Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-??B through Akt and p38 inhibition in RAW 264.7 macrophage cells

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DOI: 10.1016/j.ejphar.2006.06.047 · Source: PubMed
Cite this publication
Abstract
Cordyceps militaris, a caterpillar-grown traditional medicinal mushroom, produces an important bioactive compound, cordycepin (3'-deoxyadenosine). Cordycepin is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, anti-virus and anti-infection activities. The molecular mechanisms of cordycepin on pharmacological and biochemical actions of macrophages in inflammation have not been clearly elucidated yet. In the present study, we tested the role of cordycepin on the anti-inflammation cascades in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. In LPS-activated macrophage, nitric oxide (NO) production was inhibited by butanol fraction of C. militaris and the major component of C. militaris butanol faction was identified as cordycepin by high performance liquid chromatography. To investigate the mechanism by which cordycepin inhibits NO production and inducible nitric oxide synthase (iNOS) expression, we examined the activation of Akt and MAP kinases in LPS-activated macrophage. Cordycepin markedly inhibited the phosphorylation of Akt and p38 in dose-dependent manners in LPS-activated macrophage. Moreover, cordycepin suppressed tumor necrosis factor (TNF-alpha) expression, IkappaB alpha phosphorylation, and translocation of nuclear factor-kappaB (NF-kappaB). The expressions of cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were significantly decreased in RAW 264.7 cell by cordycepin. Taken together, these results suggest that cordycepin inhibits the production of NO production by down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB activation, Akt and p38 phosphorylation. Thus, cordycepin may provide a potential therapeutic approach for inflammation-associated disorders.
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Cordycepin inhibits lipopolysaccharide-induced inf lammation by the
suppression of NF-κB through Akt and p38 inhibition in
RAW 264.7 macrophage cells
Ho Gyoung Kim
a,f
, Bhushan Shrestha
b
, So Yeon Lim
e
, Deok Hyo Yoon
a
, Woo Chul Chang
e
,
Dong-Jik Shin
e
, Sang Kuk Han
c
, Sang Min Park
a
, Jung Hee Park
a
, Hae Il Park
d
, Jae-Mo Sung
c
,
Yangsoo Jang
e
, Namsik Chung
e
, Ki-Chul Hwang
e,
, Tae Woong Kim
a,
a
Department of Biochemistry, Kangwon National University, Chunchon 200-701, Republic of Korea
b
Green Energy Mission/Nepal, P.O. Box 10647, Kathmandu, Nepal
c
Division of Applied Biology, Kangwon National University, Chunchon 200-701, Republic of Korea
d
Division of Pharmacology, Kangwon National University, Chunchon 200-701, Republic of Korea
e
Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea
f
Mushtech Co. Ltd, Chunchon, Republic of Korea
Received 13 December 2005; received in revised form 16 June 2006; accepted 22 June 2006
Available online 28 June 2006
Abstract
Cordyceps militaris, a caterpillar-grown traditional medicinal mushroom, produces an important bioactive compound, cordycepin (3-
deoxyadenosine). Cordycepin is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, anti-virus
and anti-infection activities. The molecular mechanisms of cordycepin on pharmacological and biochemical actions of macrophages in
inflammation have not been clearly elucidated yet. In the present study, we tested the role of cordycepin on the anti-inflammation cascades in
lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. In LPS-activated macrophage, nitric oxide (NO) production was inhibited by
butanol fraction of C. militaris and the major component of C. militaris butanol faction was identified as cordycepin by high performance liquid
chromatography. To investigate the mechanism by which cordycepin inhibits NO production and inducible nitric oxide synthase (iNOS)
expression, we examined the activation of Akt and MAP kinases in LPS-activated macrophage. Cordycepin markedly inhibited the
phosphorylation of Akt and p38 in dose-dependent manners in LPS-activated macrophage. Moreover, cordycepin suppressed tumor necrosis
factor (TNF-α) expression, IκB alpha phosphorylation, and translocation of nuclear factor-κB (NF-κB). The expressions of cycloxygenase-2
(COX-2) and inducible nitric oxide synthase (iNOS) were significantly decreased in RAW 264.7 cell by cordycepin. Taken together, these results
suggest that cordycepin inhibits the production of NO production by down-regulation of iNOS and COX-2 gene expression via the suppression of
NF-κB activation, Akt and p38 phosphorylation. Thus, cordycepin may provide a potential therapeutic approach for inflammation-associated
disorders.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Cordyceps militaris; Cordycepin; Macrophage; Inflammation; NO production; Lipopolysaccharide (LPS)
1. Introduction
Cordyceps militaris is a fungus that parasitizes Lepidoptera
larvae and has benefits in the human body including circulatory,
immune, respiratory and glandular systems. Previous studies
showed various properties such as anti-angiogenesis (Yoo et al.,
2004), anti-tumor and anti-diabetic (Yun et al., 2003), anti-
European Journal of Pharmacology 545 (2006) 192 199
www.elsevier.com/locate/ejphar
Corresponding authors. Kim is to be contacted at Department of
Biochemistry, Kangwon National University, Chunchon, 200-701, Republic
of Korea. Tel.: +82 33 250 8515; fax: +82 33 242 0459. Hwang, Cardiovascular
Research Institute, Yonsei University College of Medicine, 134 Shinchon-dong,
Seodaemun-gu, Seoul, 120-752, Republic of Korea. Tel.: +82 2 2228 8523; fax:
+82 2 365 1878.
