Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3′,5′-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).
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This work was supported by The Oxnard Foundation, University of New Mexico Clinical & Translational Science Center Pilot Award 1UL1RR031977, and New Mexico Cancer Nanotechnology Training Center grant R25CA153825.
Fletcher JI, Haber M, Henderson MJ, Norris MD (2010) ABC transporters in cancer: more than just drug efflux pumps. Nat Rev Cancer 10(2):147–156CrossRefGoogle Scholar
Zinzi L et al (2014) ABC transporters in CSCs membranes as a novel target for treating tumor relapse. Front Pharmacol 5:163CrossRefGoogle Scholar
Aronsen L, Orvoll E, Lysaa R, Ravna AW, Sager G (2014) Modulation of high affinity ATP-dependent cyclic nucleotide transporters by specific and non-specific cyclic nucleotide phosphodiesterase inhibitors. Eur J Pharmacol 745:249–253CrossRefGoogle Scholar
Wu C-P, Calcagno AM, Ambudkar SV (2008) Reversal of ABC drug transporter-mediated multidrug resistance in cancer cells: evaluation of current strategies. Curr Mol Pharmacol 1(2):93–105CrossRefGoogle Scholar
Shiozawa K et al (2004) Reversal of breast cancer resistance protein (BCRP/ABCG2)-mediated drug resistance by novobiocin, a coumermycin antibiotic. Int J Cancer 108(1):146–151CrossRefGoogle Scholar
Jekerle V et al (2006) In vitro and in vivo evaluation of WK-X-34, a novel inhibitor of P-glycoprotein and BCRP, using radio imaging techniques. Int J Cancer 119(2):414–422CrossRefGoogle Scholar
Ozben T (2006) Mechanisms and strategies to overcome multiple drug resistance in cancer. FEBS Lett 580(12):2903–2909CrossRefGoogle Scholar
Zarrin A, Mehdipour AR, Miri R (2010) Dihydropyridines and multidrug resistance: previous attempts, present state, and future trends. Chem Biol Drug Des 76(5):369–381CrossRefGoogle Scholar
Copsel S et al (2011) Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation. J Biol Chem 286(9):6979–6988CrossRefGoogle Scholar
Fajardo AM, Piazza GA, Tinsley HN (2014) The role of cyclic nucleotide signaling pathways in cancer: targets for prevention and treatment. Cancers (Basel) 6(1):436–458CrossRefGoogle Scholar
Coffino P, Bourne HR, Tomkins GM (1975) Mechanism of lymphoma cell death induced by cyclic AMP. Am J Pathol 81:199–204Google Scholar
Dou A, Wang X (2010) Cyclic adenosine monophosphate signal pathway in targeted therapy of lymphoma. Chin Med J (Engl) 123(1):95–99Google Scholar
Zhang J, Chung T, Oldenburg K (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73CrossRefGoogle Scholar