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Clinical Chemistry 45: 1797-1802, 1999;
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(Clinical Chemistry. 1999;45:1797-1802.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Aminopeptidase Activities in Breast Cancer Tissue

José M. Martínez1, Isabel Prieto1, María J. Ramírez1, Cristobal Cueva2, Francisco Alba3 and Manuel Ramírez1,a

1 Área de Fisiología, Universidad de Jaén, Edif. B-3, 23071 Jaén, Spain.

2 Hospital General de Especialidades Ciudad de Jaén, 23005 Jaén, Spain.

3 Departamento de Bioquímica y Biología Molecular, Universidad de Granada, 18071 Granada, Spain.
a Author for correspondence. Fax 34-953-212141; e-mail msanchez{at}ujaen.es


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Endopeptidases such as cathepsins help determine the prognosis of breast cancer (BC). However, little information is available about the role in BC of aminopeptidases (APs), which have been implicated in the metabolism of several local hormonal factors.

Methods: Using aminoacyl-ß-naphthylamides as substrates, we measured fluorometrically alanyl-AP, arginyl-AP, cystinyl-AP, glutamyl-AP, aspartyl-AP, and pyroglutamyl-AP activities in their soluble and membrane-bound forms in surgically removed BC tissue from which we separated samples of neoplastic, adjacent tumoral, and unaffected surrounding tissue.

Results: Compared with unaffected tissue, neoplastic tissue had significantly higher activities of soluble alanyl-AP (553.9 ± 82.8 vs 1615.2 ± 183.0 pmol/mg protein; P <0.001), arginyl-AP (372.4 ± 56.6 vs 1027.2 ± 143.5 pmol/mg protein; P <0.001), and cystinyl-AP (74.8 ± 10.0 vs 282.9 ± 37.2 pmol/mg protein; P <0.001), and of membrane-bound arginyl-AP (457.7 ± 97.9 vs 886.6 ± 140.0 pmol/mg protein; P <0.01). However, membrane-bound aspartyl-AP activity was significantly lower in neoplastic tissue (17.3 ± 1.4 vs 9.2 ± 1.2 pmol/mg protein; P <0.05) and pyroglutamyl-AP activity was significantly lower in neoplastic and adjacent tissues (12.8 ± 0.9 vs 7.0 ± 1.2 and 8.0 ± 1.3 pmol/mg protein; P <0.001 for both comparisons).

Conclusions: The present results document changes in AP activities in BC tissue. These changes may reflect the functional status of the AP substrates, which can be selectively activated or inhibited locally in the affected tissue as a result of specific conditions brought about by the tumor.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Many studies have implicated endopeptidases cathepsin D, B, and L in the development, growth, and dissemination of breast cancer (BC)1 (1)(2). It has been proposed that the increased activity of these enzymes during malignant progression might be the result of changes in their regulation by endogenous endopeptidase inhibitors (1). These changes, in turn, may also result from the intracrine, autocrine, or paracrine action of several local hormonal factors (3).

Aminopeptidases (APs) generally are zinc metalloenzymes used in clinical chemistry as markers of disease, and APs also play a physiological role in the regulation of several biologically active peptides (4). Changes in these peptides thus may be reflected in enzymatic activity. However, in spite of their major role in controlling functionally active peptides (5), the possible role of APs in BC has rarely received attention (6). Therefore, to evaluate the local role of APs in BC, we performed a comprehensive study of a broad spectrum of AP activities, which included neutral, basic, acid, and omega substrates. We measured the activities of alanyl-AP (AlaAP), arginyl-AP (ArgAP), cystinyl-AP (CysAP), glutamyl-AP (GluAP), aspartyl-AP (AspAP), and pyroglutamyl-AP (pGluAP) in their soluble and membrane-bound forms, using aminoacyl-ß-naphthylamides as substrates (7), in biopsy specimens of BC from which we separated samples of neoplastic tissue, adjacent tumoral tissue, and unaffected surrounding tissue. Because there is evidence that the activities of GluAP and AspAP reflect two enzymes with different properties and roles, glutamyl-ß-naphthylamide (GluNNap) and aspartyl-ß-naphthylamide (AspNNap) were included as substrates (8).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Nine BC patients ranging in age from 47 to 63 years (mean, 54.7 years) participated in this study after giving their informed consent. All BCs were diagnosed as ductal infiltrating carcinomas. Tumor samples, always containing some contiguous healthy tissue, were obtained shortly after surgical removal, quickly separated into pieces of neoplastic tissue, nonnecrotic tissue removed from the edges of tumoral area (adjacent neoplastic tissue), and unaffected surrounding tissue, which was used as a control. Samples were quickly frozen in dry ice and stored at -80 °C until processed.

