Conjugated linoleic acid isomers decrease all-trans-retinoic acid

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Conjugated linoleic acid isomers decrease all-transretinoic acid-induced expression of sodium/iodide transporter expression in mammary epithelial cells Gaiping Wen, Julia M Fischer, Erika Most, Klaus Eder, and Robert Ringseis J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00673 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Journal of Agricultural and Food Chemistry

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Conjugated linoleic acid isomers decrease all-trans-retinoic acid-induced

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expression of sodium/iodide transporter expression in mammary epithelial

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cells

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Gaiping Wen, Julia Fischer, Erika Most, Klaus Eder, Robert Ringseis*

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Institute of Animal Nutrition and Nutrition Physiology, Justus-Liebig-University Giessen,

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Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany

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*Corresponding author address: Apl. Prof. Dr. Robert Ringseis, Institute of Animal Nutrition

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and Nutrition Physiology, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 26-32, 35392

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Giessen,

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[email protected]

Germany;

Fax:

+49-641-9939239;

Phone:

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1 ACS Paragon Plus Environment

+49-641-9939231;

E-mail:


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Abstract

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Expression of sodium/iodide symporter (NIS) is stimulated by sterol regulatory element binding

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transcription factors (SREBFs) in mammary epithelial MCF-7 cells. Since conjugated linoleic

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acid (CLA) isomers have been shown to inhibit transcriptional activity of SREBFs in the

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mammary gland, the hypothesis was tested that CLA isomers inhibit NIS expression induced

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by all-trans retinoic acid (ATRA) in MCF-7 cells through inhibiting SREBF activity. c9t11-

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CLA and t10c12-CLA decreased ATRA-induced NIS mRNA [from 1.00 (ATRA alone) to 0.80

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± 0.12 (200 µM c9t11-CLA) and 0.62 ± 0.10 (200 µM t10c12-CLA), P < 0.05] and protein

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expression [from 1.00 (ATRA alone) to 0.77 ± 0.08 (200 µM c9t11-CLA) and 0.63 ± 0.05 (200

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µM t10c12-CLA), P < 0.05] and NIS promoter activity [from 1.00 (ATRA alone) to 0.74 ± 0.13

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(200 µM c9t11-CLA) and 0.76 ± 0.13 (200 µM t10c12-CLA), P < 0.05], but increased the

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mRNA levels of SREBF isoforms and their target genes. In contrast, the mRNA level of

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peroxisome proliferator-activated receptor γ (PPARG) was strongly induced by ATRA alone,

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but decreased by CLA isomers [from 1.00 (ATRA alone) to 0.80 ± 0.06 (200 µM c9t11-CLA)

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and 0.86 ± 0.06 (200 µM t10c12-CLA), P < 0.05]. Overexpression of PPARγ in MCF-7 cells

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increased basal NIS promoter activity and treatment with the PPARγ ligand troglitazone

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stimulated ATRA-induced NIS promoter activity. In conclusion, the results suggest that CLA

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isomers exert their effect on expression of NIS by decreasing PPARG expression in MCF-7

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cells.

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Key words: conjugated linoleic acid, sodium/iodide symporter, peroxisome proliferator-

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activated receptor γ, mammary epithelial cells, retinoic acid


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INTRODUCTION

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The sodium/iodide symporter (NIS) is a transport protein facilitating cellular uptake of iodide

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from the blood stream. Due to this, NIS is essential for accumulating iodide in the thyroid, an

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important step in the synthesis of iodine-containing thyroid hormones [1]. Besides, NIS is

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functionally expressed from the end of pregnancy and throughout lactation in the mammary

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epithelium [2], where it is required for iodide secretion into the milk and thus is crucial for the

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provision of iodide for thyroid hormone biosynthesis in the neonate. Earlier studies

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demonstrated that feedstuff containing glucosinolates like coproducts from oil production

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(rapeseed meal, rapeseed press cake) lowers iodine excretion via the milk and milk iodine

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content of by 50 to 75 % in dairy cows [3-5]. This effect is attributed to specific breakdown

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products of glucosinolates in the animal´s body like thiocyanates and isothiocyanates, which

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competitively inhibit NIS-mediated uptake of iodide into the thyroid and the mammary gland

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[6, 7]. Despite this long-established knowledge about the interference of dietary glucosinolates

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with NIS-mediated iodide transport, knowledge about regulation of NIS by other dietary factors

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in mammary epithelial cells is scarce.

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We have recently demonstrated in MCF-7 cells, a human mammary epithelial cell line, that

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NIS expression and NIS promoter activity are stimulated by overexpression of sterol regulatory

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element binding transcription factors (SREBFs) [8]. In contrast, inhibition of SREBF activation

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by 25-hydroxycholesterol (25HC) was shown to decrease NIS expression following induction

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by all-trans retinoic acid (ATRA) in this mammary epithelial cell line [8]. SREBFs which

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comprise of at least two different isoforms, SREBF1 and SREBF2, have been established as

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key regulators of lipid metabolism promoting transcription of the complete set of genes

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involved in lipid synthesis [9]. Due to this, SREBFs play an important role for production of

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lipids including fatty acids, triglycerides, cholesterol and phospholipids in the lactating

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mammary gland and lipid secretion into the milk [10-12]. Specific isomers of conjugated

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linoleic acids (CLA) such as trans-10, cis-12 CLA (t10c12-CLA) were found to decrease milk 3 ACS Paragon Plus Environment


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fat content in different species such as cows [13, 14, 15], sheep [16], goats [17], and rats [18],

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and this has been explained by inhibition of the transcriptional activity of SREBFs in mammary

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epithelial cells [19, 20]. Based on this, we hypothesized that t10c12-CLA inhibits expression

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of NIS induced by ATRA in mammary epithelial cells through inhibiting activation of SREBFs.

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To test this hypothesis, we used the MCF-7 cell line which is a widely used in vitro-model to

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explore the regulation of NIS expression and function in the mammary gland [21]. In order to

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clarify if t10c12-CLA affects NIS expression in an isomer-specific manner we also studied the

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effect of another CLA isomer, namely cis-9, trans-11 CLA (c9t11-CLA). The CLA isomers

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were investigated in concentrations from 10 to 200 µM, with the lower concentration reflecting

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approximately the CLA concentration in blood plasma of cows fed fresh pasture [22].

