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

The effects of 100 mu M 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 (Sezary 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 mu M strongly inhibited proliferation and promoted apoptosis of HuT-78 cells (cultured in the presence of alpha CD3 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 '-(alpha,beta-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.