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Endocrinology, doi:10.1210/en.2007-1223
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Endocrinology Vol. 149, No. 1 389-396
Copyright © 2008 by The Endocrine Society

Increased P2X7 Receptor Expression and Function in Thyroid Papillary Cancer: A New Potential Marker of the Disease?

Anna Solini, Sabina Cuccato, Davide Ferrari, Eleonora Santini, Sara Gulinelli, Maria Giulia Callegari, Angela Dardano, Pinuccia Faviana, Stephanie Madec, Francesco Di Virgilio and Fabio Monzani

Department of Internal Medicine (A.S., S.C., E.S., A.D., S.M., F.M.) and Department of Surgical Sciences, Section of Pathology (P.F.), University of Pisa, I-56100 Pisa, Italy; and Department of Experimental and Diagnostic Medicine (D.F., S.G., G.C., F.D.V.), Section of General Pathology and Interdisciplinary Centre for the Study of Inflammation, University of Ferrara, I-44100 Ferrara, Italy

Address all correspondence and requests for reprints to: Anna Solini, M.D., Ph.D., Department of Internal Medicine, University of Pisa, Via Roma 67, I-56100 Pisa, Italy. E-mail: a.solini{at}med.unipi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nucleotides are increasingly recognized as nonredundant extracellular signals for chemotaxis, cell growth, and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X7 subtype is attracting increasing attention for its involvement in apoptosis, cell growth, and cytokine release. Recent studies showed that P2X7 is overexpressed in chronic lymphocytic leukemia and breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X7 receptor expression in normal and cancer human thyroid tissues. P2X7 receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic or papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, expresses the P2X7 receptor (P2X7R) to a much higher level than normal thyroid tissue. The P2X7R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca2+ and secretion of IL-6. Ca2+ increase was attenuated and release of IL-6 was fully blocked by P2X7R inhibitors. Finally, the thyroid carcinoma cell lines had at least a 3-fold higher intracellular ATP concentration and maintained at least a 3-fold higher extracellular ATP level, compared with control cells. These data suggest that an enhanced P2X7R function might be a feature of human thyroid cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS INCREASING awareness that the tumor microenvironment has a key role in tumor growth and invasion. The biochemical composition of this milieu is crucial for the modulation of cancer cell growth as well as the functions of infiltrating leukocytes. A major source of factors found in the tumor interstitium are the cancer cells themselves. Recent findings show that the concentration of adenosine is increased in the interstitium of tumors, and that this ATP metabolite may inhibit the antitumor immune response and thus enhance invasion and metastasis (1).

The major source of extracellular adenosine is ATP (2). It is now clear that virtually all cells, tumor cell included, release ATP via nonlytic mechanisms and that secreted ATP acts as an autocrine/paracrine stimulus by acting at P2 receptors. As extracellular mediators, nucleotides participate in the regulation of cell growth and differentiation and may also be involved in neoplastic transformation (3). The P2 receptor subtype involved in these responses is, however, unclear. Recently overexpression of the P2X7 receptor was shown in chronic lymphocytic leukemia, breast and prostate cancer (4, 5, 6), and neuroblastoma (7). In addition, there is substantial evidence that the P2X7 receptor may mediate cell survival and growth by increasing the efficiency of oxidative phosphorylation and total intracellular ATP stores (8). Finally, we have shown that P2 receptor (P2R) stimulation triggers from healthy thyrocytes release of IL-6 (9), a cytokine with differentiating and growth-promoting effects in several endocrine glands (10, 11).

Thyroid cancer appears in less than 10% of hypofunctioning thyroid nodules (12). Papillary thyroid cancer, the most common histological type, accounts for approximately 60% of all thyroid cancers. It has a variable macroscopic appearance, depending on the underlying microscopic heterogeneity and the presence or absence of degenerative changes (13). The histological variants can be challenging to the pathologist for the clinical implications; in fact, some experts contend that if these tumors are small and not invasive (the usual case), then simply removing the lobe that harbors the tumor and isthmus will provide as good a chance of cure as removing the entire thyroid.

Few studies have addressed the possible growth-promoting role of extracellular ATP in thyroid cancer and the role of the various P2 subtypes (14). In the thyroid ATP is released by the rich autonomic innervation (15), capillary endothelial cells (16), and the thyrocytes themselves (9). Several effects, ranging from stimulation of thyroglobulin secretion to mobilization of Ca2+, from mitogenesis to production of H2O2, from increased efflux of I– and Cl– to inhibition of forskolin-stimulated Na+ absorption and modulation of cytokine release (9, 17, 18, 19, 20), have been described in the Fisher rat thyroid cell line FRTL-5 (17) and cultured human thyrocytes (18). Of particular relevance is the ability of thyrocytes to actively secrete ATP because this suggests that thyroid cells may be able to create and maintain an ATP-rich microenvironment within the gland.

In the present study, we show that the P2X7 receptor is overexpressed in two cell lines of papillary and anaplastic carcinoma as well as histological samples of human thyroid neoplasms differing in histotype, staging, and grading of malignancy. In addition, we also show that thyroid cancer cells release an almost 3-fold higher amount of ATP, compared with normal thyrocytes. Thus, we suggest that the P2X7 receptor may be a candidate marker of papillary thyroid cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and tissues
Two established human thyroid cancer cell lines were used: FB1 and FB2, the former derived from anaplastic carcinoma (21) and the latter from papillary cancer (22). Cells were grown in DMEM containing 10% fetal calf serum, 100 IU/ml penicillin, and 50 µg/ml streptomycin (all from Sigma-Aldrich, Milan, Italy) and harvested in PBS containing 2 mM EDTA.

Human thyrocytes obtained from freshly isolated thyroid tissue samples from six euthyroid patients undergoing total thyroidectomy for cold nodular goiter served as control cells. Tissue samples were dissected and minced with scissors into 1- to 3-mm pieces and incubated with 200 U/ml type II collagenase (Invitrogen, Grand Island, NY) in a shaking water bath at 37 C for 4–6 h. Cells were then resuspended in DMEM supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 50 µg/ml streptomycin (Sigma-Aldrich). Cells were counted and plated in 25 cm2 Falcon primary tissue culture flasks (Becton Dickinson, Franklin Lakes, NJ), and incubated at 37 C in humidified incubator in the presence of 5% CO2. Cells were used for experiments between the second and third passage after 7–8 d from plating and at 70–80% confluence. As specific markers we used thyroglobulin mRNA expression and thyroxine release.

Thirty-seven histological samples of human papillary cancer (follicular and classical variants) were consecutively obtained. All the patients signed an informed consent allowing the use of part of the removed thyroid tissue for scientific purposes.

P2X mRNA and protein expression
P2X receptors were identified by RT-PCR and Western blot. Total RNA was isolated by cells and solid tissues using a RNeasy minikit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Total RNA was eluted with 30 µl RNase-free water. Residual genomic DNA was removed by incubating the RNA solution with 15 U of RNase-free DNase I in 2 mM MgCl2 for 15 min at 37 C, followed by 5 min at 90 C to inactivate the DNase. Reverse transcription was performed using 1 µg total RNA, random primers, and ImProm-II reverse transcriptase (Promega, Madison, WI) in a total volume of 20 µl. The samples were incubated at 25 C for 15 min and then at 42 C for 60 min followed by 5 min at 70 C to inactivate the reverse transcriptase. The cDNA samples were stored at –20 C.

PCR was performed using the Cycler thermal cycler (Bio-Rad, København, Denmark) in a standard 25-µl reaction mixture containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3), 0.2 mM deoxynucleotide triphosphates, 20 pmol of each sense and antisense primer, and 2.5 U AmpliTaq DNA polymerase (Laboratories Eurobio, Les Ulis Cedex, France). The following primers were used: P2X1, 5'-CGCCTTCTTCTTCGAGTATG-3' (forward), 5'-GGAAGACGTAGTCAGCCACA-3' (reverse) with annealing temperature of 55 C and the expected cDNA size of 248 bp; P2X2, 5'-CCACCATGGCCGCCGCCCAGCCCAAGTA-3' (forward), 5'-GGAAAGGAGCTCAGAGTTGAGCCAAACC-3' (reverse) with annealing temperature of 59 C and the expected cDNA size of 600 bp; P2X3, 5'-GAGAGTGAGGAGAAATACCG-3' (forward), 5'-CACTGGTCCCAGGCCTTG-3' (reverse) with annealing temperature of 60 C and the expected cDNA size of 437 bp; P2X4, 5'-TGCATTTATGATGCTAAAACA-3' (forward), 5'-CAAGACCCTGCTCGTAAT-3' (reverse) with annealing temperature of 60 C and the expected cDNA size of 521 bp; P2X5, 5'-CCGGGAGCGACTTCCAGGATATAG-3' (forward), 5'-GGCATGGGATCACTGGGTGCTAGAC-3' (reverse) with annealing temperature of 60 C and the expected cDNA size of 614 bp; P2X6, 5'-AAAAACAGGCCAGTGTGTGGTGTTC-3' (forward), 5'-TGCCTGCCCGGTGACGAGGATGTCGA-3' (reverse) with annealing temperature of 60 C and the expected cDNA size of 520 bp; P2X7, 5'-AGATCGTGGAGAATGGAGTG-3' (forward), 5'-TTCTCGTGGTGTAGTTGTGG-3' (reverse) with annealing temperature of 58 C and the expected cDNA size of 399 bp; and β-actin, 5'-CTCCTTAATGTCACGCACGATTTC-3' (forward), 5'-GTGGGGCGCCCCAGGCACCA-3' (reverse). Amplified PCR products were run on a 2% agarose gel containing 0.5 µg/ml ethidium bromide. The quantification of mRNA expression, relative to an internal control gene β-actin, was obtained by densitometric analysis using the system Kodak Digital Science 1D (Kodak Italia, Cinisello Balsamo, Italy).

