组织蛋白酶D和nm23基因蛋白在乳腺癌中的表达及与淋巴结转移的关系

采用免疫组织化学 ABC法检测了 43例乳腺癌中的组织蛋白酶 D和 nm 2 3基因蛋白的表达。结果显示组织蛋白酶D在乳腺癌组织中表达阳性率为 76 .7% (33/43)。 43例乳腺癌中 2 5例伴有淋巴结转移 ,18例无淋巴结转移 ,其组织蛋白酶D表达阳性率分别为 92 % (2 3/2 5 )及 5 5 .6 % (10 /18) ,两者之间具有显著性差异 (P<0 .0 1)。 nm 2 3基因蛋白在 43例乳腺癌中阳性率为 72 .1% (31/43)。在有淋巴结转移和无淋巴结转移的乳腺癌中 nm2 3基因蛋白阳性率分别为 6 0 % (15 /2 5 )及80 .9% (16 /18) ,nm2 3基因蛋白的表达与淋巴结转移呈显著负相关 (P<0 .0 5 )。结果提示对乳腺癌根治术后无淋巴结转移 ,但组织蛋白酶 D表达阳性 ,nm2 3基因蛋白表达阴性的患者 ,可能具有潜在的复发和转移倾向 ,应多加关注 ,并密切随访。     

Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound

The cysteine protease cathepsin B has been causally linked to progression and metastasis of breast cancers. We demonstrate inhibition by a dipeptidyl nitrile inhibitor (compound 1) of cathepsin B activity and also of pericellular degradation of dye-quenched collagen IV by living breast cancer cells. To image, localize and quantify collagen IV degradation in real-time we used 3D pathomimetic breast cancer models designed to mimic the in vivo microenvironment of breast cancers. We further report the synthesis and characterization of a caged version of compound 1, [Ru(bpy)₂(1)₂](BF₄)₂ (compound 2), which can be photoactivated with visible light. Upon light activation, compound 2, like compound 1, inhibited cathepsin B activity and pericellular collagen IV degradation by the 3D pathomimetic models of living breast cancer cells, without causing toxicity. We suggest that caged inhibitor 2 is a prototype for cathepsin B inhibitors that can control both the site and timing of inhibition in cancer.     

Mechanism of the overexpression of the cathepsin D gene in breast cancer and consequences in the metastatic process]

Cathepsin D, an acidic protease normally acting in lysosomes, is overproduced both in vitro and in vivo in most breast cancer cells. The mechanism of gene regulation by estrogens and the biological and clinical significance of this overexpression in metastasis are reviewed. In MCF7 cells, cathepsin D is specifically and directly induced by estrogens and also induced by growth factors (EGF, IGF-I and bFGF), but this induction is dependent upon de novo protein synthesis. The mechanism of estrogen induction involves EREs located upstream from the gene. Our laboratory cloned the promoter region (-4kb) of cathepsin D of MCF7 cells and found EREs located in the proximal 5' region of the gene. In MDA-MB231 and BT20 cells, cathepsin D is overexpressed but not regulated by estrogens. Total cathepsin D concentration were assayed by IRMA in breast cancer cytosol routinely prepared for receptor assays. Several retrospective clinical studies indicate a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and further development of clinical metastasis. High cathepsin D concentration in the primary tumor may be either a consequence, or more likely a cause, of metastasis. Transfection experiments using cDNA-cathepsin D in rat tumoral cells facilitates their metastatic potential in nude mice (Garcia et al., Oncogene, 1990, 5, 1809-1814). The mechanism of cathepsin D action in facilitating metastasis is unknown and may involve proteolytic activity in an acidic compartment, and/or interaction with the Man 6P/IGFII receptor.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Pepstatin, an inhibitor of acid kininogenases and ascites retardant in neoplastic disease.

Evidence is reviewed that pepstatin, an inhibitor of acid kininogenases such as cathepsin D, may be an effective therapeutic agent in retarding ascites accumulation in certain cancers. The evidence for this conclusion is based on the actions of pepstatin in retarding ascites in six different tumor strains inoculated into various species of mice, as well as the demonstration that cathepsin D activity is reduced in vivo in several organs following pepstatin administration. The latter is significant since we have postulated that ascites formation, in good part, is due to leukokinin formation, which is catalyzed by cellular-released cathepsin D.     

