Adenosine deaminase activity in the serum and malignant tumors of breast cancer: The assessment of isoenzyme ADA1 and ADA2 activities
Mahmood Aghaeia, Fatemeh Karami-Tehrania, , , Siamak Salamiaand Morteza Atrib
aClinical Biochemistry Department, Cancer Research Laboratory, School of Medical Science, Tarbiat Modarres University, PO Box: 14115-111, Tehran, Iran
bDepartment of Surgery, School of Medical Science, Tehran University of Medical Science, Tehran, Iran
Received 1 February 2005; revised 4 May 2005; accepted 25 May 2005. Available online 28 July 2005.
Abstract
Objective:
The potential relationship between adenosine deaminase activity and cancer progression was examined by investigating the activity of total ADA and its isoenzymes in serum and simultaneously in the cancerous tissue of each patient with breast cancer.
Methods:
Total ADA and its isoenzymes were measured using the Giusti method. ADA2 activity was measured in the presence of a specific ADA1 inhibitor, EHNA.
Results:
Our results indicated that ADA2 and total ADA activities were higher in serum and malignant tissues than those of corresponding controls (P < 0.05). Tumor ADA2 and total ADA activities were significantly (P < 0.05) correlated with lymph node involvement, histological grade and tumor size, whereas their levels in serum were significantly (P < 0.05) correlated with menopausal status and patient age.
Conclusion:
Although serum and tumor total ADA activity and its ADA2 isoenzyme were both found to be increased, distinct correlation patterns were observed with some of the prognostic factors. It can be speculated that increased ADA and isoenzyme activities in serum originated from sources other than the breast tumors.
Keywords: Adenosine deaminase; Isoenzyme; Breast cancer
Abbreviations: ADA, adenosine deaminase; EHNA, erytro-9-(2-hydroxy-3-nonyl) adenine; ADA1, adenosine deaminase isoform 1; ADA2, adenosine deaminase isoform 2
Article Outline
Serum total ADA activity and its isoenzymes
Tumor total ADA activity and its isoenzymes
Introduction
Breast cancer is the most common malignancy in women and comprises 18% of all cancers [1]. In order to characterize human breast cancer, numerous studies have focused on enzyme activities [2]and [3]. Alteration of enzyme activity has been clarified in human breast tumors. To better understand purine enzymology in breast cancer, more attention has been paid to investigate the interrelationship between the carcinogenic process and enzyme activity (de novo and salvage pathways) for purine biosynthesis.
Adenosine deaminase (ADA, E.C.3.5.4.4) is an enzyme that catalyses deamination of either adenosine or deoxyadenosine. Due to the irreversibility of the reaction catalyzed by ADA, the reaction appears to be one of the rate limiting steps in adenosine degradation [4]. There are two isoenzymes of ADA in human tissues, ADA1 and ADA2 [5]and [6]. ADA1 is present in all human tissues and the majority of ADA activity is derived from ADA1. However, ADA2 is the predominant isoenzyme in the serum of normal subjects [7]. Most human cells contain very small amounts of ADA2 and its major source is likely to be the monocyte–macrophage cell system [8]. In several studies, ADA activities were found to be increased or decreased in cancerous tissues and cells [9], [10], [11], [12], [13], [14], [15], [16], [17]and [18]. Most of these studies focused on purine enzymatic differences in cancerous tissues or cells as well as normal tissue. However, the patterns of ADA1, ADA2 and total ADA and their correlations with prognostic factors have not been elucidated in women with breast cancer.
The aim of this study was to investigate the activity of total ADA and its isoenzymes, ADA1 and ADA2, in the serum and cancerous tissues of patients with breast cancer and to evaluate the relationship between these enzyme activities and some clinicopathological factors of the disease.
Materials and methods
With local ethical approval and informed consent, 34 patients with breast cancer were included in this study. Samples of breast cancer tumors were collected at surgery between September 2003 and March 2004. Patients received no therapy prior to surgery. Patients were evaluated on the basis of age, menopausal status, tumor grade, tumor size and lymph node involvement. Normal adjacent tissue was sampled away from the edge of a tumor region, and tumor samples were taken from non-necrotic proliferative regions after their surgical removal. The fresh surgical specimens were frozen immediately and stored at −80°C until assayed. Tissue samples were washed out from contaminated blood with cold water and were homogenized in equal amounts of phosphate buffer solution (pH = 6.5). The homogenate was centrifuged at 15,000 × g for 60 min. The clear upper supernatant fluid was taken and assays were performed. All procedures were done at 4°C. Fasting blood was collected in 5-mL tubes without using any anticoagulant. The blood samples were then centrifuged at 3000 × g for 10 min at 4°C to obtain serum.
Protein was determined by the Bradford method [19]. Total ADA activity and its isoenzymes (ADA1, ADA2) were estimated spectrophotometrically by the Giusti method [20], which is based on the direct measurement of the formation of ammonia produced when ADA acts in excess of adenosine. ADA2 activity was measured in the presence of a selective ADA1 inhibitor, erytro-9-(2-hydroxy-3-nonyl) adenine (EHNA). Total ADA activity was measured in the absence of EHNA, and ADA1 activity was then calculated by subtracting the ADA2 activity from the total activity. The sensitivity of the assay was 0.5 U/L, with intra- and inter-assay coefficients of variation (CV) of 3.9 and 6.4%, respectively.
Data were expressed as mean ± standard deviation (SD). The Mann–Whitney U test and Spearman analysis were used for the analysis of differences among the groups. A P value of less than 0.05 was considered statistically significant. The statistical analysis was performed by the Statistical Package for Social Science (SPSS).
