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Lutein Activates the Transcription Factor Nrf2 in Human Retinal Pigment Epithelial Cells Katja Frede, Franziska Ebert, Anna Kipp, Tanja Schwerdtle, and Susanne Baldermann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01929 • Publication Date (Web): 30 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017
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Journal of Agricultural and Food Chemistry
Title: Lutein Activates the Transcription Factor Nrf2 in Human Retinal Pigment Epithelial Cells
Short title: Nrf2 Activation in ARPE-19 Cells by Lutein
Authors: Katja Fredeab*, (
[email protected]); Franziska Ebertb, (
[email protected]); Anna P. Kippc, (
[email protected]); Tanja Schwerdtleb, (
[email protected]); Susanne Baldermannab, (
[email protected])
Affiliations: a
Leibniz-Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Plant Quality and
Food Security, Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany b
University of Potsdam, Institute of Nutritional Science, Department of Food Chemistry, Arthur-
Scheunert-Allee 114-116, 14558 Nuthetal, Germany c
Friedrich Schiller University Jena, Institute of Nutrition, Dornburger Straße 24, 07743 Jena,
Germany
* Corresponding author: Katja Frede - Leibniz-Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Plant Quality and Food Security, Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany. Tel.: +49 (0) 33701-78313; Fax. +49 (0) 33701-55391; Email:
[email protected] 1 ACS Paragon Plus Environment
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Abstract
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The degeneration of the retinal pigment epithelium caused by oxidative damage is a stage of
3
development in age-related macular degeneration (AMD). The carotenoid lutein is a major
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macular pigment that may reduce the incidence and progression of AMD, but the underlying
5
mechanism is currently still not fully understood. Carotenoids are known to be direct
6
antioxidants. However, carotenoids can also activate cellular pathways resulting in indirect
7
antioxidant effects. Here, we investigate the influence of lutein on the activation of nuclear factor
8
erythroid 2-related factor 2 (Nrf2) target genes in human retinal pigment epithelial cells (ARPE-
9
19 cells) using lutein-loaded Tween40 micelles. The micelles were identified as a suitable
10
delivery system since they were non-toxic in APRE-19 cells up to 0.04% Tween40 and led to a
11
cellular lutein accumulation of 62 µM ± 14 µM after 24 h. Lutein significantly enhanced Nrf2
12
translocation to the nucleus 1.5 ± 0.4-fold compared to unloaded micelles after 4 h. Furthermore,
13
lutein treatment for 24 h significantly increased the transcripts of NAD(P)H:quinone
14
oxidoreductase 1 (NQO1) by 1.7 ± 0.1-fold, glutamate-cysteine ligase regulatory subunit
15
(GCLm) by 1.4 ± 0.1-fold, and heme oxygenase-1 (HO-1) by 1.8 ± 0.3-fold. Moreover, we
16
observed a significant enhancement of NQO1 activity by 1.2 ± 0.1-fold. Collectively, this study
17
indicates that lutein not only serves as a direct antioxidant, but also activates Nrf2 in ARPE-19
18
cells.
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Keywords: lutein, Nrf2, ARPE-19 cells, AMD, Tween40 micelles
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Introduction
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Carotenoids are natural yellow, orange, and red pigments that are synthesized by plants, algae,
27
bacteria, and fungi.1 In plants, they are required for photosynthesis, are attractants for pollinators
28
and seed dispersers, and serve as substrates in the biosynthesis of carotenoid-derived
29
phytohormones and plant volatile scents.1 Animals and humans cannot synthesize carotenoids,
30
and therefore, they can only obtain these compounds from their diet.1 Carotenoids are of special
31
interest because they promote health in animals and humans since they have the potential to
32
protect against cardiovascular diseases, certain types of cancer, eye-related diseases, and light-
33
induced skin damage.2
34
For the carotenoid lutein, there is evidence that it reduces the incidence and progression of age-
35
related macular degeneration (AMD).3-5 AMD is an age-related, progressive degeneration of
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photoreceptors in the macular region of the retina,6 which is becoming a global burden.7 In detail,
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it is estimated that the number of people with AMD will be 196 million in 2020 and 288 million
38
in 2040. Therefore, preventing the development of this disease is of great interest. The
39
carotenoids lutein, zeaxanthin, and meso-zeaxanthin are the only carotenoids found in the macula
40
of the retina. These macular carotenoids are important for optimal visual performance because of
41
their ability to filter blue light and consequently, they can attenuate both chromatic aberration and
42
light scatter.8 Moreover, these carotenoids protect against AMD by reducing oxidative stress in
43
the retina via different mechanisms. For example, the above-mentioned filter ability decreases the
44
amount of energy-rich light reaching the photoreceptors.2 Furthermore, carotenoids serve as
45
direct antioxidants and can activate cellular pathways resulting in indirect antioxidant effects.9
46
The cellular pathways activated by lutein remain to be fully determined to advance our current
47
understanding of the cellular signalling function of lutein in the eye.
