PURPOSE: We investigated the extent of adenosine A1 agonist-induced expression and regulation of matrix metalloproteinase 2 (MMP-2) synthesis in human trabecular meshwork cells (HTMC). METHODS: Primary HTMC cultures were exposed to 0.1 or 1.0 µM N6-cyclohexyladenosine (CHA) for 2 h in the presence or absence of an inhibitor thereof, 8-cyclopentyl-1,3-dimethylxanthine (CPT). The expression level of mRNA encoding MMP-2 was assessed via reverse transcription-polymerase chain reaction, and the levels of tissue inhibitor of metalloproteinase 2 (TIMP2) and membrane-type-1 MMP (MT1-MMP) measured by Western blotting. The permeability of the HTMC monolayer was assessed with the aid of carboxyfluorescein. RESULTS: CHA at 1.0 µM increased the permeability of the HTMC monolayer (p = 0.003) and CHA at both 0.1 and 1.0 µM significantly increased MMP-2 mRNA expression, which was inhibited by co-exposure to CPT (all p < 0.05). CHA increased MMP-2 activity, decreased that of TIMP2, and increased that of MT1-MMP (all p < 0.05). CONCLUSIONS: CHA increased the permeability of the HTMC monolayer and increased MMP-2 activity, decreased TIMP2 activity, and increased MT1-MMP activity. Thus, regulation of TIMP2 and MT1-MMP expression may be involved in the adenosine A1 agonist-induced increase in MMP-2 activity.
Adenosine ; Blotting, Western ; Humans ; Matrix Metalloproteinase 14 ; Matrix Metalloproteinase 2 ; Permeability ; RNA, Messenger ; Tissue Inhibitor of Metalloproteinase-2 ; Trabecular Meshwork

PURPOSE: hear stress (SS) and cyclic strain (CS) influence the expression of membrane type 1-matrix metalloproteinase (MT1-MMP) in microvascular endothelial cells (MVECs). It is known that changes in the level of Sp1 phosphorylation are important for MT1-MMP expression following SS and CS. However, the exact mechanism underlying this process is poorly understood. The aim of this study was to determine the effect of PKCzeta on serine phosphorylation and activation of Sp1 in response to SS and CS. METHOD: MVECs were exposed to SS or CS for up to 8 hours with or without PKCzeta inhibitors. The activity and phosphorylation of Sp1 were assessed by Western blot analysis and immunoprecipitation. MT1-MMP protein expression was assessed by Western blot analysis. RESULT: PKCzeta was phosphorylated and activated under SS, whereas no significant changes were noted under CS. SS increased Sp1 phosphorylation in a time-dependent manner, but no changes in the Sp1 phosphorylation were observed when the MVECs were pretreated with the PKCzeta inhibitors. By contrast, MVECs exposed to CS in the presence or absence of PKCzeta inhibitors showed no change in the phosphorylation of Sp1. SS decreased MT1-MMP protein expression in a time-dependent manner, but in the presence of PKCzeta inhibitors, MT1-MMP expression was not changed compared with the static levels after SS. CS increases MT1-MMP expression in a time-dependent manner. Similar expression was observed when the cells were pretreated with PKCzeta inhibitors under CS. CONCLUSION: These data demonstrate that the increased affinity of Sp1 for the MT1-MMP's promoter site occurs because of PKCzeta induced phosphorylation of Sp1 in response to SS.
Blotting, Western ; Endothelial Cells* ; Immunoprecipitation ; Matrix Metalloproteinase 14* ; Membranes ; Phosphorylation ; Serine

Membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) plays the pivotal role in tumor development and metastasis, so it is a promising drug target in malignancy. To acquire MT1-MMP specific binding peptides, we first analyzed MMPs sequences to find the divergent and specific sequence of MT1-MMP by bioinformatics approach, then set the specific sequence as the sense peptide target and designed antisense peptide library. Finally, by means of molecular docking, molecular dynamics simulation and in vitro cell assays, we screened the antisense peptide library against MT1-MMP and further studied the obtained specific peptides. Here, we identified the divergent and specific sequence of AYIREGHE (Named MT1-loop) located in MT1-MMP loop by multiple sequence alignment and established the antisense peptides library with capacity of 1 536 sequences. After two rounds of virtual screening, we obtained five antisense peptides with Rerankscores in the top for further screening. They all interacted with MT1-MMP, and docked well at the active site composed of MT1-loop sequence. Analysis of the affinities of these five antisense peptides to other MMPs (MMP1-3, MMP7-13, MMP14 HPX, MMP16) revealed that the peptide FVTFPYIR was more specific to MT1-MMP. Molecular dynamics simulation showed that the peptide FVTFPYIR might affect the stability of MT1-MMP and thus have effects on its activities. Meanwhile, the peptide FVTFPYIR could specifically inhibit the growth of MG63 and MDA-MB-231 tumor cells both of which expressed MT1-MMP. The work provides a new insight and way for the development of antitumor lead peptides targeting MT1-MMP.
Amino Acid Sequence ; Humans ; Matrix Metalloproteinase 14 ; chemistry ; Molecular Dynamics Simulation ; Neoplasms ; Peptide Library ; Peptides ; chemistry

BACKGROUND: The activation of proMMP-2 is induced by membrane type 1-matrix metalloproteinase (MT1-MMP), but inhibited by tissue inhibitors of matrix metalloproteinase type 2 (TIMP-2). This study was carried out to establish the pattern of mRNA expression of MMP-2, MT1-MMP, and TIMP-2 in breast carcinomas. METHODS: Seventy-nine cases of invasive ductal carcinoma, 10 of ductal carcinoma in situ, and 10 of fibrocystic disease as a control were analysed for the expression of MMP-2, MT1-MMP, and TIMP-2 mRNA, using in situ hybridization. Correlations of the results with the clinical stage, tumor size, nodal status, and nuclear grade were analysed. RESULTS: The expression rates of MMP-2, MT1-MMP, and TIMP-2 mRNA in invasive ductal carcinoma were 68%, 73%, and 56%, respectively. They were localized to both stromal and tumor cells, but mainly in the latter. The MMP-2 mRNA expression was significantly correlated with the clinical stage (p < 0.05), while the expression of TIMP-2 mRNA was inversely correlated with clinical stage and tumor size(p < 0.05). Significant positive correlations between MMP-2 and MT1-MMP expressions, along with inverse relationships between MMP-2 and TIMP-2, and between TIMP-2 and MT1-MMP, were also found. CONCLUSIONS: MMP-2 and TIMP-2 mRNA expressions might be useful as one of a range of prognostic parameters in breast carcinoma patients.
Breast Neoplasms* ; Breast* ; Carcinoma, Ductal ; Carcinoma, Intraductal, Noninfiltrating ; Humans ; In Situ Hybridization ; Matrix Metalloproteinase 14* ; Matrix Metalloproteinase 2 ; Membranes ; RNA, Messenger* ; Tissue Inhibitor of Metalloproteinase-2*

