Brain metastases in melanoma: roles of neurotrophins

Brain metastasis, which occurs in 20% to 40% of all cancer patients, is an important cause of neoplastic morbidity and mortality. Successful invasion into the brain by tumor cells must include attachment to microvessel endothelial cells, penetration through the blood-brain barrier, and, of relevance, a response to brain survival and growth factors. Neurotrophins (NTs) are important in brain-invasive steps. Human melanoma cell lines express low-affinity NT receptor p75NTR in relation to their brain-metastatic propensity with their invasive properties being regulated by NGF, or nerve growth factor, the prototypic NT. They also express functional TrkC, the putative receptor for the invasion-promoting NT-3. In brain-metastatic melanoma cells, NTs promote invasion by enhancing the production of extracellular matrix (ECM)-degradative enzymes such as heparanase, an enzyme capable of locally destroying both ECM and the basement membrane of the blood-brain barrier. Heparanase is an endo-beta-d-glucuronidase that cleaves heparan sulfate (HS) chains of ECM HS proteoglycans, and it is a unique metastatic determinant because it is the dominant mammalian HS degradative enzyme. Brain-metastatic melanoma cells also produce autocrine/paracrine factors that influence their growth, invasion, and survival in the brain. Synthesis of these factors may serve to regulate NT production by brain cells adjacent to the neoplastic invasion front, such as astrocytes. Increased NT levels have been observed in tumor-adjacent tissues at the invasion front of human brain melanoma. Additionally, astrocytes may contribute to the brain-metastatic specificity of melanoma cells by producing NT-regulated heparanase. Trophic, autocrine, and paracrine growth factors may therefore determine whether metastatic cells can successfully invade, colonize, and grow in the CNS.     

Modulation of tumor cell response to chemotherapy by the organ environment

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression ofmdr1 mRNA. However, the organ-specific mechanism for upregulatingmdr1 and P-glycoprotein has yet to be elucidated.     

Differential stimulation of the growth of lung-metastasizing tumor cells by lung (paracrine) growth factors: identification of transferrin-like mitogens in lung tissue-conditioned medium.

Certain metastatic tumor cells successfully form metastases at particular organ sites, and their organ colonization properties cannot be explained by mechanical or anatomic factors. These tumor cells possess the ability to colonize such sites through preferential adhesion to organ microvessel endothelial cells, preferential organ invasion by expression of particular degradative enzymes and response to organ motility factors, and preferential organ growth by response to growth factors present at relatively higher concentrations in the target organ. The likelihood that target organ-associated growth factors exist and are important in metastatic colonization has been approached by studying the mitogenic effects of target organ extracts, fragments, or conditioned media on poorly and highly metastatic tumor cells that show organ preference of metastasis. We previously described the isolation of a major organ-derived (paracrine) growth factor from lung tissue-conditioned medium. Characterization of this mitogen has demonstrated that it is a transferrin or a transferrin-like glycoprotein, and antibodies to transferrin can remove significant growth activity from lung tissue-conditioned medium. Further demonstration of the existence and characterization of metastasis-associated organ (paracrine) growth factors and their receptors will be helpful in understanding the organ preference of metastasis.     

Malignant melanoma metastasis to brain: role of degradative enzymes and responses to paracrine growth factors

Mouse and human melanoma cells metastatic to the brain express degradative enzyme activities that are used for invasion of brain basement membrane and parenchyma. Compared to poorly metastatic or lung- or ovary-metastatic murine melanoma lines, the brain-metastatic sublines secreted higher levels of a variety of degradative enzymes. Brain-metastatic murine and human melanoma cells also degraded subendothelial basement membrane and reconstituted basement membrane at rates higher than other metastatic melanoma cells. In some cases these degradative activities in mouse and human melanoma cells can be induced by paracrine factors known to be present in the brain parenchyma, such as nerve growth factor (NGF). NGF stimulates the expression of degradative enzymes, such as the endo--glucuronidase heparanase, that are important in basement membrane penetration but this factor does not stimulate melanoma cell growth. The growth of brain-metastasizing melanoma cells appears to be stimulated by other paracrine growth factors, such as paracrine transferrin. Melanoma cells metastatic to brain express higher numbers of transferrin receptors and respond and proliferate at lower concentrations of transferrin than do melanoma cells metastatic to other sites or poorly metastatic melanoma cells. The results suggest that degradation and invasion of brain basement membrane and responses to paracrine neurotrophins and paracrine transferrins are important properties in brain metastasis of murine and human malignant melanoma cells.     

