The Expression Of CD147 And MMP-2 In Human Gliomas And The Influence Of CD147 Genetic Engineering Antibody On Glioma Cells Invasion In Vitro

OBJECTIVEMatrix metalloproteinases (MMP) is a crucial enzyme in malignanttumor invasion and metastasis. CD147 could stimulate adjacent stromalcells to produce several matrix metalloproteinases (MMPs). The presentstudy was to investigate: (1)the expression level of CD147 and MMP-2 inhuman glioma cell, especially the diffirence of expression level betweenhige and low grade gliomas by using immunohistochemical localization andRT-PCR, and the relationship between the expression level and the gliomaprognosis were analyed statistically depends on the follow-up investigationof the disease development; (2)CD147 genetic engineering antibody is usedto influence the expression of CD147 on U251 cells, so that the expressionand activity of CD147 was inhibited. This study provide both rationale andexperiment evidences for designing high effectiveness way to the treatmentof glioma.MATERIALS AND METHODS1. Specimens: from 2001 to 2004, 50 surgical specimens were obtainedfrom patients with gliomas, including 28 males and 22 females, ranging inage from 22 to 67 years(mean 48.2).There were 5 gradeⅠ, 18 gradeⅡ, 9 gradeⅢand 18 gradeⅣgliomas, according to the WHO classification.There were 10 normal brain tissue as negative control. Freezing specimenswere used for RT-PCR, other specimens were fixed with 4% paraform andembedded in paraffin, and were routine serial section with the thickness of4μm. U251 globlastoma cell and NIH3T3 fibroblast were cultured andco-cultured in in-vitro study.2. Main agents: rat-anti-CD147, rat-anti-MMP-2, rat-anti-MT1-MMPand rat-anti-MT2-MMP monoclonal antibodies, SP agent box and DABagent box. CD147 genetic engineering monoclonal antibody was obtainedfrom Cell engineering centre, 4th military medicine university, Xi'an China.3. Methods: Human brain glioma tissues and normal brain tissues wereobtained after surgical removal or autopsy, stored at -80℃for RT-PCRanalysis of expression of CD147 mRNA, then other sections wereperformed HE staining to verify the pathological diagnosis and devided intolow and high grade groups. Sections from 50 specimen wereimmunohistochemically stained to observe the difference of CD147,MMP-2 expression level between low and high grade group, and theaccuracy of predicting patient prognosis was analyzed. In cell culture trial,cultures contained either U251 glioblastoma cells, NIH3T3 fibroblasts orboth, and the co-culture experiments employed different dose of CD147genetic engineering monoclonal antibody. The harvested media were usedfor zymography to detect enzyme activity of pro-form and activated-form ofMMP-2, co-culture media collected were subjected to western blot to detectexpression of CD147, MMP-2, MT1-MMP and MT2-MMP protein. Theprotein bands on the film were subjected to image analysis. The data of each group were recorded for comparison and statistical analysis.4. Statistical analysis: ANOVA were used to analysize measurement dataand x~2 test were used for numeration data. The relative coefficient adoptedSpearman correlation analysis. All data statistics were completed in theSPSS12.0 software package.RESULTS1. MMP-2 and CD147 protein positive expression were both observed inlow and high grade groups, and CD147 mRNA expression was also detectedin low and high grade groups, but their expression levels were obviouslyenhanced in high grade group. There was a significant difference betweenthese two group. The variations were significant statistically.2. In cell culture trial, Zymography showed that there were twogelatinolytic bands at 68 and 62kDa on the resulting blue background. BothU251 and NIH3T3 cells exhibited gelatinolytic bands at 68 and 62 kDa. The68-kDa band corresponds to the molecular mass of the pro-form of MMP-2in the absence of dithiothreitol and the 62-kDa band corresponds to theactivated form. These gelatinolytic activities corresponding to pro- andactivated MMP-2 were both stimulated in cocultures compared withindividual cultures of each cell type. Moreover, coculturing caused greaterincrease in the activated form of MMP-2 than the pro-form while theconcentrations of U251 cells were increase. Western blot showed that a highlevel of CD147 expression was demonstrated in the U251 cell, however,CD147 protein was not detected in NIH3T3 fibroblast, showed faint bandsat approximately 50 kDa. Then the pro-MMP-2 was detected by westernblot trial. It was found that MMP-2 production was stimulated by CD147 in cocultures compared with individual cultures of U251 and NIH3T3 cells, weperformed coculture experiments in the presence of varying concentrationsof activity-blocking CD147 genetic engineering monoclonal antibody.Stimulation was inhibited by CD147 genetic engineering monoclonalantibody in a dose-dependent manner. The same results were obtained bywestern blot trial that production of MT1-MMP and MT2-MMP werestimulated by CD147 in cocultures of U251 and NIH3T3 cells. Thesestimulatory effects were inhibited by CD147 genetic engineeringmonoclonal antibody in a dose-dependent manner. The variations mentionedabove were also significant statistically.CONCLUSIONSExpression of CD147 and MMP-2 in the glioma could be greatassociated with glioma progression and invasion, There are actually positiveexpression of CD147 and MMP-2 in human glioma, especially stronger inhigh grade gliomas which suggests: along with the malignant extent oftumor cells increases, the expression of CD147 and MMP-2 were obviouslyenhanced. In vitro trial, we have demonstrated that cocultures of U251 cellsand NIH3T3 fibroblasts produce elevated levels of MMP-2, MT1-MMP andMT2-MMP, and give rise to greatly increased activation of MMP-2, ascompared with either cell type alone. These effects were shown to bedependent on CD147 present on the surface of U251 cells. In addition,CD147 stimulates production of the MMP-2 activators, MT1-MMP andMT2-MMP. Stimulation of MMP-2, MT1-MMP and MT2-MMP productionwas inhibited by CD147 genetic engineering monoclonal antibody in adose-dependent manner, because the functional domain of cell surface CD147 has been blocked by specific antibodies. From our trials, MMP-2and its inducer CD147 maybe participate in invasion and metastasis ofglioma. CD147 may be an independent prognostic fctor. It suggests thatinterruption of tumor cell-fibroblast (or other stromal cell) interaction couldbe a new target of anti-invasion therapy in gliomas. This research provideboth rationale and experiment evidences for designing high effectivenessway to the treatment of gliomas. The detailed mechanism between CD147and MMP-2 is remain uncertain and in vivo trial is necessary to underinvestigation.