Reversal of cathepsin D routing modulation in MCF-7 breast cancer cells expressing antisense insulin-like growth factor II (IGF-II).

We previously demonstrated that expression of IGF-II modulates the routing of cathepsin D in MCF-7 cells. In our present study, we transfected antisense IGF-II into IGF-II secreting MCF-7 cells to test the hypothesis that blocking IGF-II may reduce the secretion of cathepsin D in breast cancer cells. The concentration of IGF-II in media conditioned by the antisense clone was reduced to almost undetectable levels. Likewise, Northern blotting analysis revealed that IGF-II mRNA was nearly undetectable in the antisense transfected cells. Metabolic labeling experiments performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes demonstrated that cathepsin D secretion was dramatically reduced. Similarly, a significant reduction in cathepsin D was observed when conditioned media and cell extracts were examined by Western blotting after a 48 h incubation. No changes in cathepsin D mRNA in antisense cells were detected by Northern blot analysis. We conclude that endogenous IGF-II may modulate the routing of cathepsin D by interfering with receptor trafficking in MCF-7 cells, and that this modulation is reversible. Abnormally high levels of IGF-II may alter this homeostasis, conferring on breast cancer cells an advantageous mechanism that promotes rapid growth, and may facilitate metastasis.     

Cathepsin D in breast cancer tissue

The cathepsin D concentration in 18 women with benign breast pathology has a cut-off value of 43 pmol/mg of protein. High values have been detected in two cases of chronic mastitis. These high values of cathepsin D were found in a study of 62 patients suffering from breast cancer and are independent of the hormone dependent state of the tumour. The cathepsin D concentration may have a prognostic function in breast cancer determination, as high concentrations are found in combination with other prognostic factors such as clinical stage, size of the tumour, state of the axillary lymph nodes and in the histological differentiation grade, where from a statistical point of view, the combination is important.     

Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells.

A cDNA library was constructed from the mRNA of the ZR-75 oestrogen responsive human breast cancer cell line and screened for oestrogen regulated mRNA sequences. Of the recombinants isolated, 30 contained cDNA that corresponded to a single mRNA species of 2.1 kb that was induced between 10 and 15 fold by oestradiol treatment. The sequence of the entire open reading frame and 3' non-coding region of the mRNA was determined and shown to encode the aspartyl protease cathepsin D. The induction of cathepsin D mRNA is specific for oestrogen and is maximal at 3 X 10(-10) M. Cathepsin D mRNA levels were increased by oestrogen in 3 oestrogen responsive breast cancer cell lines. Cathepsin D mRNA was expressed but not regulated in an oestrogen receptor negative breast tumour cell line, BT 20, and in 2 other malignant cell lines, Hela and A431.     

Clinical significance of cathepsin D concentration in tumor cytosol of primary breast cancer

BACKGROUND: Cathepsin D is the proteolytic enzyme most frequently implicated as a prognostic factor in primary breast cancer. In the present study we evaluated by means of an immunoradiometric assay the tumor content of this protease in primary breast cancer, its relationship with tumor-related clinical and pathological parameters, and its prognostic significance in a large series of breast cancer patients. METHOD: The study comprised 1033 women with histologically established invasive breast cancer. Cathepsin D was measured in cytosol samples by means of an immunoradiometric assay to determine the total amount of cathepsin D (52 kDa, 48 kDa and 34 kDa). Evaluation of relapse-free survival and cause-specific survival was performed in the group of 1003 patients without evidence of metastasis at the time of initial diagnosis. The median follow-up of the patients who were free of recurrence was 54 months. RESULTS: Cathepsin D levels showed a wide range among the studied tumors (n = 1033; median (range) 41 (0.9-2504) pmol/mg protein). Statistical analysis showed that the median cathepsin D levels were considerably higher in large tumors (T2-4) than in smaller ones (T1) (p = 0.017), as well as in node-positive than in node-negative tumors (p = 0.004). Cathepsin D levels were also higher in ductal tumors than in the other histological types (p = 0.001), as well as in moderately or poorly differentiated tumors (p < 0.001). Likewise, the median value of the protease was significantly higher in ER or PgR-positive tumors than in hormone receptor-negative ones (p = 0.011 and p = 0.004, respectively), as well as in aneuploid tumors than in diploid tumors (p = 0.029). Multivariate analysis demonstrated that elevated cathepsin D levels (> 59 pmol/mg protein) were notably associated with a shorter cause-specific survival in the whole group of patients with breast cancer, as well as in the subgroup of node-positive patients (p < 0.05). CONCLUSIONS: This study suggests that elevated intratumoral cathepsin D levels may identify a subset of node-positive breast cancer patients showing a high probability of earlier death.     

