ABSTRACT
Gastric carcinomas can be classified into scirrhous carcinomas (SC), i.e. 'linitis plastica' or Borrmann 4 gastric cancer, and non-scirrhous carcinomas (NSC). SC are characterized by diffuse invasive growth patterns with marked fibrosis, frequent peritoneal dissemination and lymph-node metastases and poor prognosis, while NSC show medullary growth patterns and common hematogenous metastases. To study the differences in local expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) between SC and NSC, we examined the expression of MMPs and TIMPs in human gastric carcinoma tissues by several methods including sandwich-enzyme immunoassay systems, gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR, immunoblotting, immunohistochemistry and in situ zymography. Of the seven MMPs and two TIMPs tested, only proMMP-2 levels were remarkably higher in SC than in NSC (P < 0.01), and proMMP-2 activation ratio was significantly lower in SC than in NSC (P < 0.05). TIMP-3 mRNA levels were remarkably about 2-fold higher in SC than in NSC tissues (P < 0.01). TIMP-3 production in SC was confirmed by immunoblotting and TIMP-3 was immunolocalized to stromal fibroblasts in SC. TIMP-3 mRNA levels inversely correlated with proMMP-2 activation ratios, although the expression levels of MT1-MMP and MT2-MMP were not different in SC and NSC. By in situ zymography, gelatinolytic activity appeared to be weaker in SC than in NSC. All these data suggest that proMMP-2 activation is down-regulated by TIMP-3 expressed in scirrhous gastric carcinomas. Our findings may explain the differences in clinical behaviors of SC and NSC.
Subject(s)
Adenocarcinoma, Scirrhous/pathology , Enzyme Precursors/metabolism , Gelatinases/metabolism , Metalloendopeptidases/metabolism , Stomach Neoplasms/pathology , Tissue Inhibitor of Metalloproteinase-3/metabolism , Adenocarcinoma, Scirrhous/enzymology , Aged , Aged, 80 and over , Base Sequence , Blotting, Western , DNA Primers , Enzyme Activation , Female , Gelatin/metabolism , Humans , Immunoenzyme Techniques , Immunohistochemistry , Male , Middle Aged , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stomach Neoplasms/enzymology , Tissue Inhibitor of Metalloproteinase-3/geneticsABSTRACT
A two-step sandwich enzyme immunoassay (EIA) system for the detection of human membrane Type 1-matrix metalloproteinase (MT1-MMP) was established by using two monoclonal antibodies against recombinant MT1-MMP. MT1-MMP in which samples were reacted with solid-phase antibody and then detected with peroxidase-labeled second antibody. At least 1.25 ng/mL was detected by the EIA system, and linearity was obtained between 1.25 and 160 ng/mL. This EIA system is specific for MT1-MMP and did not show cross-reactivity against several other MMP's examined. Shedding of soluble MT1-MMP into the medium by some cancer cell lines was also detected by this system. However, soluble MT1-MMP in serum from normal and cancer patients was under the detection limit. Membrane-associated MT1-MMP of cancer cell lines was also detected after solubilization of the membranes with extraction buffer containing detergent. Additionally, MT1-MMP in clinical samples was examined. Elevated levels of MT1-MMP were detected in homogenate of cancer tissue compared with the levels for normal tissue and the level of MT1-MMP in tumors correlated with the rate of metastasis to the regional lymph nodes. Thus, we demonstrated that this EIA system is the first to measure MTI-MMP in clinical specimens, thus suggesting its useful for diagnosis of cancer or prediction of malignancy.
Subject(s)
Antibodies, Monoclonal/immunology , Immunoenzyme Techniques/methods , Matrix Metalloproteinase 1/analysis , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Female , Head and Neck Neoplasms/enzymology , Humans , Mice , Mice, Inbred BALB C , Recombinant Proteins/analysis , Reproducibility of Results , SolubilityABSTRACT
BACKGROUND: Matrix metalloproteinase 9 (MMP-9) facilitates tumor invasion and metastasis via basement membrane degradation. Control of MMP-9 production by cancer-stromal cell interactions in these processes has been observed.METHODS: We measured plasma MMP-9 concentrations, using a one-step sandwich enzyme immunoassay, and also immunohistochemically localized MMP-9 in patients with small gastric cancers limited to the mucosa. The cancers were classified as intraepithelial tumor (Tis) and T1 disease according to the tumor-node-metastasis (TNM) classification of the International Union Against Cancer.RESULTS: Patients with T1 disease had a higher positivity rate for and mean value of MMP-9 than patients with Tis disease. In T1 tumors, MMP-9 expression determined immunohistochemically, was greater in cells in cancer stroma than in cells in noncancerous stroma, a situation found in only a few Tis tumors.CONCLUSIONS: These results suggest that MMP-9 is related to the initial step of gastric cancer invasion.
