Quercetin, Morin, Luteolin, and Phloretin Are Dietary Flavonoid

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Quercetin, Luteolin, Phloretin and Morin are Dietary Flavonoid Inhibitors of Monocarboxylate Transporter 6 Robert Spencer Jones, Mark Parker, and Marilyn Emily Morris Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 19, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Quercetin, Luteolin, Phloretin and Morin are Dietary Flavonoid Inhibitors of Monocarboxylate Transporter 6 Authors: Robert S. Jones1, Mark. D. Parker2, and Marilyn E. Morris1, * 1

Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences,

2

Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA

*

Correspondence should be addressed to:

Marilyn E. Morris, PhD Department of Pharmaceutical Sciences University at Buffalo State University of New York 352 Kapoor Hall Buffalo, NY 14214 Ph: (716) 645-4839 Fax: (716) 829-6569 [email protected]

Keywords: transporters, diet, flavonoids, drug-food interactions, bumetanide, monocarboxylate transporter 6, slc16a5 To be submitted to the Molecular Pharmaceutics This manuscript contains 5 figure(s), 0 table(s), and 28 references

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 28

Abstract: Monocarboxylate transporter 6 (MCT6; SLC16A5) has been recognized for its role as a xenobiotic transporter, with characterized substrates probenecid, bumetanide, and nateglinide. To date, the impact of commonly ingested dietary compounds on MCT6 function has not been investigated and therefore, the objective of this study was to evaluate a variety of flavonoids for their potential MCT6-specific interactions. Flavonoids are a large group of polyphenolic phytochemicals found in commonly consumed plant-based products that have been recognized for their dietary health benefits. The uptake of bumetanide in human MCT6 gene-transfected Xenopus laevis oocytes was significantly decreased in the presence of a variety of flavonoids (e.g. quercetin, luteolin, phloretin, and morin), but was not significantly affected by flavonoid glycosides (e.g. naringin, rutin, phlorizin). The IC50 values of quercetin, phloretin, and morin were determined to be 25.3 ± 3.36, 17.3 ± 2.37, and 33.1 ± 3.29 µM, respectively. The mechanism of inhibition of phloretin was reversible and competitive, with a Ki value of 22.8 µM. Furthermore, typical MCT substrates were also investigated for their potential interactions with MCT6. Substrates of MCTs 1, 2, 4, 8, and 10 did not cause any significant decrease in MCT6mediated bumetanide uptake, suggesting that MCT6 has distinct compound selectivity. In summary, these results suggest that dietary aglycone flavonoids may significantly alter the pharmacokinetics and pharmacodynamics of bumetanide and other MCT6-specific substrates, and may represent potential substrates for MCT6.


2

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction: The monocarboxylate transporter family (SLC16A) is composed of fourteen isoforms, with MCTs 1-4 being proton-dependent lactate transporters 1. Within the past decade, several previously uncharacterized MCT isoforms have been associated with a variety of diseases including cancer, atherosclerosis, cataract formation, and mental retardation 2. More specifically, monocarboxylate transporter 6 (MCT6; SLC16A5) has been reported to transport the organic anions bumetanide, nateglinide, and probenecid in a pH- and membrane potentialdependent manner

3, 4

; however no functional role or verified endogenous substrates have been

characterized, making MCT6 one of the few remaining orphan transporters in a family of clinically significant isoforms. To date, limited MCT6 protein expression data is available in the literature for humans; however it has been noted in two reports that MCT6 is expressed in intestinal tissues (Note that “MCT5” represents MCT6 in Gill et al. 5 due to previous differences in isoform nomenclature) 4, 5. The high expression of MCT6 in the intestine suggests that MCT6 may play an important role in not just endogenous compound absorption and distribution, but potentially the absorption of dietary compounds, such as flavonoids. Flavonoids, a naturally occurring group of polyphenolic phytochemicals, are one of the most commonly ingested group of compounds found in a plant-based diet. Responsible for a wide range of pharmacological activities, there are ~8,000 flavonoids that can be divided into several classes, including but not limited to: flavanones, flavonols, flavones, and isoflavones, based on their chemical structure. Approximately 1 g of total flavonoids are ingested from the typical U.S. diet per day, and concentrations of these flavonoids have been shown to range in the low micromolar range in human plasma

6, 7

. In addition, flavonoids are also ingested as single-

compound dietary supplements, resulting in potentially higher concentrations in humans.


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 28

Overall, flavonoids continue to be extensively studied due to their potential treatment benefits in disorders such as obesity, diabetes, cardiovascular disease, as well as cancer 8, 9. Numerous studies have been performed investigating the effects of flavonoids as potent substrates/inhibitors of a wide variety of transporters such as glucose transporters (e.g. SGLT, GLUT)

10, 11

, ATP-binding cassette (ABC) transporters (e.g. P-glycoprotein, BCRP)

wide variety of Phase I and Phase II metabolizing enzymes

14, 15

.

