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Drug Combinations Evoke Collateral Sensitivity against ABCG2 effect is called CS or hypersensitivity (recently reviewed by Pluchino et al. (18) and Szakács et al. (1)). Collateral sensitivity has been reported in cells expressing ABCB1 (P-gp) (12) and ABCC1 (MRP1) (21) transporters. For instance, Gatenby and co-workers (12, 20) recently presented a compelling potential approach to cancer therapy by combined administration of low doses of verapamil and low doses of 2-deoxyglucose to suppress resistant P-gp-expressing cells by adaptive administration of chemotherapy with the goal of keeping tumor burden constant in disseminated cancers that are typically fatal when treated with conventional chemotherapy regimen. With regard to cells expressing ABCG2 (BCRP) transporters, we found only two previous reports on CS; in both cases, a single compound induced CS (22, 23). Here, we introduce an approach that exploits the MDR phe- notype to achieve targeted ATP depletion in ABCG2-express- ing cells by variation of a strategy described by Karwatsky et al. (24) and Silva et al. (12) in cells overexpressing P-gp: rather than inhibiting ATP synthesis, we selectively stimulate ATP hydrol- ysis by ABCG2 transporters and thereby induce a lethal reduc- tion of ATP levels in MDR cells but not in parental cells. We accomplished this selective stimulation of ATP hydroly- sis in ABCG2-expressing cells by treatment with a combination of subtoxic concentration of curcumin with either gramicidin A (gA) or ouabain. Curcumin, the bioactive compound in the South Asian spice turmeric, is an effective chemosensitizer that modulates the function of ABCB1, ABCC1, and ABCG2 trans- porters presumably without being transported by these efflux pumps (6, 25). Instead, curcumin inhibits drug efflux and increases the efficacy of many anticancer agents in multidrug- resistant cancers (25, 26). Curcumin also stimulates ATP hydrolysis by these transporters, and we exploited this activity to increase consumption of ATP in ABCG2-expressing cells. To kill ABCG2 cells selectively over parental cells, we amplified the ATP depletion effect of curcumin with a second ATP-de- pleting process, the activation of the Na ,K -ATPase. To this end, we treated cells with subtoxic (micromolar) concentra- tions of curcumin in combination with subtoxic (nanomolar) concentrations of gA (27) or ouabain (28, 29). Gramicidin A is a pore-forming peptide that disrupts the homeostasis of ion gra- dients across lipid membranes and the resulting change in transmembrane potential activates the Na ,K -ATPase (30, 31). Ouabain is a cardiac glycoside that inhibits the Na ,K - ATPase at micromolar concentrations (29), whereas it stimu- lates the Na ,K -ATPase activity at nanomolar concentrations (28). Stimulating these two ATP-depleting processes together lowered the intracellular ATP levels in ABCG2-expressing cells sufficiently to kill them selectively over parental cells. MATERIALS AND METHODS Chemicals—We purchased Eagle’s minimal essential medium, DMEM, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide cell cytotoxicity assay kits from ATCC (Manassas, VA). We obtained FBS, OPTIMEM reduced serum medium, improved minimum essential medium, Dulbecco’s PBS, 0.05% (w/v) trypsin-EDTA, penicillin, streptomycin, BODIPY-FL-pra- zosin, annexin V-FITC and ethidium homodimer I from Invitro- gen. We purchased Aprotinin from Roche Diagnostics. All other chemicals were purchased from Sigma-Aldrich. Cell Lines and Culture Conditions—HEK-293 cells trans- fected with the empty pcDNA3.1 vector (HEK-293 parental cells) or pcDNA3.1 vector containing the ABCG2 gene (HEK- 293 ABCG2 cells) were maintained in Eagle’s minimal essential medium supplemented with 10% (v/v) FBS, penicillin (100 units/ml), streptomycin (100 g/ml), and geneticin disulfate (G418) at a concentration of 2 mg/ml (32). The MCF-7 human breast cancer cell line was exposed to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7/FLV1, overexpressed ABCG2, whereas neither P-gp nor MRP overexpression was detected (33). Control MCF7 and MCF7/FLV1 (482R) cells overexpressing ABCG2 were cultured in RPMI with 10% (v/v) FBS in the absence or presence of 1 g/ml flavopiridol, respectively (33). Cytotoxicity Assay—We seeded 5,000 cells/well in 96-well plates and cultured overnight. After adding various concentra- tions of compounds and incubating for an additional 72 h, we replaced the medium with OPTIMEM and determined the cell viability using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide cell cytotoxicity assay kit, according to the manufacturer’s instructions. We calculated the cell viability using the following formula: Cell survival in % (mean absorb- ance in test well)/(mean absorbance in untreated well) 100. We calculated IC50 values with their errors from best curve fits of mean values of viability as a function of the drug concentra- tion to the following equation y A2 (A1 A2)/(1 (x/x0)ˆp) using Origin software version 7.5. Fluorescence- based life versus death staining of HEK-293 cells and HEK- ABCG2 cells after 72 h of incubation with the specified com- pound/s was performed with calcein (green, representing live cells) and ethidium bromide (red, representing dead cells). Flow Cytometry Analysis of Efflux Activity by ABCG2—We trypsinized cells in phenol red-free improved minimum essen- tial medium for 30 min at 37 °C in the absence or presence of 250 nM BODIPY-prazosin alone or with various concentrations of compounds. We washed the cells twice in ice-cold PBS and incubated for an additional hour at 37 °C before washing the cells and analyzing them by flow cytometry (Beckman Coulter Quanta SC). We obtained the histograms and mean fluores- cence intensity (MFI) for each condition using WinMD soft- ware. We defined 100% efflux as the difference in MFI values between the most frequently observed fluorescence value in the presence of 250 nM BODIPY-prazosin combined with 10 M fumitremorgin C (FTC), a ABCG2 inhibitor, and that in the presence of 250 nM BODIPY-prazosin without FTC. This difference therefore quantified FTC-inhibitable efflux of BODIPY-prazosin. We calculated the BODIPY-prazosin efflux (as percentages) for each condition using the following formula: (FTC-inhibitable BODIPY-prazosin efflux in the presence of compound(s))/(FTC-inhibitable BODIPY-prazosin efflux in the absence of compound(s)) 100%. Isolation of Crude Membranes from ABCG2-expressing HEK- 293 Cells—We prepared crude membranes from HEK-ABCG2 cells as described previously (34) with some modifications. Briefly, we scraped cells from their culture dishes into ice-cold PBS containing 1% (w/v) aprotinin and centrifuged at 500 g 31398 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 45 • NOVEMBER 7, 2014PDF Image | Curcumin with Either Gramicidin or Ouabain
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