Matrix metalloproteinases (MMP) 1 and MMP10 but not MMP12 are potential oral cancer markers

The aim of this study was to investigate the mRNA performance of matrix metalloproteinases (MMP) 1, MMP10 and MMP12 as oral cancer markers. With gingiva as the control, the areas under the receiver- operating characteristic curves (AUCs) of the relative gene expressions for MMP1, MMP10 and MMP12 were 0.715, 0.727 and 0.513, respectively. With the margins or neck platysma muscles as controls, the AUCs of MMP1, MMP10 and MMP12 were 0.746 vs 0.626, 0.712 vs 0.683 and 0.697 vs 0.630, respectively. MMP10 displayed the best sensitivity for oral cancer detection with any controls. MMP1 and MMP10 were suitable markers for cancer detection with gingiva and margin as controls. Using neck tissue as the control, only MMP10 was suitable for cancer detection. With margin and neck controls, there were no significant differences for MMP1, MMP10 and MMP12 in different stages, invasion and locations or different habits. Therefore, MMP1 and MMP10 but not MMP12 are potential oral cancer markers.     

Meta-Analysis of MMP2, MMP3, and MMP9 Promoter Polymorphisms and Head and Neck Cancer Risk

Background

The 1306 C>T, 1171 5A>6A, and 1562C>T polymorphisms of matrix metalloproteinase (MMP) 2, MMP3, and MMP9 genes, respectively, have been found to be functional and may contribute to head and neck carcinogenesis. However, the results of case-control studies examining associations between MMP polymorphisms and head and neck cancer (HNC) risk remain inconclusive. Therefore, we performed a meta-analysis to further evaluate the role of these polymorphisms in HNC development.

Methods

We searched PubMed, ISI Web of Knowledge, MEDLINE, Embase, and Google Scholar to identify all published case-control studies of MMP2-1306 C>T, MMP3-1171 5A>6A, and MMP9-1562 C>T polymorphisms and HNC risk in the meta-analysis. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the association between these polymorphisms and HNC risk.

Results

Thirteen studies were included in this meta-analysis. For MMP2-1306 C>T polymorphism, significant associations were observed under three genetic models both in overall comparison and in a hospital-based subgroup, and in oral cavity cancer and nasopharyngeal cancer under dominant model as well. For MMP3-1171 5A>6A and MMP9-1562 C>T polymorphisms, no association was found in overall comparison; however, in subgroup analyses based on ethnicity and tumor site, significant associations were detected between the MMP3-1171 5A>6A polymorphism and HNC risk in a European population and pharyngeal/laryngeal cancer under two genetic contrasts.

Conclusion

This meta-analysis suggests that the MMP2-1306 C>T polymorphism is associated with HNC risk, as is the MMP3-1171 5A>6A polymorphism specifically in some subgroups. Further studies with larger sample sizes are warranted.     

Gelatinases (MMP2 and MMP9) in swine antral follicle

Follicular growth is allowed by extracellular matrix remodeling and vascular network development. Since matrix metalloproteinases (MMPs) play a crucial role in both of these processes, the aim of the present study was to evaluate the expression of gelatinases MMP2 and MMP9 in swine ovarian follicle (theca and granulosa compartments) during its development. Moreover, we measured gelatinase activities into follicular fluids (FF). Our data demonstrate for the first time that MMP2 and MMP9 are expressed in swine ovarian follicle both in theca and granulosa layers; moreover we show that the expression of both gelatinases increases in theca while decreases in granulosa during follicle growth. Additionally, MMP2 activity has been detected in FF. The spatial pattern of expression of gelatinases in swine follicle suggests a differential role during physiological ovarian events.     

Development of a receptor-based 3D-QSAR study for the analysis of MMP2, MMP3, and MMP9 inhibitors

The ability of Gold software to predict the binding disposition of matrix metalloproteinase (MMP) inhibitors was evaluated using MMP3 and MMP8. The best procedure was subsequently employed to dock into MMP2, MMP3 and MMP9 nearly 70 compounds that were tested for their inhibitory activity against the three MMP subtypes. The best binding poses were used as an alignment tool for the development of 3D-QSAR studies. Evaluation of the three resulting 3D-QSAR models allowed us to indicate the ligand properties and residues important for activity and selectivity. MMP2 is an important anticancer drug target, while MMP3 and MMP9 are considered to be anti-targets for tumor pathologies. As such, our results could predict the binding affinities of new MMP2 inhibitors, providing additional information regarding the selectivity against MMP3 and MMP9. Furthermore, this strategy may be used also for the investigation of other MMPs.     