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MATERIALS AND METHODS

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Chemicals. c9t11-CLA (> 98% pure) and t10c12-CLA (> 98% pure) were obtained from

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Biomol (Hamburg, Germany). Linoleic acid (LA) (> 98% pure), ATRA (> 98% pure) and 25HC

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(98% pure) were purchased from Sigma-Aldrich (Taufkirchen, Germany). From fatty acids

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(LA, c9t11-CLA, t10c12-CLA), 100 mM stock solutions (in ethanol) were prepared. From

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ATRA and 25 HC, 1 mM and 5 mM stock solutions [in dimethylsulfoxide (DMSO)],

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respectively, were prepared.

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Cell culture. MCF-7 cells (Cell Lines Service, Eppelheim, Germany) were grown at 37°C

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in a humidified atmosphere composed of 95 % air and 5 % CO2 in Dulbecco´s Modified Eagle

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Medium (DMEM; Gibco/Life Technologies, Darmstadt, Germany) and 10% fetal bovine serum

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(FBS) (Gibco/Life Technologies) (“growth medium”). Growth medium was replaced every 2

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days. At a confluence of 70%, MCF-7 cells were either used for experiments or sub-cultivated.

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Aliquots from stock solutions (fatty acids, ATRA, 25HC) were directly added to the culture

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medium. Control cells were incubated with the same vehicle concentration (DMSO and/or 4 ACS Paragon Plus Environment

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ethanol in the concentrations indicated in figure legends). The detailed experimental conditions

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(concentrations of fatty acids, ATRA, 25HC, FCS and duration of experiments) are indicated

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in the figure legends. All experiments were performed at least two times from a different cell

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passage number (= independent experiments). An independent experiment was defined as an

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experiment performed with cells of a specific passage number and included seeding, treatment

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and analysis.

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Cell viability assay. MCF-7 cell viability in response to ATRA in combination with

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different concentrations of CLA isomers was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT; Sigma) assay. For MTT assay, MCF-7 cells were seeded

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in 96-well culture plates at 1.2 x 104 cells/well. Cells of all treatments were incubated with the

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same vehicle (either DMSO, ethanol or both) concentration as described in the figure legend.

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The MTT assay was carried out as described recently [23], except that MCF-7 cells were treated

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with the solution containing MTT for 4 h.

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Fatty acid composition of MCF-7 total lipids. MCF-7 cells were seeded in 24-well

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culture plates (6 x 104 cells/well) and incubated as described in the figure legend. After

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removing the cell medium and washing the cell monolayer, total cell lipids were extracted by a

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3:2 (v/v)-mixture of n-hexane and isopropanol [24]. Lipids were dried (under N2 stream) and

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subsequently methylated using trimethylsulfonium hydroxide solution (Sigma-Aldrich) [25].

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Separation of fatty acid methyl esters (FAMEs) was carried out with a gas chromatography

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system (Clarus 580, Perkin Elmer, Rodgau, Germany) consisting of an on-column injector, a

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polar capillary column (Permabond FFAP, 60 m, 0.25 mm internal diameter column, 0.25 µm

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film thickness, Macherey-Nagel, Düren, Germany) and a flame ionization detector. Three

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microliters of sample were injected at a 1:20 split ratio into the column. The thermal profile

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was as follows: 200°C for 10 min; 200 to 220°C at 2°C per min; 220°C for 25 min. The injector

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and detector temperatures were both set to 260°C. Helium served as carrier gas at a flow rate



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of 0.8 mL/min. FAMEs were identified based on their retention times in comparison to a

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standard mixture (FAME Mix, C4-C24, Sigma-Aldrich).

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RNA isolation and quantitative real-time PCR. For quantitative real-time PCR (qPCR)

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analysis, MCF-7 cells were seeded in 24-well culture plates at 6 x 104 cells/well and treated as

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described in the figure legend. Total RNA extraction was carried out with TRIzol reagent

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(Invitrogen, Karlsruhe, Germany) and RNA quantity and quality were evaluated as recently

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published [26]. The cDNA was generated from an aliquot of the total RNA using M-MuLV

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Reverse Transcriptase (Thermo Fisher Scientific St. Leon-Rot, Germany), dT18 primer

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(Eurofins MWG Operon, Ebersberg, Germany), dNTP mix (GeneCraft, Lüdinghausen,

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Germany) and a Reverse Transcriptase reaction buffer (Thermo Fisher Scientific) as described

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recently [27]. qPCR was performed with KAPA SYBR FAST qPCR Mastermix (Peqlab,

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Erlangen, Germany) and a Rotor-Gene Q device (Qiagen, Hilden, Germany) as in our recent

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publication [26]. Gene-specific primer pairs for target genes (NIS, SREBF1, SREBF2, LDLR,

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HMGCR, RARA, RARG, RXRA, PPARG) and reference genes (ATP5B, CYC1, EIF4A2) and

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their characteristics are listed in Table 1. Normalization was carried out using the procedure

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from Vandesompele et al. [28] based on determination of multiple potential reference genes as

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described recently [26]. In the present study, the normalization factor was calculated from the

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abovementioned references genes.

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Immunoblotting. For Immunoblotting of NIS, MCF-7 cells were seeded in 6-well culture

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plates at 1.8 x 105 cells/well and incubated as indicated in the figure legend. Following

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treatment, total protein was isolated using RIPA lysis buffer as described in detail [26].

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Determination of protein concentration, protein separation, protein transfer onto membranes,

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blocking of membranes, incubation of membranes with primary [anti-rabbit NIS (1:2000),

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which was kindly provided by Nancy Carrasco [29]; anti-mouse β-actin (1:40000), Abcam,

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Cambridge, UK; anti-rabbit PPARγ (1:1000), Upstate Biotechnologies/Millipore, Schwalbach,

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Germany] and secondary antibodies [anti-rabbit IgG (1:10000), Sigma-Aldrich; anti-mouse 6 ACS Paragon Plus Environment

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IgG (1:10000), Santa Cruz Biotechnology, Heidelberg, Germany], developing of blots and

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quantification were performed as in our recent publication [26].

