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Apoptotic and anti-proliferative effect of guanosine and guanosine derivatives in HuT-78 T lymphoma cells.

Naunyn-Schmiedeberg's archives of pharmacology (2020-04-22)
Erich H Schneider, Olga Hofmeister, Solveig Kälble, Roland Seifert
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
1,3-Dipropyl-8-(p-sulfophenyl)xanthine, powder
Sigma-Aldrich
Guanosine, ≥98%
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Propidium iodide solution, solution (1.0 mg/ml in water)
Sigma-Aldrich
Anti-CD3, low endotoxin antibody, Mouse monoclonal, clone OKT3, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
S-(4-Nitrobenzyl)-6-thioinosine, ≥98%, solid
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5(6)-Carboxyfluorescein diacetate N-succinimidyl ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
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Trypsin-EDTA solution, 0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA, 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
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RPMI-1640 Medium, With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Guanosine 5′-monophosphate disodium salt hydrate, from yeast, ≥99%