12, 13

, and a

Flavonoids have been

demonstrated to be substrates and potent inhibitors of many members of the organic anion transporter family, including MCTs 16, 17, and cause significant drug-food interactions 18-21. More specifically, flavonoids have been reported to interact with organic anion transporters MCT1 16, OAT1

22

, and OATP1B1

23

. Flavonoids are eliminated predominantly by metabolism, resulting

in high plasma concentrations of flavonoid metabolites. In the liver, flavonoids may undergo Phase I and Phase II metabolism into metabolic derivatives, with major Phase II metabolites formed from methylation, sulfation, and glucuronidation reactions 7. It is important to note that these flavonoid metabolites vary significantly in structure and tissue distribution. Some of the conjugated metabolites of flavonoids have also been demonstrated to be substrates and potent inhibitors of many members of the organic anion transporter family

16, 17

, and may also be

responsible for significant drug-food interactions 18-21. Considering the structural similarity between flavonoids and estrone sulfate (that is a known inhibitor of MCT6 3) is relatively high, we hypothesize that some flavonoids may also inhibit MCT6. For this study, our laboratory investigated the inhibition of MCT6-mediated uptake of a probe substrate (bumetanide) using a variety of flavonoids of several structural classes in vitro using the Xenopus laevis oocyte expression system. In addition, we investigated


4

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


whether other prototypical substrates/inhibitors of other MCT isoforms have any significant interactions with MCT6.


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 28

Materials and Methods: Materials. Bumetanide, and all of the other compounds (with the exception of AR-C155858) were purchased from Sigma-Aldrich (St. Louis, MO). AR-C155858 was purchased from Tocris Bioscience (Bristol, UK). [3H]Bumetanide (15-30 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). GIBCO Leibovitz’s L-15 medium with glutamine (Cat. No. 41300-039) and all Western Blotting materials were supplied by Thermo Fisher Scientific (Rockford, IL). The pCR™2.1-TOPO®, TOPO® TA cloning kit, TRIzol® Reagent, and mMESSAGE mMACHINE T7 transcription kit were purchased from Thermo Fisher Scientific (Rockford, IL). pGH19 vector was kindly provided by Dr. Walter F. Boron (Case Western Reserve University, Department of Physiology and Biophysics, Cleveland, Ohio)

24

.

The FlashGel™ System was purchased from Lonza (Portsmouth, NH). All enzymes were purchased from New England Biotechnology (Ipswich, MA). The Gel Extraction and PCR Purification kits were purchased from Qiagen (Valencia, CA). DNA purity and concentration was verified using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Rockford, IL). A ZOE™ Fluorescent Cell Imager made by Bio-Rad (Hercules, CA) was used for fluorescent microscopy. The mouse anti-Egfp antibody (JL-8, Cat. # 632381) was purchased from Clontech (Mountain View, CA).

Generation of the pGH19-hMCT6 vector. For these studies, we generated injectable cRNA encoding for the human MCT6 protein from the in vitro transcription of a MCT6 genetic construct. Briefly, total RNA derived from human kidney cortical tissue (obtained from Ohio State University Tissue Procurement Services, National Cancer Institute Collaborative Human Tissue Network (CHTN), Columbus, OH) was isolated using TRIzol® Reagent according to the


6

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


manufacturer’s instructions. After checking the concentration, purity, and stability using the FlashGel™ System; cDNA was generated by performing RT-PCR using the BioRad CFX Connect™ RT System. Using this cDNA library, sequence specific primers were designed to amplify the specific cDNA fragment encoding for the MCT6 gene (SLC16A5) (Forward Primer: 5‘-AAGAAAAGAGCACTTCCCTGC-3‘;

Reverse

Primer:

5‘-

ACAGGGCACTCAGGTAGGGC-3). Following the successful subcloning of the MCT6 cDNA into an intermediate expression vector (pCR2.1-TOPO TA), the plasmid was linearized with BamHI, and PCR was performed using nested primers (FP: 5‘-AGCGT CCCGGG GCCACC ATGCCCCAGGCCCTGGAG-3‘; RP: 5‘-ATTA TCTAGA GGCACTCAGGTAGGGCTA-3‘). Two restriction sites (XmaI and XbaI) were incorporated into the primers which are underlined, and a Kozak consensus sequence (in bold) was incorporated into the forward primer in order to enhance translational efficiency. The pGH19 plasmid was used as the oocyte expression vector due to its translation-enhancing features (5‘/3‘ untranslated regions of the X. laevis β-globin, polyadenosine tract, etc.) 24. To insert the MCT6 cDNA into the construct, empty pGH19 vector and MCT6 PCR product were double-digested using XmaI and XbaI. The 5‘ and 3‘ phosphates of the digested vector were then removed using Antarctic Phosphatase. The digested PCR product was purified and a ligation reaction was performed using a T4 DNA ligase reaction mixture. The construct was transformed into chemically competent TOP10 E. Coli cells and the ligated product was isolated, purified, and confirmed via sequencing.

