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for 10 min to collect the pellet. We resuspended these cells in a lysis buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM MgCl2, and 1% (w/v) aprotinin and incubated on ice for 45 min. We disrupted cells using a Dounce homogenizer (30 strokes with pestle A and B). We removed undisrupted cells and nuclear debris by centrifugation at 500 g for 10 min. We diluted the supernatant 2-fold in resuspension buffer contain- ing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 250 mM sucrose, and 1% (w/v) aprotinin. Finally, we collected the membrane vesicles by centrifugation at 100,000 g for 60 min and resuspended in resuspension buffer. We stored the membranes in small ali- quots at 70 °C. Using the Amido Black protein method described by Schaffner and Weissmann (35) with BSA as a standard, we determined the protein content of each preparation. ATPase Assay—We used crude membranes from ABCG2- expressing HEK-293 cells (100 g protein/ml) and incubated them with varying concentrations of compounds in the pres- ence and absence of 300 M vanadate in ATPase assay buffer for 10 min at 37 °C. This ATPase assay buffer contained 50 M KCl, 5mM NaN3,2mM EGTA,10mM MgCl2,1mM DTT,2mM ouabain, and 50 mM Tris-HCl (pH 7.5). We started the reaction by adding 5 mM ATP and incubated for 20 min at 37 °C. We terminated the reaction with the addition of SDS solution (0.1 ml of 5% (w/v) SDS) and quantified the amount of inorganic phosphate released by a sensitive colorimetric reaction as described previously (34). We recorded the specific activity of the transporter as vanadate-sensitive ATPase activity (34). We determined the Na ,K -ATPase activity in crude mem- brane vesicles from ABCG2-expressing cells treated with the ABCG2 inhibitor FTC at a concentration of 10 M to com- pletely inhibit the ATPase activity of the ABCG2 transporter. We determined total ATP hydrolysis in crude membrane vesi- cles from ABCG2 cells in the absence of the Na ,K -ATPase inhibitor ouabain and in the absence of FTC. Determination of Cellular ATP Levels—We determined cel- lular ATP levels with a luciferase assay (36) using a biolumines- cence assay kit from Sigma-Aldrich. We seeded 5 105 cells/ well in 96-well plates and cultured overnight. We incubated the cells for 1 h at 37 °C with or without drug treatments. We lysed the cells with lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% (v/v) Triton X-100, 0.1% w/v SDS, and 1% w/v deoxycholate) and mixed 100 l (2.5 105 cells) of cell suspension with an equal volume of luciferase solution in ATP assay dilution buffer as indicated by the manufacturer. We mixed the plate for 3–5 s and immediately placed the plate in the luminometer (Fluoros- kan Ascent FL). We compared the observed light intensity from the cell samples to those from several ATP standards to deter- mine the ATP concentration of each sample. Determination of Membrane Potentials by Whole Cell Cur- rent Clamp Recordings—We plated cells on poly-L-lysine- coated 35-mm glass-bottomed Petri dishes (MatTek, Ashland, MA) before performing whole cell recordings in current clamp mode. The standard pipette solution consisted of 130 mM KCl, 1 mM MgCl2, 10 mM HEPES, 5 mM ATP, 0.2 mM GTP, and 0.5 mM EGTA (pH 7.2). The bath solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 5 mM glucose (pH 7.4). We fabricated the patch electrodes with resistances of 2.0 – 4.0 M from borosilicate glass (Sutter Instruments, Novato, CA) using a P-87 puller (Sutter Instru- ments). We recorded the membrane potentials using an Axo- patch 200B amplifier (Molecular Devices, Sunnyvale, CA) and digitized using a Digidata 1322A (Molecular Devices). Data acquisition was done using pCalmp9 software (Molecular Devices). Apoptosis/Necrosis Assay—We trypsinized and harvested cells that were incubated in the absence or presence of 35 nM gA, a combination of 35 nM gA and 2 M curcumin, 7.5 nM ouabain, and a combination of 7.5 nM ouabain and 2 M curcu- min for 24, 48, and 72 h. We washed cells with PBS and binding buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, and 2.5 mM CaCl2 with pH 7.4) before incubation with annexin V-FITC and ethidium homodimer I (EthD I) for 15 min at room tempera- ture in the dark. Then we determined the percentage of early apoptotic and late apoptotic or necrotic cells by flow cytometry. To inhibit the activity of caspase-3/7, we incubated cells in the presence of 50 M Z-VAD-FMK, a well known caspase inhibi- tor, for 2 h prior to the addition of other compounds. Caspase-3/7 Activity Assay—We trypsinized and harvested cells that were incubated for 72 h in the absence or presence of 35 nM gA, a combination of 35 nM gA and 2 M curcumin, 7.5 nM ouabain, and a combination of 7.5 nM ouabain and 2 M curcumin. We washed cells with 0.1% (w/v) BSA-PBS and stained cells with CellEvent caspase-3/7 green detection rea- gent (Invitrogen) for 30 min at 37 °C in the dark. Then we deter- mined the population of caspase-3/7-activated cells by flow cytometry. To distinguish early apoptotic cells from late apo- ptotic or necrotic cells, we labeled cells with both CellEvent caspase-3/7 green detection reagent and SYTOX AADvanced dead cell stain (Invitrogen) as instructed by the manufacturer. We subsequently determined the percentages of early apopto- tic and late apoptotic or necrotic cells by flow cytometry. Statistical Analysis—Unless indicated otherwise, we repeated experiments at least three times and performed each measure- ment in triplicate. In bar graphs, each bar represents a mean value standard error of the mean from at least two (typically three or more) separate experiments. We determined the sta- tistical significance of differences by a two-tailed Student’s t test, and a p value 0.05 was considered significant. A single asterisk indicates a p value 0.05, whereas a double asterisk indicates a p value 0.01. RESULTS AND DISCUSSION A Combination of Curcumin with Either gA or Ouabain Selectively Kills HEK-ABCG2 Cells—Fig. 1 shows that subtoxic concentrations (defined here as concentrations that killed 10% of cells, LD10) of curcumin (2 M) in combination with subtoxic concentrations of gA (35 nM) or ouabain (7.5 nM) were able to kill most of the cells with the MDR phenotype (HEK-293 ABCG2), whereas almost all parental cells (HEK-293 control) remained viable. We observed this selective cell death only in the presence of the combination of compounds (Fig. 1 and Table 1). Curcumin, gA, or ouabain by themselves were slightly less toxic to HEK-ABCG2 cells than to the parental cells (Fig. 1 and Table 1). These results suggest that the MDR phenotype turned from an advantage that incurred a slight resistance toward these compounds individually to a disadvantage that Drug Combinations Evoke Collateral Sensitivity against ABCG2 NOVEMBER 7, 2014 • VOLUME 289 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 31399PDF Image | Curcumin with Either Gramicidin or Ouabain
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