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The effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and… Sachs-Barrable, Kristina; Darlington, Jerald W; Wasan, Kishor M Oct 1, 2014

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RESEARCH Open AccessThe effect of two novel cholesterol-loweringagents, disodium ascorbyl phytostanol phosphateed aluminosilicate (NSAS)Conclusions: DAPP but not NSAS decreases P-gp mediated drug efflux through decreased mdr-1 gene expressionSachs-Barrable et al. Lipids in Health and Disease 2014, 13:153http://www.lipidworld.com/content/13/1/153and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, CanadaFull list of author information is available at the end of the articleand consequently decreased P-gp protein expression. These findings have to be taken into consideration whenDAPP is concurrently given with other drugs that are substrates for P-gp since drug-drug interactions harbour asafety issue and alter bioavailability profiles.NSAS does not have any P-gp altering properties and therefore might not affect drug-drug interactions. We concludefrom this study that NSAS might make a safer drug candidate compared to DAPP for lowering LDL-cholesterol.* Correspondence: Kishor.Wasan@usask.ca1Division of Pharmaceutics and Biopharmaceutics, Faculty of PharmaceuticalSciences, The University of British Columbia, 2405 Wesbrook Mall, VancouverV6T 1Z3, British Columbia, Canada3Drug Discovery and Development Research Group, College of PharmacyP-gp protein expression nor P-gp activity were observed.on the expression and activity of P-glycoproteinwithin Caco-2 cellsKristina Sachs-Barrable1, Jerald W Darlington2 and Kishor M Wasan1,3*AbstractBackground: Many drugs are substrates for P-glycoprotein (P-gp) and interactions involving P-gp may be relevantto clinical practice. Co-administration with P-gp inhibitors or inducers changes the absorption profile as well as therisk for drug toxicity, therefore it is important to evaluate possible P-gp alterations. The purpose of this study was toinvestigate the effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP)and nanostructured aluminium silicate (NSAS), a protonated montmorillonite clay, on mdr-1 gene expression and itsprotein, P-glycoprotein (P-gp) within Caco-2 cells.Methods: The effects of DAPP and NSAS on the regulation of mdr-1 gene, P-gp protein expression and activitywithin Caco-2 cells, were determined using cell viability and cytotoxicity tests, RT-PCR, Western Blot analysis andbi-directional transport studies.Results: We observed a significant down-regulation of mdr-1 mRNA (e.g. 38.5 ± 17% decrease vs. control at 5 μMDAPP and 61.2 ± 25% versus control at 10 μM DAPP; n = 6, P* < 0.05) within Caco-2 cells. Western Blot analysis ofP-gp expression showed that changes in mdr-1 gene expression lead to correlating changes in P-gp proteinexpression. This down-regulation of P-glycoprotein also resulted in decreased activity of P-glycoprotein comparedto untreated control. In contrast, when Caco-2 cells were treated with NSAS, no changes in mdr-1 gene expression,(DAPP) and nanostructur© 2014 Sachs-Barrable et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons PublicDomain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in thisarticle, unless otherwise stated.IntroductionIt has been estimated that by 2030 the number of deathsdue to Cardiovascular disease (CVD) will climb to 23.3million and CVD will remain the single leading cause ofdeath [1]. The World Health Organization recommendsincreased government investment in prevention and earlydetection through national programs that are aimedto prevent and control non-communicable diseaseslike CVD. The application of new research findingstowards new and better treatments, personalized medicalintestinal wall and cannot be taken up by the enterocytesfor subsequent packaging into chylomicrons. Other studiesare suggestive of alternate mechanisms contributing to theirLDL-cholesterol lowering characteristics. Kaneko et al. haveproposed that phytosterols might function as agonistsfor the liver X receptor (LXR). LXR is a nuclear receptorresponsible for