CDr10b inhibits the expression of cyclooxygenase-2 and inducible nitric oxide synthase induced by lipopolysaccharide

Article abstract of DOI:10.1016/j.ejphar.2014.08.036

The pathophysiological processes of inflammation can lead to a host of diseases, such as periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer. The dysregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activation play important roles in the development of certain inflammatory diseases. Here, we investigated the effects of CDr10b which is originally developed for a microglia staining probe on inflammation, by modulating NF-κB activation and iNOS and COX-2 expression induced by lipopolysaccharide (LPS) in murine macrophages. The CDr10b suppressed NF-κB activation and iNOS and COX-2 expression induced by LPS. All the results suggest that CDr10b is a promising novel agent for the treatment of inflammatory diseases.

Full text of DOI:10.1016/j.ejphar.2014.08.036

European Journal of Pharmacology 742 (2014) 42–46
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
CDr10b inhibits the expression of cyclooxygenase-2 and inducible
nitric oxide synthase induced by lipopolysaccharide
Gyo-Jeong Gu a,1, Se-Jin Lim b,1, Sang-il Ahn a, Sung-Chan Lee c, Young-Tae Chang d,e
,
Tae Hyun Choi f, Byoung Soo Kim f, Yong-Bin Eom b, Na Kyung Lee b, Hyung-Sun Youn a,b,
n
a Departments of Medical Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 336-745, Republic of Korea
b Department of Biomedical Laboratory Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 336-745, Republic of Korea
c Aptabio Therapeutics Inc., Kyunggi-do, Yong-in city 446-908, Republic of Korea
d Department of Chemistry and MedChem Program, Life Sciences Institute, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
e Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium (SBIC), 11 Biopolis Way, #02-02 Helios, Agency for Science, Technology
and Research (A
nSTAR), Biopolis, Singapore 138667, Singapore
f Department of Imaging, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The pathophysiological processes of inflammation can lead to a host of diseases, such as periodontitis,
atherosclerosis, rheumatoid arthritis, and even cancer. The dysregulated inducible nitric oxide synthase
(iNOS) and cyclooxygenase-2 (COX-2) activation play important roles in the development of certain
inflammatory diseases. Here, we investigated the effects of CDr10b which is originally developed for a
Received 12 May 2014
Received in revised form
19 August 2014
Accepted 24 August 2014
Available online 6 September 2014
microglia staining probe on inflammation, by modulating NF-
expression induced by lipopolysaccharide (LPS) in murine macrophages. The CDr10b suppressed NF-
κ
B activation and iNOS and COX-2
κB
Keywords:
Inflammation
activation and iNOS and COX-2 expression induced by LPS. All the results suggest that CDr10b is a
promising novel agent for the treatment of inflammatory diseases.
& 2014 Elsevier B.V. All rights reserved.
Nuclear factor-κB
Cyclooxygenase-2
Inducible nitric oxide synthase
CDr10b
1. Introduction
NOS, which catalyzes the conversion of L-arginine to nitric
oxide, is classified into several isoforms, based on the cell type or
the location and manner of expression, such as endothelial NOS
(eNOS), neuronal NOS (nNOS), and a macrophage or inducible NOS
(iNOS) (Murakami and Ohigashi, 2007). The eNOS and nNOS are
constitutively active enzymes in vascular endothelial cells and
neuronal tissues. On the other hand, iNOS enzymes are induced
under both normal and pathological conditions in a variety of cell
types, including macrophages, keratinocytes, hepatocytes, astro-
cytes, and microglial cells. Since dysregulated iNOS expression
might be intimately involved in the development of certain
inflammatory diseases, iNOS is a very important therapeutic target
in the development of anti-inflammatory drugs (Vallance, 2003).
Cyclooxygenase (COX), which catalyzes the conversion of
arachidonic acid to prostanoids, is the molecular target for anti-
inflammatory remedies (Rouzer and Marnett, 2009). The COX
consists of at least two isoforms: COX-1 and COX-2. The COX-1
enzyme is constitutively expressed in many normal tissues. In
contrast, although COX-2 is undetectable in most of the normal
mammalian tissues, it is an inducible enzyme expressed in them in
response to pathophysiological stimuli including ultraviolet light,
dioxin, and lipopolysaccharide (LPS) (Murakami and Ohigashi,
2007). It is reported that the expression of COX-2 is regulated at
Toll-like receptors (TLRs) play an important role in recognition
of conserved pathogen-associated molecular patterns (PAMPs)
derived from various microbial pathogens including viruses, bac-
teria, protozoa and fungi, and in the subsequent initiation of innate
immune responses (Akira and Takeda, 2004; Medzhitov et al.,
1997). The activation of TLRs by agonists can induce inflammatory
responses that are key etiological conditions for the development
of many chronic inflammatory diseases.
Inflammation, which is mediated by multiple molecular mechan-
isms, refers to the pathological and physiological processes known to
be involved in numerous diseases. Two of the most important
enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-
2 (COX-2), have important roles in the development of certain
inflammatory diseases (Moncada, 1999; Turini and DuBois, 2002).
n
Corresponding author at
: Department of Biomedical Laboratory Science,
College of Medical Sciences, Soonchunhyang University, Chungnam, Asan-Si
336-745, Republic of Korea. Tel.: þ82 41 530 3086; fax: þ82 41 530 3085.
E-mail address: hyoun@sch.ac.kr (H.-S. Youn).
1
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2014.08.036
0014-2999/& 2014 Elsevier B.V. All rights reserved.

