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Curcumin, bisdemethoxycurcumin and dimethoxycurcumin
complexed with cyclodextrins have structure specific effect
on the paracellular integrity of lung epithelia in vitro
Berglind Eva Benediktsdottir
a
, Olafur Baldursson
b
, Thorarinn Gudjonsson
c
,
Hanne Hjorth Tønnesen
d
, Mar Masson
a,
n
a
Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland, Hofsvallagata 53, IS-107 Reykjavik, Iceland
b
Department of Pulmonary Medicine, Landspitali-The National University Hospital of Iceland, Eiríksgata 5, IS-101 Reykjavík, Iceland
c
Biomedical Center, School of Health Sciences, University of Iceland, Vatnsmýrarvegur 16, IS-101 Reykjavík, Iceland
d
School of Pharmacy, Dept. of Pharmaceutics, University of Oslo, Blindern, 0136 Oslo, Norway
article info
Article history:
Received 18 June 2015
Received in revised form
31 October 2015
Accepted 5 November 2015
Available online 10 November 2015
Keywords:
Bronchial epithelium
Cyclodextrin
Curcumin
Epithelial integrity
TER
VA10
abstract
The phytochemical curcumin may improve translocation of the cystic fibrosis transmembrane regulatory
(CFTR) protein in lung epithelium and therefore be helpful in the treatment of cystic fibrosis (CF)
symptoms. However, previous studies often use commercial curcumin that is a combination of curcumin,
demethoxycurcumin and bisdemethoxycurcumin which could affect the investigated cells differently. In
the present study, we investigated the potential difference between curcumin, bisdemethoxycurcumin
and dimethoxycurcumin on the epithelial tight junction complex, in the bronchial epithelial cell line
VA10, by measuring transepithelial electrical resistance (TER), immunofluorescence and western blotting
of tight junction proteins. The curcuminoids were complexed with hydroxypropyl-
γ
–cyclodextrin for
increased solubility and stability. Curcumin (10 mg/ml) increased the TER significantly after 24 h of
treatment while four times higher concentration of bisdemethoxycurcumin was required to obtain si-
milar increase in TER as curcumin. Interestingly, dimethoxycurcumin did not increase TER. Curcumin
clearly affected the F-actin structures both apically and basolaterally. These results begin to define
possible effects of curcuminoids on healthy bronchial epithelia and shows that difference in the phenyl
moiety structure of the curcuminoids influences the paracellular epithelial integrity.
& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Curcumin is a phytochemical, found in the dried rhizome of the
plant Curcuma longa L. The dried rhizome, called turmeric, is often
used as spice and is a common ingredient in curry powder.
Amount of curcumin in turmeric is commonly around 2–8% [1].In
Southeast Asia, turmeric is not only used as a spice or a coloring
agent but is also used to externally treat wounds, inflammation
and tumors, liver-and gall diseases among other illnesses. Curcu-
min has been studied from a pharmaceutical perspective regard-
ing its antioxidant, anti-inflammatory and anti-cancer properties
[2]. It is currently one of nearly twenty possible therapies against
cystic fibrosis (CF) in development according to Cystic Fibrosis
Foundation. A phase I clinical trial, to assess safety and dosage
parameters when given to CF patients, has been initiated. CF is a
lethal, hereditary disease caused by a mutation in the gene that
codes for the cystic fibrosis transmembrane regulator (CFTR)
chloride channel protein [3–5] causing the misfolded CFTR protein
to be degraded [6]. This disease is characterized by chronic re-
spiratory infections and inflammation and irrespective of in-
creased knowledge of the CF pathology, the mean predicted sur-
vival of CF patients is around 40 years [7]. Studies have shown that
if the mutant CFTR protein could relocate from the endoplasmic
reticulum to the plasma membrane, it could restore the chloride
pump activity [8].
Curcumin may improve the translocation of the CFTR chloride
channel protein in lung epithelium [9] although recent studies
have not been able to confi rm those results [10–12]. Berger and
colleagues discovered that curcumin stimulated the activity of the
CFTR channels by elongating the channel opening time and these
effects were dose dependent, reversible and ATP dependent [13].
