Abstract
Purpose
We have previously demonstrated that the cytotoxicity of anthracyclines, pirarubicin (THP) and doxorubicin (DOX), is partially dominated by their intracellular amounts, which depend on the uptake efficacy of transporter(s). To clarify their transport mechanism, we examined whether or not Na+/nucleoside cotransporter (CNT) is involved in the uptake of THP by M5076 cells.
Methods
Expression of the CNT isoforms was determined by reverse-transcription PCR. We used two cell lines, intact M5076 and CNT2-transfected Cos-7 cells, to characterize the uptake of THP and [3H]uridine.
Results
The mRNA for CNT2, but not that for CNT1 or CNT3, was expressed in M5076 cells, and [3H]uridine uptake by the cells required a Na+ gradient as a driving force. THP uptake by M5076 cells depended on a Na+ gradient, and furthermore, formycin B and AZT had cis-inhibitory and trans-stimulatory effects on the uptake. The efflux of [3H]uridine from M5076 cells was stimulated by the addition of THP extracellularly, which constituted definite evidence of CNT-mediated uptake of THP. However, THP uptake by CNT2 transfectant was almost the same as that by mock cells, indicating that an unidentified CNT isoform contributes to THP uptake by M5076 cells, this being supported by the differences in transport characteristics of [3H]uridine between M5076 and CNT2-transfected cells.
Conclusion
THP is partially taken up into M5076 cells via a novel Na+-dependent transport system common to nucleosides.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barcelo F, Barcelo I, Gavilanes F, Ferragut JA, Yanovich S, Gonzalez-Ros JM (1986) Interaction of anthracyclines with nucleotides and related compounds studied by spectroscopy. Biochim Biophys Acta 884:172–181
Barthelemy-Clavey V, Maurizot JC, Dimicoli JL, Sicard P (1974) Self-association of daunorubicin. FEBS Lett 46:5–10
Blum RH, Carter SK (1974) Adriamycin. A new anticancer drug with significant clinical activity. Ann Intern Med 80:249–259
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Cabral S, Leis S, Bover L, Nembrot M, Mordoh J (1984) Dipyridamole inhibits version by thymidine of methotrexate effect and increases drug uptake in Sarcoma 180 cells. Proc Natl Acad Sci U S A 81:3200–3203
Cass CE, Young JD, Baldwin SA (1998) Recent advances in the molecular biology of nucleoside transporters of mammalian cells. Biochem Cell Biol 76:761–770
Chan TC (1989) Augmentation of 1-beta-d-arabinofuranosylcytosine cytotoxicity in human tumor cells by inhibiting drug efflux. Cancer Res 49:2656–2660
Damaraju VL, Damaraju S, Young JD, Baldwin SA, Mackey J, Sawyer MB, Cass CE (2003) Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy. Oncogene 22:7524–7536
Doyle LA, Ross DD (2003) Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 22:7340–7358
Eksborg S (1978) Extraction of daunorubicin and doxorubicin and their hydroxyl metabolites: self-association in aqueous solution. J Pharm Sci 67:782–785
Gaffen JD, Chambers EA, Bennett A (1989) The effects of dipyridamole and indomethacin on methotrexate cytotoxicity in LoVo human colon cancer cells. J Pharm Pharmacol 41:350–352
Goh LB, Mack P, Lee CW (1995) Nitrobenzylthioinosine-binding protein overexpression in human breast, liver, stomach and colorectal tumor tissues. Anticancer Res 15:2575–2579
Griffith D, Jarvis SM (1996) Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta 1286:153–181
Hazlehurst LA, Landowski TH, Dalton WS (2003) Role of the tumor microenvironment in mediating de novo resistance to drugs and physiological mediators of cell death. Oncogene 22:7396–7402
