High Cathepsin Research Articles

Clinicopathological Significance of Cathepsin D Expression in Gastric Adenocarcinoma

Objective: An estrogen-regulated lysosomal protease, cathepsin D, has been detected in a variety of tissues. This proteinase has been described as closely associated with tumor progression and metastasis in malignant tumors. The purpose of this study was to determine the clinicopathological and prognostic significance of cathepsin D expression in gastric adenocarcinoma. Methods: In a consecutive series of 478 patients with gastric carcinoma (median follow-up period: 93 months, range: 1–285 months), cathepsin D expression in tumors was quantitatively analyzed with immunohistochemistry using a monoclonal antibody against cathepsin D (clone: 1C11). The percentage of cathepsin-D-positive cancer cells (the CD index) was calculated. In addition, the amount of cathepsin-D-positive stromal cells was evaluated; three grades (high, intermediate, and low) were used for the classification. Results: The mean CD index of 478 tumors was 12.8% (range: 0–100%, median: 8%). The mean CD index of diffuse-type gastric carcinomas (14.9%) was significantly higher than that of intestinal-type carcinomas (10.1%, p < 0.0001). Cathepsin D expression of cancer cells was significantly associated with the depth of tumor invasion in both types. The percentage of tumors with high cathepsin D expression in stromal cells was significantly higher in well-differentiated tumors (25.5%) than in moderately differentiated (12.8%) or in poorly differentiated tumors (19.1%). Cathepsin D expression of stromal cells was significantly associated with the depth of tumor invasion in the intestinal type, in contrast to the diffuse type. Highly expressed cathepsin D in cancer cells was associated with a poor prognosis in both types of carcinoma, but in stromal cells highly expressed cathepsin D was associated to a poor prognosis in the intestinal type only. Conclusion: These results indicate that cathepsin D expression in cancer cells may play an important role in tumor progression in diffuse-type gastric carcinoma, whereas in the intestinal type of carcinoma, cathepsin D expression in stromal cells may play an important role in tumor progression.

Cathepsins B and L are markers for clinically invasive types of meningiomas.

Meningiomas are benign neoplasms that derive from coverings of the brain. Approximately 10% of benign tumors progress into atypical, malignant tumors, thus constituting a subset of histopathologically benign tumors that are clinically invasive. The aim of this study was to evaluate cathepsins B and L and their inhibitors as new prognostic factors that could distinguish malignant from benign forms of meningiomas. Using immunohistochemical analysis and specific monoclonal antibodies, we evaluated the levels of cathepsins B and L and the levels of the endogenous cysteine proteinase inhibitors stefin A and cystatin C in 88 meningiomas. Immunohistochemical scores were determined as the sum of the frequency (0-3) and intensity (0-3) of immunolabeling of the tumor cells. Of the 88 tumors studied, 67 were benign meningiomas and 21 were atypical meningiomas. Among the benign group, nine tumors had certain features of malignancy. These tumors were classified as border benign meningiomas, and the rest were classified as clear benign meningiomas. A high immunohistochemical score (4-6) for cathepsin B was more frequent in atypical tumors than in clear benign tumors (P < 0.001). Compared with clear benign tumors, higher cathepsin B immunohistochemical scores were found in atypical tumors (P < 0.001) and border benign tumors (P < 0.03). No statistical difference in immunohistochemical staining of cathepsin B was found between atypical meningiomas and border benign meningiomas. Higher expression of cathepsin L was found in atypical tumors as compared with clear benign tumors (P < 0.03), but it was not observed in border benign as compared with clear benign meningiomas. No immunostaining for stefin A and cystatin C was detected in any of the tumors. We show that the levels of cathepsin B and cathepsin L antigens are significantly higher in invasive types of benign meningioma. Specifically, cathepsin B may be used as a diagnostic marker to distinguish histomorphologically benign but invasive meningiomas from histomorphologically clear benign tumors.