P2X7 protein expression was analyzed by Western blot. Protein was extracted in ice-cold lysis buffer (Nonidet P-40 cell lysis buffer; Biosource, Camarillo, CA) containing 1 mM phenylmethylsulfonyl fluoride, 6 µg/ml aprotinin, and 10 µg/ml leupeptin for 30 min with vortexing at 10-min intervals. The extracts were used for electrophoresis on sodium dodecyl sulfate and 7.5% polyacrylamide gels and transferred electrophoretically to polyvinyl difluoride filters (Hybond-P; Amersham, Amersham, UK). After blocking 1 h with 10% nonfat dried milk in Tris-buffered saline containing 0.05% Tween 20, the membranes were incubated overnight at 4 C with P2X7 antibody (Chemicon International, Temecula, CA) at 1:500 dilution in 2% BSA in 10 ml of Tris-buffered saline containing 0.05% Tween 20. The filters were washed and incubated for 1 h with protein A peroxidase conjugated (Calbiochem, Cambridge, MA) at 1:3000 dilution. Bands were visualized by enhanced chemiluminescence (Amersham).

Immunohistochemistry
Ten percent formalin-fixed, paraffin-embedded blocks routinely prepared from surgical specimens of 37 cases of thyroid tumors were examined. Tissue sections 5 µm thick were deparaffinized in xylene and dehydrated in a graded ethanol series. Anti-P2X7 antibody (Sigma-Aldrich) was used at the dilution of 1:100; this marker was stained with Ventana Medical Systems and revealed with a biotin-free immunoperoxidase procedure. The evaluation of immunostaining was performed as previously reported (5), classifying the cases into four categories according to number of positive cells in the specimens as follows: –, less than 10%; +, from 10 to 25%; ++, from 25 to 75%; +++, greater than 75%.

Real-time PCR
Real-time PCR of endogenous control gene (glyceraldehyde-3-phosphate dehydrogenase) and target gene (full-length P2X7) was performed with TaqMan probes Fam dye, commercially available as Assay on Demand (Applied Biosystems, Foster City, CA), with optimized primer and probe concentrations. Quantitative PCR was performed in the iCycler iQ real-time PCR detection system (Bio-Rad) in 96-well plates, and the reaction was performed in a final volume of 25 µl. All quantitative PCR mixtures contained 2 µl of cDNA template, 1x TaqMan universal PCR master mix (two times) (Applied Biosystems, Foster City, CA) and 1x Assay on Demand gene expression assay mix (20 times). Cycle conditions were as follows: after an initial 2-min hold at 50 C to allow AmpErase-UNG activity and 10 min at 95 C, the samples were cycled 40 times at 95 C for 15 sec and 60 C for 1 min. The data obtained were analyzed using Gene Expression Macro (version 1.1; mathematical model Jo Vandesompele; Bio-Rad).

Intracellular calcium concentration
Thyrocytes were detached from flasks, seeded onto glass coverslips, and after 24–48 h, they were loaded with the Ca2+ indicator fura-2/AM (Molecular Probes, Leiden, The Netherlands) at a concentration of 2 µM for 20 min at 37 C in saline solution [125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM NaH2PO4, 20 mM HEPES, 5.5 mM glucose, 5 mM NaHCO3, 1 mM CaCl2, and 250 µM sulfinpyrazone (pH 7.4)] and then stimulated with increasing concentrations of ATP, 2', 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) or uridine 5'-triphosphate (UTP). In some experiments CaCl2 was omitted and 0.5 mM EGTA added (Ca2+ free saline solution). Coverslips were mounted in a temperature-controlled magnetically stirred cuvette at 37 C. Fluorescence was measured with a fluorometer (PerkinElmer Ltd., Beaconsfield, UK). The excitation was at 340–380 nm and emission at 510 nm. Ca2+ concentration was calculated using the FLwinlab software (PerkinElmer) (23).

IL-6 secretion
IL-6 release was measured by an ELISA using a biotin-conjugated monoclonal anti-IL-6 antibody (Bioscience, San Diego, CA). The assay sensitivity was less than 2 pg/ml, and the interassay coefficient of variation was 5.2%.

Extracellular ATP quantification
ATP levels were measured by luminometric assay, using the ATP-Lite luminescence ATP detection assay system (PerkinElmer), a high-sensitivity detection system (down to five cells in 100 µl medium) with high reproducibility and long half-life of the light emission (>5 h). Moreover, this system ensures an efficient inactivation of ecto-ATPases, thus avoiding underestimation of extracellular ATP concentration. Cells were seeded at a concentration of 5 x 103 cells/well in microtiter plastic dishes in a total volume of 100 µl of culture medium. Before ATP measurements, cells were rinsed and supplemented with 100 µl of ATP-Lite (buffer solution + substrate solution with luciferase-luciferin assay; PerkinElmer). Cells were directly placed into the test chamber of the luminometer, and light emission was recorded and converted into ATP concentration (extracellular ATP). Cells were then shaken for 5 min in the presence of lysis solution and measurements repeated to determine total ATP levels; intracellular ATP was calculated by subtraction.

