OBJECTIVETo study the anti-invasion effect of SEPT7 gene on U251MG glioma cells and its possible molecular mechanism.

METHODSRecombinant adenovirus vector carrying SEPT7 gene (rAd5-SEPT7) was transduced to human glioma cell line U251MG, and empty adenovirus vector was used as control. Tumor invasion was examined by Transwell method and 3 D-Matrigel assay, and tumor cell migration by wound-healing method and 2 D-Matrigel assay. Three major molecular events associated with cell motility and migration, including changes of expression in MMP2, MMP9, MT1-MMP, TIMP1 and TIMP2, the alteration of integrin alpha(v)beta(3) expression, and the structural change of cytoskeleton protein, tubulin-alpha, in U251 cells transduced with rAd5-SEPT7 were studied by Western blotting, immunofluorescence and laser scanning confocal microscope, respectively.

RESULTSThe invasive and migratory capabilities of cells transduced with rAd5-SEPT7 were inhibited. The expression of extracellular matrix metalloproteinases MMP-2, MMP-9, MT1-MMP and integrin alpha(v)beta(3) was significantly decreased, while the expression of matrix metalloproteinase inhibitor TIMP1, TIMP2 was upregulated. Intracellular cytoskeleton protein-tubulin-alpha in U251 cells exhibited prominent morphological changes which including the appearance of distortion and aggregation resulting from redistribution of tubulin-alpha, and this feature of alteration was similar to the tubulin-alpha structure in normal non-tumor cells.

CONCLUSIONSEPT7 gene can inhibit the invasion and migration ability of U251 glioma cells. Its molecular mechanism may include that SEPT7 gene reverses the imbalanced state of MMPs/TIMPs, downregulates the expression of integrin alpha(v)beta(3) and alters the structure of tubulin-alpha of U251MG glioma cells. It is suggested that SEPT7 gene could be a good candidate for gene therapy of gliomas.

Adenoviridae ; genetics ; Blotting, Western ; Cell Cycle Proteins ; genetics ; physiology ; Cell Line, Tumor ; Cell Movement ; genetics ; Genetic Vectors ; genetics ; Glioma ; metabolism ; pathology ; Humans ; Integrin alphaVbeta3 ; metabolism ; Matrix Metalloproteinase 14 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Microscopy, Confocal ; Neoplasm Invasiveness ; genetics ; Septins ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism

OBJECTIVETo examine the expression pattern of membrane type-1 matrix metalloproteinase (MT1-MMP) in breast carcinomas.

METHODSForty-three breast cancer tissues were collected and examined for MT1-MMP protein and mRNA expressions using immunohistochemistry, semi-quantitative RT-PCR and in situ hybridization.

RESULTSImmunohistochemistry of the breast cancer specimens showed MT1-MMP immunoreactivity on the cancer cell membrane. MT1-MMP mRNA was located in the stromal cells surrounding the breast cancer nest as shown by in situ hybridization. MT1-MMP mRNA expression was detected in all of the carcinomas, but its level was significantly lower in immunohistochemically negative specimens than in positive ones (0.547=0.0886 vs 0.759=0.0802, Plt;0.01).

CONCLUSIONMT1-MMP is very likely produced by stromal cells surrounding the breast cancer nest and anchored on the cell membrane after activation.

Breast Neoplasms ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect of membrane type-1 matrix metalloproteinase (MTI-MMP) on the invasive potential of breast cancer cell and analyze its mechanisms.

METHODSAfter treatment of breast cancer MDA-MB-453 cell line with concanavalin A ( ConA, 20 microg/ml) for 24 h, MT1-MMP protein was detected in cancer cells by Western analysis and immunocytochemistry. MDA-MB-453 cells were cultured with exogenous latent proMMP-2 and MMP-2 activity was analyzed by gelatin zymography. The invasive potential of the tumor cells was measured with a membrane invasion culture system. Cancer cells of the cell line were divided into four groups: the control group treated by neither reagent, group ConA was only treated by ConA, group MMP-2 was treated only by MMP-2, and group ConA + MMP-2 was treated by both ConA and MMP-2. RESULTS The expression of MTI-MMP protein could be detected in groups ConA and ConA + MMP-2, but nothing was detected in control and group MMP-2. There was only 72 000 precursor form of MMP-2 in group MMP-2 and there were both 72 000 precursor form and 64 000 active enzyme form of MMP-2 in group ConA + MMP-2, but there was no forms of MMP-2 in the other two groups detected by gelatin zymography. The largest amount of cells penetrated through Matrigel was observed in group ConA + MMP-2 than in the other three groups.