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Plasmids. A human NIS promoter reporter plasmid (pGL4.10-hNISp-2060) containing a

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2060 bp hNIS promoter fragment (from -1710 to +350 relative to transcription start site) was

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generated based on published sequences (cDNA: accession no. NM_000453; genomic DNA:

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NC005796; NCBI GenBank) by PCR amplification from a BAC clone RP11–343E23

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(BACPAC Resources, Oakland, CA). The following primer pair (forward, reverse) was as used:

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5’-TCGACAGGAGGTACGCTCCAGCC-3’, 5’-AGCTCATGAGGGCGGGTGCGGAG-3’).

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The PCR fragment obtained, which contained XhoI and HindIII restriction sites at the 5’ ends

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(underlined), was subcloned in front of a luciferase reporter gene into a pGL4.10 [luc2] vector

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(Promega, Mannheim, Germany), which was digested with XhoI and HindIII. A human PPARγ

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expression plasmid (pCMX-hPPARγ) was generated by PCR amplification from MCF-7

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cDNA.

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ATAGGTACCATGACCATGGTTGACACAGAG-3’,

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ATACCCGGGCTAGTACAAGTCCTTGTAGATC-3’. The PCR fragment obtained, which

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contained KpnI and SmaI restriction sites at the 5’ ends (underlined), was subcloned into a

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pCMX vector, which was digested with KpnI and SmaI. To confirm the integrity of NIS

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promoter plasmid and PPARγ expression plasmid, the plasmids were sequenced. The pCMX-

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mRXRα plasmid expressing mouse RXRα was obtained from R. M. Evans from the Salk

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Institute for Biological Studies (San Diego, CA).

The

following

primer

pair

(forward,

reverse)

was

used:

5’5’-

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PPARγ overexpression. MCF-7 cells were seeded in 6-well plates at 1.8 x 105 cells/well

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and transiently transfected with 1 µg of pCMX-hPPARγ or empty plasmid (pCMX). FuGENE

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6 (Roche Diagnostics, Mannheim, Germany) was used as transfection reagent. Following

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transfection, cells were incubated in growth medium for 24 h. Subsequently, total protein was

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extracted and immunoblotting was performed as above-described.



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Transient transfection and dual luciferase assay. For transient transfection, MCF-7 cells

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were seeded in 96-well plates at 1.2 x 104 cells/well and transiently transfected with 50 ng of

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either NIS promoter reporter construct (pGL4.10-hNISp-2060) or empty plasmid (pGL4.10

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[luc2]) and 5 ng of internal control vector (pGL4.74 [luc2], Promega) encoding Renilla

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luciferase. FuGENE 6 was used as transfection reagent. To test the effect of PPARγ/RXRα

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expression, MCF-7 cells were also co-transfected with 50 ng of either PPARγ and RXRα

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expression plasmids or empty expression plasmid (pCMX). After 12 h, cells were either

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incubated for 12 h with growth medium (PPARγ/RXRα expression experiment) or treated with

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ATRA alone or ATRA and PPARγ ligand troglitazone (TGZ) as described in the figure legend.

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After incubation, cells were washed and lysed and dual luciferase assay (DLR) was carried out

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as published recently [8]. Luciferase activities were normalized by dividing luciferase activity

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of NIS promoter by that of Renilla.

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Statistical analysis. All data shown are means and SD which were calculated from all

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replicates for the same treatments of all independent experiments. In each independent

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experiment, all treatments were represented in 1-8 wells (= technical replicates per treatment:

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immunoblotting and fatty acid composition, one; qPCR, four; DLR assay, six to eight; MTT

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assay, eight) depending on the plate format. Minitab statistical software (Rel. 13.0, State

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College, PA, USA) was used for statistical analyses. Data from qPCR, DLR assay and MTT

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assay were subjected to 2-factorial ANOVA with classification factors being treatment (T),

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experiment (E) and the interaction of both factors (T x E). Because data from immunoblotting

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and fatty acid composition included only one replicate per treatment within each independent

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experiment, treatment effects were analyzed by 1-factorial ANOVA. In case of statistically

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significant F values, the means of the treatment groups were compared using the Fisher´s

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multiple range test. Effects were considered significant if P < 0.05.

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RESULTS AND DISCUSSION 8 ACS Paragon Plus Environment

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c9t11-CLA and t10c12-CLA but not LA decrease ATRA-induced mRNA expression

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of NIS in MCF-7 cells. To study the effect of CLA isomers on expression of NIS induced by

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ATRA, MCF-7 cells were pre-treated with ATRA (1 µM) and subsequently treated either

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without (control) or with 100 µM of CLA isomers or LA in the presence of ATRA. The ATRA

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concentration of 1 µM was chosen based on recent dose-response experiments (0.25, 0.5, 1, 2,

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5 and 10 µM ATRA) demonstrating that this concentration does not reduce MCF-7 cell viability

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[8]. NIS mRNA was markedly induced by approx. 20-fold by treatment with ATRA alone

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which agrees with the known effect of ATRA to stimulate NIS gene transcription in MCF-7

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cells [8, 21, 30]. Both CLA isomers decreased ATRA-induced NIS expression by approx. 20%

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(Fig. 1). In contrast, LA did not affect expression of NIS induced by ATRA indicating that this

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effect was a specific effect of CLA isomers. In line with our recent study [8], treatment with 5

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µM 25HC decreased ATRA-induced expression of NIS by 40% (Fig. 1). The 25HC

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concentration of 5 µM was chosen based on recent dose-response experiments (2.5, 5, 10, 15,

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25 and 50 µM 25HC) showing that this concentration does not impair MCF-7 cell viability [8].