Tissue samples (mean weight, 300 mg) were homogenized in 10 volumes of 10 mmol/L Tris-HCl buffer (pH 7.4) and ultracentrifuged at 100 000g for 30 min (4 °C) to obtain the soluble fraction. The resulting supernatants were used to measure soluble enzymatic activity and protein content, assayed in triplicate. To solubilize membrane proteins, the pellets were rehomogenized in Tris-HCl buffer (pH 7.4) containing 10 mL/L Triton X-100. After centrifugation (100 000g for 30 min at 4 °C), the supernatants were used to measure membrane-bound activity and proteins, also in triplicate. To ensure complete recovery of activity, the detergent was removed from the medium by adding adsorbent polymeric Biobeads SM-2 (100 g/L) to the samples, which were then shaken for 2 h at 4 °C (9).

AlaAP, ArgAP, and CysAP were measured fluorometrically using alanyl-ß-naphthylamide (AlaNNap), arginyl-ß-naphthylamide (ArgNNap), and cystinyl-ß-naphthylamide (CysNNap) as substrates, according to the modified method of Greenberg (10). Ten microliters of each supernatant was incubated for 30 min at 25 °C with 1 mL of the substrate solution (21.4 mg/L AlaNNap, 33.5 mg/L ArgNNap, or 56.3 mg/L CysNNap), 100 mg/L bovine serum albumin (BSA), and 100 mg/L dithiothreitol (DTT) in 50 mmol/L phosphate buffer, pH 7.4, for AlaAP and ArgAP and 50 mmol/L HCl-Tris buffer, pH 6, for CysAP.

pGluAP was measured in a fluorogenic assay using pyroglutamyl-ß-naphthylamide (pGluNNap) as the substrate, according to the modified method of Schwabe and McDonald (11): 10 µL of each supernatant was incubated for 120 min at 37 °C with 1 mL of substrate solution (25.4 mg/L pGluNNap, 100 mg/L BSA, 100 mg/L DTT, and 378 mg/L EDTA in 50 mmol/L phosphate buffer, pH 7.4).

AspAP was determined fluorometrically with AspNNap as the substrate, according to the method of Cheung and Cushman (12) modified as follows: 10 µL of each supernatant was incubated for 120 min at 37 °C with 1 mL of the substrate solution (25.8 mg/L AspNNap, 100 mg/L BSA, 100 mg/L DTT, and 394 mg/L MnCl2 in 50 mmol/L Tris-HCl buffer, pH 7.4).

GluAP was also determined in a fluorometric assay using GluNNap as the substrate according to the method of Tobe et al. (13) modified as follows: 10 µL of each supernatant was incubated for 120 min at 37 °C with 1 mL of the substrate solution (27.2 mg/L GluNNap, 100 mg/L BSA, 100 mg/L DTT, and 5.55 g/L CaCl2 in 50 mmol/L Tris-HCl, pH 7.4).

All reactions were stopped by the addition of 1 mL of 0.1 mol/L acetate buffer (pH 4.2). The amount of ß-naphthylamine released as a result of enzymatic activity was measured fluorometrically at an emission wavelength of 412 nm with excitation at 345 nm. Proteins were quantified in triplicate by the method of Bradford (14), using BSA as the calibrator. Specific soluble and membrane-bound AP activities were expressed as picomoles of AlaNNap, ArgNNap, CysNNap, AspNNap, pGluNNap, or GluNNap hydrolyzed per minute per milligram of protein. Fluorogenic assays were linear with respect to time of hydrolysis and protein content.

statistical analysis
We used one-way ANOVA to analyze differences between groups. Posthoc comparisons were made using Duncan's test; P values below 0.05 were considered significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mean ± SE activities of soluble and membrane-bound AP in the three samples of tissue studied are presented in Figs. 1 and 2.



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  		Figure 1. Soluble AP activities in BC tissue.