G.-J. Gu et al. / European Journal of Pharmacology 742 (2014) 42–46
43
Fig. 1. (A) The structure of CDr10b. (B) Excitation/emission spectra of CDr10b.
transcriptional and post-transcriptional levels by pro-inflammatory
2.4. Plasmids
agents, cytokines, growth factors, and tumor-promoters (Alvarez
et al., 2005; Antman et al., 2005).
Previously, we reported the development of a Compound of
Designation red 10 binding (CDr10b) (Fig. 1) for the isolation and
imaging of live microglia, when investigating their roles in
neuroinflammatory diseases (Leong et al., 2014). Herein, we report
The NF-
Frank Mercurio (Signal Pharmaceuticals, San Diego, CA, USA). The
heat shock protein 70 (HSP70)- -galactosidase reporter plasmid
was purchased from Robert Modlin (University of California,
Los Angeles, CA, USA). The COX-2 and iNOS luciferase reporter
constructs were obtained from Dr. Daniel Hwang (University of
California, Davis, CA, USA). A wild type of MyD88 was provided by
J. Tschopp (University of Lausanne, Lausanne, Switzerland). The
κ
B (2x)-luciferase reporter construct was provided by
β
immunomodulatory activity of CDr10b in the context of NF-
activation induced by LPS (TLR4 agonist) in murine macrophages.
The CDr10b suppressed NF- B activation and iNOS and COX-2
κ
B
κ
expression induced by LPS. These results suggest that CDr10b can
be developed as a potent anti-inflammatory drug.
wild-type IKK
β
was obtained from M. Karin (University of Cali-
fornia, San Diego, CA). The wild type of p65 was obtained from
J. Ye (Pennington Biomedical Research Center, Baton Rouge, LA). All
DNA constructs were prepared in large scale for transfection using
an EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA, USA).
2. Materials and methods
2.1. Synthetic procedure of CDr10b
2.5. Transfection and reporter gene luciferase assay
CDr10b was synthesized by following the established proce-
dure (Leong et al., 2014). The 8-(4-Aminophenyl)-4,4-difluoro-1,
3-dimethyl-4-bora-3a,4a-diaza-s-indacene (aminophenyl bod-
ipy) (100 mg, 320 mmole) and 3-ethoxy-4-methoxybenzaldehyde
(86 mg, 480 mmole) were dissolved with acetonitrile (50 ml). Then,
3 eq. of pyrrolidine and acetic acid was added to the reaction mixture.
The reaction mixture was heated to 60 1C for 3 h. After completion of
the reaction, NaHCO3 saturated solution (500 ml) and chloroacetyl
chloride 200 ml were added to the reaction mixture and stirred
vigorously for 5 min. The crude compound was purified using column
chromatography with MeOH/DCM eluent system (0–5% of MeOH),
and 148 mg of product was obtained. The spectroscopic properties
and analytical data were identical as the reference.
The assays were performed as previously described (Youn et al.,
2005, 2006b). RAW264.7 cells were co-transfected with a luciferase
plasmid and a plasmid containing heat shock protein (HSP)70- -
galactosidase as an internal control, using SuperFect transfection
reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's
β
instructions and then pretreated with 20 or 50 μM CDr10b for 1 h
and then treated with LPS (10 ng/ml) for an additional 8 h. Cell
lysates were prepared and luciferase enzyme activities were deter-
mined using the Luciferase Assay System (Promega, Madison, WI,
USA) according to the manufacturer's instructions. The luciferase
activity was normalized by -galactosidase activity.
β
2.2. Reagents
2.6. Immunoblotting
LPS was obtained from List Biological Laboratories (San Jose, CA,
USA), and it was dissolved in endotoxin-free water. All other reagents
were purchased from Sigma-Aldrich, unless otherwise indicated.
Immunoblotting was performed as previously described (Youn
et al., 2006a, 2006c). Equal amounts of cell extracts were subjected to
8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and
the separated proteins were electrotransferred to a polyvinylidene
difluoride membrane. The membrane was blocked to prevent non-
specific binding of antibodies in phosphate-buffered saline contain-
ing 0.1% Tween-20 and 3% nonfat dry milk. The immunoblotting was
performed with the indicated antibodies, and the secondary anti-
bodies were conjugated to horseradish peroxidase (Amersham
Biosciences, Arlington Heights, IL, USA). The reactive bands were
visualized with the enhanced chemiluminescence Western blot
detection reagents (Intron, Seongnam, Gyeonggi-do, South Korea).
2.3. Cell culture
RAW 264.7 cells (a murine monocytic cell line; ATCC TIB71) and
293T human embryonic kidney cells were cultured in Dulbecco's
modified Eagle's medium (DMEM) containing 10% (v/v) heat-
inactivated fetal bovine serum (FBS), 100 units/ml Penicillin (Invitro-
gen, Carlsbad, CA, USA), and 100
Cells were maintained at 37 1C in a 5% CO2/95% air environment.
μ
g/ml Streptomycin (Invitrogen).