MMP2和MMP9在粘膜内胃癌中表达及其临床病理意义

目的:观察基质金属蛋白酶(MMP)家族成员MMP2和MMP9在粘膜内胃癌中的表达及其与淋巴结转移的相关性。方法:研究病例为病理诊断为粘膜内胃癌的档案病例,应用免疫组织化学技术检测MMP2和MMP9在粘膜内胃癌中表达的临床病理意义,特别是与淋巴结转移的相关性。结果:临床病理分析结果显示有淋巴结转移的IMGC病例肿块直径要显著大于无淋巴结转移的IMGC。有淋巴结转移IMGC中低分化腺癌发生率要显著高于无淋巴结转移组。有淋巴结转移IMGC中淋巴管侵犯发生率要显著高于无淋巴结转移组。免疫组化结果显示,MMP2在正常胃粘膜上皮和粘膜内胃癌中的阳性表达率分别是7%和43.93%,有显著性差异(P0.01),MMP9在正常胃粘膜上皮和粘膜内胃癌中的阳性表达率分别为和23%和48.48%,无显著性差异(P0.05)。MMP9在淋巴结转移组中的阳性率(87.5%)显著高于无淋巴结转移组(36%),在有淋巴管侵犯病例中的表达率(83.3%)显著高于无淋巴管侵犯的病例(30%),差异均有统计意义(P0.05);而MMP2的表达与有无淋巴结转移及淋巴管侵犯均无显著相关性(P0.05)。结论:MMP9可能作为预测粘膜内胃癌是否有淋巴结转移的标志物,但需要结合组织分化、肿块大小和淋巴管侵犯等临床病理特点综合判断。MMP2可能与粘膜内胃癌的发生有关而作为早期诊断的指标。     

Finasteride Inhibits Human Prostate Cancer Cell Invasion through MMP2 and MMP9 Downregulation

Introduction

The use of the 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride for prostate cancer prevention is still under debate. The FDA recently concluded that the increased prevalence of high-grade tumors among 5-ARI-treated patients must not be neglected, and they decided to disallow the use of 5-ARIs for prostate cancer prevention. This study was conducted to verify the effects of finasteride on prostate cell migration and invasion and the related enzymes/proteins in normal human and tumoral prostatic cell lines.

Materials and Methods

RWPE-1, LNCaP, PC3 and DU145 cells were cultivated to 60% confluence and exposed for different periods to either 10 µM or 50 µM finasteride that was diluted in culture medium. The conditioned media were collected and concentrated, and MMP2 and MMP9 activities and TIMP-1 and TIMP-2 protein expression were determined. Cell viability, migration and invasion were analyzed, and the remaining cell extracts were submitted to androgen receptor (AR) detection by western blotting techniques. Experiments were carried out in triplicate.

Results

Cell viability was not significantly affected by finasteride exposure. Finasteride significantly downregulated MMP2 and MMP9 activities in RWPE-1 and PC3 cells and MMP2 in DU145 cells. TIMP-2 expression in RWPE-1 cells was upregulated after exposure. The cell invasion of all four tested cell lines was inhibited by exposure to 50 µM of finasteride, and migration inhibition only occurred for RWPE-1 and LNCaP cells. AR was expressed by LNCaP, RWPE-1 and PC3 cells.

Conclusions

Although the debate on the higher incidence of high-grade prostate cancer among 5-ARI-treated patients remains, our findings indicate that finasteride may attenuate tumor aggressiveness and invasion, which could vary depending on the androgen responsiveness of a patient’s prostate cells.     

MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression.

Methods and Findings

We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins—MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A—that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%–100%) and specificity of 98.1% (95% CI 89.9%–100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%–100%) and specificity of 87.2% (95% CI 72.6%–95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DL_CO%).

Conclusions

Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.     

