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Tubular cell epithelial-mesenchymal transition (EMT) is a fundamental contributor to renal fibrosis. The aim of this study was to investigate the activity of different matrix metalloproteinases by immunohistochemistry and gel-zymography in a model of chronic canine kidney disease. Immunohistochemistry for antibodies against MMP-9, MMP-2, MMP-13, MMP-14 and TIMP-2 was performed on 28 renal biopsy specimens. Selected cases were chosen for gelatin zymography. In moderate and severe tubulo-interstitial damage, increased expression of MMP-2 was noted. A peculiar staining pattern for MMP-2 in variable-sized vesicles, corresponding to the area of basement membrane splitting, was observed. The immunoexpression of MMP-9 and TIMP-2 was reduced in the same cases, compared to control dogs. The splitting of the membrane suggests an active role of this gelatinase in the disruption of type-IV collagen, the main basement membrane component, confirmed by MMP2 gelatinolytic activity by gel-zymography. These data could provide the basis for clinical trials examining the potential benefits of selective MMP-2 inhibitors in dogs with chronic kidney disease.

The extra-cellular matrix (ECM) related antigens, type IV collagen, laminin, M(r) 68,000 laminin receptor (LR), M(r) 72,000 type IV collagenase (MMP-2), its inhibitor TIMP-2, and alpha 2-macroglobulin expression have been immunohistochemically investigated in 100 cases of human gastric carcinoma with a 5-yr follow up. Basement membranes were inversely related to tumoral differentiation. At the early intramucosal stage of both intestinal and diffuse histological types, TIMP-2 was expressed by the majority of tumor cells (60/63%), whereas MMP-2+ and LR+ cells were in the minority (24/19%, 23/0%, respectively). At the early submucosal stage, TIMP-2+ cells moderately decreased in both histological types (49/49%), whereas a consistently higher number of both MMP-2+ and LR+ cells were detected only in the diffuse carcinomas (72%). In the advanced stage, the expression of TIMP-2 further declined (22/24%), although the other two antigens increased or maintained high levels of expression. AMG+ cells never exceeded 10% in either histological type at any stage. In the liver metastases, both MMP-2+ and LR+ cells were more numerous than in the primary tumor (P < 0.002 and P < 0.01). Patients who died from their primary tumor had higher percentages of LR+, MMP-2+, and AMG+ cells and lower percentages of TIMP-2+ cells with respect to survivors. We believe evaluation of ECM-related antigens, and especially TIMP-2, may help determine a confident prognosis for gastric cancer.

The activation of zymogen and the amount of proteinase and its inhibition are important in determining the eventual activity of matrix-degrading enzymes involved in tumor aggressiveness. To evaluate a gene complement leading to matrix metalloproteinase 2 (MMP-2; Mr 72,000 gelatinase) activity, membrane type 1 MMP (MT1-MMP), urokinase-type plasminogen activator, MMP-2, and tissue inhibitor of metalloproteinase 2 transcriptional levels were measured in gastric carcinoma biopsies. Comparative tumor:normal tissue reverse transcription-PCR in a cohort of 25 patients revealed up to a 10-fold difference in the expression of MT1-MMP, a metalloproteinase that has been proposed as a membrane receptor activator of MMP-2; a 1-unit increment resulted in a 30% risk to survival. A 20% risk also resulted from a 1-unit increment in the MT1-MMP: MMP-2 ratio, which showed differences of up to 15-fold. Instead, the expression of urokinase-type plasminogen activator, which trips off a cascade ending in the activation of MMP-2, as well as the expression of MMP-2 itself and its inhibitor, tissue inhibitor of metalloproteinase 2, lacked correlation with patient follow-up. Zymography revealed MMP-2 activities that were often in conflict with the transcription results and also with follow-up. The results suggest the evaluation of MT1-MMP and/or MT1-MMP:MMP-2 transcription as a new preoperative molecular-level prognostic factor for gastric carcinoma.

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.