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G.-J. Gu et al. / European Journal of Pharmacology 742 (2014) 42–46
2.7. Cell viability test
2.8. Data analysis
The cell viability was assessed using MTS [3-(4,5-dimethylthia-
zol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet-
razolium, inner salt] based colorimetric assay. RAW264.7 cells
The data were obtained from triplicate experiments. The values
are expressed as the mean7standard error of the mean (S.E.M.).
were treated with CDr10b (20, 50, 100 M) for 4 h. Twenty
μ
3. Results
microliters of the CellTiter 96 AQueous One Solution Reagent
was added directly to culture wells. The plate was incubated at
37 1C for 4 h in a humidified, 5% CO2 atmosphere. The absorbance
was recorded at 490 nm with a 96-well plate reader.
3.1. CDr10b inhibits LPS-induced NF-κB activation
To evaluate the cytotoxic nature of CDr10b in RAW 264.7 cells,
the MTS viability assay was used. It was apparent that CDr10b was
not cytotoxic at the tested concentrations of 20–100 M. There-
μ
fore, the cellular toxicity could be excluded from the possible
reason for any observed changes in the subsequent study.
Next, we determined the effect of CDr10b on LPS-induced
NF-
The CDr10b inhibited NF-
(Fig. 2). The NF- B is activated in response to stimuli of various
pathogens (Hacker and Karin, 2006). Therefore, the suppression of
NF- B activation is considered as an important strategy for anti-
κ
B activation, by using the NF-
κ
B luciferase reporter assay.
κB activation which was induced by LPS
κ
κ
inflammation and anti-cancer therapies.
3.2. CDr10b suppresses LPS-induced iNOS expression
The next experiment assessed the capability of CDr10b to
regulate iNOS expression, which is one of the target genes
regulated through NF-κB activation in macrophages. The CDr10b
suppressed the iNOS expression that was induced by LPS in
RAW264.7 cells, as determined by iNOS-luciferase reporter assay
(Fig. 3A). The CDr10b also suppressed the iNOS protein that was
induced by LPS, as determined by iNOS immunoblotting assay
Fig. 2. CDr10b inhibits LPS-induced NF-κB activation. NF-κB luciferase assay in
nn
RAW264.7 cells. Values are expressed as mean7S.E.M. (n¼3).
denotes sig-
nificantly different from LPS alone, Po 0.01. Veh, vehicle.
Fig. 3. CDr10b inhibits LPS-induced iNOS expression. (A) iNOS luciferase assay in
Fig. 4. CDr10b inhibits LPS-induced COX-2 expression. (A) COX-2 luciferase assay in
nn
nn
RAW264.7 cells. Values represent the mean7S.E.M. (n¼3).
Significantly differ-
RAW264.7 cells. Values represent the mean7S.E.M. (n¼3).
Significantly differ-
ent from LPS alone, Po 0.01. (B) RAW264.7 cells were pretreated with 20 or 50 μM
CDr10b for 1 h and then further stimulated with LPS (10 ng/ml) for 8 h. Cell lysates
were analyzed for iNOS and β-actin protein by immunoblots. Veh, vehicle.
ent from LPS alone, Po0.01. (B) RAW264.7 cells were pretreated with 20 or 50 μM
CDr10b for 1 h and then further stimulated with LPS (10 ng/ml) for 8 h. Cell lysates
were analyzed for COX-2 and β-actin protein by immunoblots. Veh, vehicle.