Regulation of Gelatinase Production in Metastatic Renal Cell Carcinoma by Organ-specific Fibroblasts

We have recently established a human renal cell carcinoma KG-2 line that is tumorigenic in the subcutis (ectopic) and kidney (orthotopic) of nude mice but spontaneously metastasizes to the lung only after orthotopic implantation. KG-2 cells growing in the kidney (orthotopic) and lung metastases secreted higher levels of gelatinase than did cells growing in the subcutis (ectopic). We examined whether organ-specific fibroblasts play a role in the regulation of gelatinase production and invasion by renal carcinoma cells. The gelatinase level in the culture supernatants of KG-2 cells was increased by their cultivation with mouse kidney or lung fibroblasts. In contrast, cocultivation of KG-2 cells with mouse skin fibroblasts resulted in a significant reduction of gelatinase activity. Similar results were obtained by culturing KG-2 cells in the media conditioned by the different mouse fibroblasts. We, therefore, investigated effects on KG-2 cells of cytokines and growth factors known to be produced by fibroblasts of various origins. Of ten cytokines and growth factors tested, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor-β₁ (TGF-β₁) stimulated gelatinase expression by the cultured KG-2 cells. Parallel immunohistochemical analyses revealed that mouse kidney and lung fibroblasts produced higher levels of TGF-β₁ than did skin fibroblasts. These results indicate that gelatinase production by KG-2 renal cell carcinoma cells is influenced by the organ microenvironment. Specifically, organ-specific fibroblasts regulate the production of degradative enzymes by KG-2 cells and, hence, profoundly influence their invasive and metastatic capacity.     

Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of metastasis

Summary Human colon carcinomas (HCC) are heterogeneous for a varriety of biological properties that include invasion and metastasis. The presence of a small subpopulation of cells with a highly metastatic phenotype has important clinical implications for diagnosis and therapy of cancer. For this reason, it is important to develop animal models for the selection and isolation of metastatic variants from human colon concers and for testing the metastatic potential of these cells. We have implanted cells from more than 100 HCC (obtained from surgical specimens) into different organs of nude mice. Regardless of their malignant potential in the patient, the HCC did not metastasize unless they were implanted orthotopically. Only when they were injected into the cecum or spleen of nude mice did they yield hepatic metastases. These metastases consisted of highly metastatic cells. The invasive phenotype was influenced by the organ environment. HCC cells in the subcutis did not produce degradative enzymes and the cells did not metastasize. In contrast, HCC cells in the cecum did both. Collectively, the results demonstrate that the orthotopic implantation of HCC cells can yield metastatic subpopulations of cells suitable for the study of metastasis.     

Time-dependent dephosphorylation through serine/threonine phosphatases is required for stable adhesion of highly and poorly metastatic HT-29 colon carcinoma cell lines to collagen

Adhesion stabilization is a prerequisite for the long-term adhesion of circulating metastatic tumor cells, and tumor cells with different metastatic potential demonstrate distinct patterns of cell adhesion properties. An important event during formation of organ metastases is integrin-mediated extracellular matrix (ECM) binding that can initiate signal transduction events. Recently we reported that Ser/Thr kinases are involved in regulation of tumor cell adhesion. In the present study the influence of dephosphorylation by Ser/Thr protein phosphatases (PPases) on tumor cell adhesion was investigated. Pretreatment of poorly and highly metastatic human HT-29 colon carcinoma cells with the broad-range inhibitors sodium fluoride (NaF) and sodium pyrophosphate (PyroP) resulted in strong reduction in adhesion of HT-29 cells to various ECM components. Surprisingly, when specific Ser/Thr PPase inhibitors like tautomycin were used we found only a partial reduction in adhesion of highly metastatic HT-29LMM cells to collagen I but not to collagen IV. Other inhibitors did not inhibit adhesion, and poorly metastatic HT-29P were not affected by any specific Ser/Thr PPase inhibitors. Therefore, the effects of NaF on adhesion-mediated Tyr phosphorylation were investigated further. Pretreatment with this inhibitor led to a reduction in phosphorylation of focal adhesion kinase (FAK). In contrast, in cells grown adherent to tissue culture dishes, low concentrations of NaF increased FAK phosphorylation whereas high concentrations inhibited the amount of phosphorylated FAK. Although NaF inhibited adhesions it did not cause changes in cell morphology or detachment of cells from ECM. We hypothesize that dual-specific PPases may be involved in the regulation and establishment of new adhesive interactions in HT-29 cells, but they are not required for maintenance of stable adhesions to ECM.     