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The initial pathogenesis of AMD involves the degeneration of the underlying retinal pigment
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epithelium (RPE) caused by oxidative damage.6 Moreover, nuclear factor erythroid 2-related
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factor 2 (Nrf2) signaling is impaired in the aging RPE, thereby rendering it more vulnerable to
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oxidative stress.10 The transcription factor Nrf2 regulates genes encoding antioxidative and phase
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II enzymes that are involved in the maintenance of the cellular redox status as well as in the
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detoxification of xenobiotics.11,12 Nrf2 activates genes by binding to antioxidant response
54
elements (AREs).13 Under unstressed conditions, the Kelch-like ECH-associated protein 1
55
(Keap1) suppresses Nrf2 transcriptional activity, which subsequently leads to the proteasomal
56
degradation of Nrf2.14,15 Upon activation of the Nrf2 pathway, Nrf2 translocates to the nucleus,
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forms a heterodimer with a small musculoaponeurotic fibrosarcoma (Maf) protein that then binds
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to the ARE and activates genes under the control of the AREs.13,14 Enzymes regulated by Nrf2
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include - amongst others - NAD(P)H:quinone oxidoreductase 1 (NQO1), a quinone reductase that
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promotes obligatory 2-electron reductions,16 glutamate-cysteine ligase regulatory subunit
61
(GCLm), a subunit of GCL catalyzing the first, rate limiting step of glutathione synthesis,17 and
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heme oxygenase-1 (HO-1), an enzyme degrading heme to biliverdin.18 Several phytochemicals
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can increase Nrf2 target genes in RPE cells, e.g. the isothiocyanate sulforaphane, saponins, and
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phenolic compounds.19-24 Also carotenoids can activate the Nrf2 pathway in RPE cells. For
65
example, zeaxanthin and astaxanthin both lead to an activation of Nrf2 in ARPE-19 cells.9,25,26
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However, the exact role of lutein relating to Nrf2 in the RPE still remains to be determined. It is
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important to separately evaluate different carotenoids in different cell lines because the degree of
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Nrf2 activation varies. For instance, zeaxanthin activates Nrf2 more strongly than astaxanthin in
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the ARPE-19 cells.25,26 On the one hand, lutein activates the Nrf2 pathway in mouse microglial
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BV-2 cells27 and in mice liver.28 On the other hand, lutein induces antioxidative enzymes in a
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neuronal cell line (PC12D) without activating the Nrf2 pathway.29 Since current data support a 4 ACS Paragon Plus Environment
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preventive role of lutein against AMD,3-5 it is of high interest to increase macular carotenoids by
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a higher dietary uptake of lutein using lutein-rich foods or lutein supplements. Clarifying the
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protective function of lutein in the eye is important to support the need of this lutein-rich diet.
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Thus, the aim of this study is to investigate for the first time whether lutein activates the Nrf2
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pathway in ARPE-19 cells, thereby leading to the subsequent expression of Nrf2 target genes.
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Materials and Methods
79 80
Chemicals
81
Lutein was isolated from Xanthophyll pastös (Tagetes erecta oleoresin, Roth, Karlsruhe,
82
Germany) using column chromatography, followed by saponification with ethanolic KOH,
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extraction with diethyl ether and precipitation from methanol. Purity and identity were confirmed
84
by HPLC-DAD, 1H-NMR, and
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FAD (FAD-Na2*2H2O), NADP (ß-NADP-Na2), as well as neutral red and tert-butyl methyl ether
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were purchased from Roth (Karlsruhe, Germany). ARPE-19 cells were purchased from LGC
87
Standards (Teddington, UK). Dulbecco's Modified Eagle Medium (DMEM)/Ham's F-12 and L-
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glutamine were obtained from Biochrom (Berlin, Germany). Fetal calf serum (FCS) was obtained
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from PAA Laboratories (Pasching, Austria). Bovine serum albumin (BSA), dicoumarol (3,3´-
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Methylen-bis(4-hydroxycoumarin)), DMSO, EDTA, formaldehyde (36.5-38% in H2O), glucose-
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6-phosphate, glucose-6-phosphate-dehydrogenase, HEPES, hydrogen peroxide (25-35%), KCl,
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menadione,
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penicillin/streptomycin (5000 U penicillin and 5 mg streptomycin/mL), resazurin sodium salt,
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trypsin solution 10x, and Tween20 were bought from Sigma-Aldrich (St. Louis, Missouri, USA).
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The antibody anti-Nrf2 was purchased from Santa Cruz (Dallas, Texas, USA), anti-histone H2B
13
C-NMR spectroscopy (data not shown). Ammonium acetate,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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bromide,
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from Epitomics (Burlingame, California, USA), and anti-rabbit from Cell Signaling Technology
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(Danvers, Massachusetts, USA). R,S-sulforaphane was obtained from Enzo Life Sciences
98
(Farmingdale, New York, USA). The RNeasy Plus Mini Kit (50) and the RNase-free Dnase Set
99
were purchased from Qiagen (Hilden, Germany). Oligo (dT)12−18 primers and SuperScript III
100
reverse transcriptase were purchased from Thermo Fisher Scientific (Waltham, Massachusetts,
101
USA). SensiMix™ SYBR Low-ROX Kit was bought from Bioline (Luckenwalde, Germany).
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Methanol and Tween40 were purchased from Th. Geyer (Berlin, Germany). Dichloromethane,
103
HCl, n-hexane, K2HPO4, KH2PO4, Na2HPO4, phosphatase inhibitor (sodium vanadate), 2-
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propanol, protease inhibitor cocktail set III (EDTA-free), and Triton X-100 were obtained from
105
Merck (Darmstadt, Germany). Ethanol and acetic acid were bought from VWR International
106
(Radnor, Pennsylvania, USA). Tris was obtained from AppliChem (Darmstadt, Germany).
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Protein Assay Dye Reagent Concentrate and Protein Assay Standard II were purchased from Bio-
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Rad (Hercules, California, USA). Non-fat dry milk powder was bought from TSI (Zeven,
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Germany). Finally, NaCl was purchased from AppliChem (Darmstadt, Germany).
