Higher Concentrations of Extracellular ATP Suppress Proliferation of Caco-2 Human Colonic Cancer Cells via an Unknown Receptor Involving PKC Inhibition

Cellular Physiology and Biochemistry

关键词: ATP;    Protein kinase C;    Mitogen-activated protein kinase;    Caco-2 cell;    Proliferation;   

DOI  :  10.1159/000320518

学科分类：分子生物学，细胞生物学和基因

来源: S Karger AG

【 摘 要 】

Background/Aims 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 µM-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. P₂ purinoceptor agonists reduced Caco-2 cell viability with the order of potency 2-methylthio ATP>UTP>β, γ-methylene ATP, and the ATP effect was partially inhibited by suramin, a non-selective inhibitor of P₂ 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 ATPγS, 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. Conclusion 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.

【 授权许可】

CC BY-NC-ND