Ethanolic Extract of Ocimum sanctum Leaves Reduced Invasion and Matrix Metalloproteinase Activity of Head and Neck Cancer Cell Lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) has a yearly incidence of 600,000 cases worldwide with a low survival rate. Ocimum sanctum L. or Ocimum tenuiflorum L. (Holy basil; Tulsi in Hindi), is a traditional medicine herb that demonstrates numerous effects including anti-oxidant, anti-microbial, and anti-tumor effects. The aim of this study was to evaluate the anti-invasive effect of O. sanctum leaf extract on HNSCC cell lines. METHODS: Ethanolic extract of O. sanctum leaf (EEOS) was prepared and the phenolic compounds were identified using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. Genetically matched HNSCC cell lines derived from primary (HN30 and HN4) and metastatic sites (HN31 and HN12) from the same patient were used in this study. The EEOS cytotoxicity to the cell lines was determined using an MTT assay. The invasion and matrix metalloproteinase (MMP)-2 and -9 activity of EEOS-treated cells were tested using a modified Boyden chamber assay and zymography, respectively. RESULTS: We found that EEOS significantly inhibited the invasion and MMP-2 and MMP-9 activity of HN4 and HN12 cells, but not HN30 and HN31 cells. Rosmarinic acid, caffeic acid, and apigenin were detected in EEOS. Moreover, rosmarinic acid was found as the major phenolic compound. CONCLUSION: EEOS exerted its anti-invasive effect on HNSCC cells by attenuating MMP activity. The active compounds identified in EEOS might be promising as an alternative therapeutic agent for HNSCC.

Enzyme-induced in vivo assembly of gold nanoparticles for imaging-guided synergistic chemo-photothermal therapy of tumor.

This article describes a nanoplatform based on matrix metalloproteinase (MMP)-responsive gold nanoparticles (AuNPs) for tumor-targeted photoacoustic (PA) imaging-guided photothermal therapy and drug delivery. AuNPs were grafted with complementary DNA strands, tethered with doxorubicin and coated with poly(ethylene glycol) via a thermal-labile linker and a MMP-cleavable peptide, respectively. The nanoprobes remained well-isolated in healthy tissues, but formed aggregates rapidly under MMP-abundant conditions. The DNA hybridization-induced assembly of the nanoprobes led to prolonged tumor retention and strong near-infrared (NIR) absorption, which is beneficial to deep-tissue imaging and therapy. Compared with MMP-inert nanoprobes, our platform demonstrated significantly enhanced efficiency in PA imaging and photothermal conversion upon NIR irradiation. Meanwhile, doxorubicin could be released rapidly in response to the localized elevation of temperature, leading to synergistic chemo-photothermal therapy. The unique nanoplatform may find applications in effective disease control by delivering imaging and therapy to tumors with high specificity, safety, and universality.

Vascular Imaging of Matrix Metalloproteinase Activity as an Informative Preclinical Biomarker of Drug-induced Vascular Injury.

Lack of biomarkers specific to and either predictive or diagnostic of drug-induced vascular injury (DIVI) continues to be a major obstacle during drug development. Biomarkers derived from physiologic responses to vessel injury, such as inflammation and vascular remodeling, could make good candidates; however, they characteristically lack specificity for vasculature. We evaluated whether vascular remodeling-associated protease activity, as well as changes to vessel permeability resulting from DIVI, could be visualized ex vivo in affected vessels, thereby allowing for visual monitoring of the pathology to address specificity. We found that visualization of matrix metalloproteinase activation accompanied by increased vascular leakage in the mesentery of rats treated with agents known to induce vascular injury correlated well with incidence and severity of histopathological findings and associated inflammation as well as with circulating levels of tissue inhibitors of metalloproteinase 1 and neutrophil gelatinase-associated lipocalin. The weight of evidence approach reported here shows promise as a composite DIVI preclinical tool by means of complementing noninvasive monitoring of circulating biomarkers of inflammation with direct imaging of affected vasculature and thus lending specificity to its interpretation. These findings are supportive of a potential strategy that relies on translational imaging tools in conjunction with circulating biomarker data for high-specificity monitoring of VI both preclinically and clinically.