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Preparation of lutein-loaded Tween40 micelles
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The micelles were prepared in darkened tubes according to a slightly modified method previously
113
described by Lornejad-Schäfer, et al.30, which produces micelles with low cytotoxicity and good
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cellular uptake of carotenoids. A stock solution of a lutein standard of about 175 µM in
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dichloromethane was prepared, diluted in ethanol, and quantified spectrophotometrically (final
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concentration of dichloromethane ≤ 10%; specific absorption coefficient A1%,1cm = 2550 at
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445 nm in ethanol31). Aliquots were dried under a stream of N2 and stored at -80 °C until further
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use. Lutein aliquots were dissolved in dichloromethane/ethanol (1/20, v/v) to a final
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concentration of 70.5 µM and transferred with ethanol to a round bottom flask. The solvent was 6 ACS Paragon Plus Environment
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removed in a rotary evaporator. Tween40/ethanol (1/4029, v/v) was added to lutein in a ratio of
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20 µL Tween40/1 µmol lutein. After treatment in an ultrasonic bath, the solvent was evaporated
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again. Lutein was micellized by adding cell culture medium followed by sonication in an
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ultrasonic bath. The micelles were again diluted with cell culture medium before applying to the
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ARPE-19 cells. Cells were treated with concentrations ranging from 0.5 µM (0.001% Tween40)
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to 40 µM (0.08% Tween40) lutein. Finally, for the control, micelles without lutein were prepared
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in the exact same way.
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Cell culture
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The human retinal pigment epithelial cell line ARPE-19 (ATCC® CRL2302™) is a suitable
130
model for RPE cells because it expresses typical RPE markers such as cellular retinaldehyde
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binding protein (CRALBP ) and RPE-specific 65 kDa protein (RPE65).32 Cells were cultivated in
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25 cm2 cell culture flasks (passages 8-32) and were maintained at 37 °C and 5% CO2 in 100%
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humidified air. The culture medium consisted of DMEM/Ham's F-12, 10% (v/v) FCS, 100 U/mL
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penicillin, 0.1 mg/mL streptomycin, and 2 mM L-glutamine. Cells were subcultured every 2-
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3 days. Unless otherwise stated, cells were seeded at a density of 40,000 cells/cm2 in all
136
experiments.
137 138
Neutral red- and resazurin-based cytotoxicity assays
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To determine cell viability after incubation with micelles, both a neutral red- and a resazurin-
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based assay were carried out. The reduction of resazurin to the red fluorescent resorufin indicates
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enzymatic and metabolic activity of cells, while the neutral red test measures lysosomal
142
integrity.33,34
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ARPE-19 cells were seeded in 96-well plates at a density of 23,300 cells/cm2 and cultivated for
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48 h. Cells were treated with different micelles concentrations with and without lutein for 24 h.
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The neutral red-based assay was performed as described previously with neutral red-containing
146
medium at a concentration of 230 µM.35 For the resazurin based-assay, cells were incubated with
147
resazurin-containing medium at a concentration of 40 µM for 3 h at 37 °C. Fluorescence was
148
determined in a plate reader (Infinite M200 Pro, Tecan Group Ltd., Zürich, Switzerland;
149
excitation: 530 nm, emission: 590 nm). Values of untreated control wells were set to 100%
150
viability.
151 152
In vitro test of cellular lutein uptake
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ARPE-19 cells were cultivated for 2 days after seeding and incubated with lutein-loaded micelles
154
for 24 h. The final concentrations were 1 µM lutein (0.002% Tween40), 5 µM lutein (0.01%
155
Tween40), and 10 µM lutein (0.02% Tween40). Afterwards, the cell monolayers were washed
156
twice with warm (37 °C) PBS-A (pH 7.4, 100 mM NaCl, 4.5 mM KCl, 7 mM Na2HPO4, 3 mM
157
KH2PO4). Cells were detached with 2 mL trypsin, suspended in 5 mL cold FCS/PBS-A (5/95,
158
v/v), and put on ice. Cell number and cell volume were determined (CASY Model TTC 150 µm,
159
Roche Diagnostics, Mannheim, Germany). The cell suspension was centrifuged (4 °C, 5 min,
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314 x g), resuspended in 1 mL cold PBS-A, and centrifuged again (4 °C, 5 min, 2380 x g). The
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cell pellet was stored at -80 °C under N2 until analysis of lutein content.
162 163
LC-QToF-MS analysis of lutein
164
The lutein from the cell pellets was extracted by adding 250 µL water, placing in an ultrasonic
165
bath for 5 min, adding 500 µL ethanol and 700 µL n-hexane, and then shaking for 5 min at
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1400 rpm (modified after Schweigert, et al.36). After centrifugation for 10 min at 4000 x g the 8 ACS Paragon Plus Environment
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supernatant was transferred into a new tube. The extraction step was repeated twice using 700 µL
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n-hexane. After concentrating the combined supernatant under N2 to dryness, it was re-dissolved
169
in 20 μL dichloromethane and 80 µL 2-propanol. The solution was filtered (0.2 μm, PTFE) and
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kept at 10 °C in the autosampler. Lutein content was analyzed as previously described.37
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Separation was performed on a C30-column (YMC Co. Ltd., Kyoto, Japan, YMC C30, 100 x
172
2.1 mm, 3 µm) on an Agilent Technologies 1290 Infinity UHPLC. The column temperature was
173
maintained at 20 °C. The mobile phases were (A) methanol//water (96/4, v/v) and (B)
174
methanol/tert-butyl methyl ether/water (6/90/4, v/v/v). To increase the ionization, 20 mM
175
ammonium acetate was added to the mobile phases. The flow rate was 0.2 mL/min. Elution was
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carried out with the following gradient: 100% A for 10 min, 100% A to 80% A in 7 min, 80% A
177
for 28 min, 80% A to 0% A in 10 min, and 0% A to 100% A in 2 min. The samples were
178
analyzed on an Agilent Technologies 6530 QTOF LC/MS equipped with an APCI ion source in
179
positive ionization mode. The gas temperature was set to 300 °C at a flow rate of 8 L/min, the
180
vaporizer to 350 °C, and the nebulizer pressure to 35 psig. The voltage was set to 3500 V and a
181
fragmentor voltage of 175 V was applied at a corona current of 4 µA. A lutein standard was
182
prepared and the concentration was determined spectrophotometrically as aforementioned in the
183
section micelles preparation. The lutein standard was used for external calibration. Lutein
184
quantification was performed at a detection wavelength of 450 nm.