E-mail addresses: kchwang@yumc.yonsei.ac.kr (K.-C. Hwang),
tawkim@kangwon.ac.kr (T.W. Kim).
0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2006.06.047
mutagenic (Cho et al., 2003) and hypoglycemic effect (Choi
et al., 2004). Cordycepin, a major component of C. militaris,
has also been studied in anti-fungal activity (Sugar and
McCaffrey, 1998), anti-herpes activity (De Julian-Ortiz et al.,
1999), anti-metastatic action on some cell lines (Nakamura et
al., 2005), stimulating effect on interlukin-10 production as an
immune modulator (Zhou et al., 2002), effect of polyadenyla-
tion inhibition (Ioannidis et al., 1999), and anti-leukemic
activity (Koc et al., 1996).
Activation of macrophage plays an important role in the
initiation and propagation of inflammatory responses by the
production of cytokines, interleukin-1 beta (IL-1β), tumor
necrosis factor-alpha (TNF-α), granulocyte/macrophage colony
stimulating factor (GM-CSF), nitric oxide (NO), cycloxygenase-
2 (COX-2) and other inflammatory mediators. Over-expression
of the inflammatory mediators in macrophage is involved in
many diseases, such as rheumatoid arthritis (Tilg et al., 1992),
atherosclerosis (Coker and Laurent, 1998), chronic hepatitis
(Lind, 2003) and pulmonary fibrosis (Bertolini et al., 2001).
Lipopolysaccharide (LPS) is the main component of
endotoxin and is formed by a phosphoglycolipid, called lipid
A that is covalently linked to a hydrophilic heteropolysacchar-
ide (Rietschel et al., 1994). LPS-induced macrophage activation
increased the production of cytokines such as IL-1β, TNF-α,
GM-CSF and nitric oxide, which is modulated by the up-
regulation of inducible nitric oxide synthase or NOS2. Animal
models of septic shock showed that IFN-γ, TNF-α, and IL-10
were related to the regulation of LPS-induced NO release (ter
Steege et al., 1998). Especially, LPS is a potent activator for the
mitogen-activated protein kinases (MAPKs) which are
expressed in many inflammatory cells (Chan and Riches,
2001). Among MAPKs, the p38 MAPK was shown to play an
important role in the NF-κB regulation of inflammation (Kim
et al., 2004). Akt signals were thought to be p38 MAPK-
independent component of LSP-induced NF-κB activation
(Hattori et al., 2003).
In this study, we tested the role of cordycepin on the Akt
IκBNF-κB-dependent inflammation cascades as well as
inhibition of p38 phosphorylation in lipopolysaccharide
(LPS)-stimulated RAW 264.7 macrophage cells.
2. Materials and methods
2.1. Chemicals
Dulbecco's modified Eagle's medium (DMEM), penicillin,
streptomycin, and fetal bovine serum (FBS) were purchased
from Life Technology (Rockville, USA). LPS (Escherichia coli
O11:B4) was obtained from Sigma (St. Louis, USA). Mouse
iNOS (BD Biosciences 610328), Actin (SIGMA. A5441),
phospho-specific p38 MAPK (Biosciences 612280) or goat
polyclonal antibody for COX-2 (SANTA CRUZ sc-1746), 2-(4-
Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochlo-
ride (LY294002, Sigma) and curcumin (Sigma) were used.
