Inhibition of P-Glycoprotein Mediated Efflux in Caco-2 Cells by Phytic

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Inhibition of P-glycoprotein mediated efflux in Caco-2 cells by phytic acid Lujia Li, Qingxue Fu, Mengxin Xia, Lei Xin, Hongyi Shen, Guowen Li, Guang Ji, Qianchao Meng, and Yan Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04307 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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Journal of Agricultural and Food Chemistry

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Inhibition of P-glycoprotein mediated efflux in Caco-2 cells by phytic acid

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Lujia Lia,c,1, Qingxue Fua,b,1, Mengxin Xiaa, Lei Xina, Hongyi Shena, Guowen Lic, Guang Jid,

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Qianchao Menge, Yan Xiea,d,*

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a

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Medicine, Shanghai 201203, China

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b

7

Shanghai 201203, China

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c

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Traditional Chinese Medicine, Shanghai 200082, China

Research Center for Health and Nutrition, Shanghai University of Traditional Chinese

Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine,

Pharmacy Department, Shanghai TCM-integrated Hospital, Shanghai University of

10

d

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Chinese Medicine, Shanghai 200032, China

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e

13

Shanghai 201203, China

Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional

Center for Drug Safety Evaluation, Shanghai University of Traditional Chinese Medicine,

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These authors contributed equally to this work.

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*

Corresponding author:

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Yan Xie, Ph.D., Professor

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Research Center for Health and Nutrition, Shanghai University of Traditional Chinese

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Medicine

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1200 Cailun Road, Shanghai, China 201203

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E-mail: [email protected]; [email protected]

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1

ACS Paragon Plus Environment


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Phone: +86(21)51322440, Fax: +86(21)51322407

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2


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Abstract

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Phytic acid (IP6) is a natural phosphorylated inositol which abundantly present in most cereal

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grains and seeds. This study investigated the effects of IP6 regulation on P-gp and its potential

28

mechanisms using in situ and in vitro models. The effective permeability of the typical P-gp

29

substrate rhodamine 123 (R123) in colon was significantly increased from (1.69 ± 0.22) × 10-5

30

cm/s in the control group to (3.39 ± 0.417) ×10-5 cm/s (p < 0.01) in the 3.5 mM IP6 group.

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Additionally, IP6 can concentration-dependently decrease the R123 efflux ratio in both

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Caco-2 and MDCK II-MDR1 cell monolayers and increase intracellular R123 accumulation

33

in Caco-2 cells. Furthermore, IP6 noncompetitively inhibited P-gp by impacting R123 efflux

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kinetics. The noncompetitive inhibition of P-gp by IP6 was likely due to decreases in P-gp

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ATPase activity and P-gp molecular conformational changes induced by IP6. In summary, IP6

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is a promising P-gp inhibitor candidate.

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Keywords: Allosteric modulation, Efflux, P-glycoprotein inhibitor, Phytic acid, Rhodamine

38

123

39 40

Abbreviations:

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ABC, ATP-binding cassette; AP, apical; BCA, bicinchoninic acid; BL, basolateral; Caco-2,

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colon

43

6-diamidino-2-phenylindole, dihydrochloride; DMEM, dulbecco's modified eagle's medium;

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ER, efflux ratio; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate

45

dehydrogenase; HBSS, hanks’ balanced salt solution; HRP, horseradish peroxidase; IP6,

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phytic acid; MDCK II, madin-darby canine kidney II; MTS, 3-(4,5-dimethylthiazol-2-yl)-

adenocarcinoma;

CLSM,

confocal

laser

scanning

3


microscopy;

DAPI,

4’,


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5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium;

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orthovanadate; Papp, apparent permeability coefficient; PBS, phosphate-buffered saline; Peff,

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effective permeability; P-gp, P-glycoprotein; PVDF, polyvinylidene difluoride; R123,

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rhodamine 123; RNA, ribonucleic acid; RT-PCR, reverse transcriptase-PCR; SD,

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Sprague-Dawley; SPIP, single-pass intestinal perfusion; SPSS, sigmastat statistical software;

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Ver, verapamil.

53 54

4


Na3VO4,

sodium

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Introduction

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Phytic acid (IP6), is a major form of polyphosphorylated carbohydrate present in many

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cereals, legumes, grains, nuts and oil seeds.1 This compound exhibits a wide range of health

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benefits including nutritional effects,2 antioxidant effects,3 chelating functions,4 etc. IP6 is not

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only present in many foods but also used as an additive in some foods, i.e., apple juice

60

products.5 In recent, some clinical studies demonstrated that IP6 strengthened the action of

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chemotherapy drugs on breast and colon cancer and diminished their side effects.6

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P-glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) transporter

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superfamily.7 It is ubiquitously distributed among various human tissues, e.g. brain, kidneys,

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liver, and enterocytes.8 P-gp is mainly located in the apical membranes of epithelial cells,

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where it mediates the efflux of fargoing substrates,9 protects organs from the toxic effects of

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xenobiotics and regulates drug absorption and disposition.10 P-gp substrates can specifically

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bind with P-gp and be transported out of the cell by P-gp,11 whilst this efflux can be inhibited

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by P-gp inhibitors.12 Many therapeutic drugs, such as vincristine (anticancer), HIV protease

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inhibitors (antiviral), perospirone (antipsychotic) and digoxin (antiarrhythmic) are substrates

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of P-gp. They pose a significant pharmaceutical challenge in clinical drug applications

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because they are pumped out of the cell, which caused the insufficient intracellular drug

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concentrations and, consequently, decreased therapeutic efficacy.10,13 To solve this problem, it

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is necessary to identify a P-gp inhibitor that can be co-administered with P-gp substrate drugs

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to enhance their therapeutic effects.