MMP inhibition by barbiturate homodimers

We have studied some homodimeric compounds derived from 5-homopiperazine substituted pyrimidine triones (barbiturates) with linkers in the range 2–20 carbon atoms. The compounds were designed to be capable of resisting absorption, to be stable in the gut and to maintain inhibitory potency against gelatinases and related function.The compounds were then assessed for inhibitory potency against a panel of MMPs (1, 2, 8, 9 and 13). The dimer compounds had similar potency and selectivity to the homopiperazine barbiturate monomer class. At 100 nM, selected dimers significantly inhibited cancer cell invasion in a matrigel assay using Caco-2 cells stimulated by hepatic growth factor. Finally, selected dimers showed adequate stability in simulated intestinal fluid to suggest the capacity to transit to the colon.     

Polymorphism in promoter regions of matrix metalloproteinases (MMP1, MMP9, and MMP12) in chronic obstructive pulmonary disease patients

Our studies have shown that the genotype and allele frequencies of polymorphisms G(-1607)GG of MMPI gene, C(-1562)T of MMP9 gene and A(-82)G of MMP12 gene do not significantly differ in the samples of chronic obstructive pulmonary disease (COPD) patients (N = 318) and healthy controls (N = 319) dwelling in Bashkortostan Republic. However, association of (-1562)T allele of the MMP9 gene with the severity of COPD disease progression has been revealed. In COPD patients at stage 4 of the disease, the frequency of allele T was significantly higher that in patients with the stages 2 and 3 (15.89% versus 8.38%; chi2 = 7.804, d.f. = 1, P = = 0.005; OR = 2.06 95% CI 1.22-3.49). The distribution of the genotype frequencies of C(-1562)T polymorphism of MMP9 gene significantly differed between the patients with various COPD severity (chi2 = 9.849, d.f. = 2, P = 0.007). The individuals with rare genotype TT were revealed only among patients with severe COPD form (3.97% versus 0%; chi2 = 4.78, P = 0.029, Pcor = 0.058). Analysis of this polymorphism in patients with early COPD onset (younger than 55 years old) has shown a significant increase in the allele Tfrequency in the group of patients with severe COPD (stage 4 according to GOLD) compared to the patients of the same age but with less severe COPD progression (chi2 = 5.26, d.f. = 1, P = 0.022). As the major clinical characteristics of stage 4 COPD is the development of pulmonary emphysema as well as bronchial walls deformation, we suggest that the increased expression of MMP9 gene caused by genetic polymorphism in the gene promoter is important in the early development of serious complications of the disease.     

Proteomic approaches to uncover MMP function

Proteomics has revolutionized protease research and particularly contributed to the identification of novel substrates and their sites of cleavage as key determinants of protease function. New technologies and rapid advancements in development of powerful mass spectrometers allowed unprecedented insights into activities of matrix metalloproteinases (MMPs) within their complex extracellular environments. Mass spectrometry-based proteomics extended our knowledge on MMP cleavage specificities and will help to develop more specific inhibitors as new therapeutics. Quantitative proteomics and N-terminal enrichment strategies have revealed numerous novel MMP substrates and shed light on their modes of action in vitro and in vivo. In this review, we provide an overview of current proteomic technologies in protease research and their application to the functional characterization of MMPs.     

Matrix Metalloproteinases MMP2 and MMP9 Are Produced in Early Stages of Kidney Morphogenesis but Only MMP9 Is Required for Renal Organogenesis In Vitro

We analyzed matrix metalloproteinase (MMP) production by 11-d embryonic mouse kidneys and the effects of these enzymes on subsequent renal organogenesis. In vivo, immunolocalization of metalloproteinases by laser scanning confocal microscopy and zymograms of kidney lysates showed that the mesenchyme of embryonic kidneys synthesized both MMP9 and MMP2 enzymes. In vitro, embryonic kidneys also secreted both enzymes when cultured in a medium devoid of hormone, growth factor, and serum for 24 h during which T-shaped branching of the ureter bud appeared. We then evaluated the role of MMP2 and MMP9 in kidney morphogenesis by adding anti-MMP2 or anti-MMP9 IgGs to the culture medium of 11-d kidneys for 24 or 72 h. Although it inhibited activity of the mouse enzyme, anti-MMP2 IgGs had no effect on kidney morphogenesis. In contrast, anti-MMP9 IgGs with enzyme-blocking activity impaired renal morphogenesis, in a concentration-dependent manner, by inhibiting T-shaped branching and further divisions of the ureter bud. This effect was irreversible, still observed after inductive events and reproduced by exogenous tissue inhibitor of metalloproteinase 1 (TIMP1), the natural inhibitor of MMP9. These data provide the first demonstration of MMP9 and MMP2 production in vivo by 11-d embryonic kidneys and further show that MMP9 is required in vitro for branching morphogenesis of the ureter bud.     

