The discovery of shorter synthetic proteolytic peptides derived from Tob1 protein.

We screened nearly 1000 synthetic peptides and found that JAL-AK22 (KYEGHWYPEKPYKGSGFRCIHI), which is derived from the BoxA domain in the Tob1 protein, activates both unfolded and folded proMMP-7. Interestingly, the smaller derivative of JAL-AK22, termed JAL-TA9 (YKGSGFRMI) possessed auto-proteolytic activity and cleaved three synthetic peptides fragment (MMP18-33, MMP18-40, and Aß11-29) under physiological conditions. The kcat of JAL-TA9 was 4.58 × 10-4 min-1 against MMP18-33 and 6.5 × 10-4 min-1 against MMP18-40. These kinetic parameters are lower than those of general proteinases like trypsin, for which the kcat is 247.2 × 105 min-1 against benzoyl-l-arginine ethyl ester. In addition, a 5-mer peptide derived from JAL-TA9, GSGFR also cleaved Aß11-29. These proteolytic activities were inhibited by AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), a serine protease inhibitor. Our results demonstrate that some small synthetic peptides have protease activity. Thus, we propose calling small peptides possessing with protease activity Catalytides (catalytic peptides). We expect that our findings will stimulate the development of novel Catalytides and related applications such as the development of strategic peptide drugs.

CoCl2 , a mimic of hypoxia, enhances bone marrow mesenchymal stem cells migration and osteogenic differentiation via STAT3 signaling pathway.

Mesenchymal stem cells homing and migration is a crucial step during bone fracture healing. Hypoxic environment in fracture site induces bone marrow mesenchymal stem cells (BMSCs) migration, but its mechanism remains unclear. Our previous study and studies by other groups have reported the involvement of signal transducer and activator of transcription 3 (STAT3) pathway in cell migration. However, the role of STAT3 pathway in hypoxia-induced cell migration is still unknown. In this study, we investigated the role of STAT3 signaling in hypoxia-induced BMSCs migration and osteogenic differentiation. BMSCs isolated from C57BL/6 male mice were cultured in the presence of cobalt chloride (CoCl2 ) to simulate intracellular hypoxia. Hypoxia enhanced BMSCs migration, and upregulated cell migration related gene expression, that is, metalloproteinase (MMP) 7, MMP9, and C-X-C motif chemokine receptor 4. Hypoxia enhanced the phosphorylation of STAT3, and cell migration related proteins: c-jun n-terminal kinase (JNK), focal of adhesion kinase (FAK), extracellular regulated protein kinases, and protein kinase B 1/2 (ERK1/2). Moreover, hypoxia enhanced expression of osteogenic differentiation marker. Inhibition of STAT3 suppressed the hypoxia-induced BMSCs migration, cell migration related signaling molecules phosphorylation, and osteogenic differentiation related gene expression. In conclusion, our result indicates that hypoxia-induced BMSCs migration and osteogenic differentiation is via STAT3 phosphorylation and involves the cooperative activity of the JNK, FAK, and MMP9 signaling pathways.

Soluble factors from the notochordal-rich intervertebral disc inhibit endothelial cell invasion and vessel formation in the presence and absence of pro-inflammatory cytokines.

BACKGROUND: Chronic low back pain can be associated with the pathological ingrowth of blood vessels and nerves into intervertebral discs (IVDs). The notochord patterns the IVD during development and is a source of anti-angiogenic soluble factors such as Noggin and Chondroitin sulfate (CS). These factors may form the basis for a new minimally invasive strategy to target angiogenesis in the IVD. OBJECTIVE: To examine the anti-angiogenic potential of soluble factors from notochordal cells (NCs) and candidates Noggin and CS under healthy culture conditions and in the presence of pro-inflammatory mediators. DESIGN: NC conditioned media (NCCM) was generated from porcine NC-rich nucleus pulposus tissue. To assess the effects of NCCM, CS and Noggin on angiogenesis, cell invasion and tubular formation assays were performed using human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor alpha (TNFα [10 ng/ml]). vascular endothelial growth factor (VEGF)-A, MMP-7, interleukin-6 (IL-6) and IL-8 mRNA levels were assessed using qRT-PCR. RESULTS: NCCM (10 & 100%), CS (10 and 100 µg) and Noggin (10 and 100 ng) significantly decreased cell invasion of HUVECs with and without TNFα. NCCM 10% and Noggin 10 ng inhibited tubular formation with and without TNFα and CS 100 µg inhibited tubules in Basal conditions whereas CS 10 µg inhibited tubules with TNFα. NCCM significantly decreased VEGF-A, MMP-7 and IL-6 mRNA levels in HUVECs with and without TNFα. CS and Noggin had no effects on gene expression. CONCLUSIONS: We provide the first evidence that soluble factors from NCs can inhibit angiogenesis by suppressing VEGF signaling. Notochordal-derived ligands are a promising minimally invasive strategy targeting neurovascular ingrowth and pain in the degenerated IVD.