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Over the years, numerous P-gp inhibitors have witnessed rapid progress. At present, the

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P-gp inhibitors were roughly divided into three generations: verapamil (Ver) and felodipine 5



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are examples of the first generation, dexverapamil and PSC 833 are examples of the second

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generation inhibitors, which have more effective targeting and fewer adverse effects, and the

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third generation, which includes tariquidar and laniquidar, exhibits the highest affinity for

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P-gp.14 Even so, still lack of P-gp inhibitors can be used in clinical practice because the

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existing inhibitors altered the absorption characteristics of P-gp substrate drugs and increased

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their side effects.15 For example, the toxicity of Ver has been observed in clinical

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manifestations with arterio-ventricular block and hypotension;13 PSC 833 co-administered

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with P-gp substrate drugs vincristine and doxorubicin16 or daunorubicin and etoposide17 has

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been shown to cause acute myeloid leukemia. In recent years, some excipients, such as

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poloxamer 235 and cremophor EL, were found to enhance the bioavailability of docetaxel and

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fexofenadine (P-gp substrate drugs) through their interactions with P-gp,18,19 suggesting a new

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direction for developing novel non-toxic P-gp inhibitors. Interestingly, IP6 enhanced the oral

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absorption of isorhamnetin, quercetin, and kaempferol in our previous study,20 while these

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compounds were substrates of P-gp confirmed by using original and transfected

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Bcap37/MDR1 cell models.21 Presumably, IP6 regulates the absorption of these drugs via a

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P-gp mediated mechanism. So, the present study was designed to clarify the mechanisms of

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P-gp regulation by IP6 to determine whether IP6 can serve as a potential P-gp inhibitor.

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To date, various methods including intestinal perfusion, everted gut sac, cell models,

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Ussing chamber and intestinal loops, have been used to investigate the mechanisms of

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potential P-gp inhibitors. Among these methods, single-pass intestinal perfusion (SPIP) is a

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well-established technique for determining the permeability characteristics of drugs in a

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manner that mimics physiological conditions.22 The human colon adenocarcinoma (Caco-2) 6


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cell line provides an in vitro cell culture model for simulating the absorption process in the

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small intestine and is extensively used to estimate the properties and potential mechanisms of

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drug transport.23 Madin-Darby canine kidney II (MDCK II) cells grow and differentiate

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quickly, thus greatly shorten the cycle of in vitro transport studies, while MDCK II-MDR1

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cells, which stably overexpress MDR1 gene, are utilized in directly probing P-gp-mediated

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efflux and its related mechanisms.24 Furthermore, there is a strong correlation between the in

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situ perfusion model and in vitro cell models (Caco-2/MDCK II-MDR1 cell lines).22 The three

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absorption models based on SPIP, Caco-2 and MDCK II-MDR1 cell lines with their

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respective characteristics are feasible and comprehensive methods for investigating the

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interaction between xenobiotics and translocators such as P-gp. In addition, rhodamine 123

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(R123), a representative P-gp substrate, was a sensitive dye for indicating the P-gp function,25

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and thus R123 was chosen as a marker for evaluating P-gp activity in the following

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experiments.

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In current study, the inhibitory effects of IP6 on P-gp and the related mechanisms were

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investigated using SPIP and monolayer cell models. Specifically, the effects of IP6 on P-gp

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efflux function, i.e., the absorption of P-gp substrate R123, were investigated using SPIP in

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situ and in vitro models, transport and uptake experiments, etc. Furthermore, the regulation of

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protein expression by IP6, efflux inhibition kinetics, ATPase activity and P-gp conformation

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were explored using Caco-2 and/or MDCK II-MDR1 cell lines to elucidate the possible

118

mechanisms. This study provides a theoretical support for the regulation of IP6 on P-gp and

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provides valuable information for the potential application of IP6 combination therapy with

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P-gp-mediated therapeutic drugs in the clinic. 7



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Materials and Methods

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Chemicals

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IP6 (90% purity, for molecular biology) and sodium orthovanadate (Na3VO4, 99.99% purity)

124

were purchased from Aladdin Industrial Corporation (Shanghai, China). R123 was obtained

125

from Sigma Aldrich Company (St. Louis, MO, USA). Ver (>99% purity) was provided by

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Dalian Meilun Biotech Co., Ltd (Dalian, China). All other reagents and solvents were of

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analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

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China).

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Materials

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MDCK II and MDCK II-MDR1 cells were kindly provided by the Netherlands Cancer

131

Institute (Amsterdam, Netherlands). Caco-2 cells were obtained from the American Type

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Culture Collection (ATCC, Manassas, VA, USA). Sprague-Dawley (SD) rats were supplied by

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the Shanghai Laboratory Animal Co., Ltd. (Certificate no.: SCXK; Shanghai, China;

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2013-0016). Dulbecco’s modified eagle’s medium (DMEM), SuperScript®II kit and

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SuperScript®III One-Step RT-PCR System were purchased from Thermo-Fisher

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Biochemical Product (Waltham, MA, USA). Fetal bovine serum (FBS) and Hanks’ balanced

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salts solution (HBSS) were purchased from Gibco Laboratory (Grand Island, NY, USA).

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RIPA lysis buffer and goat horseradish peroxidase (HRP)-conjugated anti-rabbit and

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anti-mouse IgG antibodies were purchased from Beyotime Institute of Biotechnology

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(Shanghai, China). Polyvinylidene difluoride (PVDF) membranes with a 0.4 µm mean pore

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size were purchased from the Millipore Company (Bedford, MA, Germany). Rabbit anti-P-gp

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antibody, P-gp monoclonal antibody (UIC2-PE) and mouse control IgG2a isotype were 8


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obtained from Abcam (Cambridge, UK). PCR primer was provided by Shanghai Generay

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Biotech Co., Ltd. (Shanghai, China). The CellTiter 96® AQueous One Solution Cell

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Proliferation Assay and P-gp-GloTM assay system with a P-gp membrane was purchased

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from the Promega Corporation (Madison, WI, USA). Mouse anti-β-actin antibody was

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provided by Abgent (San Diego, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase

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(GAPDH) was provided by Bioworld (St. Louis, MN, USA).

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Single-pass Intestinal Perfusion

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The single-pass intestinal perfusion model was performed as described in the Supporting

151

Information. For this experiment, a solution containing 10 µM R123 solution with or without

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IP6 (0.5, 2, 3.5 mM) or Ver (50 µM, positive control) was perfused through the intestinal

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segment with a constant flow rate of 0.2 mL/min. The samples were then periodically

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collected both from the inlet and the outlet at intervals of 20 min for 80 min to determine the

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concentration of R123 and subsequently calculate the effective permeability (Peff).

