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Biochimica et Biophysica Ac
Enzymes that hydrolyze adenine nucleotides in platelets from
breast cancer patients
Maria do Carmo Araujoa, Joao Batista Teixeira Rochab, Andre Morschb, Rafael Zaninb,
Rita Bauchspiessa, Vera Maria Morschb, Maria Rosa Chitolina Schetingerb,*
aHospital Universitario de Santa Maria–HUSM, Centro de Ciencias da Saude, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, BrasilbDepartamento de Quımica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, 97105-900, RS, Brasil
Received 28 May 2004; received in revised form 14 October 2004; accepted 1 November 2004
Available online 18 November 2004
Abstract
The activities of NTPDase (EC 3.6.1.5, apyrase, CD39) and 5V-nucleotidase (EC 3.1.3.5, CD73) enzymes were analyzed in platelets from
breast cancer patients. Initially, patients were compared in terms of length (years) of tamoxifen use. The following groups were studied:
breast cancer patients who did not use tamoxifen, patients using tamoxifen for 1–48 months, patients using tamoxifen for 49–84 months, and
controls (healthy subjects). Results demonstrated that adenosine triphosphate (ATP) hydrolysis was enhanced (F(3,114)=8.53; Pb0.001) and
adenosine diphosphate (ADP) hydrolysis was reduced (F(3,106)=5.09, P=0.002) as a function of tamoxifen use, while adenosine
monophosphate (AMP) hydrolysis was unchanged. Next, patients were compared statistically according to disease stage, determined by the
tumor–node–metastasis (TNM) staging system for classifying breast tumor. ATP hydrolysis was significantly elevated in patients with stage I
and II breast cancer (F(4,113)=4.35; P=0.003), but was normal in patients with stage III and IV cancer. ADP hydrolysis was reduced in
stages II to IV (F(4,105)=3.88, P=0.006) and AMP hydrolysis was elevated in stage II (F(4,105)=3.45 P=0.01), but was normal in stages III
and IV. Platelet aggregation time was similar in all patients regardless of tamoxifen use or disease stage. Prothrombin time (PT) and activated
partial thromboplastin time (APTT) were also within the normal range and similar among all groups. Similarly, fibrinogen and fibrin
degradation product (FDP) were unchanged in all groups. In conclusion, our study demonstrated for the first time that hydrolysis of adenine
nucleotides is modified in platelets from breast cancer patients taking tamoxifen.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Breast cancer; Tamoxifen; NTPDase; 5V-nucleotidase; Platelet
1. Introduction
Thromboregulation is a process or group of processes
by which circulating blood cells and vessel wall cells
interact to regulate or inhibit thrombus formation [1,2].
Platelets are one of the most important blood components
that participate in and regulate thrombus formation by
releasing active substances such as ADP [1,3]. It is known
0925-4439/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2004.11.001
* Corresponding author. Fax: +55 552208978.
E-mail address: [email protected]
(M.R. Chitolina Schetinger).
that micromolar concentrations of ADP are sufficient to
induce human platelets aggregation, whereas adenosine
(the final product of ATP hydrolysis) can inhibit platelet
aggregation [4–6]. Furthermore, the roles of nucleotides
and nucleosides as extracellular signaling molecules have
been well established. More recently, there is growing
interest in the long-term trophic actions of extracellular
nucleotides and nucleosides on cell growth, proliferation
and death [7].
NTPDase (EC 3.6.1.5, CD39, ecto-apyrase, ATP
diphosphohydrolase) is a glycosylated membrane-bound
enzyme that hydrolyzes ATP and ADP to AMP, which is
subsequently converted to adenosine by 5V-nucleotidase(EC 3.1.3.5, CD73). Both NTPDase and 5V-nucleotidase
ta 1740 (2005) 421–426
M. do Carmo Araujo et al. / Biochimica et Biophysica Acta 1740 (2005) 421–426422
are located in the platelet membrane [5,8,9]. NTPDase and
CD73 play an important role in the regulation of blood
flow and thrombogenesis by regulating ADP catabolism
[6,9].
