Self-Assembly-Induced Enhancement of Cathodic Electrochemiluminescence of Copper Nanoclusters for a Split-Type Matrix Metalloproteinase 14 Sensing Platform.

The unique optoelectronic and tunable luminescent characteristics of copper nanoclusters (Cu NCs) make them extremely promising as luminophores. However, the limited luminescence intensity and stability of Cu NCs have restricted their application in the field of electrochemiluminescence (ECL). Herein, a self-assembly-induced enhancement strategy was successfully employed to enhance the cathodic ECL performance of flexible ligand-stabilized Cu NCs. Specifically, Cu NCs form ordered sheetlike structures through intermolecular force. The restriction of ligand torsion in this self-assembled structure leads to a significant improvement in the ECL properties of the Cu NCs. Experimental results demonstrate that the assembled nanoscale Cu NC sheets exhibit an approximately three-fold increase in cathodic ECL emission compared to the dispersed state of Cu NCs. Furthermore, assembled nanoscale Cu NCs sheets were utilized as signal probes in conjunction with a specific short peptide derived from the catalytic structural domain of matrix metalloproteinase 14 (MMP 14) as the identification probe, thereby establishing a split-type ECL sensing platform for the quantification of NMP 14. The investigation has revealed the exceptional performance of assembled nanoscale Cu NCs sheets in ECL analysis, thus positioning them as novel and promising signal probes with significant potential in the field of sensing.

Effects of adrenocorticotrophic hormone on the expression of matrix metalloproteinases and their inhibitors in the bovine ovary.

Cattle undergo numerous environmental and management stressors that reduce fertility and affect ovulation. The extracellular matrix of the follicle wall can be altered by matrix metalloproteinases (MMPs), the activities of which are regulated by interleukins and tissue-specific inhibitors of metalloproteinases (TIMPs), especially during ovulation. The aims of the present study were to: (1) evaluate changes in the hormone milieu, the localisation and activity of MMP2 and MMP9 and the localisation of MMP14, TIMP1 and TIMP2 in response to adrenocorticotrophic hormone (ACTH) during the preovulatory period in cows; and (2) determine the direct effects of ACTH on the mRNA expression of MMP2 and MMP9 in the cultured follicle wall of bovine ovaries obtained from an abattoir. 100IU ACTH was administered during pro-oestrus every 12h until ovariectomy, which was performed before ovulation. Cortisol concentrations in the plasma and follicular fluid (FF) of preovulatory follicles were higher in ACTH-treated than control cows. Progesterone presented subluteal concentrations in plasma of ACTH-treated cows (P<0.05). MMP2 immunostaining and activity in ovaries were higher in ACTH-treated than control cows (P<0.05), whereas MMP9 immunostaining was similar between the two groups. However, unlike in control cows, MMP9 activity was absent in the FF of ACTH-treated cows. These results suggest that the administration of ACTH during the preovulatory period in cows could cause changes that culminate in modifications in the content and activation of MMPs and TIMPs in the ovary, which could interfere with the ovulation process.

Metalloproteinase 14 (MMP-14) and hsa-miR-410-3p expression in human inflamed dental pulp and odontoblasts.

The objective of this study is to evaluate MMP-14 expression in odontoblasts and in the bulk of dental pulp of teeth with pulpitis; to determine the expression of microRNA-410 (miR-410) in pulp tissue, since sequence analysis suggests that miR-410 has potential binding site on MMP-14's 3'UTR, and hence, can regulate expression of the latter one. Tissue samples of dental pulp from teeth with pulpitis and healthy (control) were formalin fixed and paraffin embedded (FFPE). Samples were examined using immunohistochemical staining for MMP-14 and the expression of miR-410 was evaluated using qRT-PCR. In both, healthy and inflamed pulp odontoblasts stained more intensively than remaining pulp tissue, but this difference was not statistically significant. More positive staining was observed in inflamed pulps compared to healthy pulps. Expression of miR-410 was found significantly lower in inflamed pulps than in healthy ones. In the two examined zones, odontoblasts and remaining pulp, miR-410 was expressed on a similar level. No statistically significant correlation of miR-410 and MMP-14 expression was found. We showed that inflammation changes the MMP-14 expression in pulp tissue and odontoblasts. This study demonstrates for the first time miR-410 expression in human dental pulp and that expression of this microRNA was downregulated in inflamed dental pulp and odontoblasts.

iTRAQ-Based Membrane Proteomics Reveals Plasma Membrane Proteins Change During HepaRG Cell Differentiation.

