Effect of Hops Derived Prenylated Phenols on TNF-α Induced Barrier Dysfunction in Intestinal Epithelial Cells

Article abstract of DOI:10.1021/acs.jnatprod.6b00869

For the prenylated hops phenols 6- and 8-prenylnaringenin (1 and 2), xanthohumol (3), and isoxanthohumol (4), a variety of biological activities has been described. In the current study, a transwell based in vitro model using the human intestinal epithelial cell line Caco-2 was developed to assess potential beneficial effects of compounds 1-4 on TNF-α-induced impairment of tight junction (TJ) permeability. Transepithelial electrical resistance (TEER) was measured using the latest cellZScope online monitoring device. TNF-α treatment (25 ng/mL) induced a significant decrease in TEER values (204.71 ± 4.57 at 72 h) compared to that in control values (245.94 ± 1.68 at 72 h). To determine preventive effects on TNF-α-induced impairment of TJ permeability, 1-4 were added to the apical compartment of Caco-2 monolayers 1 h before TNF-α treatment; afterward, TNF-α was added to the basolateral compartment to induce TJ dysfunction and incubated for a further 72 h. Using this setting, only 1 and 2 prevented epithelial disruption induced by TNF-α. To evaluate restorative effects of 1-4, TNF-α was added to the basolateral compartment of Caco-2 cell monolayers. After 48 h of incubation, 1-4 were added to the apical side, and TEER values were monitored online for a further 72 h. Under these experimental conditions, only 2 restored TNF-α induced barrier dysfunction.

Full text of DOI:10.1021/acs.jnatprod.6b00869

Article
pubs.acs.org/jnp
Effect of Hops Derived Prenylated Phenols on TNF‑α Induced Barrier
Dysfunction in Intestinal Epithelial Cells
Sandro Luescher, Corinna Urmann, and Veronika Butterweck*
†
‡
,†
†Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland,
Gruendenstrasse 40, 4132 Muttenz, Switzerland
‡
Hochschule Weihenstephan Triesdorf, University of Applied Sciences, Schulgasse 16, 94315 Straubing, Germany
ABSTRACT: For the prenylated hops phenols 6- and 8-prenylnaringenin
(
1 and 2), xanthohumol (3), and isoxanthohumol (4), a variety of
biological activities has been described. In the current study, a transwell
based in vitro model using the human intestinal epithelial cell line Caco-2
was developed to assess potential beneficial effects of compounds 1−4 on
TNF-α-induced impairment of tight junction (TJ) permeability. Trans-
epithelial electrical resistance (TEER) was measured using the latest
cellZScope online monitoring device. TNF-α treatment (25 ng/mL)
induced a significant decrease in TEER values (204.71 ± 4.57 at 72 h)
compared to that in control values (245.94 ± 1.68 at 72 h). To determine
preventive effects on TNF-α-induced impairment of TJ permeability, 1−4
were added to the apical compartment of Caco-2 monolayers 1 h before
TNF-α treatment; afterward, TNF-α was added to the basolateral
compartment to induce TJ dysfunction and incubated for a further 72 h.
Using this setting, only 1 and 2 prevented epithelial disruption induced by TNF-α. To evaluate restorative effects of 1−4, TNF-α
was added to the basolateral compartment of Caco-2 cell monolayers. After 48 h of incubation, 1−4 were added to the apical
side, and TEER values were monitored online for a further 72 h. Under these experimental conditions, only 2 restored TNF-α
induced barrier dysfunction.
he intestinal epithelium is a single layer of columnar
epithelial cells that separates the intestinal lumen from the
underlying lamina propria. These essential epithelial cells are
injury is essential for the prevention of diseases. Unfortunately,
the global immunomodulation caused by anti-TNF treatment
puts patients at increased risk for potentially fatal infec-
T
13−15
bound together by intercellular junctional complexes that
tions
because many other aspects of the immune response
1
,2
regulate the paracellular permeability. Intercellular junctional
complexes consist of the tight junctions (TJs), gap junctions,
adherence junctions, and desmosomes, of which TJs constitute
are targeted.
Interestingly, some spices such as black pepper, green
pepper, nutmeg, extracts of bay leaf and linden flowers, star
anise, and black tea were found to decrease paracellular flux and
increase transepithelial electrical resistance (TEER) values in
1
,2
the major determinant of the intestinal physical barrier.