Extracellular matrix destruction by invasive tumor cells

Summary The invasion of normal tissues and penetration of basement membranes by malignant cells is likely to require the active participation of hydrolytic enzymes. The four major groups of connective tissue proteins, glycoproteins, proteoglycans, collagen and elastin, vary in their quantitative distributions between different tissues. With the exception of elastin, they also vary qualitatively within each class, so that there are no typical connective tissue barriers to tumor cell penetration. The matrix constituents are stabilized and organized by a variety of covalent and noncovalent interactions between the connective tissue proteins. These interactions play important roles in matrix integrity and may alter the susceptibilities of the constituents to degradative enzymes. It is likely that the complete degradation of the matrix will require the action of more than one enzyme because of differing susceptibilities to tissue proteinases. Primary and transplantable tumors produce well-characterized enzymes which may participate in invasion. These enzymes may also be involved in connective tissue turnover in other normal and pathological situations. The use of long-term tumor cell cultures has verified that tumor cells themselves are capable of producing these enzymes. However, there are many potential modulating influences opperative in vivo which are absent in culture so that details of actual mechanisms and control of digestion of complex substrates are not well understood. Recent work on the degradation by tumor cells of extracellular matrices previously produced by cultured cells is likely to shed more light on pathways of tissue destruction in vivo. Experiments with tumor cell variants of defined metastatic potentials will also be useful, but invasive and metastatic abilities are not necessarily correlated. It is unlikely that simple correlations can be drawn between the production of one particular degradative enzyme by all tumor cells and the complex biological mechanisms operative during tumor invasion.     

Expression of heparanase by platelets and circulating cells of the immune system: possible involvement in diapedesis and extravasation.

Interaction of T and B lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated with degradation of heparan sulfate (HS) by a specific endoglycosidase (heparanase) activity. The enzyme is released from intracellular compartments (i.e., lysosomes, specific granules) in response to various activation signals (i.e., thrombin, calcium ionophore, immune complexes, antigens, mitogens), suggesting its regulated involvement in inflammation and cellular immunity. In contrast, various tumor cells appear to express and secrete heparanase in a constitutive manner, in correlation with their metastatic potential. Heparanase enzymes produced by different cell types may exhibit different molecular properties and substrate cleavage specificities. The platelet enzyme appears also in a latent form. It can be activated by tumor cells and thereby facilitate their extravasation in the process of metastasis. Degradation of ECM-HS by all cell types was facilitated by a proteolytic activity residing in the ECM and/or expressed by the invading cells. This proteolytic activity produced a more accessible substrate for the heparanase enzymes. Heparanase-inhibiting, nonanticoagulant species of heparin markedly reduced the incidence of lung metastasis in experimental animals. These species of heparin also significantly impaired the traffic of T lymphocytes and suppressed cellular immune reactivity and experimental autoimmune diseases. Heparanase activity expressed by intact cells (i.e., platelets, mast cells, neutrophils, lymphoma cells) was found to release active HS-bound basic fibroblast growth factor from ECM and basement membranes. Heparanase may thus elicit an indirect neovascular response in processes such as wound repair, inflammation and tumor development. The significant anticancerous effect of heparanase-inhibiting molecules may therefore be attributed to their potential inhibition of both tumor invasion and angiogenesis. Both normal leukocytic cells and metastatic tumor cells can enter the bloodstream, travel to distant sites and extravasate to the parenchyma at these sites. We suggest that heparanase is utilized for this purpose by both types of cells. Other functions (i.e., enzyme activities, adhesive interactions, chemotactic and proliferative responses) of metastatic tumor cells seem to mimic the equivalent functions of leukocytes as they migrate across blood vessels to gain access to sites of inflammation.     