Samples were obtained in the same patient from neoplastic tissue (Tumoral), nonnecrotic tissue removed from the edges of the tumoral area (Adjacent), and unaffected surrounding tissue (Control). Values represent mean ± SE of specific AP activities expressed as picomoles of the corresponding aminoacyl-ß-naphthylamide hydrolyzed per minute per milligram of protein. ***, P <0.001 vs control and adjacent tissue.

The highest activities were observed for AlaAP, ArgAP, and CysAP, and the lowest for AspAP, GluAP, and pGluAP. In comparison with unaffected tissue, the results showed highly significant differences in specific soluble and membrane-bound AP activities, especially in neoplastic tissue. Whereas comparisons between adjacent tumoral and unaffected tissue showed no significant differences, in samples of neoplastic tissue, the soluble activities of AlaAP (P <0.001), ArgAP (P <0.001) and CysAP (P <0.001) were increased threefold, and membrane-bound ArgAP (P <0.01) was increased twofold. However, the membrane-bound AspAP activity was significantly lower in neoplastic tissue (P <0.05), and pGluAP activity was significantly lower in neoplastic and adjacent tissues (P <0.001).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The etiology of BC remains unknown, and changes in hormonal status during its development are not totally understood. Proteases are particularly interesting in cancer research because they may play a part in the metastatic process (15). AP activity not only reflects tissue damage but also participates in the functional control of local factors that act through intracrine, autocrine, or paracrine mechanisms. Therefore, AP activity reflects the functional status of its peptidergic substrates. AlaAP, which exhibits broad substrate specificity, may hydrolyze bradykinins (4) and enkephalins (16), and may also act as an angiotensinase (17). ArgAP activity specifically hydrolyzes basic N-terminal residues from peptides and arylamide derivatives (7). Because of its exopeptidase activity, it has been implicated in the metabolism of met-enkephalin (18) and angiotensin (Ang) III (17); its endopeptidase activity is also thought to be involved in neurotensin metabolism (19). CysAP activity has been reported to hydrolyze oxytocin (OT) and vasopressin (VP) (20). AP A activity, including GluAP and AspAP, displays restricted specificity that may be responsible for cleaving aspartic acid from Ang II to produce Ang III (7), from Ang I to produce des-Asp1 Ang I (21), and from the cholecystokinin octapeptide (22). pGluAP reportedly is able to remove the NH2-terminal pyroglutamic acid residue from peptides such as thyrotropin-releasing hormone or gonadotropin-releasing hormone (GnRH) and artificial substrates (23) (Table 1 ).


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  		Table 1. Peptide hydrolysis by APs.1

AP activity not only reflects tissue damage but is also indicative of local hormonal status. In fact, the changes observed in the present study are not unidirectional: some activities increased, whereas others decreased. These results illustrate the functional complexity of local factors, which are selectively activated or inhibited by the tumoral process in the affected tissue.

The present results demonstrate a highly significant increase in soluble AlaAP activity (AP M) in neoplastic tissue. The CD13 molecule has been reported as identical to AlaAP (24), and immunocytochemical findings have shown that it is present on healthy breast epithelium and in breast carcinomas (25). This enzymatic activity is also increased in the serum of BC patients and has been proposed as a marker for cancer (26)(27). These findings, especially the study of Dixon et al. (25) in BC specimens, are in agreement with our present results.

Several AP activities (AlaAP, ArgAP, and AspAP), which were significantly modified in the material we analyzed, possess angiotensinase activity. Ang III is produced from Ang II by AP A or A-like activity. Ang III is also produced from Ang I through the production of des-Asp1-Ang I, which is further converted to Ang III by the action of angiotensin-converting enzyme. GluAP has been ruled out as the particulate enzyme that degrades Ang I to des-Asp1-Ang I; another enzyme (AspAP) with higher affinity for AspNNap than GluNNap must be responsible (21). Ang III is further converted to Ang IV by AP B or AP M. In accordance with this scheme, our findings of a decrease in AP A activity and an increase in AP M and AP B activities suggest that the metabolism of Ang II to Ang III is slow, whereas the metabolism of Ang III to Ang IV is rapid. As a result, Ang II action predominates. Ang II has been demonstrated to stimulate cellular growth, proliferation, and differentiation as well as angiogenesis (28). In this connection, the expression of the Ang II type 1 receptor has been demonstrated in healthy and diseased human breast tissues (29). Ang II stimulates hypertropic growth of vascular smooth muscle cells. Accompanying this growth is the induction of the expression of growth-related protooncogenes as well as the synthesis of several autocrine growth factors. An imbalance in the signals activated by Ang II may produce abnormal vascular growth (30). The angiotensin-converting enzyme inhibitor captopril inhibits proliferation of human ductal carcinoma cells in culture (31); in addition, angiotensin-converting enzyme and enkephalinase activities have been detected in human breast cyst fluid (32). Therefore, modified local angiotensinase activity, reported for the first time in this study, may also reflect changes in the renin-angiotensin system in neoplastic cells and hence changes in the functions in which this system is involved. Our results, together with earlier findings, support that the renin-angiotensin system may be involved in normal and abnormal breast tissue function. This is important in view of the broad current knowledge of the renin-angiotensin system and the possibilities for pharmacological intervention at several levels of this system.