Effect of green tea as a protective measure against dental erosion in coronary dentine.

The aim of this study was to evaluate the effect of green tea as a protective measure on eroded dentin. Disks of human coronary dentin were selected based on surface hardness and randomly assigned to 3 groups (n = 10): DW - distilled water, CHX - 0.2% chlorhexidine digluconate, and GT - green tea. The disks were allowed to acquire pellicle for 2 hours and were then subjected to 3 cycles per day of demineralization (C6H8O7 0.05 M, pH 3.75, 60 s), treatment (DW or CHX or GT, 5 min) and remineralization (artificial saliva, 60 min) over a period of 3 days. Changes in the dentin were determined by loss of surface hardness (%SHL) and mechanical profilometry analysis at the end of each day. Data were analyzed by two-way ANOVA followed by Tukey's test for %SHL and profilometry (p < 0.05). Significant reductions in dentin hardness loss were observed only for the CHX group when compared to the DW group (p < 0.05). However, there was no significant difference between the CHX and GT groups (p > 0.05). A significant difference was observed between DW and GT treatments for wear and roughness measurements (p < 0.05). The green tea extract solution was able to reduce the wear and roughness caused by dentin erosion under the conditions of this study.

Effect of green tea as a protective measure against dental erosion in coronary dentine

Abstract The aim of this study was to evaluate the effect of green tea as a protective measure on eroded dentin. Disks of human coronary dentin were selected based on surface hardness and randomly assigned to 3 groups (n = 10): DW - distilled water, CHX - 0.2% chlorhexidine digluconate, and GT - green tea. The disks were allowed to acquire pellicle for 2 hours and were then subjected to 3 cycles per day of demineralization (C6H8O7 0.05 M, pH 3.75, 60 s), treatment (DW or CHX or GT, 5 min) and remineralization (artificial saliva, 60 min) over a period of 3 days. Changes in the dentin were determined by loss of surface hardness (%SHL) and mechanical profilometry analysis at the end of each day. Data were analyzed by two-way ANOVA followed by Tukey’s test for %SHL and profilometry (p < 0.05). Significant reductions in dentin hardness loss were observed only for the CHX group when compared to the DW group (p < 0.05). However, there was no significant difference between the CHX and GT groups (p > 0.05). A significant difference was observed between DW and GT treatments for wear and roughness measurements (p < 0.05). The green tea extract solution was able to reduce the wear and roughness caused by dentin erosion under the conditions of this study.

Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive. RESULTS: A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells. CONCLUSION: VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

In vitro inhibition of matrix metalloproteinases, invasion and growth of Fanconi anemia human FANCA and FANCC lymphoblasts by nutrient mixture.

AIM: Fanconi anemia is a rare genetic disorder with high propensity for development of cancers, such as aplastic anemia, leukemia and head and neck cancers. Collagen digesting matrix metalloproteinase (MMP) enzymes have been implicated in for their role in various malignancies and to promote metastasis. Biological agents that prevent extracellular matrix digestion by the MMPs have been shown to be promising therapeutic approaches to cancer. In this study, we investigated effects of a nutrient mixture (NM) containing, ascorbic acid, lysine, proline and green tea extract, on human FANCA and FANCC lymphoblasts for viability, MMP secretion and invasion. METHODS: Human FANCA lymphoblasts GM13022 and HCS536 were challenged with NM at concentration range within 10-1000 µg/ml. Cell toxicity was assessed by Trypan blue dye exclusion test. Invasion was evaluated through Matrigel and gelatinase zymography for MMP activity. RESULTS: NM was toxic in dose dependent mode to HCS536 cells but not to GM13022 cells. GM13022 cells but not HCS536 cells exhibited MMP-9 secretion, which was inhibited by NM. Matrigel invasion was inhibited in HCS536 cells at 100 and 500 µg/ml by 27% and 93%, respectively. In GM13022 cells, the NM showed completely blocked Matrigel invasion at 500 µg/ml. CONCLUSION: NM inhibited MMP secretion and Matrigel invasion in FANCA and inhibited invasion and induced toxicity in FANCC lymphoblasts. These results suggest that the NM may have therapeutic potential in Fanconi anemia associated neoplasia.

