein (P = 0.006) and mRNA (P = 0.002) in invasive ductal than in invasive lobular carcinoma. VEGF protein (rs = 0.32, P = 0.047) and mRNA (rs = 0.56, P = 0.04) correlated with vascular density in invasive ductal carcinoma. In invasive lobular carcinoma, vascular density did not correlate with VEGF mRNA (rs = 0.15, P = 0.35) and was inversely related to VEGF protein (rs = -0.57, P = 0.04). There were no significant differences in vascular density between the two types of invasive carcinoma, suggesting that VEGF is important in angiogenesis in invasive ductal carcinoma, but that other angiogenic factors are important in invasive lobular carcinoma. Although VEGF protein was frequently expressed in ductal carcinoma in situ, no relationship was found between VEGF and the two patterns of angiogenesis previously described.     

Activation of stress-activated protein kinases by hepatocyte isolation induces gene 33 expression

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by acting as a potent inducer of vascular permeability as well as serving as a specific endothelial cell mitogen. The importance of angiogenic factors such as VEGF, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. We examined the expression of mRNA and protein for VEGF in 12 human hematopoietic tumor cell lines, representing multiple lineages and diseases, including leukemia, lymphoma, and multiple myeloma. Our results revealed that VEGF message was expressed in these cells and that the corresponding protein was secreted into the extracellular environment. Five of the 12 cell lines were also found to express the Flt-1 receptor for VEGF at a moderate to strong level, suggesting an autocrine pathway. When human vascular endothelial cells were exposed to recombinant human VEGF, there was an increase in the mRNA for several hematopoietic growth factors including macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 6. Plasma cells in the bone marrow from patients diagnosed with multiple myeloma were found to express VEGF, whereas both the Flt-1 and KDR high affinity VEGF receptors were observed to be markedly elevated in the normal bone marrow myeloid and monocytic cells surrounding the tumor. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through either a paracrine or an autocrine mechanism.     

Proliferative activity of gastric cancer assessed by immunostaining for proliferating cell nuclear antigen

The growth activity of 107 gastric carcinomas was assessed by immunohistochemical staining for formalin-fixed, paraffin-embedded tissue with a monoclonal antibody against proliferating cell nuclear antigen (PCNA). When the tumor doubling times (Tds) of 10 patients were estimated from the serum levels of carcinoembryonic antigen and carbohydrate antigen 19-9, there was an inverse correlation between the Tds and PCNA labeling index (LI) at P = 0.055. Flow-cytometric analysis was carried out by double staining for PCNA and DNA using fresh materials from 14 patients. The PCNA-positive cell fraction revealed by flow cytometry showed a good linear correlation with PCNA LI in routinely stained tissue. The LI of well-differentiated adenocarcinoma was significantly higher than that of the poorly differentiated type. When the LI was analyzed in well- or poorly differentiated adenocarcinoma, the value was significantly higher in the well-differentiated type with hepatic metastasis and in the poorly differentiated type with lymph node metastasis.     

Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker

Hypoxia in human tumors is associated with poor prognosis, but the molecular mechanisms underlying this association are poorly understood. One possibility is that hypoxia is linked to malignant progression through vascular endothelial growth factor (VEGF) induction and the associated angiogenesis and metastasis. The present clinical study measures hypoxia and VEGF expression on a cell-by-cell basis in human squamous cell carcinomas to test the hypothesis that hypoxia and VEGF protein expression are coupled in human tumors. Eighteen patients with invasive squamous cell carcinoma of the uterine cervix and head and neck have been investigated by a quantitative image analysis of immunostained sections from their tumors. The hypoxia marker pimonidazole was used to measure tumor hypoxia, and a commercially available antibody was used to measure VEGF protein expression. A quantitative immunohistochemical comparison of hypoxia and VEGF protein expression revealed no correlation between the two factors.