Extracellular matrix-resident growth factors and enzymes: possible involvement in tumor metastasis and angiogenesis

Summary Neoplastic cells require an appropriate pericellular environment and new formation of stroma and blood vessels in order to constitute a soilid tumor. Tumor progression also involves degradation of various extracellular matrix (ECM) constituents. In this review we have focused on the possible involvement of ECM-resident growth factors and enzymes in neovascularization and cell invasion. We demonstrate that the pluripotent angiogenic factor, basic fibroblast growth factor (bFGF) is an ECM component required for supporting cell proliferation and differentiation. Basic FGF has been identified in the subendothelial ECM producedin vitro and in basement membranes of the cornea and blood vesselsin vivo. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Our results indicate that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by cellular heparanase. We propose that restriction of bFGF bioavailability by binding to ECM and local regulation of its release, provides a novel mechanism for regulation of capillary blood vessel growth in normal and pathological situations. Heparanase activity correlates with the metastatic potential of various tumor cells and heparanase inhibiting molecules markedly reduce the incidence of lung metastasis in experimental animals. Heparanase may therefore participate in both tumor cell invasion and angiogenesis through degradation of the ECM-HS and mobilization of ECM-resident EC growth factors. The subendothelial ECM contains also tissue type- and urokinase type- plasminogen activators (PA), as well as PA inhibitor which may regulate cell invasion and tissue remodeling. Heparanase and the ECM-resident PA participate synergistically in sequential degradation of HS-proteoglycans in the ECM. These results together with similar observations on the properties of other ECM-immobilized enzymes and growth factors, suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized, regulated and persistent mode of action, as compared to the same molecules in a fluid phase.     

Tumor and host molecules important in the organ preference of metastasis

Many cancers metastasize nonrandomly to particular distant sites, and their colonization properties cannot be explained by mechanical considerations, such as arrest of tumor cells in the first microcirculatory network encountered. Metastatic cells that show a high propensity to metastasize to certain organs adhere at higher rates to microvessel endothelial cells isolated from these target sites, invade into target tissue at higher rates and respond better to paracrine growth factors from the target site. These properties depend on multiple tumor cell, host cell, and stromal molecules that are differentially expressed by particular tumor and organ cells and by the extracellular matrix. For example, some of the adhesion molecules involved in tumor cell-endothelial cell adhesion have been identified on both tumor and host cells. Among them are: integrins, endogenous lectins, annexins, and other molecules. The invasive properties of particular tumor cells are dependent on the production of degradative enzymes, such as metalloproteinases, heparanase and cys and ser proteinases, and on responses to organ-derived paracrine and autocrine motility factors. The subsequent growth of particular tumor cells at certain organ sites is determined, in part, by their responses to organ paracrine growth factors and the organ extracellular matrix. Collectively these factors appear to determine the organ colonization properties of blood-borne metastatic cells.     

TNF-alpha increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes

Inflammatory mediators have been reported to promote malignant cell growth, invasion and metastatic potential. More specifically, we have recently reported that tumour necrosis factor alpha (TNF-alpha) increases melanoma cell attachment to extracellular matrix (ECM) substrates and invasion through fibronectin. In this study, we extend these investigations asking specifically whether the TNF-alpha effect on cell invasion and migration involves activation of proteolytic enzymes. We examined the effect of TNF-alpha on melanoma expression/activation of type IV gelatinases matrix metalloproteinases 2 and 9 (MMPs -2 and -9) and general proteolytic enzymes. Stimulation with TNF-alpha significantly increased both melanoma cell migration at 24 h (+21%) and invasion through fibronectin (+35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity. However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-alpha were inhibited by the general protease inhibitor alpha(2) macroglobulin. These findings suggest that the promigratory and proinvasive effect of TNF-alpha on this melanoma cell line may be mediated to some extent by induction of localised cell membrane-bound degradative enzyme activity, which is not readily detected in biochemical assays.     