Although the intercellular space between adjacent epithelial
cells prevents translocation of microorganisms (commensals
and pathogens) and antigens (including bacterial toxins and
16,17
intestinal cells.
3
,4
The female cones of the hop plant (Humulus lupulus L.,
Cannabaceae) contain the prenylflavonoids 6- and 8-prenylnar-
ingenin (1 and 2), their common precursor compound
xanthohumol (a chalcone) (3), and the corresponding isomeric
prenylflavanone isoxanthohumol (4). In vivo, as well as in the
brewing process, 3 is converted into 4, which represents the
food components) across the epithelium, some pathogens
(
e.g., Salmonella spp.) impair or subvert the intestinal epithelia5l
TJ barrier and subsequently cause acute inflammation.
Moreover, chronic inflammation is often associated with
defective intestinal epithelial TJ barrier function that allows
luminal antigens to stimulate underlying immune cells in
various diseases, including celiac disease, inflammatory bowel
18
major prenylflavonoid in beer. Further, 3 can be demethy-
6
−8
disease (IBD), Crohn’s disease, diabetes, and food allergies.
19
lated in the human intestine to 2. While for 2 potent
In Crohn’s disease, TNF-α is thought to be a critical
determinant for chronic intestinal inflammation. Treatment of
Crohn’s disease patients with anti-TNF-α improved impaired
20
phytoestrogenic effects were shown, 1 has been reported to
21
be nonestrogenic. On the other hand, 1−4 exhibited
9
antiproliferative, antiangiogenic, tumor-preventive, anti-inflam-
intestinal barrier function. TNF-α acts both as a pro-
22−26
matory, and antiviral activities.
However, for 1 and 2, poor
inflammatory cytokine that activates the endogenous inflam-
matory cascade and as a highly potent inducer of epithelial TJ
1
0−12
disruption.
The maintenance of an intact epithelial barrier
Received: September 23, 2016
is a prerequisite for normal health, and rapid resealing after
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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bioavailability, high metabolization rates, and intracellular
27−31
accumulation have been reported.
Figure 1. Effect of 1−4 on cell viability in Caco-2 cells. Caco-2 cells
were treated with the hops derived prenylflavonoids for 24 h. ATP
levels were measured using the CellTiter-Glo luminescent cell viability
assay. All data are expressed as percentage of control (0.5% DMSO)
and represent the mean ± SEM (n = 4). Asterisks indicate significant
difference from the control group (*p < 0.05; **p < 0.01; ***p <
On the basis of the known biological characteristics of the
hop-derived prenylated phenols, a transwell based in vitro
model using the human intestinal epithelial cell line Caco-2 was
developed to assess potential beneficial effects of these
compounds on TNF-α-induced impairment of TJ permeability.
The aim was to evaluate preventive and reparative abilities of
0
.001).
decreased for a period of 72 h, indicating a decrease in TJ
integrity (Figure 2A). The integrity of the Caco-2 cell
monolayer was further confirmed using the Lucifer yellow
1
−4 after causing an epithelial barrier disruption in Caco-2 cells
using low, physiologically relevant levels of TNF-α.
(
LY) assay. In this experimental setting, Caco-2 cells were
RESULTS AND DISCUSSION
■
pretreated on the apical side for 1 h with 1−4 (10 μM)
followed by a 48 h exposure to TNF-α (25 ng/mL) on the
This proof of concept study deals with the evaluation of the
effects of hop derived prenylflavonoids 1−4 on TJ function of
intestinal epithelial Caco-2 cells with a special emphasis on
preventive and restorative effects. To find appropriate, nontoxic
doses of 1−4, a luminescent cell viability assay was performed
by homogeneously measuring the presence of metabolically
active cells via quantification of the ATP content. 1−4 in
concentrations between 1 and 100 μM were added to the Caco-
2
cells for 24 h. 1 showed a significant decrease in metabolism
(
18%) in the highest applied concentration of 100 μM, whereas
the range between 1 and 50 μM was comparable to the
untreated control (Figure 1A). 2 did not affect cell viability over
the entire concentration range (1−100 μM) (Figure 1B). 3
provoked significantly higher metabolism at 10 μM in
comparison to that of control cells, followed by a significant
ATP loss applying 50 and 100 μM with a 55.3% reduction
against control at the highest concentration (Figure 1C). 4
induced (equally to 3) a significant decrease in ATP levels at
1
00 μM (13.2%) (Figure 1D). On the basis of these results, a
test concentration of 10 μM for 1−4 was selected for further
assessment of restorative and protective effects of flavonoids on
intestinal TJ barrier function.