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Transport Studies

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The effects of IP6 on R123 transport across the Caco-2 cell monolayer and MDCK

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II-MDR1 cell monolayer were studied as described in the Supporting Information. For the

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bidirectional transport experiments, 10 µM R123 solution alone or supplemented with varying

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concentrations of IP6 (0.5, 2, 3.5 mM) or Ver (50 µM) was added to the donor compartments.

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The apparent permeability coefficient (Papp) and the efflux ratio (ER) of R123 were measured

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in each experimental group to investigate the effects of IP6 on P-gp efflux.

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Uptake Studies

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The Uptake of R123 in Caco-2 Cells. For the uptake assays, Caco-2 cells were seeded on 9



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6-well plates with the density of 2 × 105 cells /mL (1 mL/well) and cultured for 6-7 days. The

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cells were washed twice with warm HBSS and then pre-incubated with HBSS containing Ver

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or blank HBSS for 15 min at 37°C. Cells were treated with R123 (10 µM) and varying

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concentrations of IP6 (0 mM, 0.5 mM, 2 mM, and 3.5 mM) or Ver (50 µM) for 2 hours. After

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incubation, the cells sample were washed twice with cold PBS and scraped with 1 mL 1%

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Triton-X 100. The 100 µL of each sample was thoroughly mixed with 100 µL of acetonitrile

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and then centrifuged at 16,200 g for 10 min at 4°C for protein precipitation. Finally, cell

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supernatant containing R123 was analyzed using a Multiskan spectrum microplate reader

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(Thermo, Waltham, MA, USA) with λex = 485 nm and λem = 535 nm. Intracellular R123

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concentration was normalized with respect to the total protein content in each well measured

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using the bicinchoninic acid (BCA) assay.

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Flow Cytometry. The cell samples were prepared as described in “The Uptake of R123 in

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Caco-2 Cells”. After incubation, the cells were washed twice with cold PBS and collected

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with trypsin. Then, the cells were harvested using centrifugation (800 g, 3 min). Subsequently,

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the cells were resuspended and fixed with 4% paraformaldehyde for 10 min. Then, the cells

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were resuspended in 0.5 mL PBS. Finally, samples containing 20,000 cells each were

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analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San José, CA, USA), with

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λex = 485 nm and λem = 535 nm.

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Confocal Laser Scanning Microscopy (CLSM). Cells were grown on a Lab-Tek®

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chamber slide with 2 × 105 cells/well and cultured for 6 days. After pre-incubation, Caco-2

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cells were treated with R123 (10 µM) together with 0 mM, 0.5 mM, 2 mM, and 3.5 mM of

186

IP6 or Ver (50 µM) for 2 h at 37°C. Then, the Caco-2 cells were washed three times with PBS 10


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and subsequently fixed with 4% paraformaldehyde for 10 min. After staining with 200 µL of

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DAPI (4’, 6-diamidino-2-phenylindole) solution for 2-3 min, the cells were washed three

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times with PBS and then incubated with 0.5 mL of PBS to maintain the morphology of the

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cells. Finally, all images were taken at 63 × magnification in oil using CLSM (Leica, Wetzlar,

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HE, Germany). Intracellular R123 was determined from the fluorescence intensity with λex =

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485 nm and λem = 535 nm, respectively; DAPI was observed (λex/λem = 364/454 nm).

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Western blot Analysis

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Caco-2 cells were grown on a 6-well plate with 2 × 105 cells/well and cultivated for 6-7

195

days. MDCK II and MDCK II-MDR1 cells were grown on a 6-well plate with 2 × 105

196

cells/mL and cultivated for 3-4 days. After removal of the medium, the cells were treated with

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HBSS containing varying concentrations of IP6 (0, 0.5, 2, 3.5 mM) or Ver (50 µM) for 2 h at

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37°C. After trypsinization, the cells were harvested using centrifugation (800 g, 3 min).

199 200 201 202

Colon segments excised in the previous SPIP experiment were collected from SD rats and were stored at -80°C for later use. The western blot analysis methods are detailed in the Supporting Information. RT-PCR

203

Caco-2 cells were seeded onto a 6-well plate with 2 × 105 cells/well and cultivated for 6-7

204

days. After washed by PBS, the cells were treated with varying concentrations of IP6 (0, 0.5,

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2, 3.5 mM) or Ver (50 µM) for 2 h at 37°C. Total RNA was extracted from the Caco-2 cells

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using Trizol reagent. Isolated P-gp RNA was converted into cDNA using a SuperScript®II kit

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and thereafter subjected to PCR analysis using the SuperScript®III One-Step RT-PCR

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System. The reaction mix was prepared under the following conditions: denaturation at 37°C 11



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for 60 min, 85°C for 5 min and 4°C for 5 min, followed by 40 cycles of 95°C for 15 s, 60°C

210

for 45 s; followed by 95°C for 10 s, 65°C for 45 s, and 40 °C for 60 s. The primer sequences

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used for the RT-PCR were as follows: P-gp (forward, 5’ CGAAGAGTGGGCACAAAC 3’;

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reverse,

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CACCCACTCCTCCACCTTTG 3’; reverse, 5’ CCACCACCCTGTTGCTGTAG 3’). The

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relative quantification was obtained using the comparative threshold cycle (∆∆Ct) method.

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The genes of all samples were normalized to that of GAPDH.

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Inhibition Kinetics

5’

GCTATCGTGGTGGCAAAC

3’)

and

GAPDH

(forward,

5’

217

MDCK II-MDR1 cells were seeded onto a 24-well plate with 2 × 105 cells/well and

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cultivated in the medium for 4-5 days. The cells were washed with warm HBSS and

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subsequently exposed to HBSS solution containing 10 µM, 12 µM, 20 µM, or 30 µM R123

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for 30 min. After being washed with PBS, the cells were allowed to efflux R123 after adding

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1 mL of HBSS with or without IP6 for 40 min at 37°C. Efflux samples (200 µL) were took

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out the plate for fluorescence analysis of R123 (λex = 485 nm and λem = 535 nm) using a

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Multiskan spectrum microplate reader. The R123 concentration was normalized with respect

224

to the total protein content in each well measured using the BCA assay.