HepaRG cell, a stabilized bipotent liver progenitor cell line, exhibits hepatocyte functions only after differentiation. However, the mechanism of transition from nondifferentiated to differentiated states, accompanied by proliferation migration and differentiation, remains poorly understood, particularly those proteins residing in the plasma membrane. In this study, the membrane protein expression change of HepaRG cell during differentiation were systematically analyzed using an iTRAQ labeled quantitative membrane proteomics approach. A total of 70 membrane proteins were identified to be differentially expressed among 849 quantified membrane proteins. Function and disease clustering analysis proved that 11 of these proteins are involved in proliferation, migration, and differentiation. Two key factors (MMP-14 and OCLN) were validated by qRT-PCR and Western blot. Blockade of MMP-14 further demonstrated its important function during tumor cell migration. The data sets have been uploaded to ProteomeXchange with the identifier PXD004752.

Interaction between hepatic membrane type 1 matrix metalloproteinase and acireductone dioxygenase 1 regulates hepatitis C virus infection.

Membrane type 1 matrix metalloproteinase (MT1-MMP) binds to and regulates the function of tetraspanin-enriched microdomains. It also physically interacts with claudin-1 and acireductone dioxygenase 1 (ADI1), both associated with hepatitis C virus (HCV) cell entry. Here, we examined hepatic expression of MT1-MMP, ADI1 and claudin-1 as well as their physical interaction in relation to serum or intrahepatic HCV-RNA levels. A total of 104 liver biopsies obtained from chronic hepatitis C patients and 84 liver tissues obtained from noncancerous parts of surgically removed HCV-related hepatocellular carcinoma were analysed. Positive cytoplasmic ADI1 in liver biopsies was associated with higher serum HCV-RNA levels (P = 0.009). Positive MT1-MMP and ADI1 interaction assessed by co-immunoprecipitation was associated with lower tissue HCV-RNA levels (P = 0.009). Hepatic HCV-RNA levels were positively associated with ADI1 levels in the MT1-MMP and ADI1 co-immunoprecipitates (P = 0.030). Overexpression of MT1-MMP in Huh7.5 cells suppressed cell entry of HCV pseudoparticles as well as HCVcc infection. The suppression effect could be reversed by co-expression of ADI1 in a dose-dependent manner. In summary, clinical and cell-based experiments suggested that physical interaction between MT1-MMP and ADI1 led to suppression of HCV infection. This inhibitory effect could be reversed by ADI1 overexpression.

Activatable and Cell-Penetrable Multiplex FRET Nanosensor for Profiling MT1-MMP Activity in Single Cancer Cells.

We developed a quantum-dot-based fluorescence resonance energy transfer (QD-FRET) nanosensor to visualize the activity of matrix metalloproteinase (MT1-MMP) at cell membrane. A bended peptide with multiple motifs was engineered to position the FRET pair at a close proximity to allow energy transfer, which can be cleaved by active MT1-MMP to result in FRET changes and the exposure of cell penetrating sequence. Via FRET and penetrated QD signals, the nanosensor can profile cancer cells.

MMPs-2 and -14 Are Elevated in Eosinophilic Esophagitis and Reduced Following Topical Corticosteroid Therapy.

OBJECTIVES: Eosinophilic esophagitis (EoE) is a chronic, antigen-mediated disease in children and adults associated with substantial esophageal remodeling and fibrosis. The expression of the remodeling-associated matrix metalloproteinases (MMPs) has not been previously detailed in EoE. METHODS: MMP-2 and -14 expression and cellular localization were assessed using real-time quantitative polymerase chain reaction and immunohistochemistry/immunofluorescence in EoE fibroblasts, active and inactive pediatric EoE biopsies, and nondiseased control biopsies. The effect of transforming growth factor (TGF)-ß1 treatment on MMP-2 expression in cultured esophageal epithelial (HET1A) cells was analyzed. RESULTS: MMP-2 and -14 mRNA were expressed in EoE fibroblasts and biopsies. Proliferating epithelial cells produced MMP-14 more abundantly in EoE than in controls (P < 0.001) and the degree of epithelial MMP-14 expression correlated positively with basal zone hyperplasia (r = 0.65, P = 0.002). EoE lamina propria had higher numbers of MMP-2- and -14-positive cells (906 ±â€Š167 and 701 ±â€Š93 cells/mm²) as compared with controls (258 ±â€Š93 cells/mm², P < 0.01 and 232 ±â€Š54 cells/mm², P < 0.01), and MMP-14 expression correlated with the severity of fibrosis. Following therapy with topical corticosteroids, MMP-14 and -2 were significantly diminished (P < 0.01). TGF-ß1 increased the expression and secretion of MMP-2 from esophageal epithelial HET1A cells. CONCLUSIONS: MMP-2 and -14 are elevated in pediatric patients with EoE and significantly decrease following topical corticosteroid therapy. TGF-ß1 increases MMP-2 in esophageal epithelial cells. This alludes to previously unappreciated role for MMPs in EoE-associated esophageal remodeling and a potential positive feedback loop via TGF-ß1.