CONCLUSIONMTI-MMP can remarkably promote the invasive potential of breast cancer cells mainly through its ability of activating latent proMMP-2 to degrade

Blotting, Northern ; Blotting, Western ; Breast Neoplasms ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Concanavalin A ; pharmacology ; Female ; Humans ; Immunohistochemistry ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Invasiveness ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo observe the expression of the matrix metalloproteinase 2 (MMP-2) and membrane-type 1 metalloproteinase (MT1-MMP) in lung of rats exposed to paraquat (PQ) and the effects of Salvia miltiorrhiza monomer IH764-3 on above expression.

METHODSNinety adult healthy Sprague-Dawley (SD) rats were randomly divided into the control group (group A, 6 rats), the exposure group (group B, 42 rats) and the group treated by Salvia miltiorrhiza monomer IH764-3 (group C, 42 rats). The group B and C were treated intragastrically with 1ml of PQ (50 mg/kg), and the group A was treated intragastrically with normal saline. The group C was treated intraperitoneally with 1 ml Salvia miltiorrhiza monomer IH764-3 at the dose of 40 mg/kg a day. The group A and B were treated intraperitoneally with 1 ml normal saline day. The expression of MMP-2 and MT1-MMP was detected on the 1st, 3rd, 7th, 14th, 21st, 28th and 35th days after exposure for all groups.

RESULTSAs compared with the expression level (0.305 ± 0.045) of MMP-2 mRNA in group A, the expression levels of MMP-2 mRNA in Group B significantly increased, which were 0.654 ± 0.077, 0.623 ± 0.051, 0.637 ± 0.024, 0.533 ± 0.043 and 0.552 ± 0.050 on the 1st, 3rd, 7th, 14th, 21st days after exposure, respectively (P < 0.01). As compared with group A, the the expression levels of MMP-2 mRNA on the 1st, 3rd, 7th days in Group C slightly increased, but the expression levels of MMP-2 mRNA on the 1st, 3rd, 7th, 14th, 21st days in Group C were 0.523 ± 0.074, 0.567 ± 0.097, 0.514 ± 0.058, 0.359 ± 0.018 and 0.374 ± 0.020, respectively, which were significantly lower than those in group B (P < 0.01). As compared with the expression level (0.391 ± 0.058) of MT1-MMP mRNA in group A, the expression levels of MT1-MMP mRNA in Group B significantly increased, which were 0.796 ± 0.021, 0.762 ± 0.043, 0.590 ± 0.010, 0.803 ± 0.076 and 0.680 ± 0.034 on the 1st, 3rd, 7th, 14th and 21st days after exposure, respectively (P < 0.01). As compared with group A, the expression levels of MT1-MMP mRNA in Group C significantly increased, which were 0.594 ± 0.010, 0.653 ± 0.044 and 0.564 ± 0.009 on the 1st, 3rd and 21st days after exposure, respectively (P < 0.01). The expression levels of MT1-MMP mRNA in Group C were significantly lower than those in group B (P < 0.05 or P < 0.01).

CONCLUSIONThe expression changes of MMP-2 and MT1-MMP genes of lungs in rats intragastrically exposed to PQ could result in the unbalance the synthesis and degradation of ECM, which may be a cause of lung fibrosis. The Salvia miltiorrhiza monomer IH764-3 could affect the expression of MMP-2 and MT1-MMP genes to a certain extent, resulting in the reduction of lung fibrosis.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Lung ; drug effects ; metabolism ; Male ; Matrix Metalloproteinase 14 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Paraquat ; toxicity ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza

OBJECTIVETo study location of MT1-MMP and effect of its change in expression on rabbit VX2 tumor tissues after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol.