upregulating cholesterol efflux pathwaysthroughout the body [12]. Additionally, suppression ofde novo cholesterol synthesis has been shown in a ratmodel [13].Sachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 2 of 10http://www.lipidworld.com/content/13/1/153care and interventions as well as the implementation ofupdated and improved guidelines are necessary for betteroutcomes for patients.Substantial scientific evidence highlights elevatedcholesterol levels as a risk factor for coronary arterydisease. Despite much clinical success, statins are not welltolerated by all patients. Sufficient LDL-cholesterol loweringcannot be achieved by statin monotherapy in every patient[2-4] and reduction of cardiovascular events can only bereduced by 33% in the most responsive patients [5].In about 2-10% of patients on statins the side effectsare very severe and therefore the statin therapy has tobe discontinued and replaced with alternative therapies [6].An alternative approach to decreasing LDL-cholesterol isinhibiting the absorption of dietary cholesterol using plantsterols and plant stanols (phytosterols and phytostanols),either as as dietary supplements or as additives in foodproducts including margarine, cereals, juices and yogurt[7]. The ability of plant sterols to reduce serum cholesterollevels has been known since the 1920s and was confirmedby studies in animals and humans in the 1950s [8-10].Despite considerable research, the mechanism by whichthey reduce cholesterol is a topic of debate. Phytosterolsare poorly absorbed in the intestines (between 0.4%and 3.5%), and as with cholesterol, they are poorlywater soluble [11].One explanation is that phytosterols compete withcholesterol for being incorporated into mixed micelleswhich are made of dietary fat, bile acids and sterols.Cholesterol not incorporated into the micelle phase isunable to cross the unstirred water layer that lines theFigure 1 Chemical structure of DAPP. The two major components of DAascorbyl sitostanol phosphate, each covalently linked to ascorbic acid by aPhytosterols can be modified to increase their micellarincorporation capacity by esterification with fatty acidsand many different modified phytosterols have been testedfor activity. One such well characterized phytosterol isdisodium ascorbyl phytostanol phosphate (DAPP), alsoknown as FM-VP4 (Figure 1). It is a water-solublederivative of sitostanol and campestanol linked byesterification with an ascorbyl-phosphate group [14,15].In animal studies with rats, gerbils and mice FM-VP4effectively reduces dietary cholesterol absorption [16,17].This cholesterol lowering effect also leads to a reductionof atherosclerotic lesion formation in apo E knockoutmice, a model that represents atherosclerosis [18].FM-VP4 has also shown a decrease in body masswithout any toxic effects [19]. This dose dependentreduction of mass in mice treated with FM-VP4 was notdue to an increase in resting metabolic rate or decreasedfood or water intake, but through decreased absorption orincreased excretion of lipids.Potent cholesterol-lowering characteristics without anysignificant toxic effects was also shown in preclinicaland clinical studies [15,16]. A clinical study in 30 mendemonstrated that up to 800 mg/day of DAPP is safeand well tolerated for at least 4 weeks. LDL-cholesterolwas significantly reduced by 6.6% (p = 0.02) in the 400 mgper day group [20].Another compound of interest is nanostructuredaluminosilicate (NSAS). NSAS is a protonated montmoril-lonite (bentonite) clay. Bentonites are naturally occurringcompounds and recent studies have demonstrated choles-terol lowering effects and define them as a new class ofPP are disodium ascorbyl campestanol phosphate and disodiumphosphodiester bond [14,15].cholesterol absorption inhibitors. NSAS can reduce theabsorption of cholesterol by 39%, similar to an identicaldose of stigmasterol, by competing with cholesterol forabsorption in the intestine after ingestion [21]. The firstreported medical use of bentonite clay goes back toancient Mesopotamia where it was applied externallyas a mud bath and was touted for anti-inflammatoryand antiseptic characteristics.NSAS