Comparative expression of matrix metalloproteinases in internal malignancies and paired cutaneous metastatic lesions.

BACKGROUND: Matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP), and membrane-type 1 matrix metalloproteinase (MT1-MMP) are thought to be involved in the destruction of basement membrane and stromal invasion by cancer cells. OBJECTIVE: The aim of this study was to identify and compare MMP and TIMP expression in internal malignancies and paired cutaneous metastatic lesions. MATERIALS AND METHODS: We compared the expression of MMP-2, MMP-9, MT1-MMP, and TIMP-2 in the internal malignancy and paired cutaneous metastatic lesion using immunohistochemical stains. RESULTS: The cutaneous metastatic lesions expressed significantly more MMP-2, MMP-9, and MT1-MMP and significantly less TIMP-2 than did the paired internal malignancies. In breast cancer, cutaneous metastatic lesions expressed significantly more MMP-9 and significantly less TIMP-2 than did the primary breast cancer lesion. In lung cancer, the cutaneous metastatic lesion expressed significantly more MMP-2 and MT1-MMP than did the primary lesion. In stomach cancer, the cutaneous metastatic lesion expressed significantly less TIMP-2 than did the primary lesion. CONCLUSIONS: Our study demonstrates that cutaneous metastatic lesions have different MMPs and TIMP-2 expression patterns compared with their paired internal malignancies. Also, MMPs and TIMP-2 expression differs according to the type of primary cancer.

In vivo imaging of membrane type-1 matrix metalloproteinase with a novel activatable near-infrared fluorescence probe.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a protease activating MMP-2 that mediates cleavage of extracellular matrix components and plays pivotal roles in tumor migration, invasion and metastasis. Because in vivo noninvasive imaging of MT1-MMP would be useful for tumor diagnosis, we developed a novel near-infrared (NIR) fluorescence probe that can be activated following interaction with MT1-MMP in vivo. MT1-hIC7L is an activatable fluorescence probe comprised of anti-MT1-MMP monoclonal antibodies conjugated to self-assembling polymer micelles that encapsulate NIR dyes (IC7-1, λem : 858 nm) at concentrations sufficient to cause fluorescence self-quenching. In aqueous buffer, MT1-hIC7L fluorescence was suppressed to background levels and increased approximately 35.5-fold in the presence of detergent. Cellular uptake experiments revealed that in MT1-MMP positive C6 glioma cells, MT1-hIC7L showed significantly higher fluorescence that increased with time as compared to hIC7L, a negative control probe lacking the anti-MT1-MMP monoclonal antibody. In MT1-MMP negative MCF-7 breast adenocarcinoma cells, both MT1-hIC7L and hIC7L showed no obvious fluorescence. In addition, the fluorescence intensity of C6 cells treated with MT1-hIC7L was suppressed by pre-treatment with an MT1-MMP endocytosis inhibitor (P < 0.05). In vivo optical imaging using probes intravenously administered to tumor-bearing mice showed that MT1-hIC7L specifically visualized C6 tumors (tumor-to-background ratios: 3.8 ± 0.3 [MT1-hIC7L] vs 3.1 ± 0.2 [hIC7L] 48 h after administration, P < 0.05), while the probes showed similarly low fluorescence in MCF-7 tumors. Together, these results show that MT1-hIC7L would be a potential activatable NIR probe for specifically detecting MT1-MMP-expressing tumors.

Visual and automated assessment of matrix metalloproteinase-14 tissue expression for the evaluation of ovarian cancer prognosis.