Statistical analysis
Results are expressed as mean ± SD. Statistical comparisons between treatments were performed by ANOVA; comparisons between groups were performed by t test for paired data or two-way ANOVA. Post hoc comparisons were carried out with the use of the Bonferroni-Dunn test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P2X7 receptor is abundantly represented in human papillary thyroid carcinoma
Several reports over the last few years have associated the expression of the P2X7 receptor to malignant tumors (4, 5, 6, 7). Figure 1AGo shows RT-PCR amplification of P2X7 receptor message from two specimens of thyroid papillary carcinomas and healthy contralateral nonneoplastic lobe. The P2X7 receptor message was well amplified from the tumor samples, much less so from the healthy lobe. A total of 37 samples of papillary cancer (26 classical variant and 11 follicular variant) were screened for P2X7 expression. Summary densitometry data are also shown. Intensity of the P2X7 receptor band was almost twice as strong in the tumors, compared with healthy tissue. Tissue specimens were processed in parallel for immunohistochemistry with anti-P2X7 antibodies. As shown in Fig. 1Go, B and C, there was a strong P2X7 reactivity in the cancerous tissue (Fig. 1CGo) but not in normal thyroid tissue of the same individual (Fig. 1BGo). Malignant thyrocytes showed a diffuse staining throughout the cytoplasm, at times with an intensification over the cell periphery. A few cells, likely infiltrating immune cells, also looked positive in the stroma. A faint positivity was also seen in the stroma of the healthy sample, in this case also likely due to infiltrating inflammatory elements. In Fig. 1DGo, P2X7 expression from cancerous and healthy tissue was quantitated by real-time PCR. Like the densitometric analysis shown in Fig. 1AGo, real-time PCR revealed that the P2X7 receptor message was about twice more abundant in the tumor than in healthy tissue. This difference was also maintained when the tumor population was split into the follicular and classical variants, and each variant individually compared with healthy tissue.


Figure 1
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  		FIG. 1. A, Qualitative RT-PCR of P2X7 receptor in two samples of human papillary carcinoma (T) and contralateral normal tissue (C) and semiquantitative evaluation [expressed in arbitrary units (AU)] of PCR from 37 samples of papillary thyroid cancer tissues (T, gray bars) and nonneoplastic tissue (C, hatched bars). B and C, Sample of an immunohistochemical determination of P2X7 receptor in normal (B) and neoplastic (C) thyroidal tissue. D, P2X7 receptor expression (measured by real-time PCR and expressed as ratio between gene of interest and housekeeping gene) in 37 cases of human papillary cancer considered as a whole or divided into two subgroups on the basis of the histological characteristics (classical variant, n = 26; follicular variant, n = 11). *, P < 0.001 vs. C.

 
Characterization of P2X receptors in tumoral thyroid cell lines
Despite anecdotal evidence of association with the P2X7 receptor to the cancerous phenotype, the underlying biochemical factor that might provide a mechanistic link between this receptor and malignancy are unknown. To tackle this issue, we turned to two widely used neoplastic thyroid cell lines derived from anaplastic carcinoma (FB1) or papillary cancer (FB2) and compared them with human primary normal thyrocytes. Micrographs of monolayers from the three cell lines are shown in Fig. 2AGo. Figure 2Go, B and C, shows that all three cell types express P2X7 receptor message and protein but the cancerous cell lines to higher level. Besides P2X7, the FB2 cells also express all other P2X receptor subtypes, with the exception of P2X1 (data not shown).


Figure 2
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  		FIG. 2. RNA (B) and protein expression (C) of P2X7 receptor in normal thyrocytes (NT) and cell lines of human thyroidal anaplastic carcinoma (FB1) and human thyroidal papillary carcinoma (FB2). D, Mean densitometric analysis of six Western blots is reported. A, Samples of culture of the three cell types are reported.

 
P2X7 receptor is functionally active in tumoral thyroid cell lines
To explore thyroid cell responses to nucleotide stimulation, we performed a thorough investigation of Ca2+ changes triggered by ATP, BzATP, and UTP. ATP, which is a pan-P2R agonist, caused a large increment in intracellular free calcium concentration ([Ca2+]i), whether in the presence or absence of extracellular Ca2+ (Fig. 3Go), in both FB1 and FB2 cell lines. In the absence of extracellular Ca2+, however, the fast early spike (due to both Ca2+ release from intracellular stores and influx across the plasma membrane) was reduced by 50%, and the delayed increase (almost exclusively due to influx across the plasma membrane) was obliterated. The ATP threshold for the [Ca2+]i increase was about 10–20 and 100–200 nM in the presence or absence of extracellular Ca2+, respectively, in both FB1 and FB2 cell lines. Pretreatment of the cells with the P2X covalent inhibitor oxidized ATP (oATP) reduced by 60 ± 12% in FB1 and 71 ± 15% in FB2 the maximal response to ATP.


