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Background: Leukocytes plays crucial role in breast cancer progression and lymph node metastasis. Veins draining the breast through the axillary tributaries contain tumor-associated leukocytes migrating away from tumor microenvironment. Collecting tumor-associated leukocytes and characterizing their immunophenotype and biological properties in positive and negative lymph node patients may elucidate their role in lymph node metastasis. Aims: Using an innovative surgical approach previously described, we collected tumor-associated leukocytes from venous drainage of the breast during breast cancer surgery of positive and negative lymph node (LN)metastasis patients. Isolated leukocytes were subjected to: 1) immunophenotyping; 2) cytokine profiling; and 3) assessment of the expression level of total and phospho NF-kβ. Material and Methods: We enrolled 40 patients diagnosed with breast cancer from Ain Shams University Hospitals. Patients were sub-grouped into negative (zero) and positive (4) LN metastasis patients. All enrolled patients had not received neo-adjuvant chemotherapy. During modified radical mastectomy or conservative breast surgery, 15--20 ml of blood were collected from axillary vein tributaries for the isolation of tumor-associated leukocytes by Ficoll- Hypaque density gradient centrifugation. The immunophenotype of the isolated mononuclear cells was assessed using flow cytometry to identify the percentage of T lymphocytes (CD3+) and their subpopulations (CD4+ and CD8+), NK cells (CD56+) and B-lymphocytes (CD19+). Tumor-associated leukocytes isolated from positive and negative LN patients were seeded overnight in appropriate culture media at 37°C in a humidified CO2 incubator. Conditioned media containing secreted cytokines/chemokines were profiled using RayBio human cytokine antibody arrayIIIand then analyzed and quantified using ImageJ software. In addition, using immunoblotting we assessed the level of expression of total and phospho NF-kβ in leukocyte lysates from positive and negative LN patients. Result: Flow cytometric analysis revealed a significant increase in the percentage of T lymphocytes and T helper(CD3+/CD4+) cells collected from axillary tributaries of positive LN patients as compared to negative LN patients. Leukocytesisolated from positive LN patients were characterized by more than five-foldincreases in IL-12, Thrombopoietin, IL-13, INF-γ , TARC, IGF-1, IL-3, TNF-α, IL-4, GCSF, IL-5 and IL-1α. Moreover, we detected an increase in cellular expression of total and phospho NF-kβ in leukocytes isolated from positive LN as compared to negative LN breast cancer patients. Conclusion: Our results demonstrate for the first time that tumor-associated leukocytes of positive LN patients possess different cytokine patterns than those of negative LN patients. Moreover, we identified major cytokines that may contribute to LN metastasis and could be therapeutically targeted. [ABSTRACT FROM AUTHOR]

The skeleton is the most common site of metastasis in patients with advanced prostate cancer. Despite many advances in targeting skeletal metastases, the mechanisms behind the attraction of prostate cancer cells to the bone are not known. Osteoclast cathepsin K, due to its ability to effectively degrade bone matrix collagen I, has been implicated in colonization and growth of prostate tumours in the bone. Identification of new cathepsin K substrates in the bone microenvironment and the recent findings demonstrating its involvement in obesity and inflammation suggest additional roles for this enzyme in skeletal metastases of prostate cancer.

C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection.