Doxycycline stabilizes vulnerable plaque via inhibiting matrix metalloproteinases and attenuating inflammation in rabbits.

Enhanced matrix metalloproteinases (MMPs) activity is implicated in the process of atherosclerotic plaque instability. We hypothesized that doxycycline, a broad MMPs inhibitor, was as effective as simvastatin in reducing the incidence of plaque disruption. Thirty rabbits underwent aortic balloon injury and were fed a high-fat diet for 20 weeks. At the end of week 8, the rabbits were divided into three groups for 12-week treatment: a doxycycline-treated group that received oral doxycycline at a dose of 10 mg/kg/d, a simvastatin-treated group that received oral simvastatin at a dose of 5 mg/kg/d, and a control group that received no treatment. At the end of week 20, pharmacological triggering was performed to induce plaque rupture. Biochemical, ultrasonographic, pathologic, immunohistochemical and mRNA expression studies were performed. The results showed that oral administration of doxycycline resulted in a significant increase in the thickness of the fibrous cap of the aortic plaque whereas there was a substantial reduction of MMPs expression, local and systemic inflammation, and aortic plaque vulnerability. The incidence of plaque rupture with either treatment (0% for both) was significantly lower than that for controls (56.0%, P<0.05). There was no significant difference between doxycycline-treated group and simvastatin-treated group in any serological, ultrasonographic, pathologic, immunohistochemical and mRNA expression measurement except for the serum lipid levels that were higher with doxycycline than with simvastatin treatment. In conclusion, doxycycline at a common antimicrobial dose stabilizes atherosclerotic lesions via inhibiting matrix metalloproteinases and attenuating inflammation in a rabbit model of vulnerable plaque. These effects were similar to a large dose of simvastatin and independent of serum lipid levels.

Matrix metalloproteinase sensitive gold nanorod for simultaneous bioimaging and photothermal therapy of cancer.

Herein, we developed matrix metalloprotease (MMP) sensitive gold nanorods (MMP-AuNR) for cancer imaging and therapy. It was feasible to absorb NIR laser and convert into heat as well as visualize MMP activity. We showed the possibility of gold nanorods as a hyperthermal therapeutic agent and MMP sensitive imaging agent both in vitro and in vivo condition. The results suggested potential application of MMP-AuNR for simultaneous cancer diagnosis and therapy.

Matrix metalloproteinase inhibitors: a critical appraisal of design principles and proposed therapeutic utility.

Matrix metalloproteinases (MMPs) play an important role in tissue remodelling associated with various physiological and pathological processes, such as morphogenesis, angiogenesis, tissue repair, arthritis, chronic heart failure, chronic obstructive pulmonary disease, chronic inflammation and cancer metastasis. As a result, MMPs are considered to be viable drug targets in the therapy of these diseases. Despite the high therapeutic potential of MMP inhibitors (MMPIs), all clinical trials have failed to date, except for doxycycline for periodontal disease. This can be attributed to (i) poor selectivity of the MMPIs, (ii) poor target validation for the targeted therapy and (iii) poorly defined predictive preclinical animal models for safety and efficacy. Lessons from previous failures, such as recent discoveries of oxidative/nitrosative activation and phosphorylation of MMPs, as well as novel non-matrix related intra- and extracellular targets of MMP, give new hope for MMPI development for both chronic and acute diseases. In this article we critically review the major structural determinants of the selectivity and the milestones of past design efforts of MMPIs where 2-/3-dimensional structure-based methods were intensively applied. We also analyse the in vitro screening and preclinical/clinical pharmacology approaches, with particular emphasis on drawing conclusions on how to overcome efficacy and safety problems through better target validation and design of preclinical studies.