The association between clinical venous thromboemb-
olism (VTE) and cancer has been recognized in the
medical literature [2,10,11]. The interactions between
components of the hemostatic system and cancer cells
are multifaceted. Patients with cancer may exhibit
increased platelet activation. Cancer cells also activate
platelets in vitro by contact, releasing ADP, thromboxane
A2 and generating thrombin through the activity of tumor-
associated procoagulants [10]. The magnitude of the
problem of VTE in cancer is related to various therapeutic
interventions [12].
Breast cancer is the most frequent female cancer in the
world [13]. TNM staging system has become the accepted
method for classifying breast tumors. According to TNM,
adapted by the International Union against Cancer (IUCC),
patients are assigned to stages I, II, III, IV [14]. There is
evidence indicating that the amount of estrogen available
to breast tissue is a critical factor in the cause of human
breast cancer [15]. Tamoxifen is a drug of choice for the
adjuvant treatment of breast cancer and its ability to
prevent growth of cancer cells is due to a broader range of
effects on cancer cells than just its blocking of estrogen
[16]. However, it is known that tamoxifen can increase the
chance of developing blood clots in veins (phlebothrom-
bosis) [17].
Although thrombosis is one of the most severe
problems in patients with cancer [2,10], many aspects of
prothrombotic conditions in cancer still need to be
clarified. Another aspect that must be emphasized here
is the fact that adenine nucleotides and nucleosides,
particularly adenosine and AMP, play a complex role in
breast cancer growth [18,19]. Of particular importance,
adenosine stimulates the proliferation of certain tumor
cells [20], whereas AMP inhibits the growth of breast
cancer cells [21]. In an attempt to investigate the potential
role of enzymes that participate in the hydrolysis of ATP,
ADP and AMP in the etiology of breast cancer, as well as
the possible interference of tamoxifen with adenine
nucleotide hydrolysis, we examined the influence of the
breast cancer stage and of the use of tamoxifen on platelet
NTPDase and 5V-nucleotidase, two enzymes involved in
thrombus regulation.
2. Patients and methods
2.1. Materials
Nucleotides, sodium azide, HEPES, and Trizma base
were purchased from Sigma (St. Louis, USA). All other
reagents used in the experiments were of analytical grade
and of the highest purity.
2.2. Patients
The sample consisted of female patients with breast
cancer, histologically identified as infiltrating ductal breast
carcinoma, aged 20 to 85 years, under treatment at the
Federal University of Santa Maria Hospital.
The sample was first divided into four groups: The
group not treated with tamoxifen (No Tam) consisted of
15 patients (49.1F12.64 years old) with diagnosed breast
cancer who did not receive any interventions such as
chemotherapy, hormone therapy, surgery or radiotherapy.
Group tamoxifen 1–48 mo consisted of 65 patients
(56F12.59 years old) submitted to adjuvant hormonal
therapy with tamoxifen (20 mg/day) for 1 to 48 months.
Group tamoxifen 49–84 mo consisted of 11 patients
(68F13.03 years old) submitted to adjuvant hormonal
therapy with tamoxifen (20 mg/day) for 49 to 84 months.
The control group, selected by clinical evaluation,
consisted of 30 women (52.42F2.63 years old) who
did not present any disease and had not been submitted
to any pharmacological therapy during the collection
period. All subjects gave written informed consent to
participate in the study. The protocol was approved by
the Human Ethics Committee of the Health Science
Center, Federal University of Santa Maria (Protocol
number: 100/02). Eight milliliters of blood was obtained
from each participant and used for platelet-rich plasma
preparations, biochemical determinations and hematolog-
ical determinations.
Patients were further subdivided according to the stage
of breast cancer. The TNM staging system has become the
accepted method for classifying breast tumors. According
to the TNM, adapted from the IUCC, patients are assigned
to stages I, II, III, IV [14]. All patients from stages I, II, III
and IV were estrogen receptor (ER)-positive and, con-
sequently, were treated with tamoxifen. The total score
(intensity score+proportionality score) for ER from stage I
patients ranged from 5/8 to 8/8; for stage II patients, the
score ranged from 2/8 to 8/8; for stage III patients the
score varied from 5/8 to 8/8; and for stage IV the score
varied from 3/8 to 8/8.
2.3. Estrogen receptor (ER) staining
ER in tumors was determined by immunocytochemical
assay using the Novocastra 6F11 mouse monoclonal anti-
body [22].