Figure 3
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  		FIG. 3. [Ca2+]i changes induced by extracellular ATP in FB1 (A and B) and FB2 (C and D) cells. A and C, Representative traces in the presence and absence of extracellular Ca2+. B and D, Dose-response variations in [Ca2+]i induced by increasing concentrations of extracellular ATP in the presence and absence of extracellular Ca2+. Data are mean ± SD of five experiments for any nucleotide concentration in each experimental condition.

 
We also tested the effect of BzATP, an agonist at P2X1 and P2X7 as well as P2Y11 receptors. As shown in Fig. 4Go, BzATP triggered a [Ca2+]i increase, whether in the presence or absence of extracellular Ca2+. UTP, which is an agonist at the P2Y2, and P2Y4, and P2Y6 level, in the presence of extracellular Ca2+ triggered a biphasic [Ca2+]i increase (Fig. 5Go), suggestive of the involvement of at least two P2Y receptor subtypes. On the contrary, in Ca2+-free medium, the [Ca2+]i increase was monophasic. The UTP threshold for the [Ca2+]i rise was 10–30 nM, whether in the presence or absence of extracellular Ca2+.


Figure 4
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  		FIG. 4. [Ca2+]i changes induced by BzATP in FB1 (A and B) and FB2 (C and D) cells. A and C, Representative traces in the presence and absence of extracellular Ca2+. B and D, Dose-response variations in [Ca2+]i induced by increasing concentrations of BzATP in the presence of extracellular Ca2+. Data are mean ± SD of five experiments for any nucleotide concentration in each experimental condition.

 

Figure 5
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  		FIG. 5. [Ca2+]i changes induced by UTP in FB1 (A and B) and FB2 (C and D) cells. A and C, Representative traces in the presence and absence of extracellular Ca2+. B and D, Dose-response variations in [Ca2+]i induced by increasing concentrations of UTP in the presence and absence of extracellular Ca2+. Data are mean ± SD of five experiments for any nucleotide concentration in each experimental condition.

 
Extracellular nucleotides induce IL-6 release from tumoral thyroid cells
In our previous work (9), we reported that extracellular nucleotides triggered release of IL-6 from normal thyrocytes. IL-6 is an important cytokine in thyroid pathophysiology, also mediating interactions between thyrocytes and infiltrating immune cells (24, 25). As reported in Fig. 6Go, basal IL-6 release from FB1 and FB2 cell lines was about 2- to 4-fold higher than from freshly isolated normal thyrocytes. However, as in normal thyrocytes, incubation in the presence of apyrase decreased IL-6 release about 20% (all P < 0.05 vs. unstimulated cells), hinting at the operation of a purinergic loop-controlling basal IL-6 release. Exogenous ATP increased the spontaneous IL-6 release in a dose-dependent fashion in the three cell types; pretreatment of the cells with oATP completely abrogated ATP-stimulated IL-6 release (Fig. 6AGo). Different from ATP, BzATP was inactive in nonneoplastic thyrocytes, strongly suggesting lack of coupling of the P2X7 receptor to release of this cytokine, whereas it was a potent stimulus for IL-6 secretion in both cancer lines (Fig. 6BGo). The P2X7 antagonist 4[2-[(5-isoquinolinylsulfonyl)methylamino]3-oxo-3-(4-phenyl-1-piperazinyl) propyl]phenyl ester (KN62) blocked the release of the inflammatory cytokine in these cell lines, a finding that further reinforces the participation of this receptor in IL-6 release.


Figure 6
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  		FIG. 6. Il-6 release in the unstimulated state and after 3 h of treatment with increasing concentrations of ATP (A) and BzATP (B), alone or in cells pretreated for 1 h with the ATP hydrolyzer apyrase (1 U/ml), the P2X receptors antagonist oATP (300 µM) (A) and the selective human P2X7 blocker KN62 (50 nM) (B). Black symbols, Normal thyrocytes; dark gray symbols, FB1 cells; light gray symbols, FB2 cells. Data are mean ± SD of four experiments, each performed in duplicate. *, P < 0.001 vs. unstimulated cells; °, P < 0.001 vs. FB2.

