Study of the effects of FPMINT on human equilibrative nucleoside transporters and cell growth

Abstract

Equilibrative nucleoside transporters (ENTs) are a group of proteins which facilitate the nucleosides transport across the membrane. They control the intracellular and extracellular concentrations of physiological nucleosides such as adenosine, as well as the entry and efflux of anticancer nucleoside analogues. ENT1 plays a crucial role in cardioprotection but the physiological role of ENT2 remains unclear. Development of ENT2-selective inhibitors can facilitate the research on ENT2 but all the commercially available inhibitors of ENTs such as nitrobenzylthioinosine and dipyridamole are ENT1-selective. In this study, we characterized the effects of a novel compound known as 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT). The results of radioactive nucleoside uptake study showed that FPMINT was an inhibitor with a higher selectivity to ENT2 than ENT1. Kinetic study showed that FPMINT inhibited the maximum velocity but had no effect on Michaelis constant of nucleoside uptake, indicating that the inhibitory effect of FPMINT was irreversible and through a non-competitive mechanism. The chemical structure of FPMINT contains a piperzine moiety, which is know to be cytotoxic. Therefore, the cytotoxic effect of FPMINT and the mechanism behind were also studied. FPMINT possessed toxic effect to cardiovascular cells such as H9c2, which is rat embryonic cardiomyoblasts. As reflected from the dihydroethidium fluorescence, FPMINT induced the production of superoxide. The origin of superoxide was mainly from mitochondria since the cytotoxic effect of FPMINT could be reversed by mitochondrial targeted superoxide inhibitor Mito-TEMPO. With the use of biochemical assay and fluorescent probe which is sensitive to mitochondrial potential change, our results showed that FPMINT increased the intracellular adenosine triphosphate level and mitochondrial membrane potential. The results of MDA assay demonstrated that FPMINT caused lipid peroxidation. FPMINT-induced cytotoxicity could be inhibited by apoptosis inhibitor Z-VAD(OMe)-FMK. Taken together, FPMINT could induce apoptotic cell death through the mitochondrial damage and oxidative stress. It is hoped that FPMINT can serve as a lead compound and ENT2-elective inhibitor with minimal cytotoxic effect can be developed after the structural modification of FPMINT.