Role of Carboxypeptidase N Invasion and Migration in Breast Cancer.

BACKGROUND: Carboxypeptidase N (CPN) is highly expressed in breast cancer and plays an important role in cleaving specific polypeptide fragments within the tumor microenvironment, so here we studied the important role of its invasion and migration in breast cancer. METHODS: MDA-MB-231, MDA-MB-468, and MCF-7 cells were selected for cell culture. We used real-time polymerase chain reaction (PCR) and western blotting to determine CPN gene and protein expression. If CPN was obviously expressed, we designed and synthesized a molecular sequence using an RNA interference approach to remove the CPN and observed its proliferation, migration, and invasion within tumor cells. RESULTS: Real-time PCR and western blotting show the following CPN expression: minimal in MDA-MB-468 cells but obvious in MDA-MB-231 and MCF7 cells. MDA-MB-231 breast cancer cell lines were selected for the control group and CPN was knocked out for the experimental group. Compared to the control group, the experimental group had significantly less migration and invasion. CONCLUSION: CPN may play an important biological function in breast cancer and will provide a new target for the effective diagnosis and treatment of breast cancer.

Characterization of kininases in testicular cells.

Kininases are an important part of the kallikrein-kinin system. We investigated the pattern of kininases in rat Sertoli cells. Sertoli cells are found in the seminifereous tubule of the testis and play a key role in spermatogenesis. Bradykinin was actively cleaved by cultivated Sertoli cells at the Pro7-Phe8, Phe5-Ser6 and Gly4-Phe5 bonds as demonstrated by high performance liquid chromatography analysis. Addition of phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase 3.4.24.11 (NEP), strongly inhibited the degradation of bradykinin. In contrast, the kininase type II-specific inhibitors captopril and enalapril were only partially effective in preventing peptidolysis. NEP and kininase type II were shown to be located in Sertoli cell membranes. The action of kininase type I leads to the formation of the metabolite bradykinin (1-8) which could be detected in small amounts by HPLC analysis. Cleavage of the Ph5-Ser6 bond might be caused by the action of the endopeptidases 24.15 and 24.16, which are phosphoramidon-insensitive. Our results indicate that neutral metalloendopeptidase 24.11 is the main kininase responsible for rapid bradykinin inactivation in Sertoli cells. Further kininases with minor activities are the kininases type I and II and probably the metalloendopeptidases 24.15 and 24.16.

[The effect of diazepam on the activity of carboxypeptidase N and the angiotensin-converting enzyme in sections of the brain of rats with differing normal emotionality and in emotional stress]. / Vliianie diazepama na aktivnost' karboksipeptidazy N i angiotenzinprevrashchaiushchego fermenta v otdelakh mozga krys s razlichnoi émotsional'nost'iu v norme i pri émotsional'nom stresse.

Diazepam increased the carboxypeptidase H activity and decreased the angiotensin converting enzyme activity in the rat brain. Joint action of diazepam and emotional stress changed the carboxypeptidase H activity to the least extent and the angiotensin converting enzyme activity to the utmost extent as compared to the agents' separate effects. The role of both enzymes in the diazepam effect is discussed.

Immunological localization of neuropeptide-degrading enzymes in the nasal mucosa.