2.4. Platelet-rich plasma (PRP) preparation
Platelet-rich plasma was prepared from human donors
by the methods of Pilla et al. [8] and Lunkes et al. [23].
Briefly, blood was collected into 0.129 M citrate and
centrifuged at 160�g for 10 min. The platelet-rich plasma
was centrifuged at 1400�g for 15 min and washed twice
with 3.5 mM HEPES isosmolar buffer containing 142
Table 1
Blood parameters
Groups/Tam Platelet
aggregation (s)
Groups/stage Platelet
aggregation (s)
A (n=30) 35.0F0.34 I (n=14) 35.0F0.50
B (n=15) 34.1F0.98 II (n=36) 33.0F0.68
C (n=11) 33.1F1.23 III (n=17) 33.8F0.87
D (n=65) 33.5F0.48 IV (n=21) 33.4F1.10
Platelet aggregation consisted of the in vitro macroscopic visualization of
aggregates at intervals of 15 to 50 s by the addition of ADP to platelet-rich
preparation (PRP). Values represent meanFstandard error. Groups:
A=control; B=no tam; C=1–48 mo; D=49–84 mo.
M. do Carmo Araujo et al. / Biochimica et Biophysica Acta 1740 (2005) 421–426 423
mM NaCl, 2.5 mM KCl, and 5.5 mM glucose. The
washed platelets were resuspended in HEPES buffer and
protein was adjusted to 0.3–0.5 mg/ml where 6–10 Ag of
protein was used per tube to ensure linearity in the
enzyme assay. NTPDase is an ecto-enzyme and thus
platelet viability and integrity were confirmed by the
measurement of lactate dehydrogenase (LDH) activity
using the enzymatic Cobas Integra 400 method (Cobas,
Basel, Switzerland).
2.5. NTPDase and 5V-nucleotidase determinations
Twenty microliters of the PRP preparation (10–15 Agprotein) was added to the reaction mixture of NTPDase or 5V-nucleotidase and preincubated for 10 min at 37 8C, to a finalvolume of 200 AL. NTPDase activity was determined by the
method of Pilla et al. [8], in a reaction medium containing 5.0
mM CaCl2, 100 mM NaCl, 4.0 mM KCl, 50 mM glucose,
and 50 mM Tris–HCl buffer, pH 7.4. The reaction was
started by the addition of ATP or ADP as substrate at a final
concentration of 1.0 mM. 5V-Nucleotidase was determined
by the method of Heymann et al. [24] in a reaction medium
containing 10 mMMgCl2, 100 mM Tris–HCl buffer, pH 7.4.
NTPDase and 5V-nucleotidase reactions were stopped by the
addition of 200 AL of 10% trichloroacetic acid (TCA) to
provide a final concentration of 5%. The inorganic phosphate
(Pi) released by ATP, ADP and AMP hydrolysis was
measured by the method of Chan et al. [25] using KH2PO4
as standard. Controls were prepared to correct for non-
enzymatic hydrolysis by adding PRP after TCA addition. All
samples were run in triplicate. Enzyme activities are reported
as nmol Pi released/min/mg protein.
2.6. Hematological determinations
Quantitative determinations of platelets obtained by
venipuncture were performed using a Coulter-STKS analyzer
(Miami, USA). Platelet aggregation was performed by the
technique of Biggs [26], consisting of the in vitro macro-
scopic visualization of aggregates at intervals of 15 to 50 s by
the addition of ADP to platelet-rich preparation (PRP). PT,
APTT and fibrinogen were determined with a Coag-a-mate-
MTX apparatus (Organon Teknika, Durham, NC, USA). The
presence of fibrin degradation products (FDP) was detected
by a quantitative and semiquantitative latex slide test using
the Hemoliance–Dimertest (Behring, Marburg, Germany).
2.7. Protein determination
Protein was determined by the Coomassie blue method
using bovine serum albumin as standard [27].
2.8. Statistical analysis
Data were analyzed statistically by one-way ANOVA
followed by the Duncan test when the F-test was significant
(Pb0.05) to determine the differences between the study
groups and the control.