185 186
Western blot analysis of nuclear Nrf2 protein levels
187
Cells were cultivated for 68 h before stimulation with 10 µM lutein (0.02% Tween40), unloaded
188
micelles (0.02% Tween40), 10 µM sulforaphane (positive control for Nrf2 activation), or water
189
for 4 h. Cells were washed twice with PBS-B (pH 7.4, 140 mM NaCl, 10 mM Na2HPO4, 3 mM
190
KH2PO4), harvested with PBS-B, and centrifuged at 250 x g for 5 min at 4 °C. The supernatant 9 ACS Paragon Plus Environment
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was discarded. Cell pellets were resuspended in 350 µL hypotonic homogenization buffer
192
(20 mM HEPES, 1 mM EDTA, pH 7.5, containing 1 µL/mL protease inhibitor cocktail set III,
193
EDTA-free, and 2 µL/mL phosphatase inhibitor (sodium vanadate)). After homogenization with
194
a Potter homogenizer (Potter S, Typ 8533024, B. Braun Biotech International, Melsungen,
195
Germany), the lysate was centrifuged at 720 x g for 15 min at 4 °C. The precipitated nuclei were
196
washed in 200 µL homogenization buffer, resuspended in 100 µL homogenization buffer, and
197
sonicated. The nuclear extract was centrifuged at 20,000 x g for 15 min at 4 °C. The protein
198
content of the supernatant was determined according to Bradford.38
199
Samples (10 µg protein) were separated on a gradient SDS-PAGE gel (7.5-15%) and transferred
200
to nitrocellulose (2 h, 1.2 mA/cm2, 4 °C). Blots were blocked in 5% non-fat dry milk in Tris-
201
buffered saline with 0.1% Tween20 at room temperature for 1 h. Western blot analysis was
202
performed with a rabbit-anti-human-Nrf2 antibody (1:1000; Santa Cruz, sc-13032) and a rabbit-
203
anti-human-histone-H2B antibody (1:15000; Epitomics, 1810-1). A horseradish peroxidase-
204
conjugated anti-rabbit antibody (1:2000) was used as secondary antibody. Bands were visualized
205
by chemiluminescence imaging (Intelligent Dark Box LAS 3000, FUJIFILM, Tokyo, Japan). The
206
relevant band for Nrf2 is at 95-110 kDa.39
207 208
RT-qPCR analysis of Nrf2 target gene transcripts
209
The cells were cultivated for 48 h or 68 h after seeding. The incubation with lutein-loaded
210
micelles for 24 h (long-term effect) or 4 h (short-term effect) was performed with 10 µM lutein
211
(0.02% Tween40). Furthermore, cells were treated with micelles without lutein as control. Cells,
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which were grown for 48 h and treated with 10 µM sulforaphane for 24 h, served as positive
213
control for Nrf2 pathway activation. The control cells for the sulforaphane treatment were
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incubated with water. Cells were harvested as described in the section ‘In vitro test of cellular
215
lutein uptake’.
216
RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) as described by
217
the
218
spectrophotometrically at 260 nm (Nanodrop ND1000, Thermo Fisher Scientific, Waltham,
219
Massachusetts, USA). The purity was checked using the ratios of absorbance 260/280 and
220
260/230. cDNA was synthesized from 1 µg total RNA using SuperScript III reverse transcriptase
221
(Thermo Fisher Scientific, Waltham, Massachusetts, USA) with oligo (dT)12−18 primers according
222
to the manufacturer’s instructions. Each transcript quantification was carried out in triplicate. The
223
reaction mix per well consisted of 3 µL cDNA (diluted 1:10), 5 µL 2x SensiMix™ SYBR Low-
224
ROX, and 2 µL 2 µM primer (forward and reverse primer). The RT-qPCR was carried out in 96-
225
well reaction plates on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.,
226
Hercules, California, USA). The thermal cycling conditions were as follows: 50 °C for 2 min,
227
95 °C for 10 min, 39 cycles of 95 °C for 15 s, and 60 °C for 1 min. Afterwards, a melt curve
228
analysis was performed. The amplification efficiency (E) for each primer pair was determined
229
using a cDNA dilution series and used for calculation of gene expression. The expression was
230
calculated as n-fold induction of the gene of interest in treated compared to control cells by the
231
∆∆Cq method using the geometric mean of three reference genes.40,41 Reference genes were beta-
232
actin (βAct), hypoxanthine phosphoribosyl transferase 1 (HPRT1) and peptidylprolyl
233
isomerase A (PPIA). No-template controls were included. Primers and amplification efficiencies
234
were
235
CCTTAGGGCAGGTAGATTCAG
236
ATGTTGGGATACTGTGGGCTCTGG (forward) and CCCTTTCTGGCTTTTAGTCCTTCCT
237
(reverse),
manufacturer,
as
including
follows:
E
=
on-column
NQO1
100%;
HO-1
-
DNase
I
digestion.
RNA
TGGCTAGGTATCATTCAACTC (reverse),25
-
E
=
100%;
GCCAGCAACAAAGTGCAAGAT 11
ACS Paragon Plus Environment
was
quantified
(forward) GCLm
(forward)
and -
and
Journal of Agricultural and Food Chemistry
(reverse),25
GGTAAGGAAGCCAGCCAAGAG
239
CCACACCTTCTACAATGAGC (forward) and GGTCTCAAACATGATCTGGG (reverse)25,
240
E = 96%;
241
CCTGACCAAGGAAAGCAAAG
242
AGACAAGGTCCCAAAGAC (forward) and ACCACCCTGACACATAAA (reverse),42 E =
243
100%.