The purpose of this study was to evaluate whether the membrane type 1 matrix metalloproteinase-14 (or MT1-MMP) tissue expression, as assessed visually on digital slides and by digital image analysis, could predict outcomes in women with ovarian carcinoma. Tissue microarrays from a cohort of 211 ovarian carcinoma women who underwent a debulking surgery between 1993 and 2006 at the CHU de Québec (Canada) were immunostained for matrix metalloproteinase-14. The percentage of MMP-14 staining was assessed visually and with the Calopix software. Progression was evaluated using the CA-125 and/or the RECIST criteria according to the GCIG criteria. Dates of death were obtained by record linkage with the Québec mortality files. Adjusted hazard ratios of death and progression with their 95% confidence intervals were estimated using the Cox model. Comparisons between the two modalities of MMP-14 assessment were done using the box plots and the Kruskal-Wallis test. The highest levels of MMP-14 immunostaining were associated with nonserous histology, early FIGO stage, and low preoperative CA-125 levels (P<0.05). In bivariate analyses, the higher level of MMP-14 expression (>40% of MMP-14-positive cells) was inversely associated with progression using visual assessment (hazard ratio=0.39; 95% confidence interval: 0.18-0.82). A similar association was observed with the highest quartile of MMP-14-positive area assessed by digital image analysis (hazard ratio=0.48; 95% confidence interval: 0.28-0.82). After adjustment for standard prognostic factors, these associations were no longer significant in the ovarian carcinoma cohort. However, in women with serous carcinoma, the highest quartile of MMP-14-positive area was associated with progression (adjusted hazard ratio=0.48; 95% confidence interval: 0.24-0.99). There was no association with overall survival. The digital image analysis of MMP-14-positive area matched the visual assessment using three categories (>40% vs 21-40 vs <20%). Higher levels of MMP-14 immunostaining were associated with standard factors of better ovarian carcinoma prognosis. In women with serous carcinoma, high expression of MMP-14 was associated with lower progression.

EMMPRIN co-expressed with matrix metalloproteinases predicts poor prognosis in patients with osteosarcoma.

Several studies have focused on the relationships between the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and the prognosis of patients with malignant tumors. However, few of these have investigated the expression of EMMPRIN in osteosarcoma. We examined expression levels of EMMPRIN immunohistochemically in 53 cases of high-grade osteosarcoma of the extremities and analyzed the correlation of its expression with patient prognosis. The correlation between matrix metalloproteinases (MMPs) and EMMPRIN expression and the prognostic value of co-expression were also analyzed. Staining positivity for EMMPRIN was negative in 7 cases, low in 17, moderate in 19, and strong in 10. The overall and disease-free survivals (OS and DFS) in patients with higher EMMPRIN expression (strong-moderate) were significantly lower than those in the lower (weak-negative) group (0.037 and 0.024, respectively). In multivariate analysis, age (P=0.004), location (P=0.046), and EMMPRIN expression (P=0.038) were significant prognostic factors for overall survival. EMMPRIN expression (P=0.024) was also a significant prognostic factor for disease-free survival. Co-expression analyses of EMMPRIN and MMPs revealed that strong co-expression of EMMPRIN and membrane-type 1 (MT1)-MMP had a poor prognostic value (P=0.056 for DFS, P=0.006 for OS). EMMPRIN expression and co-expression with MMPs well predict the prognosis of patients with extremity osteosarcoma, making EMMPRIN a possible therapeutic target in these patients.

MMP-14 overexpression correlates with poor prognosis in non-small cell lung cancer.

Matrix metalloproteinase-14 (MMP-14) has been demonstrated to play an important role in tumor progression. The aim of this study was to analyze the correlation between MMP-14 expression and clinicopathologic features and its prognostic significance in non-small cell lung cancer (NSCLC). Immunohistochemical staining for MMP-14 protein was performed in 104 patients with NSCLC. High levels of MMP-14 protein were positively correlated with the status of clinical stage (I-II vs. III-IV; P < 0.001), N classification (N0-N1 vs. N2-N3; P < 0.001), distant metastasis (no vs. yes; P = 0.014), and differentiated degree (high vs. low or undifferentiated; P = 0.001). The patients with higher MMP-14 expression of protein had shorter survival time than patients with low MMP-14 expression. Multivariate analysis indicated that the level of MMP-14 expression was an independent prognostic indicator (P < 0.001) for the survival of patients with NSCLC. In conclusion, MMP-14 is a potential unfavorable prognostic factor for patients with NSCLC.