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In order to ensure that inhibition of NIS expression by CLA isomers was not the

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consequence of an impairment of cell viability, an MTT assay was carried out. As illustrated in

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Fig. 2A+B, viability of MCF-7 cells was not impaired by 24 h-pre-treatment with ATRA and

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subsequent 24 h-treatment with increasing concentrations of c9t11-CLA (Fig. 2A) and t10c12-

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CLA (Fig. 2B) up to a concentration of 100 µM in the presence of ATRA. At 200 µM, cell

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viability was strongly reduced by t10c12-CLA, but not by c9t11-CLA. Because c9t11-CLA was

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not toxic up to a concentration of 200 µM, both CLA isomers were tested in the further

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experiments up to a concentration of 200 µM, still keeping in mind that t10c12-CLA impaired

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cell viability at 200 µM. The potential of CLA isomers to affect viability of MCF-7 cells has

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been investigated also in other studies, with however inconsistent outcomes. While 24 h-

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treatment with 5-40 µM of c9t11-CLA did not affect viability of MCF-7 cells in one study [31], 9 ACS Paragon Plus Environment


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MCF-7 cell viability was reduced by 24 h-treatment with 40 µM of both c9t11-CLA and t10c12-

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CLA in another study [32]. In a further study, both c9t11-CLA and t10c12-CLA decreased

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MCF-7 cell viability through apoptosis at a concentration 50 µM [33]. Similar as in our study,

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Amaru et al. [34] demonstrated that c9t11-CLA has no effect on the proliferation of MCF-7

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cells even at high concentrations (128 and 256 µM), whereas t10c12-CLA decreased

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proliferation of MCF-7 cells at these concentrations. Although the reason for the partially

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conflicting results between our study and those of others remain a matter of speculation, it is

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likely that slight differences in the experimental setup (e.g., different cell media, co-treatment

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with ATRA, addition of albumin, …) are causative.

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We also studied whether the CLA isomers were efficiently taken up by MCF-7 cells.

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Evaluation of the composition of fatty acids of cellular total lipids following 24 h-treatment

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with increasing CLA concentrations (in the absence of ATRA) revealed that both c9t11-CLA

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(Fig. 2C) and t10c12-CLA (Fig. 2D) were dose-dependently incorporated into MCF-7 total

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lipids at the expense of C16:1, C18:1c9 and C18:1c11. The concentration of LA (C18:2c9c12)

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was not influenced by incubation with increasing concentrations of both CLA isomers. Similar

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results with regard to CLA incorporation into MCF-7 cell lipids and the modulation of the

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composition of fatty acids of MCF-7 cell lipids have been reported from Amaru et al. [34].

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Incubation of MCF-7 cells with 128 µM of c9t11-CLA and t10c12-CLA caused comparable

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proportions of CLA isomers in MCF-7 cell lipids when compared to MCF-7 cells treated with

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100 µM of these two CLA isomers in our study.

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c9t11-CLA and t10c12-CLA decrease ATRA-induced protein expression of NIS and

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transcriptional activity of NIS promoter in MCF-7 cells. Further dose-response experiments

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confirmed that both c9t11-CLA (Fig. 3A) and t10c12-CLA (Fig. 3B) decrease NIS mRNA

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expression at a concentration of ≥ 100 µM. In addition, these experiments revealed that t10c12-

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CLA reduced ATRA-induced NIS mRNA level even at 50 µM indicating that t10c12-CLA was

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more potent than c9t11-CLA with regard to this effect. To clarify if the CLA effect on NIS 10 ACS Paragon Plus Environment

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mRNA expression is also translated to the protein level, western blotting was carried out

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following 24 h-pre-treatment with ATRA and subsequent 24 h-treatment with CLA isomers

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and ATRA. NIS protein expression was reduced by c9t11-CLA only at 200 µM (Fig. 3C), while

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t10c12-CLA decreased NIS protein expression at 100 and 200 µM (Fig. 3D). At 50 µM, none

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of the two CLA isomers decreased NIS protein expression. The lack of effect on protein

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expression at this concentration might be attributed to the fact that the 24 h-incubation time was

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too short to translate the moderate effect on the mRNA level at lower CLA concentrations to

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the protein level.

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To next study whether the CLA effect on expression of NIS is mediated at the

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transcriptional level, the effect of increasing concentrations of CLA isomers on ATRA-induced

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transcriptional activation of NIS promoter was investigated. Following transient transfection of

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MCF-7 cells with a 2 kb human NIS promoter reporter construct (from -1709 to +350 relative

255

to transcription start site), we determined the reporter activity in response to 24 h-treatment

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with either ATRA alone or increasing concentrations of CLA isomers in the presence of ATRA.

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As shown in Fig. 3E+F, the activity of NIS promoter was increased by ATRA alone and the

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activity of NIS promoter induced by ATRA was reduced by c9t11-CLA (at ≥ 50 µM, Fig. 3E)

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and t10c12-CLA (at ≥ 100 µM, Fig. 3F).

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Inhibition of ATRA-induced NIS expression by c9t11-CLA and t10c12-CLA does not

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involve inhibition of SREBF transcriptional activities in MCF-7 cells. To clarify if the

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inhibitory effect of CLA isomers involves an inhibition of SREBP activity, the action of fatty

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acids (each 100 µM) and 25HC (5 µM) on transcript levels of SREBF1 and SREBF2 and some

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of their target genes (SREBF1 target gene: LDLR [35], SREBF2 target gene: HMGCR [36])

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was determined. In agreement with findings from Wen et al. [8], 25HC markedly decreased

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mRNA levels of SREBF1, SREBF2 and their target genes compared to MCF-7 cells incubated

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with ATRA alone (Fig. 4A). The effect of 25HC is explained by intracellular formation of

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25HC 3-sulfate [37], which inhibits proteolytic processing of SREBF1 and 2 and thereby 11 ACS Paragon Plus Environment


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inhibits SREBF-dependent gene transcription [38]. In contrary to our hypothesis, both isomers

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of CLA increased the mRNA levels of SREBF1, SREBF2 and t10c12-CLA also the mRNA

271

levels of their target genes compared to cells treated with ATRA alone (Fig. 4A). These results

272

were also confirmed in dose-response experiments demonstrating that c9t11-CLA and t10c12-

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CLA increased mRNA levels of SREBF1, SREBF2 and their targets genes at concentrations ≥

274

100 µM (Fig. 4B) and ≥ 50 µM (Fig. 4C), respectively. Our findings in MCF-7 cells are in

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contrast to recent reports showing that t10c12-CLA specifically inhibits the transcriptional

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activity of SREBFs in different mammary epithelial cells, such as bovine MAC-T cells [19, 20,

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39], goat primary epithelial cells [40] and MCF-7 cells [33]. Although we cannot provide a

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mechanistic explanation for the lack of inhibitory effect of t10c12-CLA on expression of

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SREBF1 in our study, it is a matter of fact that the main difference between these studies and

280

our study is the presence of ATRA in the culture medium (our cell model), and ATRA had a

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consistent inhibitory effect on SREBF1 expression in our experiments. Regarding this, we

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propose that ATRA prevented the known effect of t10c12-CLA to inhibit SREBF1 expression.