Venom neutralization by purified bioactive molecules: Synthetic peptide derivatives of the endogenous PLA(2) inhibitory protein PIP (a mini-review).

Envenomation due to snakebite constitutes a significant public health problem in tropical and subtropical countries. Antivenom therapy is still the mainstay of treatment for snake envenomation, and yet despite recent research focused on the prospects of using antivenom adjuncts to aid in serotherapy, no new products have emerged so far for therapeutic use. Various methodologies including molecular biology, crystallography, functional and morphological approaches, etc., are employed in the search for such inhibitors with a view to generate molecules that can stop partially or completely the activities of toxic phospholipase A(2) (PLA(2)) and snake venom metalloproteinase (SvMPs) enzymes at the molecular level. Herein, both natural and synthetic inhibitors derived from a variety of sources including medicinal plants, mammals, marine animals, fungi, bacteria, and from the venom and blood of snakes have been briefly reviewed. Attention has been focused on the snake serum-based phospholipase A(2) inhibitors (PLIs), particularly on the PLI derived from python snake serum (PIP), highlighting the potential of the natural product, PIP, or possible derivatives of it, as a complementary treatment to serotherapy against the inflammation and/or muscle-damaging activity of snake venoms. The data indicate a more efficient pathway for inhibition and blocking the activity of PLA(2)s and matrix metalloproteinases (MMPs), thus representing a feasible complementary treatment for snakebites. Such information may be helpful for interfering on the biological processes that these molecules are involved in human inflammatory-related diseases, and also for the development of new drugs for treatment of snake envenomation.

Identification of novel non-hydroxamate anthrax toxin lethal factor inhibitors by topomeric searching, docking and scoring, and in vitro screening.

Anthrax is an infectious disease caused by Bacillus anthracis, a Gram-positive, rod-shaped, anaerobic bacterium. The lethal factor (LF) enzyme is secreted by B. anthracis as part of a tripartite exotoxin and is chiefly responsible for anthrax-related cytotoxicity. As LF can remain in the system long after antibiotics have eradicated B. anthracis from the body, the preferred therapeutic modality would be the administration of antibiotics together with an effective LF inhibitor. Although LF has garnered a great deal of attention as an attractive target for rational drug design, relatively few published inhibitors have demonstrated activity in cell-based assays and, to date, no LF inhibitor is available as a therapeutic or preventive agent. Here we present a novel in silico high-throughput virtual screening protocol that successfully identified 5 non-hydroxamic acid small molecules as new, preliminary LF inhibitor scaffolds with low micromolar inhibition against that target, resulting in a 12.8% experimental hit rate. This protocol screened approximately 35 million nonredundant compounds for potential activity against LF and comprised topomeric searching, docking and scoring, and drug-like filtering. Among these 5 hit compounds, none of which has previously been identified as a LF inhibitor, three exhibited experimental IC(50) values less than 100 microM. These three preliminary hits may potentially serve as scaffolds for lead optimization as well as templates for probe compounds to be used in mechanistic studies. Notably, our docking simulations predicted that these novel hits are likely to engage in critical ligand-receptor interactions with nearby residues in at least two of the three (S1', S1-S2, and S2') subsites in the LF substrate binding area. Further experimental characterization of these compounds is in process. We found that micromolar-level LF inhibition can be attained by compounds with non-hydroxamate zinc-binding groups that exhibit monodentate zinc chelation as long as key hydrophobic interactions with at least two LF subsites are retained.

Screening of matrix metalloproteinases available from the protein data bank: insights into biological functions, domain organization, and zinc binding groups.

A total of 142 matrix metalloproteinase (MMP) X-ray crystallographic structures were retrieved from the Protein Data Bank (PDB) and analyzed by an automated and efficient routine, developed in-house, with a series of bioinformatic tools. Highly informative heat maps and hierarchical clusterograms provided a reliable and comprehensive representation of the relationships existing among MMPs, enlarging and complementing the current knowledge in the field. Multiple sequence and structural alignments permitted better location and display of key MMP motifs and quantification of the residue consensus at each amino acid position in the most critical binding subsites of MMPs. The MMP active site consensus sequences, the C-alpha root-mean-square deviation (RMSd) analysis of diverse enzymatic subsites, and the examination of the chemical nature, binding topologies, and zinc binding groups (ZBGs) of ligands extracted from crystallographic complexes provided useful insights on the structural arrangements of the most potent MMP inhibitors.