Immunohistochemical expression of MMP-14 and MMP-2, and MMP-2 activity during human ovarian follicular development.

BACKGROUND: The aim of this study was to investigate the presence of MMP-14 and MMP-2 during human ovarian follicular development using immunohistochemistry, and the activity of MMP-2 in follicular fluid using zymography. METHODS: Ovarian tissue collected from the archives of the Department of Pathology was examined and medical records and histopathology were reviewed. Follicular fluids were collected at the IVF-department and analyzed using zymography. RESULTS: MMP-14 and MMP-2 were increasingly found in the growing follicles and MMP-2 was highly expressed in the corpus luteum. Pro-MMP-2 was present in follicular fluid of IVF-patients. CONCLUSIONS: The presence of MMP-14 and MMP-2 during human ovarian follicular development from the primordial follicle to the tertiary follicle and corpus luteum is confirmed, as was indicated by earlier animal studies following stimulation with gonadotrophins.

Calcifying Cystic Odontogenic Tumour: immunohistochemical expression of matrix metalloproteinases, their inhibitors (TIMPs and RECK) and inducer (EMMPRIN).

BACKGROUND: Calcifying cyst odontogenic tumour (CCOT) is a rare benign cystic neoplasm of odontogenic origin. MMPs are responsible for extracellular matrix remodelling and, together their inhibitors and inducer, determinate the level of its turnover in pathological processes, leading to an auspicious microenvironment for tumour development. Thus, our goal was to evaluate matrix metalloproteinases (MMPs-2, -7, -9 and -14), their inhibitors (TIMPs-2, -3, -4 and RECK) and its inductor (EMMPRIN) expression in CCOT. MATERIALS AND METHODS: We used 18 cases of CCOT submitted to immunolocalization of the target proteins and analysed in both neoplastic odontogenic epithelial and stromal compartments. RESULTS: All molecules evaluated were expressed in both compartments in CCOT. In epithelial layer, immunostaining for MMPs, TIMPs, RECK and EMMPRIN was found in basal, suprabasal spindle and stellate cells surrounding ghost cells and ghost cells themselves, except for MMP-9 and TIMP-2 which were only expressed by ghost cells. In stromal compartment, extracellular matrix, mesenchymal (MC) and endothelial cells (EC) were positive for MMP-2, -7, TIMP-3 and -4, while MMP-9, TIMP-2 and RECK were positive only in MC and MMP-14 only in EC. Statistical significance difference was found between both compartments for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001), being more expressed in epithelium than in stroma. Positive correlation between both stromal EMMPRIN and RECK expression was found (R = 0.661, P = 0.003). CONCLUSIONS: We concluded that these proteins/enzymes are differentially expressed in both epithelium and stroma of CCOT, suggesting an imbalance between MMPs and their inducer/inhibitors may contribute on the tumour behaviour.

Matrix metalloproteinases and myeloperoxidase in gingival crevicular fluid provide site-specific diagnostic value for chronic periodontitis.

AIM: To identify the diagnostic accuracy of gingival crevicular fluid (GCF) candidate biomarkers to discriminate periodontitis from the inflamed and healthy sites, and to compare the performance of two independent matrix metalloproteinase (MMP)-8 immunoassays. MATERIALS AND METHODS: Cross sectional study. GCF (N = 58 sites) was collected from healthy, gingivitis and chronic periodontitis volunteers and analysed for levels of azurocidin, chemokine ligand 5, MPO, TIMP-1 MMP-13 and MMP-14 by ELISA or activity assays. MMP-8 was assayed by immunofluorometric assay (IFMA) and ELISA. Statistical analysis was performed using linear mixed-effects models and Bayesian statistics in R and Stata V11. RESULTS: MMP-8, MPO, azurocidin and total MMP-13 and MMP-14 were higher in periodontitis compared to gingivitis and healthy sites (p < 0.05). A very high correlation between MPO and MMP-8 was evident in the periodontitis group (r = 0.95, p < 0.0001). MPO, azurocidin and total levels of MMP-8, MMP-13 and MMP-14 showed high diagnostic accuracy (≥0.90), but only MMP-8 and MPO were significantly higher in the periodontitis versus gingivitis sites. MMP-8 determined by IFMA correlated more strongly with periodontal status and showed higher diagnostic accuracy than ELISA. CONCLUSIONS: MPO and collagenolytic MMPs are highly discriminatory biomarkers for site-specific diagnosis of periodontitis. The comparison of two quantitative MMP-8 methods demonstrated IFMA to be more accurate than ELISA.