Previous studies have demonstrated that TNF-α could
32
induce barrier dysfunction in Caco-2 cells. In the present
study, an in vitro model was developed similar to others
reported in the literature in which low, physiologically relevant
levels of TNF-α were required to decrease barrier
Figure 2. (A) TEER measurement of Caco-2 cell monolayers treated
with TNF-α. TNF-α treatment at a dose of 25 ng/mL showed time-
dependent TEER loss. Values represent mean ± SEM (n = 3).
Asterisks indicate significant differences from control (***p < 0.01).
1
0,33,34
function.
TEER was measured using the latest cellZScope
online monitoring device, which had the advantage of
measuring 24 wells automatically at a constant 37 °C.
Therefore, an adapted protocol was established to reproduce
cytokine induced barrier dysfunction as discussed in the
literature, of which the majority was measured by traditional
(
B) Preventive effect of 1−4 against TNF-α induced barrier
dysfunction in Caco-2 cells. 1−4 (10 μM) were added to the apical
compartment 1 h prior to the addition of TNF-α (25 ng/mL) to the
basolateral compartment. The absolute amount of LY was monitored
after 48 h of basolateral TNF-α (25 ng/mL) exposure. Values are
shown as mean ± SEM (n = 3).
6
,35,36
EVOM (epithelial voltohmmeter) instruments.
After
addition of 25 ng/mL TNF-α, TEER values significantly
B
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basolateral side with subsequent LY administration on the
apical side. As shown in Figure 2B, TNF-α caused a significant
increase in the absolute amount of LY transported into the
basolateral compartment in comparison to that of control cells.
Pretreatment with 1−4 (10 μM) alone did not notably increase
the paracellular LY transport rate, indicating no barrier
disruption.
In another set of experiments, 1−4 were evaluated for
potential TJ barrier-strengthening effects. After the fully
differentiated monolayers reached a TEER plateau in the
measuring device (∼24 h), the cells were exposed to 10 μM of
1−4 from the apical side for 72 h (Figure 3). Interestingly, 2
Figure 4. Preventive effect of hops compounds against TNF-α-
induced TJ impairment. (A) 1 and 2 and (B) 3 and 4 were added to
the apical side of Caco-2 monolayers 1 h prior to TNF stimulation.
After 1 h, Caco-2 monolayers were stimulated on the basolateral side
with TNF-α (25 ng/mL) and incubated for a further 72 h. Data
represent mean ± SEM (n = 3); *p < 0.05 vs control; **p < 0.01 vs
+
2
control; and p < 0.01 vs TNF-α.
(
Figure 4A). On the other hand, 3 and 4 did not prevent the
TNF-α-induced impairment of TJ function (Figure 4B).
The ability of 1−4 to restore TJ function of Caco-2 cell
monolayers damaged by previous TNF-α exposure was
examined after 24 h of pro-inflammatory cytokine pretreatment
(
Figure 5). Twenty-four hours after TNF-α addition on the
basolateral side, 1−4 were then added apically. TEER values
were measured over time to assess the restoration of TJ
function. Compound 2 facilitated the restoration of TJ function
after 72 h, whereas 1 was comparable to the TNF-α treated
negative control (Figure 5A). In contrast, 3 and 4 further
impaired the spontaneous recovery of the Caco-2 monolayer
similarly (Figure 5B).
Figure 3. TJ barrier-strengthening ability of (A) 1 and 2 and (B) 3 and
in Caco-2 cells. TEER assessment in Caco-2 cell monolayers
4
incubated with 10 μM of 1−4. TEER values were monitored over a
period of 72 h. Values are presented as mean ± SEM (n = 3); **p <
0.01.
Previous studies indicated that TNF-α caused morphological
disruption and delocalization of tight junction proteins in
induced an appreciable increase in TEER starting after 36 h of
treatment, leading to a significant increase after 48, 60, and 72 h
32,37−39
intestinal cells.