Pharmacogenetic associations of MMP9 and MMP12 variants with cardiovascular disease in patients with hypertension

Objectives

MMP-9 and -12 function in tissue remodeling and may play roles in cardiovascular disease (CVD). We assessed associations of four MMP polymorphisms and three antihypertensive drugs with cardiovascular outcomes.

Methods

Hypertensives (n = 42,418) from a double-blind, randomized, clinical trial were randomized to chlorthalidone, amlodipine, lisinopril, or doxazosin treatment (mean follow up, 4.9 years). The primary outcome was coronary heart disease (CHD). Secondary outcomes included combined CHD, all CVD outcomes combined, stroke, heart failure (HF), and mortality. Genotype-treatment interactions were tested.

Results

There were 38,698 participants genotyped for at least one of the polymorphisms included here. For MMP9 R668Q (rs2274756), lower hazard ratios (HRs) were found for AA subjects for most outcomes when treated with chlorthalidone versus amlodipine (eg., CCHD: GG = 1.00, GA = 1.01, AA = 0.64; P = 0.038). For MMP9 R279Q (rs17576), modest pharmacogenetic findings were observed for combined CHD and the composite CVD outcome. For MMP12 N122S (rs652438), lower HRs were observed for CHD in subjects carrying at least one G allele and being treated with chlorthalidone versus lisinopril (CHD: AA = 1.07, AG = 0.80, GG = 0.49; P = 0.005). In the lisinopril-amlodipine comparison, higher HRs were observed for participants having at least one G allele at the MMP12 N122S locus (CHD: AA = 0.94, AG = 1.19, GG = 1.93; P = 0.041). For MMP12 −82A>G (rs2276109), no pharmacogenetic effect was found for the primary outcome, although lower HRs were observed for AA homozygotes in the chlorthalidone-amlodipine comparison for HF (P = 0.015).

Conclusions

We observed interactions between antihypertensive drugs and MMP9 and MMP12 for CHD and composite CVD. The data suggest that these genes may provide useful clinical information with respect to treatment decisions.     

Development and in vitro efficacy of novel MMP2 and MMP9 specific doxorubicin albumin conjugates.

Two doxorubicin albumin conjugates (A-DP1 and A-DP2), which differ in their substrate specificity for the matrix metalloproteinases MMP2 and MMP9, were prepared by binding maleimide doxorubicin peptide derivatives to the cysteine-34 position of human serum albumin. The incorporated octapeptide, Gly-Pro-Gln-Arg-Ile-Ala-Gly-Gln, in A-DP2 is not cleaved by activated MMP2 and MMP9 in contrast to Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln incorporated in A-DP1 that is cleaved efficiently by activated MMP2 and MMP9 liberating a doxorubicin tetrapeptide. A-DP1 showed antiproliferative activity in a murine renal cell carcinoma line in the low micromolar range (IC(50) value approximately 0.2 microM).     

Regulation of MMP2 and MMP9 metalloproteinases by FSH and growth factors in bovine granulosa cells

Matrix metalloproteinases (MMP) are key enzymes involved in tissue remodeling. Within the ovary, they are believed to play a major role in ovulation, and have been linked to follicle atresia. To gain insight into the regulation of MMPs, we measured the effect of hormones and growth factors on MMP2 and MMP9 mRNA levels in non-luteinizing granulosa cells in serum-free culture. FSH and IGF1 both stimulated estradiol secretion and inhibited MMP2 and MMP9 mRNA abundance. In contrast, EGF and FGF2 both inhibited estradiol secretion but had no effect on MMP expression. At physiological doses, none of these hormones altered the proportion of dead cells. Although we cannot link MMP expression with apoptosis, the specific down regulation by the gonadotropic hormones FSH and IGF1 in vitro suggests that excess MMP2 and MMP9 expression is neither required nor desired for follicle development.     