The chemokine receptors CXCR1 and CXCR2 regulate oral cancer cell behaviour.

BACKGROUND: Chemokines regulate physiological and pathological leucocyte trafficking, and chemokine receptors play a role in tumorigenesis. Expression of interleukin-8 (IL-8) receptors CXCR1 and CXCR2 has been shown in oral squamous cell carcinoma (OSCC) but remains poorly characterised. This aim of this study was to investigate CXCR1 and CXCR2 expression on normal oral keratinocytes (NOKs) and oral cancer cell lines (OCCL) and their relative response when exposed to IL-8 and growth-related oncogene-α (which selectively binds CXCR2). METHODS: mRNA and protein expression was studied using RT-PCR, immunocytochemistry and flow cytometry. ELISAs were used to investigate ERK1/2 phosphorylation and MMP production, whereas a MTS-based assay was employed to study proliferation. Migration assays were carried out using modified Boyden chambers with a matrigel coating used for invasion assays. RESULTS: mRNA expression of CXCR1 and CXCR2 was seen in both NOKs and OCCL with significantly higher protein expression in OCCL. Exposure to IL-8 and GROα increased intracellular ERK phosphorylation, proliferation, migration and invasion with OCCL showing a greater response than NOKs. These effects were mediated through CXCR1 and CXCR2 (for IL-8) and CXCR2 (for GROα) as receptor-blocking antibodies significantly inhibited the responses. IL-8 and GROα also increased MMP-9 release from NOKs and OCCL with significantly higher amounts released by OCCL. However, an increase in MMP-7 production was only seen in OCCL. CONCLUSIONS: Functional CXCR1 and CXCR2 exist on normal and cancerous oral epithelial cells, and our data suggests a role for these receptors in oral cancer biology.

Dual-luminophore-labeled gold nanoparticles with completely resolved emission for the simultaneous imaging of MMP-2 and MMP-7 in living cells under single wavelength excitation.

Although considerable effort has been devoted to the design of various nanoprobes for the fluorescent detection of multiple biomarkers in a single assay, they often suffer from emission-overlapping, owing to small Stokes shifts and wide emission spectra, which results in cross-talk and inaccurate quantification. Herein, we report the design and synthesis of a new nanoprobe for multienzyme detection with completely resolved emission peaks under single-wavelength excitation. The probe was assembled by attaching a cleavable peptide spacer, which was comprised from a matrix metalloproteinase-2 (MMP-2) substrate and a MMP-7 substrate, onto the surface of gold nanoparticles (AuNPs) through cysteine residues. A lanthanide complex, BCTOT-Eu(III) (BCTOT=1,10-bis(5'-chlorosulfo-thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone), and 7-amino-4-methylcoumarin (AMC) were attached to the N terminus and the C terminus of the peptide, respectively. In the presence of one or both targeting enzymes, the substrate was cleaved and fluorescence resonance energy transfer (FRET) between the dyes and AuNPs was prohibited, thereby resulting in the dramatic fluorescence emission of dyes. Importantly, there was no emission cross-talk between the two dyes, thereby ensuring accurate detection of each enzyme. Based on this, the simultaneous fluorescence image of MMP-2 and MMP-7 was accomplished in living cells under single wavelength excitation. The apparent differences in the fluorescence imaging indicated distinct differences between the expression levels of MMPs between the human normal liver cells and the human hepatoma cells.

Polyozellin blocks tumor necrosis factor α-induced interleukin 8 and matrix metalloproteinase 7 production in the human intestinal epithelial cell line HT-29.