Cathepsin L activity and its inhibitor in human otitis media.

We examined cathepsin L activity, expression of cystatin A, and copper- and zinc-containing superoxide dismutase in human chronic otitis media. The relationships of our findings to clinical findings (e.g., grade of bone destruction) were also studied. Retrospective basic and clinical study. Department of Otolaryngology and First Department of Biochemistry, Kinki University School of Medicine, Osaka, Japan. The human middle ear tissues evaluated in this study were surgically obtained from seven patients with cholesteatoma epithelium, three patients with granulation tissues in cholesteatoma, three patients with granulation tissues in noncholesteatoma, and three patients with intact mucous membrane of the middle ear. Cathepsin L activities in cholesteatoma epithelium, granulation tissues in cholesteatoma, or granulation tissues in noncholesteatoma were measured using Barrett's method. Cystatin A expressions were observed by Western blot analysis. Copper- and zinc-containing superoxide dismutase in cholesteatoma was examined immunohistochemically. Mean cathepsin L activity was higher in diseased tissues than in intact mucous membranes of the middle ear. Granulation tissues with high cathepsin L activity resulted in extensive bone destruction in both cholesteatomas and noncholesteatomas of the middle ear. All cases with intact mucous membrane of the middle ear exhibited no expression of cystatin A. Seven of 10 cases with diseased tissues expressed cystatin A in cholesteatoma epithelium, granulation tissues in cholesteatoma, or granulation tissues in noncholesteatoma. No relationships were found between cystatin A expression and grade of cathepsin L activity. Copper- and zinc-containing superoxide dismutase was more strongly positive in cholesteatoma epithelium regions than in granulation tissues. These results suggest that copper- and zinc-containing superoxide dismutase in cholesteatoma epithelium prevents complications by suppressing cathepsin L activity.

Cystatin C and cathepsin B in CSF from patients with inflammatory neurologic diseases.

In CSF, proteolytic enzymes are believed to have crucial roles in the initiation and progression of inflammatory neurologic diseases (IND). Cystatin C, a major cysteine protease inhibitor in CSF, is tightly bound to cathepsin B and H. To determine if cystatin C is involved in the disease process of IND, the authors measured the cystatin C concentration by ELISA method and cathepsin B and H activities in the CSF of patients with acute IND. Cystatin C concentration and cathepsin B and H activities were measured in CSF samples taken from patients during the acute phase of their disease. Subjects studied were 8 patients with Guillain-Barré syndrome (GBS), 5 with chronic inflammatory demyelinating polyneuropathy (CIDP), 12 with MS, 16 with aseptic meningitis, 15 with neurodegenerative diseases as disease controls, and 35 healthy controls. A significant decrease in CSF cystatin C level was seen in the patients with GBS, CIDP, and MS compared to the control subjects. High cathepsin B activity, but not cathepsin H activity, was also observed in the patients with GBS, CIDP, and MS. Cystatin C levels in CSF measured by ELISA may help the physician recognize GBS, CIDP, and MS. Decreased levels of cystatin C may be related to the high levels of cathepsin B activity seen in the CSF of patients with GBS, CIDP, and MS.

ORIGINAL ARTICLE H-ras and Nm23-H1 Gene Expression and Proteolytic Activity in Squamous Cell Carcinoma of the Uterine Cervix