Miniaturized antibodies for imaging membrane type-1 matrix metalloproteinase in cancers.

Since membrane type-1 matrix metalloproteinase (MT1-MMP) plays pivotal roles in tumor progression and metastasis and holds great promise as an early biomarker for malignant tumors, a method of evaluating MT1-MMP expression levels would be valuable for molecular biological and clinical studies. Although we have previously developed a (99m) Tc-labeled anti-MT1-MMP monoclonal IgG ((99m) Tc-MT1-mAb) as an MT1-MMP imaging probe by nuclear medical techniques for this purpose, slow pharmacokinetics were a problem due to its large molecular size. Thus, in this study, our aim was to develop miniaturized antibodies, a single chain antibody fragment (MT1-scFv) and a dimer of two molecules of scFv (MT1-diabody), as the basic structures of MT1-MMP imaging probes followed by in vitro and in vivo evaluation with an (111) In radiolabel. Phage display screening successfully provided MT1-scFv and MT1-diabody, which had sufficiently high affinity for MT1-MMP (KD  = 29.8 and 17.1 nM). Both (111) In labeled miniaturized antibodies showed higher uptake in MT1-MMP expressing HT1080 cells than in non-expressing MCF7 cells. An in vivo biodistribution study showed rapid pharmacokinetics for both probes, which exhibited >20-fold higher tumor to blood radioactivity ratios (T/B ratio), an index for in vivo imaging, than (99m) Tc-MT1-mAb 6 h post-administration, and significantly higher tumor accumulation in HT1080 than MCF7 cells. SPECT images showed heterogeneous distribution and ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles in tumors corresponded to MT1-MMP-positive areas. These findings suggest that the newly developed miniaturized antibodies are promising probes for detection of MT1-MMP in cancer cells.

Differentiated expression of membrane type metalloproteinases (MMP-14, MMP-15) and pro-MMP2 in laryngeal squamous cell carcinoma. A novel mechanism.

Cancer progression involves multiple proteolytic interactions, with metalloproteinases (MMPs) performing a crucial role. MMP-2, a major MMP, plays a key role in the degradation of basement membranes. Mechanisms underlying MMP-2 activation had to be investigated. Membrane-type matrix metalloproteinases are not only responsible for the regulation of extracellular matrix remodeling, but also involved in the activation of several inactive MMPs. The aim of this study was to evaluate the expression of pro-MMP2, MMP-14, and MMP-15 in tumor cells and tumor stroma. Immunohistochemical studies were performed on paraffin-embedded tissue sections including laryngeal squamous cell carcinoma (SCC). We found the expression of pro-MMP2 in 58% of cases, MMP-14 in 78%, and MMP-15 in 98% of cases of SCC. In all tumor cases, we revealed a higher expression of pro-MMP2 in tumor stoma than in tumor cells. The expression of MMP-14 and MMP-15 was higher in tumor cells than in the stroma. Moreover, we found a statistically significant difference between the expression of MMP-14 and MMP-15 in the tumor in comparison with the surrounding stroma (P < 0.05). An analysis of expression levels of MT-MMPs by classification trees showed that the probability of metastases was related to decreased expression of MMP-14 and increased expression of MMP-15. Our results may suggest that tumor cells with low MMP-14 expression invade tumor stroma and form metastases. Probably, in such cases, tumor progression is stimulated by MMP-15 in an MMP-14 independent pathway, a novel (alternative) mechanism.

All-trans-retinoic acid and the glycolipid α-galactosylceramide combined reduce breast tumor growth and lung metastasis in a 4T1 murine breast tumor model.

Vitamin A compounds are promising for cancer prevention and reducing risk of recurrence. Herein we have evaluated the combination of all-trans-retinoic acid (RA), a vitamin A metabolite, and alpha-galactosylceramide (αGalCer), a lipid immune activator, in Balb/C mice inoculated with syngeneic 4T1 breast tumor cells on reduction in breast tumor growth and lung metastasis. In Balb/c inoculated with the syngenic 4T1 primary tumor, and administered dendritic cells treated with RA + αGalCer, the size of the primary tumor and the number of lung metastatic foci were reduced. When 4T1 cells were introduced into the circulation as a model of hematogenous spread of tumor cells and RA and αCalCer were administered directly to mice without dendritic cells, lung metastatic foci were reduced 70% (P < 0.05), whereas each agent alone resulted in an intermediate decrease. Concomitantly, the expression of matrix metalloproteinases (MMP), membrane type-1 (MT1)-MMP and MMP3, were reduced by RA + αGalCer in lung. MMP3 protein was also reduced in plasma and culture supernatants from RA + αGalCer-treated 4T1 cells. Together, our results provide new evidence that a nutritional-immunological combination of RA + αGalCer may be promising for preventing or slowing the growth of metastatic foci, and suggest reduced MMP production as a possible mechanism.