-
=
100%;
βAct
238
HPRT1
E
Page 12 of 37
GACCAGTCAACAGGGGACAT (reverse),42
E
=
97%;
(forward) and
PPIA
-
and -
244 245
Measurement of NQO1 activity
246
ARPE-19 cells were seeded and cultivated for 48 h. Cells were treated with 10 µM lutein (0.02%
247
Tween40), unloaded micelles (0.02% Tween40), 10 µM sulforaphane, or water for 24 h.
248
Cells were washed twice with PBS-B (pH 7.4, 140 mM NaCl, 10 mM Na2HPO4, 3 mM
249
KH2PO4) and transferred to a new tube with 150 µL/well homogenization buffer (100 mM Tris-
250
HCl, 300 mM KCl, 0.01% Triton X-100, pH 7.6, containing 1 µL/mL protease inhibitor cocktail
251
set III, EDTA-free, and 2 µL/mL phosphatase inhibitor (sodium vanadate)). Cells were
252
homogenized with an ultrasonic homogenizer and centrifuged for 15 min at 21000 x g and 4 °C
253
before the protein contents of the supernatants were measured according to Bradford.38
254
NQO1 activity was determined as described previously.43 In principle, reduction of 3-(4,5-
255
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) mediated by menadione was
256
measured on a microtiter plate absorbance reader at 590 nm (Synergy 2, Biotek Instruments, Bad
257
Friedrichshall, Germany). For the assay, 50 µL water and 190 µL reaction buffer (25 mM Tris-
258
HCl, pH 7.4, 0.665 mg/mL BSA, 0.01% Tween20, 5 µM FAD, 1 mM glucose-6-phosphate,
259
30 µM
260
dehydrogenase) were added to 10 µL lysate per well before measurement of the kinetics in the
261
microtiter plate absorbance reader. Another plate was measured with 50 µL NQO1 inhibitor
NADP,
0.72 mM
MTT,
50 µM
menadione,
0.3 U/mL
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glucose-6-phosphate
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dicoumarol (0.3 mM dicoumarol, 0.5% DMSO, 5 mM potassium phosphate buffer (pH 7.4))
263
instead of water. NQO1 activity was calculated as the difference between the rates of MTT
264
reduction with and without dicoumarol (ɛreduced MTT = 11.961 mM-1 cm-1 at 590 nm).
265 266
Statistical analyses
267
Data are presented as the means ± SD. For statistical analyses IBM SPSS Statistics 22 (IBM
268
Deutschland, Ehningen, Germany) was used. Results were analyzed using one-way ANOVA
269
followed by Tukey HSD post hoc test. For comparison of two samples, significant differences
270
were determined by Student’s t-test for unpaired samples. A p-value ≤ 0.05 was considered as
271
statistically significant.
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Results
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Examination of micelles cytotoxicity
276
To test the suitability of micelles for delivering lutein to ARPE-19 cells, the effect of Tween40
277
micelles with and without lutein on cell viability was tested in a neutral red- and a resazurin-
278
based assay (Figure 1). A 24 h treatment with Tween40 micelles ranging from 0.001% to 0.04%
279
neither reduced lysosomal integrity nor dehydrogenase activity, no matter whether they contained
280
lutein (0.5-20 µM) or not. At 0.08% Tween40, lysosomal integrity significantly decreased to 60%
281
± 20% without lutein and to 53% ± 24% with 40 µM lutein. The same concentration of Tween40
282
led to a dehydrogenase activity of 71% ± 17% without lutein and 75% ± 19% with 40 µM lutein.
283 284
Cellular lutein uptake
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Cellular lutein uptake using several non-toxic concentrations was tested. In this experiment, we
286
could not detect a significant reduction in cell number or cell volume by the micelles which
287
confirmed the results of the above-mentioned viability assays. We found that the used micelles
288
were a suitable delivery system for lutein to ARPE-19 cells. The cells were incubated with 1 µM
289
lutein (0.002% Tween40). After 24 h, the measured cellular lutein concentration was 31 µM
290
± 8 µM (Figure 2) which is equivalent to 102 pmol/1x106 cells ± 24 pmol/1x106 cells (Figure S1
291
in Supporting Information). The cellular lutein uptake increased in parallel to the increased lutein
292
concentration. After incubating the cells with 10 µM lutein (0.02% Tween40) for 24 h, the
293
cellular lutein concentration reached a plateau at 60 µM (200 pmol/1x106 cells). A concentration
294
of 10 µM lutein (0.02% Tween40) was defined as working concentration for the subsequent
295
experiments.
296 297
Determination of Nrf2 translocation by lutein
298
Western blot analysis of nuclear fractions showed that lutein at 10 µM (0.02% Tween40) for 4 h
299
enhanced the Nrf2 translocation 1.5 ± 0.4-fold compared to unloaded micelles (Figure 3). The
300
positive control sulforaphane (10 µM, 4 h) increased the nuclear translocation of Nrf2 by
301
4.9 ± 0.5-fold.
302 303
Induction of Nrf2 target genes by lutein in ARPE-19 cells
304
ARPE-19 cells were incubated with 10 µM lutein (0.02% Tween40) for 4 h or 24 h to analyze the
305
effect on Nrf2 target genes (Figure 4). After treatment with lutein-loaded micelles for 24 h,
306
1.7 ± 0.1-fold higher transcript levels of NQO1 compared to unloaded micelles were detected.