METHODSSixty rabbits implanted with tumor tissue of cell line VX2 were divided into three groups (control group, lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group). The transarterial embolization was performed super-selectively via gastro- duodenal artery of rabbits, each rabbit in control group was inserted with 1 ml normal saline,that in lipiodol group was inserted with 0.3 lipiodol ml/kg, also 0.3 ml hydroxyapatite nanoparticles loaded with lipiodol per kg for that in the last group. Results of embolization were detected by using CT scanning 3 days after operation. After two weeks, all tumors were took out as specimens to investigate location of MT1-MMP in VX2 tumor tissues,and also to determine the change of its expression in tumor tissues after embolization with different medicines, with three-step immunohistochemical technique (S-P). MT1-MMP mRNA was measured by RT-PCR to determine whether there were differences in three groups. Western blot technique was performed to determine difference of MT1-MMP protein expression of in three groups.

RESULTSImmunohistochemical results exposed that MT1-MMP was expressed on membrane of tumor cells and in extracellular matrix of tumor cells. Comparison of MT1-MMP expression in control group with that in other two groups, showed a significant lower level in control group (P < 0.05). There was no difference in MT1-MMP expression between lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group (P > 0.05). Western blot supported this conclusion. RT-PCR detecting MT1-MMP mRNA was found no differences among three groups (P > 0.05).

CONCLUSIONSMT1-MMP was mainly expressed on membrane of tumor cells and in extracellular matrix of tumor cells. There was an increasing tendency on expression of MT1-MMP in tumor tissues and extracellular matrix after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol,it might be one of important mechanisms provoking high recurrence rate for hepatocellular carcinoma after treatment embolization.

Animals ; Durapatite ; Embolization, Therapeutic ; Iodized Oil ; Liver Neoplasms, Experimental ; enzymology ; pathology ; therapy ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; Nanoparticles ; RNA, Messenger ; genetics ; Rabbits

OBJECTIVETo examine the expression of membrane-type-1 matrix metalloproteinase (MT1-MMP) and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in gastric carcinoma, and investigate its clinical significance, at the same time analyze the correlation between MT1-MMP and RECK expression.

METHODSMT1-MMP and RECK expression in surgically resected tissue samples of gastric carcinoma was examined by immunohistochemical method (two-step method) , and its correlation with clinicopathological factors was analyzed.

RESULTSAmong the 44 gastric carcinoma samples, 37 (84.1%) were stained positive for MT1-MMP, and 31 (70.5%) for RECK. The expression of MT1-MMP was much higher in poorly differentiated gastric carcinoma samples than moderately and well-differentiated samples (P = 0.015). The expression level of MT1-MMP was associated with invasive depth of tumor cells (P = 0.007), but no difference between sex and lymph node metastasis. On the contrary, the well-differentiated samples showed higher expression of RECK than poorly and moderately differentiated gastric carcinoma samples (P = 0.006). The expression level of RECK did not correlate with sex, lymph node metastasis and invasive depth of tumor cells. RECK expression showed no relation to MT1-MMP expression in the gastric carcinoma.

CONCLUSIONOverexpression of MT1-MMP in gastric carcinoma may play an important role during tumor differentiation and metastasis, the RECK protein may have positive effects on the tumor differentiation.

Adult ; Aged ; Aged, 80 and over ; Carcinoma ; enzymology ; genetics ; metabolism ; pathology ; Female ; GPI-Linked Proteins ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; Middle Aged ; Stomach Neoplasms ; enzymology ; genetics ; metabolism ; pathology

Adult ; Aged ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; Cervix Uteri ; metabolism ; pathology ; Disease Progression ; Female ; Furin ; genetics ; metabolism ; Humans ; Matrix Metalloproteinase 14 ; metabolism ; Middle Aged ; RNA, Messenger ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; pathology ; Vascular Endothelial Growth Factor C ; metabolism ; Young Adult

