pepstatin and Breast-Neoplasms

Topics: alpha 1-Antitrypsin; Animals; Aprotinin; Breast Neoplasms; Cathepsin B; Cathepsins; Cell Transformation, Neoplastic; Female; Humans; Laryngeal Neoplasms; Lung Neoplasms; Mice; Neoplasms; Ovarian Neoplasms; Pancreatic Neoplasms; Pepstatins; Peptide Hydrolases; Protease Inhibitors; Rats; Stomach Neoplasms

(1) Background: Nanomedicine has recently emerged as a new area of research, particularly to fight cancer. In this field, we were interested in the vectorization of pepstatin A, a peptide which does not cross cell membranes, but which is a potent inhibitor of cathepsin D, an aspartic protease particularly overexpressed in breast cancer. (2) Methods: We studied two kinds of nanoparticles. For pepstatin A delivery, mesoporous silica nanoparticles with large pores (LPMSNs) and hollow organosilica nanoparticles (HOSNPs) obtained through the sol⁻gel procedure were used. The nanoparticles were loaded with pepstatin A, and then the nanoparticles were incubated with cancer cells. (3) Results: LPMSNs were monodisperse with 100 nm diameter. HOSNPs were more polydisperse with diameters below 100 nm. Good loading capacities were obtained for both types of nanoparticles. The nanoparticles were endocytosed in cancer cells, and HOSNPs led to the best results for cancer cell killing. (4) Conclusions: Mesoporous silica-based nanoparticles with large pores or cavities are promising for nanomedicine applications with peptides.

Topics: Breast Neoplasms; Cell Line, Tumor; Drug Delivery Systems; Female; Humans; Nanoparticles; Pepstatins; Porosity; Silicon Dioxide

Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) breast cell lines has been characterized with regard to its kinetic properties, activity levels, precursor and processed M(r) forms, and isoform composition. Normal cell cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the cancer cell line (pH 3.0-3.2) and greater activity between pH values of 4.0 to 4.5. The two cancer cell lines have approximately 1.5 to 2.0-fold increased total acid protease activity and 2 to 3-fold increased pepstatin-inhibitable protease activity (i.e. cathepsin D) when compared to the normal breast cell line. Western blotting indicates that a major processed form of cathepsin D for all three cell lines occurs at 31 kDa. The cancer cell lines contain significant amounts of cathepsin D precursors of 47 and 42 kDa whereas the normal cell line contains little if any of these precursors. Isoelectric focusing indicates that the normal cell line contains approximately 50% of its total acid protease activity at pIs above 4 whereas the cancer cell lines contain 70-80% of their protease activity at such pIs. In addition, the cancer cell lines contain two to three major isoforms between pIs of 5.5 and 6.3 which were not present in the normal cell line. The isoforms from pI values of 5.5 to 7.3 for all three cell lines are 100% pepstatin-inhibitable. In addition, Western blot analysis indicates that these isoforms contain the processed 31 kDa form of cathepsin D. The combined results indicate that the two breast cancer cell lines are similar to biopsied malignant breast tissue in exhibiting altered acid protease isoform profiles with increased relative amounts of pepstatin-inhibitable and immunoreactive acid protease activity (cathepsin D) compared to normal breast tissue or cells.

Topics: Aspartic Acid Endopeptidases; Blotting, Western; Breast Neoplasms; Cathepsin D; Cells, Cultured; Enzyme Precursors; Female; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Isomerism; Kinetics; Pepstatins; Tumor Cells, Cultured

A simple procedure for the affinity purification of procathepsin D from tissue culture medium conditioned by breast-cancer cells is described. This procedure yielded 2 micrograms of procathepsin D/100 ml medium. The procathepsin D was approximately 95% pure as judged by silver staining of polyacrylamide gels, the major contaminant being mature cathepsin D. The ability of procathepsin D to stimulate the proliferation of oestrogen-responsive MCF-7 breast cancer cells was determined. The purified procathepsin D had no mitogenic effect alone or in combination with oestradiol or other growth factors. These data suggest that procathepsin D does not act as an oestrogen-regulated autocrine growth factor for malignant breast epithelial calls.

Topics: Breast Neoplasms; Cathepsin D; Cell Division; Chromatography, Affinity; Enzyme Precursors; Estradiol; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Mitogens; Pepstatins; Tumor Cells, Cultured

The aspartyl protease cathepsin D has been shown to be a marker of poor prognosis when found at high levels in primary breast tumors. It has been suggested that this is because the production of cathepsin D increases the invasive potential of the tumor cells, thus increasing the probability of metastasis. We have therefore conducted experiments to determine if secreted cathepsin D makes a significant contribution to the invasive phenotype of breast cancer cells in the Boyden chamber assay of invasion, which measures the ability of a cell to invade through an artificial basement membrane. Cathepsin D secretion and Boyden chamber invasiveness were measured in nine clones of the breast cancer cell line MCF-7, and no correlation was found between cathepsin secretion and invasive behavior. Invasion assays were also conducted in the presence of the aspartyl protease inhibitor pepstatin A, and no inhibition of the invasive behavior of cells was seen. Since low-pH environments are required for both the activation of pro-cathepsin D and the activity of the mature enzyme, assays were also conducted in the presence of chloroquine to neutralize the pH in the acidic compartments of the cells. This treatment did not inhibit invasiveness. Cathepsin D secretion by the breast cancer cell lines MDA-MB-231, MDA-MB-435, MDA-MB-435s, MDA-MB-468, SK-Br-3, and MCF-7-ADRr was also measured. Again, there was no correlation with invasion. In fact, cathepsin D levels were inversely correlated with aggressive behavior in vivo and in vitro in previously reported studies. These data suggest that cathepsin D secretion by tumor cells is not an important determinant of the invasiveness of the tumor cells per se. These data also reinforce the view that the poor prognosis in clinical breast cancer linked to high tumor levels of cathepsin D is probably due to high levels of cathepsin D in the stromal components of the tumor such as infiltrating inflammatory cells.