is composed of layers of aluminium octahedralsheets sandwiched between two silicon-oxygen tetrahedrallayers (Figure 2). NSAS has a high surface area(200-800 m2/g) and shows high water and organic materialabsorption characteristics either by adsorption onto itsexternal surface or into its interlaminar space. It isnegatively charged in contrast to positively charged bileacids and surface protons are incorporated to counterbal-ance platelet surface negative charge [22]. Additionally,NSAS appears relatively safe for oral administration and isminimally absorbed.Oral administration of NSAS significantly inhibits chol-esterol absorption in mice. Apo E knockout mice fed a diethigh in cholesterol and fat were treated for 12 weeks withNSAS (1.4% w/w) or stigmastanol (2% w/w) and showedsignificant reductions in plasma cholesterol concentrationsrelative to control animals in both groups. No changes infood and water intake or body weight were observed.Atherosclerotic lesion formation at the aortic rootwas also reduced [23]. In vitro lipolysis studies adapted tostimulate intraluminal processing of triglycerides andcholesterol in the presence of NSAS showed an inhibitionof cholesterol absorption either by direct binding tocholesterol or by other mechanisms like incorporationinto micelles. Protonated NSAS adsorbs and sequesterscholesterol from the aqueous phase in the digestive tractresulting in precipitation and excretion with feces [24].Potent inhibition of cholesterol absorption and minimalsystemic exposure make NSAS a considerable candidatefor treatment of hypercholesterolemia, either as adjunctiveSachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 3 of 10http://www.lipidworld.com/content/13/1/153Figure 2 Structure of montmorillonite clay. Montmorillonite has a layerenclosing a sheet of octahedral aluminium crystals. Water and surface cations-lattice structure consisting of two sheets of tetrahedral siliconcrystalsenter between adjacent silicon sheets causing the material to expand.Sachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 4 of 10http://www.lipidworld.com/content/13/1/153therapy with statin or as monotherapy in patients unableto tolerate statins.Most cardiovascular patients require multiple medicationsin addition to lipid lowering therapy, creating the possibilityfor drug interactions. Many of these drugs are substrates forP-glycoprotein (P-gp), which mitigates cellular exposure tovarious hydrophobic compounds thereby protectingcells from xenobiotics and other potential toxins. P-gp isexpressed in numerous tissues including the small intestine,blood–brain barrier, hepatocytes, and kidney proximaltubule which affects intracellular and systemic drugconcentrations; therefore, it is important to understandthe behaviour of new drugs and therapies in regards tomodification of P-gp.P-gp belongs to the ATP-binding cassette (ABC)superfamily, which in humans, consists of severalhundred transmembrane transport proteins which belongto 7 distinct subfamilies named ABCA – ABCG. Theyimport and export a broad variety of substrates acrosscellular membranes including amino acids, lipids, drugs,and proteins. ABC genes are essential for many processesin the cell and mutations cause or contribute to severalgenetic disorders such as cystic fibrosis, neurologicaldisease, retinal degeneration, Stargardt disease, cholesteroland bile transport defects, anemia, and abnormal drugresponses). P-gp was the first identified human ABC trans-porter gene [25] and was first described in drug-resistantcells with a defined pattern of multidrug resistance[26]. P-gp relies on ATP as an energy source for drugefflux. Humans have two multidrug resistance (mdr)genes mdr-1 and mdr-2 (also called mdr-3), whereasrodents have three mdr genes mdr-1A (also calledmdr-3), mdr-1B (also called mdr-1) and mdr-2. Onlymdr-1 in humans and mdr-1A & B in rodents are involvedin drug transport and drug-resistance [27,28]. Expressionof P-gp in intestinal epithelial cells is responsible for effluxthat limits cellular uptake into enterocytes and drugabsorption. Drugs can be defined as P-gp inhibitors, P-gpenhancers or P-gp