Syntenin is a PDZ domain-containing adaptor protein that has been recently shown to regulate migration and invasion in several tumors. Small cell lung cancer (SCLC) is notorious for its invasiveness and strong potential for metastasis. We therefore studied the influence of syntenin on the invasiveness of SCLC. Immunohistochemistry in tumor tissues showed that syntenin was more frequently expressed in small cell carcinomas than other neuroendocrine tumors, such as carcinoids and neuroblastomas, suggesting that syntenin expression may be related to more aggressive forms of neuroendocrine tumors. In SCLC patients, syntenin overexpression in tumor cells was significantly associated with more extensive and advanced disease at the time of diagnosis (P=0.029). Overexpression of syntenin in SCLC cells that were intrinsically syntenin-low increased the invasiveness of cells and led to the induction of extracellular matrix (ECM)-degrading membrane type 1-matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase 2 (MMP2). In contrast, suppression of syntenin in syntenin-high cells was associated with the downregulation of MT1-MMP. Contrary to the results of previous studies using malignant melanomas and breast carcinomas, signaling cascades were shown to be further transduced through p38 MAPK and PI3K/AKT, with activation of SP1 rather than NF-kappaB, under circumstances not involving ECM interaction. In addition, the upstream molecule focal adhesion kinase was induced by syntenin activation, in spite of the absence of ECM interaction. These results suggest that syntenin might contribute to the invasiveness of SCLC and could be utilized as a new therapeutic target for controlling invasion and metastasis in SCLC.
Cell Line, Tumor ; Focal Adhesion Protein-Tyrosine Kinases/*metabolism ; Humans ; Matrix Metalloproteinase 14/genetics/metabolism ; Matrix Metalloproteinase 2/genetics/metabolism ; Neoplasm Invasiveness ; Phosphatidylinositol 3-Kinases/*metabolism ; Proto-Oncogene Proteins c-akt/*metabolism ; Signal Transduction ; Small Cell Lung Carcinoma/*metabolism/pathology ; Sp1 Transcription Factor/*metabolism ; Syntenins/genetics/*metabolism ; p38 Mitogen-Activated Protein Kinases/*metabolism

OBJECTIVETo study the function of MT1-MMP in tumor angiogenesis and elucidate the possible way of action that MT1-MMP contributes to angiogenesis.

METHODSMT1-MMP was transfected into human breast carcinoma cell line MCF-7 cells. Semi-quantitative RT-PCR and immunofluorescence staining were employed to detect the expression of VEGF in the transfected and non-transfected MCF-7 cells. Tumor growth, microvessel density and expression of VEGF in nude mice were detected through in vivo tumorigenicity assay.

RESULTSIn MT1-MMP stable transfected MCF-7 cells, mRNA expression of VEGF(189), VEGF(165), and VEGF(121) and immunofluorescence intensity were significantly elevated (P < 0.001). In vivo tumorigenicity assay in the nude mice showed that MT1-MMP promoted tumor growth. The MVD in the MT1-MMP-transfected cells-transplanted tumor tissue was significantly elevated (P < 0.05). Immunohistochemical assay showed that there was a strong immunostaining of VEGF in those tumor tissues.

CONCLUSIONMT1-MMP can induce tumor angiogenesis through up-regulation of VEGF expression. This function of MT1-MMP may open a new approach for clinical anti-tumor research and anti-tumor drug development.

Animals ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; physiology ; Mice ; Mice, Nude ; Microvessels ; pathology ; Neoplasm Transplantation ; Neovascularization, Pathologic ; pathology ; RNA, Messenger ; metabolism ; Tumor Burden ; Up-Regulation ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

One of the 14-3-3 protein isoforms, 14-3-3epsilon, was previously shown to be increased during skin aging. We suggest here a possible role for the 14-3-3epsilon protein in skin aging by providing evidence that 14-3-3epsilon increases the expression of the matrix-metalloproteinase (MMP)-2 gene in NIH3T3 fibroblast cells. Expression of the 14-3-3epsilon gene in NIH3T3 cells primarily up-regulated the expression of the MMP-2 gene at the transcriptional level by inducing specific DNA binding proteins bound to an upstream region of the MMP-2 promoter from -1,629 to -1,612. Inhibition of endogenous 14-3-3epsilon gene expression by RNA interference also decreased endogenous MMP-2 gene expression. Furthermore, up-regulation of the MMP-2 gene by 14-3-3epsilon was suppressed by expression of a dominant-negative mutant of p38 MAP kinase. These findings strongly suggest that increased expression of 14-3-3epsilon contributes to remodeling of extracellular matrix in skin through increasing MMP-2 gene expression via p38 MAP kinase signaling.
14-3-3 Proteins/*physiology ; Animals ; Electrophoretic Mobility Shift Assay ; Gene Expression Regulation, Enzymologic/*physiology ; Matrix Metalloproteinase 2/antagonists & inhibitors/*genetics/metabolism ; Mice ; NIH 3T3 Cells ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; *Signal Transduction ; Transfection ; p38 Mitogen-Activated Protein Kinases/*metabolism