Correlation of serum metalloproteinase levels with lung cancer metastasis and response to therapy.

Cancer cells elaborate metalloproteinases which may play a role in invasion and metastasis. The serum level of the M(r) 72,000 type IV collagenase (MMP-2) was measured in 87 lung cancer patients. Stage IV cancer levels were significantly elevated (P less than 0.0001) compared to normal sera. A significant difference (P less than 0.01) was found between enzyme levels in the presence versus the absence of distant metastasis. For 29 patients treated with combination chemotherapy, a positive relationship was noted between response failure and elevated enzyme levels. Serum metalloproteinase levels may provide information relevant to prognosis as well as treatment decisions.     

Heparin modulates proliferation and proteoglycan biosynthesis in murine mesangial cells: molecular clues for its activity in nephropathy

Glycosaminoglycan administration has favourable effects on morphologicaland functional renal abnormalities in different models. Thepossibility that exogenous glycosaminoglycans modulate glomerularmatrix synthesis was explored in both primary and SV40-MES13murine mesangial cell cultures. On both cell types, both low-molecular-weightheparin and different glycosaminoglycans showed dose-dependentinhibition of proliferation and increase of ³⁵SO^2–₄ uptake.After 36 h the cell compartment contained a spectrum of ³⁵S-moleculesof less than 200 kDa; under heparin treatment, the two main³⁵SO^2–₄ components (high and medium MW) increased by 16and 37% respectively. Susceptibility to glycosidases revealedthat heparin promotes the expression of heparan sulphate andincreases that of chondroitin sulphate. Moreover, heparin modifiesthe expression of decorin and bigly-can, involved in adhesionand fibrillogenesis, while not affecting perlecan. The extracellularmatrix modulation in renal cells, for which the sulphation typeand ratio of heparin are crucial, may thus explain the beneficialrenal effects of heparin.     

Correlation between differentiation and lung colonization by retinoic acid-treated F9 cells as revealed by the expression pattern of extracellular matrix and cell surface antigens.

For study of the correlation between differentiation and organ colonization properties of tumor cells, F9 embryonal carcinoma (EC) cells were treated with retinoic acid, an inducer of differentiation; and their organ colonization pattern was assessed by the experimental metastasis assay. Untreated cells were found to colonize the liver, whereas treated cells colonized the lungs. This pattern held true when metastases were scored after spontaneous death or after a careful microscopic search for micrometastases. Histologic examination revealed that both the tumor nodules produced by the untreated and the treated cells had the characteristics of EC devoid of any evidence of differentiation. The immunohistochemical study of the expression of markers typical of embryonal carcinoma cells or of the extracellular matrix components laminin and collagen type IV, typical of differentiated cells, confirmed these results. However, the lack of expression of stage-specific embryonal antigen 1 (SSEA-1), a marker generally associated with the undifferentiated state, observed only in the tumors obtained after injection of treated cells, indicates that the lung nodules probably derive from cells that have responded to the induction in vitro but have dedifferentiated in vivo.     

Dual Action of NAMI-A in inhibition of solid tumor metastasis: selective targeting of metastatic cells and binding to collagen.

NAMI-A is a ruthenium complex endowed with a selective effect on lung metastases of solid metastasizing tumors. The aim of this study is to provide evidence that NAMI-A's effect is based on the selective sensitivity of the metastasis cell, as compared with other tumor cells, and to show that lungs represent a privileged site for the antimetastatic effects. The transplantation of Lewis lung carcinoma cells, harvested from the primary tumor of mice treated with 35 mg/kg/day NAMI-A for six consecutive days, a dose active on metastases, shows no change in primary tumor take and growth but a significant reduction in formation of spontaneous lung metastases. Transmission electron microscopy examination of lungs and kidney shows NAMI-A to selectively bind collagen of the lung extracellular matrix and also type IV collagen of the basement membrane of kidney glomeruli. The half lifetime of NAMI-A elimination from the lungs is longer than for liver, kidney, and primary tumor. NAMI-A bound to collagen is active on tumor cells as shown in vitro by an invasion test, using a modified Boyden chamber and Matrigel, and it inhibits the matrix metallo-proteinases MMP-2 and MMP-9 at micromolar concentrations, as shown in vitro by a zimography test. These data show NAMI-A to significantly affect tumor cells with metastatic ability. Binding to collagen allows NAMI-A to exert its selective activity on metastatic cells during dissemination and particularly in the lungs. These data also stress the wide spectrum of daily doses and treatment schedules at which NAMI-A is active against metastases.     

