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THEJOURNALOFBIOLOGICALCHEMISTRY VOL.289,NO.45,pp.31397–31410,November7,2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. A Combination of Curcumin with Either Gramicidin or Ouabain Selectively Kills Cells That Express the Multidrug Resistance-linked ABCG2 Transporter* Received for publication, April 28, 2014, and in revised form, September 12, 2014 Published, JBC Papers in Press, September 24, 2014, DOI 10.1074/jbc.M114.576819 Divya K. Rao‡1, Haiyan Liu‡1, Suresh V. Ambudkar§, and Michael Mayer‡¶2 From the Departments of ‡Biomedical Engineering and ¶Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109 and the §Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 Background: The ABCG2 transporter is an ATP-dependent efflux pump that contributes to multidrug resistance. Results: Curcumin in combination with gramicidin or ouabain reduces intracellular ATP levels in ABCG2-expressing cells and selectively kills these cells over parental cells. Conclusion: ABCG2-expressing cells display collateral sensitivity toward these combinations of compounds. Significance: Understanding ABCG2-mediated collateral sensitivity is helpful in finding ways to combat multidrug resistance. This paper introduces a strategy to kill selectively multidrug- resistant cells that express the ABCG2 transporter (also called breast cancer resistance protein, or BCRP). The approach is based on specific stimulation of ATP hydrolysis by ABCG2 transporters with subtoxic doses of curcumin combined with stimulation of ATP hydrolysis by Na ,K -ATPase with subtoxic doses of gramicidin A or ouabain. After 72 h of incubation with the drug combinations, the resulting overconsumption of ATP by both pathways inhibits the efflux activity of ABCG2 trans- porters, leads to depletion of intracellular ATP levels below the viability threshold, and kills resistant cells selectively over cells that lack ABCG2 transporters. This strategy, which was also tested on a clinically relevant human breast adenocarcinoma cell line (MCF-7/FLV1), exploits the overexpression of ABCG2 transporters and induces caspase-dependent apoptotic cell death selectively in resistant cells. This work thus introduces a novel strategy to exploit collateral sensitivity (CS) with a combi- nation of two clinically used compounds that individually do not exert CS. Collectively, this work expands the current knowledge on ABCG2-mediated CS and provides a potential strategy for discovery of CS drugs against drug-resistant cancer cells. This paper reports a collateral sensitivity (CS)3 strategy (1) to kill selectively resistant cells that overexpress the ABCG2 trans- This is an open access article under the CC BY license. * This work was supported, in whole or in part, by National Institutes of Health Grant 1R01GM081705 (to M. M.) and by the Intramural Research Program of the NCI National Institutes of Health Center for Cancer Research (to S. V. A.). 1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed: Depts. of Biomedical Engi- neering and Chemical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109. Tel.: 734-763-4609; Fax: 734-763-4371; E-mail: mimayer@umich.edu. 3 The abbreviations used are: CS, collateral sensitivity; MDR, multidrug resistance; MFI, mean fluorescence intensity; Bp, BODIPY-prazosin; gA, gramicidinA; CCCP, carbonyl cyanide m-chlorophenylhydrazone; 2-DG, 2-deoxyglucose; FTC, fumitremorgin C; Z-VAD-FMK, benzyloxycarbonyl- Val-Ala-Asp(OMe)-fluoromethylketone; ABC, ATP-binding cassette; P-gp, P-glycoprotein; EthD I, ethidium homodimer I; RMP, resting membrane porter. This transporter is an ATP-binding cassette (ABC) pro- tein (2) that is located in the plasma membrane of cells in the blood-brain barrier, intestines, and placenta (2– 6). Expression of ABCG2 transporters is a marker for stem cells (7) and is being discussed as a functional marker of cancer stem cells (8). ABCG2 transporters, together with multidrug resistance pro- tein 1 (MDR1/ABCB1, also called P-glycoprotein (P-gp)) and multidrug resistance-associated protein 1 (MRP1/ABCC1), prevent accumulation of xenobiotics and steroids in the human body (3, 6). This efflux activity is also believed to render stem cells resistant to drugs and oxidative stress, as well as maintain stem cells in an undifferentiated state (7, 8). Overexpression of efflux pumps in malignant cells can, however, lead to a multi- drug resistance (MDR) phenotype that results in the failure of cancer chemotherapy (4, 9). This efflux-induced drug resis- tance has motivated attempts to circumvent ABC transporter- mediated MDR in cancer cells. Inhibitors of MDR transporters, however, have had limited success in clinical trials because of excessive systemic side effects when administered in combina- tion with chemotherapeutic drugs (10 –12). One alternative strategy to address MDR is the so-called ATP depletion strategy that takes advantage of increased metabolic needs of cancer cells (13, 14). This approach kills tumor cells by inhibiting ATP synthesis, which leads to apoptosis and necrosis (15) in fast growing cells (16). All previously explored approaches have in common that they achieved ATP depletion by inhibiting ATP synthesis. Despite the potential of this strategy for cancer therapy, it appears difficult to inhibit the energy metabolism of tumor cells selectively because host cells are dependent on the same ATP generating pathways (13). An emerging strategy to address MDR is to exploit mecha- nisms of drug resistance to target these resistant cells (17). Sev- eral research groups reported that resistant cells were more sensitive to certain compounds than their parental cells (12, 18–20). This little known and mechanistically underexplored potential; ROS, reactive oxygen species; TISAD, transporter-induced, syn- ergistic ATP depletion. NOVEMBER 7, 2014 • VOLUME 289 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 31397PDF Image | Curcumin with Either Gramicidin or Ouabain
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