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increased the rate of ATP hydrolysis by the Na ,K -ATPase by a factor of 2.6. Because ouabain is known to have both stimula- tory and inhibitory effects on the Na,K-ATPase (28, 29, 38, 39), we determined the effect of various concentrations of oua- bain on the Na,K-ATPase (Fig. 5A). We found that ouabain concentrations of 1 M and above inhibited the Na ,K - ATPase in crude membranes from HEK-ABCG2 cells, whereas ouabain concentrations between 1 pM and 10 nM activated the Na,K-ATPase relative to its basal activity. A similar activa- tion/inactivation response was reported for the ATPase activity of P-gp as a function of various verapamil concentrations (24). Maximal activation of the Na,K-ATPase in membrane vesi- cles occurred at 10 pM ouabain, leading to a 2.3 0.1-fold increase in the rate of ATP hydrolysis compared with the absence of ouabain (Figs. 4 and 5A). We found that 2 M cur- cumin also increased ATP hydrolysis by the Na,K-ATPase by a factor of 1.8 compared with basal activity (Fig. 4). The combination of gA with curcumin, however, did not exert an additive effect; ATP hydrolysis by the Na,K-ATPase was similar to that of gA alone. With regard to the total ATP hydrolysis rate by ABCG2 transporters and the Na,K-ATPase together, Fig. 4 shows that the experimentally measured total ATP hydrolysis rate induced by gA, ouabain, or curcumin by themselves was approximately the sum of the ATP hydrolysis rate by ABCG2 transporters and the Na ,K -ATPase, as expected. In contrast, the combination of curcumin with gA or ouabain caused a syn- ergistic effect on the rate of ATP hydrolysis, leading to 5- and 4-fold increases compared with the basal hydrolysis rate, respectively. Therefore, we hypothesized that the resulting ATP turnover of 140 –170 nmol Pi/(min mg protein) may be sufficiently fast to deplete intracellular ATP levels in HEK- ABCG2 cells below the viability threshold. Gramicidin A and Ouabain Alter the Resting Membrane Potential of Cells—Gramicidin A affects energy metabolism by forming pores in cellular membranes and thereby altering their resting membrane potential (30, 31). This membrane depolar- ization stimulates the Na ,K -ATPase, leading to an increased rate of ATP hydrolysis and a decrease in intracellular ATP con- tent (30, 31). In the case of ouabain, the mechanism of activa- tion of the Na ,K -ATPase is not well understood. Ouabain binds to the Na ,K -ATPase, but Oselkin et al. (40) proposed that its stimulatory effect at pico- and nanomolar concentra- tions may be exerted indirectly by a cascade of intracellular signaling events. To explore the effect of gA and ouabain on the resting membrane potential (RMP) of HEK-ABCG2 and paren- tal cells, we performed whole cell current clamp experiments. Fig. 5B shows that even in the absence of curcumin, gA, or ouabain, the RMP of HEK-ABCG2 cells was approximately half that of HEK-293 parental cells. Hoffman et al. (41) have previ- ously linked membrane depolarization to an ABC-transporter mediated phenomenon in MDR cells. Fig. 5B also shows that gA, curcumin, ouabain, or a combination of curcumin with either gA or ouabain induced further depolarization of the RMP in both cell types. Because gA is a pore-forming peptide (27, 42), we expected a strong effect on the RMP as shown in Fig. 5B. Surprisingly, however, ouabain at concentrations in the low nanomolar FIGURE 6. Effect of subtoxic concentrations of curcumin (2 M), gA (35 nM), or ouabain (7.5 nM) and combinations of curcumin with either gA or ouabain on intracellular ATP levels in HEK-293 control (black bars) and HEK-ABCG2 cells (white bars) in the absence (A) and presence (B) of 10 M FTC. A double asterisk indicates a difference compared with untreated cells with a p value 0.01. range depolarized both the HEK-ABCG2 and parental cells to a similar extent as gA. In light of the result that ouabain stimu- lated ATP hydrolysis by the Na ,K -ATPase (Fig. 5A), this depolarization suggests one plausible mechanism for gA- and ouabain-induced stimulation of the Na ,K -ATPase as part of a cellular regulation response to restore the RMP. Because oua- bain does not form pores in membranes (43), the mechanism for ouabain-induced depolarization remains unclear. A Combination of Curcumin with Either gA or Ouabain Selectively Depletes Intracellular ATP Levels in HEK-ABCG2 Cells—To determine whether the increased ATP turnover may reduce the intracellular ATP level, we measured intracellular ATP concentrations in HEK-ABCG2 cells and parental cells. Fig. 6A shows that the basal intracellular ATP levels in HEK- ABCG2 and parental cells were similar. Treatments with cur- cumin, gA, or ouabain individually decreased the intracellular ATP levels up to 20%. In contrast, exposure to a combination of curcumin with gA or ouabain depleted intracellular ATP levels by 40–50% in HEK-ABCG2 cells, whereas these levels remained almost unaffected in the parental cells. These results hence demonstrate the synergistic effects by the drug combina- tions on intracellular ATP depletion in HEK-ABCG2 cells. Drug Combinations Evoke Collateral Sensitivity against ABCG2 NOVEMBER 7, 2014 • VOLUME 289 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 31403PDF Image | Curcumin with Either Gramicidin or Ouabain
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