substrates. P-gp inhibitors impair P-gpmediated efflux while inducers enhance P-gp activity.A multitude of drugs, including several with a narrowtherapeutic index, interact with P-gp and drug-druginteractions must be considered given their impact onbioavailability and therapeutic outcome. Hence, the FDAnow advises drug developers to characterize interactionsbetween new drugs and P-gp, as stated in guidelinesreleased in 2012 concerning drug-drug interactions. Thisjust signifies the importance for evaluating P-gp interac-tions during novel drug development and to recommendclinical guidelines for clinicians when administering P-gpsubstrate drugs.We studied the in vitro interactions between two novelcholesterol lowering agents, DAPP and NSAS, and theP-gp transporter protein using Caco-2 cells.Materials and methodsMaterialsNSAS was provided by AMCOL International Corporation(Chicago, USA). Triton X-100, Tween-80, HEPES, Proteaseinhibitor cocktail, Na-deoxycholate, EDTA and NaCl wereobtained from Sigma-Aldrich (St. Louis, MO, USA).All tissue culture reagents were from Invitrogen/LifeTechnologies (Grand Island, NY, USA). T-75 flasks,tissue culture treated plates and Transwell® insertswere from Corning Incorporated (Corning, NY, USA).CytoTox96® Non-Radioactive Cytotoxicity Assay, MTSCellTiter 96® AQueous One Solution Cell Proliferation Assayand Pgp-GloTM Assay were from Promega Corporation(Madison, WI, USA). BCATM Protein Assay Kit wasobtained from Pierce Biotechnology, Inc. (Rockford, IL,USA). NP-40 was purchased from Roche Applied Scienceand Trans-Blot® Transfer medium (nitrocellulose membrane0.45 μM) from Bio-Rad (Hercules, CA, USA).MethodsCell cultureCaco-2, human colon adenocarcinoma cells, were pur-chased from ATCC (Rockville, MD, USA) and passagenumbers between 20 and 35 were used. Cells werecultured in Dulbecco’s modified Eagle’s medium (DMEM),supplemented with 10% fetal bovine solution (FBS),292 μg mL−1 L-glutamine, 0.1 mM non-essential aminoacids, 100 U/mL penicillin and 100 μg/mL streptomycincontaining 1.5 g/L NaHCO3 at 37°C in humidified air con-taining 5% CO2. Stock cultures were grown in T-75 flasks.Once the cells reached a confluency of about 90%, theywere then split by using 0.25% trypsin containing 1.0 mMEDTA. Cells were seeded in 96-well, 48-well, 12-well, orTranswell plates depending on the type of experiment.The medium was changed every other day.Cytotoxicity measurementsTo determine non-cytotoxic concentrations, the followingmarkers of toxicity were employed: 1) cell plasmamembrane integrity as determined by lactate dehydrogenase(LDH) release and 2) mitochondrial respiration asmeasured by the reduction of a tetrazolium compound(MTS) to a soluble formazan product. Caco-2 cells wereseeded onto 96-well plates and were kept at 37°C inhumidified air containing 5% CO2 until the cells reached90% confluency. On the day of the experiment, the culturemedium was exchanged for treatment solutions of 0 to1000 μg/mL NSAS or 0 to 500 μM DAPP and 1% TritonX-100 (as positive control for cytotoxicity) in Hanks’Balanced Salt Solution without phenol (HBSS) containing10 mM HEPES, pH 7.4. LDH release (CytoTox96®Non-Radioactive Cytotoxicity Assay), MTS reduction(CellTiter 96® AQueous One Solution Cell proliferationAssay) and bicinchoninic acid (BCATM Protein Assay Kit)Sachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 5 of 10http://www.lipidworld.com/content/13/1/153assays were then performed, and cell viability was calcu-lated relative to the 100% control (MTS-Assay). Cytoxicitywas calculated relative to 100% cytotoxicity obtained fromthe Triton X-100 group (LDH-Assay).Protein expression: western blottingProtein levels were qualitatively observed by Westernblot techniques. Caco-2 cells were treated with culturemedium (control) or culture medium containing non-cytotoxic concentrations of NSAS or DAPP. Cellswere washed three times with PBS and harvested withRIPA lysis buffer (50 mM HEPES, 150 mM NaCl,2 mM EDTA, 0.5% Na-deoxycholate, 1% NP-40) containingprotease inhibitor cocktail (1:100 dilution). Protein contentwas determined by BCA protein assay and the cellmembrane proteins (20 