Interaction of human breast fibroblasts with collagen I increases secretion of procathepsin B

Interactions of stromal and tumor cells with the extracellular matrix may regulate expression of proteases including the lysosomal proteases cathepsins B and D. In the present study, we determined whether the expression of these two proteases in human breast fibroblasts was modulated by interactions with the extracellular matrix component, collagen I. Breast fibroblasts were isolated from non-malignant breast tissue as well as from tissue surrounding malignant human breast tumors. Growth of these fibroblasts on collagen I gels affected cell morphology, but not the intracellular localization of vesicles staining for cathepsin B or D. Cathepsins B and D levels (mRNA or intracellular protein) were not affected in fibroblasts growing on collagen I gels or plastic, nor was cathepsin D secreted from these cells. In contrast, protein expression and secretion of cathepsin B, primarily procathepsin B, was induced by growth on collagen I gels. The induced secretion appeared to be mediated by integrins binding to collagen I, as inhibitory antibodies against alpha(1), alpha(2), and beta(1) integrin subunits prevented procathepsin B secretion from fibroblasts grown on collagen. In addition, procathepsin B secretion was induced when cells were plated on beta(1) integrin antibodies. To our knowledge, this is the first examination of cathepsin B and D expression and localization in human breast fibroblasts and their regulation by a matrix protein. Secretion of the cysteine protease procathepsin B from breast fibroblasts may have physiological and pathological consequences, as proteases are required for normal development and for lactation of the mammary gland, yet can also initiate and accelerate the progression of breast cancer.     

Exploration of subsite binding specificity of human cathepsin D through kinetics and rule-based molecular modeling.

The family of aspartic proteinases includes several human enzymes that may play roles in both physiological and pathophysiological processes. The human lysosomal aspartic proteinase cathepsin D is thought to function in the normal degradation of intracellular and endocytosed proteins but has also emerged as a prognostic indicator of breast tumor invasiveness. Presented here are results from a continuing effort to elucidate the factors that contribute to specificity of ligand binding at individual subsites within the cathepsin D active site. The synthetic peptide Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu has proven to be an excellent chromogenic substrate for cathepsin D yielding a value of kcat/Km = 0.92 x 10(-6) s-1 M-1 for enzyme isolated from human placenta. In contrast, the peptide Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu and all derivatives with Ala-Lys in the P3-P2 positions are either not cleaved at all or cleaved with extremely poor efficiency. To explore the binding requirements of the S3 and S2 subsites of cathepsin D, a series of synthetic peptides was prepared with systematic replacements at the P2 position fixing either Ile or Ala in P3. Kinetic parameters were determined using both human placenta cathepsin D and recombinant human fibroblast cathepsin D expressed in Escherichia coli. A rule-based structural model of human cathepsin D, constructed on the basis of known three-dimensional structures of other aspartic proteinases, was utilized in an effort to rationalize the observed substrate selectivity.     

A lysosomal pepstatin-insensitive proteinase as a novel biomarker for breast carcinoma

Lysosomal proteinases play an important role in the turnover of intracellular proteins, and acidic proteinases such as cathepsin D are known to be increased in breast carcinoma. In the present study the activity of a newly discovered acidic lysosomal pepstatin-insensitive proteinase (CLN2p) was measured in breast tissues by the most sensitive and highly specific assay that we had developed for the diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) (2). Samples from eight normal subjects undergoing reductive mammoplasty and 200 patients with primary breast carcinoma were analyzed. The results suggest a two- to seventeen-fold higher CLN2p activity in tumors, which was significantly and positively correlated with already known breast cancer biomarkers such as levels of cathepsin D, estrogen receptor and progesterone receptor. These results suggest a diagnostic and prognostic potential for this novel acid proteinase in breast cancer.     

