Abstract
The effects of 100 μM of 3′,5′-cGMP, cAMP, cCMP, and cUMP as well as of the corresponding membrane-permeant acetoxymethyl esters on anti-CD3-antibody (OKT3)-induced IL-2 production of HuT-78 cutaneous T cell lymphoma (Sézary lymphoma) cells were analyzed. Only 3′,5′-cGMP significantly reduced IL-2 production. Flow cytometric analysis of apoptotic (propidium iodide/annexin V staining) and anti-proliferative (CFSE staining) effects revealed that 3′,5′-cGMP concentrations > 50 μM strongly inhibited proliferation and promoted apoptosis of HuT-78 cells (cultured in the presence of αCD3 antibody). Similar effects were observed for the positional isomer 2′,3′-cGMP and for 2′,-GMP, 3′-GMP, 5′-GMP, and guanosine. By contrast, guanosine and guanosine-derived nucleotides had no cytotoxic effect on peripheral blood mononuclear cells (PBMCs) or acute lymphocytic leukemia (ALL) xenograft cells. The anti-proliferative and apoptotic effects of guanosine and guanosine-derived compounds on HuT-78 cells were completely eliminated by the nucleoside transport inhibitor NBMPR (S-(4-Nitrobenzyl)-6-thioinosine). By contrast, the ecto-phosphodiesterase inhibitor DPSPX (1,3-dipropyl-8-sulfophenylxanthine) and the CD73 ecto-5′-nucleotidase inhibitor AMP-CP (adenosine 5′-(α,β-methylene)diphosphate) were not protective. We hypothesize that HuT-78 cells metabolize guanosine-derived nucleotides to guanosine by yet unknown mechanisms. Guanosine then enters the cells by an NBMPR-sensitive nucleoside transporter and exerts cytotoxic effects. This transporter may be ENT1 because NBMPR counteracted guanosine cytotoxicity in HuT-78 cells with nanomolar efficacy (IC50 of 25–30 nM). Future studies should further clarify the mechanism of the observed effects and address the question, whether guanosine or guanosine-derived nucleotides may serve as adjuvants in the therapy of cancers that express appropriate nucleoside transporters and are sensitive to established nucleoside-derived cytostatic drugs.
Highlights
IntroductionCyclic nucleotides (cNMPs), cAMP and cGMP, are well-established second messengers
Cyclic nucleotides, cAMP and cGMP, are well-established second messengers
The effect of 100 μM of the purine cNMPs 3′,5′-cAMP, 3′,5′cGMP, and 3′,5′-cIMP as well as of the pyrimidine cNMPs 3′,5′-cUMP and 3′,5′-cCMP on αCD3-antibody-induced IL-2 production of HuT-78 lymphoma cells was analyzed by ELISA
Summary
Cyclic nucleotides (cNMPs), cAMP and cGMP, are well-established second messengers. 3′,5′-cAMP is exported into the extracellular space, followed by enzymatic degradation to adenosine, which in turn activates G protein-coupled receptors (Godinho et al 2015, Jackson and Raghvendra 2004). The non-canonical cyclic nucleotide cUMP, which is currently discussed as a potential second messenger (Seifert et al 2015; Berrisch et al 2017; Ostermeyer et al 2018; Scharrenbroich et al 2019), may as well be exported and degraded to biologically active products. Preliminary results suggest that first- and second messenger effects of cyclic nucleotides are highly dependent on the investigated cell type (Schneider et al 2015)
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