Hyde RJ, Cass CE, Young JD, Baldwin SA (2001) The ENT family of eukaryote nucleoside and nucleobase transporters: recent advances in the investigation of structure/function relationships and the identification of novel isoforms. Mol Membr Biol 18:53–63
Krishna R, Mayer LD (2001) Modulation of P-glycoprotein (PGP) mediated multidrug resistance (MDR) using chemosensitizers: recent advances in the design of selective MDR modulators. Curr Med Chem Anti-Canc Agents 1:163–174
Mcclean S, Hill BT (1992) An overview of membrane, cytosolic and nuclear proteins associated with the expression of resistance to multiple drugs in vitro. Biochim Biophys Acta 1114:107–127
Micha P, Naz C, Irina S, Ying Z, Imogen RC (2001) Differential expression of human nucleoside transporters in normal and tumor tissue. Biochem Biophys Res Commun 280:951–959
Nagai K, Nagasawa K, Sadzuka Y, Tsujimoto M, Takara K, Ohnishi N, Yokoyama T, Fujimoto S (2002) Relationships between the in vitro cytotoxicity and transport characteristics of pirarubicin and doxorubicin in M5076 ovarian sarcoma, and comparison with those in Ehrlich ascites carcinoma cells. Cancer Chemother Pharmacol 49:244–250
Nagai K, Nagasawa K, Ishimoto A, Fujimoto S (2003) Pirarubicin is taken up by uridine-transportable sodium-dependent concentrative nucleoside transporter in Ehrlich ascites carcinoma cells. Cancer Chemother Pharmacol 51:512–518
Nagasawa K, Natazuka T, Chihara K, Kitazawa F, Tsumura A, Takara K, Nomiyama M, Ohnishi N, Yokoyama T (1996) Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells. Cancer Chemother Pharmacol 37:297–304
Nagasawa K, Ohnishi N, Yokoyama T (1998) Possibility of contribution of nucleoside transport systems to pirarubicin uptake by HL60 cells but not mononuclear cells. Jpn J Cancer Res 89:673–680
Nagasawa K, Nagai K, Ohnishi N, Yokoyama T, Fujimoto S (2001) Contribution of specific transport systems to anthracycline transport in tumor and normal cells. Curr Drug Metab 2:355–366
Nagasawa K, Nagai K, Ishimoto A, Fujimoto S (2003) Transport mechanism of lovastatin acid in bovine kidney NBL-1 cells: kinetic evidences imply involvement of monocarboxylate transporter 4. Int J Pharm 262:63–73
Patel S, Sprung AU, Keller BA, Heaton VJ, Fisher LM (1997) Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity. Mol Pharmacol 52:658–666
Ritzel MW, Ng AM, Yao SYM, Graham K, Loewen SK, Smith KM, Ritzel RG, Mowles DA, Carpenter P, Chen XZ, Karpinski E, Hyde RJ, Baldwin SA, Cass CE, Young JD (2001) Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). J Biol Chem 276:2914–2927
Shan K, Lincoff AM, Young JB (1996) Anthracycline-induced cardiotoxicity (see comments). Ann Intern Med 125:47–58
Tsuruo T, Iida H, Tsukagoshi S, Sakurai Y (1982) 4′-O-Tetrahydropyranyladriamycin as a potential new antitumor agent. Cancer Res 42:1462–1467
Wijnholds J (2002) Drug resistance caused by multidrug resistance-associated proteins. Novartis Found Symp 243:69–79
Yao SY, Cass CE, Young JD (1996) Transport of the antiviral nucleoside analogs 3′-azido-3′-deoxythymidine and 2′,3′-dideoxycytidine by a recombinant nucleoside transporter (rCNT) expressed in Xenopus laevis oocytes. Mol Pharmacol 50:388–393
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Nagai, K., Nagasawa, K. & Fujimoto, S. Uptake of the anthracycline pirarubicin into mouse M5076 ovarian sarcoma cells via a sodium-dependent nucleoside transport system. Cancer Chemother Pharmacol 55, 222–230 (2005). https://doi.org/10.1007/s00280-004-0861-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00280-004-0861-7