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Drug Combinations Evoke Collateral Sensitivity against ABCG2 To answer this question, we conducted an apoptosis/necro- sis assay with both HEK-ABCG2 and parental cells that were exposed to curcumin in combination with either gA or ouabain. After only 24 h of treatment, a significant fraction (5%) of resistant cells was already identified as late apoptotic or necrotic. After 72 h, this fraction reached over 50% in the pres- ence of curcumin and gA and 15% in the presence of curcu- min and ouabain. In comparison, less than 5% of parental cells that were treated identically or ABCG2-expressing cells that were treated with a single compound (i.e. gA or ouabain) were identified as late apoptotic or necrotic after 72 h (Fig. 9). To clarify whether the drug combinations induce apoptosis or necrosis, we measured the activation of the effector caspases (i.e. caspase-3 and -7), the hallmark of apoptosis (54), in both HEK-ABCG2 and parental cells. After 72 h of incubation with curcumin and gA, nearly 50% of resistant cells showed a signif- icant increase in caspase-3/7 activation. This fraction was 35% when these cells were treated with curcumin and oua- bain (Fig. 10A). In contrast, the activity of caspase-3/7 in paren- tal cells remained at the basal level under all conditions (Fig. 10A). In addition, we distinguished early apoptotic cells from late apoptotic or necrotic cells using the caspase-3/7 activity assay similar to the apoptosis/necrosis assay. After 72 h, 40% of resistant cells were late apoptotic or necrotic when treated with curcumin and gA; this fraction was 30% when these cells were treated with curcumin and ouabain (Fig. 10B). These results are consistent with those obtained from the apoptosis/ necrosis assay (Fig. 9), suggesting that HEK-ABCG2 cells treated by the drug combinations undergo apoptosis instead of necrosis involving the activation of caspases. Furthermore, we explored the effect of a caspase-3/7 inhibitor, Z-VAD-FMK, on the selective killing of HEK-ABCG2 cells using the apoptosis/necrosis assay. Interestingly, 50 M Z-VAD-FMK completely eliminated the selective cell death of ABCG2 cells caused by the drug combinations after 72 h (Fig. 10C). Z-VAD- FMK is not a substrate of ABCG2 transporters in the sense that treatment with only the inhibitor induced no significant difference in viability between HEK-293 parental cells (IC50 255 M) and ABCG2 cells (IC50 226 3.8 M). Consistently, results from cytotoxicity assays showed that 50 M Z-VAD-FMK protected HEK-ABCG2 cells against the toxicity of gA in the presence of 2 M curcumin (IC50 47 5.6 nM in HEK-293 parental and 50 3.8 nM in HEK-ABCG2 cells). Z-VAD-FMK had a similar effect on cells treated with ouabain in the presence of 2 M curcumin (IC50 29 1.2 nM in HEK-293 parental cells and 33 4.9 nM in HEK-ABCG2 cells). Together, these results indicate that the com- bination of curcumin with either gA or ouabain selectively kills HEK-ABCG2 cells by a caspase-dependent pathway, which ulti- mately leads to apoptotic cell death. Effect of Curcumin in Combination with gA or Ouabain on the Human Breast Adenocarcinoma Cell Lines Expressing ABCG2 Transporters (MCF-7/FLV1)—To test whether this approach would have similar effects on a clinically relevant cell line, we conducted cytotoxicity assays with human breast adenocarci- noma parental cells (MCF-7) and flavopiridol-selected daugh- ter cells that express ABCG2 transporters (MCF-7/FLV1) (33). Surprisingly, the IC50 value of gA in MCF-7/FLV1 cells was FIGURE 9. A combination of curcumin (Cur) with either gA or ouabain (Oua) stimulates selective cell death of HEK-ABCG2 cells over parental cells. Cells were incubated in the absence (A) or presence of various com- pounds: 35 nM gA (B), a combination of 35 nM gA and 2 M curcumin (C), 7.5 nM ouabain (D), and a combination of 7.5 nM ouabain and 2 M curcumin (E) for 24, 48, and 72 h and were subsequently stained with annexin V-FITC/EthD I for analysis using a flow cytometer. The dark gray bars indicate the population of early apoptotic cells (annexin V-FITC /EthD I ), whereas the light gray bars indicate the population of late apoptotic or necrotic cells (annexin V-FITC /EthD I ). significantly higher (4.5 0.8-fold) than in the MCF-7 parental cell line (Table 5 and Fig. 11A). This result contrasts with Table 1, which shows that the IC50 values for gA in HEK-293 parental control and ABCG2 cells were not significantly different (p 0.14). To explore whether this resistance toward gA was either a consequence of ABCG2 efflux activity or of a pleiotropic effect 31406 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 45 • NOVEMBER 7, 2014PDF Image | Curcumin with Either Gramicidin or Ouabain
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