Neutral endopeptidase (NEP, EC 3.4.24.11), angiotensin-converting enzyme (ACE, EC 3.4.15.1) and carboxypeptidase N (CPN, EC 3.4.17.3) are potentially important enzymes which regulate the degradation of neuropeptides, such as bradykinin (BK) and substance P (SP), in the respiratory mucosa. Some neuropeptides are also degraded by these enzymes in vitro and in vivo. We investigated the localization of these enzymes in the human nasal mucosa by an indirect immunohistochemical technique (immunogold silver staining). NEP-immunoreactive areas were present in the epithelium, the serous cells of the submucosal glands, and the endothelial cells of small vessels. The epithelium and the serous cells were the predominant areas of NEP immunoreactivity in the nasal mucosa. ACE-immunoreactive areas were seen in the outer layer of the epithelium, the endothelial cells of vessels, and widely distributed in the superficial lamina propria. The endothelial cells of the vessels showed maximum positive intensity to ACE. CPN-immunoreactive areas were observed in the epithelium, the endothelium of vessels and the superficial lamina propria, except for the gland cells. The superficial lamina propria exhibited maximum immunoreactivity for CPN. We observed that the enzymes were widely distributed in the nasal mucosa. The epithelium, including the epithelial cells and glycocalyx, contains all three enzymes. These enzymes play an important role in the mucosal immunity of the respiratory mucosa by degrading active neuropeptides. These results show that NEP secretion is regulated by a glandular, cholinergic control. On the other hand, ACE and CPN secretion are regulated by vascular permeability.

[The effect of emotional-pain stress on the activity of carboxypeptidase N--the enzyme of neuropeptide processing in the rat brain]. / Vliianie émotsional'no-bolevogo stressa na aktivnost' karboksipeptidazy N--fermenta protsessinga neiropeptidov golovnogo mozga krys.

Activity of carboxypeptidase N [correction of H] increased under emotional-algic stress in the rat pituitary gland and hypothalamus. Carboxypeptidase N [correction of H] seems to be involved in development of the stress response, and soluble forms of the enzyme take part in processing of secretory peptides. Membrane-bound forms take part in processing of the neuropeptides specific by their central action.

[The subcellular localization of carboxypeptidase N in the substantia grisea of the cat brain]. / Subkletochnaia lokalizatsiia karboksipeptidazy N v serom veshchestve golovnogo mozga koshki.

The activity of carboxypeptidase H in the cat grey matter is established to be connected with microsomes. A possibility to involve carboxypeptidase H to the processing of neuropeptide predecessors in the encephalon is discussed.

Rat aortic smooth muscle cells in culture express kallikrein, kininogen, and bradykininase activity.

We have studied rat vascular smooth muscle (VSM) cells in culture for the presence of key elements of the glandular kallikrein-kinin system. Direct radioimmunoassay (RIA) using antiserum against rat urinary kallikrein detected a glandular kallikrein-like enzyme (GKLE) in VSM cells and in media. VSM homogenates and culture media had kininogenase activity, generating kinins from dog kininogen. About half of the GKLE was enzymatically inactive which could be activated with trypsin. Kininogenase activity was inhibited completely by aprotinin but only 20% by soybean trypsin inhibitor (SBTI). Trypsin liberated kinins from homogenates and media, demonstrating that VSM cells contain kininogen. Homogenates and media rapidly degrade bradykinin. GKLE, kininogen, and bradykininase activity were all present in VSM cells grown in defined media that contain no serum, thus eliminating any contamination or artefacts from fetal calf serum in standard culture media. Blood vessels of the rat have been reported to contain GKLE. Our observations indicate that GKLE is synthesized by VSM cells, not deposited from plasma. Furthermore, VSM cells synthesize kininogen and bradykininase(s), the other key elements of the glandular kallikrein-kinin system. Thus it is possible that the system functions as an autocoid mechanism that regulates local vascular tone.

[Studies on the quality of enzyme preparations (IX)--kallidinogenase preparations].

Kallidinogenase preparations were investigated with a view of comparing their quality by the enzymological method. These studies were carried out on 13 types of preparations (tablet: 7 kinds, capsule: 3 kinds, ampoule: 3 kinds) of commercially available kalliginogenase preparations. The kinin-liberating activity per one international unit (IU) of kallidinogenase was approximately 400 ng bradykinin/min/IU. The contents of other enzymes, i.e., kininase, trypsin and protease, were determined as the impurities. Kininase, trypsin and protease activities in the preparations for injection were very low. However, one preparation for internal use contained large amounts of these enzyme as impurities. It was presumed that contamination with large amounts of kininase, trypsin and protease have an adverse influence upon the assay and stability and efficacy of these preparations.