Polyozellin isolated from Polyozellus multiplex (Thelephoraceae) displays potent anti-inflammatory effects in murine macrophages. Here we evaluated whether polyozellin has the potential to ameliorate diseases characterized by mucosal inflammation in intestinal epithelial HT-29 cells. Polyozellin significantly inhibited tumor necrosis factor (TNF)-α-induced interleukin-8 secretion and mRNA expression. Moreover, polyozellin suppressed the expression of matrix metalloproteinase-7 mRNA and extracellular pro-matrix metalloproteinase-7 secretion. A signal transduction study revealed that polyozellin significantly attenuates TNF-α-mediated p38 phosphorylation, inhibitory factor κBα degradation, and nuclear factor-κB-mediated transcriptional activation. Collectively, these results suggest that polyozellin has the potential to attenuate intestinal inflammation and shed light on the novel signal pathway evoked by TNF-α during intestinal inflammation.

Effects of Monascus-fermented rice extract on malignant cell-associated neovascularization and intravasation determined using the chicken embryo chorioallantoic membrane model.

The traditional Chinese medicine, Hong-Qu, also called red mold rice in the United States and Europe, is used for treating blood stasis, a disorder related to hyperlipidemia and atherosclerosis. In addition to improving metabolic syndrome, extracts from Monascus-fermented rice inhibit the proliferation of various cancer cells in vitro and in vivo. The objective was to examine the effect of red mold rice ethanol extract (RMRE) on angiogenesis, invasion, and metastasis during tumor progression. RMRE significantly inhibited the proliferation of SW480 and SW620 human colorectal carcinoma cells in a dose- and time-dependent manner by using the MTT assay. A capillary-like network morphology was observed after the addition of 20 ng/mL vascular endothelial growth factor or SW620 culture-conditional medium, which was not seen after RMRE treatment. Moreover, spontaneous intravasation into Matrigel grafts of SW620 cells from the upper to the lower layers in the chick embryo chorioallantoic membrane (CAM) model was detected by the polymerase chain reaction (PCR) amplification of human Alu genomic DNA from the lower CAMs in the RMRE-untreated group. Neovascularization increased to 75.3% +/- 11.6% by SW620 cells onplant with Matrigel grafts in the CAM model. However, RMRE significantly reduced CAM neovascularization in a dose-dependent manner. Finally, RMRE effectively decreased the activity of matrix metalloproteinase (MMP)-7 as determined by reverse transcription PCR (RT-PCR), Western blotting, and casein zymography assays. In summary, Monascus-fermented products exert a potent effect on tumor growth and activation, suggesting that they may serve as supplementary agents in adjuvant cancer therapy.

Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase-3 in human gingival fibroblasts via extracellular signal-regulated kinase.

BACKGROUND AND OBJECTIVE: Periodontal disease is characterized by increased expression and activity of matrix metalloproteinases (MMPs) and insufficient expression/activity of their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). This altered MMP-TIMP balance results in progressive destruction of gingival and periodontal extracellular matrix. Enamel matrix derivative (EMD), clinically used for periodontal regeneration in a device called Emdogain, has been suggested to enhance gingival healing following periodontal procedures in humans. We previously showed that EMD increases the proliferation of human and rat gingival fibroblasts and protects them from tumor necrosis factor-induced apoptosis. In the present study, the modulation of MMP and TIMP expression by EMD was investigated. MATERIAL AND METHODS: Primary human gingival fibroblasts were treated in vitro with tumor necrosis factor, EMD or both in serum-free conditions, and RNA was analyzed with an extracellular matrix-focused microarray and quantitative real-time polymerase chain reaction. RESULTS: Microarray analysis showed detectable expression of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-13, as well as TIMP-1 and TIMP-3 in untreated cells. There was no apparent regulation of the expression of MMP-2, MMP-7, MMP-13 and TIMP-1 by either tumor necrosis factor or EMD. In contrast, tumor necrosis factor significantly increased MMP-1 expression, and EMD reduced it when both agents were present. Also, EMD significantly induced TIMP-3 expression, an effect which was dependent on activation of extracellular signal-regulated kinase 1/2, since it was totally abolished by a selective extracellular signal-regulated kinase pathway inhibitor. CONCLUSION: These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, i.e. by stimulation of TIMP-3 production, which could improve the MMP-TIMP balance in gingival tissue and curb extracellular matrix destruction.

MMP7 expression regulated by endocrine therapy in ERbeta-positive colon cancer cells.