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Kinetic parameters were calculated by applying nonlinear and linear regression analysis using Origin 8.0 (Origin lab, MA, USA) according to the following equations:

227

V=(Vmax×C)/(Km+C)

(1)

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1/V=(Km/Vmax)×(1/C)+(1/Vmax)

(2)

229 230

where V and Vmax represent the efflux rate and the maximal efflux rate, respectively; Km is the Michaelis constant; C is the concentration of P-gp substrate R123. 12


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P-gp ATPase Activity Assays

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P-gp ATPase activity was measured using the P-gp-Glo assay system (Promega, Madison,

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WI, USA); Na3VO4 (P-gp noncompetitive inhibitor) served as the inhibitor and Ver as the

234

positive control.26 The experiment procedure was based on the manufacturer’s instruction.

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Specifically, membranes (25 µg/well) prepared in P-gp-Glo assay buffer were incubated at

236

37°C in a 96-well plate for 5 min with varying concentrations of IP6 (0 mM, 0.5 mM, 2 mM,

237

3.5 mM), Ver (0.2 mM), Na3VO4 (0.2 mM), or buffer alone. Then 25 mM Mg-ATP (10 µL)

238

was added to initiate the reaction and incubated 40 minutes at 37°C. After the ATP detection

239

reagent (50 µL/well) was added, the mixture was maintained at room temperature for another

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20 min. The ATPase activity was defined as the difference of Pi liberation measured in the

241

presence and absence of 0.2 mM Na3VO4 (vanadate-sensitive ATPase activity) and expressed

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as pmol ATP consumed/mg protein/40 min. The ATP standard curve was constructed for each

243

plate, and the ATPase activity for each sample was calculated using the following equations:

244

Basal P-gp ATPase activity = (ATPNa3VO4-ATPcontrol)/ (25 µg×40 min)

(3)

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Treatment P-gp ATPase activity = (ATPNa3VO4-ATPtreament)/ (25 µg×40 min)

(4)

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In addition, the buffer in the P-gp-Glo kit contains sodium azide and EGTA, which can

247

effectively inhibit the activity of mitochondrial type ATPase,27,28 and Ca2+-independent

248

ATPase 29,30 to eliminate the interference of other ATPase activity during the assay.

249

UIC2 Shift Assay

250 251 252

UIC2 is a P-gp specific antibody, thus the UIC2 shift assay was used to analyze the P-gp conformational change.31 MDCK II-MDR1 cells were cultivated on 6-well plates and exposed to 0.5 mM, 2 mM, and 13



253

3.5 mM IP6, as described in Section of “Western blot Analysis”, and 500 µM Ver was used

254

as the positive control and 100 µM Na3VO4 as the negative control. A suspension containing

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approximately 1 × 106 MDCK II-MDR1 cells was subsequently washed and resuspended in

256

0.1 mL of UIC2 binding buffer (1 × PBS, 3% FBS). Cells were then incubated with UIC2

257

antibody (1 mg per 200,000 cells) 1 h at 37°C. The isotype control cells treated with

258

fluorescent IgG2a (20 µL) were detected to eliminate the background interference. Then, the

259

samples were washed and resuspended in 0.5 mL of UIC2 binding buffer to analyzed with a

260

FACSCalibur flow cytometer (λex = 488 nm and λem = 575 nm).

261

Statistical Analysis

262

All data were presented as the mean ± SD of three independent experiments. Significant

263

differences in the Papp values, relative fluorescence intensity, consumed ATP, relative mRNA

264

expression and relative P-gp protein expression were evaluated by performing a repeated

265

measure 1-way ANOVA followed by Tukey’s post hoc test using Sigmastat statistical software

266

(SPSS). P < 0.05 and p < 0.01 were considered to be statistically significant.

267

Results

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Regulation of IP6 on P-gp in Rat Intestinal Segments

269

Effects of IP6 on R123 Intestinal Absorption by SPIP. In the ileum (Figure 1A), the

270

R123 Peff of the 3.5 mM IP6 group was significantly higher than that in the control group (p
3.5 mM IP6 (6.69) > Ver (3.78), suggesting that IP6 might inhibit

303

P-gp efflux with the increase of IP6 concentration.

304

Effects of IP6 on R123 Uptake in Caco-2 Cells. To examine the effects of IP6 on the

305

uptake activity of P-gp, the intracellular accumulation of R123 was assayed using the

306

following three methods: qualitative imaging, quantitative flow cytometry and classic uptake

307

experiments.

308

The uptake result obtained using CLSM is shown in Figure 3C. After treatment with 0.5

309

mM, 2 mM and 3.5 mM IP6, the fluorescence intensity of R123 exhibited a continuous

310

increase around the nucleus of the cells compared to the control group. This result indicated

311

that IP6 could increase intracellular R123 accumulation in the cytoplasm (Figure 3C, a, f, k; c,

312

h, m; d, i, n; e, j, o). Among the treatment groups, the fluorescence intensity of R123 in the

313

3.5 mM IP6 group exhibited the most obvious increase (Figure 3C, e, j, o) compared to the

314

control group. The extent of the increase was nearly equal to that of the Ver treatment group

315

(Figure 3C, b, g, l), suggesting that IP6 can increase intracellular R123 accumulation and is a

316

potential P-gp inhibitor similar to Ver. Additionally, DAPI staining revealed that the

317

morphology of the cell nuclei remained unchanged after treatment with either IP6 or Ver

318

(Figure 3C, f, g, h, i, j), indicating that concentrations of IP6 in the 0.5-3.5 mM range were 16


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non-cytotoxic and did not change the morphology of the cells.

320

The effect of IP6 on intracellular R123 accumulation was quantified using FACSCalibur

321

flow cytometry (Figure 3A and 3B). The cell fluorescence intensity increased with increasing

322

concentration of IP6, suggesting that IP6 enhances intracellular accumulation of R123. This

323

result was consistent with the results from the uptake study using CLSM in Caco-2 cells.

324

Specifically, there were 1.41-fold and 2.19-fold increases in R123 uptake after treatment with

325

2 mM and 3.5 mM IP6, respectively, relative to the control group (p < 0.05, p < 0.01). In

326

addition, the Ver group increased R123 uptake 2.22-fold compared to the control group. The

327

magnitude of this increase was almost equal to that of the 3.5 mM IP6 group, demonstrating

328

the strong inhibitory effects of IP6 on R123 efflux. The uptake of R123 in Caco-2 cells was

329

quantitatively measured after treatment with IP6 for 2 h (Figure 2B). The results showed that

330

the amount of intracellular R123 significantly increased from 100% in the control group to

331

281.69% in 3.5 mM IP6 group; this result was consistent with the flow cytometry results.

