2-ClATP exerts anti-tumoural actions not mediated by P2 receptors in neuronal and glial cell lines.
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. EC(50) was in the 5-25 microM 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 (P1 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.
D'Ambrosi
Nadia
N
Fondazione Santa Lucia, Cellular Neurobiology Unit, Rome, Italy.
Costanzi
Stefano
S
Angelini
Daniela F
DF
Volpini
Rosaria
R
Sancesario
Giuseppe
G
Cristalli
Gloria
G
Volonté
Cinzia
C
D015762:2-Chloroadenosine / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D009457:Neuroglia / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects*; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism*
D000970:Antineoplastic Agents; D018047:Receptors, Purinergic P1; D018048:Receptors, Purinergic P2; D015762:2-Chloroadenosine; C006979:2-chloro-ATP; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases