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FIGURE 10. Selective cell death of HEK-ABCG2 cells occurs through caspase-3/7-dependent apoptosis. Cells were incubated in the absence or presence of 35 nM gA, a combination of 35 nM gA and 2 M curcumin (Cur), 7.5 nM ouabain (Oua), and a combination of 7.5 nM ouabain and 2 M curcumin for 72 h. A, cells were stained with CellEvent caspase-3/7 green detection reagent and analyzed subsequently by flow cytometry. Bars represent the percentages of caspase-3/7 activated cells of the total population. B, cells were stained with both CellEvent caspase-3/7 green detection reagent and SYTOX AADvanced dead cell stain and analyzed subsequently by flow cytom- etry. The dark gray bars indicate the population of early apoptotic cells (Cel- lEvent caspase-3/7 green /SYTOX AADvanced ), whereas the light gray bars indicate the population of late apoptotic or necrotic cells (CellEvent caspase-3/7 green /SYTOX AADvanced ). C, cells were incubated with 50 M Z-VAD-FMK for 2 h prior to the addition of various compound(s) for 72 h and subsequently stained with annexin V-FITC/EthD I for flow cytometry anal- ysis. The dark gray bars indicate the population of early apoptotic cells (annexin V-FITC /EthD I ), whereas the light gray bars indicate the popula- tion of late apoptotic or necrotic cells (annexin V-FITC /EthD I ). p values indicate no significant difference between HEK-control and ABCG2 cells in the presence of Z-VAD-FMK. (55, 56) as a result of stepwise selection with flavopiridol, we exposed MCF-7/FLV1 cells to gA in the presence of FTC. Despite inhibition of ABCG2 transporters, these cells retained their resistance toward gA (IC50 5.9 M in the absence of FTC and 5.2 M in the presence of FTC), providing strong evidence for a pleiotropic effect. Fig. 11A shows that in the presence of 2 M curcumin, the resistance of MCF-7/FLV1 cells to gA was reversed by 22.5 9-fold and slightly inversed compared with MCF-7 parental cells (Table 5). Therefore, the CS effect on ABCG2-expressing MCF-7/FLV1 cells by curcumin with gA was similar, albeit smaller, than that observed in HEK-ABCG2 cell lines (Fig. 1). With regard to ouabain, Fig. 11B shows that in the presence of curcumin, MCF-7/FLV1 cells showed CS to increasing con- centrations of ouabain and died at 2.8 times lower concentra- tions of ouabain than MCF-7 parental cells. Treatment with curcumin or ouabain individually showed no significant differ- ences in viability between parental cells and the resistant MCF- 7/FLV1 cells (Fig. 11B and Table 5). Similar to results in Fig. 2, the presence of FTC restored the viability of MCF-7/FLV1 cells to that of MCF-7 parental cells when we incubated cells with varying concentrations of gA or ouabain in combination with 2 M curcumin (Table 5 and Fig. 11, C and D). Furthermore, we found that gA, ouabain, or the combination of curcumin with gA or with ouabain induced membrane depolarization in MCF-7 and MCF-7/FLV1 similar to that observed in HEK-293 parental cells and ABCG2 cells (Fig. 11E). These results show that the principle idea of inducing selec- tive cytotoxicity in ABCG2-transfected cells by stimulated depletion of ATP levels could be replicated in the clinically relevant human breast adenocarcinoma cell line MCF-7/FLV1 that expresses ABCG2 transporters. This approach may there- fore be generally applicable to resistant cells with significant expression of ABCG2 transporters. In summary, the CS approach of intracellular ATP depletion by treatment with agents that stimulate ATP hydrolysis by ABC transporters and ion motive pumps presented here provides a strategy to target drug resistant cells with minimal effects on nonresistant cells. This method utilizes curcumin in combina- tion with either gA or ouabain, all of which have previously been tested clinically (6, 57, 58). Therefore, the transporter- induced, synergistic ATP depletion (TISAD) strategy intro- duced here could inspire a fresh approach for discovery of CS agents against MDR cells. For this TISAD approach to be applicable to efflux pumps other than ABCG2 transporters, it will be necessary to identify modulators or substrates of these pumps that are able to induce significantly increased ATP hydrolysis rates in MDR cells with- out inducing significant toxicity in nonresistant cells. In addi- tion, co-administration of a second molecule may be necessary to induce further ATP depletion by stimulating major ATPases In contrast to the ATP starvation therapy, the TISAD approach introduced here provides targeting of MDR cells by selectively stimulating ATP hydrolysis rather than indiscrimi- nately inhibiting its synthesis. The results presented here sug- gest that severe ATP depletion and the resulting induction of cellular apoptosis may be one of the mechanisms by which Drug Combinations Evoke Collateral Sensitivity against ABCG2 such as the Na ,K -ATPase. More generally, any physical or chemical process that induces significant ATP consumption in combination with stimulated ATP hydrolysis by MDR trans- porters might have the potential to induce CS. NOVEMBER 7, 2014 • VOLUME 289 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 31407PDF Image | Curcumin with Either Gramicidin or Ouabain
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