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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) 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 phototoxicity of liposome-incorporated Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) was studied in the tumorigenic but nonmetastatic (RE4) and the highly metastatic (4R) transformed rat embryo fibroblasts. Upon irradiation with 585–605 nm light in the presence of ZnPc, the cell survival drastically decreased, while it was unaffected by ZnPcTS. Enzymatic assays showed that ZnPc induced about a 60% decrease in the activity of the mitochondrial enzymes NADH and succinate dehydrogenase after 3 min of irradiation, while no significant reduction in the activity of lactate dehydrogenase and lysosomal N-acetyl-β-glucosaminidase was observed. The transport of thymidine, deoxyglucose and α-aminoisobutyric acid through the plasma membrane was strongly inhibited after irradiation. Similarly, the intracellular ATP content was significantly reduced. The reduction of DNA biosynthesis showed a time dependence quite similar to the photo-induced decrease in cell survival. No repair of cellular functions affected by ZnPc was observed in the 2 cell lines. These results indicate that, under our experimental conditions, hydrophobic ZnPc exerts its cytotoxic activity mainly by impairing those functions localized in the plasma membrane of the cells. Int. J. Cancer 75:412–417, 1998. © 1998 Wiley-Liss, Inc.

Treatment of mouse Lewis lung carcinoma with razoxane or dacarbazine was protracted for 10 transplant generations. While the capacity of the treated tumors to grow locally in immuno-competent or in immuno-depressed hosts was retained and not significantly modified, the metastatic phenotype was eliminated when the treated tumor cells were transplanted into immuno-competent hosts. The reduction in metastatic potential was slightly less pronounced, in terms of both number and volume of metastases, when the treated tumor cells were transplanted into immuno-depressed hosts. These properties were retained after 3 transplant generations without treatment. Northern blotting and zymography of primary-tumor crude extracts revealed that treatment with either razoxane or dacarbazine for one generation approximately doubled the expression of MMP-2 and MMP-9, while lacking any effect on that of 1.0 and of 3.5 kb TIMP-2. When the treatment was maintained for 10 generations, the expression of MMP-2 and MMP-9 for both drugs showed up-regulation of approximately 10- and 2-fold respectively. TIMP-2 mRNA of 1.0 kb doubled its expression, while that of 3.5 kb registered just above the control. Dacarbazine doubled the expression of uPA after 10 generations, while razoxane boosted it approximately 3-fold after either 1 or 10 generations. The permanent loss of metastatic phenotype induced in Lewis lung carcinoma by dacarbazine and razoxane is thus attributable to biological mechanisms independent of downregulation of expression and/or activation of the 2 gelatinases. Int. J. Cancer 72:1056‐1061, 1997. r 1997 Wiley-Liss, Inc.

The anti-metastatic ruthenium complex NaCtransRuCI4(DMSO)lm] was given i.p. at 22 and 44 mg/kg/day, on days 8-13 after tumour implantation, to mice carrying S.C. implants of MCa mammary carcinoma. The aim ofthe study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT-PCR analysis for the type-IV collagenases MMP-9 and MMP-2 and their respective inhibitors TIMP-I and TIMP-2 mRNAs. NactransRuCl,(DMSO)lm] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans-RuCI~(DMSO)lm] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose-dependent manner, MMP-2/TIMP-2 balance, but not that of MMP-9/TIMP-I. The different enzyrne/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumourinfiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in conbols. o 1996 Wiley-Liss, Inc. Basic research on synthetic drugs, effective against tumour metastases, has recently highlighted the effects of a ruthenium complex, namely sodium trans-rutheniumtetrachloridedimethylsulphoxideimidazole (hereafter indicated by Na[transRuCI4(DMSO)Im]) (Sava er al., 1992a, b, 1993, 1994). The effects of this new generation ruthenium(II1) complex on solid metastasizing tumour are particularly evident on the formation of spontaneous metastases. The selectivity of Na[transRuCI4(DMSO)Im] on lung metastases is also marked on advanced metastases and accounts for a significant prolongation of the host's survival time; combined with surgical removal of primary tumour, Na[trans-RuC14(DMSO)Im] prevents the formation of metastases and inhibits the growth of those already formed (Sava et al., 1994). The histological analysis of tumour growth and of healthy host tissues such as lung and kidney epithelia, muscle and liver cells, splenocytes and bone-marrow cells, by light microscopy and by SEM, shows a lack of significant cytotoxicity (Gagliardi et al., 1994). It thus appears that the selective anti-metastatic effects do not result directly from histological modification of the primary tumour structure.

In 187 node-negative breast cancers, the expression of laminin receptors and collagenase IV was directly related in 52% of cases, independently of pathological (tumor size and histology) and biological (estrogen receptors and proliferative activity) features. Moreover, the presence of laminin receptors and collagenase IV did not appear to influence tumor proliferative activity, evaluated as 3H-thymidine labelling index. In this case series, relapse-free survival and overall survival at 6 years were significantly affected by tumor size, hormone receptor status and proliferative activity. Conversely, high levels of laminin receptors and collagenase IV failed to influence relapse-free or overall survival, whereas they were strong indicators of local-regional diffusion of the disease.

Prostate-specific antigen (PSA) is a serine-protease that, in addition to cleaving semenogelins in the seminal coagulum, is able to cleave extracellular matrix glycoproteins, thereby affecting cell migration and metastasis. We here report some new activities of PSA that deserve careful consideration in the cancer context: degradation of gelatin, degradation of type IV collagen in reconstituted basement membrane (Matrigel) and activation of progelatinase A (MMP-2), but not pro-MMP-9, in a cell-free system. Since consumption of green tea has been reported to lower the risk of prostate cancer, we investigated the effects of the major flavanol of green tea, (-)epigallocatechin-3-gallate (EGCG), on expression and activity of PSA by prostate carcinoma cells. In addition to restraint of PSA expression, EGCG was found to inhibit in a dose-dependent manner all the above PSA activities, at concentrations lower than the cytotoxic serine-protease inhibitor PMSF and close to levels measured in the serum following ingestion of green tea. The activity of PSA was suppressed also by the elastase released by the inflammatory leukocytes. These results highlight new PSA activities, suggest gelatin zymography as a new convenient assay for PSA, propose EGCG as natural inhibitor of prostate carcinoma aggressiveness, but also stimulate further investigation on the role of prostatic inflammation.

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.

Over-production of gelatinase A (MMP-2) or under-production of its inhibitor (TIMP-2) may result in the matrix degradation crucial for metastasis, and early evaluation of their expression in primary tumor would offer important prognostic informations. RT-PCR amplicons of MMP-2 and TIMP-2 mRNA from tissue biopsies of 13 breast carcinomas and one fibrocystic mastopathy were quantitated. In comparison with their normal-tissue counterparts, their expression trends were not uniform: in some cases MMP-2 increased in the tumor without changes in TIMP-2, in others TIMP-2 expression also increased, although to a lesser extent than MMP-2; only in 2 cases was it slightly lower in the tumor tissue. Nevertheless, clearer insights were gained from the comparison of the ratio (R) between MMP-2tumor/normal and TIMP-2tumor/norma: as in the fibrocystic mastopathy, the R in carcinomas without lymph-node involvement (LN−) was usually lower than 1 in most cases. In contrast, in 5 out of 6 patients with lymph-node metastasis (LN+), the ratio ranged between 2 and 4. While the R magnitude was not related to the frequency of positive lymph nodes out of the total analyzed, nor to relapse status at follow-up (all relapse-free), the clear-cut difference between the LN− and LN+ groups was statistically significant. Results suggest that evaluation of MMP-2/TIMP-2 mRNA balance may constitute an early prognostic approach, which may also be more reliable concerning cancer aggressiveness as compared with the MMP-2 alone, and that boosting TIMP-2 expression may be a therapeutic strategy to prevent metastasis. © 1995 Wiley-Liss, Inc.

It is widely accepted that collagenolytic enzymes are required to facilitate the invasion and spread of tumour cells into host tissues. Immunohistochemical, zymographic and PCR analyses have produced evidence that the recently established human mammary carcinoma cell line, 8701-BC, expresses several metalloproteinases (MMP-1, -2, -9 and -10) and their tissue inhibitors (TIMP-1 and -2). Application of these different techniques has led to several observations, both complementary and dissimilar. Whereas PCR analysis showed that mRNA was detected for each of the proteins, the immunolocalization study demonstrated that MMP-1, MMP-2, MMP-9 and TIMP-1 production was restricted to only a proportion of the tumour cells, with no evidence of MMP-3 or TIMP-2 synthesis. Such observations suggested phenotypic heterogeneity within the cell line, which was further examined by use of the tumour cell clones BC-3A and BC-61 derived from the parental 8701-BC line. Comparative studies using zymography and PCR analysis demonstrated differences in MMP-2 and MMP-10 expression between the 3 cultures. The data indicate that the 8701-BC cell line retains an inherent capacity for metalloproteinase and TIMP expression, with the production of both interstitial collagenase (MMP-1) and the 2 basement-membrane-degrading enzymes (MMP-2 and MMP-9) representing an aggressive collagenolytic phenotype. The concomitant production of TIMP-1 by these cell cultures, and the apparent phenotypic heterogeneity displayed by these lines, suggest that metalloproteinase dysregulation may represent an important feature of clonal heterogeneity. Although the 8701-BC and BC-61 cells were much more invasive than those of the BC-3A clone, as judged by the penetration of "Matrigel", it has not yet been possible to relate this invasive potential to the metalloproteinase and TIMP profiles reported here for each cell line.

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.