Expression of gelatinases (MMP-2, MMP-9) and gelatinase activator (MMP-14) in actinic keratosis and in in situ and invasive squamous cell carcinoma.

Given the established role of matrix metalloproteinases (MMPs) in physiological processes in the skin, we investigated the expression of MMP-2, MMP-9, and MMP-14 to evaluate their role in the grading and development of atypical epithelial lesions. Immunohistochemistry was performed using antibodies against these MMPs in actinic keratosis (AK; n = 24), squamous cell carcinoma (SCC) in situ (SCCIS; n = 27), SCC well differentiated (SCCWD; n = 28), and SCC moderately to poorly differentiated (SCCMPD; n = 20). Tumoral and stromal expression was assessed by intensity (SI) and percentage positivity (PC). The mean of the total score, calculated by adding intensity and percentage positivity, was used for statistical analyses. In AK, SCCIS, SCCWD, and SCCMPD, mean tumoral MMP-2 expression was 3.33, 4.07, 4.46, and 3.40, respectively (P = NS for all) and stromal expression was 1.42, 3.26, 3.07, and 1.55 respectively (P < 0.05 for AK vs. SCCIS/SCCWD and SCCMPD vs. SCCIS/SCCWD); mean tumoral MMP-9 expression was 4.33, 4.11, 4.46, and 3.35, respectively, and stromal expression was 4.29, 4.41, 4.75, and 4.60, respectively (P = NS for all) and, mean tumoral MMP-14 expression was 1.58, 2.41, 0.32, and 0.35, respectively (P < 0.05 AK vs. SCCWD and SCCIS vs. SCCWD/SCCMPD) and stromal expression was 3.04, 3.52, 0.46, and 0.60, respectively (P < 0.05 for AK vs. SCCWD/SCCMPD). Only MMP-14 showed a statistically significant linear trend with decreasing values for tumoral and stromal expression with invasion suggesting that it might be of use as a prognosticator. Enhanced stromal MMP-2 expression in SCCIS and SCCWD relative to AK suggests that it may be of relevance to disease progression.

Proteolytic activation of heparin-binding EGF-like growth factor by membrane-type matrix metalloproteinase-1 in ovarian carcinoma cells.

Increased expression of heparin-binding EGF-like growth factor (HB-EGF) and membrane-type matrix metalloproteinase-1 (MT1-MMP) is frequently associated with various types of malignant tumor. HB EGF-like growth factor has been reported to promote the malignant progression of ovarian carcinoma. Based on this finding, inhibition of HB-EGF activity with CRM197 is now under phase I clinical evaluation. On the other hand, MT1-MMP expressed in ovarian carcinoma cells is thought to promote invasion and growth of tumor cells by degrading the extracellular matrix. However, we recently demonstrated that co-expression of MT1-MMP and HB-EGF in gastric carcinoma cells leads to cleavage of HB-EGF within its N-terminal heparin-binding region, converting it into a potent heparin-independent growth factor. In this study, we evaluated the importance of regulation of HB-EGF by MT1-MMP in clinical samples of ovarian carcinoma. We detected co-expression of HB-EGF and MT1-MMP in clear cell ovarian carcinoma tissues, particularly at the invasion front and in tumor cells that had disseminated into the ascites, whereas HB-EGF alone was expressed in non-invasive borderline ovarian tumor tissue. Furthermore, a soluble HB-EGF fragment that corresponds to that processed by MT1-MMP was detected in malignant ascites obtained from patients with metastatic ovarian carcinoma. Ovarian carcinoma cells that express MT1-MMP and HB-EGF exhibited enhanced cell growth in a 3D-collagen matrix and anchorage-independent growth in suspension. These results indicate that MT1-MMP co-expressed with HB-EGF in ovarian carcinoma cells potentiates the activity of HB-EGF to promote invasive tumor growth and spreading in vivo.