Hormonal and basement membrane markers for immunoidentification of cultured human trophoblast cells

Improvement of human chorionic villi cells in vitro culture has been obtained by supplementing the medium with 30% human fetal cord serum, instead of fetal calf serum. Expression of human chorionic gonadotropin, estrogen and progesterone receptors, laminin, laminin receptors, and type IV collagen has been studied on first subculture passages (4-7 weeks) by immunoperoxidase method. A minority of the cultured cells were positive for estrogen receptors, the majority were positive for progesterone receptors, while all cells were negative for human chorionic gonadotropin. Cultured cells showed variable positive immunostaining for basement membrane markers like laminin, and type IV collagen, and for laminin receptors. Detection of both progesterone receptors and laminin, or type IV collagen, excluded fibroblast contamination and could then be useful for quick identification of cultured trophoblast cells.     

Inhibition of matrix-proteases by polyphenols: chemical insights for anti-inflammatory and anti-invasion drug design

Flavanols--a class of plant polyphenols abundant in tea leaves and grape seeds and skins--have been found to inhibit some matrix-proteases instrumental in inflammation and cancer invasion, such as leukocyte elastase (LE) and gelatinases. In order to establish the relationship between chemical structure and activity, 27 different flavonoids (antocyanidins, dihydrochalcones, dihydroflavonols, flavanolignans, flavanols, flavones, flavonols and isoflavones) and other compounds with anti-oxidant properties were evaluated for their potential in blocking LE and gelatinase activities. LE activity was measured using a chromogenic substrate: from comparison of the different levels of inhibition, it was deduced that a crucial role in inhibition might be played by a galloyl moiety or hydroxyl group at C3, three hydroxyl groups at B ring, one hydroxyl group at C4', and a 2,3-double bond. Gelatinase activity was measured using the gelatin-zymography assay, and its inhibition showed that three hydroxyl groups at the A or B ring, or, for non-planar molecules, a galloyl moiety at C3 could be determinant. This comparative study is proposed as a basis for designing new molecules with enhanced anti-proteolytic activities, and no or reduced side-effects, for use in hindering inflammation, cancer invasion and angiogenesis.     

Inhibition of endothelial cell functions and of angiogenesis by the metastasis inhibitor NAMI-A

NAMI-A is a ruthenium-based compound with selective anti-metastasis activity in experimental models of solid tumours. We studied whether this activity was dependent on anti-angiogenic ability of NAMI-A. We thus investigated its in vitro effects on endothelial cell functions necessary for angiogenesis to develop, as well as its in vivo effects in the chick embryo chorioallantoic membrane model. Endothelial cell proliferation, chemotaxis, and secretion of the matrix-degrading enzyme metalloproteinase-2 were inhibited by NAMI-A in a dose-dependent manner, and without morphologic signs of cell apoptosis or necrosis. Lastly, NAMI-A displayed a dose-dependent in vivo anti-angiogenic activity in the chorioallantoic membrane model. These data suggest that the anti-angiogenic activity of NAMI-A can contribute to its anti-metastatic efficacy in mice bearing malignant solid tumours.     