Background The invasive and metastatic potential of malignant cells results from complex interactions of numerous factors not yet fully understood. Genomic alterations such as ras overexpression and nm23-H1 inhibition have been found to be frequently associated with increased invasiveness in various cancers. On the other hand, secretion of different proteinases are necessary for malignant cells to traverse a network of matrix macromolecules, but the relationship between the genomic alterations and the proteolytic phenotype is still unclear. Our aim was to investigate whether the appearance of the proteolytic phenotype had any correlation with the expression of H-ras and nm23-H1 genes in carcinoma of the uterine cervix. Methods Twenty-five samples from patients with carcinoma of the uterine cervix at different clinical stages were studied. Cathepsin B1, plasminogen activator, and collagenase activity were assessed in tissue cytosols using specific synthetic oligopeptides as substrates. The expression of H-ras and nm23-H1 was investigated by means of immunohistochemistry and in situ hybridization. Results Our results showed that cathepsin B1 was the most consistently elevated proteinase, demonstrating a linear correlation with clinical staging. H-ras expression was found elevated in 40% of the cases. Nm23-H1 protein immunoreactivity was positive in 40% of the cases. No correlation was found among H-ras, cathepsin B1 activity, and survival rate. Among cases with high cysteine proteinase activity, a different clinical behavior depending on the expression of Nm23-H1 was observed. The cases with Nm23-H1 protein had a markedly better survival rate than those lacking this protein. In contrast, the absence of Nm23-H1 in association with high cathepsin B1 activity was a clear indicator of a poor prognosis. Conclusions These findings suggest a complex interaction between the proteolytic phenotype and the expression of H-ras and nm23-H1 genes in carcinoma of the cervix that influences the clinical behavior of the tumor.

Cathepsin B in infiltrated lymph nodes is of prognostic significance for patients with nonsmall cell lung carcinoma

Tumor cells require specific proteolytic enzymes for invasion and metastasis, including lysosomal peptidases--cathepsins. Cathepsin B is a lysosomal cysteine peptidase, which appears to play a major role in invasion and metastasis of human tumors. In this study, the authors focused on the possible role of cathepsin B in lymphogenic metastasis by investigating the enzyme localization and its activity in lung tumors and corresponding tumor-infiltrated lymph nodes. Cathepsin B activity was determined in lung tumors, lung parenchyma, and tumor cell-infiltrated and noninfiltrated regional lymph nodes of the same patient. The authors investigated 35 cancer patients suffering from nonsmall cell lung carcinoma. Cathepsin B throughout activity was measured by cleavage of the fluorogenic substrate Z-Arg-Arg-AMC at pH 6.0. The median specific cathepsin B activity was highest in tumors, followed by the infiltrated lymph nodes, noninfiltrated lymph nodes, and lung parenchyma. The authors showed a significant 1.8-fold increase in cathepsin B activity in tumor-infiltrated lymph nodes compared with noninfiltrated regional lymph nodes and a 4.5-fold increase in lung tumor tissue compared with lung parenchyma. High cathepsin B activity, both in tumors and tumor cell-infiltrated lymph nodes, indicated poor prognosis for overall survival. Immunohistochemical analysis showed the presence of cathepsin B in histiocytes and tumor cells but not in lymphocytes of lymph node tissue. The authors' findings on higher cathepsin B levels in tumor cell-infiltrated lymph nodes show that increased level of cathepsin B activity is characteristic of the invasive tumor cell phenotype. This corroborates the hypothesis, that tumor cell associated cathepsin B may play a role in lymphogenic metastasis. The authors' results support the use of lymph node associated cathepsin B as a prognostic factor for survival of patients with lung carcinoma.

慢性中耳炎のカテプシンＬとシスタチンＡ

We examined the cathepsin L activity and expression of cystatin A (an inhibitor of cathepsin L) in 7 cases of cholesteatoma epithelia, 3 cases of cholesteatoma subepithelial granulation, 3 cases of non-cholesteatoma granulation and 3 cases with an intact mucous membrane in the middle ear. The mean cathepsin L activity was higher in the diseased tissues than in the intact mucous membrane of the middle ear. Those granulation tissues with high cathepsin L activity resulted in extensive bony destruction in both cholesteatomas and non choleateatomas of the middle ear. Cystatin A expression was assessed by Western blotting. All of the cases with an intact mucous membrane in the middle ear exhibited no expression of cystatin A. 7 of the 10 cases with diseased tissues expressed cystatin A in their cholesteatoma epithelia, cholesteatoma subepitheial granulation or non cholesteatoma granulation tissues. No relationship between cystatin A expression and the level of cathepsin L activity could be observed.

