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Abstract

BACKGROUND:

The key role in carcinogenesis with destruction of the extracellular matrix is played by proteases released by invasive cancer cells. Cysteine peptidases, such as cathepsin B and L, take an important role in cancer progression and metastasis.

OBJECTIVES:

Cysteine peptidase-like activity (CPA) in sera of patients with breast cancer at different stages of disease and the influence of genetic predisposition associated with BRCA-1 gene mutations were analysed.

METHODS:

CPA in serum was determined with the spectrofluorometric technique using Z-Phe-Arg-AMC as a substrate. Determination was carried out in 111 breast cancer patients in comparison to a control group of 50 healthy subjects.

RESULTS:

The highest CPA was found in breast cancer patients with a hereditary predisposition bearing BRCA1 gene mutations, and the lowest activity was found in patients who had a tumour surgically removed and before adjuvant therapy. The differences in the activities between control group and cancer groups were statistically significant ( $p<$ 0.05), except from group of cancer patients in complete remission ( $p<$ 0.52).

CONCLUSIONS:

Serum CPA in patients with breast cancer differs depending on the cancer stage and treatment methods. Our study demonstrate the correlation between BRCA-1 gene mutations and the increased level of CPA.

Keywords

Breast cancer serum biomarkers cysteine peptidases-like activity cathepsins

1. Introduction

Carcinogenesis is a process associated with the transformation of normal cells into cancer cells that form a tissue with a potential for invasion and metastasis. The destruction of the extracellular matrix (ECM) is a vital stage of these processes. The key role in the degradation is played by proteases released by invasive cancer cells. Increased expression and/or activity were observed for serine, aspartyl, cysteine proteases and metallopeptidases. These enzymes may lead directly to the degradation of ECM, or indirectly through the activation of other proteases, acting in a specific sequence. It starts most likely with the activation of aspartyl protease cathepsin D (CatD), which is able to activate cysteine peptidases – cathepsin B and L (CatB, CatL), which can activate pro-urokinase type plasminogen activator (UPA) in the next step. Then the active UPA can convert plasminogen to plasmin. Cathepsin B and plasmin are able to degrade several components in the stromal tumor and activate metallopeptidase zymogens, the major group of ECM-degrading proteases. These processes are regulated by a complex system of cell inhibitors and activators. Under physiological conditions the activities of proteases and their inhibitors are balanced. Disorders of this balance may be important for carcinogenesis, usually leading to proteolysis and degradation of ECM proteins, and facilitating invasion and metastasis. CatB and L involvement at different stages of oncogenic process is particularly important for carcinogenesis in humans. Many studies demonstrated that overexpression of these enzymes is correlated with invasive metastasis in various types of cancer [1, 33, 36]. Furthermore, some studies suggested that disorders in their regulation could trigger itself the transformation of normal cells into cancer cells [15]. The proteases are normally located in lysosomes and are involved in the processes of immune response and autophagy. It was shown that in cancer they are released to the extracellular matrix (ECM), facilitating its degradation [19, 43]. The inhibition of the proteolytic activity of the enzyme delays the migration and infiltration of cancer cells in tissue. Proteases are synthesized as biologically inactive precursors and become activated in endosomal/lysosomal system or extracellulary after release from the cell, thus gaining the potential for the degradation of basal membranes in normal tissues [42]. Furthermore, some researchers found that active cysteine cathepsins suppress anticancer immune response, depending on the cell type and location [28]. It was also proposed that the lack of balance between cysteine cathepsins and cystatins might suppress immune cell functions and facilitate the invasion of cancer cells. Other studies indicated that the proteolytic enzymatic cascades associated with the activity of cysteine, serine and metallopeptidases are involved in the degradation of extracellular matrix by breast cancer cells [37]. It was also demonstrated that the combined inhibition of various proteases, including CatB and L, could significantly limit tumor growth. Therefore, the inhibition of the activity or expression of these proteases can be a promising target in cancer therapy [13].

The direct activity of proteolytic enzymes involved in progression and metastasis suggests that they can be used as potential prognostic markers in cancer diagnostics. Previous studies in cancer patients demonstrated a correlation between the high serum levels of proteases and poor prognosis [9, 34]. Determination of levels of urokinase-type plasminogen activator and CatD was found to be a useful prognostic factor in breast cancer [8, 13]. Similarly, levels of CatB and CatL and some metallopeptidases were confirmed as useful prognostic factors in the assessment of metastasis in certain types of cancer. It was also found that the serum levels of cathepsins B, H and X, and their endogenic inhibitor, cystatin C could be used as prognostic parameters for the assessment of health/disease status [7, 30].