Prostate carcinoma and green tea: (-)epigallocatechin-3-gallate inhibits inflammation-triggered MMP-2 activation and invasion in murine TRAMP model

Green tea infusion has been shown to inhibit metastatic spreading of the transgenic adenocarcinoma of mouse prostate (TRAMP). Investigation on the molecular mechanisms triggered by the main green tea flavonoid, (-)epigallocatechin-3-gallate (EGCG), shows that EGCG restrains TRAMP-C1 cell proliferation in a dose-dependent manner, at concentrations (IC(50) < 0.2 microM) equivalent to those measured in the plasma of moderate green-tea drinkers. Up to 10 microM, EGCG does not modify the cell-surface immuno-localization of MMP-2, one of the invasion-instrumental proteinases; but while in default culture conditions these cells secrete mainly pro-MMP-2, in the presence of reconstituted basement membrane (Matrigel) they release almost exclusively pro-MMP-9. In contrast, when stimulated to traverse Matrigel toward a chemo-attractant, in addition to pro-MMP-9, they secrete pro-MMP-2. In the presence of 0.2 microM EGCG, only the level of the latter is markedly lowered in the conditioned medium, in parallel with the invasive behavior (>50%). In vivo, s.c. injection of TRAMP-C1 cells dispersed in Matrigel gives origin to a tumor mass, whose growth is not inhibited by green-tea regimen. This growth is contained greater than two-thirds by LPS-triggered polymorpho-nuclear phagocyte (PMN) recruitment but this effect is abolished by green tea. Nevertheless, while tumor-released pro-MMP-2 is activated by co-incubation of TRAMP-C1 cells with PMNs, in the presence of 10 microM EGCG the activation is almost abolished. These results suggest that inflammatory involvement of prostate carcinoma could be efficaciously prevented by green tea with a concomitant lowering of the invasive potential.     

The capsule surrounding primary liver tumors: Wherefrom its prognostic significance?

Primary liver neoplasms in cirrhotic and non-cirrhotic patients were studied by electron microscopy and immunohistochemical methods for extracellular matrix (ECM) antigens. A capsule of variable thickness was present in many expanding hepatocellular carcinomas, while it was absent in those of small size, and either fragmented or absent in the infiltrating ones. In the capsules of early onset, fibronectin was the most frequent stromal glycoprotein. In the completely formed capsular structures, fibronectin, type-V collagen and laminin were the most common macromolecules seen. No differences were evident in the pattern of ECM macromolecules in the capsules surrounding hepatocellular carcinomas compared with those found in benign lesions, such as hepatocellular adenomas or focal nodular hyperplasia. The possibility is discussed that the capsule could be a result of the following sequence: tissue with expansive (not infiltrative) growth-mechanical compression-ischemic necrosis of surrounding tissues-repair process with ECM deposition.     

Role of collagenases in tumor cell invasion

Summary Collagenases are a family of metalloproteinases which may play a role in facilitating tumor cell invasion of the extracellular matrix. Tumor cells traverse two types of extracellular matrix: basement membranes and interstitial stroma, at multiple stages of the metastatic process. The matrix is a dense meshwork of collagen, proteoglycans, elastin and glycoproteins. Normally the matrix does not contain open spaces large enough for cell movement. Therefore numerous investigators have postulated that collagenolytic proteases, secreted by tumor cells or associated host cells, breakdown the extracellular matrix during tumor cell invasion. A large number of animal and human tumors have been shown to contain collagenase at a higher level than corresponding benign tissues. Separate collagenolytic metalloproteinases have been identified which degrade specific types of collagen. A basement membrane collagenolytic protease was shown to be elevated in a series of metastatic murine tumor cells. Immunologic studies using antibodies specific for collagenase have demonstrated that in vivo, tumor cells can produce collagenase. Therefore identification of collagenase in cultured lines of tumor cells is not an artifact of in vitro cultivation. In some cases, tumor cells may induce host cells to produce collagenase. The best evidence to date that collagenases actually play a role in invasion is derived from experiments in which natural collagenase inhibitors block tumor cell invasion of extracellular matrix in vitro.