307
Treatment with lutein-loaded micelles increased GCLm-mRNA by 1.6 ± 0.3-fold after 4 h and by
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1.4 ± 0.1-fold after 24 h. In the presence of lutein-loaded micelles, HO-1 mRNA was induced
309
significantly 3.5 ± 0.7-fold after 4 h. After 24 h, the mRNA level was 1.8 ± 0.3-fold higher
310
compared to unloaded micelles. Similar results for the analyzed Nrf2 target genes were obtained
311
after treatment with only 5 µM lutein (0.01% Tween40) (Figure S2). A treatment with 10 µM
312
sulforaphane for 24 h served as positive control. As expected, the mRNA expression of NQO1
313
increased by 6.8 ± 1.3-fold, of GCLm by 2.5 ± 0.2-fold, and of HO-1 by 1.5 ± 0.2-fold in the cells
314
treated with sulforaphane.
315
In addition to transcript levels, we also investigated the effect of lutein on NQO1 activity
316
(Figure 5). Lutein (10 µM, 0.02% Tween40) led to a significant enhancement of activity by
317
1.2 ± 0.1-fold from 113.0 mU/mg protein ± 4.5 mU/mg protein in cells incubated with unloaded
318
micelles to 137.9 mU/mg protein ± 7.0 mU/mg protein in cells treated with lutein-loaded micelles
319
for 24 h. The positive control sulforaphane (10 µM, 24 h) showed a significant increase of
320
specific activity by 2.3 ± 0.03-fold from 99.9 mU/mg protein ± 4.9 mU/mg protein in untreated
321
cells to 225.7 mU/mg protein ± 3.3 mU/mg protein in sulforaphane-treated cells.
322 323
Discussion
324
The Nrf2 pathway regulates the transcription of phase II genes that process and eliminate
325
carcinogens and toxins as well as the transcription of many genes with direct or indirect
326
antioxidant effects such as enzymes related to glutathione or to the thioredoxin metabolism,
327
superoxide dismutase, NQO1, HO-1, and catalase.12,19 In our study, we focused on typical Nrf2
328
target genes with antioxidant effects: GCLm is a subunit of the glutamate-cysteine ligase that
329
catalyzes the rate-limiting step in glutathione synthesis. NQO1 and HO-1 degrade quinones and
330
heme, respectively. Quinones and heme both transfer electrons and are therefore direct sources of 15 ACS Paragon Plus Environment
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free radicals.12 The goal of our study was to investigate whether the macular pigment lutein is
332
able to activate Nrf2 and enhances the expression of Nrf2 target genes in ARPE-19 cells.
333
In cell culture, it is often a problem to apply lipophilic carotenoids to the cells. We used a system
334
for delivering lipophilic phytochemicals to ARPE-19 cells which was previously described by
335
Lornejad-Schäfer, et al.30. Lutein-loaded micelles were produced by dissolving lutein in ethanol
336
and using Tween40 as a non-toxic surfactant. In their study, they showed that a Tween40
337
concentration of 0.1% in the culture medium reduces cell viability in a MTT assay, whereas
338
0.01% Tween40 has no damaging effect. Our results confirmed this study. With up to 0.04%
339
Tween40, we observed no reduction in lysosomal integrity or dehydrogenase activity. We next
340
evaluated the cellular lutein uptake to confirm that the used micelles are a suitable delivery
341
system of lutein to the cells. The cellular lutein concentration in the ARPE-19 cells ranged from
342
31 µM ± 8 µM to 62 µM ± 14 µM after incubating the cells with 1-10 µM lutein for 24 h. The
343
incubation concentrations were higher than the usual serum concentrations which can reach
344
2.7 µM after supplementation with 30 mg lutein per day.44 However, the cellular lutein
345
concentration is more crucial for the activation of Nrf2. The concentration of carotenoids in the
346
macular region of the retina is estimated to range between 0.3 mM and 1.3 mM.4 Studies on the
347
carotenoid distribution in the eye described a lower amount of lutein in the underlying RPE. In
348
detail, Sommerburg, et al.45 detected about a sixth and Bernstein, et al.46 about an eighteenth of
349
the amount of lutein in the submacular RPE compared to the macular retina (Sommerburg, et
350
al.45: 0.85 ng/mm2 in the macular retina, 0.15 ng/mm2 in the submacular RPE; Bernstein, et al.46:
351
0.70 ng/mm2 in the macular retina, 0.04 ng/mm2 in the submacular RPE). Thus, the cellular lutein
352
levels determined in our study did not yet reach the highest possible concentrations in the RPE.
353
Nevertheless, the measured cellular lutein concentrations were higher than the applied
354
concentrations indicating an accumulation mechanism. We assume that carotenoid-binding 16 ACS Paragon Plus Environment
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proteins are involved in this process. It is known that in the retina glutathione S-transferase pi
356
(GSTP1) serves as a zeaxanthin-binding protein and the steroidogenic acute regulatory domain
357
protein 3 (StARD3) as a lutein-binding protein.47,48 Tubulin is a less specific carotenoid-binding
358
protein of the retina.47 It still has to be determined which carotenoid-binding proteins may play a
359
role in the RPE. Since there is a higher diversity of carotenoids present in the RPE than in the
360
retina,46,49 other carotenoid binding proteins might be involved. Furthermore, our data indicate
361
that lutein uptake could be saturable in ARPE-19 cells, which might be a result of a protein-
362
mediated uptake. It is known that ARPE-19 cells absorb lutein and zeaxanthin via the scavenger
363
receptor class B1 (SR-B1) and the LDL receptor.50-52 In the study of During, et al.52, Tween40
364
micelles were used to deliver zeaxanthin to ARPE-19 cells. Knockdown of SR-B1 led to a
365
decreased uptake of zeaxanthin from the micelles. Thus, it seems that SR-B1 also plays a role
366
when Tween40 micelles are used for delivery.
367
Importantly, this is the first study to show the activation of Nrf2 by lutein in ARPE-19 cells. In
368
addition, we observed an induction of three Nrf2 target genes, namely NQO1, GCLm, and HO-1.