3. Results
3.1. Influence of tamoxifen on coagulation parameters
The levels of platelets obtained from all patients were
within normal limits, with values ranging from 200,000 to
400,000 platelets/mm3. Platelet integrity was determined by
comparing the LDH activity obtained after lysis with Triton
X-100 with that of intact platelets. Less than 4% of platelets
were disrupted (data not shown), indicating that the PRP
preparation was predominantly intact. By the absence of an
inhibitory effect of levamisole and a modest hydrolysis of h-glycerolphosphate (less than 2%), the presence of non-
specific phosphatase in the PRP preparation was excluded
(data not shown).
Platelet aggregation time was similar in the tamoxifen
groups (Table 1). PT and APTT were also within the
normal range and similar among groups (data not shown).
FDP was not detected in subjects from all groups (data not
shown).
3.2. Influence of length of tamoxifen use on platelet ATP,
ADP and AMP hydrolysis
ATP hydrolysis was enhanced as a function of length of
tamoxifen use (F(3,114)=8.53; Pb0.001) and post-hoc
comparisons by Duncan’s test revealed that ATP hydrolysis
was significantly higher in patients receiving tamoxifen
from 1 to 48 and 49 to 84 months (Fig. 1A).
ADP hydrolysis was reduced as a function of tamoxifen
use (F(3,106)=5.09, P=0.002) and post-hoc comparisons by
Duncan’s multiple range test revealed that tamoxifen groups
had a significant reduction in platelet ADP hydrolysis
compared to control subjects (Fig. 1B).
AMP hydrolysis was not significantly altered by tamox-
ifen use but tended to increase as a function of the length of
drug use (F(3,106)=2.10; P=0.10; Fig. 1C).
Fig. 1. Effect of tamoxifen on ATP (A), ADP (B) and AMP (C) hydrolysis
in platelets from breast cancer patients. NTPDase is specified by ATP and
ADP hydrolysis and 5V-nucleotidase by AMP hydrolysis. The group was
divided in No Tam (n=15), group 1–48 mo (n=65), group 49–84 mo (n=11)
and control group (n=30) as described in Patients and methods. (a,b)
Indicates a significant difference at Pb0.05 from columns not labeled with
these letters (Duncan’s multiple range test).
Fig. 2. ATP (A), ADP (B) and AMP (C) hydrolysis in the different breast
cancer stages. NTPDase is specified by ATP and ADP hydrolysis and 5V-nucleotidase by AMP hydrolysis. Patients were subdivided according to
TNM, adapted by the International Union against Cancer (IUCC), assigned
to stages I (n=14), II (n=36), III (n=17), IV (n=21) as described in Patients
and methods. (a) Indicates a significant difference at Pb0.05 from columns
not labeled with this letter (Duncan’s multiple range test).
M. do Carmo Araujo et al. / Biochimica et Biophysica Acta 1740 (2005) 421–426424
3.3. Influence of breast cancer stage on platelet ATP, ADP
and AMP hydrolysis
ATP hydrolysis was modified by the breast cancer stage
(F(4,113)=4.35; P=0.003) and post-hoc comparisons by
Duncan’s test revealed that ATP hydrolysis was signifi-
cantly higher in patients with stage I and II cancer, but
returned to normal values in patients with stage III and IV
cancer (Fig. 2A).
ADP hydrolysis was reduced in breast cancer patients
(F(4,105)=3.88, P=0.006) and post-hoc comparisons by
Duncan’s multiple range test revealed that patients with stage
II to IV cancer had a significant reduction in platelet ADP
hydrolysis when compared to control subjects (Fig. 2B).
AMP hydrolysis was modified by breast cancer staging
(F(4,105)=3.45 P=0.01) and post-hoc comparisons by
Duncan’s test revealed that AMP hydrolysis was significantly
higher in patients with stage II cancer, but returned to normal
values in patients with stage III and IV cancer (Fig. 2C).
Platelet aggregation time was similar in patients in the
different cancer stages (Table 1). PT and APTT were also
within the normal range and similar among groups (data
not shown). Similarly, FDP analysis indicated negative
results for all groups (data not shown).
4. Discussion
The present results clearly demonstrate that hydrolysis of
nucleotides by platelets is changed in breast cancer patients.