332

Combined, these results using the three different methods demonstrate that the amount of

333

intracellular P-gp substrate R123 was increased with the increasing concentrations of IP6 in

334

the range of 0.5-3.5 mM, suggesting that IP6 is a potential P-gp inhibitor.

335

Effects of IP6 on the Protein and mRNA Expression of P-gp in Caco-2 Cells. The

336

regulation of P-gp protein and mRNA expression by IP6 in Caco-2 cells was evaluated using

337

western blot and RT-PCR. As shown in Figure 4A and 4B, P-gp protein expression was

338

significantly decreased in the presence of IP6 compared to that of the control group (p < 0.05)

339

and reached its maximal extent at 3.5 mM (p < 0.01), demonstrating that IP6 can

340

down-regulate P-gp expression in Caco-2 cells. P-gp mRNA levels (Figure 4C) were 17



341

incrementally expressed with increasing concentration of IP6, demonstrating that IP6

342

concentration-dependently up-regulates P-gp mRNA levels in Caco-2 cells. In particular, P-gp

343

mRNA levels in the 3.5 mM IP6 group significantly increased to 141% compared to that in

344

the control group (p < 0.01). Combined with the results described above, it can be inferred

345

that the down-regulation of P-gp expression induced by IP6 was likely unrelated to the altered

346

P-gp mRNA expression levels regulated by IP6.

347

Effects of IP6 on P-gp Regulation in MDCK II-MDR1 Cells

348

Validation of MDCK II-MDR1 cell line. The P-gp expression in MDCK II and MDCK

349

II-MDR1 cells was evaluated using western blot and RT-PCR, respectively. Figure 5A shows

350

that P-gp was highly expressed in the MDCK II-MDR1 cell line but was almost undetectable

351

in the parental (MDCK II) cells. P-gp mRNA levels (Figure 5B) in the MDCK II-MDR cell

352

line were significantly higher than those in the MDCK II cell line (p < 0.01), suggesting that

353

P-gp was stably overexpressed in MDCK II-MDR1 cells. Therefore, MDCK II-MDR1 cell

354

line, as the P-gp overexpression model, could be used to investigate the P-gp-mediated drug

355

efflux and the related properties.

356

Effects of IP6 on the Transport of the P-gp Substrate R123 in MDCK II-MDR1 Cell

357

Monolayers. In the MDCK II-MDR1 cell monolayers (Figure 6A), the Papp (A-B) values of

358

R123 with 0.5 mM and 2 mM IP6 were (0.84 ± 0.001) × 10-6 cm/s and (1.36 ± 0.14) × 10-6

359

cm/s, respectively, while the Papp (A-B) value of R123 in the presence of 3.5 mM IP6

360

significantly increased to (15.43 ± 0.48) × 10-6 cm/s (p < 0.01) compared with the control

361

group (0.87 ± 0.001) × 10-6 cm/s, suggesting that high concentrations of IP6 (i.e., 3.5 mM)

362

could substantially enhance the transport of the P-gp substrate R123 in the AP→BL direction. 18


Page 18 of 44

Page 19 of 44


363

For BL→AP transport (Figure 6A), the Papp (B-A) value of R123 in the 0.5 mM IP6 group

364

was nearly equal to that in the control group. In the 2 mM and 3.5 mM IP6 groups, the Papp

365

(B-A) values of R123 significantly increased 1.41-fold and 4.56-fold, respectively, compared

366

to those of the control group (p < 0.05, p < 0.01), implying that 2 mM and 3.5 mM IP6 can

367

increase the transport of R123 in the BL→AP direction. Similar to the results obtained from

368

the Caco-2 cell transport experiments, the order of R123 ER in MDCK II-MDR1 cells was

369

control (9.15) ≈ 0.5 mM IP6 (9.58) > 2 mM IP6 (8.23) > Ver (2.71) > 3.5 mM IP6 (2.40).

370

These results consistently indicated that IP6 can effectively inhibit the efflux transport of the

371

P-gp substrate in a concentration-dependent manner and that 3.5 mM IP6 has a strong

372

inhibitory effect on P-gp efflux. Moreover, the ER was reduced to 59.36% relative to that of

373

the control after treatment with 3.5 mM IP6 in Caco-2 cells (Figure 2A). In MDCK II-MDR1

374

cells, the ER was reduced to 26.23%, indicating that the inhibitory action of IP6 on P-gp in

375

MDCK II-MDR1 cell monolayers was stronger than that in Caco-2 cell monolayers. This

376

result indirectly implies that P-gp overexpression in the former cell line mainly contributed to

377

the efflux.

378

Effects of IP6 on P-gp Expression in MDCK II-MDR1 Cells. The protein regulation by

379

IP6 in MDCK II-MDR1 cells is shown in Figure 6B and 6C. The protein expression of P-gp

380

in MDCK II-MDR1 cells significantly decreased in the presence of 2 mM and 3.5 mM IP6

381

compared to the control group (p < 0.05, p < 0.01), while 0.5 mM IP6 treatment resulted in

382

only a slight decrease. These results suggest that P-gp expression levels were effectively

383

down-regulated with the increasing concentrations of IP6. In addition, the P-gp protein

384

expression levels were down-regulated by 25.71% after treatment with 3.5 mM IP6 in Caco-2 19



385

cells (Figure 4B), while P-gp protein expression in the 3.5 mM IP6 group was down-regulated

386

by 45.94% in MDCK II-MDR1 cells (Figure 6C). These results revealed that the inhibition of

387

P-gp expression by IP6 in MDCK II-MDR1 cells was greater than that in Caco-2 cells, further

388

confirming that IP6 was responsible for the inhibition of P-gp protein expression levels.

389

Effects of IP6 on the Efflux Kinetics of R123 in MDCK II-MDR1 Cells. The terms Km

390

and Vmax are often used to describe inhibition kinetics. As shown in Figure 7A, the amount of

391

R123 efflux increased then plateaued at a higher concentration range, indicating that R123

392

efflux in MDCK II-MDR1 cells was saturable; the data fit a Michaelis-Menten distribution.