369
Thus, our results broaden our current knowledge about possible mechanisms by which lutein
370
modulates signaling pathways in the RPE. Other studies show that lutein has similar effects in
371
other tissues. For example, lutein led to an activation of Nrf2 in mouse microglial BV-2 cells.
372
The nuclear Nrf2 increased by 1.7-fold and the protein levels of NQO1 were upregulated 4-fold
373
and of HO-1 5-fold after incubation with 50 µM lutein for 4 h.27 Another study showed a nuclear
374
Nrf2 accumulation in mice liver after treatment with 40 mg/kg lutein for 5 weeks leading to a
375
1.6- and 1.4-fold enhancement of NQO1 and HO-1 mRNA, respectively.28 These values are
376
similar to our findings in RPE cells. Other carotenoids are also able to activate Nrf2 in the RPE.
377
Astaxanthin led to an increase (2.3-fold) of nuclear Nrf2 after incubation of ARPE-19 cells with
378
20 µM astaxanthin for 24 h.26 This carotenoid also induced transcript levels of NQO1 by 1.3-fold, 17 ACS Paragon Plus Environment
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of GCLm by 1.9-fold, and of HO-1 by 2-fold.26 Zeaxanthin (10 µM, 6 h) was also observed to
380
increase nuclear Nrf2 in ARPE-19 cells and upregulated Nrf2 target genes even more strongly.
381
The transcript levels of NQO1 increased by 3.7-fold, of GCLm by 3.2-fold, and of HO-1 by 4.5-
382
fold.25 The same study also showed that zeaxanthin induces Nrf2 target genes and GSH levels in
383
rats and reduces the oxidative damage in the retina of these animals.25
384
It was shown that carotenoid degradation products can activate Nrf2.53 Carotenoid degradation
385
products can be formed from chemical or enzymatic degradation. However, initial experiments
386
performed by our group could not detect any degradation of lutein in the medium under our cell
387
culture conditions for 24 h. Therefore, we assume that lutein is not degraded chemically in our
388
experiments. Concerning enzymatic degradation, the enzyme β,β-carotene-9′,10′-dioxygenase
389
(BCO2) is able to cleave lutein. However, Li, et al.54 showed that BCO2 is expressed in the
390
human retina, but the enzyme is inactive. A study with ARPE-19 cells could neither detect the
391
BCO2 protein nor any cleavage products.50
392
The goal of our study was to test whether lutein can activate Nrf2 in a RPE cell line thereby
393
protecting the cells against oxidative stress. Oxidative stress in the RPE plays a major role in the
394
development of AMD in older people.6 Reduction of oxidative damage in the RPE could thus be
395
an important mechanism to protect the overlying retina. How important it is to activate the Nrf2
396
pathway in the RPE in older people by applying phytochemicals like lutein is illustrated by the
397
following studies. Nrf2-deficient mice were observed to develop AMD-like retinal pathology
398
with aging.55 In another study, the induction of Nrf2 target genes, such as NQO1, GCLm, and
399
HO-1, in the RPE following oxidative stress induced with sodium iodate was impaired in older
400
compared to younger mice.10 Yet another study indicated that the HO-1 expression in the RPE
401
seems to decline with aging.56 Consistently, the glutathione redox system is affected by age as the
402
GSH level in the blood decreases.57 Consequently, older people seem to be more susceptible to 18 ACS Paragon Plus Environment
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oxidative stress because their defense system is impaired. Thus, it is of considerable interest to
404
increase cellular resistance against oxidative damage by upregulating Nrf2 target genes with
405
antioxidant properties. Of note is that there is evidence that lutein is protective against AMD. In
406
the Age-Related Eye Disease Study 2, participants with low dietary intake of lutein and
407
zeaxanthin prior to the start of the study, but who took lutein and zeaxanthin in the study, showed
408
a lower probability to develop advanced AMD compared to participants with similar dietary
409
intake who did not take additional lutein and zeaxanthin.5 However, by which mechanism lutein
410
protects the retina and how important the activation of Nrf2 is are both issues that remain to be
411
evaluated. In this context, there are already some evidences that lutein can reduce oxidative
412
damage and inflammation in the RPE. For example, lutein was observed to protect the
413
proteasome against photooxidative damage and to modulate the expression of inflammation-
414
related genes like IL-8 in ARPE-19 cells.58 Treatment of human D407 RPE cells with lutein
415
reduced hydrogen peroxide-induced cytotoxicity, ROS generation, and lipid peroxidation. Lutein
416
also reduced hydrogen peroxide-induced decrease in GSH levels and in the activity of superoxide
417
dismutase and glutathione peroxidase.59 However, lutein alone was not able to enhance GSH
418
levels and the aforementioned enzyme activities in unstressed conditions.59 The activation of
419
Nrf2 in RPE cells shown in our study could be another mechanism by which lutein reduces
420
oxidative damage in the RPE, thereby preventing the development of AMD.
421
In addition to carotenoids, other phytochemicals are known to activate Nrf2 in ARPE-19 cells.
422
Our study confirmed that the isothiocyanate sulforaphane upregulates Nrf2 target genes in
423
ARPE-19 cells as also shown in an earlier study of Gao and Talalay20. Moreover, escin (a
424
mixture of triterpene-saponines) was reported to increase nuclear Nrf2 levels and induce NQO1
425
mRNA 9-fold and HO-1 mRNA 17-fold after incubation of ARPE-19 cells with 10 µM escin for
426
2 h. Thus, escin is a stronger inducer than lutein.21 Further, different phenolic compounds are 19 ACS Paragon Plus Environment
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known to enhance Nrf2 target genes in the RPE. For example, treatment of ARPE-19 cells with
428
50 µM canolol for 24 h led to a 1.5-fold induction of HO-1 mRNA.22 Incubation of ARPE-19
429
cells with 5 µM 4-acetoxyphenol for 24 h increased nuclear Nrf2 and upregulated NQO1 mRNA
430
3.3-fold and HO-1 mRNA 2.5-fold.23 Finally, the flavonoid eriodictyol induced the nuclear
431
translocation of Nrf2 and enhanced HO-1 protein levels, NQO1 protein levels and glutathione
432
after incubating ARPE-19 cells with eriodictyol at a concentration of 0-100 μM for 2-24 h.24
433
Collectively, these studies show that the Nrf2 pathway is upregulated by various phytochemicals.