Preparation of cRNA and verification of protein expression in X. laevis oocytes. The cRNA encoding for MCT6 was in vitro transcribed from NotI-linearized pGH19-hMCT6 vector using


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 28

the mMESSAGE mMACHINE T7 transcription kit. The oocytes were isolated from resected and collagenase-treated X. laevis ovaries. The oocytes that were injected ranged from Dumont stages IV-VI and approximately 13.8 nL of cRNA or water was injected into each oocyte (~20 ng). The oocytes were then incubated in OR3 medium (which was prepared as described previously 24) at 18°C for 3 to 4 days. All cRNA was checked for purity, concentration, stability, and correct size utilizing a NanoDrop 1000 and FlashGel™ System. Due to the inability of a variety of antibodies against human MCT6 to verify positive protein expression 3 – 4 days post-injection in our oocytes, our lab developed an MCT6 Egfp-tagged construct using similar methods as described previously in order to verify the protein expression and localization to the membrane using a fluorescent imager and an anti-Egfp antibody. Briefly, 3 days post-injection, oocytes were washed with 200 mOsm Tris-buffered saline (TBS) and visualized using a fluorescence microscope. In addition, Western Blotting was performed from water-injected, MCT6 cRNAinjected, Egfp cRNA-injected, and MCT6-Egfp cRNA-injected oocytes from day 1 to day 4 post-injection. Briefly, 5 oocytes from each group each day were washed three times in 200 mOsm TBS and homogenized using in 500 µL lysis buffer (200 mOsm TBS, 1% Triton X-100, with protease inhibitor). The insoluble fraction was removed via centrifugation at 4ᵒC at 3,000 g for 10 min. The soluble fraction was then spun in a Spin-X® centrifuge tube filter (Corning® Costar®, 0.45 µm) at 4ᵒC at 16,000 g for 10 min. For each soluble fraction, 25 µL of lysate was added to 12.5 µL of LDS, 10 µL of lysis buffer, 2.5 µL of 1 M DTT, and heated at 37ᵒC for 10 min. An aliquot of each of these samples (20 µL, equivalent to 0.25 oocytes) was loaded in a NuPAGE 4-12% Bis-Tris protein gel. The gel ran for 1.5 hours at 100 V using 1X MOPS as running buffer. The gel was then transferred to a PVDF membrane at 100 V for 2.5 hours. After washing the membrane three times in 1X TBST, the membrane was blocked for in 5% non-fat


8

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


milk overnight. The membrane was then washed three times in 1 X TBST and incubated in primary antibody (1:200, anti-Egfp, 5% non-fat milk) on a rocker overnight at 4ᵒC. After washing the membrane for an additional three times in 1X TBST, the membrane was incubated in secondary antibody (1:10,000 anti-mouse, HRP-linked, 5% non-fat milk) for 1 hour at room temperature. After a final three washes in 1X TBST, the membrane was developed using ECL substrate (Bio-Rad) and visualized on a ChemiDoc™ XRS+ System (Bio-Rad).

Uptake studies. The uptake studies were performed similarly to the methods published by Murakami et al. 3. Briefly, the uptake studies were performed using groups of 4 to 10 oocytes that were transferred into 24-well multi-dishes and preincubated in uptake buffer for 30 min (15 mM HEPES, 82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, and 1 mM MgCl2, adjusted to pH 7.4 with Tris). The oocytes were then transferred to 400 µL of uptake buffer containing 0.1 µM radiolabeled bumetanide ([3H]bumetanide), and the oocytes were incubated at pH 7.4 at room temperature (~20–23°C). The uptake was stopped by the addition of ice-cold uptake buffer, and the oocytes were washed three times. Individual oocytes were placed in separate scintillation vials and dissolved in 250 µl of 10% sodium lauryl sulfate by slowly shaking for 1.5 h. Radioactivity was determined by liquid scintillation counting following the addition of scintillation cocktail. For these studies, a range of typical MCT substrates/inhibitors were investigated for the inhibitory effects on MCT6-mediated bumetanide uptake, as well as a variety of different flavonoids that we had previously investigated in our laboratory