In our study, we determined cysteine peptidases-like activity, employing the spectrofluorometric method using the Z-Phe-Arg-AMC substrate. This substrate marks the activity of both cathepsins B and L, but not their concentration, which could be established with the use of ELISA. In that way only the active form of enzymes was measured – and not the passive ones.

Some genetic markers appeared to be useful in breast cancer diagnostics too. BRCA-1 and BRCA-2 gene mutations are recognized as valuable molecular trait. BRCA gene products are proteins responsible for the repair of damaged DNA during replication in the cell cycle. BRCA mutation carriers have a very high risk of disease by the age of 70, in the range 47%–66%. Additionally, women with BRCA mutation-associated breast cancer also have an elevated risk of other or secondary malignancies [4, 12].

This paper presents findings on cysteine peptidases-like activity (CPA) in sera of 111 patients with breast cancer at different stages of treatment in comparison to a group of healthy women. Interestingly, the highest CPA was found in patients with a hereditary predisposition to breast cancer (BRCA 1 gene mutations).

2. Materials and methods

2.1 Clinical material

Serum samples were taken from patients with diagnosed breast cancer. The patients were referred for routine tests to the Department of Oncology, Regional Hospital in Swidnica for the years 2007–2009. Blood for biochemical tests was taken in parallel with blood taken for routine follow-up tests. For the study we recruited 111 patients with breast cancer and the decision on inclusion was made based on the results of preliminary chest radiographs, mammography and USG. Patients were diagnosed with various stages of breast cancer (IIA – IIIA TNM). Biochemical tests were performed at the International Hereditary Cancer Center, Molecular Laboratory Services of the Department of Genetics and Pathology, Pomeranian Medical University.

The patients were divided into four groups: I – patients after surgical procedure and before adjuvant treatment (mean age 56.36 $\pm$ 9.9 years), II – patients after completed adjuvant treatment and in full remission (mean age 57.93 $\pm$ 11.1 years), III – patients with disseminated cancer (mean age 57.3 $\pm$ 10.8 years), IV – cancer patients with a hereditary predisposition to breast cancer (BRCA-1 gene mutations, mean age 51.7 $\pm$ 8.56 years) and V (control group) – healthy women (mean age 52.77 $\pm$ 8.41 years). There were no statistical differences in mean age between groups I-IV and control group V. Full remission was assumed as 5-year recurrence-free survival. Sera were kept at $-$ 20 ${}^{\circ}$ C for 1–2 months and then used for the determination of CPA. The activities were calculated per ml of serum.

The experiments were done in accord with the Helsinki Declaration (1964), a written informed consent has been taken from patients. The investigation protocol was approved by the local Bioethics Committee at Wroclaw Medical University.

2.2 Determination of cysteine peptidases-like activity

Cysteine peptidases-like activity was measured spe-ctrofluorometrically using a Z-Phe-Arg-AMC (Sigma-Aldrich) as a substrate by the method of Barrett and Kirschke [3] with the modification of Lah et al. [25]. Briefly, 20 $\mu$ l of samples were added to 500 $\mu$ l of activation buffer 0.4 M sodium phosphate (pH 6.0), containing 4 mM EDTA and 2.5 mM DTT. Samples were preincubated for 5 min at 37 ${}^{\circ}$ C before addition of 100 $\mu$ l of 200 $\mu$ M Z-Phe-Arg-AMC. In control assay (blank) the sample was replaced with the activation buffer. The incubation was carried out for 30 minutes and stopped by addition of 2 ml of 1 mM iodoacetic acid. Fluorescence was measured in a luminescence spectrometer, Perkin Elmer LS 50 B at 370 nm excitation and 440 nm emission wavelengths, respectively. One unit of enzymatic activity (U) was defined as the amount of enzyme able to catalyse the release of 1 nM 7-AMC per one minute.

2.3 Identification of BRCA-1 gene mutations

Three mutations in the BRCA1 gene (5382insC, C61G and 4153delA), most frequent in Polish population, were identified using the PCR technique at the Department of Genetics and Pathology, Pomeranian Medical University in Szczecin according to previously described protocol [14]. Genomic DNA was prepared from peripheral blood leukocytes, by use of the nonenzymatic, rapid method described by Lahiri and Nurnberger [27]. The entire coding region of BRCA1 was screened for the presence of germline mutations in BRCA1, by use of SSCP followed by direct sequencing of variants. The primers used for PCR amplification of the BRCA1 gene have been described in the Breast Cancer Information Core (BIC) database. Samples were sequenced with the use of fluorescently labeled dideoxy-chain terminators from the ABI Prism Kit (PE Biosystems), in a model 373 automated DNA sequencer (PE Biosystems).

