Higher concentrations of extracellular ATP suppress proliferation of Caco-2 human colonic cancer cells via an unknown receptor involving PKC inhibition.
OBJECTIVE
Adenosine 5'-triphosphate (ATP) mediates a variety of signal transductions via ATP receptors such as P2X and P2Y receptors. The present study aimed at understanding the mechanism underlying extracellular ATP-induced suppression of Caco-2 human colonic cancer cell proliferation.


METHODS
Caco-2 cells were cultured. To examine cell viability and cell cycling, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, fluorescent cytochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and flow cytometry were carried out. To see mRNA expression of ATP receptors, reverse transcription-polymerase chain reaction (RT-PCR) was performed. To examine PKC activity and mitogen-activated protein (MAP) kinase activity, in situ PKC assay and Western blotting using an anti-extracellular signal-regulated kinase 1 (ERK1)-antibody and an anti-phospho-ERK antibody were carried out.


RESULTS
Extracellular ATP or the unhydrolyzed ATP analogue 5'-adenylyimido-diphosphate (AMP-PNP) reduced Caco-2 cell viability in a concentration (10 microM-10 mM)-dependent manner at 48-h treatment, and the effect was not affected by caspase inhibitors. Caco-2 cells were little reactive to propidium iodide and Hoechst 33342 or little positive to TUNEL after 48-h treatment with ATP (1 mM). In the flow cytometry, 48-h treatment with ATP (1 mM) arrested cell cycling at the S phase in Caco-2 cells. P(2) purinoceptor agonists reduced Caco-2 cell viability with the order of potency: 2-methylthio ATP>UTP>beta, gamma-methylene ATP, and the ATP effect was partially inhibited by suramin, a non-selective inhibitor of P(2) purinoceptors. The PKC inhibitor GF109203X or the MAP kinase kinase inhibitor PD98059 reduced Caco-2 cell viability to an extent similar to that achieved by ATP (1 mM), and no further reduction was obtained with co-treatment with ATP. ATP and its ATP analogues such as AMP-PNP and ATPgammaS, at higher concentrations (1-10 mM), inhibited PKC activation in Caco-2 cells in a fashion that mimics the effect of GF109203X, but PD98059 exhibited no effect on PKC activation. The inhibitory effect of ATP on PKC activation was not found with SK-N-SH cells, a human neuroblastoma cell line, but the cells expressed all the mRNAs for P2X and P2Y receptors that Caco-2 cells did. ATP (10 mM) or GF109203X inhibited activation of ERK, a MAP kinase, in Caco-2 cells.


CONCLUSIONS
Extracellular ATP, at higher concentrations, suppresses Caco-2 cell proliferation at the S phase of cell cycling by inhibiting PKC, possibly as mediated via an unknown ATP receptor, followed by MAP kinase.
Yaguchi
Takahiro
T
Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan.
Saito
Masaru
M
Yasuda
Yoshiyuki
Y
Kanno
Takeshi
T
Nakano
Takashi
T
Nishizaki
Tomoyuki
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D018938:Caco-2 Cells; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists / Q000494:pharmacology; D058469:Receptors, Purinergic P2X / Q000737:chemistry / Q000378:metabolism; D058470:Receptors, Purinergic P2Y / Q000737:chemistry / Q000378:metabolism
D000970:Antineoplastic Agents; D005419:Flavonoids; D007211:Indoles; D008301:Maleimides; D058910:Purinergic P2 Receptor Agonists; D058469:Receptors, Purinergic P2X; D058470:Receptors, Purinergic P2Y; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D048052:Mitogen-Activated Protein Kinase 3; C070515:bisindolylmaleimide I; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one