13, 16, 22, 23

. Lastly, the

mechanism of inhibition was investigated for a potent flavonoid inhibitor of MCT6, phloretin.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

Data analysis. The uptake by oocytes (µl/oocyte) was calculated as the ratio of radioactivity in each sample (dpm/oocyte) to the initial concentration in the uptake buffer (dpm/µl). MCT6mediated uptake was calculated as the difference between the MCT6 cRNA-injected oocytes and the water-injected oocytes. Statistical analysis was performed using the one-way unpaired analysis of variance (ANOVA) followed by Dunnett’s test to test for multiple comparisons. Differences were considered statistically significant when p < 0.05. Data analysis was performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego CA). The MCT6-specific uptake rates of bumetanide were obtained from the uptake value at 30 min which was previously shown to be in the linear range 3. The inhibition of bumetanide uptake by flavonoids was calculated by fitting with the equation below (Eq. 1) using weighted nonlinear regression analysis (ADAPT 5; Biomedical Simulations Research, University of South California, Los Angeles, CA). ∙஼ ം

ூ

‫ = ܨ‬100 ∙ ൬1 − ூ஼೘ೌೣ ൰ ം ା஼ ം

(1)

ఱబ

Where ‫ ܨ‬is the percentage of uptake rate of bumetanide in the presence of flavonoids compared with the control, C is the concentration of flavonoids, Imax is the maximal inhibition, and IC50 is the inhibitor concentration at 50% inhibition. After comparisons of competitive, noncompetitive, and uncompetitive inhibition models, the mechanism of inhibition was best fitted to the competitive inhibition equation displayed below (Eq. 2): ‫=ܬ‬

௃೘ೌೣ ∙஼

௄೟ ∙൬ଵା

(2)

ሾ಺ሿ ൰ା஼ ಼೔

Where J is the MCT6-mediated bumetanide uptake rate (pmol/oocyte/30 min), C is the concentration of bumetanide (µM), Jmax is the maximum uptake rate (pmol/oocyte/30min), Kt is the substrate concentration at the half-maximal uptake rate (µM), and Ki is the inhibition


10

Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


constant (µM). The goodness of fit for all model-fitting was determined by the sum of the squared derivatives, the residual plots, and the AIC values.


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 28

Results: Verification of MCT6 localization and protein expression in X. laevis oocytes. In order to verify the positive protein expression of MCT6-Egfp in oocytes, a protein kinetics study was performed to evaluate the protein expression over 4 days post-injection in the oocytes. As shown in Figure 1, MCT6-Egfp protein expression was apparent in the membrane of the oocytes, demonstrating successful protein translation and trafficking. MCT6 cRNA- and water-injected oocytes demonstrated minimal background fluorescence in the oocytes. Egfp cRNA-injected oocytes demonstrated positive protein expression in the oocytes, however, this was mostly restricted to the cytosol of the cells. The western blot results (Figure 1E) using an anti-Egfp antibody demonstrated apparent positive bands at the molecular weight corresponding to MCT6Egfp protein (~85 KDa) and Egfp alone (~30 KDa). The MCT6-Egfp band was stable and did not appear to lose intensity for up to 4 days post-injection, in contrast to the Egfp cRNA-injected oocytes which appeared to be largely expressed at day 1 post-injection in comparison to day 4 post-injection.

Effects of MCT Substrates and Inhibitors on MCT6. Previously, it has been demonstrated that bumetanide is a suitable substrate for measuring MCT6-mediated uptake 3. It was demonstrated that the uptake of bumetanide was shown to be in the linear range for 30 min, at a concentration of 0.1 µM; therefore, for the purpose of our studies we used the same concentration and uptake time. To investigate the effects of other MCT substrates and inhibitors on the activity of MCT6, cis-inhibition assays were performed as done previously using bumetanide as the probe substrate. The uptake of bumetanide in the absence of any other compounds was significantly higher in the MCT6 cRNA-injected oocytes in comparison to the


12

Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


water-injected control oocytes (Figure 2A), demonstrating that human MCT6 was actively transporting bumetanide. As shown in Figure 2B, probenecid inhibited MCT6-mediated bumetanide uptake (~75%), at the concentration previously used by Murakami et al. which was consistent with probenecid being a MCT6 substrate and inhibitor 3. Most inhibitors were evaluated at concentrations that were >10-fold higher than their reported plasma concentrations. Interestingly, taurocholate, an OAT/OATP substrate, also significantly inhibited bumetanide uptake (~50%). Substrates/inhibitors for other MCTs, namely pyruvate (MCT1-4 substrate), Ltyrosine (MCT10 substrate), L-thyroxine (MCT8 substrate), and AR-C155858 (MCT1/2 inhibitor), did not significantly affect MCT6-mediated bumetanide uptake.