 
Thyroid carcinoma lines show high extracellular ATP in the pericellular space
Autocrine/paracrine stimulation by extracellular ATP may promote cytokine secretion or cell growth; thus, we investigated thyroid cell lines and normal thyrocytes for nonlytic ATP release. Both thyroid carcinoma lines maintained in the pericellular space an at least 3-fold higher ATP concentration, compared with healthy thyrocytes (255 ± 26, 222 ± 31, and 80 ± 18 nmol/liter in FB1, FB2, and normal thyrocytes, respectively; P < 0.001 vs. normal thyrocytes for both FB1 and FB2). The difference in the intracellular ATP concentration was even more striking, because whereas normal thyrocytes had a total ATP content of about 2.6 ± 0.4 mmol/liter, i.e. within the usual range typical of mammalian cells, FB1 and FB2 carcinomas showed an intracellular ATP level of 13.4 ± 1.2 and 12.1 ± 1.0 mmol/liter, respectively, i.e. about 4-fold higher than normal thyrocytes (both P < 0.001 vs. normal thyrocytes).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thyroid cancer accounts for less than 1% of all cancer deaths; however, some patients develop local recurrences and distant metastases. Patients at high risk are usually diagnosed on the basis of clinical evaluation (age, local invasion, metastases), and the histological finding of poorly differentiated subtypes (trabecular, insular, solid). However, the relative weight of these features and the prognostic significance of additional parameters, such as lymph nodal metastases, is controversial (26). One of the most serious challenges in presurgical screening of thyroid nodules is the differential diagnosis between adenomas and carcinomas, as witnessed by the common observation that almost 20% of nodules with follicular pattern at cytological examination turn out to be cancers after histological analysis. Availability of cytological markers of malignancy would be of great help to avoid unnecessary surgical operations and subject cancer patients to proper therapy as early as possible.

In a previous study, we found that extracellular nucleotides acting at P2 receptors promote release of IL-6 from normal thyrocytes. In addition, the inhibitory effect of the enzyme apyrase on basal, nonstimulated IL-6 release suggested that thyrocytes spontaneously released ATP (9). An overwhelming body of literature data show that purinergic signaling supports embryogenesis, cell growth, and differentiation and is even involved in cell death. In addition, expression of functional P2 receptors has been documented in several malignant cell lines, including MCF-7 breast, endometrial, prostate, and colorectal cancer cells; ovarian cells; and HL-60 leukemia cells (27, 28, 29).

Among P2 receptors, P2X7 is of unusual interest because it is overexpressed by several tumors (4, 6, 30) but surprisingly barely detectable in normal thyrocytes (9). Very interestingly, PCR and histochemistry data show high P2X7 expression in samples from thyroid papillary carcinomas, whether of the classical or follicular variant. Cell lines established from thyroid cancers also showed P2X7 overexpression. The P2X7 receptor was not only expressed in thyroid cancers but also functional, at further variance with nonneoplastic thyrocytes. In fact, whereas, in our previous study in normal thyrocytes we were unable to detect functional responses linked to P2X7 receptor stimulation, in cancer thyrocytes activation of the P2X7 receptor is coupled to [Ca2+]i changes and IL-6 secretion. In the FB1 and FB2 cell lines, the Ca2+ response to the global P2 agonist ATP is brilliant; the reduced response, in terms of both early spike and delayed increase, in the absence of external Ca2+, suggests a strong contribution of extracellular Ca2+ influx (either capacitative or via P2 receptors) to intracellular Ca2+ levels. In contrast to that previously described in normal thyrocytes (9), BzATP triggers a large Ca2+ influx in both FB1 and FB2 cells, confirming the functional activity of the P2X7 receptor subtype in such cells. Moreover, the ability of BzATP to mobilize Ca2+ from the intracellular stores is a clear indication of expression of the P2Y11 in FB1 and FB2 cells because P2XRs do not trigger release of Ca2+ from the intracellular stores. Also, the biphasic shape of the UTP-dependent [Ca2+]i increase (see Fig. 5Go) is intriguing because it suggests expression by cancer thyrocytes of at least two different P2Y receptor subtypes with very different affinities.

The striking [Ca2+]i response triggered by BzATP in tumor thyrocytes is paralleled by the effect on IL-6 release. As reported previously, normal thyrocytes are unable to secrete this cytokine in response to BzATP; on the contrary, in FB1 and FB2 cells, BzATP is a powerful stimulus, causing an IL-6 release about half of that caused by the pan-P2 stimulus ATP. The specific role of P2X7 receptor in mediating this response is strongly supported by the lack of such effect in cells pretreated with the selective human P2X7 blocker KN62. The IL-6-secreting activity of BzATP appears particularly important, given the documented role of this cytokine in the pathogenesis of malignancies. The IL-6 ability to induce paraneoplastic syndromes is well known (31), and its local expression correlates with degree of aggressiveness in papillary and medullary thyroid cancers (32).

Assignment of a functional role to receptors for extracellular ATP implies by necessity the presence of the physiological agonist in the extracellular milieu. Therefore, an obvious, but as-yet-unproved, assumption is that the tumor microenvironment contains enough ATP to activate P2 receptors, including perhaps the low-affinity P2X7. Although we did not measure the actual ATP levels in the thyroid gland interstitium, we found that the ATP concentration in the supernatants of FB1 and FB2 cells was about 3-fold higher than in the supernatant from normal thyrocytes. This observation shows that cancer cells keep a high extracellular ATP concentration and suggests that this might be a feature of the tumor microenvironment. Recent data show that malignant tumors have a higher extracellular adenosine level (1) that might function as an actual immunosuppressive factor. There are two main avenues that drive accumulation of extracellular adenosine: direct transport by plasma membrane carriers and generation from extracellular ATP thanks to the sequential activity of ecto-ATP/ADPases and 5'-nucleotidase. Thus, the high intratumor adenosine level strongly suggests that the ATP concentration of the tumor interstitium might also be high, in keeping with our in vitro measurements. Autocrine/paracrine stimulation of IL-6 release by extracellular ATP provides a stimulatory loop that might have a relevant role in supporting thyroid cancer cell growth.