Curcumin also increased the chloride transport of CFTR channels
in normal lung epithelia but was unsuccessful in the defected CFTR
channels [13]. Similarly, it has been reported that curcumin opens
CFTR channels but unlike the study by Berger, the CFTR opening
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/bbrep
Biochemistry and Biophysics Reports
http://dx.doi.org/10.1016/j.bbrep.2015.11.004
2405-5808/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Abbreviations: CDs, cyclodextrins; CF, cystic fibrosis; CFTR, cystic fibrosis trans-
membrane regulator; TER, transepithelial electrical resistance; TJs, tight junctions
n
Corresponding author. Fax: þ 354 525 4071.
E-mail address: mmasson@hi.is (M. Masson).
Biochemistry and Biophysics Reports 4 (2015) 405–410
was not dependent on ATP [14].
Tight junctions (TJs) control paracellular ion- and water
transport and are necessary for the tightness of the epithelium and
are a key part in lung defenses [15]. CF patients often acquire
chronic pulmonary infections by the bacteria Pseudomonas aeru-
ginosa that disrupts the epithelial barrier integrity [16,17]. The
macrolide antibiotic azithromycin has proven beneficial with CF
patients in concentrations not affecting the bacterial count [18,19].
Interestingly, this macrolide also increased the paracellular in-
tegrity in normal bronchial epithelial cells [20] and protected the
epithelium during P. aeruginosa infection at concentrations not
affecting the bacterial count [21]. When considering the potential
role of curcumin in CF pathogenesis, it is important not only to
consider the CFTR translocation but also the effect on paracellular
integrity of normal epithelium.
Most studies use commercial grade curcumin [14,22,23] which
is composed of curcumin (75–85%), demethoxycurcumin (10–20%)
and bisdemethoxycurcumin (5%) [24]. As a result, valid concerns
arise regarding the use of this curcumin combination since the
effects of one curcuminoid could be masked by other curcumin
components. Additionally, previous limitations with the use of
curcumin is its poor solubility at acid and physiological pH and
rapid hydrolysis at basic pH [25]. This results in the use of DMSO
and/or ethanol as a solvents [22,23,26], which can have adverse
effects on the investigated cells [27,28]. By complexing curcumins
with cyclodextrins (CDs) in aqueous solutions [29,30], the solubi-
lity and stability can be increased [30,31]. Here, we investigate
whether curcumin and the curcuminoids dimethoxycurcumin and
bisdemethoxycurcumin (Fig. 1 ), complexed with CDs, increase the
paracellular epithelial integrity and if those effects are different
between derivatives.
2. Materials and methods
2.1. Curcuminoid solutions
Curcumin (diferuloylmethane) and curcuminoids (bisde-
methoxycurcumin and dimethoxycurcumin) were synthesized and
characterized as previously described [31,32]. The hydroxypropyl-
γ
–CD (HP
γ
CD) improves the solubility of curcumin and the
curcuminoids dimethoxycurcumin and bisdemethoxycurcumin
[31] and was therefore chosen as a complexing agent. The stock
solutions of the curcumin and the curcuminoids contained 200 m g/
ml curcumin compound, 5% v/v dimethylsulfoxide (DMSO) and
15% w/v hydroxypropyl-
γ
-cyclodextrin (HP-
γ
–CD, Cavasol
s
WB
HP Pharma, Wacker- Fine Chemicals, Germany) in phosphate
buffer saline (PBS) and were all stored away from light in at 5–8 °C.
Test solutions were prepared by dissolving appropriate amounts of
stock solutions into the DMEM-F12þ Ultroser-G cell culture med-
ium. Amount of CD/DMSO solution that curcumin and curcumi-
noids were dissolved in was also tested for possible effects on TER
and TJs.
2.2. Cell culture
The newly established and validated bronchial epithelial cell
line, VA10 [33,34] was used between passages 13–20. The cells
were maintained in 75 cm
2
flasks in a humidified incubator at
37 °C (5% CO
2
) containing bronchial epithelial growth medium
(BEGM, Cambrex, East Rutherford, NJ, USA). Medium was aspirated
and changed every other day with a fresh, prewarmed medium.