Results
Serum total ADA activity and its isoenzymes
As illustrated in Fig. 1, serum total ADA and ADA2 activities in patients with breast cancer were significantly higher (P < 0.0001) than healthy subjects but ADA1 activity was not significantly increased (P > 0.05). Serum total ADA and ADA2 activities were significantly higher in post-menopausal than pre-menopausal patients (Fig. 2). No significant correlation was found between serum total ADA, ADA1 and ADA2 activities and other prognostic factors such as a tumor's size or grade, histological types and lymph node involvement.
Fig. 1. Serum total ADA, ADA1 and ADA2 activities in breast cancer patients and healthy controls.
Fig. 2. Serum total ADA, ADA1 and ADA2 activities in patients with breast cancer according to the menopausal status.
Tumor total ADA activity and its isoenzymes
Tumor total ADA, ADA1 and ADA2 activities were significantly higher (P < 0.0001) than non-cancerous adjacent breast tissues (Fig. 3). No significant correlation was found between tumor total ADA, ADA1 and ADA2 activities and menopausal status, but tumor total ADA activity and its ADA2 isoenzyme were significantly higher in grade III (Fig. 4), lymph node involvement (Fig. 5) and larger tumor size (Fig. 6). No correlations were found between tumor and serum ADA activities (r = 0.08, P > 0.05).
Fig. 3. Total ADA, ADA1 and ADA2 activities in breast tumor tissues in comparison to normal tissues.
Fig. 4. Total ADA, ADA1 and ADA2 activities in breast tumor tissues according to the histological grade.
Fig. 5. Total ADA, ADA1 and ADA2 activities in tumor tissues according to the lymph node involvement.
Fig. 6. Total ADA, ADA1 and ADA2 activities in tumor tissues according to the tumor size.
Discussion
Several studies have been carried out to assess the role of various enzymes in breast cancer and it has long been known that there are important relationships between the carcinogenic process and the activities of some enzymes in cancerous tissues and cells.
Total ADA activity has been studied in patients with various tumors. Some authors suggest that high ADA activities take an important part in the salvage pathway activity of cancerous tissues and cells [10]and [21], while others propose that increased ADA activity may be a compensatory mechanism against toxic accumulation of its substrates due to accelerated purine and pyrimidine metabolism in the cancerous tissues and cells [22]and [23]. High ADA activity in cancerous tissues and cells might give selective advantage to the cancer cells by causing production of high amounts of hypoxanthine, which is one of the substrates of hypoxanthine guanine phosphoribosyl transferase (HGPRT), a key enzyme of the salvage pathway. Accelerated salvage pathway activity provides more mononucleotides to cancerous tissues to continue DNA synthesis needed by rapidly proliferating cancer cells. In particular, ADA might be induced by high concentrations of its substrates since the high ATP requirement of cancer cells leads to the production of toxic adenosine and deoxyadenosine substrates in high amounts, the accumulation of which causes inhibition of ribonucleotide reductase and results in some aberration in DNA synthesis [24].
Although in several studies ADA activity was found to be increased in cancerous tissue, some authors obtained low activity values in tumor tissues or lymphocytes from cancer patients [14], [15], [16]and [24]. In order to explain the discrepancy, further investigation is required.
There are several reports that serum ADA1 and ADA2 activities have been increased or decreased in several diseases where cellular immunity is stimulated [25], [26], [27]and [28]. However, the patterns of ADA1 and ADA2 activity in serum or malignant breast tumors have not been studied.
This study sought to elucidate the status of ADA activity and its isoenzymes, ADA1 and ADA2, in the serum and malignant tissues from patients with primary breast cancer and revealed that total ADA activities of tumors and serum are significantly elevated, which is mainly due to increased ADA2 isoenzyme. Although Canbolat et al. demonstrated that ADA activity in breast cancer tumors is higher than normal tissue [13]and an increased total ADA activity in the serum of breast cancer patients has been reported by Mini Valia et al. [29], the relationship between serum and tumor ADA activity, the patterns of its isoenzymes and their correlation with clinicopathological parameters have not been examined.
Our results reveal that both total ADA and its ADA2 isoenzyme activities were increased in the serum of patients with breast cancer. This increase is an age-dependent event, which correlated with menopausal status, i.e., post-menopausal age may influence both enzyme activities, but no relation to other studied prognostic factors has been found.
On the other hand, elevated tumor ADA activity (total ADA and ADA2) revealed a significant correlation with prognostic factors such as tumor grade, size and lymph node involvement in an age-independent manner. It can be speculated that increased total ADA and ADA2 activities in serum originated from sources other than the tumor.
Furthermore, correlation between tumor ADA activity and prognostic factors proves that this activity and preferentially its ADA2 isoenzyme might be due to the cancer bearing status and is proposed as an index of breast tumor proliferation, i.e., their lower activities are in favor of better prognosis. It has been reported that normal serum ADA2 may originate from monocyte/macrophage cells [8]and also it has been showed that macrophage infiltration may be found in malignant breast tumors [30]. Therefore, it would be worth evaluating ADA and its isoenzymes within homogenous selected malignant breast cells using laser microdissection or related techniques to prove or exclude a macrophage origin of increased ADA2 levels. The precise aspects of the prognostic significance of ADA and its isoenzyme activities may require further assessment.
Acknowledgments
We thank the cancer surgery department of Iran Day Hospital and Dr. Sharifian (Tehran University of Medical Science) for their kind assistance.
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