Abstract
In this study, we investigated the dosage effect of gemcitabine, an inhibitor of ribonucleotide reductase (RR), on cellular levels of ribonucleotides and deoxyribonucleotides using high performance liquid chromatography-electrospray ionization tandem mass spectrometric method. As anticipated, after 4-h incubation of non-small cell lung cancer (A549) cells with gemcitabine at 0.5 and 2 μM, there were consistent reductions in levels of deoxyribonucleoside diphosphates (dNDP) and their corresponding deoxyribonucleoside triphosphates (dNTP). However, after 24-h exposure to 0.5 μM gemcitabine, the amounts of dNTP were increased by about 3 fold, whereas cells after 24-h 2 μM gemcitabine treatment exhibited deoxycytidine diphosphate (dCDP), deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) levels less than 50% of control values, with deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphate (dGTP) returning to the control level. Using cell cycle analysis, we found that 24-h incubation at 0.5 μM gemcitabine resulted in a significant increase in S phase arrest, while 2 μM treatment increased G0/G1 population. Our data demonstrated the correlation between the level of RR and the increased levels of dNTPs in the group of 0.5 μM treatment for 24-h with a markedly reduced level of dFdCTP. Accordingly, we proposed that the dosage of dFdC could determine the arrested phase of cell cycle, in turn affecting the recovery of dNTPs pools.
Highlights
Was increased about 2-fold in 12 out of 21 cancer cell lines, while 1.6–1.9 fold increases in adenosine triphosphate (ATP), uridine triphosphate (UTP) and guanosine triphosphate (GTP) pools were observed in 19–20 cell lines[14]
After 24-h incubation, 2 μM dFdC reduced cellular AMP and GMP, whereas both doses of dFdC increased CMP, but less than 1.5-fold. dFdC including gemcitabine monophosphate (dFdCMP) is phosphorylated to dFdCDP by UMP/CMP kinase (UMP/CMPK), which belongs to bifunctional nucleoside monophosphate kinases (NMPKs) family and plays an important role in phosphorylation of UMP, CMP, and dCMP33,34
There is no evidence to demonstrate the association of genetic polymorphisms or expression of UMP/CMPK with clinical outcome of dFdC, reduction of UMP/CMPK activity might lead to the decrease of active metabolites of dFdC and subsequently reduced cytotoxic effect
Summary
Was increased about 2-fold in 12 out of 21 cancer cell lines, while 1.6–1.9 fold increases in adenosine triphosphate (ATP), uridine triphosphate (UTP) and guanosine triphosphate (GTP) pools were observed in 19–20 cell lines[14]. It has been reported that dFdC caused a significant depletion of cellular dNTP with the most pronounced reduction in the dCTP pool[15,16,17]. Despite of these previous studies, little information regarding the alteration in monophosphate (dNMP) and diphosphate deoxyribonucleotides (dNDP) is available, because their amounts are much lower than the respective triphosphate metabolites. Considering the importance of dFdC as the most effective agents for treating early and advanced stage NSCLC during the last twenty years[20,21,22], a major goal of this study was to investigate the interaction of dRN and dFdC intracellular metabolites in non-small cell lung cancer (NSCLC) cells upon treatment with gemcitabine, The information obtained from this study should facilitate animal experiments and clinical trials to assess the efficacy and toxicity of dFdC for developing the individualized chemotherapy
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