The cells were seeded at the density of 1 10
5
cells/cm
2
on the
upper chamber of Transwell filters (pore size 0.4 mm, 12 mm dia-
meter, polyester membrane, Corning Costar Corporation) and
cultured in BEGM medium for 5-6 days, with 0.5 ml medium ad-
ded to the apical side and 1.5 ml medium to the basolateral side.
Subsequently, the cells were cultured in Dulbecco’s minimum es-
sential medium Ham’s F12 1:1 (DMEM/F-12) medium (Gibco,
Burlington, Canada) supplemented with 2% Ultroser G serum
substitute (Pall Life Sciences, Cergy-Saint-Christophe, France) and
penicillin/streptomycin. Medium was changed every other day. For
morphological analysis and western blot proteins analysis, the
cells were seeded on a 6 well plates (Falcon Multiwell 6 Well,
Becton Dickinson, NJ, USA) at 2 10
5
cells/well and cultured in
BEGM.
2.3. Transepithelial Electrical Resistance (TER) Measurement
TER of VA10 cell layers was measured with Millicell-ERS
volthometer (Millipore, MA, USA). The corrected TER value was
obtained after subtraction of the background from the cell-free
culture insert.
2.4. Immunocytochemistry
VA10 cells were fixed for 10 min with 3.7% formaldehyde,
permeabilized with 0.1% Triton X-100 for 5 min and then blocked
with 10% fetal bovine serum for 5 min. The following primary
antibodies were used (diluted in PBS): Mouse anti-human claudin-
1 (IgG
1
, 1:125), mouse anti-human ZO-1 (IgG
1
,1:50) and rabbit
anti-human occludin (1:20) and were all purchased from Zymed
(CA, USA). Cells were incubated with primary antibodies for
30 min followed by incubation with isotype specific Alexa Fluor
secondary antibodies (Invitrogen, Oregon, USA,1:1000) and To-
Pro-3 (Invitrogen) for nuclear staining (1:500) for 30 min. Alexa
Fluor 488 phallotoxin (Invitrogen) was used for F-actin staining
(1:40), incubated for 30 min.
2.5. Confocal microscope
Immunofluorescence images were obtained using Zeiss LSM
5 Pa confocal laser scanning microscope (CLSM, Carl Zeiss AG,
Munich, Germany) with Plan-Neofluar 20 ,40 and Plan-
Apochromat 63 oil immersion lenses. VA10 cell layers were
mounted with Fluoromount-G (SouthernBiotech, Birmingham,
USA) and coverslips before visualization.
Fig. 1. The curcuminoids curcumin, bisdemethoxycurcumin and dimethox-
ycurcumin have different substitutions on the phenyl ring.
B.E. Benediktsdottir et al. / Biochemistry and Biophysics Reports 4 (2015) 405–410406
2.6. Quantification
For quantification of F-actin fluorescence, images were cap-
tured with confocal microscopy at the focal plane where F-actin
apical staining was most prominent. All images used for quanti-
fication were acquired using the same confocal settings. Quanti-
fication using immunofluorescence images was performed using
Fiji (ImageJ) software.
2.7. Western blotting
After the cells grown in 6 well culture plates were treated with
PBS, curcumin or curcuminoids, they were lysed in RIPA buffer
containing a protease inhibitor cocktail (Aprotinin, PMSF and
Na
3
VO
4
). The cells were then scraped from the filters and soni-
cated for 2 min followed by centrifugation at 12,000 g for 20 min
at 4 °C. The supernatant was collected and the protein con-
centration determined with the Bradford assay. Equal amounts of
proteins, as determined by the Bradford method [35], were loaded
and run on a NuPAGE 10% Bis-Tris gel (Invitrogen, Carlsbad, USA)
and transferred to a polyvinylidine difluoride membrane (In-
vitrogen). The membranes were blocked for 1 h with 5% skimmed
milk in 0.25% Tween/PBS and incubated with the primary anti-
bodies mouse anti-E-cadherin (BD Bioscience, IgG2a 1:330),
mouse anti-claudin-1 (IgG
1
,1:500) or rabbit anti-occludin (1:1000)
overnight, followed by incubation with secondary antibodies
(1:2000) for 1 h. Protein bands were visualized using enhanced
chemiluminescence system and Hyperfilm (Amersham Bioscience,
England).