Molecular characterization of porcine MMP19 and MMP23B genes and its association with immune traits

MMP19 and MMP23B belong to the Matrix metalloproteases (MMPs) family, which are zinc-binding endopeptidases that are capable of degrading various components of the extracellular matrix. They are thought to play important roles in embryonic development, reproduction and tissue remodeling, as well as in cell proliferation, differentiation, migration, angiogenesis, apoptosis and host defense. However, they are poorly understood in pigs. Here, we obtained the full length coding region sequence and genomic sequence of the porcine MMP19 and MMP23B genes and analyzed their genomic structures. The deduced amino acid sequence shares similar precursor protein domains with human and mouse MMP19 and MMP23B protein, respectively. Using IMpRH panel, MMP19 was mapped to SSC5p12-q11 (closely linked to microsatellite DK) and MMP23B was mapped to SSC8q11-q12 (linked to microsatellite Sw2521). Quantitative real-time PCR showed that MMP19 was abundantly expressed in the liver, while MMP23B was strongly expressed in the ovarian and heart. Furthermore, both genes were all expressed increasingly in prenatal skeletal muscle during development. Three SNPs were detected by sequencing and PCR-RFLP methods, and association analysis indicated that C203T at exon 5 of MMP19 has a significant association with the blood parameters WBC (G/L) and IgG2 (mg/mL) (P<0.05), SNP C131T at exon 3 of MMP23B is significantly associated with the blood parameters HGB (g/L) and MCH (P<0.05), and A150G in exon 4 has no significant association with the economic traits in pigs.     

Epb41l3 suppresses esophageal squamous cell carcinoma invasion and inhibits MMP2 and MMP9 expression

EPB41L3 may play a role as a metastasis suppressor by supporting regular arrangements of actin stress fibres and alleviating the increase in cell motility associated with enhanced metastatic potential. Downregulation of epb41l3 has been observed in many cancers, but the role of this gene in esophageal squamous cell carcinoma (ESCC) remains unclear. Our study aimed to determine the effect of epb41l3 on ESCC cell migration and invasion. We investigated epb41l3 protein expression in tumour and non‐tumour tissues by immunohistochemical staining. Expression in the non‐neoplastic human esophageal cell line Het‐1a and four ESCC cell lines – Kyse150, Kyse510, Kyse450 and Caes17 – was assessed by quantitative Polymerase Chain Reaction (qPCR) and Western blotting. Furthermore, an EPB41L3 overexpression plasmid and EPB41L3‐specific small interfering RNA were used to upregulate EPB41L3 expression in Kyse150 cells and to downregulate EPB41L3 expression in Kyse450 cells, respectively. Cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The expression levels of p‐AKT, matrix metalloproteinase (MMP)2 and MMP9 were evaluated. Expression of epb41l3 was significantly lower in tumour tissues than in non‐tumour tissues and in ESCC cell lines compared with the Het‐1a cell line. Kyse450 and Caes17 cells exhibited higher expression of epb41l3 than Kyse150 and Kyse510 cells. Overexpressing epb41l3 decreased Kyse150 cell migration and invasion, whereas EPB41L3‐specific small interfering RNA silencing increased these functions in Kyse450 cells. Furthermore, overexpressing epb41l3 led to downregulation of MMP2 and MMP9 in Kyse150 and Kyse510 cells. Our findings reveal that EPB41L3 suppresses tumour cell invasion and inhibits MMP2 and MMP9 expression in ESCC cells. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular Characterization, Mapping, and Haplotype Analysis of Porcine Matrix Metalloproteinase Genes MMP1 and MMP10

Matrix metalloproteinases (MMPs) are a family of enzymes that cleave protein components of the extracellular matrix, such as collagens, laminin, fibronectin, and proteoglycans, playing a role in degradation of the matrix of the uterus and in embryo implantation. We report the identification of two members of the MMP gene family in swine. The porcine MMP1 and MMP10 genes comprise 10 exons and 9 introns spanning approximately 8,460 and 7,030 bp. Of 28 potential single nucleotide polymorphisms found in the genomic region, five polymorphic positions were analyzed using PCR-RFLP. Allelic frequencies and haplotypes were analyzed in five pig breeds (n = 280). The AC haplotype of MMP1 and ATG haplotype of MMP10 were not detected in two foreign pig breeds. Association analysis in a French Large White population (n = 164, total four traits) showed no association between haplotypes and reproduction performance. In addition, porcine MMP1 and MMP10 were mapped on SSC9p¹³ and SSC2q²¹, respectively, in agreement with comparative mapping data.