Modulatory effects of plant phenols on human multidrug-resistance proteins 1, 4 and 5 (ABCC1, 4 and 5)

Abstract

Plant flavonoids are polyphenolic compounds, commonly found in vegetables, fruits and many food sources that form a significant portion of our diet. These compounds have been shown to interact with several ATP-binding cassette transporters that are linked with anticancer and antiviral drug resistance and, as such, may be beneficial in modulating drug resistance. This study investigates the interactions of six common polyphenols; quercetin, silymarin, resveratrol, naringenin, daidzein and hesperetin with the multidrug-resistance-associated proteins, MRP1, MRP4 and MRP5. At nontoxic concentrations, several of the polyphenols were able to modulate MRP1-, MRP4- and MRP5-mediated drug resistance, though to varying extents. The polyphenols also reversed resistance to NSC251820, a compound that appears to be a good substrate for MRP4, as predicted by data-mining studies. Furthermore, most of the polyphenols showed direct inhibition of MRP1-mediated [3H]dinitrophenyl S-glutathione and MRP4-mediated [3H]cGMP transport in inside-out vesicles prepared from human erythrocytes. Also, both quercetin and silymarin were found to inhibit MRP1-, MRP4- and MRP5-mediated transport from intact cells with high affinity. They also had significant effects on the ATPase activity of MRP1 and MRP4 without having any effect on [32P]8-azidoATP[alphaP] binding to these proteins. This suggests that these flavonoids most likely interact at the transporter's substrate-binding sites. Collectively, these results suggest that dietary flavonoids such as quercetin and silymarin can modulate transport activities of MRP1, -4 and -5. Such interactions could influence bioavailability of anticancer and antiviral drugs in vivo and thus, should be considered for increasing efficacy in drug therapies.

Figure. 1. Characterization of expression of selected ABC transporters in HEK293 transfectants

Real time RT-PCR using SYBR green was performed on all of the cell lines utilized in this work. The mRNA expression values for MDR1 (ABCB1), MRP1 (ABCC1), MRP4 (ABCC4), MRP5 (ABCC5), and MRP8 (ABCC11) were determined for each cell line. Following normalization to GAPDH, the expression values for each transfectant was compared to the expression of each transporter within the parental HEK293 cells. The values represent the mean, and the error bars are standard deviation (n=4).

Figure. 2. Sensitivity of control HEK293 and MRP4- and 5-expressing cells to thioguanine and NSC251820

Cytotoxicity assays were used to determine the sensitivity of control HEK293 (filled circles), MRP4-expressing HEK293/4.63 (open squares) and MRP5-expressing HEK293/5I (open triangles) to (A) thioguanine and predicted substrate of MRP4 based on data-mining NSC 251820 (C) as described previously [25]. The structure of NSC251820 is shown in (B). Cells (5.0 x 10³ cells) were plated into 96 well plates, cultured overnight and then exposed to thioguanine for 72 h. Viable cells were determined by the Cell Counting Kit (CCK) technique as detailed in the Material and Methods section. The mean values from three independent experiments are shown with error bars as S.D.

Figure. 3. Plant polyphenols inhibited uptake of [³H]-DNP-SG and [³H]cGMP into membrane vesicles prepared from human erythrocytes

ATP-dependent uptake at 37°C for 30 minutes in erythrocytes membrane vesicles using 3 μM [³H]-DNP-SG or 3.3 μM [³H]-cGMP was carried out as described in Materials and Methods. (A) [³H]-DNP-SG and (B) [³H]-cGMP uptake, quercetin (filled squares), hesperetin (open diamonds), daidzein (filled diamonds), silymarin (open squares), resveratrol (open circle) and narigenin (filled circle). The mean values from six independent experiments are shown with error bars as S.E.M.

Figure. 3. Plant polyphenols inhibited uptake of [³H]-DNP-SG and [³H]cGMP into membrane vesicles prepared from human erythrocytes

ATP-dependent uptake at 37°C for 30 minutes in erythrocytes membrane vesicles using 3 μM [³H]-DNP-SG or 3.3 μM [³H]-cGMP was carried out as described in Materials and Methods. (A) [³H]-DNP-SG and (B) [³H]-cGMP uptake, quercetin (filled squares), hesperetin (open diamonds), daidzein (filled diamonds), silymarin (open squares), resveratrol (open circle) and narigenin (filled circle). The mean values from six independent experiments are shown with error bars as S.E.M.

Figure. 4. Effect of selected polyphenols on calcein accumulation in MRP1-HEK293 cells

Cells (control pcDNA-HEK293 and MRP1-transfected MRP1-HEK293) were resuspended in IMDM supplemented with 5% FBS. 0.25 μM calcein-AM was added to 3x10⁵ cells in 4 ml of IMDM in the presence or absence of MK-571 and polyphenols. The cells were incubated at 37°C in the dark for 10 minutes. The cells were pelleted by centrifugation at 500 x g and resuspended in 300 μl of PBS containing 0.1% BSA. Samples were analysed immediately by using flow cytometry. Panel A shows that except 50 μM of silymarin (dotted line), MK571 and other polyphenols had no effect on control HEK293 cells. Panel B-H, thin solid line represent MRP1-overexpressing MRP1-HEK293 cells, dotted line represents MRP1-HEK293 cells in the presence of 25 μM of MK-571 and bold solid line represents MRP1-HEK293 cells in the presence of various polyphenols. Panel B: 25 μM of MK-571, Panel C: 50 μM quercetin, Panel D: 50 μM silymarin, Panel E: 50 μM hesperetin, Panel F: 50 μM resveratrol, Panel G: 50 μM daidzein and Panel H: 50 μM naringenin. Representative histograms of three independent experiments are shown.