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However, the mechanism beyond this phenomenon deserves future studies. ATRA is a known

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inducer of NIS gene transcription and it is required in the culture medium because NIS mRNA

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is only barely detectable in MCF-7 cells incubated without ATRA [21, 30]. ATRA-dependent

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gene transcription is mediated via binding to retinoic acid receptors (RARs). The RARs form

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heterodimers with retinoid X receptors (RXR) to regulate gene transcription through binding to

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specific cis-acting elements, called retinoic acid response elements (RAREs), which are located

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in the regulatory region of target genes [41, 42]. Alotaibi et al. [30] demonstrated that ATRA-

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induced expression of NIS in MCF-7 cells involves the interaction of several intronic RAREs

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in the NIS gene. Thus, it is likely that RAR and RXR signaling stimulated by ATRA interfered

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with the transcriptional effects of both isomers of CLA in the present study. Because CLA

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isomers increased transcriptional activities of SREBFs, which were recently reported to

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stimulate promoter activity and expression of NIS in MCF-7 cells under basal conditions (no 12 ACS Paragon Plus Environment

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ATRA) [8], the effect of isomers of CLA on gene expression of NIS induced by ATRA likely

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does not involve a SREBF-dependent mechanism. The observation that NIS gene expression

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induced by ATRA was not further stimulated by CLA isomers, despite activation of SREBFs,

298

indicates that activation of SREBFs does not amplify ATRA-stimulated promoter activity and

299

gene expression of NIS in our mammary epithelial cell model used.

300

c9t11-CLA and t10c12-CLA decrease ATRA-induced expression of PPARγ in MCF-

301

7 cells. To elucidate the role of further regulatory proteins (i.e. transcription factors) that may

302

be involved in the inhibition of ATRA-induced transcriptional activity of NIS promoter, we

303

investigated the effect of ATRA alone and the combination of ATRA and CLA isomers on

304

mRNA levels of RAR isoforms (RARA, RARG, RARB), RXRA, which acts as the main

305

heterodimerization partner of RARs, and peroxisome proliferator-activated receptor γ

306

(PPARG). A previous study revealed that incubation of MCF-7 cells with ligands of these

307

nuclear hormone receptors including ATRA but also PPARγ-specific ligand troglitazone (TGZ)

308

increases NIS mRNA expression [43, 44]. Fig. 5A+B shows that treatment with ATRA alone

309

either did not alter or even decreased the expression of RARA, RARG and RXRA. The RARB

310

isoform was only barely detectable in MCF-7 cells, which concurs with previous observations

311

[45, 46]. In addition, c9t11-CLA (Fig. 5A) and t10c12-CLA (Fig. 5B) did not decrease

312

expression of RARA, RARG and RXRA. These findings suggested that RAR isoforms and

313

RXRA are not involved in the reduction of the ATRA-induced promoter activity of NIS by

314

CLA isomers in our mammary epithelial cell model used. In contrast, the mRNA level of

315

PPARG was strongly increased by ATRA alone but decreased by c9t11-CLA at ≥ 100 µM (Fig.

316

5A) and by t10c12-CLA at ≥ 50 µM (Fig. 5B). This observation indicated that reduction of

317

PPARG expression by CLA isomers might play a role in the inhibition of ATRA-induced

318

transcriptional activity of NIS promoter. The observation that the expression of other PPAR

319

isoforms, such as PPARA and PPARD, was not induced by ATRA and not reduced by CLA

320

isomers (data not shown) suggested that the effect of isomers of CLA was specific to the 13 ACS Paragon Plus Environment


321

PPARG isoform. The molecular mechanism beyond the inhibition of PPARG expression by

322

CLA isomers in MCF-7 cells has not been further addressed in this study, but it might be caused

323

by the ability of isomers of CLA to act as a PPARγ modulator. At least in 3T3-L1 adipocytes,

324

t10c12-CLA was found to inhibit expression of PPARG and its target genes through acting as

325

a PPARγ modulator/antagonist which selectively and competitively blocks PPARγ ligand-

326

induced PPARγ activation [47]. Apart from this, several other in vitro-studies showed that CLA

327

isomers are able to act as PPARγ agonists thereby inducing specific PPARγ-dependent effects

328

[48, 49]. This indicates that isomers of CLA may exert their biological effects in a cell type-

329

dependent manner. Cell type-specific effects of isomers of CLA in this regard may attributed

330

to differences in the expression levels of PPARγ, its heterodimerization partner RXRα and the

331

presence or absence of transcriptional co-stimulators or co-repressors of the PPARγ/RXRα

332

heterodimer amongst different cell types.

333

Transcriptional activity of NIS promoter is regulated by PPARγ in MCF-7 cells. To

334

strengthen our suggestion that NIS promoter activity is regulated by CLA isomers via PPARγ,

335

we investigated whether NIS promoter activity is directly activated by PPARγ expression. For

336

this end, PPARG cDNA was subcloned into a commercial expression plasmid (pCMX) and

337

subsequently the PPARγ expression plasmid and a RXRα expression plasmid were transfected

338

together with the above NIS promoter reporter construct into our mammary epithelial cell

339

model used. Successful overexpression of PPARγ in this cell model following transfection with

340

the PPARγ expression plasmid was demonstrated by immunoblotting (Fig. 6A). Fig. 6B shows

341

that PPARγ/RXRα co-expression increased basal NIS promoter activity by approx. 60%

342

compared to cells transfected with empty expression plasmids (P < 0.05). This clearly indicated

343

direct regulation of transcriptional activity of NIS promoter by PPARγ in MCF-7 cells, despite

344

this effect was only moderate.

345

Because the abovementioned study has reported that PPARγ ligands and RAR and RXR

346

ligands can synergistically induce the mRNA level of NIS in MCF-7 cells [43], the regulation 14 ACS Paragon Plus Environment

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of ATRA-induced NIS promoter activity by PPARγ ligand TGZ was investigated next. To test

348

this, cells were transfected with the NIS promoter reporter construct and subsequently treated

349

with ATRA alone or with ATRA and TGZ for 24 h. Fig. 6C shows that combined treatment

350

with ATRA and TGZ (“TGZ”) compared with ATRA alone (“vehicle”) moderately but

351

significantly increased NIS promoter activity (P < 0.05). Our observations indicated that TGZ

352

amplifies the action of ATRA on NIS promoter activation.

