A. Buffon et al. / Life Sciences 86 (2010) 435–440
439
adhesion protein 2 identifies it as CD73. Journal of Experimental Medicine 182 (5),
1603–1608, 1995.
et al. 1999), which can be hydrolyzed by NPP2. Acting together with
other ectonucleotidases as well as ecto-5′-nucleotidase, previously
described by us in Walker 256 tumor (Buffon et al. 2007a,b), the NPPs
could participate in ATP hydrolysis to adenosine. ATP is released by solid
tumors and it is described as an inhibitor of the tumoral proliferation
(Agteresch et al. 1999). Thus, these enzymes in Walker 256 tumor
would terminate the actions induced by this nucleotide, protecting the
tumoral cells from ATP-mediated cytotoxic effects, facilitating solid
tumor growth and producing adenosine, which has been shown to have
a tumor-promoting action (Spychala 2000; Spychala et al. 2004).
Previously, we have also shown that Walker 256 tumor expresses
mRNA for ecto-5′-nucleotidase/CD73 and E-NTPDases (Buffon et al.
2007a,b). The presence of several ectoenzymes sharing the capability
to hydrolyze nucleotides with overlapping enzymatic functions is
poorly understood. But, the co-existence of several members of
NTPDase and NPP families has been observed in cancer cells.
Ectonucleotidases seem to have functions that are not related to its
catalytic activity, being also involved in adhesion (Kansas et al. 1991;
Airas et al. 1995). The CD39/NTPDase1 is a marker of activated
immunocompetent cells and somehow involved in adhesion (Kansas
et al. 1991). Besides, the overexpression of this enzyme in human
melanomas was related to the escape of tumor cells from immuno-
logical effector mechanisms at early steps of tumor progression, by
regulating homotypic adhesion (Dzhandzhugazyan et al. 1998). Then,
considering that cell adhesion molecules are critical components in
processes such as tumor growth and metastasis (Cavenagh et al. 1998),
the co-expression of NTPDases and NPPs could be due to their
functions on the surface of the cell not related to their catalytic activity.
Bours MJ, Swennen EL, Di Virgilio F, Cronstein BN, Dagnelie PC. Adenosine 5′-
triphosphate and adenosine as endogenous signaling molecules in immunity and
inflammation. Pharmacology & Therapeutics 112 (2), 358–404, 2006.
Bradford MM. A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-bye binding. Analytical
Biochemistry 72, 248–254, 1976.
Buffon A, Ribeiro VB, Wink MR, Casali EA, Sarkis JJ. Nucleotide metabolizing ecto-
enzymes in Walker 256 tumor cells: molecular identification, kinetic character-
ization and biochemical properties. Life Sciences 80 (10), 950–958, 2007a.
Buffon A, Wink MR, Ribeiro BV, Casali EA, Libermann TA, Zerbini LF, Robson SC, Sarkis JJ.
NTPDase and 5′ ecto-nucleotidase expression profiles and the pattern of
extracellular ATP metabolism in the Walker 256 tumor. Biochimica et Biophysica
Acta 1770 (8), 1259–1265, 2007b.
Burnstock G. Pathophysiology and therapeutic potential of purinergic signaling.
Pharmacological Reviews 58 (1), 58–86, 2006.
Cavenagh JD, Cahill MR, Kelsey SM. Adhesion molecules in clinical medicine. Critical
Reviews in Clinical Laboratory Sciences 35 (5), 415–459, 1998.
Clark CM, Goodlad GA. Protein and enzyme content of plasma membranes derived from
Walker 256 carcinoma cells grown as ascitic or solid tumours. Cancer Letters 68 (2–
3), 91–94, 1993.
Dzhandzhugazyan KN, Kirkin AF, thor Straten P, Zeuthen J. Ecto-ATP diphosphohy-
drolase/CD39 is overexpressed in differentiated human melanomas. FEBS Letters
430 (3), 227–230, 1998.
Goding JW, Grobben B, Slegers H. Physiological and pathophysiological functions of the
ectonucleotide pyrophosphatase/phosphodiesterase family. Biochimica et Biophy-
sica Acta 1638 (1), 1–19, 2003.
Guaitani A, Recchia M, Carli M, Rocchtti M, Bartŏsek I, Garattini S. Walker carcinoma
256: a model for studies on tumor induced anorexia and cachexia. Oncology 39 (3),
173–178, 1982.
He YC, Wang YH, Cao J, Chen JW, Pan DY, Zhou YK. Effect of complex amino acid
imbalance on growth of tumor in tumor-bearing rats. World Journal of
Gastroenterology 9 (12), 2772–2775, 2003.
Hosoda N, Hoshino SI, Kanda Y, Katada T. Inhibition of phosphodiesterase/pyropho-
sphatase activity of PC-1 by its association with glycosaminoglycans. European
Journal of Biochemistry 265 (2), 763–770, 1999.
Huang R, Rosenbach M, Vaughn R, Provvedini D, Rebbe N, Hickman S, Goding J,
Terkeltaub R. Expression of the murine plasma cell nucleotide pyrophosphohy-
drolase PC-1 is shared by human liver, bone, and cartilage cells. Journal of Clinical
Investigation 94 (2), 560–567, 1994.