Because these TJ proteins regulate
(
Figure 3A). 1 (Figure 3A) and 3 (Figure 3B) showed
paracellular permeability, it was of interest to examine the
organization of TJ proteins by immunofluorescence labeling
and confocal microscopy. The tight junction protein Zonula
occludens-1 (ZO-1) was localized. ZO-1 is a member of the
membrane-associated guanylate kinase homologues and a linker
between the transmembrane protein occludin and the actin
cytoskeleton. Because 2 attenuated pro-inflammatory cyto-
kine induced TJ impairment in Caco-2 cells (Figure 4A) and
facilitated the restoration of epithelial barrier function after
TNF-α-induced impairment of TJ permeability (Figure 5A), it
was of interest to further investigate tight junction morphology
after addition of 2. In addition, the effects of 3 on TJ
impairment were assessed because it was the least active of all
tested compounds. In control treated cells, the TJ distribution
of ZO-1 had a “chickenwire” pattern (Figure 6). TNF-α
treatment induced discontinuities in TJ distribution of ZO-1,
changing the pattern from regularly smooth arcs into irregularly
comparable TEER values during the tested time period.
Overall, the effects of 1, 3, and 4 were only marginal and
therefore considered irrelevant in comparison to the TEER of
the control (Figure 3B).
In additional experiments, the preventive effects of 1−4
40
against TNF-α induced impairment of TJ permeability in Caco-
2
cells were examined. For the prevention screening, 1−4 were
added to the apical side of Caco-2 cells 1 h before the addition
of TNF-α (25 ng/mL) on the basolateral side. TEER values
were measured to assess epithelial barrier function during a
total incubation time of 72 h. As displayed in Figures 4A and B,
TNF-α treatment induced a significant decrease in TEER
values. Interestingly, 1 almost fully prevented the TNF-α-
induced drop in TEER values (Figure 4A). Pre-exposure to 2,
which strengthened Caco-2 cells’ TJs (Figure 2B), demon-
strated a slower recovery of the TNF-α-induced TEER drop
C
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preventive effect against the pro-inflammatory cytokine (Figure
6
).
A number of intestinal diseases mediated by inflammation,
such as ulcerative colitis, Crohn’s disease and IBD, involve
intestinal epithelial barrier dysfunction, which is characterized
by increased paracellular permeability as well as the changes of
41,42
tight junction protein expression and organization.
A
number of research groups demonstrated that proinflammatory
cytokines disrupt intestinal epithelial barrier function both in
32,38,39,43−45
46
vitro
and in vivo. Furthermore, it is known that
treatment of intestinal epithelial cells with TNF-α promotes the
redistribution of several TJ proteins (including claudin-1,
47
occludin, and ZO-1) and impairs epithelial barrier function.
Thus, targeting the restoration of intestinal barrier function in
acute or chronic enteropathies still depicts a worthwhile
therapeutic strategy.
In the present study, we demonstrated that 1 and 2
attenuated intestinal epithelial barrier dysfunction caused by
TNF-α treatment. The most pronounced effects were observed
for 2, which alleviated both TEER decrease and paracellular
permeability increase and preserved the morphological
distribution of the TJ protein ZO-1. The molecular mechanism
by which 2 ameliorates the intestinal epithelial barrier
dysfunction induced by proinflammatory cytokines is currently
unknown.
Figure 5. Restorative effect of 1−4 after TNF-α-induced TJ
disruption. (A) 1 and 2 and (B) 3 and 4 were added to the apical
side of Caco-2 monolayers after 24 h of TNF-α stimulation (25 ng/
mL) from the basolateral side. TEER measurements were taken over a
total time period of 96 h. Data represent TEER values as mean ± SEM
It has been shown that 2 is highly metabolized in Caco-2
cells to the corresponding sulfate and glucuronide conju-
27,28
gates,
and it needs to be determined in future studies if and
(
n = 3); **p < 0.01 vs control.
how these metabolites contribute to the observed effect.
20
Because 2 has potent estrogenic activities, it is also possible
that improvement of intestinal barrier function could be
mediated by activation of estrogenic receptors (ER). ERβ, a
ligand-activated nuclear transcription factor producing genomic
effects, is the predominant ER subtype in colon tissue where it
is responsible for growth, organization, and maintenance of
4
8,49
normal epithelial architecture.
Reduced ERβ mRNA
expression and increased gut permeability were observed in
48
mouse models of intestinal inflammation and colitis was more
severe in ERβ knockout mice as compared to wild-type
50
littermates. Beneficial effects of ERβ agonists and estrogens
51,52
have been shown in various models of chronic colitis.