BACKGROUND: A number of reports have been published regarding the use of imiquimod for the treatment of melanoma in situ and metastatic melanoma. Essential steps in the process of melanoma invasion and metastasis include degradation of basement membranes and remodeling of the extracellular matrix by proteolytic enzymes, including matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). OBJECTIVE: To evaluate the antiinvasive effect of imiquimod in human malignant melanoma cell lines, SK-MEL-2 and SK-MEL-24, in vitro, and to investigate imiquimod-induced changes in the expression of MMPs and TIMPs. METHODS: Invasiveness of melanoma cell lines following imiquimod treatment was evaluated by invasion assays. In order to investigate the mechanism of the anti-invasive effect of imiquimod, mRNA and protein levels of MMP-2, -9, membrane type 1 (MT1)-MMP, TIMP-1, and -2 were assessed by real-time reverse transcription-polymerase chain reaction, gelatin zymography, and western blotting. RESULTS: Imiquimod treatment decreased in vitro viability of melanoma cells in a concentration-dependent manner. Imiquimod also elicited a concentration-dependent suppression of invasion in both melanoma cell lines. A concentration-dependent decrease in MMP-2 and MT1-MMP protein levels and a concentration-dependent increase in TIMP-1 and -2 protein levels by imiquimod was observed in both melanoma cell lines. However, expression of MMP-9 protein was increased in SK-MEL-2 but decreased in SK-MEL-24 with increasing imiquimod concentrations. Imiquimod elicited alterations in MMPs and TIMPs mRNA levels that parallel the observed changes in protein levels. CONCLUSION: Imiquimod may elicit an anti-invasive effect on human melanoma cells by regulating MMPs and TIMPs.
Basement Membrane ; Blotting, Western ; Cell Line ; Extracellular Matrix ; Gelatin ; Humans ; Matrix Metalloproteinase 14 ; Matrix Metalloproteinases* ; Melanoma* ; Membranes ; Metalloproteases* ; Neoplasm Metastasis ; Peptide Hydrolases ; RNA, Messenger ; Tissue Inhibitor of Metalloproteinase-1

OBJECTIVE: Threre are several proteolytic enzymes such as Matrix Metalloproteinases (MMP), which are involved in tumor invasion and metastasis. The aim of this study was to determine the exprssion of Membranous Type MMPs (MT-MMPs) and investigate the relationship between their expression and questioned whether their expression is related to stages and other prognostic factors of cervical cancer. METHODS: The cervical and cervical cancer tissues were taken from the patients; healthy women (n=14), and the patients with cervical cancer (n=35). The protein expression of MT1, 2, 3-MMP with MMP-2 was examined using immunohistochemical staining and western blotting. MMP-2 activity was measured by zymogram. RESULTS: The expression of MT1, 2, 3-MMP was higher in cervical cancer than that of normal cervix (p<0.05). No significant association was found between MT-MMPs and clinicopathologic factors, such as age, grade, stage, tumor sizes, and Squamous cell carcinoma-Ag (SCC-Ag) (p>0.05). But there were significant correlations between MT1-MMP, MT3-MMP and lymph node involvemen t (p<0.05). There was significant correlation between MT-MMPs and MMP-2 (p<0.05), too. CONCLUSION: According to the results, MT-MMP expression could be associated with the pathogenesis of cervical cancer. In addition, the evaluation fo MT-MMPs expression might be helpful to predict lymph node metastasis in cervical cancer. Further prospective study with a large number of cases is needed in future.
Blotting, Western ; Cervix Uteri ; Female ; Humans ; Lymph Nodes ; Matrix Metalloproteinase 14 ; Matrix Metalloproteinase 16 ; Matrix Metalloproteinases ; Neoplasm Metastasis ; Peptide Hydrolases ; Uterine Cervical Neoplasms*

PURPOSE: Matrix metalloproteinases (MMPs) have been reported to play critical roles in the endothelial cell migration and matrix remodeling during angiogenic process. To investigate the roles of the membrane type MMP (MT1-MMP) by the matrix remodeling of endothelial cells, MT1-MMP expression vector was transfected into bovine aortic endothelial cells (BAECs). Increased ex+pression of MT1-MMP in BAECs enhanced the activation of MMP-2, invasion and migration of BAECs. Moreover, the capacity of tube formation was increased by MT1-MMP transfectants. These observations indicate that MT1-MMP is involved in the angiogenic process of endothelial cells in vitro. In this study, we attempted these effects were confirmed in vivo system. MATERIALS AND METHODS: In this study, we used MT1- MMP or Antisense MT1-MMP stable transfectants in HT1080 human fibrosarcoma cells. Chorioallantoic membrane (CAM) assay was used for the detection of angiogenesis in vivo and modified CAM assay for quantification of invasion of MT1-MMP transfected cells. RESULTS: In CAM assay, the formation of microvessels was stimulated by MT1-MMP transfectants. Invasive capacity of HT1080 cells was also increased in a novel in vivo metastasis model, PCR based CAM assay. CONCLUSION: These results identify the function of MT1- MMP during the neovascularization process.
Chorioallantoic Membrane* ; Endothelial Cells ; Fibrosarcoma ; Humans ; Matrix Metalloproteinase 1* ; Matrix Metalloproteinase 14 ; Matrix Metalloproteinases ; Membranes ; Microvessels ; Neoplasm Metastasis ; Polymerase Chain Reaction