G.-J. Gu et al. / European Journal of Pharmacology 742 (2014) 42–46
45
Fig. 5. CDr10b suppresses MyD88-dependent signaling pathway of TLR4. (A–C) 293T cells were transfected with NF-κB luciferase reporter plasmid and the expression
plasmid of MyD88 (A), IKKβ (B) or p65 (C). Cells were further treated with 20 or 50 μM CDr10b for 18 h. Relative luciferase activity was normalized with β-gal activity. Values
represent mean7S.E.M. (n¼3). þdenotes a result that is significantly different from MyD88 alone, Po0.01 (þ þ) (A). # denotes a result that is significantly different from
n
nn
IKKβ alone, Po0.01 (##) (B). denotes a result that is significantly different from p65 alone, Po0.01 ( ) (C). Veh, vehicle.
(Fig. 3B). All of the results suggested that CDr10b inhibited iNOS
expression induced by LPS.
the overexpression of MyD88, IKK
suppressed the agonist-independent activation of NF-
by MyD88 (Fig. 5A), IKK (Fig. 5B) or p65 (Fig. 5C), demonstrating
β
, or p65 in 293T cells. CDr10b
κB induced
β
that CDr10b suppresses MyD88-dependent signaling pathways.
3.3. CDr10b suppresses LPS-induced COX-2 expression
Next, we determined whether CDr10b could regulate COX-2
4. Discussion
expression, which is another target gene regulated through NF- B
κ
activation. The CDr10b suppressed the COX-2 expression that was
induced by LPS in RAW264.7 cells, as determined by COX-2-
luciferase reporter assay (Fig. 4A). The CDr10b also suppressed the
COX-2 protein that was induced by LPS, as determined by COX-2
immunoblotting assay (Fig. 4B).
Toll-like receptor 4 (TLR4) is the first TLR identified that
recognizes LPS, which is a cell wall component of gram-negative
bacteria (Miyake, 2004). The LPS-induced TLR4 activation leads
to both early and late NF-
ential factor 88 (MyD88)- or Toll/iInterleukin-1 receptor (TIR)
domain-containing adapter inducing interferon- (TRIF)-depen-
κB activation through myeloid differ-
3.4. CDr10b suppresses MyD88-dependent signaling pathway
of TLRs
β
dent signaling pathways, respectively (Takeda and Akira, 2005).
MyD88 is the immediate adapter molecule which is common to all
TLRs, with the exception of TLR3. The MyD88 recruits the IL-1
receptor-associated kinase (IRAK), tumor necrosis factor (TNF), and
To further investigate the regulation of MyD88-dependent
signaling pathways by CDr10b, NF- B activation was induced by
κ

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G.-J. Gu et al. / European Journal of Pharmacology 742 (2014) 42–46
receptor-associated factor 6 (TRAF6), leading to the activation of
the canonical IKK complex. In the un-stimulated cells, NF- B is
sequestered in the cytoplasm as an inactive form by interaction
with an inhibitor I proteins, preventing its nuclear accumula-
tion and transcriptional activation of the target genes. The IKK
complex phosphorylates I which is degraded by the 26S
proteasome, allowing NF- B to translocate into the nucleus. The
translocated NF- B binds to DNA and up-regulates the expressions
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Acknowledgments
This study was supported by Soonchunhyang University
research fund and by Basic Science Research Program, through
the National Research Foundation of Korea, funded by the Ministry
of Education, Science, and, Technology (2010-0023637).
Youn, H.S., Lee, J.Y., Saitoh, S.I., Miyake, K., Kang, K.W., Choi, Y.J., Hwang, D.H., 2006b.
Suppression of MyD88- and TRIF-dependent signaling pathways of Toll-like
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