In conclusion, our findings show that human thyroid cancer cell lines, in contrast to normal nonneoplastic thyroid tissue, express high levels of functionally active P2X7 receptor coupled to Ca2+ fluxes and IL-6 release; moreover, high levels of these receptors are found in histological samples of human papillary cancer. Further studies in isolated primary cells from fine-needle aspirations are needed to confirm whether the P2X7 receptor might be a novel biomarker to differentiate normal from cancerous thyroid tissue, thus opening the way for a future use of P2X7 agonists/antagonists in the clinical practice.


   Footnotes
 
This work was supported by Grant PRIN 2004 2004061208 from the Italian Ministry for University and Scientific Research, Italian Association for Cancer Research, and Telethon of Italy.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 18, 2007

Abbreviations: BzATP, 2', 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate; Ca2+]i, intracellular free calcium concentration; KN62, 4[2-[(5-isoquinolinylsulfonyl)methylamino]3-oxo-3-(4-phenyl-1-piperazinyl) propyl]phenyl ester; oATP, oxidized ATP; P2R, P2 receptor; UTP, uridine 5'-triphosphate.

Received September 4, 2007.

Accepted for publication October 11, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ohta A, Gorelik E, Prasad SJ, Ronchese F, Lukashev D, Wong MK, Huang Y, Caldwell S, Liu K, Smith P, Chen JF, Jackson EK, Apasov S, Abrams S, Sitkowsky M 2006 A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci USA 103:13132–13137[Abstract/Free Full Text]
  2. Burnstock G 2006 Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol Rev 58:58–86[Abstract/Free Full Text]
  3. White N, Burnstock G 2006 P2 receptors and cancer. Trends Pharmacol Sci 27:211–217[CrossRef][Medline]
  4. Adinolfi E, Melchiorri L, Falzoni S, Chiozzi P, Morelli A, Tieghi A, Cuneo A, Castoldi G, Di Virgilio F, Baricordi OR 2002 P2X7 receptor expression in evolutive and indolent forms of chronic B lymphocytic leukemia. Blood 99:706–708[Abstract/Free Full Text]
  5. Slater M, Danieletto S, Pooley M, Cheng Teh L, Gidley-Baird A, Barden JA 2004 Differentiation between cancerous and normal hyperplastic lobules in breast lesions. Breast Cancer Res Treat 83:1–10[CrossRef][Medline]
  6. Slater M, Danieletto S, Gidley-Baird A, Cheng Teh L, Barden JA 2004 Early prostate cancer detected using expression of nonfunctional cytolytic P2X7 receptors. Histopathology 44:206–215[CrossRef][Medline]
  7. Raffaghello L, Chiozzi P, Falzoni S, Di Virgilio F, Pistoia V 2006 The P2X7 receptor sustains the growth of human neuroblastoma cells through a substance P-dependent mechanism. Cancer Res 66:907–914[Abstract/Free Full Text]
  8. Adinolfi E, Callegari MG, Ferrari D, Bolognesi C, Minelli M, Wieckowski MR, Pinton P, Rizzuto R, Di Virgilio F 2005 Basal activation of the P2X7 ATP receptor elevates mitochondrial calcium and potential, increases cellular ATP levels, and promotes serum-independent growth. Mol Biol Cell 16:3260–3272[Abstract/Free Full Text]
  9. Caraccio N, Monzani F, Santini E, Cuccato S, Ferrari D, Callegari MG, Gulinelli S, Pizzirani C, Di Virgilio F, Ferrannini E, Solini A 2005 Extracellular adenosine 5'-triphosphate modulates interleukin-6 production by human thyrocytes through functional purinergic P2 receptors. Endocrinology 146:3172–3178[Abstract/Free Full Text]
  10. Petersen C, Froysa B, Soder O 2004 Endotoxin and proinflammatory cytokines modulate Sertoli cell proliferation in vitro. J Reprod Immunol 61:13–30[CrossRef][Medline]
  11. Willenberg HS, Path G, Vogeli TA, Scherbaum WA, Bornstein SR 2002 Role of interleukin-6 in stress response in normal and tumorous adrenal cells and during chronic inflammation. Ann NY Acad Sci 966:304–314[Medline]
  12. Dean DS, Hay ID 2000 Prognostic indicators in differentiated thyroid carcinoma. Cancer Control 7:229–239[Medline]
  13. Al-Brahim N, Asa SL 2006 Papillary thyroid carcinoma: an overview. Arch Pathol Lab Med 130:1057–1062[Medline]