434
Further studies are now needed to determine the contribution of individual phytochemicals as
435
well as their synergistic or antagonistic effects.
436
In conclusion, our study demonstrates that lutein activates the Nrf2 pathway in ARPE-19 cells
437
and upregulates the antioxidant system, thereby leading to an enhancement of cellular resistance
438
of RPE cells against oxidative damage. We propose that better understanding of this mechanism
439
may lead to new therapeutic strategies as regards AMD.
440 441
Abbreviations used
442
AMD, age-related macular degeneration; ARE, antioxidant response element; βAct, beta-actin;
443
BCO2, β,β-carotene-9′,10′-dioxygenase; BSA, bovine serum albumin; CRALBP, cellular
444
retinaldehyde binding protein; DMEM, Dulbecco's Modified Eagle Medium; E, amplification
445
efficiency; FCS, fetal calf serum; GCLm, glutamate-cysteine ligase regulatory subunit; GSTP1,
446
glutathione S-transferase P 1; HO-1, heme oxygenase-1; HPRT1, hypoxanthine phosphoribosyl
447
transferase 1; Keap1, Kelch-like ECH-associated protein 1; LDL, low-density lipoprotein; Maf,
448
musculoaponeurotic fibrosarcoma; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
449
bromide; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor erythroid 2-related
450
factor 2; PPIA, peptidylprolyl isomerase A; Prdx1, peroxiredoxin 1; RPE, retinal pigment 20 ACS Paragon Plus Environment
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epithelium; RPE65, RPE-specific 65 kDa protein; SOD, superoxide dismutase; SR-B1, scavenger
452
receptor class B1; StARD3, steroidogenic acute regulatory domain protein 3
453 454
Acknowledgements
455
The authors would like to thank the Department of Food Chemistry, S. Deubel, and the
456
Department of Plant Quality and Food Security for their assistance.
457 458
Supporting Information
459
-
Figure S1. Cellular lutein uptake of ARPE-19 cells in pmol/1x106 cells.
460
-
Figure S2. Effect of lutein (5 μM lutein (0.01% Tween40)) on transcript levels of Nrf2
461
target genes.
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Figure captions
628 629
Figure 1. Effect of Tween40 micelles without (T40) and with (L) lutein on (I) lysosomal
630
integrity and (II) dehydrogenase activity. ARPE-19 cells were treated with different micelles
631
concentrations for 24 h. Cell viability was measured using (I) neutral red- and (II) resazurin-
632
based assays. Lysosomal integrity and dehydrogenase activity of untreated control cells (C) were
633
set at 100% (dashed line). Values (%) are expressed as mean ± SD (n = 5: untreated cells, 0.001-
634
0.02% Tween40; n = 4: 0.04-0.08% Tween40). Significant difference (p ≤ 0.05) was determined
635
compared to untreated control: * micelles without lutein, # micelles with lutein.
636 637
Figure 2. Cellular lutein uptake of ARPE-19 cells. Cells were incubated with lutein-loaded
638
micelles for 24 h. Lutein was extracted from cells and quantified by LC-ToF-MS. Values are
639
presented as mean ± SD (n = 3).
640 641
Figure 3. Effect of lutein on nuclear Nrf2 protein levels. ARPE-19 cells were treated with (I)
642
10 µM lutein (0.02% Tween40) (L) or (II) 10 µM sulforaphane (SFN) for 4 h. Cells which were
643
treated with unloaded micelles (T40) or water-treated (C) served as controls. Nuclear cell
644
fractions were isolated. Nrf2 protein content was analyzed and normalized to histone H2B by
645
western blot analysis. The control cells were set at 1. Values are expressed as mean ± SD (I: n =
646
6; II: n = 3). Significant difference (* p ≤ 0.05, ** p ≤ 0.01) was determined compared to control
647
cells.
648 649
Figure 4. Effect of lutein on transcript levels of Nrf2 target genes. ARPE-19 cells were incubated
650
with 10 µM lutein (0.02% Tween40) (L) for 4 h and 24 h or with 10 µM sulforaphane (SFN) for 30 ACS Paragon Plus Environment
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651
24 h. The mRNA levels of (I) NQO1, (II) GCLm and (III) HO-1 were analyzed by RT-qPCR. All
652
values were normalized to the expression of the gene of interest in control cells (C, represented
653
by a dashed line): with unloaded micelle-treated cells for lutein-loaded micelles, water-treated
654
control cells for sulforaphane. Data are shown as mean ± SD (n = 3). Significant difference (* p
655
≤ 0.05, ** p ≤ 0.01) was determined compared to control cells.
656 657
Figure 5. Effect of lutein on relative NQO1 activity. ARPE-19 cells were treated with (I) 10 µM
658
lutein (0.02% Tween40) (L) or (II) 10 µM sulforaphane (SFN) for 24 h. Cells that were treated
659
with unloaded micelles (T40) or water (C) served as controls. NQO1 activity was analyzed by
660
measuring MTT reduction. Activities were determined as specific activities (mU/mg protein) and
661
normalized to control cells. Data are shown as mean ± SD (n = 3). Significant difference (** p
662
≤ 0.01, *** p ≤ 0.001) was determined compared to control cells.
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