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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: mariarosa@smail.ufsm.br

(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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