Background: Cathepsin B (CB), a cysteine protease, is usually found in perinuclear lysosomes of epithelial cells of normal organs and non-malignant tumors, but is associated with the plasma membranes of many solid organ malignant tumors. Plasma membrane localized CB facilitates degradation of extracellular matrix proteins and progression of tumor cells from one biological compartment to another. The activities of CB and its subcellular distribution have not been investigated in malignant prostate. Our objective was to examine the subcellular distribution of CB by determining the activities of CB in lysosome and plasma membrane/endosome subcellular fractions and its subcellular localization by immunogold electron microscopy., Methods: Prostate tissue pieces obtained immediately after prostatectomy were homogenized and fractionated into subcellular components for determining biochemical activities of CB and cysteine protease inhibitors (CPIs). Distribution of CB was compared with that of prostate specific antigen (PSA, a serine protease), which is abundant in secretory vesicles and granules of normal prostate, benign prostatic hyperplasia (BPH) and malignant prostate cells. Localization of CB was investigated in resin embedded lysosomes and plasma membrane/endosome subcellular fractions and in prostate tissue sections by immunogold electron microscopy., Results: We have demonstrated the specificity of CB activity in human prostate homogenates by using a variety of inhibitors in our assay. We did not find any difference in the specific activity of CB based on protein or DNA content in homogenates of malignant prostate (Gleason histologic scores 5-7) and BPH (no histological evidence of cancer) whether it was measured by chromogenic or fluorogenic peptide substrate assay techniques. We found significantly higher activities of CB in the plasma membrane/endosome fractions of malignant prostate than in BPH. In contrast, CPI activity was increased relative to CB activity in plasma membrane/endosome fraction of BPH versus prostate cancer. Our data indicated a shift in the balance of enzyme to inhibitor that would favor increased activities of CB in prostate cancer. The immunogold microscopic study showed specific localization of CB in plasma membrane. They also showed localization of CB in lysosomes that were often adjacent to luminal and/or basal surfaces of malignant cells in contrast to the usual perinuclear distribution of lysosomes in hyperplastic prostate glands. PSA was localized in secretory granules and vesicles, including the plasma membranes and secretory blebs in malignant prostate cells. Occasional PSA positive secretory vesicles or membrane profiles were seen in the plasma membrane/endosomal and lysosomal fractions., Conclusions: The increased activity of CB in plasma membrane/endosomal fractions is associated with malignant prostate and not with BPH or normal prostate. Morphologic distribution CB is associated with the plasma membranes or lysosomes adjacent to apical and basal cell surfaces. This distribution is characteristic feature prostate cancer cells, but not in BPH or normal prostate cells. Subcellular distribution of PSA occurs in secretory vesicles and granules of the cytoplasm, but not in lysosomes. Our biochemical and morphological data could be used to distinguish malignant prostates from non-malignant tumors., (Copyright 2001 Wiley-Liss, Inc.)

Background: Cathepsin B (CB), a lysosomal cysteine protease, is involved in degradation of extracellular matrix proteins and progression of tumor cells from one biological compartment to another in many solid organ cancers, including prostate cancer. Our objective was to identify patterns of distribution of CB and its endogenous cellular inhibitor stefin A in cryostat sections of frozen BPH and prostate cancer tissue samples and to define these patterns in relation to Gleason histologic scores, clinical stages, and serum total PSA levels., Methods: We localized CB and stefin A in the same sections using polyclonal and monoclonal antibody immunoglobulin G (IgGs) against CB and stefin A using immunofluorescence and confocal microscopic techniques. Only cryostat sections of frozen prostates were used in localizations of CB and stefin A., Results: Benign prostatic hyperplasia (BPH) showed similar localization patterns for CB and stefin A and a ratio of 1 was indicated by CB = stefin A. Confocal studies indicated that most CB and stefin A sites in BPH glandular cells overlapped as shown by the yellow fluorescence of their co-localization. We found considerable variability in individual localization of CB and stefin A within and between Gleason histologic scores for prostate cancers. This variability was also found in Gleason score 6 tumors that are otherwise considered similar histologically and morphologically. Negative control sections did not show localization of CB by FITC, stefin A by Cy3 or yellow fluorescence for co-localization. Our analysis of the ratio of CB to stefin A showed three patterns, namely CB = stefin A, CB > stefin A, and CB < stefin A, within each Gleason score evaluated by us. Confocal microscopy showed more sites of yellow fluorescence when the ratio was CB = stefin A than those found in CB > stefin A or CB < stefin A. Statistical analyses showed prostate cancer cases with ratios of CB > stefin A (P < 0.05) and CB < stefin A (P < 0.05) significantly different from normal prostate and BPH which had ratios of CB = stefin A. Regression analysis did not show any specific relationship between the ratio of CB to stefin A and Gleason scores, clinical stages, and serum total prostate specific antigen (PSA) levels in prostate cancers. Analysis of our data indicates that the homeostatic balance between the enzyme and inhibitor was altered even in Gleason histologic score 6 tumors that are usually considered histologically similar by glandular differentiation., Conclusions: We have shown that prostate cancer is a heterogeneous tumor within each Gleason histological score regardless of the progression indicated by lower to higher Gleason score tumors. The ratio of CB > stefin A would indicate a preponderance of enzyme that would favor degradation of extracellular matrix proteins and progression of tumor cells in biological compartments. These tumors are expected to be aggressive prostate cancers. In contrast, prostate tumors showing ratios of CB < stefin A and CB = stefin A are expected to be less aggressive prostate cancers. This is the first report to define heterogeneity within any Gleason score for prostate cancers by the ratios of CB to stefin A., (Copyright 2001 Wiley-Liss, Inc.)