The tumor microenvironment in hepatocellular carcinoma: current status and therapeutic targets

A growing body of literature highlights the cross-talk between tumor cells and the surrounding peri-tumoral stroma as a key modulator of the processes of hepatocarcinogenesis, epithelial mesenchymal transition (EMT), tumor invasion and metastasis. The tumor microenvironment can be broadly classified into cellular and non-cellular components. The major cellular components include hepatic stellate cells, fibroblasts, immune, and endothelial cells. These cell types produce the non-cellular components of the tumor stroma, including extracellular matrix (ECM) proteins, proteolytic enzymes, growth factors and inflammatory cytokines. The non-cellular component of the tumor stroma modulates hepatocellular carcinoma (HCC) biology by effects on cancer signaling pathways in tumor cells and on tumor invasion and metastasis. Global gene expression profiling of HCC has revealed that the tumor microenvironment is an important component in the biologic and prognostic classification of HCC. There are substantial efforts underway to develop novel drugs targeting tumor-stromal interactions. In this review, we discuss the current knowledge about the role of the tumor microenvironment in pathogenesis of HCC, the role of the tumor microenvironment in the classification of HCC and efforts to develop treatments targeting the tumor microenvironment.     

Platelet-tumor cell interaction with the subendothelial extracellular matrix: relationship to cancer metastasis

Dissemination of neoplastic cells within the body involves invasion of blood vessels by tumor cells. This requires adhesion of blood-borne cells to the luminal surface of the vascular endothelium, invasion through the endothelial cell layer and local dissolution of the subendothelial basement membrane. Platelets may participate in each of these steps and thus play a role in the pathogenesis of tumor cell metastasis. To learn more about the possible involvement of platelets we studied the interaction of platelets and tumor cells with cultured vascular endothelial cells and their secreted basement membrane-like extracellular matrix (ECM). Whereas the apical surface of the vascular endothelium lacks adhesive glycoproteins and hence protect the vessel wall against platelet and tumor cell adhesion, the underlying ECM constitute a highly adhesive and thrombogenic surface. Interaction of platelets with this ECM was associated with platelet activation, aggregation and degradation of heparan sulfate in the ECM by means of the platelet heparitinase. The activity of a similar enzyme has been previously correlated with the metastatic potential of various tumor sublines. Biochemical and scanning electron microscopy (SEM) studies have demonstrated that platelets may detect even minor gaps between adjacent endothelial cells and degrade the ECM heparan sulfate. This may expose a larger area of the subendothelium and facilitate subsequent adhesion of blood borne tumor cells. Platelets were also shown to recruit lymphoma cells into minor gaps in the vascular endothelium, that otherwise do not constitute a preferential site of invasion. It is suggested that the platelet heparitinase is involved in the impairment of the integrity of the vessel wall and thus play a role in tumor cell metastasis.     

Antisense-mediated suppression of Heparanase gene inhibits melanoma cell invasion

Cancer metastasis, is a frequent manifestation of malignant melanoma progression. Successful invasion into distant organs by tumor cells must include attachment to microvessel endothelial cells, and degradation of basement membranes and extracellular matrix (ECM). Heparan sulfate proteoglycans (HSPG) are essential and ubiquitous macromolecules associated with the cell surface and ECM of a wide range of cells and tissues. Heparanase (HPSE-1) is an ECM degradative enzyme, which degrades the heparan sulfate (HS) chains of HSPG at specific intrachain sites. To investigate effects of changes in heparanase gene expression in metastatic melanoma cells, we constructed adenoviral vectors containing the full-length human HPSE-1 cDNA in both sense (Ad-S/hep) and antisense orientations (Ad-AS/hep). We found increased HPSE-1 expression and activity in melanoma cell lines following Ad-S/hep infection by Western blot analyses and specific HPSE-1 activity assay. Conversely, HPSE-1 content was significantly inhibited following infection with Ad-AS/Hep. Importantly, HPSE-1 modulation by these adenoviral constructs correlated with invasive cellular properties in vitro and in vivo. Our results suggest that HPSE-1 not only contributes to the invasive phenotype of melanoma cells, but also that the Ad-AS/hep-mediated inhibition of its enzymatic activity can be efficacious in the prevention and treatment of melanoma metastasis.     