High performance liquid chromatographic quantitation of carboxypeptidase activity secreted by human hep G2 cells.

A new high performance liquid chromatographic method has been developed for the determination of carboxypeptidase N activity which quantitates the furylacryloyl-alanine released by enzymatic cleavage of furylacryloyl-alanyl-lysine or furylacryloyl-alanyl-arginine. A short isocratic gradient elutes the substrate and product in less than 7 min and multiple analyses are facilitated by an automatic sample injector. The microassay readily detects and quantitates carboxypeptidase N activity secreted into culture medium. It was determined that approximately 1 X 10(6) Hep G2 cells at early confluence secreted 1 ng of carboxypeptidase N in 24 h. The microassay will also detect as little as 51 pg of purified carboxypeptidase N or 8 pg of carboxypeptidase B.

Hydrolysis of opioid hexapeptides by carboxypeptidase N. Presence of carboxypeptidase in cell membranes.

Carboxypeptidase N, purified to homogeneity from human plasma, rapidly hydrolyzed Lys6- or Arg6-enkephalins when measured by high pressure liquid chromatography. Comparison of the kinetics of hydrolysis of the enkephalin hexapeptides and bradykinin by carboxypeptidase N revealed the following values for the Km and kcat: Arg6-Met5-enkephalin, 49 microM, 1024 min-1; Arg6-Leu5-enkephalin, 57 microM, 375 min-1; Lys6-Met5-enkephalin, 216 microM, 6204 min-1; bradykinin, 19 microM, 58 min-1. Thus, while bradykinin had the lowest Km, the specificity constants (kcat/Km) for all the enkephalin hexapeptides were higher than that of bradykinin due to their high turnover numbers. Preincubation of the enzyme with 0.1 mM CoCl2 increased both the kcat and Km of bradykinin and Arg6-Met5-enkephalin. Similar results were obtained when the above experiments were conducted with the active 48,000 dalton subunit of carboxypeptidase N. Basic carboxypeptidase activity was found in the amniotic fluid, in membrane fractions of various human and bovine tissues, and in cultured cells in the following order of decreasing specific activity: human placental microvilli, human kidney, human amniotic fluid, human lung, bovine lung, bovine pulmonary artery, human foreskin fibroblasts, human pulmonary arterial endothelial cells, and human lung fibroblasts. The membrane-bound carboxypeptidase activity had a neutral pH optimum and behaved similarly to plasma carboxypeptidase N in the presence of various inhibitors and activators. It was different from the carboxypeptidase activity in bovine adrenal chromaffin granules which had an acid pH optimum and was inhibited by sulfhydryl reagents. These studies show that human carboxypeptidase N, an enzyme found in high concentration in blood, readily hydrolyzes Arg6- or Lys6-enkephalins. It could thus control the levels of these peptides if they are released into the circulation from the adrenal gland. In addition, a membrane-bound carboxypeptidase N-like enzyme in various tissues may regulate the local levels of biologically active peptides containing C-terminal basic amino acids such as hexapeptide enkephalins, kinins, anaphylatoxins or fibrinopeptides.

Properties of kininase in rat dental pulp.

A kininase, capable of degrading bradykinin, was partially purified from the dental pulp of rats, and its properties were investigated. Chromatography on both Sephadex G-200 and DEAE Sephadex A-50 columns gave a single peak of kininase activity. The molecular weight of the enzyme, estimated by gel filtration, was about 67,000, and the optimum pH for the enzymatic reaction was about 7.5. The enzyme appears to contain a labile SH group(s) that is essential for its activity, because CdCl2, HgCl2 (0.1 mM each), and p-chloromercuric benzoate (0.05 mM) inhibited the enzyme completely, while dithiothreitol retarded the loss of activity during storage. Of various peptides tested, bradykinin was the substrate most sensitive for the enzyme. The enzyme released several amino acids located in the C-terminal regions of bradykinin--angiotensin I and neurotensin--but only one C-terminal amino acid from des-Arg9--bradykinin and angiotensin II. In contrast, the enzyme did not release any amino acids from substance P, of which only the two amino acids in the N-terminal region are the same as those of bradykinin, but its C-terminal is blocked by an amino group. Although the enzyme was not so highly purified as to rule out the contribution of other peptidases, these results suggest that the dental pulp of rats may contain a single enzyme that degrades bradykinin, and the enzyme may be a type of carboxypeptidase, differing from known kininases from other animal sources.