Nitric oxide regulates prolidase activity by serine/threonine phosphorylation.

Prolidase [E.C. 3.4.13.9], a member of the matrix metalloproteinase (MMP) family, is a manganese-dependent cytosolic exopeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. It plays an important role in collagen metabolism, matrix remodeling and cell growth. Nitric oxide (NO), a versatile signaling molecule, regulates many processes including collagen synthesis and matrix remodeling and, thereby, may modulate angiogenesis, tumor invasiveness, and metastasis. Thus, we considered that prolidase may be an important target of NO regulation. In our study, SIN I and DETA/NO were used as NO donors. Both donors increased prolidase activity in a time-dependent and dose-dependent manner. Prolidase activity increased not only with NO donors but also with endogenous NO in cells transfected with iNOS. The effect of iNOS was abolished by treatment with S-methylisothiourea (SMT), a selective inhibitor of iNOS. However, with either exogenous or endogenous sources of NO, the increase in prolidase activity was not accompanied by increased prolidase expression. Therefore, we suspected phosphorylation of prolidase as a potential mechanism regulating enzyme activation. We observed increased serine/threonine phosphorylation on prolidase protein in cells treated with NO donors and in cells transfected with iNOS. To determinate the pathways that may mediate prolidase induction by NO, we first used 8-Br-cGMP, a cGMP agonist, and found that 8-Br-cGMP strongly and rapidly stimulated prolidase activity accompanied by increased phosphorylation. Rp-8-Br-pCPT-cGMP, an inhibitor of cGMP, reduced NO donor-stimulated prolidase activity to control levels. To test whether the MAPK pathway is involved in this NO-dependent activation, we used an ERK1/2 inhibitor and found that it had no effect on prolidase activity increased by NO donors. These results demonstrate that NO stimulates prolidase activity by increasing serine/threonine phosphorylation through PKG-cGMP pathway, but independent of MAPK and suggest an interaction between inflammatory signaling pathways and regulation of the terminal step of matrix degradation.

Prevention of progressive joint destruction in collagen-induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031.

FR255031 (2-[(7S)-7-[5-(4-ethylphenyl)-2-thienyl]-1,1-dioxido-4-(2-pyridinylcarbonyl)hexahydro-1,4-thiazepin-7-yl]-N-hydroxyacetamide) is a novel synthetic matrix metalloproteinase (MMP) inhibitor that inhibits human collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9) and membrane type 1 MMP (MT1-MMP/MMP-14). FR255031 also inhibits rat collagenase and gelatinase. We studied the effect of FR255031 and Trocade, an inhibitor of collagenase and MMP-14, on a rat collagen-induced arthritis (CIA) model. Rat CIA was induced by intradermal injection of type II collagen (IIC) and oral administration of FR255031 or Trocade was performed for 28 days. Body weight loss, hind paw swelling, elevation of serum anti-IIC antibody, and histological and radiographic scores were evaluated. FR255031 markedly inhibited cartilage degradation in a dose-dependent manner in the CIA model, but Trocade failed to prevent the degradation. FR255031 at a dose of 100 mg kg(-1) also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. These results indicate that FR255031 is effective for rat CIA, especially on joint cartilage destruction. These data suggest that as well as collagenases or MT-MMP, gelatinases are also involved in joint destruction in arthritis.

Unconventional activation mechanisms of MMP-26, a human matrix metalloproteinase with a unique PHCGXXD cysteine-switch motif.