Effects of Flavonoids on Bumetanide Uptake via MCT6. The effects of flavonoids (50 µM) on MCT6-mediated bumetanide uptake were investigated in this study. [3H]Bumetanide (0.1 µM) uptake was measured for 30 min at pH 7.4 at room temperature in the presence and absence of a variety of flavonoids (Figure 3). Bumetanide uptake was significantly inhibited by the aglycone flavonoids: quercetin, morin, luteolin, and phloretin, but not naringenin, genistein, and biochanin a. The flavonoid glycosides tested did not have any effect on bumetanide uptake (i.e. naringin, rutin, and phlorizin).

Concentration-Dependent Inhibition of Bumetanide Uptake by Flavonoids. Concentrationdependent inhibition of bumetanide (0.1 µM) uptake by three aglycone flavonoids (i.e. quercetin, morin, phloretin) is depicted in Figure 4. Luteolin was initially investigated, however its limited solubility in aqueous uptake buffer restricted our analysis at higher concentrations. Using Eq. 1,


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 28

the IC50 values were calculated for quercetin, morin, and phloretin (25.3, 33.1, and 17.3 µM, respectively; shown in Figure 4).

Mechanism of Inhibition of Phloretin on MCT6-mediated Bumetanide Uptake. The mechanism of phloretin inhibition on MCT6-mediated bumetanide uptake was investigated in this study. Concentration-dependent inhibition was examined in the absence and presence of phloretin (5 µM and 25µM) using a range of bumetanide concentrations (0.3 – 100 µM) at pH 7.4, room temperature, for 30 min. Goodness of fitting parameters including the sum of the squared derivatives, the residual plots, and the AIC values were used to determine which inhibition mechanism model was most appropriate. The data best fit a model of competitive inhibition (equation displayed in Eq. 2). The Lineweaver-Burk plot is shown in Figure 5 to illustrate the inhibition mechanism, In addition, preincubation of MCT6-transfected oocytes with inhibitor and subsequent washing did not result in inhibition of bumetanide uptake; this inhibition was therefore deemed reversible (data not shown). Using the Michaelis-Menten equation in Eq. 2 for competitive inhibition yielded a Kt value of 60.5 ± 14.1 µM (estimate ± S.D.) for bumetanide, a Ki value of 22.8 ± 6.95 µM, and a Jmax value of 36.1 ± 6.82 pmol/oocyte/30 min.


14

Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Discussion: In this study, we investigated the effects of a variety of flavonoids on the transport of bumetanide in human MCT6-transfected X. laevis oocytes. Flavonoids from five structurally distinct classes were investigated in this study, which included: flavonones, flavonols, a flavone, isoflavones, and dihydrochalcones. There are few compounds confirmed as MCT6 substrates or inhibitors, and there is little information regarding the structural characteristics and physiochemical motifs which govern their degree of interaction. As discussed previously, only two publications are available to date for the characterization of MCT6 function, and inhibitor/substrate identification 3, 4. Interestingly in these studies, MCT6-mediated bumetanide uptake was not inhibited by prototypical MCT 1-4 ligands, such as the short-chain monocarboxylic acids L-lactic acid and pyruvic acid, which agrees with our experimental data. And unsurprisingly, various sulfur-containing loop diuretics that are structural analogs of bumetanide (i.e. furosemide, piretanide, torasemide, and azosemide) have also been reported to have significant interactions with MCT6-mediated bumetanide uptake, with IC50 values ranging from 13 – 163 µM 3. Thiazide diuretics and members of the meglitinide class of hypoglycemic agents have demonstrated MCT6 inhibition and affinity. Interestingly, prostaglandin F2α demonstrated significant MCT6-mediated uptake, however, further kinetic studies are necessary in order to characterize eicosanoids as physiological substrates for MCT6. The degree of interaction of flavonoids with MCT6-mediated bumetanide uptake appears to be highly structure-dependent. While the majority of aglycone flavonoids appear to show inhibition of MCT6, specific aglycones of the isoflavone family (i.e. genistein and biochanin A), as well as naringenin of the flavonone family did not significantly inhibit MCT6, at the high concentration of 50 µM. This may potentially be due to the steric hindrance caused by the phenyl


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 28

group at the 3-position, resulting in the inability of this class of molecules to interact with the binding site. Also, the absence of any inhibition caused by glycoside moieties appears to be due to the same cause. However, considering there is no information known regarding the binding pocket of MCT6, further studies are required to validate this theory. The aglycone flavonoids quercetin, morin, phloretin, and luteolin appeared to have the greatest inhibitory potential of MCT6, suggesting that these compounds may cause significant drug-flavonoid interactions. Physiological systemic concentrations of flavonoids from a typical U.S. diet of ≤ 1 g total flavonoids per day

25

range from 0.1 – 10 µM; however these estimates are highly variable

26

.

