New insights into the pharmacology and cytotoxicity of gemcitabine and 2′,2′-difluorodeoxyuridine

Molecular Cancer Therapeutics - Tập 7 Số 8 - Trang 2415-2425 - 2008
Stephan A. Veltkamp1,2, Dick Pluim1, Maria A.J. van Eijndhoven1, Maria J. Bolijn1, Felix H.G. Ong1, Rajgopal Govindarajan3, Jashvant D. Unadkat3, Jos H. Beijnen2,4,5, Jan H.M. Schellens1,2,5
11Division of Experimental Therapy,
22Department of Clinical Pharmacology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital,
34Department of Pharmaceutics, University of Washington, Seattle, Washington; and
43Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute/Slotervaart Hospital, Amsterdam, the Netherlands;
55Faculty of Science, Department of Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands

Tóm tắt

Abstract In a clinical study with oral gemcitabine (2′,2′-difluorodeoxycytidine, dFdC), 2′,2′-difluorodeoxyuridine (dFdU) was extensively formed and accumulated after multiple oral dosing. Here, we have investigated the in vitro cytotoxicity, cellular uptake, efflux, biotransformation, and nucleic acid incorporation of dFdC and dFdU. Short-term and long-term cytotoxicity assays were used to assess the cytotoxicity of dFdC and dFdU in human hepatocellular carcinoma HepG2, human lung carcinoma A549, and Madin-Darby canine kidney cell lines transfected with the human concentrative or equilibrative nucleoside transporter 1 (hCNT1 or hENT1), or empty vector. Radiolabeled dFdC and dFdU were used to determine cellular uptake, efflux, biotransformation, and incorporation into DNA and RNA. The compounds dFdC, dFdU, and their phosphorylated metabolites were quantified by high-performance liquid chromatography with UV and radioisotope detection. dFdU monophosphate, diphosphate, and triphosphate (dFdU-TP) were formed from dFdC and dFdU. dFdU-TP was incorporated into DNA and RNA. The area under the intracellular concentration-time curve of dFdC-TP and dFdU-TP and their extent of incorporation into DNA and RNA inversely correlated with the IC50 of dFdC and dFdU, respectively. The cellular uptake and cytotoxicity of dFdU were significantly enhanced by hCNT1. dFdU inhibited cell cycle progression and its cytotoxicity significantly increased with longer duration of exposure. dFdU is taken up into cells with high affinity by hCNT1 and phosphorylated to its dFdU-TP metabolite. dFdU-TP is incorporated into DNA and RNA, which correlated with dFdU cytotoxicity. These data provide strong evidence that dFdU can significantly contribute to the cytotoxicity of dFdC. [Mol Cancer Ther 2008;7(8):2415–25]

Từ khóa


Tài liệu tham khảo

Hertel LW, Boder GB, Kroin JS, et al. Evaluation of the antitumor activity of gemcitabine (2′,2′-difluoro-2′-deoxycytidine). Cancer Res 1990;50:4417–22.

Noble S, Goa KL. Gemcitabine. A review of its pharmacology and clinical potential in non-small cell lung cancer and pancreatic cancer. Drugs 1997;54:447–72.

Braakhuis BJ, van Dongen GA, Vermorken JB, Snow GB. Preclinical in vivo activity of 2′,2′-difluorodeoxycytidine (Gemcitabine) against human head and neck cancer. Cancer Res 1991;51:211–4.

Boven E, Schipper H, Erkelens CA, Hatty SA, Pinedo HM. The influence of the schedule and the dose of gemcitabine on the anti-tumour efficacy in experimental human cancer. Br J Cancer 1993;68:52–6.

Heinemann V, Hertel LW, Grindey GB, Plunkett W. Comparison of the cellular pharmacokinetics and toxicity of 2′,2′-difluorodeoxycytidine and 1-β-d-arabinofuranosylcytosine. Cancer Res 1988;48:4024–31.

Grunewald R, Kantarjian H, Du M, Faucher K, Tarassoff P, Plunkett W. Gemcitabine in leukemia: a phase I clinical, plasma, and cellular pharmacology study. J Clin Oncol 1992;10:406–13.

Mackey JR, Yao SY, Smith KM, et al. Gemcitabine transport in xenopus oocytes expressing recombinant plasma membrane mammalian nucleoside transporters. J Natl Cancer Inst 1999;91:1876–81.

Huang P, Chubb S, Hertel LW, Grindey GB, Plunkett W. Action of 2′,2′-difluorodeoxycytidine on DNA synthesis. Cancer Res 1991;51:6110–7.

Heinemann V, Xu YZ, Chubb S, et al. Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2′,2′-difluorodeoxycytidine. Mol Pharmacol 1990;38:567–72.

Plunkett W, Huang P, Xu YZ, Heinemann V, Grunewald R, Gandhi V. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin Oncol 1995;22:3–10.

Heinemann V, Xu YZ, Chubb S, et al. Cellular elimination of 2′,2′-difluorodeoxycytidine 5′-triphosphate: a mechanism of self-potentiation. Cancer Res 1992;52:533–9.

Camiener GW, Smith CG. Studies of the enzymatic deamination of cytosine arabinoside. I. Enzyme distribution and species specificity. Biochem Pharmacol 1965;14:1405–16.

Pastor-Anglada M, Molina-Arcas M, Casado FJ, Bellosillo B, Colomer D, Gil J. Nucleoside transporters in chronic lymphocytic leukaemia. Leukemia 2004;18:385–93.