μg/lane) were separated byelectrophoresis through a 10% SDS-polyacrylamide geland then electroblotted onto a nitrocellulose membrane.A pre-stained protein standard from Bio-Rad was used toidentify the P-gp protein band at 170kD and actin at42kD. The membrane was incubated overnight at 4°C inblocking buffer (1 × PBS, 2.5% nonfat dried milk, 0.1%Tween-20), and probed with a 1:300 dilution of primaryantibody (C219 from Signet Pathology System Dedlam) todetect P-gp and a 1:1000 dilution of I-19 (Santa CruzBiotechnology) to detect actin as an internal control. Themembranes were washed 3 times with PBS and 1%Tween-20 (PBS-T) and the membrane was incubatedin a 1:5000 dilution of anti-mouse IgG rabbit horse-radish peroxidase (HRP)-conjugated antibody (JacksonImmunoResearch Laboratories) and a 1:3000 dilutionof anti-goat IRP bovine HRP (Santa Cruz Biotechnology)for P-gp and Actin respectively. Three washing steps withPBS-T followed. Bands were visualized with ECL andquantified with Labworks software (UVP).RNA-isolation and RT-PCRCaco-2 cells were harvested and total RNA was isolatedwith TRIzol® Reagent (Invitrogen) according to the man-ufactures instruction. RNA was reverse transcribed intocDNA. The concentration of cDNA reaction product wasmeasured by using Oligreen-Assay (Molecular Probes). Theprimers were synthesized at the Oligonucleotide SynthesisLaboratory at UBC. Parameters and conditions for thetested primers were optimized. The following primerswere used for the described studies. Mdr-1 (5′- GTC-ATT-GTG-GAG-AAA-GGA-AAT-CAT-G-3 and 5′- ATT-CCA-AGG-GCT-AGA-AAC-AAT-AGT-G-3′ and GAPDH (5′- TGA-AGG-TCG-GAG-TCA-ACG-GAT-3′ and5′- TCG-CTC-CTG-GAA-GAT-GGT-GAT-3′). A samplefrom each PCR product was subjected to electrophoresison a 1.5% agarose gel (containing Ethidium bromide). A100 bp ladder was used to identify the size of PCRproducts. The fluorescent bands were imaged under UVlight (UV-Epi Chemi II) and quantified with UVP-labworkssoftware.Transmembrane transport of rhodamine 123Caco-2 cells form monolayers and expressexpress P-gpon their apical membranes thereby differentiating intoa highly functionalized epithelial barrier that is morpho-logically and biochemically similar to small intestine col-umnar epithelium. A good system to measure apical andbasolateral transport across monolayers and to determinethe permeability of a certain substance is a Transwell®plate. Cells are grown on a semi-permeable membranewhich is placed between two chambers. Donor andacceptor chambers are determined by experiment type. Tomodel intestinal drug absorption, the substance is giveninto the apical side and the concentration in the basolateralside is measured. Secretory transport can be determined ifthe substance is added onto the basolateral side and theconcentration in the apical chamber is measured. P-gpmediated transport is characterized by basolateral to apicaltransport greater than apical to basolateral transport.Caco-2 cells were seeded in polycarbonate membraneTranswell® plates. at a densitiy of 40.000 cells/cm2 andgrown in a humidified chamber (at 37°C, of 5% CO2.)with media changes every 2 days. The growth media(Dulbecco’s minimal essential medium-DMEM) contained10% heat-activated fetal bovine serum, 292 μg/ml glutamine,0.1 mM non-essential amino acids, 100U/ml penicillinand 100 mg/ml glutamine. For the treatment experiments,NSAS or DAPP in different concentrations was addedto the media and either applied onto apical and/or thebasolateral side of the Transwell plate.Transepithelial electrical resistance (TEER) of the mono-layers was measured to confirm monolayer integrity with aMillicell Electrical Resistance System (Milipore Corp.,Bedford, MA). Caco-2 cells with TEER Values above300 Ω · cm2 were used for transport studies. Cellswere washed 3 times with PBS before treatment solutionswere loaded on the apical side. Plates were incubatedand TEER values were measured before and after thetreatment to ensure integrity of the monolayer andtight junctions.Rhodamine 123 (Rh123) is a fluorescent dye and P-gpsubstrate and has been used as a probe to measurethe functional activity of P-gp. It has a molar extinc-tion