PURPOSE: Among the theories in the pathogenesis of abdominal aortic aneurysms, matrix metalloproteinase (MMP)- induced excessive degradation of extracellular matrix protein has been widely recognized. Normally, MMPs keep a balance with their endogenous tissue inhibitors of metalloproteinases (TIMPs). Recently, the MMP-TIMP imbalance has been investigated for potential etiological role in AAA formation. The aim of this study was to define the role of the imbalance between the expressions of the specific MMPs and their physiologic inhibitors (TIMPs) in the formation of human abdominal aortic aneurysm (AAA). METHODS: Aortic tissues from 12 patients with AAAs, 4 age-matched patients with aortoiliac occlusive diseases (AODs), and 6 cadaveric organ donors, as normal controls, were obtained and prepared. The productions and expressions of MMP-2, 9, MT1-MMP and TIMP-1, 2, and 4 were analyzed using gelatin zymography, Western blotting, and immunohistochemistry. RESULTS: In the gelatin zymography, the net matrix-degrading activities were higher in the AAAs and AODs than in the normal control group due to the higher presence of MMP-2 and 9. From the Western blot analysis and immunohistochemistry, those with AAAs and AODs showed significantly higher expressions of MMP-2, 9, and MT1-MMP than the normal control group. However, no differences in the TIMP-1 and 2 expressions were found between the all groups. In contrast, TIMP-4 protein was expressed at a significantly lower level in the AAAs and AODs than in the normal control group. MMP-9/TIMP-1, MMP-2/TIMP-2, and MMP-2/TIMP-4 were significantly different between AAAs and normal control group. From the densitometric analysis of the Western blotting, no significant differences were found in tissue expressions of MMPs and TIMPs between the AAAs and AODs groups, but, in the immunohistochemistry, the AAAs group showed a different distribution of the MMP expression confined to sites of overt medial damage compared with that in the intimal plaque in AODs. CONCLUSION: The imbalance of expression between specific MMPs and their endogenous inhibitors plays an etiological role in the formation of AAAs. In addition, TIMP-4 may suppress the MMP-induced aneurysmal formation. Our results suggest that the eventual formation of aneurysms or occlusive lesions appears not to result from an ongoing difference in the proteolytic activities, but from differences in other factors, as-yet-undefined, including the distribution of MMPs expression within the aortic walls.
Aneurysm ; Aortic Aneurysm, Abdominal* ; Blotting, Western ; Cadaver ; Extracellular Matrix ; Gelatin ; Humans* ; Immunohistochemistry ; Matrix Metalloproteinase 14 ; Matrix Metalloproteinases* ; Metalloproteases ; Tissue Donors ; Tissue Inhibitor of Metalloproteinase-1