Cloning of a major human retinal pigment epithelial lysosomal aspartic protease and mapping its transcriptional start sites.

It has been proposed that the major proteolytic enzyme responsible for the proteolysis of photoreceptor outer segments is an aspartic protease similar or identical to cathepsin D. The aim of this study was to determine if the major retinal pigment epithelial (RPE) aspartic protease was cathepsin D, to study its distribution and investigate its transcription start sites (TSS). An RPE cDNA library was screened at low stringency for the presence of aspartic proteases. DNA sequencing was used to analyze the identified clones. The expression of cathepsin D mRNA was analyzed by Northern blot while immunohistochemistry was used to demonstrate cathepsin D related immunoreactivity in an eye section. RNase protection assay was used to map the Cathepsin D TSS in RPE cells. The aspartic protease identified in the RPE cDNA library was identical to the DNA sequence of cathepsin D found in other tissues. Northern blot analysis of a wide range of cell types showed that the highest level of cathepsin D mRNA expression was found in RPE cells which was similar only to cathepsin D expression in MCF7 breast cancer cells. Immunohistochemical staining of a human retina confirmed the inherent nature of high cathepsin D expression in RPE cells. An RNase protection assay demonstrated two major cathepsin D TSS in RPE cells. One of them was identical to the TSS responsible for the constitutive expression of cathepsin D and the other, a TATA box-controlled TSS, was identical to a TSS found in MCF7 cells. Cathepsin D expression was not enhanced by the phagocytosis and digestion of rod outer segments (ROS) and ROS phagocytosis did not induce the use of other cathepsin D TSS in the RPE cells. The major aspartic protease identified in RPE cells was cathepsin D. In addition, it was demonstrated that the high level of cathepsin D expression in RPE cells is the intrinsic nature of these cells and that it is linked to a TATA box-controlled TSS. This TSS has previously been described as an estrogen regulated TSS and further studies are required to identify the RPE-specific inducer or indirect factors that may be responsible for the enhanced expression of cathepsin D in the RPE cells.

Study of the frequencies of CYP1A1 gene polymorphisms and glutathione S-transferase mu1 gene in primary breast cancers: an update with an additional 114 cases

We studied the polymorphisms m1 ( Msp1 restriction site) and m2 (codon Va1 substitution) of CYP1A1 gene and the copy number of glutathione S-transferase mu1 ( GSTM1) gene on 487 DNA of breast cancer primary tumours from Caucasian group. Tumours of patients aged 55 years and under at diagnosis presented a great proportion of wild m1 (-/-) genotype; 83.6% vs. 69.5% ( p < 0.0006), and a higher percentage of copy number of GSTM1 equal or under one copy; 65.2% vs. 53.4% ( p < 0.011) for older patients. m1 and m2 variants are closely linked ( p < 0.01) and high Cathepsin D concentrations ( p < 0.02). The combinations of different genotypes showed that association wild m1 (-/-) genotype and copy number of GSTM1 inferior or equal to one copy is correlated with an early onset of breast cancer primary tumour 44% vs. 6.4% for m1 (-/+) or (+/+) genotype and copy number of GSTM1 superior to one ( p < 0.0000). The CYP1A1 gene wild form seems to be associated with an early cancer development in Caucasian patients.