2.4 Statistics

Standard statistical Student $t$ -test was applied for comparison of the two variables. The values are presented as the mean $\pm$ SD, and a level of statistical significance was set at $p<$ 0.05. Statistical differences between the groups were assessed using one-way analysis of variance (ANOVA). Tukey’s test was used for the post-hoc analysis.

3. Results

We compared differences in the of CPA levels between the groups of patients at various stages of breast cancer treatment in comparison to healthy subjects. The clinical characteristic of patients and CPA values were presented in Table 1.

Table 1
Clinical characteristic of breast cancer patients and CPA level in serum

Clinical characteristic	Group I $N=$ 30	Group II $N=$ 28	Group III $N=$ 27	Group IV $N=$ 26	Group V $N=$ 50
Age (Mean $\pm$ SD)	56.36 $\pm$ 9.9	57.93 $\pm$ 11.1	57.3 $\pm$ 10.8	51.7 $\pm$ 8.56	52.77 $\pm$ 8.41
Statistical significance	I:V – NS	II:V – NS	III:V – NS	IV:V – NS
Tumor size
T0–T1	22 (73.3%)	8 (28.6%)	3 (11.1%)	18 (69.2%)	Not
T2	8 (26.7%)	15 (53.6%)	9 (33.3%)	6 (23.1%)	applicable
T3	0 (0.0%)	5 (17.8%)	15 (55.6%)	2 (7.7%)
Metastases	Not present	Not present	Present	Not present	Not applicable
ER ${}^{{\#}}$
Negative	11 (37.9%)	12 (48.0%)	4 (30.8%)	9 (69,2%)	Not
Positive	18 (62.1%)	13 (52.0%)	9 (69.2%)	4 (30.8%)	Applicable
Serum CPA*
Mean	310.36	717.33	448.11	1192.82	621.02
$\pm$ SD	$\pm$ 137.94	$\pm$ 420.44	$\pm$ 315.31	$\pm$ 680.91	$\pm$ 293.36

ER – estrogen receptor, ${}^{\#}$ not all data were available. NS – non-statistical significance. *CPA – cysteine peptidase-like activity. Group I – cancer patients without metastases after radical surgery and before adjuvant therapy. Group II – cancer patients without metastases after radical surgery and adjuvant therapy in complete remission. Group III – cancer patients with disseminated disease and during palliative therapy. Group IV – cancer patients without metastases with mutations in BRCA-1. Group V – healthy subjects (control group)

The highest activities were found in cancer patients with a hereditary predisposition to breast cancer associated with BRCA 1 gene mutations (group IV; CP $=$ 1192.82 $\pm$ 680.91 U/ml). High activities were detected in sera of patients after surgery and adjuvant therapy (group II; CP $=$ 717.33 $\pm$ 420.44 U/ml). Lower activities were observed in patients with disseminated cancer under palliative treatment (group III; CP $=$ 448.11 $\pm$ 315.31 U/ml). The lowest activities were found in the serum from patients after surgical removal of a tumor before adjuvant therapy, samples were taken two weeks after the surgery (group I; CP $=$ 310.36 $\pm$ 137.94 U/ml). The comparison of CPA levels in sera of studied patients and control group V (CP $=$ 621.01 $\pm$ 293.36 U/ml) are presented in Table 1. The differences in the activities between control group V and cancer groups were statistically significant ( $p<$ 0.05), except from group II ( $p<$ 0.52). Statistical differences between groups were summarized in Table 2.

Table 2

Statistical differences in CPA level between examined groups of patients

Group	Group I	Group II	Group III	Group IV	Group V
Group I	–	$p<$ 0.001	$p<$ 0.21	$p<$ 0.001	$p<$ 0.001
Group II	$p<$ 0.001	–	$p<$ 0.002	$p<$ 0.002	$p<$ 0.52
Group III	$p<$ 0.21	$p<$ 0.002	–	$p<$ 0.001	$p<$ 0.016
Group IV	$p<$ 0.001	$p<$ 0.002	$p<$ 0.001	–	$p<$ 0.001
Group V	$p<$ 0.001	$p<$ 0.52	$p<$ 0.016	$p<$ 0.001	–

In the studied series of breast cancer patients, there was no statistically significant association between the level of proteolytic enzymes in serum and tumor size (TNM) or estrogen receptor expression in tumor cells.

4. Discussion

Over recent years, many diagnostic methods have been developed to support the early detection of breast cancer. In some cases, malignant neoplasms are now diagnosed in very early stages, and in some cases, diagnosis is possible even for pre-neoplastic lesions. Information on such lesions can be obtained from screening studies and clinical assessment.

