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Drug Combinations Evoke Collateral Sensitivity against ABCG2 FIGURE 7. Cell viability of HEK-293 control and HEK-ABCG2 cells as a function of increasing concentrations of CCCP, and 2-DG. The sensitivity to CCCP (A) and 2-DG (B) in HEK-293 control (black) and HEK-ABCG2 (red) cells was determined in the absence (solid curves) or presence (dashed curves) of 2 M curcumin (see Table 3 for a statistical analysis of differences in IC50 values). TABLE 3 IC50 values, relative resistance, and reversal of resistance of CCCP and 2-DG in HEK-293 parental cells and in HEK-ABCG2 cells Compound CCCP alone CCCP 2 M curcumin 2-DG alone 2-DG 2 M curcumin HEK-293 control 2.3 0.4 M 1.8 0.2 M 4.0 0.6 mM 2.1 0.3 mM HEK- ABCG2 2.40.3M 1.70.2M 4.20.9mM 2.00.4mM Relative resistanceb 1.0 0.2-fold 0.9 0.2-fold 1.0 0.3-fold 1.0 0.2-fold Reversal of resistancec 1.1 0.3-fold 1.0 0.4-fold IC50 p value of IC50 from HEK-ABCG2 cells relative to IC50 from control cellsa 0.32 0.16 0.27 0.18 a To determine p values, we used a two-tailed Student’s t test based on log(IC50) values. We considered p values 0.05 as statistically significant. b Expressed as the ratio of the IC50 value of ABCG2-expressing cells to that of the control cells. c Expressed as the ratio of the relative resistance value of ABCG2-expressing cells in the absence of the second compound to the relative resistance in its presence. To determine whether the selective ATP depletion in resis- tant cells in the presence of the drug combinations was depen- dent on active ABCG2 transporters, we repeated the experi- ments in the presence of 10 M FTC. In this case, the intracellular ATP concentration in HEK-ABCG2 cells was the same as in untreated cells for all tested conditions (Fig. 6B). These results show that curcumin-stimulated ATPase activity of ABCG2 transporters combined with the gA- or ouabain- stimulated ATPase activity of the Na ,K -ATPase were responsible for depleting the intracellular ATP levels in cells with ABCG2 transporters below the viability threshold (15), while leaving ATP levels in the parental cells unaffected. Inhibitors of Oxidative Phosphorylation and Glycolysis in Combination with Curcumin Do Not Preferentially Kill HEK- ABCG2 Cells—Gramicidin A is an uncoupler of oxidative phos- phorylation (44) and inhibits glycolysis in prokaryotes (45); both of these activities inhibit ATP synthesis (44). To dissect whether inhibition of oxidative phosphorylation and glycolysis might be relevant for the results reported here, we tested the effect of carbonyl cyanide meta-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation (46), and 2-deoxyglucose (2-DG), an inhibitor of glycolysis (47), in com- bination with curcumin. We selected these two compounds because, like gA, they were not substrates of the ABCG2 trans- porter in the sense that the IC50 values from cytotoxicity assays with HEK-293 parental cells and HEK-ABCG2 cells were not significantly different for these two compounds (Fig. 7 and Table 3). We found that the combination of curcumin with CCCP or 2-DG killed HEK-ABCG2 cells and parental cells alike, indicating that inhibiting oxidative phosphorylation or glycolysis was not sufficient to selectively kill resistant cells. Therefore, these results provide further evidence that gA and ouabain enhance the cytotoxicity of curcumin in HEK- ABCG2 cells by stimulating ATP turnover by the Na ,K - ATPase rather than by inhibiting oxidative phosphorylation or glycolysis. Inhibitors of the Electron Transport Chain Do Not Affect CS by Curcumin with gA or Ouabain—To further investigate the mechanism by which stimulated ATP hydrolysis and the result- ing low intracellular ATP levels might kill HEK-ABCG2 cells, we investigated a previously proposed mechanism of CS. Lab- erge et al. (48) showed that cells expressing P-gp exhibited CS toward the P-gp substrate verapamil. In this case, verapamil by itself killed the resistant cells preferentially over the parental cells. Along with others before (49, 50), the authors showed that low concentrations of verapamil (10 M) increased the ATPase activity of P-gp and that this effect could reduce intra- cellular ATP levels by 50%. Laberge et al. proposed that hyper- sensitivity of P-gp-expressing cells toward verapamil was caused by this increased demand for ATP, which caused an increased rate of oxidative phosphorylation and concomitant increased production of reactive oxygen species (ROS) (51). They hypothesized that accumulation of ROS ultimately initi- ated apoptosis of the resistant cells. The authors showed that two inhibitors of the electron transport chain, rotenone and antimycin A (52), amplified the collateral sensitivity to vera- pamil in the P-gp-expressing cell line, whereas the same con- centration of these compounds had no effect on the parental cell line. Based on findings by Li et al. (52), Laberge et al. pro- posed that rotenone and antimycin A exerted their effect through ROS-mediated toxicity and subsequent apoptosis. 31404 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 45 • NOVEMBER 7, 2014PDF Image | Curcumin with Either Gramicidin or Ouabain
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