2.8. Morphological analysis
After reaching confluency the cells were incubated with cur-
cumin or curcuminoids. Images were captured on day 1, 4 and
6 after beginning of treatment using Leica DFC320 digital imaging
system.
2.9. Calculations and data analysis
All data were reported as mean7standard deviation with n
representing number of experiments. Unpaired, two tailed Stu-
dent's t-test was done in GraphPad to compare two means with
the difference considered to be statistically significant when
po 0.05.
3. Results and discussion
3.1. Curcumin and bisdemethoxycurcumin increase transepithelial
electrical resistance (TER) significantly in human airway epithelia in
vitro
To determine if the curcuminoids increase the paracellular in-
tegrity of epithelia and consequently be beneficial for haltering the
CF progression, TER was measured. Indeed, addition of 10 mg/mL
curcumin to the basolateral side of the epithelium every other day
resulted in significant increase in TER after 24 h treatment
(Fig. 2A), 3-fold increase compared to initial value. The increase in
TER was not observed in the initial period of the measurements of
60 min (data not shown). After 6 days of treatment, the TER in-
crease had leveled off at around 10-fold increase from initial value.
Curcumin at 1 mg/mL did not increase TER as can be seen in Fig. 2A.
The increase in TER was not as apparent with 40 mg/mL curcumin,
reaching significant higher TER levels compared to control after
5 days of treatment (Fig. 2B). The CD/DMSO control solution did
not affect the TER values compared to normal control epithelium
(Fig. 2A).
To explore whether the effects of curcumin on TER were spe-
cific to the curcumin structure, two other curcuminoids, bisde-
methoxycurcumin and dimethoxycurcumin, were investigated
(Fig. 2C and D). Interestingly, these curcuminoids had markedly
different effects on the bronchial epithelia. Two days of treatment
with 40 mg/mL bisdemethoxycurcumin resulted in significant
Fig. 2. Different effects of curcumin and curcuminoids on TER in human airway epithelia in vitro. Human airway epithelial cells were cultured on Transwell permeable
support filters. After reaching confluency, curcumin (A, B), bisdemethoxycurcumin (C) or dimethoxycurcumin (D) were added to the basolateral side of the epithelia. TER was
measured using a Millicell-ERS electrical resistance system. Data are given as mean 7 SD (n¼ 3).
B.E. Benediktsdottir et al. / Biochemistry and Biophysics Reports 4 (2015) 405–410 407
increase in TER that leveled off after 4 days of treatment (Fig. 2C).
Conversely, dimethoxycurcumin did not affect the TER values in
any of the concentrations investigated (Fig. 2D). After 6 days of
treatment with this curcuminoid, the epithelial lining appeared to
be less continuous than the control epithelium (Fig. A1), sug-
gesting possible adverse effects on the epithelium. The dose de-
pendent effect of curcumin and the different effects of curcumin
and the other curcuminoids on TER measurements indicate a
possible agonistic/antagonistic activity that warrants further
exploration.
3.2. Curcumin affects F-actin localization but not expression of the TJ
proteins claudin-1 and occludin and the adherens protein E-cadherin
The TJs are membrane bound proteins that produce apical to
basolateral polarity [36] and form a paracellular permeability
barrier that limits the permeation to small, uncharged solutes [37].