393

To investigate the kinetics of P-gp mediated R123 efflux, the kinetic parameters (Km and

394

Vmax) for R123 efflux were computed using Lineweaver-Burk plots (Figure 7B). After

395

treatment of IP6 with 0.5-3.5 mM, the Km (14.60-14.86 µM) was nearly equal to that of the

396

control group (14.68 µM), suggesting that IP6 did not impact the affinity of P-gp substrate

397

(R123) for P-gp and that the effects of IP6 on the kinetics of R123 efflux were unrelated to the

398

physical competition at the P-gp substrate binding site. However, Vmax (9.30-10.48 pmol/mg

399

protein/40 min) decreased drastically compared to the control group (16.53 pmol/mg

400

protein/40 min), indicated that IP6 could decrease the capacity of P-gp efflux. Based on the

401

results described above, it can be speculated that IP6 was likely a noncompetitive inhibitor of

402

P-gp.

403

Effects of IP6 on P-gp ATPase Activity. By observing whether the P-gp ATPase activity is

404

activate or suppress, the P-gp ATPase assay can be used to identify P-gp substrates or P-gp

405

inhibitors.32 During the assay, the activity of mitochondrial ATPase and Ca2+-independent

406

ATPase were inhibited to study the P-gp ATPase activity in isolation. 20


Page 20 of 44

Page 21 of 44


407

As shown in Figure 8A, as the IP6 concentration increased from 0.5 mM to 2 mM and 3.5

408

mM, the amount of ATP consumed decreased to 34.40 ± 2.30 pmol/µg P-gp/min, 28.30 ± 1.59

409

pmol/µg P-gp/min (p < 0.05) and 4.65 ± 1.92 pmol/µg P-gp/min (p < 0.01) compared with

410

that in the control (35.69 ± 2.33 pmol/µg P-gp/min). These results indicated that IP6 can

411

inhibit P-gp ATPase activity. In contrast, treatment with Ver resulted in a significant increase

412

in the amount of ATP (50.94 ± 0.27 pmol/µg P-gp/min) (p < 0.01) compared to the control,

413

suggesting that Ver can stimulate P-gp ATPase activity by competing for P-gp binding sites,

414

which is consistent with previous reports.9,33 The mechanisms of P-gp ATPase modulation by

415

IP6 differed from those for Ver (a typical competitive inhibitor). From this result, it can be

416

inferred that IP6 was not a competitive inhibitor of P-gp.

417

Effects of IP6 on P-gp Conformation in MDCK II-MDR1 Cells. The UIC2 reactivity

418

shift assay can be used to distinguish between competitive inhibition and noncompetitive

419

inhibition, as P-gp substrates and/or competitive inhibitors will significantly stimulate UIC2

420

reactivity, whereas allosteric modulators will reduce UIC2 reactivity.26 To investigate whether

421

IP6 has an effect on P-gp protein conformation, the MDCK II-MDR1 cells were treated with

422

the conformation-sensitive antibody UIC2 (Figure 8B).

423

The fluorescence intensity of the IgG2a group, an isotype-matched negative control, was

424

only 2.78% that of the control group, indicating that there was some interference during this

425

experiment. As shown in Figure 8B, a decrease in fluorescence intensities was observed as the

426

concentration of IP6 increased. The fluorescence intensities significantly decreased to

427

82.759%, 79.79%, and 72.94% (p < 0.01), in the presence of 0.5 mM, 2 mM, and 3.5 mM IP6,

428

respectively, compared to the control group (100%), suggesting that IP6 could markedly 21



429

reduce UIC2 binding reactivity. Compared to the control group, Na3VO4 (a noncompetitive

430

inhibitor and allosteric modulator) significantly decreased UIC2 binding (p < 0.01), whereas

431

Ver (a competitive inhibitor) significantly increased UIC2 binding. These results imply that

432

similar to the allosteric modulators, such as Na3VO4, IP6 can inhibit P-gp efflux by changing

433

the conformation of P-gp molecules. The mechanisms of IP6 inhibition were distinct from

434

those of competitive inhibitors, such as Ver, which inhibit P-gp via competition at P-gp

435

binding sites. Combined with the above results, it can be inferred that IP6 inhibited P-gp

436

efflux via allosteric modulation.

437

Discussion

438

The present study investigated the regulation of IP6 on P-gp and its underlying mechanisms.

439

The results demonstrated that IP6 inhibits P-gp efflux function via noncompetitive inhibition,

440

which is likely due to IP6 modulation of P-gp substrate efflux kinetics, P-gp ATPase activity

441

and P-gp molecular conformation. Meanwhile, IP6 can down-regulate the P-gp expression.

442

IP6 increased the intestinal absorption of P-gp substrate R123 (Figure 1A), decreased the

443

ER of R123 (Figure 2A and 6A) in both Caco-2 and MDCK II-MDR1 cell monolayers and

444

increased R123 intracellular accumulation in Caco-2 cells (Figure 3 and Figure 2B).

445

Interestingly, the Papp of R123 increased in both AP→BL and BL→AP direction in

446

Caco-2/MDCK II-MDR1cell monolayers with the increase of IP6 concentration (Figure 2A

447

and Figure 6A), which probably can be explained by the reason that IP6 opened the tight

448

junction34 and thus increased the Papp values in both directions to some extent.35 Based on the

449

above mentioned, it can be concluded that IP6 inhibits P-gp efflux and is thereby a potential

450

P-gp inhibitor. Zhu et al. suggested that cannabidiol was a potent P-gp inhibitor employing 22


Page 22 of 44

Page 23 of 44


451

similar experimental approaches. For example, they found that cannabidiol robustly enhanced

452

the intracellular retention of R123 and doxorubicin in Caco-2 and LLC-PK1/MDR1 cells and

453

decreased the efflux ratio of R123 and even that the IC50 value of cannabidiol (8.44 µM) was

454

lower than that of the typical P-gp inhibitor Ver.36 Although Ver has many side effects in

455

clinical manifestations such as arterio-ventricular block,13 no cell toxicity and membrane

456

damage to Caco-2 and MDCK II-MDR1 cells by lactate dehydrogenase (LDH) assay (Figure

457

S2) were observed in the used concentration, thus Ver was still used as a typical P-gp

458

inhibitor37 in the present study.