However, there is a need to find additional analytical tests, which would complement mammography. The diversity of breast cancer, including subclones of cells with different epigenetic and phenotypic characteristics, is particularly complicating reliable prognosis and monitoring of the therapy of the disease. This heterogeneity has also significant clinical and therapeutic consequences in terms of patient response to hormonal and targeted therapies.

Therefore, there is a great need for reliable diagnostic markers both for early detection and prognosis, as well as to individualize the treatment and identify new target molecules for future therapies.

The overexpression of cysteine peptidases in serum suggests that these enzymes are actively involved in carcinogenesis. Many studies demonstrate that the enzymes from cysteine peptidases group play a key role in a wide range of carcinogenesis processes and they are described as a potential target in cancer therapy [32]. This process has been investigated in detail using cancer cell lines and in experimental animal models. Much effort has been devoted to elucidate their role in breast cancer progression and metastasis, as well as to assess their diagnostic utility [23, 33]. High levels of cathepsin B and L expression at both transcriptional and protein levels have been observed. Several studies reported striking imbalance between their levels and endogenous inhibitor concentrations. Prognostic value of the enzyme levels for certain subsets of patients have been postulated [1, 39].

Most of the collected data have been based on immunohistochemical or immunoenzymatic determinations of the expression of cathepsins. These changes do not, however, necessarily translate into elevated activity of the enzymes as they are synthesized in an inactive proenzyme forms that require activation. In addition, in cancer state there are changes in the expression of endogenous cysteine protease inhibitors that impact the activity of mature cathepsins [2].

The present study compares the CPA levels in sera of patients with breast cancer at the different stage of treatment and in respect to the basic parameters that are routinely assessed during diagnosis and therapy of the disease. CPA assay, that was applied in this study, is based on determination of an peptidase activity against a synthetic substrate Z-Phe-Arg-AMC in a sensitive fluorimetric assay, as originally described by Barrett and Kirschke [3] and later modified by Lah et al. [25].

In biological fluids, the majority of activity measured using this substrate reflects CatB and L proteolytic capacity, although other proteases, such as CatS and certain non-cysteine peptidases may be also involved to minor extent. The method was previously used by Lah et al. [24] for combined Cat B and L activity determination in cytosol fractions of less abundant in sera of patients with malignant tumors than those tissues obtained from breast cancer patients.

In our study, we did not observe statistically significant differences in CPA with established prognostic parameters, such as age, tumor size or estrogen receptor status. Similarly, only limited associations between those parameters and serum CatB concentration measured using ELISA were detected in the studies of Decock et al. [7] and Foekens et al. [10]. However, it should be stressed that a correlation between serum CatB activity and its concentration is not obvious because of the presence of circulating endogenous inhibitors, such as cystatin C and kininogens.

Significantly lower level of CPA was observed in patients after radical surgery as compared to preoperative patients, indicating that the tumor tissue is a main source of the activity in serum. This underscores a role of surgical intervention in eradication of metastatic environment in circulation. In concordance, several studies showed that mastectomy is one of the most important and effective therapeutic maneuvers in the treatment of breast cancer [29]. Slightly higher CPA was observed in sera of patients after adjuvant chemo- or radiotherapy, suggesting that these interventions may raise the level of circulating cathepsins or influence cathepsins/endogenous inhibitors balance [32]. Neither serum proteolytic capacity associated with cathepsins nor their concentrations in respect to applied adjuvant therapy were compared in the other studies so far. Elevated CPA level may be associated with plasma release of cathepsins from other non-cancer tissues in the course of treatment due to general chemotherapeutics’ toxicity. On the other hand, cathepsins levels in the tumor tissue might have been reduced, despite of their serum elevation [38]. Data from tumor extracts show that low cathepsin L concentration is a predictive factor for response to chemotherapy [18].

An interesting finding of this study is that patients with disseminated cancer have relatively low serum CPA levels as compared to other groups. Compatible observations were reported by Leto et al. [26], who measured serum CatL using ELISA. Their results showed a decline in the circulating levels of CatL running in parallel with the progression of this malignant disease. The level of cathepsin L, cystatin C and other proteins in the serum of female patient with breast cancer was determined and the correlation of these proteins with some clinical-pathological parameters of breast cancer progression was assessed, using the ELISA test. The results which were obtained proved these proteins to be markers of breast cancer and bone metastasis. The authors suggest that these proteins should play an important role as circulating biomarkers in the diagnostics and monitoring of patients with breast cancer. Therefore, our findings complement the results which were obtained by Leto et al. [26].