The present results show different profiles for GluAP and AspAP. Previous evidence supports the existence of two enzymes with presumably different roles in the regulation of susceptible substrates (8)(12)(21). At present, GluAP, which removes the NH2-terminal Asp residue, is considered responsible for the rapid metabolism of Ang II (7). In addition, AspAP, also acts rapidly on the NH2-terminal aspartic acid residues of Ang analogs, which points to a physiological role in Ang metabolism (7). For this reason we use the expression "aminopeptidase A activity" here to refer to the activity that separates NH2-terminal acid residues (Asp or Glu), keeping in mind that these residues may be released through the action of at least two different enzymes, GluAP and AspAP (8). In this connection, Wilk et al. (33) have recently purified from rabbit brain cytosol an AspAP with preference for NH2-terminal Asp residues but which is distinct from GluAP.

The highly significant increase in CysAP activity in tumoral tissue is particularly interesting in view of its role in the hydrolysis of OT and VP (20). Although VP is not detected in healthy breast tissue, immunohistochemical methods have revealed the presence of VP in neoplastic cells. Moreover, OT and OT-associated human neurophysin have been reported to be common in cells of healthy breast tissue but are rarely or never detected in BC (34). In addition, OT inhibits proliferation of human BC cell lines (35) and thus may play a role in preventing this disease (36). Our results confirm the involvement of these peptides and suggest a major role for CysAP in the development of BC. However, because leucine aminopeptidase is able to cleave OT (37), this enzyme may also be involved in BC.

The highly significant decrease in membrane-bound pGluAP activity in tumoral and adjacent tissue suggests that modifications in this enzyme or its putative substrates also play a major role in BC pathogenesis. In this connection, GnRH receptors and GnRH mRNA have been found in breast tissue, raising the possibility of a local role for GnRH in the human mammary gland (38). The fact that of the 12 activities measured, only membrane-bound pGluAP activity was significantly modified in tumoral and adjacent tissue suggests a role for this activity in tumor progression.

In conclusion, the present results document changes in AP activities in BC tissue; we interpret these changes to reflect the functional status of their peptidergic substrates. Our findings emphasize the importance of local intracrine, autocrine, and paracrine hormonal factors in the pathogenesis of BC and suggest that they play a role in the tumoral process.



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  		Figure 2. Membrane-bound AP activities in BC tissue.

Samples were obtained in the same patient from neoplastic tissue (Tumoral), nonnecrotic tissue removed from the edges of the tumoral area (Adjacent), and unaffected surrounding tissue (Control). Values represent mean ± SE of specific AP activities expressed as picomoles of the corresponding aminoacyl-ß-naphthylamide hydrolyzed per minute per milligram of protein. *, P <0.05 vs control and adjacent tissue for AspAP; **, P <0.01 vs control and adjacent tissue for ArgAP; ***, P <0.001 vs control for pGluAP.


   Acknowledgments
 
We thank Karen Shashok for revising the English translation of the manuscript.


   Footnotes
 
1 Nonstandard abbreviations: BC, breast cancer; AP, aminopeptidase; pGlu, pyroglutamyl; NNap, ß-naphthylamide; BSA, bovine serum albumin; DTT, dithiothreitol; Ang, angiotensin; OT, oxytocin; VP, vasopressin; and GnRH, gonadotropin-releasing hormone.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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