In a
53
recent study, Kiatpraser et al. showed that the soy isoflavones
genistein and daidzein increased tight junction gene expression
in procine glanduar endometrial cells. In our present study,
only 2, a compound with known potent estrogenic effects,
prevented and restored endothelial barrier disruption induced
by TNF-α. The onset of activity was observed after 48 h, which
could be explained by genomic effects via activation of ERs.
Several studies reported that various food components
provide beneficial anti-inflammatory effects in the intes-
Figure 6. Confocal laser scanning microscopy images showing the
localization of ZO-1. Compounds 2 or 4 were added to the Caco-2
monolayers and after 1 h; Caco-2 monolayers were stimulated with
TNF-α (25 ng/mL) for 48 h. Two suppressed the TNF-α caused
disruption, which resulted in irregularly undulating profiles along the
TJs. Four showed a poor preventive effect against the stimulation of
the pro-inflammatory cytokine. Scale bar = 20 μm.
54−61
tine.
Although the information regarding these effects on
intestinal TJ barrier function is quite limited, results for some
flavonoids are available. To date, only quercetin, kaempferol,
myricetin, and naringenin have been reported to enhance
56,58−60,62
intestinal barrier function.
However, in these studies,
intestinal cells were not pretreated with proinflammatory
cytokines. Thus, no conclusion regarding possible preventive/
restorative effects of these flavonoids can be drawn. When
enteric epithelial permeability was induced by IFN-γ,
epigallocatechin gallate (EGCG) from green tea blocked
undulating profiles along the tight junctions. Pretreatment with
2
largely prevented these morphological changes of the TJ
6
1
protein ZO-1. As expected, cells pretreated with 3 showed
distinct morphological disruption after cytokine exposure,
comparable to that of the TNF-α group, indicating a poor TJ
epithelial barrier dysfunction. Besides the mentioned
flavonoids, alkaloids such as berberine also have been reported
to attenuate TEER decrease and paracellular permeability
D
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increase in Caco-2 monolayers treated with IFN-γ and TNF-
experiments, monolayers were either pretreated for 24 h with 25 ng/
mL TNF-α (BioLegend, Luzern, Switzerland), and compounds were
added subsequently (restorative effect), or TNF-α was added after a 24
h pretreatment period with all test compounds (preventive effect).
TNF-α was added to the basal chamber without manipulating the
apical media, whereas the test compounds were added to the apical
3
7,55
α.
It was also shown that fermented soy milk prevented
63
IFN-γ induced decrease in TEER in Caco-2 cells.
Interestingly, this effect was not observed with nonfermented
soy milk. The authors showed that the protective effect of
fermented soy milk was attributed to isoflavone aglycones and
side only. The concentrations of TNF-α was based on effective
63
equol occurring in the fermented milk. As mentioned
10,33,34
concentrations previously described in the literature.
All test
previously, extensive Phase II conjugation occurs for 2 during
compounds were dissolved in 100% DMSO and diluted 1/2000 for
treatments, leading to a final concentration of 0.05% DMSO in the
apical DMEM media. To facilitate comparisons between experiments,
the mean values of TEER of the different treated monolayers were
typically compared to the mean of the control cell monolayers in the
same experiment.
Cell Viability Assay. The ATP assay was employed to evaluate the
toxicity of 1−4 and to determine the concentration for further
experiments. To determine ATP levels, the CellTiter-Glo luminescent
cell viability assay (Promega, Madison, WI, United States) was used.
One hundred microliters of the Caco-2 cell suspension was seeded
into 96-well plates at a concentration of 28 000 cells/well followed by
27,28
intestinal absorption.
It therefore needs to be determined
in future studies of how much metabolites of 2 such as
glucuronides and sulfates contribute to the improvement of
endothelial disruption.