OBJECTIVE: Treatment with cyclosporine(CsA) for a long-term period may induce renal glomerulosclersosis and interstitial fibrosis. Reactive oxygen species(ROS) seems to be involved in the process of glomerulosclersosis and interstitial fibrosis. We investigated the effect of CsA on the generation of ROS and extracellular matrix accumulation in cultured human mesangial cells. We also studied the relationship between ROS formation and extracellular matrix and the effect of antioxidant on ROS formation and/or extracellular matrix degradation. METHODS: Mesangial cells were treated with varying dose of Cyclosporine(0, 2.5, 5, 7.5 and 10microgram/ mL) and also with cyclosporine(5microgram/mL) plus N- acetylcysteine(1mM). ROS was measured by flow cytometric analysis. mRNA expression of MMP-2, TIMP-2, MT1-MMP and collagen III was assessed by RT-PCR method. MMP-2 activity was measured by gelatin zymography. RESULTS: No significant difference was noted in cell viability with each CsA concentration. CsA inhibited the cell proliferation in a dose dependent manner and induced the expression of ROS. Antioxidant NAC reversed the effect of cyclosporine. CsA had no effect on the mRNA expression of collagen III, MMP-2, TIMP-2, MT1-MMP. However CsA decreased the MMP-2 activity in a dose dependent manner, which was also reversed by NAC. CONCLUSION: Cyclosporine-induced ROS may be associated with the extracellular matrix accumulation, that is glomerulosclersosis and interstitial fibrosis by inhibiting the cell proliferation and by decreasing the degradation of extracellular matrix. Antioxidant, at least in vitro, may prevent some of the adverse effects of CsA on renal function.
Cell Proliferation ; Cell Survival ; Collagen ; Cyclosporine* ; Extracellular Matrix* ; Fibrosis ; Gelatin ; Humans* ; Matrix Metalloproteinase 14 ; Mesangial Cells* ; Oxygen ; RNA, Messenger ; Tissue Inhibitor of Metalloproteinase-2

OBJECTIVE: Cyclosporine(CsA) and tacrolimus, albeit different in structure, exert immunosuppressive effect by similar mechanism. Although most of clinical manifestations, including nephrotoxicity, are similar in the patients using these drugs, there are some differences including gum hyperplasia, neurotoxicity, and hepatic fibrosis between two drugs. There are several reports about association between reactive oxygen species(ROS) and CsA. In contrast, tacrolimus is known to decrease ROS in central nervous system. Thus, we investigated the possibility of different effects of tacrolimus and CsA on the generation of ROS, on the synthesis and degradation of collagen. METHODS: Experiments were done in primary cultured mesangial cells between 4th and 8th passages. CsA was added to the culture dishes in different concentration(making final CsA concentration of 0, 2, 4, 8 microgram/milliliter) and N-acetylcysteine(NAC) was also added in another mesangial cell culture at 4 microgram/milliliter of CsA concentration; tacrolimus was added in similar pattern(making final tacrolimus concentration of 0, 0.1, 0.2, 0.4 microgram/milliliter, NAC in 0.2 microgram/milliliter of tacrolimus concentration). RESULTS: No significant decrease in viability was noted in both cell groups, but growth retardation was weak in tacrolimus treated cells comparing with CsA treated cells. By flow cytometry, we could find the generation of ROS in CsA treated cells, but not in tacrolimus treated cells. In RT-PCR, there is no significant difference in m-RNA expression for a number of molecules including collagen III, MMP-2, TIMP-2, MT1-MMP in either CsA treated cells or tacrolimus cells. But in zymogram, MMP-2 activities were decreased at higher CsA concentration, then increased with addition of NAC. In tacrolimus cells, MMP2 activity was not changed at 0.1 and 0.2 microgram/milliliter; but, at the concentration of 0.4 microgram/milliliter, changed and not reversed by NAC. MMP-9 activity was similar in both cells. CONCLUSION: We could find ROS generation in CsA treated human mesangial cells, but not in tacrolimus treated cells. We think this difference resulted in the decrease of post-transcriptional MMP-2 activity in CsA treated cells and we also think tacrolimus cells in our experiments were not influenced by ROS. From these results, tacrolimus and CsA are different in the generation of ROS that have some effects in the matrix accumulation in mesangial cells. These result does not mean that tacrolimus is superior to CsA in nephrotoxicity, because nephrotoxicity is similar between two drugs. In conclusion, the mechanisms of nephrotoxicity are different between CsA and tacrolimus.
Central Nervous System ; Collagen ; Cyclosporine* ; Extracellular Matrix* ; Fibrosis ; Flow Cytometry ; Gingiva ; Humans* ; Hyperplasia ; Matrix Metalloproteinase 14 ; Mesangial Cells* ; Oxygen ; Tacrolimus* ; Tissue Inhibitor of Metalloproteinase-2