ProMMP-26 has the unique Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp cysteine-switch motif that discriminates this protease from all other matrix metalloproteinases (MMPs) known so far. The conserved, free cysteine residue of the conventional PRCXXPD sequence interacts with the zinc ion of the catalytic domain and provides the fourth coordination site for the catalytic zinc, thereby preventing latent proMMPs from becoming active. MMPs become functionally active when proteolytic cleavage releases the prodomain and the PRCXXPD sequence and exposes the zinc atom. Here, we report that the Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp motif is not functional in proMMP-26 and consequently is not involved in the activation mechanisms. Organomercurial treatment failed to activate proMMP-26. The autolytic Lys-Lys-Gln(59) downward arrow Gln(60)-Phe-His cleavage upstream of the Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp motif induced the proteolytic activity of recombinant proMMP-26 whereas any further cleavage inactivated the enzyme. The His(81) --> Arg(81) mutation restored the conventional cysteine-switch sequence in the prodomain but failed to induce the cysteine-switch activation mechanism. These data and computer modeling studies allowed us to hypothesize that the presence of His(81) significantly modified the fold of proMMP-26, abolished the functionality of the cysteine-switch motif, and stimulated an alternative intramolecular activation pathway of the proenzyme.

MMP-28, a new human matrix metalloproteinase with an unusual cysteine-switch sequence is widely expressed in tumors.

We report the discovery, cloning, and characterization of a novel human matrix metalloproteinase (MMP-28) cDNA gene. The deduced 520-amino-acid sequence of MMP-28 includes a signal peptide, a prodomain with an unusual cysteine-switch PRCGVTD motif followed by the furin cleavage RRKKR site, a catalytic domain, a hinge-region and a hemopexin-like domain. On the basis of their structural characteristics, MMP-28 belongs to the MMP-19 subfamily. The genomic MMP-28 gene uniquely mapped to chromosome 17q11.2 includes eight exons and seven introns. The broad range of expression in carcinomas as well as normal adult and fetal tissues suggests an important functional role for MMP-28.

Critical appraisal of the use of matrix metalloproteinase inhibitors in cancer treatment.

Experimental studies performed prior to 1990 led to the widely held belief that matrix metalloproteinases (MMPs) produced by cancer cells are of critical importance in tumor invasion and metastasis. Based on this evidence, the pharmaceutical industry produced several well tolerated, orally active MMP inhibitors (MMPIs) which demonstrated efficacy in mouse cancer models. Phase III clinical trials initiated in 1997-98 using marimastat, prinomastat (AG3340), and BAY 12-9566 alone or in combination with standard chemotherapy in patients with advanced cancers (lung, prostate, pancreas, brain, GI tract) have recently been reported; no clinical efficacy was demonstrated. Bayer and Agouron have discontinued their ongoing Phase III drug trials of MMPIs in advanced cancer. In retrospect, the failure of MMPIs to alter disease progression in metastatic cancer might have been anticipated since MMPs appear to be important in early aspects of cancer progression (local invasion and micrometastasis) and may no longer be required once metastases have been established. Our understanding of MMP pathophysiology in cancer has expanded considerably in the past 10 years. Current views indicate that: (1) most MMPs in tumors are made by stromal cells, not carcinoma cells; (2) cancer cells induce stromal cells to synthesize MMPs using extracellular matrix metalloproteinase inducer (EMMPRIN) and cytokine stimulatory mechanisms; and (3) MMPs promote cell migration and the release of growth factors sequestered in the extracellular matrix. MMPs have a dual function in tumor angiogenesis: MMP-2 and MT1-MMP are required in breaking down basement membrane barriers in the early stage of angiogenesis, while other MMPs are involved in the generation of an angiogenic inhibitor, angiostatin. In spite of considerable recent progress in identifying multiple roles of MMPs in disease, our understanding of MMP function in cancer is far from complete (see Table 1). Based on accumulated data, it is recommended that future MMPI trials focus on: (1) patients with early stage cancer; (2) the use of MMPIs along with chemotherapy; (3) the measurement of MMPs in tumor tissue and blood as a means of identifying patients who are more likely to respond to MMPI therapy; and (4) identification of biomarkers that reflect activation or inhibition of MMPs in vivo.