In conclusion, the present in vitro study demonstrates that,
of the tested compounds, 1 and 2 prevented epithelial
disruption induced by TNF-α. Further, 2 was also able to
restore TNF-α induced barrier dysfunction. Our results show
for the first time that 2 from hops attenuated a cytokine-
induced increase in intestinal epithelial TJ permeability. As it is
known that TJ barrier defects are involved in several diseases
such as IBD or colitis, dietary supplementation with hop
derived prenylflavonoids might be an effective tool for the
prevention and treatment of those diseases.
an incubation period of 24 h (37 °C, 8% CO ). Then, the cells were
2
treated for 24 h with different test substances dissolved in DMEM and
a maximum of 0.5% of DMSO. Control cells were treated with 0.5%
DMSO in DMEM medium. Finally, 100 μL of CellTiter-Glo reagent
was added to each well and shaken for 2 min on an orbital shaker (IKA
Labortechnik, Staufen, Germany) at 300 rpm. After another 10 min,
EXPERIMENTAL SECTION
General Experimental Procedures. Xanthohumol (3) was a
kind gift of Hallertauer Hopfenveredelungsgesellschaft m.b.H
1
00 μL of each well was transferred to a white 96-well plate, and
■
luminescence was measured at 470 nm using a SpectraMax L
(
Molecular Devices, Sunayvale, CA, United States).
Lucifer Yellow Assay. The integrity of the monolayer was
(
Mainburg, Germany). Isoxanthohumol (4) was synthesized using
additionally examined by assessing paracellular permeability via LY as
xanthohumol as starting material via ring closure in aqueous sodium
hydroxide solution (1% w/w) at 0 °C. After 2 h of stirring, sulfuric acid
was added, and the yellow precipitate was filtered off (yield 80%).
Demethylation of 3 resulted in 6-prenylnaringenin (1) and 8-
prenylnaringenin (2). Therefore, 1.0 mmol of xanthohumol and
lithium chloride (2 equiv) in 3 mL of dry dimethylformamide (DMF)
was heated in a synthesis microwave (CEM Discover) at 200° for 30
min. The reaction mixture was poured into water and extracted with
ethyl acetate, and afterward, the solvent was removed from the
combined organic fractions. All products were purified using
6
5
previously described. For the prevention study, the medium of the
fully differentiated Caco-2 monolayer was changed after 21 days to
DMEM (without FBS) for 24 h. Afterward, the cells were exposed to
test substances on the apical side for 1 h, followed by an addition of 25
ng/mL TNF-α on the basolateral side. Forty-eight hours later, the cells
were washed with DPBS containing Ca2 and Mg (Thermo Fisher
Scientific, Reinach, Switzerland). Subsequently, 1.6 mL of HBSS
(Thermo Fisher Scientific, Reinach, Switzerland) 1% DMSO
containing 60 μM LY (Sigma-Aldrich, Buchs, Switzerland) was
added to the apical side, and 2.8 mL of HBSS 1% DMSO was
added to the basolateral compartment. The plate was shaken on an
orbital shaker at 37 °C in a water-saturated atmosphere at 50 rpm.
After 1 h, samples were taken from both compartments, and
fluorescence was measured using a microplate reader SpectraMax
M2e (Molecular Devices, Sunayvale, CA, United States) at 428 nm
excitation and 540 nm emission. The absolute amount of Lucifer
yellow transported into the basolateral compartment was extrapolated
from a standard curve.
+
2+
1
preparative HPLC, and hence, the purity was >95% The H NMR
data of all compounds were similar to those data reported in the
57,64
literature.
The human colon adenocarcinoma cell line Caco-2 was a gift of
Prof. Dr. H.P. Hauri (University of Basel, Switzerland) and originated
from the American Type Culture Collection (Rockville, United
States). Caco-2 cells between passage numbers 60−70 were
maintained in cell culture petri dishes (Nunc, Roskilde, Denmark)
in 12 mL of culture medium composed of DMEM, high glucose
supplemented with 1% MEM non-essential amino acids solution, 1.8
mM L-glutamine, 10% fetal bovine serum (FBS), 50 U/mL penicillin,
and 50 μg/mL streptomycin. All ingredients were from Gibco (Grand
Island, NY, United States) The cells were cultivated at 37 °C in a
Immunofluorescence Staining. For immunofluorescence stain-
ing, Caco-2 cells were grown on a Nunc Lab-Tek Chamber Slide
system (8-well, Permanox) (Thermo Fisher Scientific, Reinach,
Switzerland). Several days after confluence, the cells were incubated
for 24 h in DMEM without FBS. Afterward, the cells were exposed to
test substances for 1 h before 25 ng/mL TNF-α was added. After 48 h
of cytokine incubation, the cells were washed in PBS and fixed/
permeabilized with ice cold methanol at −20 °C for 10 min. The
nonspecific binding was blocked by 5% BSA in PBS for 1 h at room
temperature. Then, the monolayers were incubated with primary ZO-1
polyclonal antibody (Thermo Fisher Scientific, Reinach, Switzerland)
1/50 in PBS 1% BSA for 2−3 h at room temperature (RT) and
subsequently probed with a secondary Alexa Fluor 488 antibody
(Abcam) 1/500 in PBS 1% BSA for 45 min in the dark at RT. Then, a
Texas Red-X Phalloidin (Thermo Fisher Scientific, Reinach, Switzer-
land) stain (1/500) in PBS 1% BSA was held for 20 min at RT in the
dark. Finally, DAPI staining (0.95 μM) in PBS 1% BSA was performed
for 2 min at RT. The slides were sealed using nail polish and kept in
the dark at 4 °C. The location of ZO-1 protein in Caco-2 cells was
visualized by an inverted fluorescent microscope (IX-81, Olympus,
Volketswil, Switzerland) equipped with a confocal scanning unit
humidified atmosphere of 8% CO until 80−90% confluency was
2
achieved. Culture medium was changed every two days. For
subcultivation, the cell monolayer was washed with DPBS (Gibco,
Grand Island, NY, United States) and treated for 3 min with Trypsin-
EDTA (Gibco, Grand Island, NY, United States).