It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general natural inhibitor of cysteine proteinases, (82.8+/-1.6% inhibition of cell invasion), followed by the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). CLIK-148, a specific inhibitor of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.

Degradation of basement membrane is an essential step for tumor invasion. In order to study degradation in real time as well as localize the site of proteolysis, we have established an assay with living human cancer cells in which we image cleavage of quenched-fluorescent basement membrane type IV collagen (DQ-collagen IV). Accumulation of fluorescent products is imaged with a confocal microscope and localized by optically sectioning both the cells and the matrix on which they are growing. For the studies described here, we seeded U87 human glioma cells as either monolayers or spheroids on a 3-dimensional gelatin matrix in which DQ-collagen IV had been embedded. As early as 24 hours after plating as monolayers, U87 cells were present throughout the 3-dimensional matrix. Cells at all levels had accumulated fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Similar observations were made for U87 spheroids and the individual cells migrating from the spheroids into the gelatin matrix. Both the migrating cells and those within the spheroid contained fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Thus, glioma cells like breast cancer cells are able to degrade type IV collagen intracellularly, suggesting that this is an important pathway for matrix degradation.

Cathepsins B and S (CatB, CatS) are lysosomal cysteine proteases which, among other functions, appear to play a role in cancer progression in different tumor models due to their matrix-degrading properties. To investigate their possible involvement in the development of prostate carcinoma, we immunohistochemically analyzed CatB and CatS in 38 primary human prostatic adenocarcinomas, as well as concomitant high-grade prostatic intra-epithelial neoplasia, nodular hyperplasia and normal tissue. CatB expression was observed in 28 (74%) and CatS in 32 (84%) carcinomas, being concomitant in 24 cases (63%). High-grade intra-epithelial neoplasia expressed CatB in 20/23 cases (87%), and a similar result was obtained for CatS, with expression of both coinciding in 18 cases (78%). In non-neoplastic tissue, strong expression of both proteases was observed in macrophages, inflamed glands and transitional metaplasia, whereas atrophic glands and basal cells of normal glands displayed intense CatB positivity. We conclude that CatB and CatS are often expressed together in neoplastic prostatic cells from pre-invasive to invasive and clinically detectable stages, suggesting a putative role in local invasion, though other functions cannot be ruled out., (Copyright 2001 Wiley-Liss, Inc.)

Transfection of Rat1 fibroblasts with an activated form of rac1 (V12rac1) stimulated cell migration in vitro compared to transfection of Rat1 fibroblasts with vector only or with dominant negative rac1 (N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal assay to evaluate the ability of the Rat1 transfectants to degrade a quenched fluorescent protein substrate (DQ-green bovine serum albumin) embedded in a three-dimensional gelatin matrix. Cleavage of the substrate results in fluorescence, thus enabling one to image extracellular and intracellular proteolysis by living cells. The Rat1 transfectants accumulated degraded substrate intracellularly. V12rac1 increased accumulation of the fluorescent product in vesicles that also labeled with the lysosomal marker LysoTracker. Treatment of the V12rac1-transfected cells with membrane-permeable inhibitors of lysosomal cysteine proteases and a membrane-permeable selective inhibitor of the cysteine protease cathepsin B significantly reduced intracellular accumulation of degraded substrate, indicating that degradation occurred intracellularly. V12rac1 stimulated uptake of dextran 70 (a marker of macropinocytosis) and polystyrene beads (markers of phagocytosis) into vesicles that also labeled for cathepsin B. Thus, stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 may be responsible for the increased internalization and subsequent degradation of extracellular proteins., (Copyright 2000 Academic Press.)

Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549) through the use of quenched-fluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin B-selective cysteine protease inhibitor, intracellular fluorescence was decreased approximately 90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence approximately 50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1) a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2) the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.

Cathepsin B and in particular cell-surface and secreted cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for cathepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Intact human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a Km of 460 microM at pH 7.0 and 37 degrees C. This is nearly the same as the Km of 430 microM obtained with purified cathepsin B assayed under the same conditions. The pericellular (i.e. both cell-surface and released) cathepsin B activity was inhibited by the cysteine protease inhibitors E-64, leupeptin, Mu-Np2-HphVS-2Np, Mu-Leu-HpHVSPh and the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver cathepsin B. Other human cancer cell lines with measurable pericellular cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlated with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.