Abstract 838: A metabolomic analysis revealed the involvement of deoxycytidine kinase and CTP synthase in the sensitivity of human pancreatic cancer cells to gemcitabine

Abstract

Abstract Gemcitabine (GEM), a pyrimidine nucleoside analogue, is used in patients with pancreatic cancer as the standard chemotherapeutic agent. However, pancreatic cancer cells often show resistance to GEM in vivo and vitro. In this study, we explored the factors involved in the sensitivity of GEM in human pancreatic cancer cells, Miapaca-2 and AsPC-1, by a metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The sensitivity of Miapaca-2 (IC50: 66 nM) to GEM was higher than that of AxPC-1 (IC50: 16 nM), whereas there were little differences in the growth rates of these cells. This result indicates that the difference in the sensitivities between Miapaca-2 and AsPC-1 is not dependent on their growth rates. At 12 hr after adding GEM, intracellular level of GEM in Miapaca-2 was higher than that in AsPC-1, whereas levels of GEM monophosphate, diphosphate and triphosphate in Miapaca-2 were lower than that in AsPC-1. In addition, expression level of deoxycytidine kinase (dCK), which catalyzes the reaction from GEM to GEM monophosphate, in Miapaca-2 was lower than that in AsPC-1. Expression levels of human equilibrium transporter (hENT1), which is known to major transporter of GEM, were similar in both cells. These results suggest that dCK is rate-limiting enzyme in GEM phosphorylation and one of the factors involved in the sensitivity. In pyrimidine metabolism, GEM was observed to inhibit CTP synthase, which converts UTP to CTP, resulting increase in UTP level, decrease in CTP level and depletion of dCTP in both cells at 12 hr after adding GEM. The degree of increase in UTP and decrease in CTP was lower in Miapaca-2 than that of AsPC-1. Indeed, the ratio of UTP to CTP in Miapaca-2 was about 2.5, whereas that of AsPC-1 was about 11. In addition, the expression level of CTP synthase in Miapaca-2 was higher than that in AsPC-1. Ribonucleotide reductase, which catalyzes the conversion of CDP to dCDP, is known to be inhibited by GEM diphosphate and be involved in the sensitivity of pancreatic cancer cells to GEM. GEM decreased CDP in AsPC-1. This may be caused by inhibiting CTP synthase. Expression level of ribonucleotide reductase in Miapaca-2 was higher than that in AxPC-1. Therefore, ribonucleotide reductase also may be involved in the sensitivity to GEM. Furthermore, we previously found that GEM mainly inhibited CTP synthase in pyrimidine metabolism, resulting in depletion of dCTP. Our current study suggests that dCK, CTP synthase and ribonucleotide reductase are involved in the sensitivity of pancreatic cancer cells to GEM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 838. doi:1538-7445.AM2012-838