Intestinal concentrations of these flavonoids are expected to be much higher, especially if flavonoids are ingested as a dietary supplement, suggesting that these compounds could play a major role in governing MCT6-substrate interactions. Previous studies performed in our lab investigating the interactions of flavonoids on MCT1-mediated γ-hydroxybutyric acid (GHB) uptake in rat MCT1-transfected MDA-MB231 cells also demonstrated significant inhibition of MCT1 activity 16. However, in comparing inhibition of MCT1 and MCT6, aglycone isoflavones and flavonones were able to inhibit MCT1, but not MCT6. In another study in Caco-2 cells, Shin et al. demonstrated that aglycone flavonoids naringenin and silybin had a competitive inhibitory interaction on benzoic acid uptake, with Ki values ranging from 15-20 µM 17, similar to that seen for phloretin in this study where phloretin has been characterized to be a competitive inhibitor, with a Ki value of ~23 µM. Phloretin is a dihydrochalcone, commonly found in apples, that has been investigated for its antioxidant effects and inhibition of lipid peroxidation 27, 28. It is unknown, however, whether MCT6 plays a role in the transport of these compounds, and more detailed quantitative data is required in order to determine the relevance of MCT6 for the membrane transport of flavonoids.


16

Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In summary, the results of this study provide further evidence that MCT6 is distinct from other MCT isoforms in that it does not seem to share overlapping ligand specificity. In contrast, our findings suggest that MCT6 could share more similarities to the OAT (SLC22A) family of transporters, in contrast to its MCT (SLC16A) family. Structurally-specific classes of aglycone flavonoids (flavonols, flavones, dihydrochalcones) were shown to potently inhibit MCT6mediated transport in contrast to flavonoid glycosides. IC50 values were obtained and the mechanism of inhibition of a potent dihydrochalcone inhibitor (phloretin) was shown to be competitive, and reversible in nature. This study was the first to investigate flavonoids and their inhibition potential on MCT6, and the first to report that taurocholate is a MCT6 inhibitor. To date, it is unclear what effect flavonoids have on MCT6-mediated transport in vivo and potential diet-drug interactions. With further characterization of MCT6 in future studies, we may be able to elucidate the contribution of flavonoids on overall drug exposure and tissue distribution.


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 28

Acknowledgments. The technical assistance from Evan Myers, and Aniko Marshall in the training and assistance in preparation of the oocytes is acknowledged. Funding support was from the National Institutes of Health National Institute on Drug Abuse [Grant R01DA023223]. Robert S. Jones was supported in part by a PhRMA Pre-Doctoral Graduate Fellowship. Dr. Mark D. Parker was supported by start-up funding from the Dean of the School of Medicine and Biomedical Sciences and the Department of Physiology and Biophysics as well as by a Carl W. Gottschalk Research Scholar Grant from the American Society of Nephrology Foundation for Kidney Research.