M. do Carmo Araujo et al. / Biochimica et Biophysica Acta 1740 (2005) 421–426 425
The changes seem to depend on both the stage of the disease
and on whether the patient was receiving adjuvant therapy
with tamoxifen. In fact, ATP hydrolysis was increased during
the initial stages of cancer (I and II) and in patients treated
with tamoxifen. In sharp contrast, platelet ADP hydrolysis
was reduced both as a function of breast cancer stage and of
tamoxifen use. The influence of the breast cancer stage or
tamoxifen on AMP hydrolysis tended to be similar to the
effects observed on platelet ATP hydrolysis. However, a
parallelism was observed for the breast cancer stage, with the
detection of a significant increase in AMP hydrolysis in
patients with stage II cancer. AMP hydrolysis tended to be
increased as a function of length of tamoxifen use. Taken
together, these results suggest that the breast cancer stage and
tamoxifen use have a rather complex influence on nucleotide
hydrolysis by platelets. Recently, Spychala et al. [19]
demonstrated that tamoxifen increases 5V-nucleotidaseexpression in breast cancer cells. This study has reinforced
the importance of the study of nucleotidase activitiy in
cancer. Also, they demonstrated that ER-negative cells
express high 5V-nucleotidase protein and mRNA levels and
produce much more adenosine from AMP and ATP.
Literature data indicate that adenine nucleotides and
adenosine have an important role in tumor growth
[18,19,28]. Of particular importance, there are lines of
evidence showing that adenosine can function as a stimulant
of tumor growth, whereas AMP has the opposite effect [19].
Since the breast cancer stage and length of tamoxifen use
are associated with changes in the hydrolysis of all adenine
nucleotides, it is difficult to link these changes to tumor
progression in these patients. Recently, Spychala and
Kitajewski [29] suggested that the increase in the generation
of adenosine by tumors could be correlated with the
development of drug resistance and the more aggressive
course of the disease. They reported that specific oncogenic
alteration causes increased expression of 5V-nucleotidase,thus increasing the ability of cells to generate adenosine
during tumorigenesis. However, the results of ATP hydrol-
ysis agree with previously published data showing that
expression of ecto-ATPases is increased during cancer
progression [20,30]. One interesting observation made in
the present study was that ATP and ADP hydrolysis was
modulated differently in relation to the stage of cancer or
use of tamoxifen. In fact, ATP hydrolysis increased, whereas
ADP hydrolysis decreased. This may indicate that more than
one enzymatic activity is being affected differently by these
conditions.
Cancer has been reported to be associated with problems
in platelet aggregation and, most frequently, with blood
coagulation [10,11]. The general tendency towards an
increase in ATP hydrolysis and a decrease in ADP
hydrolysis could locally generate an augmented ADP
concentration, which in turn could facilitate platelet
activation in patients with breast cancer or those using
tamoxifen. In fact, there are some indications in the
literature that tamoxifen could lead to an increase in platelet
activity [2,17,22,30] and that most human solid tumors can
actively release ATP [19,28]. Thus, it is plausible to suggest
that changes in activities that metabolize adenine nucleo-
tides on the surface of platelets may be involved in platelet
activation.
Another important factor to be addressed is that the
activity measured on platelets is low compared to that of
endothelial cells and leucocytes which importantly interact
with platelets in vivo and influence platelet aggregation and
thus the formation of thrombus [1,6]. Therefore, the
combined activities on the surface of these cells may have
an important role in the local regulation of nucleotide levels
in the blood.
In conclusion, our study demonstrated for the first time
that the hydrolysis of adenine nucleotides is modified in
platelets from breast cancer patients by the use of tamoxifen,
the most common adjuvant in breast cancer therapy. It is still
premature to propose the implications of these changes for
cancer development, but we may suggest that these may be
related to the changes in platelet activity observed in cancer
patients using tamoxifen.
Acknowledgements
The authors wish to thank Conselho Nacional de
Desenvolvimento Cientıfico e Tecnologico (CNPq), Funda-
cao de Amparo a Pesquisa de Rio Grande do Sul
(FAPERGS) and Fundacao Coordenacao de Aperfeicoa-
mento de Pessoal de Nıvel Superior (CAPES).
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