353

In conclusion, the two key observations from the present study that NIS promoter

354

activity is stimulated by PPARγ expression and PPARG mRNA level is decreased by CLA

355

isomers suggest that CLA isomers exert their effect on gene expression of NIS in MCF-7 cells

356

by decreasing expression of PPARG. A decreased expression of PPARG in the mammary gland

357

has been observed in two recent studies with lactating mice and lactating goats in response to

358

feeding t10c12-CLA [50, 51] indicating that reduction of PPARG expression in mammary

359

epithelial cells is a consistent biological effect of CLA, at least of t10c12-CLA. In addition,

360

several studies have demonstrated that t10c12-CLA decreases PPARγ expression in vitro [47,

361

52]. Obviously, c9t11-CLA has the same effect as t10c12-CLA at least in MCF-7 cells, even

362

though the latter CLA isomer appeared to be more potent, because inhibition of PPARG

363

expression by t10c12-CLA occurred already at 50 µM. Despite showing that NIS promoter

364

activity is regulated by PPARγ expression being indicative of direct transcriptional regulation

365

of NIS, we cannot exclude the possibility that regulation of NIS expression by CLA isomers

366

also involves indirect regulatory mechanisms or even post-translational mechanisms. Recently,

367

it has been demonstrated that histone post-translational modifications such as lysine 27

368

trimethylation affects NIS gene expression in breast cancer cells [53]. Future studies have to

369

elucidate if such mechanisms play a role in the regulation of the NIS gene by CLA isomers.

370

Because NIS in the lactating mammary gland is essential for iodide secretion into the milk and

371

the supply of the suckling newborn with iodine required for thyroid hormone biosynthesis, the

372

herein identified inhibitory effect of CLA isomers on mammary epithelial cell NIS expression 15 ACS Paragon Plus Environment


373

may be considered critical. As far as we know, no studies have been published dealing with the

374

effect of CLA feeding on iodine transfer into the milk in any species. Thus, future studies should

375

address if CLA feeding affects iodine content in the milk, even though the extent of lowering

376

milk iodine content is probably not critical to health of the suckling newborn considering the

377

decrease of NIS expression by no more than 20 to 30%. In an attempt to address this issue, the

378

iodine content of milk from ruminants like cows and goats which received a diet rich in forages

379

and supplemented with plant oils rich in polyunsaturated fatty acids (soybean oil, linseed oil)

380

and appropriate control animals (e.g. ruminants receiving a low-forage/high-concentrate diet)

381

could be analyzed, because feeding fresh grass or plant oils/oilseed supplements enhances the

382

supply of CLA to the mammary gland and milk CLA content [13]. For instance, feeding

383

calcium salts of fatty acids from soybean oil and linseed oil to Holstein cows increased CLA

384

concentration in the milk fat by four- to six-fold to approximately 20 mg/g milk total fatty acids

385

[54]. Even greater increases in the CLA concentration of milk fat were observed when plant

386

oils were directly added to the diet of cows [55].

387

With regard to the physiological relevance of our finding, we have to critically address

388

one important limitation of our study, which is the use of a human breast adenocarcinoma cell

389

line, in which regulation of NIS is partially different from that in normal mammary epithelial

390

cells. While NIS expression in both MCF-7 cells and normal human and murine mammary

391

epithelial cells exhibits regulation by important lactogenic hormons including oxytocin and

392

prolactin [2, 7, 56], differences exist between normal and neoplastic mammary epithelial cells

393

with regard to stimulation of expression of NIS and iodide uptake in response to retinoic acid

394

(RA), which is the case only in neoplastic mammary epithelial cells [21]. Also, differences exist

395

between normal breast tissue and breast cancer tissue regarding the abundance of NIS

396

expression being higher in cancer tissues (both in estrogen receptor (ER)-negative and ER-

397

positive) than in adjacent non-cancer tissues [57]. Moreover, RA markedly induces endogenous

398

expression of NIS in different malignant cells, especially in breast cancer cells being positive 16 ACS Paragon Plus Environment

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399

for ER such as MCF-7 cells, but not in cells being negative for ER which exhibit very poor NIS

400

expression [57]. Any studies about NIS gene regulation in bovine mammary epithelial cells or

401

bovine mammary gland are completely lacking, but we propose at least a similar regulation of

402

NIS expression by lactogenic hormones amongst different mammalian species. Despite the

403

abovementioned differences between MCF-7 cells and normal mammary epithelial cells, we

404

utilized the MCF-7 cell line, because this cell line is a widely used mammary epithelial cell

405

model to investigate the regulation of mammary gland NIS [21], whereas different available

406

bovine mammary epithelial cell lines such as BMEG + HM, HH2a, ET-C and Mac-T [58-61],

407

have not been characterized yet for iodide uptake and/or NIS expression. However, in view of

408

specific differences between MCF-7 and normal mammary epithelial cells, future studies have

409

to show that regulation of mammary gland NIS by CLA isomers is also the case in the lactating

410

bovine mammary epithelium.



411

Abbreviations used

412

ATRA, all-trans retinoic acid; CLA, conjugated linoleic acids; HMGCR, 3-hydroxy-3-

413

methylglutaryl-CoA reductase; LA, linoleic acid; LDLR, low density lipoprotein receptor; NIS,

414

sodium/iodide symporter; PPARG, peroxisome proliferator activated receptor gamma; RARA,

415

retinoic acid receptor alpha; RARG, retinoic acid receptor gamma; RXRA, retinoid X receptor

416

alpha; SREBF, sterol regulatory element binding transcription factor; TGZ, troglitazone; 25HC,

417

25-hydroxycholesterol.


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418

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Histone post-translational modifications induced by histone deacetylase inhibition in

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transcriptional control units of NIS gene. Mol. Biol. Rep. 2014, 41, 5257-5265.

588

(54) Chouinard, P. Y.; Corneau, L.; Butler, W. R.; Chilliard, Y.; Drackley, J. K.; Bauman, D.