18

Page 19 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References: 1. Halestrap, A. P.; Wilson, M. C. The monocarboxylate transporter family--role and regulation. IUBMB Life 2012, 64, (2), 109-19. 2. Jones, R. S.; Morris, M. E. Monocarboxylate Transporters: Therapeutic Targets and Prognostic Factors in Disease. Clinical pharmacology and therapeutics 2016, 100, (5), 454-463. 3. Murakami, Y.; Kohyama, N.; Kobayashi, Y.; Ohbayashi, M.; Ohtani, H.; Sawada, Y.; Yamamoto, T. Functional characterization of human monocarboxylate transporter 6 (SLC16A5). Drug Metab Dispos. 2005, 33, (12), 1845-51. 4. Kohyama, N.; Shiokawa, H.; Ohbayashi, M.; Kobayashi, Y.; Yamamoto, T. Characterization of Monocarboxylate Transporter 6: Expression in Human Intestine and Transport of the Antidiabetic Drug Nateglinide. Drug Metab Dispos. 2013, 41, (11), 1883-1887. 5. Gill, R. K.; Saksena, S.; Alrefai, W. A.; Sarwar, Z.; Goldstein, J. L.; Carroll, R. E.; Ramaswamy, K.; Dudeja, P. K. Expression and membrane localization of MCT isoforms along the length of the human intestine. American journal of physiology. Cell physiology 2005, 289, (4), C846-52. 6. Kuhnau, J. The flavonoids. A class of semi-essential food components: their role in human nutrition. World review of nutrition and dietetics 1976, 24, 117-91. 7. Scalbert, A.; Williamson, G. Dietary intake and bioavailability of polyphenols. The Journal of nutrition 2000, 130, (8S Suppl), 2073S-85S. 8. Hossain, M. K.; Dayem, A. A.; Han, J.; Yin, Y.; Kim, K.; Saha, S. K.; Yang, G. M.; Choi, H. Y.; Cho, S. G. Molecular Mechanisms of the Anti-Obesity and Anti-Diabetic Properties of Flavonoids. International journal of molecular sciences 2016, 17, (4). 9. Moon, Y. J.; Wang, X.; Morris, M. E. Dietary flavonoids: effects on xenobiotic and carcinogen metabolism. Toxicology in vitro : an international journal published in association with BIBRA 2006, 20, (2), 187-210. 10. Walgren, R. A.; Lin, J. T.; Kinne, R. K.; Walle, T. Cellular uptake of dietary flavonoid quercetin 4'-beta-glucoside by sodium-dependent glucose transporter SGLT1. The Journal of pharmacology and experimental therapeutics 2000, 294, (3), 837-43. 11. Kwon, O.; Eck, P.; Chen, S.; Corpe, C. P.; Lee, J. H.; Kruhlak, M.; Levine, M. Inhibition of the intestinal glucose transporter GLUT2 by flavonoids. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007, 21, (2), 366-77. 12. Bansal, T.; Jaggi, M.; Khar, R. K.; Talegaonkar, S. Emerging Significance of Flavonoids as P-Glycoprotein Inhibitors in Cancer Chemotherapy. J Pharm Pharm Sci 2009, 12, (1), 46-78. 13. Zhang, S. Z.; Yang, X. N.; Morris, M. E. Flavonoids are inhibitors of breast cancer resistance protein (ABCG2)-mediated transport. Molecular pharmacology 2004, 65, (5), 12081216. 14. Jiang, W.; Hu, M. Mutual interactions between flavonoids and enzymatic and transporter elements responsible for flavonoid disposition via phase II metabolic pathways. Rsc Adv 2012, 2, (21), 7948-7963. 15. Zheng, J.; Zhou, H. H. [Effects of the flavonoids on cytochrome P-450 CYP1, 2E1, 3A4 and 19]. Yao xue xue bao = Acta pharmaceutica Sinica 2007, 42, (1), 8-12. 16. Wang, Q.; Morris, M. E. Flavonoids modulate monocarboxylate transporter-1-mediated transport of gamma-hydroxybutyrate in vitro and in vivo. Drug metabolism and disposition: the biological fate of chemicals 2007, 35, (2), 201-8.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 28

17. Shim, C. K.; Cheon, E. P.; Kang, K. W.; Seo, K. S.; Han, H. K. Inhibition effect of flavonoids on monocarboxylate transporter 1 (MCT1) in Caco-2 cells. The Journal of pharmacy and pharmacology 2007, 59, (11), 1515-9. 18. Wong, C. C.; Botting, N. P.; Orfila, C.; Al-Maharik, N.; Williamson, G. Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6). Biochemical pharmacology 2011, 81, (7), 942-9. 19. Morris, M. E.; Zhang, S. Flavonoid-drug interactions: effects of flavonoids on ABC transporters. Life sciences 2006, 78, (18), 2116-30. 20. Nigam, S. K.; Bush, K. T.; Martovetsky, G.; Ahn, S. Y.; Liu, H. C.; Richard, E.; Bhatnagar, V.; Wu, W. The organic anion transporter (OAT) family: a systems biology perspective. Physiological reviews 2015, 95, (1), 83-123. 21. Wu, W.; Jamshidi, N.; Eraly, S. A.; Liu, H. C.; Bush, K. T.; Palsson, B. O.; Nigam, S. K. Multispecific drug transporter Slc22a8 (Oat3) regulates multiple metabolic and signaling pathways. Drug metabolism and disposition: the biological fate of chemicals 2013, 41, (10), 1825-34. 22. An, G.; Wang, X.; Morris, M. E. Flavonoids are inhibitors of human organic anion transporter 1 (OAT1)-mediated transport. Drug metabolism and disposition: the biological fate of chemicals 2014, 42, (9), 1357-66. 23. Wang, X. D.; Wolkoff, A. W.; Morris, M. E. Flavonoids as a novel class of human organic anion-transporting polypeptide OATP1B1 (OATP-C) modulators. Drug Metabolism and Disposition 2005, 33, (11), 1666-1672. 24. Musa-Aziz, R.; Boron, W. F.; Parker, M. D. Using fluorometry and ion-sensitive microelectrodes to study the functional expression of heterologously-expressed ion channels and transporters in Xenopus oocytes. Methods 2010, 51, (1), 134-45. 25. Chun, O. K.; Chung, S. J.; Song, W. O. Estimated dietary flavonoid intake and major food sources of U.S. adults. The Journal of nutrition 2007, 137, (5), 1244-52. 26. Peng, I. W.; Kuo, S. M. Flavonoid structure affects the inhibition of lipid peroxidation in Caco-2 intestinal cells at physiological concentrations. Journal of Nutrition 2003, 133, (7), 21842187. 27. Rezk, B. M.; Haenen, G. R. M. M.; van der Vijgh, W. J. F.; Bast, A. The antioxidant activity of phloretin: the disclosure of a new antioxidant pharmacophore in flavonoids. Biochemical and biophysical research communications 2002, 295, (1), 9-13. 28. Nakamura, Y.; Watanabe, S.; Miyake, N.; Kohno, H.; Osawa, T. Dihydrochalcones: Evaluation as novel radical scavenging antioxidants. Journal of agricultural and food chemistry 2003, 51, (11), 3309-3312.