The TJ complex is dynamic in nature, with its junctional proteins
affected by various internal and external stimuli [38]. TER is con-
sidered a good indicator of the functional activity of the tight
junctions [39]. Since curcumin increased TER, it could possibly
affect the expression or localization of TJ proteins or related
components. Intracellularly, there are TJ associated proteins such
as the members of the ZO family that connect to the actin cytos-
keleton which is often affected when the TJ complex is altered
(reviewed in [40]). In particular, the actin cytoskeleton is involved
in modifications of the tight junction barrier [38] with redis-
tribution of actin filaments been observed to be crucial to the in-
duced barrier formation of endothelial cells by sphingosine
1-phosphate [41]. As can be seen in Fig. 3A and B, altered staining
patterns of both apical and basolateral F-actin was observed after
treatment with curcumin. The total amount of apical F-actin fibers,
as determined by quantification of fluorescence, was significantly
reduced after treatment with 1
μ
g/ml curcumin and this reduction
was highly significant after treatment with 10
μ
g/ml curcumin as
Fig. 3C shows. Additionally, basolateral actin was not as fila-
mentous compared to the control epithelium (Fig. 3B). Curcumin is
a known upstream inhibitor of NF-
κ
B [42], a transcription factor
that has been shown to interact with the actin cytoskeleton
[43,44]. A possible relationship between the actin rearrangement
and increased TER observed in the current study with the known
inhibition of the NF-
κ
B pathway could therefore be possible.
Curcumin affects the localization of F-actin filaments. Bronchial
epithelial cells were treated with control, CD/DMSO vehicle or
curcumin (1 and 10 mg/mL) for 14 days on Transwell filters. Cur-
cumin at 10 mg/mL clearly affects both apical (A) and basolateral
(B) actin filaments. C) Quantification of the F-actin fluorescent
intensity at the apical sites shows significant decrease in apical
F-actin staining after treatment of 1
μ
g/ml and 10
μ
g/ml curcumin.
Values represent relative fluorescent intensity in arbitrary units
(AU), n¼ 5–6 and are expressed as mean7 SD. * po 0.05 compared
to control, ** po 0.005. D) Expression of occludin, claudin-1 and
E-cadherin after curcumin treatment. Bronchial epithelial cells
were treated with control, CD/DMSO vehicle or curcumin
(10 mg/mL), for 4, 10 and 14 days and the expression determined by
western blotting. Claudin-1 expression appears to be gradually
changed for both the CD/DMSO control and curcumin treatment.
To further explore these possible effects on the TJ complex, the
expression of occludin, claudin-1 and E-cadherin was investigated
after treatment with 10 mg/ml curcumin (Fig. 3B). Western blotting
of occludin revealed a double band at 60 kDa but no alterations
in the expression at the different time points investigated.
E-cadherin is a membrane spanning adhesion protein, essential for
formation and maintenance of normal epithelium [45]. Although
investigations have indicated that curcumin may down-regulate
its expression [46] this was not observed in the current study.
Claudins are one of the main components of the tight junction
complex that decide both TER and charge specificity [47,48]. Over
twenty claudins have been identified and their expression pattern
is specific for each epithelial type [49,50]. Thus it is indicated that
different composition of claudins attribute to different properties
of epithelial tissues [47]. Watari and colleagues reported that
curcumin (3.7 mg/ml) was a claudin-4 inducer, with concomitant
increase in TER of 170% of initial value after 48 h of treatment
[51]. In the current study, both the CD/DMSO vehicle and curcumin
(10 mg/ml) appeared to reduce the expression of claudin-1, there-
fore the direct effects of curcumin on the claudin-1 expression
remain inconclusive.
Fig. 3. The effects of curcumin on adherens and tight junction proteins.
B.E. Benediktsdottir et al. / Biochemistry and Biophysics Reports 4 (2015) 405–410408
All the curcuminoids had different effect on TER in the bron-
chial epithelia. Curcumin was more efficient in increasing TER
compared to bisdemethoxycurcumin, while dimethoxycurcumin
did not affect TER. Rearrangement of the basolateral F-actin stress
fibers and its decreased staining at the apical surface was observed
after curcumin treatment. These results show that different cur-
cuminoids can have different effects on the epithelia, indicating
that future studies would benefit from using pure curcuminoids.
The effects of curcumin towards the increased bronchial para-
cellular integrity could be a part of its beneficial effects in treat-
ment of CF.
Acknowledgment
Financial support from the University of Iceland Research fund
is gratefully acknowledged.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bbrep.2015.11.004.
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