Conclusion
Ikeda Y, Hayashi I, Kamoshita E, Yamazaki A, Endo H, Ishihara K, Yamashina S, Tsutsumi
Y, Matsubara H, Majima M. Host stromal bradykinin B2 receptor signaling
facilitates tumor-associated angiogenesis and tumor growth. Cancer Research 64
(15), 5178–5185, 2004.
Kansas GS, Wood GS, Tedder TF. Expression, distribution, and biochemistry of human
CD39. Role in activation-associated homotypic adhesion of lymphocytes. Journal of
Immunology 146 (7), 2235–2244, 1991.
The data presented in this study suggest the participation of the
NPP2 and NPP3 enzymes on Walker 256 tumoral cells. In addition, the
data demonstrate that the NPPs, mainly NPP3, can be important for
solid tumor growth, once these enzymes are expressed more during
tumor development, when compared with ascitic cells.
Taken together, these results demonstrate that Walker tumor cells
contain all the components for the enzymatic cascade necessary for
the complete metabolism of extracellular nucleotides to nucleosides.
Subsequently, by considering that either ATP or adenosine has been
implicated in tumor biology, these ectonucleotidases may be
controlling the rate of proliferation and invasion of Walker 256
tumor cells. Hence, the co-existence of these enzymes is very
important for their participation in an “enzyme chain” for the
sequential hydrolysis of ATP to adenosine.
Kelly SJ, Dardinger DE, Butler LG. Hydrolysis of phopshonate esters catalyzed by 5′-
nucleotide phosphodiesterase. Biochemistry 14 (22), 4983–4988, 1975.
Moolenaar WH. Lysophospholipids in the limelight: autotaxin takes center stage.
Journal of Cell Biology 158 (2), 197–199, 2002.
Nam SW, Clair T, Campo CK, Lee HY, Liotta LA, Stracke ML. Autotaxin (ATX), a potent
tumor motogen, augments invasive and metastatic potential of ras-transformed
cells. Oncogene 19 (2), 241–247, 2000.
Nam SW, Clair T, Kim YS, Mcmarlin A, Schiffmann E, Liotta LA, Stracke ML. Autotaxin
(NPP-2), a metastasis-enhancing motogen, is an angiogenic factor. Cancer Research
61 (18), 6938–6944, 2001.
Piffar PM, Fernandez R, Tchaikovski O, Hirabara SM, Folador A, Pinto GJ, Jakobi S, Gobbo-
Bordon D, Rohn TV, Fabricio VE, Moretto KD, Tosta E, Curi R, Fernandes LC.
Naproxen, clenbuterol and insulin administration ameliorates cancer cachexia and
reduce tumor growth in Walker 256 tumor-bearing rats. Cancer Letters 201 (2),
139–148, 2003.
Thus, the ability of the ectonucleotidases to control the extracellular
nucleotide/nucleoside ratios in the immediate tumor environment may
provide important therapeutic targets for anticancer therapy.
Rettori O, Vieira-Matos NA, Tahin QS. Variability and discontinuity of the pathogno-
monic systemic effects caused by the Walker 256 tumor progression in rats. Tumori
81 (5), 370–377, 1995.
Conflict of interest statement
Robson S, Sévigny J, Zimmermann H. The E-NTPDase family of ectonucleotidases:
structure function relationship and pathophysiological significance. Purinergic
Signalling 2 (2), 409–430, 2006.
The authors declare that there are no conflicts of interest.
Sakura H, Nagashima S, Nakashima A, Maeda M. Characterization of fetal serum 5′-
nucleotide phosphodiesterase: a novel function as a platelet aggregation inhibitor
in fetal circulation. Thrombosis Research 91 (2), 83–89, 1998.
Acknowledgments
Spychala J. Tumor-promoting functions of adenosine. Pharmacology & Therapeutics 87
(2–3), 161–173, 2000.
Spychala J, Lazarowski E, Ostapkowicz A, Ayscue LH, Jin A, Mitchell BS. Role of estrogen
in the regulation of ecto-5′-nucleotidase and adenosine in breast cancer. Clinical
Cancer Research 10 (2), 708–717, 2004.
Stefan C, Stalmans W, Bollen M. Growth-related expression of the ectonucleotide
pyrophosphatase PC-1 in rat liver. Hepatology 28 (6), 1497–1503, 1998.
Stefan C, Gijsbers R, Stalmans W, Bollen M. Differential regulation of the expression of
nucleotide pyrophosphatases/phosphodiesterases in rat liver. Biochimica et
Biophysica Acta 1450 (1), 45–52, 1999.
This work was supported by grants from CNPq-Brazil and CAPES.
The authors are very grateful to Dr. JJF Sarkis for his supervision
during this work and for dedicating his professional life to study the
purinergic system.
References
Agteresch HJ, Dagnelie PC, van den Berg JW, Wilson JH. Adenosine triphosphate:
established and potential clinical applications. Drugs 58 (2), 211–232, 1999.
Airas L, Hellman J, Salmi M, Bono P, Purune Smith DJ, Jalkanen S. CD73 is involved in
lymphocyte binding to the endothelium: characterization of lymphocyte-vascular
Stefan C, Jansen S, Bollen M. NPP-type ectophosphodiesterases: unity in diversity.
Trends in Biochemical Sciences 30 (10), 542–550, 2005.
Stefan C, Jansen S, Bollen M. Modulation of purinergic signaling by NPP-type
ectophosphodiesterases. Purinergic Signalling 2 (2), 361–370, 2006.