coefficient of 85,200 M−1 cm−1 at 511 nm. Rh123was added to either the apical or basolateral cham-ber, with transport buffer in the corresponding re-ceiver chamber. In a time dependent manner, samples(50 μl) were collected with media replacement fromreceiver chamber and transferred into a 96-well plate.Rh123 concentrations were measured with a FluoroskanAscent fluorometer (excitation = 485 nm and emission =538 nm).The apparent permeability Papp can be determined byusing the following equation:Papp ¼ dQdt C∩ ⋅IAA is the area of absorption (cell monolayer), dQ/dt isthe cumulative amount of test compound appearing inthe receiver compartment of the assay system versustime, and Co is the initial concentration of the testcompound in the donor compartment.Statistical analysisAll data sets were analyzed for statistical significance byparametric methods using SigmaStat version 3.5. Whencomparisons were made between two groups, unpairedtwo-tailed t tests were used. All data are expressed asmean ± standard deviation.ResultsMeasurement of toxicity of NSAS and DAPP in Caco-2subsequently used to treat cells for cholesterol uptake,transporter protein expression, Rh123 accumulation andtransport studies across the Caco-2 cell monolayer.P-gp protein expressionNo changes in protein expression for P-glycoprotein weremeasured when Caco-2 cells were incubated for 24 hourswith NSAS at 10 μg/mL, 100 μg/mL, and 500 μg/mL(Figure 5). However when Caco-2 cells were treated for thesame length of time with DAPP a significant change wasobserved (Figure 6). Incubation with DAPP 10 μM leads toa lesser expression of P-gp (41 ± 6% versus untreatedcontrol cells). We performed PCR reactions for those testedtime points. A significant decrease in mdr-1 gene expres-sion was observed when cells were treated with DAPP. Alldata obtained for mdr-1 gene expression were normalizedfor GAPDH gene concentration (Figures 5C and 6C).In summary, down-regulation of mdr-1 gene expressionwas shown, complementing the results for decreased P-gpprotein expression (38.5 ± 17% for treatment with 5 μMDAPP and 61.2 ± 25% for treatment with 10 μMDAPP ver-effect was seen for a concentration of 10 μM of DAPP.EffeSachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 6 of 10http://www.lipidworld.com/content/13/1/153cellsTreatment with concentrations between 0 and 500 μg/mlNSAS and 0 and 250 μM DAPP showed no significantdifference in cell viability compared to untreated controlas measured by MTS assay (Figures 3A and 4A). Resultsfrom the LDH assay showed no significant cytoxicityfor concentrations between 0 and 300 μg/mL NSASand 0 and 100 μM DAPP compared to untreated control(Figures 3B and 4B). These nontoxic concentrations wereFigure 3 Cell viability and cytotoxicity after treatment with NSAS.with different concentrations of NSAS for 24 hours. Results are compared to uthe mean ± SD. *p < 0.05. N = 3.ct on Caco-2 cells. MTS-Assay (A) and LDH-Assay (B) after incubationTransmembrane transport of rhodamine 123Experiments were performed to measure the activity ofP-gp in Caco-2 cells after treatment with DAPP.sus untreated control cells). Figure 6 shows the correlationfrom a decreased gene expression into decreased proteinexpression even after one week of treatment. The largestntreated control cells (in A) and Triton X-100 (in B). Each bar representsEffeo uSachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 7 of 10http://www.lipidworld.com/content/13/1/153Figure 4 Cell viability and cytotoxicity after treatment with DAPP.with different concentrations of DAPP for 24 hours. Results are compared tthe mean ± SD. *p < 0.05. N = 4.We observed a significant down-regulation of mdr-1m-RNA (e.g. 38.5 ± 17% for treatment with 5 μM DAPPand 61.2 ± 25% for treatment with 10 μM DAPP; n = 6,P* < 0.05) within Caco-2 cells. Western Blot analysis ofP-glycoprotein expression showed that changes in mdr-1gene expression lead to correlating changes in P-gp proteinFigure 5 P-gp protein expression in Caco-2 cells after treatment with10 μg/mL, 100 μg/mL and 500 μg/mL NSAS compound A. (A) Representatof P-gp protein normalized by the protein expression of actin. There