Mackey JR, Mani RS, Selner M, et al. Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines. Cancer Res 1998;58:4349–57.

Garcia-Manteiga JM, Molina-Arcas M, Casado F, Mazo A, Pastor-Anglada M. Nucleoside transporter profiles in human pancreatic cancer cells: role of hCNT1 in 2′,2′-difluorodeoxycytidine-induced cytotoxicity. Clin Cancer Res 2003;9:5000–8.

Spratlin J, Sangha R, Glubrecht D, et al. The absence of human equilibrative nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine-treated pancreas adenocarcinoma. Clin Cancer Res 2004;10:6956–61.

Huang QQ, Yao SY, Ritzel MW, Paterson AR, Cass CE, Young JD. Cloning and functional expression of a complementary DNA encoding a mammalian nucleoside transport protein. J Biol Chem 1994;269:17757–60.

Govindarajan R, Bakken AH, Hudkins KL, et al. In situ hybridization and immunolocalization of concentrative and equilibrative nucleoside transporters in the human intestine, liver, kidneys, and placenta. Am J Physiol Regul Integr Comp Physiol 2007;293:R1809–22.

Veltkamp SA, Jansen RS, Callies S, et al. Oral administration of gemcitabine in patients with refractory tumors: a clinical and pharmacological study. Clin Cancer Res 2008;14:3477–86.

Lai Y, Bakken AH, Unadkat JD. Simultaneous expression of hCNT1-CFP and hENT1-YFP in Madin-Darby canine kidney cells. Localization and vectorial transport studies. J Biol Chem 2002;277:37711–7.

Voigt W. Sulforhodamine B assay and chemosensitivity. Methods Mol Med 2005;110:39–48.

Skehan P, Storeng R, Scudiero D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990;82:1107–12.

Begg AC, Moonen L, Hofland I, Dessing M, Bartelink H. Human tumour cell kinetics using a monoclonal antibody against iododeoxyuridine: intratumour sampling variations. Radiother Oncol 1988;11:337–47.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.

Cohen RM, Wolfenden R. Cytidine deaminase from Escherichia coli. Purification, properties and inhibition by the potential transition state analog 3,4,5,6-tetrahydrouridine. J Biol Chem 1971;246:7561–5.

Stoller RG, Myers CE, Chabner BA. Analysis of cytidine deaminase and tetrahydrouridine interaction by use of ligand techniques. Biochem Pharmacol 1978;27:53–9.

Laliberte J, Marquez VE, Momparler RL. Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2′-deoxycytidine by human cytidine deaminase. Cancer Chemother Pharmacol 1992;30:7–11.

Lai Y, Tse CM, Unadkat JD. Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs. J Biol Chem 2004;279:4490–7.

Ruiz van Haperen V, Veerman G, Boven E, Noordhuis P, Vermorken JB, Peters GJ. Schedule dependence of sensitivity to 2′,2′-difluorodeoxycytidine (Gemcitabine) in relation to accumulation and retention of its triphosphate in solid tumour cell lines and solid tumours. Biochem Pharmacol 1994;48:1327–39.

Pauwels B, Korst AE, Pattyn GG, et al. Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro. Int J Radiat Oncol Biol Phys 2003;57:1075–83.

Van Putte BP, Hendriks JM, Romijn S, et al. Pharmacokinetics after pulmonary artery perfusion with gemcitabine. Ann Thorac Surg 2003;76:1036–40.

Reid G, Wielinga P, Zelcer N, et al. Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Mol Pharmacol 2003;63:1094–103.

Chen ZS, Lee K, Walther S, et al. Analysis of methotrexate and folate transport by multidrug resistance protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system. Cancer Res 2002;62:3144–50.

Pratt S, Shepard RL, Kandasamy RA, Johnston PA, Perry W III, Dantzig AH. The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites. Mol Cancer Ther 2005;4:855–63.

Riva C, Barra Y, Carcassonne Y, Cano JP, Rustum Y. Effect of tetrahydrouridine on metabolism and transport of 1-β-d-arabinofuranosylcytosine in human cells. Chemotherapy 1992;38:358–66.

Ritzel MW, Yao SY, Ng AM, Mackey JR, Cass CE, Young JD. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine. Mol Membr Biol 1998;15:203–11.

Wang J, Su SF, Dresser MJ, Schaner ME, Washington CB, Giacomini KM. Na(+)-dependent purine nucleoside transporter from human kidney: cloning and functional characterization. Am J Physiol 1997;273:F1058–65.

Gutierrez MM, Brett CM, Ott RJ, Hui AC, Giacomini KM. Nucleoside transport in brush border membrane vesicles from human kidney. Biochim Biophys Acta 1992;1105:1–9.

Patil SD, Unadkat JD. Sodium-dependent nucleoside transport in the human intestinal brush-border membrane. Am J Physiol 1997;272:G1314–20.

Chandrasena G, Giltay R, Patil SD, Bakken A, Unadkat JD. Functional expression of human intestinal Na+-dependent and Na+-independent nucleoside transporters in Xenopus laevis oocytes. Biochem Pharmacol 1997;53:1909–18.

Wang J, Su C, Neuhard J, Eriksson S. Expression of human mitochondrial thymidine kinase in Escherichia coli: correlation between the enzymatic activity of pyrimidine nucleoside analogues and their inhibitory effect on bacterial growth. Biochem Pharmacol 2000;59:1583–8.

Thông tin
Thông tin xuất bản

Molecular Cancer Therapeutics

Tập 7 Số 8

2415-2425

Thông tin tác giả