459

On the other hand, IP6 induced a significant decrease in P-gp expression in rat intestinal

460

segments (Figure 1B and 1C), Caco-2 cells (Figure 4A and 4B) and MDCK II-MDR1 cells

461

(Figure 6B and 6C) at 2 h. These results suggest that the inhibitory effects of IP6 on P-gp

462

efflux are likely the result of the effects of IP6 on protein expression. Similarly, it was

463

speculated that cetirizine and disodium ascorbyl phytostanol (DAPP) also decreased

464

P-gp-mediated drug efflux via the down-regulation of P-gp gene and protein expression.38,39

465

Interestingly, an increase in P-gp mRNA levels contradicts the observed P-gp protein level

466

decreases (Figure 4C), and P-gp protein expression remain unchanged with increasing

467

concentrations of IP6 when pre-treated with cyclohexamide (an inhibitor of translation)40

468

(data not shown), suggesting that IP6 might arrest the P-gp translation process to

469

down-regulate the P-gp expression. Meanwhile, IP6 likely activated P-gp proteins or other

470

substances, such as steroid xenobiotic receptors,41 which subsequently resulted in feedback

471

regulation of P-gp mRNA.

23



472

According to the Michaelis-Menten and Lineweaver-Burk plots, IP6 inhibition of P-gp

473

(Figure 7) occurred via noncompetitive mechanisms. P-gp contains two cytosolic

474

nucleotide-binding domain (NBD) and two transmembrane domain (TMD),42 which

475

constitute two homologous halves of P-gp. ATP hydrolysis of the NBDs drives

476

conformational changes in the TMDs, resulting in the efflux of P-gp substrate.43 Thus, NBDs

477

were mainly responsible for ATP binding and hydrolysis.44 The inhibition of ATPase activity

478

(Figure 8A) by IP6 observed in the current study demonstrated that IP6 likely inhibits ATP

479

hydrolysis in the P-gp NBDs.45 In addition, a conformational change in P-gp (Figure 8B) was

480

detected by the conformation sensitive antibody UIC2, suggesting that IP6 can directly

481

interfere with substrates at the P-gp binding site on TMDs.46 Therefore, IP6 functioned as a

482

noncompetitive inhibitor by inhibiting ATP hydrolysis and changing the conformation of P-gp

483

molecules.

484

Some attempts have been made to investigate the inhibition of P-gp using natural

485

compounds.47 This strategy possesses numerous advantages including low cost, lower toxicity,

486

enhancement of intracellular drug accumulation and improved bioavailability of P-gp

487

substrates, such as anticancer drugs.48 For example, natural lamellarins isolated from marine

488

organisms were used as inhibitors of P-gp in SW620 Ad300 cell;49 Su et al. reported that the

489

natural lignans from Arctium lappa L. inhibited P-gp activity in Caco-2 cell and CEM/ADR

490

5000 cells; indicating that they might be good novel adjuvants to increase the intracellular

491

uptake of some anticancer drugs.50 IP6 is also a naturally occurring polyphosphorylated

492

carbohydrate that is widely present in plant and mammalian cells.1 IP6 has low cytotoxicity

493

and is considered to be biological safe.20 Additionally, IP6 was found to inhibit P-gp efflux in 24


Page 24 of 44

Page 25 of 44


494

this study. Therefore, IP6 is a potential P-gp inhibitor, and further exploration into its utility in

495

drug delivery (combination therapy or pharmaceutical adjuvants) applications is warranted.

496

Further clinical studies should be considered to avoid potential risks after codelivery of IP6

497

and P-gp substrate drugs.

498

In summary, combined with the increased intestinal absorption, decreased ER, and

499

increased intracellular accumulation of the P-gp typical substrate R123, it can be concluded

500

that IP6 is a potential P-gp inhibitor, likely due to its down-regulation of P-gp expression

501

levels both in vitro and in situ. Furthermore, IP6 inhibits P-gp noncompetitively by decreasing

502

P-gp ATPase activity and changing the P-gp molecular conformation. This study provides, for

503

the first time, insights into the inhibitory effects of IP6 on P-gp and supports the potential of

504

IP6 as an agent for increasing the pharmacological effects of P-gp substrate drugs.

505

Conflict of Interest

506 507

The authors declare that there is no conflict of interest. Acknowledgements

508

This work was sponsored by the National Science Foundation of China (81303304), the

509

Shanghai Talent Development Fund (201565), the “Shu Guang” project supported by

510

Shanghai Education Development Foundation and Shanghai Municipal Education

511

Commission (15SG39), the Shanghai Pujiang Program (16PJD044) and the scientific and

512

technological innovation project of traditional Chinese Medicine supported by Shanghai

513

Health and Family Planning Commission (ZYKC201603008).

514

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515

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[42] Loo, T. W.; Clarke, D. M. Tariquidar inhibits P-glycoprotein drug efflux but activates

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ATPase activity by blocking transition to an open conformation. Biochem Pharmacol.

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2014, 92, 558-66.

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[43] Wilkens, S. Structure and mechanism of ABC transporters. F1000Prime Rep. 2015, 7, 14.

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[44] Pérez-Victoria, J. M.; Di Pietro, A.; Barron, D.; Ravelo, A. G.; Castanys, S.; Gamarro, F.

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Multidrug resistance phenotype mediated by the P-glycoprotein-like transporter in 31



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Leishmania: a search for reversal agents. Curr Drug Targets. 2002, 3, 311-33.

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[45] Loo, T. W.; Bartlett, M. C.; Detty, M. R.; Clarke, D. M. The ATPase activity of the

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P-glycoprotein drug pump is highly activated when the N-terminal and central regions of

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the nucleotide-binding domains are linked closely together. Journal Biol Chem. 2012,

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287, 26806-16.

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[46] Ghosh, R. D.; Chakraborty, P.; Banerjee, K.; Adhikary, A.; Sarkar, A.; Chatterjee M.; Das,

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T.; Choudhuri, S. K. The molecular interaction of a copper chelate with human

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P-glycoprotein. Mol Cell Biochem. 2012, 364, 309-20.

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[47] Abdallah, H. M.; Al-Abd, A. M.; El-Dine, R. S.; El-Halawany, A. M. P-glycoprotein

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inhibitors of natural origin as potential tumor chemo-sensitizers: A review. J Adv Res.

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2015, 6, 45-62.