BACKGROUND: Many studies have shown that colon cancer is an estrogen-dependent carcinoma. This study explored the efficacy of endocrine therapy in colon cancer cells with high metastatic potential (HT29). We investigated the proliferation of HT29 cells after exposure to endocrine therapy (tamoxifen) and 5-FU. METHODS: Apoptosis was evaluated using flow cytometry. The expression of matrix metalloproteinases 7 (MMP-7) and estrogen receptor beta (ERbeta) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The migration capability of treated cells was determined with wound scratch assay. RESULTS: Tamoxifen alone, 5-FU alone, and the combination of the two drugs can significantly inhibit HT29 cell proliferation and migration, block the cells in G2/M phase and induce cell apoptosis. These drugs also can down-regulate MMP7 and ERbeta expression. CONCLUSION: Our findings suggest that endocrine therapy is an efficient therapy for inhibiting ERbeta-positive colon cancer cell proliferation and migration via down-regulation of MMP7.

The effect of epidermal growth factor on matrix metalloproteinases and tissue inhibitors of metalloproteinase gene expression in cultured human gingival fibroblasts.

OBJECTIVE: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a role in the breakdown of the extracellular matrix during normal physiological processes, and in pathological processes, including periodontitis. The aim of this study was to evaluate the effect of epidermal growth factor (EGF) on the expression of MMPs and TIMPs in cultured human gingival fibroblasts. METHODS: Fibroblasts were stimulated with 10(-3), 10(-6) or 10(-12)M EGF for 24h; untreated fibroblasts served as controls. Alterations in the expression of MMP-1, 2, 3, 7, 11, TIMP-1 and 2 were evaluated using real-time PCR and Western blotting. beta-Actin expression was used as a reference to normalize gene expression. RESULTS: Increased MMP-1, 3, 7 and 11 expressions were observed at all EGF concentrations (p<0.05). At the lowest EGF concentration, MMP-1, 3 and 7 presented the lowest expression and MMP-11 presented the greatest expression; at higher EGF concentrations, MMP-1, 3 and 7 presented greater up-regulation, and MMP-11 lower up-regulation (p<0.05). Protein expression was similarly regulated by EGF: increased up-regulation of MMP-1, 3 and 7 was observed with increasing EGF concentrations, except for MMP-11 that exhibited greater up-regulation at the lower EGF concentration. The gene expression of MMP-2, TIMP-1 and 2 was not affected by EGF (p<0.05). CONCLUSIONS: We conclude that EGF regulates expression for MMP-1, 3, 7 and 11 in a dose-dependent manner, suggesting that EGF may play a role in periodontal destruction and wound repair.

Inhibition of matrilysin expression by antisense or RNA interference decreases lysophosphatidic acid-induced epithelial ovarian cancer invasion.

Our previous reports show that matrilysin [matrix metalloproteinase (MMP)-7] is overexpressed in epithelial ovarian cancer (EOC) and recombinant MMP-7 promotes EOC invasion in vitro. In the present study, we further evaluated the correlation of MMP-7 expression to EOC invasiveness and examined its role in lysophosphatidic acid (LPA)-induced invasion. By sense and antisense gene transfection in vitro, we show that overexpression of MMP-7 in all MMP-7 stably transfected DOV13 clones significantly enhanced their invasiveness, although MMP-7 antisense transfection caused a 91% decrease of MMP-7 expression (P < 0.01) and 87% decrease of invasion (P < 0.05) in geneticin (G418)-selected DOV13 clone P47-M7As-3 compared with vector-transfected control. As assessed by MMP-7 ELISA, LPA treatment at 10 to 80 micromol/L significantly stimulated the secretion of total MMP-7 in DOV13 conditioned medium (P < 0.01). In addition, LPA apparently induced the activation of MMP-7 in DOV13 cells as detected by gelatin zymography. In the antisense MMP-7-transfected DOV13 clone (P47-M7As-3), LPA-increased invasion was significantly decreased compared with vector control. Moreover, knocking down of MMP-7 by small interfering RNA also suppressed LPA-induced invasion in two EOC cell lines (DOV13 and R182). Altogether, our results show that MMP-7 expression is correlated with EOC invasiveness and LPA-induced MMP-7 secretion/activation may represent a new mechanism that facilitates ovarian cancer invasion besides the well-known induction of MT1-MMP-mediated proMMP-2 activation by LPA.

Involvement of MMP-7 in invasion of pancreatic cancer cells through activation of the EGFR mediated MEK-ERK signal transduction pathway.