is no stati(C) Expression profile of mdr-1 gene expression in Caco-2 cells. A sample fromand the fluorescent bands were quantified with UVP-Labworks software. Eachct on Caco-2 cells. MTS-Assay (A) and LDH-Assay (B) after incubationntreated control cells (in A) and Triton X-100 (in B). Each bar representsexpression. This down-regulation of P-glycoprotein alsoresulted in a decreased activity of P-glycoprotein comparedto untreated control.Treatment with 5 and 10 μM DAPP resulted in a signifi-cant increase in Rh123 accumulation by two-fold relative tountreated control cells (Figure 7B). Accumulation of Rh123NSAS. Cells were exposed for 24 hours to media alone (control),ive Western Blot. (B) The data represent the mean ± standard deviationstically significant difference between treatment groups and control. N = 3.each PCR product was subjected to electrophoresis on a 1.5% agarose gelvalue represents the mean ± standard deviation of N = 3.Figure 6 P-gp protein expression in Caco-2 cells after treatment with DAPP. Cells were exposed for 24 hours to media alone (control),1 μM, 10 μM and 500 μM DAPP. (A) Representative Western Blot. (B) The data represent the mean ± standard deviation of P-gp protein normalized bydr-ndSachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 8 of 10http://www.lipidworld.com/content/13/1/153in those Caco-2 cells is similar to Rh123 accumulation incells treated with the positive control for P-gp inhibition,verapamil.These findings suggest that DAPP not only hascholesterol-lowering properties but also decreases P-pgmediated drug efflux and might reverse multi-drugresistance.DAPP treatment lead to a statistically significantdecrease in secretory flux compared with untreated controlcells (Figure 8), while TEER values stayed unchanged.Bentonite on the other hand had shown cholesterollowering characteristics without influencing transporterthe protein expression of actin. *p < 0.05. N = 6. (C) Expression profile of msubjected to electrophoresis on a 1.5% agarose gel and the fluorescent bamean ± standard deviation of N = 6. *p < 0.05.proteins. Also, no changes for Rh123 accumulationwere observed (Figure 7A) when compared to untreatedcontrol cells.Figure 7 Effect of NSAS and DAPP on Rhodamine 123 uptake in Cacoincreasing concentrations of NSAS (A) or DAPP (B). Cellular accumulation oin each well. Data is presented as mean ± standard deviation with N = 6. *pIn Summary DAPP has an influence on the expressionof mdr-1.; a decrease of the mRNA level lead to a decreasein P-glycoprotein expression. A corresponding decreasedP-gp activity was also observed. None of those changes inmdr-1 gene expression, P-gp protein expression andactivity were seen with NSAS.ConclusionsSince the introduction of statins in the 1980s, they havebecome a golden standard of lipid-lowering therapy. Theyare prescribed for treatment of hypocholesterolemiaand to reduce LDL-cholesterol and therefore reducing1 gene expression in Caco-2 cells. A sample from each PCR product wass were quantified with UVP-Labworks software. Each value represents thecoronary artery disease-related morbidity and mortality.Over the past years statins have been proved to be safeand well-tolerated drugs. However there are more and-2 cells. Cells were exposed for 24 h to media alone (control), or tof Rhodamine 123 was normalized with respect to the protein content< 0.001 vs. control (A) and *p < 0.05 vs. control (B).hoeerewitSachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 9 of 10http://www.lipidworld.com/content/13/1/153Figure 8 Effect of pre-incubation with DAPP on P-gp transport of Rtransport) and apparent Permeability Papp. Data represent the averagtreatment and at the end of the experiment. No changes in TEER values wDAPP lead to a statistically significant decrease in secretory flux comparedmore reports on patients who either have an intoleranceto statin treatment or experience minor to severe adversereactions. The need for new and alternative therapiesis more present and prominent. Studies of either newpharmacological agents or treatments in combinationwith established therapies are under way.The study here