Several studies demonstrated overexpression of ca-thepsins in cancer tissues, including very invasive and highly metastatic inflammatory breast cancer [31, 41]. In some reports, however, no or inverse correlation was observed. Chen and Platt found higher levels of tissue CatL in patients from early stages of the disease than those measured in tissues from the late stages [5]. Accordingly, CatB/cystatin C complex determined by ELISA was found to be less abundant in sera of patients with malignant tumors then those with benign diseases and healthy controls in the study of Kos et al. [22]. This is also consistent with the observation of Harbeck et al. [17], who demonstrated that tissue CatB level determined by ELISA was significantly lower in patients with lymph node positive status as compared to negative ones. There was no correlation between both CatB, CatL activities or concentrations and lymph node status in the study of Lah et al. [24]. Thus, these and our studies suggest a preferential role of cathepsins in early phases of breast cancer progression.

A striking observation of this study is very high CPA level in sera of patients with BRCA-1 mutations. There are no reports concerning cysteine peptidases activity or concentration in serum in respect to inherited predispositions to breast cancer. Nuclear CatL was shown to be upregulated in patients with a either germline or somatic mutations in BRCA1 tumor suppressor gene [16]. It is suggested that nuclear CatL confers a survival advantage on BRCA1 deficient tumor cells by improving their DNA repair capacities. The mechanism might be also a cause of the resistance to radiation and genotoxic chemotherapeutics [40]. The parameter was a predictive marker for treatment response in this subset of breast cancer patients [6]. An aspartyl peptidase D and cysteine cathepsin L expression was examined in cancer tissue specimens from patients with BRCA-1 and BRCA-2 mutations but no associations were found [11]. While, determining cathepsins L and S-like activity, they were established as prognostic markers [16, 21, 34].

The aim of investigating the cysteine peptidases-like activity was to prove that in the body of patients with cancer two dynamic processes occur: cancer aggression against healthy tissues/organs, which has been demonstrated in the present study, and the ability of the organism to defend itself against this aggression with autogenic peptide inhibitors. We presented the activity of inhibitors in the serum of female patients with breast cancer in our study from 2015 [20]. Many researchers, including the authors of the present work, suggest that the intensity of cancer progress is related to two opposite to each other key processes: cancer aggression and organism defence. In many cases, intervention, or therapy, consists in direct or indirect intervention in these processes, i.e. aggression. The subject of our study is the recognition of the correlations between the level of active cysteine peptidases and active inhibitors which are able to control the activity of these peptidases. Therefore, our present and previous works are interconnected. Still, the occurrence of the activity of both cysteine peptidases-like and autogenic peptide inhibitors, which are able to inhibit plant cysteine peptidase, or papain, has not been yet explained.

It is worth noting that a direct comparison of antigen content and activities of cathepsins is not feasible as immunological methods detect not only the active molecules but also the enzymes preforms and their complexes with inhibitors. Furthermore, we consider CPA as an independent parameter to measure for diagnostic purposes rather than a parameter strictly reflecting a subset of particular cysteine peptidases derived activity or their expression levels [13, 35].

The presented findings, in which we determine cathepsins B and L – like activity did not always coincide with the ones obtained when cathepsins B and L determining with the use of available ELISA tests to establish their level. Establishing de facto different parameters of the same enzymes does not always lead to similar findings. The ensuing various results cannot be compared, because of differences in measurement.

In our study, the activity (not concentrations) of cathepsin B and L was measured by means of specific fluorogenic substrate. The aim of the study was to focus on cancer therapies, in which the immediate inhibition of the activity of the tumor–associated cysteine peptidases is applied. This method turned out to be an effective approach to the selective, clinical treatment of cancer and metastases. Contrary to the evaluation of cathepsin B and L concentration using ELISA assay, this method of diagnosing breast cancer has not been investigated in detail as yet [35].

Taking into account, our findings and the simplicity of the CPA assay it is tempting to refine its diagnostic utility for breast cancer patients in more detail. The studies should involve an expanded patients’ characteristic and a larger study group. A panel of specific peptidase inhibitors might be also introduced in the assay in order to more closely delineate its molecular meaning. Our study was the first to demonstrate the correlation between BRCA-1 gene mutations and the increased level of CPA. However, the molecular mechanisms of this process are unknown and require further research.

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Differences in cysteine peptidases-like activity in sera of patients with breast cancer

Abstract

BACKGROUND:

OBJECTIVES:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Clinical material

2.2 Determination of cysteine peptidases-like activity

2.3 Identification of BRCA-1 gene mutations

2.4 Statistics

3. Results

Table 1 Clinical characteristic of breast cancer patients and CPA level in serum

References

Table 1
Clinical characteristic of breast cancer patients and CPA level in serum