Hyaluronan facilitates invasion of colon carcinoma cells in vitro via interaction with CD44

Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in vitro. To assess HA functionality, HAS expression was inhibited in SW620 cells by transfection with antisense HAS constructs. Decreased HA secretion and retention in the transfectants were confirmed using competitive binding and particle exclusion assays. SW620 cells demonstrated greater invasion through Matrigel than did SW480 cells. Antisense transfection decreased Matrigel invasion by SW620 cells by >60%; addition of exogenous HA restored invasion. Because the cell-surface HA receptor CD44 has been implicated in cancer progression, HA-CD44 interaction was then inhibited by incubation with an anti-CD44 antibody. Anti-CD44 antibody impaired invasion into Matrigel by 95%. Taken together, these data suggest that pericellular HA is critical for colon carcinoma cell invasion and that this invasive capability is dependent on interaction with CD44.     

Human ovarian carcinoma beta-N-acetylglucosaminidase isoenzymes and their role in extracellular matrix degradation

Degradation and invasion of basement membrane by tumor cells involves the cooperative hydrolysis of proteoglycans, collagens, and glycoproteins mediated by a number of enzymes including proteases, collagenases, and glycosidases. In order to study these processes in vitro, a tissue culture system was developed in which bovine corneal endothelial cell extracellular matrix (ECM) serves as a substrate for attachment and degradation by human ovarian carcinoma cells. Using this system, a correlation was observed between solubilization of glycoconjugates present in ECM and extracellular levels of beta-N-acetylglucosaminidase (EC 3.2.1.30). To determine the role of individual isoenzymes of beta-N-acetylglucosaminidase (beta-NAG) in ECM degradation, the cellular and secreted forms of the enzyme were fractionated and characterized. Three intracellular isoenzyme forms A, I, and B, were isolated from invasive human ovarian carcinoma cell line A-121. In cell homogenate, forms A and B corresponded to 65 and 33% of total beta-NAG activity, respectively. Form I was found to be localized in the plasma membrane fraction of these cells. Two secreted forms of beta-NAG (As and Bs) were detected in serum-free medium. The separated intracellular and secreted isoenzymes demonstrated similar Km values, ranging from 1 to 5 mM, with p-nitro-B-N-acetylglucosaminide substrate. Treatment of [3H]glucosamine-labeled ECM with the separated isoenzymes of beta-NAG resulted in time- and concentration-dependent releases of radioactivity with potency of 1 greater than B much greater than A. These results suggest that human ovarian carcinoma cell beta-N-acetylglucosaminidase isoenzymes (forms B and A) contribute to ECM degradation as secreted enzymes and form I as a membrane enzyme.     

Matrix metalloproteinases and oral cancer

For tumours to invade and metastasise, neoplastic cells must be capable of degrading the extracellular matrix (ECM), and accessing blood vessels and lymphatics. This process is mediated in the pericellular environment and is a highly controlled cascade of events utilising the same mechanisms that normal cells use for migrating through tissue barriers, for example, in development and wound healing. Proteolytic enzymes from several families, including matrix metalloproteinases (MMPs), are involved in ECM remodelling. Increased production of these enzymes has been associated with the invasive and/or metastatic phenotype in many tumours. Several MMPs have been shown to play a role in the invasion and metastasis of oral carcinoma, and it is increasingly apparent that tumour cells, as well as producing endogenous MMP, are capable of utilising MMP produced by tumour stromal cells, indicating an active role for stroma in tumour invasion. It is not clear whether a particular invasive system is favoured by oral carcinoma, but it is likely that further understanding of the interactions between carcinoma and stromal cells will provide an opportunity to refine the therapeutic interventions that are currently being tested.