Pro-cathepsin D can activate in vitro pro-cathepsin B secreted by ovarian cancers

A precursor form of cathepsin B (Mr 45-47 kd) was purified from ascitic fluids of patients with ovarian adenocarcinomas. Following pepsin activation, this precursor produced a 33 kd cathepsin B-like proteinase closely related to lysosomal cathepsin B. A similar activation was found using the 52 kd pro-cathepsin D secreted by the MCF7 human breast cancer cells. This activation was a time, dose and pH dependent process. These results suggest that the 52 kd pro-cathepsin D may be involved in the early steps of the "metastatic cascade", activating pro-cathepsin B in an acidic environment.     

Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.     

Detection of procathepsin D in rat milk

The presence of procathepsin D, a zymogen of the soluble lysosomal aspartic proteinase cathepsin D, was detected in rat milk using Western blot analysis and assay of proteolytic activity in acidic buffers. No other forms of cathepsin D were found. Two different polyclonal anti-procathepsin D antibodies were used for immunochemical detection of procathepsin D. Both antibodies we found to recognize rat procathepsin D. Proteolytic activity in acidic buffers was detected using a fluorogenic substrate specific for cathepsin D and was abolished by pepstatin A, a specific inhibitor of aspartic proteinases. This study represents third demonstration of presence of procathepsin D in mammal breast milk. Potential sources and physiological functions are discussed.     

Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells

Angiostatin, a potent endogenous inhibitor of angiogenesis, is generated by cancer-mediated proteolysis of plasminogen. The culture medium of human prostate carcinoma cells, when incubated with plasminogen at a variety of pH values, generated angiostatic peptides and miniplasminogen. The enzyme(s) responsible for this reaction was purified and identified as procathepsin D. The purified procathepsin D, as well as cathepsin D, generated two angiostatic peptides having the same NH(2)-terminal amino acid sequences and comprising kringles 1-4 of plasminogen in the pH range of 3.0-6.8, most strongly at pH 4.0 in vitro. This reaction required the concomitant conversion of procathepsin D to catalytically active pseudocathepsin D. The conversion of pseudocathepsin D to the mature cathepsin D was not observed by the prolonged incubation. The affinity-purified angiostatic peptides inhibited angiogenesis both in vitro and in vivo. Importantly, procathepsin D secreted by human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that by human prostate carcinoma cells. Since deglycosylated procathepsin D from both prostate and breast carcinoma cells exhibited a similar low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in carbohydrate structures of procathepsin D molecules between the two cell types. The seminal vesicle fluid from patients with prostate carcinoma contained the mature cathepsin D and procathepsin D, but not pseudocathepsin D, suggesting that pseudocathepsin D is not a normal intermediate of procathepsin D processing in vivo. The present study provides evidence for the first time that cathepsin D secreted by human prostate carcinoma cells is responsible for angiostatin generation, thereby causing the prevention of tumor growth and angiogenesis-dependent growth of metastases.     

Purification and Characterization of Cathepsin D from Normal Human Breast Tissue

The lysosomal aspartyl protease cathepsin D is present in most mammalian cells and is active in the catabolism of intracellular and endocytosed proteins. It appears to be overexpressed and abnormally secreted in breast cancer cells, and may contribute to the process of tumor metastasis. In the present study, cathepsin D was purified 4500-fold from normal human breast tissue using pepstatin-agarose, DEAE Sephadex, and Sephadex G-75 chromatography. The resulting enzyme on SDS-PAGE contained five protein bands (47, 31, 29, 13, and 12kDa) which were all immunoreactive on western blot analysis using anti-cathepsin D polyclonal antibodies. The isoform profile of purified cathepsin D consisted of three major peaks at approximate pI 7.3, 6.8, and 6.3, and a broad area of lower activity between pI of 5.0 and 2.0. The purified enzyme had a broad pH optimum centered around pH 3.3. Lectin blotting indicated that cathepsin D is a glycoprotein which is recognized by Galanthus nivalis agglutinin and concanavalin A, suggesting the presence of mannose residues. However, Sambucus nigra agglutinin, Tetragonolobus purpureas agglutinin, Triticum vulgaris agglutinin, and Erythrina cristagalli agglutinin failed to recognize cathepsin D, suggesting a lack of lectin-available sialic acid, fucose, N-acetylglucosamine, and galactose residues, respectively.