Elevations in Cathepsin B Protein Content and Enzyme Activity Occur Independently of Glycosylation during Colorectal Tumor Progression

Western blots, enzyme assays, protein glycosylation studies, and immunohistochemical staining were used to characterize cathepsin B expression at successive stages of colorectal tumor progression. In normal colon mucosa and premalignant adenomas, cathepsin B expression was predominantly due to mature two-chain protein detected on Western blots as the nonglycosylated 27-kDa form, with overexpression of this protein occurring in only 4 of 18 adenomas. Overexpression increased significantly in Dukes A and B carcinomas (26 of 37 cases), with cathepsin B protein generally detectable in carcinomas as a combination of both 27-kDa nonglycosylated and 28-kDa glycosylated mature two-chain forms. Glycosylated cathepsin B protein in carcinoma extracts was sensitive to PNGase F but resistant to Endo H, indicating a pattern consistent with complex rather than high mannose type glycosylation. When sorted by advancing tumor stage, peak expression of cathepsin B protein occurred in carcinomas involved in local invasion compared with adenomas or metastatic cancers. At all stages, cathepsin B activity correlated significantly with the levels of heavy chain mature cathepsin B protein (r = 0.6682, p < 0.0001) irrespective of glycosylation. Immunohistochemical staining of cathepsin B protein revealed fine diffuse cytoplasmic staining in both adenomas and carcinomas compared with coarse granular cytoplasmic staining (typical of lysosomes) seen in matched normal mucosa. Our results demonstrate several sequential, apparently independent changes in cathepsin B expression during colorectal tumor progression including early changes in subcellular localization, up-regulation of cathepsin B protein and activity in invasive cancers, and altered protein glycosylation detected in malignant tumors at all stages.

Lack of prognostic value of cathepsin D levels for predicting short term outcomes of breast cancer patients.

The value of cathepsin D determinations done on tumor cytosols in evaluating the prognosis of breast cancer patients has been debated in the literature. Our previous work suggested that cathepsin D determinations were not of prognostic value, but in that study we used immunoblotting and immunohistochemical methods rather than the more widely used double antibody immunoradiometric (IRMA) assay for measuring cathepsin levels. Here we report our results determining cathepsin D using components of a commercially available IRMA system on a large patient sample (n = 1984). Reagents from a commercially available IRMA kit were used to analyze cathepsin D levels in the cytosols of 1984 patients with breast cancer. All patients had invasive breast cancer with known tumor size and with some axillary nodes pathologically examined. Only patients with T1 and T2 tumor sizes were included. Median follow-up was 37 months. The hypothesis that high cathepsin D levels correlated with poorer outcome (poorer DFS or OS) was not confirmed, either in all patients, or in node-positive or node-negative subsets. Only in patients treated with adjuvant therapy were higher cathepsin D levels correlated with negative outcome (worsened OS, but not DFS), although given the large number of subsets analyzed this correlation may be spurious. Multivariate analyses using interaction terms did not support the concept that high cathepsin D levels correlate with resistance to adjuvant therapy. In this study evaluating the value of cathepsin D using components from a kit widely used for measuring cathepsin D levels, we conclude that cathepsin D is of doubtful value in predicting risk of early relapse or death for patients with newly diagnosed invasive breast cancer.

Cathepsin A activity of normal bovine ocular tissues and pathological human intraocular fluids.

Cathepsin A activity assayed with N-Cbz-Phe-Ala, N-Cbz-Glu-Tyr and N-Cbz-Glu-Phe as substrates, was measured in fresh corneas, lenses, aqueous humor, vitreous humor and choroid plus retinal pigment epithelium taken from normal bovine eye balls and in human intraocular fluids from the eye balls in various ocular diseases (cataract, glaucoma, diabetes, intraocular tumors). Cathepsin A exhibited a pH optimum at 5.0 and showed the highest specificity towards N-Cbz-Phe-Ala as a substrate. In bovine ocular tissues high cathepsin A activity was found in the choroid plus retinal pigment epithelium and in cornea. The lens and the vitreous humor showed low enzyme activity and the aqueous humor none at all. In the human aqueous humor of the eye with cataract cathepsin A activity was more than three times higher then in the eye with choroid tumor. In human vitreous humor in absolute glaucoma the activity was twice as high as in melanoma and almost three times higher than in the case of lung metastatic tumor. Diabetes in glaucoma increased seven fold cathepsin A activity in the vitreous humor.