Topics: Breast Neoplasms; Cathepsin D; Chloroquine; Diffusion Chambers, Culture; Female; Humans; Molecular Weight; Neoplasm Invasiveness; Pepstatins

High and low affinity receptors for basic fibroblast growth factor (bFGF) were detected by binding experiments on MCF7 breast cancer cells. These cells were stimulated for growth by physiological concentrations of bFGF. However, in contrast to endothelial cells, these MCF7 cells did not produce detectable amounts of biologically active bFGF or related heparin-binding growth factor(s) of the FGF family. In vitro, the cathepsin D (cath-D) secreted by MCF7 cells was able to digest extracellular matrix (ECM) and to release ECM-bound 125I-bFGF. When MCF7 cells were cultured on ECM containing bound bFGF, they internalized bFGF, which was slowly and partially proteolyzed in the cells. Processing occurred in acidic compartments and was inhibited by leupeptin. Pepstatin A, an inhibitor of aspartyl proteases, had no effect on the processing but reduced internalization of matrix-bound bFGF by MCF7 cells. Taken together, these results suggest a cooperation between cath-D and bFGF, by which the protease could facilitate the release of bFGF from ECM and its subsequent use by breast cancer cells and/or adjacent cells involved in angiogenesis.

Topics: Breast Neoplasms; Cathepsin D; Cell Division; Cell Line; Extracellular Matrix; Female; Fibroblast Growth Factor 2; Humans; Kinetics; Pepstatins; Protease Inhibitors; Receptors, Cell Surface; Receptors, Fibroblast Growth Factor

It has been proposed that proteases secreted by cancer cells facilitate metastasis by degrading extracellular matrix. Estrogen receptor-positive breast cancer cells secrete a Mr 52,000 pro-cath-D under estrogen stimulation, whereas this protease is produced constitutively by estrogen receptor-negative cancer cells. We report on the degradation in vitro of extracellular matrix by purified Mr 52,000 cathepsin D (cath-D) and by conditioned media prepared from different cell lines. The purified Mr 52,000 pro-cath-D was autoactivated at pH 4.5 into a Mr 51,000 cath-D and found to digest the extracellular matrix of endothelial bovine corneal cells labeled with [3H]proline or [35S]methionine. Culture medium conditioned by estrogen-treated MCF7 cells had a similar effect at pH 4.5 but not at pH 7.4. Matrix degradation was totally inhibited by pepstatin. Other breast cancer cells (BT20, MDA-MB231, T47D cells, etc.) and other cancer cells also secreted a pepstatin-sensitive proteinase able to degrade extracellular matrix. By contrast, the U2 variant of MCF7 cells, which lacks the Mr 52,000 cath-D gene, and the nontumoral epithelial mammary cells secreted a negligible amount of this proteinase. In all conditioned media, the pepstatin-dependent extracellular matrix degrading activity was highly correlated to the Mr 52,000 cath-D concentration measured by immunoenzymatic assay. We conclude that the Mr 52,000 cath-D is the major acidic protease secreted by mammary cancer cells. We suggest that this protease may degrade basement membrane and consequently facilitate tumor invasion when it is released in an acidic microenvironment.

Topics: Breast Neoplasms; Cathepsin D; Collagen; Enzyme Precursors; Extracellular Matrix; Extracellular Space; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Pepstatins; Proteoglycans; Tumor Cells, Cultured

An estrogen-induced 52-kDa glycoprotein secreted by human breast cancer cells and able to autostimulate the growth of MCF7 cells has been purified, using monoclonal antibodies, and characterized. The protein contains mannose 6-phosphate signals on its N-linked high-mannose chains, suggesting that it is a lysosomal enzyme. Both the secreted 52-kDa protein and its processed cellular forms (52-, 48- and 34-kDa) were identified as carboxyl proteinases having an optimal activity at pH 3.5 and being specifically inhibited by pepstatin. This protease is characterized by its inducibility by estrogens and its high concentration in proliferative benign and malignant mammary tissue, when detected by immunohistochemistry. The estrogen-induced secretion of this protease may help to understand how estrogens stimulate mammary tumor growth and/or invasion.

Topics: Antibodies, Monoclonal; Aspartic Acid Endopeptidases; Breast Neoplasms; Cathepsin D; Cell Line; Electrophoresis, Paper; Endopeptidases; Estrogens; Female; Humans; Hydrogen-Ion Concentration; Lysosomes; Mannosephosphates; Molecular Weight; Pepstatins; Protease Inhibitors