Caco-2 Cell Culture. The TEER of filter-grown monolayers was
̈
measured using a cellZScope (nanoAnalytics, Munster, Germany)
optimized for use with large 6-well-type inserts placed inside a
5
standard incubator. Cells (5.37 × 10 ) were seeded and grown as
monolayers on 24 mm polycarbonate membrane Transwell supports
(
Corning Costar, Kaiserslautern, Germany) with 0.4 μm pore size. On
days 19−21, the transwell inserts were transferred to the cellZScope,
and the growth medium was switched to DMEM (without FBS).
Readings were taken until the cells stabilized after approximately 24 h
2
at ±240 Ω·cm . At this point, the cells were deemed fully
differentiated. A final TEER reading was taken immediately before
the cells were exposed to cytokines or test substances. For the
E
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(
FluoView FV1000, Olympus, Volketswil, Switzerland) using the
(22) Cook, M. R.; Luo, J.; Ndiaye, M.; Chen, H.; Kunnimalaiyaan, M.
Am. J. Surg. 2010, 199, 315−318 discussion 318..
software FV10-ASW Ver. 04.01 (Olympus, Volketswil, Switzerland).
The ImageJ Software (Ver. 1.49, NIH, Bethesda, MD, United States)
was used for picture postprocessing.
Statistical Analysis. Data are shown as mean ± SEM. All
experiments were performed at least in triplicate. Statistical analysis
was carried out by a one- or two-way analysis of variance (ANOVA),
followed by a Dunnett’s multiple comparisons procedure using the
software package GraphPad Prism (version 6.01, GraphPad Software,
Inc., La Jolla, CA, United States). A p-value < 0.05 was considered as
statistically significant.
(23) Delmulle, L.; Bellahcene, A.; Dhooge, W.; Comhaire, F.;
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AUTHOR INFORMATION
Corresponding Author
Tel: +41 61 467 46 89. Fax: +41 61 467 47 01. E-mail:
veronika.butterweck@fhnw.ch.
(27) Nikolic, D.; Li, Y.; Chadwick, L. R.; Grubjesic, S.; Schwab, P.;
■
*
Metz, P.; van Breemen, R. B. Drug Metab. Dispos. 2004, 32, 272−279.
(28) Nikolic, D.; Li, Y.; Chadwick, L. R.; van Breemen, R. B. Pharm.
Res. 2006, 23, 864−872.
29) Pang, Y.; Nikolic, D.; Zhu, D. W.; Chadwick, L. R.; Pauli, G. F.;
Farnsworth, N. R.; van Breemen, R. B. Mol. Nutr. Food Res. 2007, 51,
72−879.
30) van Breemen, R. B.; Yuan, Y.; Banuvar, S.; Shulman, L. P.; Qiu,
(
ORCID
8
(
Veronika Butterweck: 0000-0001-7516-8789
Notes
The authors declare no competing financial interest.
X.; Alvarenga, R. F.; Chen, S. N.; Dietz, B. M.; Bolton, J. L.; Pauli, G.
F.; Krause, E.; Viana, M.; Nikolic, D. Mol. Nutr. Food Res. 2014, 58,
1
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