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Drug Combinations Evoke Collateral Sensitivity against ABCG2 TABLE 2 Mean fluorescence intensity (MFI) and percentage of efflux of Bp in HEK-ABCG2 cells treated with the specified compound(s) Distribution Treatment 1 250 nM Bp (substrate) 2 Bp 10 M FTC (inhibitor) 3 Bp 2 M curcumin 4 Bp35nM gA 5 Bp35nM gA2M curcumin 6 Bp 7.5 nM ouabain 7 Bp 7.5 nM ouabain 2 M curcumin MFIa Effluxb % 10566 100 5464 0 13960 9219 10871 9921 5402 1.31 10642 10019 37632 3812 a Mean values of the mode (i.e. the most frequently measured fluorescence inten- sity) of each distribution standard deviation of the mean from two indepen- dent experiments. b The difference in MFI values in the presence of 250 nM Bp alone to that in the presence of 250 nM Bp with 10 M FTC was defined as 100% efflux activity. FIGURE 4. Effect of gA, curcumin (cur), ouabain (oua), and a combination of curcumin and gA, or curcumin and ouabain on ATP hydrolysis by the ABCG2 transporter, on ATP hydrolysis by the Na,K-ATPase, and on total ATP hydrolysis by both transporters in crude membrane vesicles from HEK-ABCG2 cells. Single, double, and triple asterisks indicate a differ- ence compared with the basal activity with a p value 0.05, 0.01, and 0.001 respectively. the inhibition by FTC. Similarly, the combination of curcumin with ouabain reduced efflux by 60%. For comparison, the accumulation of BODIPY-prazosin remained unaffected under all conditions in parental cells (Fig. 3). These results demon- strate a strong effect on the efflux activity of ABCG2 transport- ers by the combined treatments. A Combination of Curcumin with Either gA or Ouabain Increases ATP Turnover in HEK-ABCG2 Cells—Because simul- taneous treatment with subtoxic concentrations of curcumin with gA or ouabain inhibited the transport of BODIPY-prazo- sin by ABCG2 transporters, we hypothesized that selective killing of ABCG2 cells after exposure to the drug combina- tions was not caused by transport activity (Table 2) but rather by increased ATP hydrolysis by the ABCG2 trans- porter and Na ,K -ATPase. This hypothesis was based on previous observations that curcumin stimulates ATP hydroly- sis by ABCG2 transporters presumably without being a sub- strate (26) and that nanomolar concentrations of gA and oua- bain can increase Na,K-ATPase activity (27, 30). To quantify ATP hydrolysis by the ABCG2 transporter, we performed experiments with crude membranes from HEK- ABCG2 cells in the presence of 2 mM ouabain (to inhibit ATP hydrolysis by the Na ,K -ATPase) (26). Fig. 4 shows that cur- FIGURE 5. Effects of gA and ouabain on the ATP hydrolysis rate by the Na ,K -ATPase in crude membrane fragments from HEK-ABCG2 cells and on the RMP of HEK-293 control and HEK-ABCG2 cells. A, stimulatory and inhibitory effect of various concentrations of ouabain (red) and gA (blue) on the ATP hydrolysis rate by the Na,K-ATPase in crude membrane frag- ments from HEK-ABCG2 cells. A single asterisk indicates a difference com- pared with the basal hydrolysis rate (black horizontal line) with a p value 0.05; a double asterisk indicates a p value 0.01. B, effect of curcumin (cur), gA, and ouabain (oua) on the RMP of HEK-293 control (black bars) and HEK-ABCG2 cells (white bars). The cells were incubated under various conditions as indi- cated. The bars labeled with RMP represent the control without addition of compounds. FTC was used at a concentration of 10 M. Triple asterisks indicate a difference compared with the control (RMP) with a p value 0.001. The values in brackets indicate the numbers of measurements. cumin-induced stimulation of ATP hydrolysis by ABCG2 transporters was 3.7 times higher than the basal rate of ATP hydrolysis. Gramicidin A or ouabain, in contrast, had no effect on the ATP hydrolysis rate by ABCG2 transporters. For com- parison, the maximal stimulation of ATP hydrolysis by 20 M mitoxantrone was double the basal rate. These results demon- strate that curcumin increases ATP hydrolysis by ABCG2 transporters approximately twice as strongly as mitoxantrone, whereas gA or ouabain exerts its cytotoxic effect without directly modulating ATP hydrolysis by ABCG2 transporters. To quantify ATP hydrolysis by the Na,K-ATPase, we performed experiments with crude membranes from HEK- ABCG2 cells in the presence of 10 M FTC (to inhibit all ATP hydrolysis by the ABCG2 transporter) (26). Fig. 4 shows that gA 31402 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 45 • NOVEMBER 7, 2014PDF Image | Curcumin with Either Gramicidin or Ouabain
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