A possible application of cathepsin B activity determination for estimating the spread of the cervix uteri carcinoma.

The value of cathepsin B activity determination for evaluation of the extent of disease was investigated in 98 patients with the cervix uteri carcinoma and 25 women with cervix uteri dysplasia. The measurements were performed before treatment. Cathepsin B activity was estimated in serum using Z-Phe-Arg-NMec, and in tumor tissue using Z-Arg-Arg-pNA . HCl as substrates. The mean activity of the enzyme increased both in serum and tumor tissue with progression of neoplastic disease and was dependent on the clinical stage of cervical carcinoma. It should be stressed, however, that among the patients with the clinically observed early stage of the disease, higher cathepsin B activity was observed in those in whom metastases to pelvic lymph nodes were detected than in those in whom the disease was limited to cervix uteri.

Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha.

A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon-gamma- and Fas/APO-1-induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF-alpha-induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady-state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well-known endoprotease plays an active role in cytokine-induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.

Cathepsin A Deficiency in Galactosialidosis: Studies of Patients and Carriers in 16 Families

Deficiency of lysosomal protective protein/cathepsin A in humans is the primary cause of galactosialidosis, a lysosomal storage disease characterized by combined deficiency of beta-galactosidase and neuraminidase. We have investigated 20 galactosialidosis patients and nine of their obligate heterozygous parents. A group of 12 patients with the early infantile type of the disease exhibited practically complete absence of cathepsin A activity, whereas eight patients with either the late infantile or the juvenile/adult type had 2-5% residual activity. Highest levels (5%) were present in two patients with milder clinical manifestations and later onset of the disease. In most fibroblast strains, beta-galactosidase activity was 10-15% of normal levels, whereas neuraminidase was reduced to less than 4%. Interestingly, a substantial residual activity (10%) of the latter enzyme was detected in the patient with the mildest phenotype and the highest cathepsin A activity. Heterozygous values for cathepsin A were reduced on average to half of normal levels. However, in two cell strains, the activity was far below control range, and in these cases, neuraminidase activity was severely depressed. Finally, we showed that cathepsin A had considerable activity in chorionic villi and amniocytes, but was deficient in amniocytes from a pregnancy with an affected fetus, indicating the relevance of cathepsin A assay for prenatal diagnosis of galactosialidosis.

Endogenous Proteolytic Enzymes in Chicken Muscles. Differences Among Strains with Different Growth Rates and Protein Efficiencies

The theory that net muscle growth is, at least partly, regulated by catabolic factors has been tested in order to set up an animal model to study meat aging and post-mortem tenderization. Male and female chickens of a layer strain (White Leghorn), a commercial broiler strain (Ross), and two experimental broiler lines (designated GL and FC) were used to estimate differences in proteolytic enzyme activities in the breast muscles. The GL and the FC lines were selected for high body weight gain and high feed efficiency, respectively. At 6 wk of age the birds were slaughtered and the activities of endogenous proteinases and their specific inhibitors in breast muscles measured.The Leghorns showed significant differences in all traits compared with the three broiler genotypes. Within the broiler types, FC birds tended in the direction of the Leghorns and GL birds in the opposite direction. Ross birds were intermediate between FC and GL birds. All types and sexes differed significantly in slaughtering weight. Feed conversion ratio and protein conversion ratio were highest for Leghorns. The FC birds showed the lowest feed conversion. Ross and GL birds showed intermediate values. The Leghorns showed higher calpain activities and lower calpastatin activity than the three broiler genotypes. The FC broilers showed intermediate calpain and calpastatin activities but higher cathepsin H and total cystatin values. The GL broilers showed lower cathepsin B, D, and H activities. In all cases the Ross broilers showed intermediate values.From these figures it is concluded that the strains of birds used in this study can be used as a natural source of variability to study the mechanisms involved in post-mortem proteolytic degradation and thus in the study of muscle tenderization and meat aging. It is also concluded that it could be very interesting to study the behavior of the different proteolytic systems more carefully in relation to muscular growth characteristics and compare them to anabolic factors involved in muscle growth.