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[48] Yu, J.; Zhou, P.; Asenso J.; Yang, X. D.; Wang, C.; Wei, W. Advances in plant-based inhibitors of P-glycoprotein. J Enzyme Inhib Med Chem. 2016, 2, 1-15.

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[49] Plisson, F.; Huang, X. C.; Zhang, H.; Khalil, Z.; Capon, R. J. Lamellarins as inhibitors of

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P-glycoprotein-mediated multidrug resistance in a human colon cancer cell line. Chem

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Asian J. 2012, 7, 1616-23.

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[50] Su, S.; Cheng, X.; Wink, M. Natural lignans from Arctium lappa modulate

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P-glycoprotein efflux function in multidrug resistant cancer cells. Phytomedicine. 2015,

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22, 301-7.

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668

Figure captions

669

Figure 1. (A) The effective permeability (Peff) value of R123 in the rat ileum and colon in

670

combination with varying concentrations of IP6 or Ver (50 µM) using the SPIP model. R123

671

(10 µM), a substrate of P-gp, was measured at the time points of 0, 20, 40, 60, and 80 min

672

after incubation. Data are expressed as the mean ± SD (n = 4).

673

different from the control group (p < 0.01). (B) Effect of IP6 on the expression of P-gp in rat

674

colon using western blot. The whole samples of intestinal tissue extracts were prepared 80

675

min after the start of incubation with or without IP6 or Ver. The samples were immunoblotted

676

with anti-P-gp antibody. (C) Specific bands for P-gp were quantified using Image J software

677

and were normalized to GAPDH in each lane. Data are expressed as the mean ± SD (n = 3). **

678

denotes results that significantly differ from the control group (p < 0.01).

679

Figure 2. (A) The bidirectional Papp values of R123 across Caco-2 cell monolayers. Caco-2

680

cell monolayers were incubated with 10 µΜ R123 with or without IP6 either in the apical or

681

basolateral side. The A-B and B-A represent the R123 transport in the AP→BL direction and

682

BL→AP direction, respectively. (B) The uptake of R123 in Caco-2 cells was measured after

683

incubation with varying concentrations of IP6 for 2 h. The value of each group was calculated

684

as a percentage of the control group, and 50 µM Ver was used as the positive control. Data are

685

expressed as the mean ± SD (n = 3). * and

686

control group (p < 0.05 and p < 0.01).

687

Figure 3. Effects of IP6 on intracellular R123 accumulation in Caco-2 cells. (A) The

688

accumulation of R123 was measured after incubation with varying concentrations of IP6 for 2

689

h using FACSCalibur flow cytometry. (B) The relative fluorescence intensity was quantified

**

**

denotes results significantly

denote results that significantly differ from the

33



690

using Image J software and normalized to the control value. Data are expressed as the mean ±

691

SD (n = 3). * and

692

and p < 0.01). (C) The accumulation of R123 (green) was measured after incubation with

693

varying concentrations of IP6 for 2 h using CLSM. The bars in all panels are equal to 50 µm.

694

All images were taken at 63 × magnification in oil.

695

Figure 4. Effects of IP6 on P-gp protein and mRNA expression in Caco-2 cell monolayers.

696

The whole cell extracts were prepared 2 h after the start of incubation with or without IP6 or

697

Ver. (A) P-gp protein expression was analyzed using western blot. (B) Specific bands for P-gp

698

were quantified using Image J software and normalized to β-actin in each lane. (C) The

699

mRNA expression of P-gp was calculated using RT-PCR analysis. The amount of loaded P-gp

700

relative to the control was calculated using the comparative ∆∆Ct method. GAPDH served as

701

the loading control.

702

Data are expressed as the mean ± SD (n = 3). * and

703

from the control group (p < 0.05 and p < 0.01).

704

Figure 5. Immunoblot analysis (A) of P-gp expression and RT-PCR analysis (B) of P-gp

705

mRNA expression in MDCK II and MDCK II-MDR1 cells. Data are expressed as the mean ±

706

SD (n = 3). ** denotes results that significantly differ from the parental cell line (p < 0.05 and

707

p < 0.01).

708

Figure 6. Effects of IP6 on the function and expression of P-gp in MDCK II-MDR1 cell

709

monolayers. (A) The bidirectional R123 Papp values across MDCK II-MDR1 cell monolayers.

710

The A-B and B-A represent the R123 transport in the AP→BL direction and BL→AP

711

direction, respectively. (B) P-gp protein expression was analyzed using western blot. (C)

**

denote results that significantly differ from the control group (p < 0.05

**

denote results that significantly differ

34


Page 34 of 44

Page 35 of 44


712

Specific bands for P-gp were quantified using Image J software. Each lane was normalized to

713

GAPDH.

714

Data are expressed as the mean ± SD (n = 3). * and

715

from the control group (p < 0.05 and p < 0.01).

716

Figure 7. R123 efflux after treatment with different IP6 concentrations in MDCK II-MDR1

717

cell monolayers. (A) R123 efflux was measured after incubation with 0 (control), 0.5, 2, and

718

3.5 mM IP6 for 40 min. The panels show the nonlinear regression analysis of R123 efflux. (B)

719

The panels summarize the results from the Lineweaver-Burk plot analysis of R123 efflux.

720

Data are expressed as the mean ± SD (n = 3).

721

Figure 8. (A) Effect of IP6 on P-gp ATPase activity. Human P-gp overexpressing membranes

722

were incubated with varying concentrations of IP6 or 200 µM Ver. (B) Effects of IP6, 500 µM

723

Ver and 100 µM Na3VO4 on the binding of the conformation-sensitive antibody UIC2 to P-gp.

724

Cells incubated with normal IgG2a were included as a background control. Relative

725

fluorescence intensities for the detection of the UIC2 shift were determined using a

726

FACSCalibur flow cytometer.

727

Data are expressed as the mean ± SD (n = 3).

728

the control group (p < 0.01).

**

**

denote results that significantly differ

denotes results that significantly differ from

35



729

Figure 1.

36


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Figure 2.

37



Figure 3.

38


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Page 39 of 44


Figure 4.

39



Figure 5.

40


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Page 41 of 44


Figure 6.

41



Figure 7.

42


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Figure 8.

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Table of Contents (TOC) Graphic

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Inhibition of P-Glycoprotein Mediated Efflux in Caco-2 Cells by Phytic

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