investigates the effect on P-gp expressionand activity of two substances, DAPP and NSAS, of thenew class of cholesterol lowering agents.This is of relevance for drug toxicity and efficacy sincemost patients take several medications simultaneouslyand many of the commonly prescribed drugs aresubstrates for P-gp. Drugs with narrow therapeuticindexes have shown large increases in concentrationwhen co-administred with potent P-gp inhibitors orwhen the drug itself functions as P-gp inhibitor.In previously published studies both agents (DAPP andNSAS) have shown a significant decrease in cholesterolabsorption. This was tested in different models andno severe side effects were observed.We found that unlike DAPP, NSAS did not changeP-gp expression in our Caco-2 cell model. From thatperspective NSAS may be a safer candidate for cholesterol-lowering treatment with regards to drug-drug interactionsand adverse effects. Given our observation, DAPP hasthe potential to modulate P-gp mediated drug transport.Phytostanols and phytosterols have been widely used ascontrol for P-gp inhibition.damine 123 across Caco-2 cell monolayer (basolateral to apicalof N = 6 ± SD. *p < 0.025. TEER values were measured before and after theseen which indicates the integrity of the monolayers. Treatment withh untreated control cells. Treatment with Verapamil represents a positiveadditives to cereals, margarine, drinks and other foodsources or are given as supplements to reduce serumcholesterol levels. Given the new findings of loweringP-gp protein expression and activity, this observationraises the possibility that when given with other drugs,they might impact uptake, bioavailability, and therapeuticoutcomes.There are several limitations to this set of studies.Further studies are required to determine the interactionsbetween these two agents and other ABC transporterproteins. Though P-gp is the most significant one,there are several other transporter proteins whichmay impact drug absorption such as multidrug resist-ance associated protein (MRP), breast cancer resist-ance protein (BCRP), organic anion transporter (OAT),organic anion transporting polypeptide (OATP), organiccation transporter (OCT) and oligopeptide transporter(PEPT). Additionally, these finding need to be confirmedin an animal model to evaluate clinical significance ofour findings.Data presented here further substantiate the need foraddressing and investigating potential transporter proteininteractions when developing new drug therapies.Competing interestsKishor Wasan and Kristina Sachs Barrable declare that they have nocompeting interests. However, Jerry Darlington is a Vice President ofAMCOL Int.20. Vissers MN, Trip MD, Pritchard PH, Tam P, Lukic T, de Sain-van der Velden MG,Sachs-Barrable et al. Lipids in Health and Disease 2014, 13:153 Page 10 of 10http://www.lipidworld.com/content/13/1/153Authors’ contributionsKW designed and implemented the experiments and wrote and edited themanuscript. KS-B completed the experiments and wrote and edited themanuscript. JD was involved in the design of the experiments and editedthe manuscript. All authors read and approved the final manuscript.AcknowledgementsThe authors would like to thank Ankur Midha and Jacob Gordon from TheUniversity of British Columbia, Faculty of Pharmaceutical Sciences, for theircritical feedback and discussions.FundingThis work was supported by a University/Industry grant from the CanadianInstitutes of Health Research (CIHR) and Amcol International Corporation.Author details1Division of Pharmaceutics and Biopharmaceutics, Faculty of PharmaceuticalSciences, The University of British Columbia, 2405 Wesbrook Mall, VancouverV6T 1Z3, British Columbia, Canada. 2AMCOL International Corporation,Chicago, USA. 3Drug Discovery and Development Research Group, College ofPharmacy and Nutrition, University of Saskatchewan, Saskatoon,Saskatchewan, Canada.Received: 27 May 2014 Accepted: 24 September 2014Published: 1 October 2014References1. Mathers CD, Loncar D: Projections of global mortality and burden ofdisease from 2002 to 2030. PLoS Med 2006, 3:e442.2. 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