Study on Cathepsin B Activity in Human Thyroid Tumors

Cathepsin B activity was measured in human thyroid tissue obtained surgically from 2 patients with Grave's disease, 3 with follicular adenoma, 4 with papillary carcinoma, and 4 with follicular carcinoma. Three normal thyroid tissues were also studied. Comparisons were made between cathepsin B activity and clinical findings, including histopathological diagnosis and the presence or absence of extra-capsular invasion and metastasis. The abilities of original tumors to degrade type I and type IV collagen were also measured. Mean cathepsin B activities of both specimens with benign and those with malignant disease were significantly higher than those of normal thyroid. On cases of thyroid carcinoma, those with extra-capsular invasions and metastasis had the highest cathepsin B activities. Cases with high cathepsin B activities also tended to show high type I and IV collagen degrading abilities. These findings suggest that cathepsin B plays a role in the development of extra-capsular invasion and lymph node metastasis in human thyroid tumors.

Stromal cell cathepsin D expression and long-term survival in breast cancer.

Breast cancers with an increased level of cathepsin D in tumour tissue extract have been found to have poor prognosis, but studies performed with immunohistochemistry have produced variable results. We analysed 213 primary invasive breast cancers for cathepsin D expression from archival tissue with immunohistochemistry. The minimum follow-up of the patients still alive was 26 years. Women with ductal cancer that lacked cathepsin D expression in stromal macrophage-like cells had a 75% 5 year and 55% 30 year survival rate as compared with only a 40% 5 year and 20% 30 year survival rate if stromal cells expressed cathepsin D (P = 0.0003), whereas cathepsin D expression of cancer cells was associated with neither survival nor the several prognostic factors investigated. Stromal cell cathepsin D was more often present in the ductal than in the lobular histological type (80% vs 54%, P = 0.002), and its expression was strongly associated particularly with a high cell proliferation rate. However, in a multivariate analysis stromal cell cathepsin D expression did not have independent influence on survival in the entire series. We conclude that high stromal cell cathepsin D expression is associated with a poor short- and long-term outcome in breast cancer.

Prognostische Bedeutung von Kathepsin D beim primären Mammakarzinom

The prognostic significance of the lysosomal protease cathepsin D in breast cancer was evaluated in a retrospective study. Cathepsin D was measured in 346 deep-frozen (-70 degrees C) cytosol specimens of primary breast carcinomas (1982-1990). Among the established prognostic factors, only axillary lymph node involvement correlated with the expression of cathepsin D (> 40/60 pmol/mg) (p = 0.04-0.05). Univariate analyses of disease-free survival (DFS) and overall survival (OS) showed, that the expression of cathepsin D had no effect on the prognosis either in the whole population of breast cancer patients during long-term follow-up (n = 302; median observation time 51 months) or in the group of women with positive lymph nodes (n = 157; 46 months). However, within the group of N0 patients (n = 145; 57 months), high cathepsin D levels were associated with an unfavourable OS, but this relationship was statistically insignificant (p = 0.08-0.13). A similar influence of cathepsin D on DFS could not be demonstrated. Compared to tumour size, grading and receptor status in multivariate analysis, cathepsin D was a more indicative, but finally insignificant prognostic factor for OS. According to these results, cathepsin D may contribute only in combination with other prognostic factors to identify those 20-30% of node-negative patients with unfavourable prognosis, who may benefit from adjuvant therapy.

Characterization of very acidic phagosomes in breast cancer cells and their association with invasion

Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 microns) in which endocytosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcourrier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epifluorescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 microns diameter latex beads and fluorescence Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are heterophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H(+)-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (< 4), lower than the lysosomal value (pH approximately 5) in the same cells and in specialized phagocytotic cells such as macrophages. We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.