589

E. Effect of dietary lipid source on conjugated linoleic acid concentrations in milk fat. J.

590

Dairy Sci. 2001, 84, 680-690.

591

(55) Dhiman, T. R.; Satter, L. D.; Pariza, M. W.; Galli, M. P.; Albright, K.; Tolosa, M.X.

592

Conjugated linoleic acid (CLA) content of milk from cows offered diets rich in linoleic

593

and linolenic acid. J. Dairy Sci. 2000, 83, 1016-1027. 25 ACS Paragon Plus Environment


594

(56) Arturi, F.; Ferretti, E.; Presta, I.; Mattei, T.; Scipioni, A.; Scarpelli, D.; Bruno, R.;

595

Lacroix, L.; Tosi, E.; Gulino, A.; Russo, D.; Filetti, S. Regulation of iodide uptake and

596

sodium/iodide symporter expression in the MCF-7 human breast cancer cell line. J. Clin.

597

Endocrinol. Metab. 2005, 90, 2321-236.

598

(57) Yao, C.; Pan, Y.; Li, Y.; Xu, X.; Lin, Y.; Wang, W.; Wang, S. Effect of sodium/iodide

599

symporter (NIS)-mediated radioiodine therapy on estrogen receptor-negative breast

600

cancer. Oncol. Rep. 2015, 34, 59-66.

601

(58) Schmid, E.; Franke, W. W.; Grund, C.; Schiller, D. L.; Kolb, H.; Paweletz, N. An

602

epithelial cell line with elongated myoid morphology derived from bovine mammary

603

gland. Expression of cytokeratins and desmosomal plaque proteins in unusual arrays. Exp.

604

Cell. Res. 1983, 146, 309-328.

605

(59) Huynh, H.; Pollak, M. HH2A, an immortalized bovine mammary epithelial cell line,

606

expresses the gene encoding mammary derived growth inhibitor (MDGI). In Vitro Cell.

607

Dev. Biol. Anim. 1995, 31, 25-29.

608

(60) Zavizion, B.; van Duffelen, M.; Schaeffer, W.; Politis, I. Establishment and

609

characterization of a bovine mammary epithelial cell line with unique properties. In Vitro

610

Cell. Dev. Biol. Anim. 1996, 32, 138-148.

611

(61) Huynh, H. T.; Robitaille, G.; Turner, J. D. Establishment of bovine mammary epithelial

612

cells (MAC-T): an in vitro model for bovine lactation. Exp. Cell. Res. 1991, 197, 191-

613

199.

614


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615


Figure legends

616 617

Figure 1

618

Effect of ATRA alone and ATRA in combination with different fatty acids or 25HC on

619

relative mRNA level of NIS in MCF-7 cells. MCF-7 cells were incubated in growth medium

620

(DMEM with 10% FBS) with either vehicle alone (DMSO) or 1 µM all-trans-retinoic acid

621

(ATRA dissolved in DMSO) for 24 h. Both treatments contained the same vehicle (DMSO)

622

concentration (0.1% v/v). Subsequently, cells were incubated in DMEM with 1% FBS with

623

either vehicle alone (DMSO and ethanol), 1 µM ATRA or 1 µM ATRA and 100 µM of either

624

LA, c9t11-CLA or t10c12-CLA (each dissolved in ethanol) or 1 µM ATRA and 5 µM 25HC

625

(dissolved in DMSO) for additional 24 h. Ethanol was added to vehicle control cells and cells

626

treated with ATRA alone or ATRA and 25HC to reach the same vehicle concentration in all

627

treatments (0.1% DMSO v/v, 0.1% ethanol v/v). Bars represent relative mRNA levels expressed

628

as fold of ATRA alone and are means ± SD from three independent experiments. a,b,cBars with

629

unlike letters are significantly different (P < 0.05). 2-factorial ANOVA classification factors:

630

treatment (T), experiment (E), interaction (T x E).

631 632

Figure 2

633

Effect of ATRA alone and ATRA in combination with different fatty acids on viability of

634

MCF-7 cells and incorporation of CLA isomers in MCF-7 total lipids. For cell viability

635

testing, MCF-7 cells were incubated in DMEM with 1% FBS with either vehicle alone (DMSO

636

and ethanol), 1 µM all-trans-retinoic acid (ATRA dissolved in DMSO) or 1 µM ATRA and

637

different concentrations (10, 50, 100 and 200 µM) of either c9t11-CLA or t10c12-CLA (each

638

dissolved in ethanol). Ethanol was added to vehicle control cells and cells treated with ATRA

639

alone or ATRA and CLA isomers to reach the same vehicle concentration in all treatments

640

(0.1% DMSO v/v, 0.2% ethanol v/v). For fatty acid incorporation studies, MCF-7 cells were 27 ACS Paragon Plus Environment


641

incubated in DMEM with 1% FBS with either vehicle alone (ethanol) or different

642

concentrations (10, 50, 100 and 200 µM) of either c9t11-CLA or t10c12-CLA (each dissolved

643

in ethanol). Ethanol was added to vehicle control cells and cells treated with CLA isomers to

644

reach the same vehicle concentration in all treatments (0.2% ethanol v/v). A, B: Bars represent

645

relative cell viability expressed as percent of ATRA alone and are means ± SD from three

646

independent experiments. C, D: Bars represent means ± SD from three independent

647

experiments. A-D: Bars with unlike letters are significantly different (P < 0.05). A, B: 2-

648

factorial ANOVA classification factors: treatment (T), experiment (E), interaction (T x E).

649 650

Figure 3

651

Effect of ATRA alone and ATRA in combination with increasing concentrations of CLA

652

isomers and NIS expression and NIS promoter activity in MCF-7 cells. MCF-7 cells were

653

incubated in growth medium (DMEM with 10% FBS) with either vehicle alone (DMSO) or 1

654

µM all-trans-retinoic acid (ATRA dissolved in DMSO) for 24 h. Both treatments contained the

655

same vehicle (DMSO) concentration (0.1% v/v). Subsequently, cells were incubated in DMEM

656

with 1% FBS with either vehicle alone (DMSO and ethanol), 1 µM all-trans-retinoic acid

657

(ATRA dissolved in DMSO) or 1 µM ATRA and different concentrations (10, 50, 100 and 200

658

µM) of either c9t11-CLA or t10c12-CLA (each dissolved in ethanol). Ethanol was added to

659

vehicle control cells and cells treated with ATRA alone or ATRA and CLA isomers to reach

660

the same vehicle concentration in all treatments (0.1% DMSO v/v, 0.2% ethanol v/v). Bars

661

represent relative mRNA levels (A, B), relative protein levels (C, D) and relative luciferase

662

activities (E, F) expressed as fold of ATRA alone and are means ± SD from three independent

663

experiments. C, D: One representative immunoblot for NIS and β-Actin is shown. A-F: Bars

664

with unlike letters are significantly different (P < 0.05). A, B, E, F: 2-factorial ANOVA

665

classification factors: treatment (T), experiment (E), interaction (T x E).