Figure. 5. Effect of various polyphenols on BCECF accumulation and MRP5-HEK293 cells

Cells (control HEK293 and MRP5-transfected HEK293/5I) were resuspended in IMDM supplemented with 5% FBS. 0.25 μM BCECF-AM was added to 3x10⁵ cells in 4 ml of IMDM in the presence or absence of MK-571 and polyphenols. The cells were incubated at 37°C in the dark for 10 min and pelleted by centrifugation at 500 x g and resuspended in 300 μl of PBS containing 0.1% BSA. Samples were analysed immediately by flow cytometry. Panel A shows that all polyphenols and MK-571 had no effect on control HEK293 cells. Panel B-H, thin solid line and bold solid line represent MRP5-overexpressing HEK293/5I cells in the absence and presence of drugs tested, respectively. Panel B: 25 μM of MK-571 (dotted line), Panel C: 50 μM quercetin, Panel D: 50 μM silymarin, Panel E: 50 μM hesperetin, Panel F: 50 μM resveratrol, Panel G: 50 μM daidzein and Panel H: 50 μM naringenin. Representative histograms of three independent experiments are shown.

Figure. 6. Effect of various polyphenols on MRP1-mediated ATP hydrolysis

Crude membranes of MRP1 baculovirus infected High Five insect cells (100 μg/ml protein) were incubated at 37°C for 5 minutes with polyphenols in the presence and absence of BeFx. The reaction was initiated by addition of 5 mM ATP and terminated with SDS (2.5% final concentration) after 20 min incubation at 37°C. The amount of P_i released was quantitated using a colorimetric method [30, 34]. MRP1-specific activity was recorded as the BeFx-sensitive ATPase activity. Top panel: quercetin (filled squares), silymarin (open squares) and naringenin (filled circle); Bottom panel: hesperetin (open diamonds), daidzein (filled diamonds) and resveratrol (open circle). Values represent mean ± S.D from at least three independent experiments.

Figure. 7. Effect of various bioflavonoids on basal and PGE1-stimulated MRP4 ATPase activity

(A) Crude membranes of MRP4 baculovirus infected High Five insect cells (100 μg protein/ml) were incubated at 37°C for 5 min with polyphenols in the presence and absence of BeFx. The reaction was initiated by addition of 5 mM ATP and terminated with SDS (2.5% final concentration) after 20 min incubation at 37°C. The MRP4-specific activity was determined as described in the legend to Fig. 7. (B) MRP4 substrate PGE1 stimulates MRP4 ATPase activity [30]. Briefly, crude membranes were incubated with 20 μM of PGE1 in the absence or presence of polyphenols at indicated concentrations, and the ATPase assay was carried out as described above. In A and B panels: quercetin (filled squares), silymarin (open squares), naringenin (filled circle), hesperetin (open diamonds), daidzein (filled diamonds), and, resveratrol (open circle). Values represent mean ± S.D from at least three independent experiments.

Figure. 7. Effect of various bioflavonoids on basal and PGE1-stimulated MRP4 ATPase activity

(A) Crude membranes of MRP4 baculovirus infected High Five insect cells (100 μg protein/ml) were incubated at 37°C for 5 min with polyphenols in the presence and absence of BeFx. The reaction was initiated by addition of 5 mM ATP and terminated with SDS (2.5% final concentration) after 20 min incubation at 37°C. The MRP4-specific activity was determined as described in the legend to Fig. 7. (B) MRP4 substrate PGE1 stimulates MRP4 ATPase activity [30]. Briefly, crude membranes were incubated with 20 μM of PGE1 in the absence or presence of polyphenols at indicated concentrations, and the ATPase assay was carried out as described above. In A and B panels: quercetin (filled squares), silymarin (open squares), naringenin (filled circle), hesperetin (open diamonds), daidzein (filled diamonds), and, resveratrol (open circle). Values represent mean ± S.D from at least three independent experiments.

Figure. 8. Quercetin and silymarin do not inhibit photoaffinity labelling of MRP1 or MRP4 with [α-³²P]8-azidoATP

Crude membranes (50–75 μg protein) of MRP1 or MRP4 baculovirus infected High Five insect cells were incubated at 4°C for 5 minutes with 10 μM [α-³²P]8-azidoATP (10 μC_i/nmole) in the presence and absence of quercetin or silymarin. The photocrosslinking with 365 nm UV light was carried out on ice for 10 minutes as described previously [30]. Incorporation of [α-³²P]8-azidoATP detected by phosphorimaging and by exposure to X-ray film at −70C for 2-8 h after gel electrophoresis. A, Photolabeling of MRP1 and B, MRP4, respectively. In both panels, lane 1 and 5, membranes exposed to [α-³²P]8-azidoATP alone, lanes 2, 3 and 4 membranes treated with 10 nM, 50 μM and 100 nM quercetin, respectively and lanes 6, 7 and 8, with 10 nM, 50 μM and 100 nM silymarin, respectively. Lane 9, crude membranes were incubated with 10 μM [α-³²P]8-azidoATP in the presence of 10 mM ATP-Mg²⁺.