AIMS: To clarify the involvement of matrix metalloproteinase-7 (MMP-7) in cell dissociation and the subsequent invasion of pancreatic cancer cells. METHODS: Western blotting, in vitro invasion assay, immunocytochemistry, and immunohistochemistry were performed in pancreatic cancer cell lines or pancreatic cancer tissue. RESULTS: The active form of the MMP-7 protein was expressed exclusively in the conditioned medium of dissociated (PC-1.0 and AsPC-1) pancreatic cancer cells, whereas proMMP-7 protein was only detected in the conditioned medium of non-dissociated (PC-1 and Capan-2) cells. Both intracellular and conditioned medium localised MMP-7 was greatly reduced by treatment with the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the mitogen activated protein kinase kinase (MEK) inhibitor U0126 in pancreatic cancer cells. MMP-7 treatment significantly induced the disruption of tight junction (TJ) structures and subsequent cell dissociation, and activation of the EGFR mediated MEK- ERK (extracellular signal regulated protein kinase) signalling pathway in the non-dissociated pancreatic cancer cells. Moreover, the strong in vitro invasiveness of dissociated cells was inhibited by AG1478 and U0126 treatment, whereas the weak invasiveness of non-dissociated cells was apparently induced by MMP-7 treatment. In addition, MMP-7 expression was stronger at the invasive front than at the centre of human pancreatic tumours. CONCLUSION: MMP-7 is involved in cell dissociation and the subsequent invasion of pancreatic cancer cells. It induces the disruption of TJ structures and forms a positive feedback loop with activation of the EGFR mediated MEK-ERK signalling pathway.

Rosuvastatin reduces MMP-7 secretion by human monocyte-derived macrophages: potential relevance to atherosclerotic plaque stability.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to reduce cardiovascular morbidity and mortality by their actions on atherogenic lipid profiles and by pleiotropic effects. In this study, we have investigated the effect of a new statin, rosuvastatin (Crestor), on sterol synthesis and the expression of metalloproteinases (MMPs) in human monocyte-derived macrophages (HMDM). Rosuvastatin dose-dependently inhibited sterol synthesis from acetate with an IC(50) of 70 nM. In addition, MMP-7 levels were reduced in a dose-dependent manner with maximal inhibition of 50% (P < 0.01) at 1 microM. Also, addition of isoprenoids such as farnesyl pyrophosphate (Fpp) or geranylgeranyl pyrophosphate (GGpp) fully overcame the inhibitory effect of rosuvastatin on MMP-7. Neither quantitative PCR nor transient transfection of HMDM with a luciferase reporter construct under the control of human MMP-7 promoter (2300 bp of the 5' region on MMP-7 gene) showed a decrease in MMP-7 mRNA following treatment with rosuvastatin (10(-6)M). However, the inhibitory effect of the statin occurred at the post-transcriptional level as determined by actinomycin D experiment. In conclusion, several studies have reported a high expression of active MMP-7 in human atherosclerotic plaques indicating a potential role in the weakening of the fibrous cap, predisposing it to rupture. The effect of rosuvastatin in reducing MMP-7 might protect fibrous caps from degradation and in turn stabilize atheromatous plaques.

Relaxin enhances in-vitro invasiveness of breast cancer cell lines by up-regulation of matrix metalloproteases.

Until recently, relaxin (RLX) has been known predominantly for its effects on the reproductive system, where it induces remodelling of the extracellular matrix and up-regulation of matrix metalloproteases (MMPs). In solid cancers, tissue remodelling and MMP activation are essential for invasion and metastasis. We therefore investigated the effect of RLX on invasiveness and MMP expression of human breast cancer cell lines. Upon incubation with porcine RLX, the invasiveness of SK-BR3 cells was significantly increased. Similar effects could be achieved in MCF-7 cells, especially when RLX was combined with epidermal growth factor. Enhanced invasiveness was accompanied by up-regulation of MMP production and could be almost completely blocked by the MMP inhibitor FN 439. Zymography revealed increased secretion of MMP-2, -7 and -9, associated with up-regulated mRNA concentrations of MMP-2, -9, -13 and -14. mRNA expression levels of MMP-1, -3, -7, -8, -10, -11, -12 and of tissue inhibitors of metalloproteases-1, -2, -3 and -4 were either very low or not detectably influenced by RLX. Taken together, RLX enhances in-vitro invasiveness of breast cancer cell lines by induction of MMP expression. It remains to be clarified whether RLX might play a similar role in vivo and promote tumour progression.