20

Page 21 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure captions: Fig. 1. Immunofluorescence analysis of water-injected (A), MCT6 cRNA-injected (B), MCT6Egfp cRNA-injected (C), and Egfp cRNA-injected (D) (three days post-injected, 13.8 nL of respective cRNA or water injected per oocyte). A white arrow in panel C depicts the apparent positive MCT6-Egfp protein expression at the oocyte membrane. A western blot using an antiEgfp mouse antibody is shown in panel E. No bands were visualized for water and MCT6 cRNA-injected oocytes. Fig. 2. Uptake of [3H]bumetanide by MCT6 in the absence (A) and presence of MCT substrates/inhibitors (B). Uptake of 0.1 µM [3H]bumetanide into MCT6-cRNA injected (red) or water-injected control oocytes (blue) was measured at room temperature and pH 7.4 (A and B) for 30 min. MCT6-mediated uptake (black) was calculated by subtracting the uptake amount of water-injected control oocytes from that of MCT6-expressing oocytes. Each bar represents the mean ± S.E.M. with three replicates (n = 7–10 oocytes/replicate) and 2–3 separate ovaries from separate frogs. One-way ANOVA followed by Dunnett’s test was used for statistical analysis. (A) ****p < 0.0001 versus water-injected oocytes; (B) *p < 0.05, ***p < 0.001 versus control. Fig. 3. Uptake of [3H]bumetanide by MCT6 in the absence and presence of flavonoids (50 µM). Uptake of 0.1 µM [3H]bumetanide into MCT6-cRNA injected or water-injected control oocytes was measured at room temperature and pH 7.4 for 30 min. MCT6-mediated uptake was calculated by subtracting the uptake amount of water-injected control oocytes from that of MCT6-expressing oocytes. Flavonones (red), flavonols (green), flavone (brown), isoflavones (blue), and dihydrochalcones (purple) are plotted as mean ± S.E.M. with three replicates (n = 8– 10 oocytes/replicate) and 2–3 separate ovaries from separate frogs. One-way ANOVA followed


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

by Dunnett’s test was used for statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Fig. 4. Concentration-dependent inhibition of MCT6-mediated bumetanide transport by (A) quercetin, (B) morin, and (C) phloretin. The lines represent the fitted IC50 curves using Eq. 1. The results were normalized by the control uptake (absence of flavonoids). Uptake by waterinjected oocytes has been subtracted from the uptake of MCT6-transfected oocytes. The studies were performed at pH 7.4, room temperature, and for 30 min. The concentration of bumetanide used was 0.1 µM. Data is plotted as mean ± S.E.M. with three replicates (n = 4–10 oocytes/replicate) and 2–3 separate ovaries from separate frogs. Fig. 5. Lineweaver-Burk plot of the uptake of bumetanide in the absence and presence of phloretin. The concentration of substrate (S, bumetanide) was 0.3, 1.0, and 100 µM. Uptake by water-injected oocytes cells has been subtracted from the uptake of MCT6-transfected oocytes. The studies were performed at pH 7.4, room temperature, and for 30 min. Data is plotted as mean ± S.E.M. with three replicates (n = 4–10 oocytes/replicate) and 2–3 separate ovaries from separate frogs.


22

Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

Figure 2.


24

Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

Figure 4.

Figure 5.


26

Page 27 of 28


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Flavonoids inhibit MCT6-mediated transport 255x191mm (96 x 96 DPI)


Page 28 of 28

Quercetin, Morin, Luteolin, and Phloretin Are Dietary Flavonoid

Recommend Documents