666


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667

Figure 4

668

Effect of ATRA alone and ATRA in combination with different fatty acids or 25HC on

669

relative mRNA levels of SREBFs and their target genes in MCF-7 cells. A: MCF-7 cells

670

were incubated identically as in Figure 1. B, C: MCF-7 cells were incubated identically as in

671

Figure 3. Bars represent relative mRNA levels expressed as fold of ATRA alone and are means

672

± SD from three independent experiments. A-C: Bars with unlike letters are significantly

673

different (P < 0.05). 2-factorial ANOVA classification factors: treatment (T), experiment (E),

674

interaction (T x E).

675 676

Figure 5

677

Effect of ATRA alone and ATRA in combination with increasing concentrations of CLA

678

isomers on relative mRNA levels of specific nuclear hormone receptors in MCF-7 cells. A,

679

B: MCF-7 cells were incubated identically as in Figure 1. B, C: MCF-7 cells were incubated

680

identically as in Figure 3. Bars represent relative mRNA levels expressed as fold of ATRA

681

alone and are means ± SD from three independent experiments. A, B: Bars with unlike letters

682

are significantly different (P < 0.05). 2-factorial ANOVA classification factors: treatment (T),

683

experiment (E), interaction (T x E).

684 685

Figure 6

686

Effect of PPARγ expression and PPARγ ligand troglitazone on NIS promoter activity in

687

MCF-7 cells. A: Protein expression of PPARγ in MCF-7 cells transiently transfected with either

688

empty plasmid (pCMX) or PPARγ expression plasmid (pCMX-hPPARγ) and subsequently

689

incubated in DMEM with 10% FBS for 24 h. Left: Bars represent data from densitometric

690

analysis of band intensities for PPARγ normalized by β-Actin. Right: One representative

691

immunoblot for PPARγ and β-Actin is shown. B: MCF-7 were transiently transfected with

692

either empty plasmid (pCMX) or expression plasmids for PPARγ (pCMX-hPPARγ) and its 29 ACS Paragon Plus Environment


693

heterodimerization partner RXRα (pCMX-mRXRα) together with the NIS promoter reporter

694

construct and Renilla plasmid pGL4.74 [luc2] for 24 h. C: MCF-7 were transiently transfected

695

with NIS promoter reporter construct and Renilla plasmid pGL4.74 [luc2] for 24 h, and

696

subsequently incubated in DMEM with 1% FBS in the presence of 1 µM ATRA and either

697

vehicle alone (DMSO) or 10 µM troglitazone (dissolved in DMSO) for 24 h. Both treatments

698

contained the same vehicle (DMSO) concentration (0.1% v/v). B, C: After cell lysis, luciferase

699

activities from NIS promoter construct and Renilla plasmid were determined by dual luciferase

700

assay and NIS luciferase activity was normalized by Renilla luciferase activity. Bars represent

701

relative normalized luciferase activities expressed as fold of control (B: empty plasmid, C:

702

vehicle) are means ± SD from at least two independent experiments. A-C: Bars with unlike

703

letters are significantly different (P < 0.05). B, C: 2-factorial ANOVA classification factors:

704

treatment (T), experiment (E), interaction (T x E).


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Page 31 of 38

705


Table 1

Chacteristics of gene specific primers used for qPCR

Gene symbol

Primer sequence (5´-3´, forward, reverse)

Product size NCBI GenBank (bp)

accession number

Reference genes ATP5B

TCGCGTGCCATTGCTGAGCT, CGTGCACGGGACACGGTCAA

218

NM_001686

CYC1

TTCGCTTCGCGGGGTAGTGTTGG, GACAAGGCCACTGCCTGAGGT

126

NM_991916

EIF4A2

GCGCAAGGTGGACTGGCTGA, GCACATCAATCCCGCGAGCC

170

NM_001967

NIS

TGCGGGACTTTGCAGTACATT, TGCAGATAATTCCGGTGGACA

133

NM_000453

SREBF1

CGGAGCCATGGATTGCACTTTC, GATGCTCAGTGGCACTGACTCTTC

328

NM_001005291

SREBF2

TAGGCAGTCTGGTGGACAATG, GTCTGGCTCATCTTTGACCTTTG

179

NM_004599

GPAM

AATGGTGAACAACTGGGCAAAC, ATCCACTCGGACACAACCATAG

105

NM_001244949

HMGCR

GACAGGATGCAGCACAGAATG, TTGAACACCTAGCATCTGCAAAC

179

NM_000859

LDLR

GTCAGCTCCACAGCCGTAAG, CCCAGAGCTTGGTGAGACATTG

128

NM_000527

PPARG

GCAGGAGCAGAGCAAAGAGGTG, AAATATTGCCAAGTCGCTGTCATC

352

XM_011533842

RARA

ACCCCCTCTACCCCGCATCTACAAG, CATGCCCACTTCAAAGCACTTCTGC

226

XM_005257552

RARG

TACCACTATGGGGTCAGC, CCGGTCATTTCGCACAGCT

195

NM_001243730

RXRA

TTCGCTAAGCTCTTGCTC, ATAAGGAAGGTGTCAATGGG

113

NM_001291921

Target genes




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Conjugated linoleic acid

Na+ I-

• c9t11 (100-200 µM) • t10c12 (50-200 µM)

Sodium/iodide symporter • promoter activity • mRNA • protein

Na+ I-

Page 38 of 38

Peroxisome proliferator-activated receptor γ mRNA

Human mammary epithelial cell line MCF-7

ACS Paragon Plus Environment

Conjugated linoleic acid isomers decrease all-trans-retinoic acid

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