Characterization of the carboxypeptidase involved in the proteolytic cleavage of the influenza haemagglutinin.

The arginine carboxypeptidase involved in the proteolytic cleavage of the haemagglutinin of influenza A virus has been analysed by an assay employing a Sepharose-bound peptide containing radioactive arginine as a substrate. The enzyme activity has been extracted from purified virus with non-ionic detergents and has been separated from the haemagglutinin and from the neuraminidase by isoelectric focusing and by affinity chromatography. The carboxypeptidase present in virus grown in different host cells shows variations in its isoelectric point. It can be concluded from these observations that the carboxypeptidase is a host component incorporated into the virus envelope. When the enzyme is inhibited by 2-mercaptomethyl-3-guanidinoethyl-thiopropanoic acid, haemagglutinin with the arginine attached to the carboxy terminus of HA1 can be obtained. The observation that under these conditions the haemagglutinin has retained its haemolytic activity indicates that the carboxypeptidase does not play an essential role in the activation process.

Fluorometric assay of kininase activities in rat tissues.

A simple method for the assay of bradykinin (BK)-degrading enzymes was investigated. The procedure of the method includes enzymatic degradation of BK, separation of the residual BK on a small P-cellulose column (0.6 X 3 cm), and its fluorometrical determination based on the reaction with fluorescamine. BK was separated completely from its fragments produced during enzymatic reaction by the column chromatography. The recovery rate of BK was 96 +/- 3%. Quantitative determinations could be carried out on 0.2 nmol of BK, at least in the fluorometry. This method was available for the assay of the enzymes in tissue homogenates as well as in purified preparations, and its usefulness for the study of the enzymes is presented.

Alterations of exocrine pancreatic enzymes in virus-induced diabetic cattle as revealed by immunohistochemistry.

The pancreatic tissue of normal and virus-induced diabetic cattle was investigated by the indirect immunofluorescence technique. Seven secretory proteins (chymotrypsinogen A, trypsinogen, carboxypeptidase A, RNase, DNase, alpha-amylase and lipase) were localized in normal bovine pancreatic acinar cells but in diabetic animals amylase, lipase and carboxypeptidase were either not detectable or markedly diminished. Decrease in amylase content has been reported previously in other diabetic animals. The diminution of the three pancreatic enzymes may be related to the destruction of pancreatic endocrine tissue that occurs in these diabetic animals.

Isolation of membrane-bound renal enzymes that metabolize kinins and angiotensins.

Cortex of rat kidney was homogenized and fractions enriched in plasma membrane, endoplasmic reticulum or brush border were prepared by several techniques of differential centrifugation. The identity and homogeneity of the membrane fragments were investigated by assaying marker enzymes and by transmission and scanning electron microscopy. Kallikrein was present in both plasma-membrane- and endoplasmic-reticulum-enriched fractions isolated by two fractionation procedures. Kallikrein was highly concentrated in a plasma-membrane fraction but was absent from the brush-border membrane of proximal tubular cells. Cells of transplanted renal tumours of the rat, originating from the proximal tubule, had no kallikrein activity. Kininase activity, angiotensin I-converting enzyme (kininase II) and angiotensinase were found in a plasma-membrane-enriched fraction and especially in the fraction containing isolated brush border. It is suggested that after renal kallikrein is synthesized on endoplasmic reticulum, it is subsequently reoriented to a surface membrane for activation and release. Renal kallikrein may enter the tubular filtrate distal to the proximal tubules. The brush-border membrane of proximal tubule is the major site of inactivation of kinins and angiotensin II..