  14. Pines A, Bivi N, Vascotto C, Romanello M, D’Ambrosio C, Scaloni A, Damante G, Morisi R, Filetti S, Ferretti E, Quadrifoglio F, Tell G 2006 Nucleotide receptors stimulation by extracellular ATP controls Hsp90 expression through APE1/Ref-1 in thyroid cancer cells: a novel tumorigenic pathway. J Cell Physiol 209:44–55[CrossRef][Medline]
  15. Green ST 1987 Intrathyroidal autonomic nerves can directly influence hormone release from rat thyroid follicles: a study in vitro employing electrical field stimulation and intracellular microelectrodes. Clin Sci (Lond) 72:233–238[Medline]
  16. Schwiebert LM, Rice WC, Kudlow BA, Taylor AL, Schwiebert EM 2002 Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells. Am J Physiol Cell Physiol 282:C289–C301
  17. Kochukov MY, Ritchie AK 2004 A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line. Am J Physiol Cell Physiol 287:C992–C1002
  18. Schofl C, Rossig L, Potter E, von zur Muhlen A, Brabant G 1995 Extracellular ATP and UTP increase cytosolic free calcium by activating a common P2u-receptor in single human thyrocytes. Biochem Biophys Res Commun 213:928–934[CrossRef][Medline]
  19. Di Jeso B, Laviola L, Liguoro D, Formisano S, Consiglio E 1993 P2 purinergic agonists and 12-O-tetradecanoylphorbol-13-acetate, as well as protein kinase A activators, stimulate thyroglobulin secretion in FRTL-5 cells. Biochem Biophys Res Commun 191:385–391[CrossRef][Medline]
  20. Ekokoski E, Webb TE, Simon J, Tornquist, K 2001 Mechanisms of P2 receptor-evoked DNA synthesis in thyroid FRTL-5 cells. J Cell Physiol 187:166–175[CrossRef][Medline]
  21. Fiore L, Pollina LE, Fontanini G, Casalone R, Berlingieri MT, Giannini R, Pacini F, Miccoli P, Toniolo A, Fusco A, Basolo F 1997 Cytokine production by a new undifferentiated human thyroid carcinoma cell line, FB-1. J Clin Endocrinol Metab 82:4094–4100[Abstract/Free Full Text]
  22. Basolo F, Giannini R, Toniolo A, Casalone R, Nikiforova M, Pacini F, Elisei R, Miccoli P, Berti P, Faviana P, Fiore L, Monaco C, Pierantoni GM, Fedele M, Nikiforov YE, Santoro M, Fusco A 2002 Establishment of a non-tumorigenic papillary thyroid cell line (FB-2) carrying the RET/PTC1 rearrangement. Int J Cancer 97:608–614[CrossRef][Medline]
  23. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
  24. Hidaka Y, Okumura M, Shimaoka Y, Takeoka K, Tada H, Amino N 1998 Increased serum concentration of interleukin-5 in patients with Grave’s disease and Hashimoto’s thyroiditis. Thyroid 8:235–239[Medline]
  25. Bartalena L, Brogioni S, Grasso L, Martino E 1995 Interleukin-6 and the thyroid. Eur J Endocrinol 132:386–393[Abstract/Free Full Text]
  26. Watkinson JC, Franklyn JA, Olliff JF 2006 Detection and surgical treatment of cervical lymph nodes in differentiated thyroid cancer. Thyroid 16:187–194[Medline]
  27. Wagstaff SC, Bowler WB, Gallagher JA, Hipskind RA 2000 Extracellular ATP activates multiple signalling pathways and potentiates growth factor-induced c-fos gene expression in MCF-7 breast cancer cells. Carcinogenesis 21:2175–2181[Abstract/Free Full Text]
  28. Coutinho-Silva R, Stahl L, Cheung KK, de Campos NE, de Oliveira Souza C, Ojcius DM, Burnstock G 2005 P2X and P2Y purinergic receptors on human intestinal epithelial carcinoma cells: effects of extracellular nucleotides on apoptosis and cell proliferation. Am J Physiol Gastrointest Liver Physiol 288:G1024–G1035
  29. Yoon MJ, Lee HJ, Kim JH, Kim DK 2006 Extracellular ATP induces apoptotic signaling in human monocyte leukemic cells, HL-60 and F-36P. Arch Pharm Res 29:1032–1041[Medline]
  30. Wang Q, Wang L, Feng YH, Li X, Zeng R, Gorodeski GI 2004 P2X7 receptor-mediated apoptosis of human cervical epithelial cells. Am J Physiol Cell Physiol 287:C1349–C1358
  31. Barton BE 2005 Interleukin-6 and new strategies for the treatment of cancer, hyperproliferative diseases and paraneoplastic syndromes. Expert Opin Ther Targets 9:737–752[CrossRef][Medline]
  32. Ruggeri RM, Villari D, Simone A, Scarfi R, Attard M, Orlandi F, Barresi G, Trimarchi F, Trovato M, Benvenga S 2002 Co-expression of interleukin-6 (IL-6) and interleukin-6 receptor (IL-6R) in thyroid nodules is associated with co-expression of CD30 ligand/CD30 receptor. J Endocrinol Invest 25:959–966[Medline]



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