We investigated the effects of the ATP analogue and P2 receptor agonist 2-ClATP on growth and survival of different neuronal (PC12, PC12nnr5 and SH-SY5Y) and glial (U87 and U373) cell lines, by the use of direct count of intact nuclei, fluorescence microscopy, fluorescence-activated cell sorter analysis (FACS) and high pressure liquid chromatography (HPLC). 2-ClATP lowered the number of cultured PC12nnr5, SH-SY5Y, U87 and U373 cells to almost 5%, and of PC12 cells to about 35% after 3-4 days of treatment. EC50 was in the 5-25 muM range, with 2-ClATP behaving as a cytotoxic or cytostatic agent. Analysis of the biological mechanisms demonstrated that pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (P2 receptor antagonist and nucleotidases inhibitor), but not Caffeine or CGS-15493 (PI receptor antagonists) effectively prevented 2-ClATP-induced toxicity. 2-ClATP metabolic products (2-ClADP, 2-ClAMP, 2-Cladenosine) and new synthesis derivatives (2-CldAMP, 2-Cldadenosine-3',5'-bisphosphate and 2-CldATP) exerted similar cytotoxic actions. Inhibition of both serum nucleotidases and purine nucleoside transporters strongly reduced 2-ClATP-induced cell death, which was conversely increased by the nucleotide hydrolyzing enzyme apyrase. The adenosine kinase inhibitor 5-iodotubericidin totally prevented 2-ClATP or 2-Cladenosine-induced toxicity. In summary, our findings indicate that 2-ClATP exerts either cell cycle arrest or cell death, acting neither on P2 nor on P1 receptors, but being extracellularly metabolized into 2-Cladenosine, intracellularly transported and re-phosphorylated. (C) 2003 Elsevier Inc. All rights reserved.
D'Ambrosi, N., Costanzi, S., Angelini, D., Volpini, R., Sancesario, G., Cristalli, G., et al. (2004). 2-ClATP exerts anti-tumoural actions not mediated by P2 receptors in neuronal and glial cell lines. BIOCHEMICAL PHARMACOLOGY, 67(4), 621-630 [10.1016/j.bcp.2003.09.015].
2-ClATP exerts anti-tumoural actions not mediated by P2 receptors in neuronal and glial cell lines
D'Ambrosi, N;SANCESARIO, GIUSEPPE;
2004-01-01
Abstract
We investigated the effects of the ATP analogue and P2 receptor agonist 2-ClATP on growth and survival of different neuronal (PC12, PC12nnr5 and SH-SY5Y) and glial (U87 and U373) cell lines, by the use of direct count of intact nuclei, fluorescence microscopy, fluorescence-activated cell sorter analysis (FACS) and high pressure liquid chromatography (HPLC). 2-ClATP lowered the number of cultured PC12nnr5, SH-SY5Y, U87 and U373 cells to almost 5%, and of PC12 cells to about 35% after 3-4 days of treatment. EC50 was in the 5-25 muM range, with 2-ClATP behaving as a cytotoxic or cytostatic agent. Analysis of the biological mechanisms demonstrated that pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (P2 receptor antagonist and nucleotidases inhibitor), but not Caffeine or CGS-15493 (PI receptor antagonists) effectively prevented 2-ClATP-induced toxicity. 2-ClATP metabolic products (2-ClADP, 2-ClAMP, 2-Cladenosine) and new synthesis derivatives (2-CldAMP, 2-Cldadenosine-3',5'-bisphosphate and 2-CldATP) exerted similar cytotoxic actions. Inhibition of both serum nucleotidases and purine nucleoside transporters strongly reduced 2-ClATP-induced cell death, which was conversely increased by the nucleotide hydrolyzing enzyme apyrase. The adenosine kinase inhibitor 5-iodotubericidin totally prevented 2-ClATP or 2-Cladenosine-induced toxicity. In summary, our findings indicate that 2-ClATP exerts either cell cycle arrest or cell death, acting neither on P2 nor on P1 receptors, but being extracellularly metabolized into 2-Cladenosine, intracellularly transported and re-phosphorylated. (C) 2003 Elsevier Inc. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