Cordycepin was purchased from Sigma (St. Louis, USA). All
other reagents were purchased from Sigma unless indicated
otherwise.
2.2. Extraction and fractionation from C. militaris and
identification of cordycepin
Fresh fruiting bodies of C. militaris grown on brown rice and
silkworm pupae were obtained from Mushtech Co. (Chunch-
eon, Korea). The fruiting bodies were dried at 50 °C and
crushed in a blender and the crude powder was extracted with
methanol at 70 °C for 3 h. The extracts were evaporated at 60 °C
under pressure and resuspended in distilled water. The aqueous
layer was mixed with hexane, butanol and ethyl acetate. The
hexane layer was evaporated to dryness under pressure. Ethyl
acetate, butanol, and water fractions were progressed following
the same method. To further identify the major component of
C. militaris butanol fraction, silica gel 60 (0.20.5) (Merk,
Germany) was packed into 7.5 cm × 30 cm column (Korea
Chemistry, Korea) and was gradiently eluted with chloroform
and methanol. Sephadex LH20 was packed into 2.5 cm ×100 cm
open column (Korea chemistry, Korea) and eluted with
methanol. First, C. militaris butanol fraction (CMBF) was
fractionated by 1st silica gel open column chromatography with
step gradient (methanol/chloroform = 0/100, 5/95, 10/90, 15/85,
20/80, 30/70, 40/60, 70/30 100/0). SI-10 (methanol 10%)
fraction showed highest anti-inflammation activities. Then, SI-
10 was separated by 2nd sephadex LH 20 chromatography that
was eluted with 100% methanol. Through 2nd chromatography,
we obtained 2 fractions. In these fractions, first and second
fractions showed anti-inflammation activities. Next purification
steps were salting out methods and high performance liquid
chromatography (HPLC) (Hattori et al., 2003). Thin layer
chromatography (TLC) plates were developed in chloroform/
methanol/water (64:14:1) and were stained with 10% sulfuric
acid solution (in ethanol). Finally, cordycepin was identified by
high performance liquid chromatography (HPLC) analysis; the
system was equipped with KNAUER (Wellchrom HPLC-pump,
K-1001, Wellchrom fast scanning spectrophotometer K-2600,
and 4 channel degasser K-500). Elution solvents were distilled
water and acetonitrile. The gradient step of the solvent was
water to acetonitrile 1%/minand (Vydac C18) Column was
used. Cordycepin purchased from Sigma (USA) was used for
the following tests.
2.3. Macrophage cell culture
Murine macrophage cell line RAW 264.7 (American Type
Culture Collection, Bethesda, USA) was cultured in DMEM
(Dulbecco's modified Eagle's medium) including 2 mM
L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and
10% fetal bovine serum (HyClone Labs, USA). Macrophages
were cult ured in six-well plates (4 × 10
6
cells /well) at 37 °C in
5% CO
2
/95% air. Cells were washed twice with fresh medium
and stimulated with 1 μg/ml LPS.
2.4. Determination of NO concentration
RAW 264.7 cells(4 × 10
6
cells/well) were incubated for 16 h
with 1 μg/ml LPS. The presence of nitrite, a stable oxidized
product of NO, was determined in cell culture media by Griess
193H.G. Kim et al. / European Journal of Pharmacology 545 (2006) 192199
reagent. Briefly, 100 μl of culture supernatant was removed and
combined with 100 μl Griess reagent (mixture of equal volume
of 1% sulfanilamide in 5% H
3
PO
4
and 0.1% N-(1-naphthyl)
ethylenediamine dihydrochloride in H
2
O) in a 96-well plate,
followed by spectrophotometric measurement at 550 nm. Nitrite
concentrations in the supernatants were determined by com-
parison with a sodium nitrite standard curve.
2.5. Preparation of nuclear extracts
Nuclear extracts were prepared by a modified method of
Wadsworth and Koop (Wadsworth and Koop, 1999). Treated
cells were washed, then scraped into 1.5 ml of ice-cold Tris-
buffered saline (pH 7.9), and centrifuged at 12,000 gfor 30 s.
The pellet was suspended in 10 mM HEPES [(N-[2-hydro-
xyethyl]piperazine-N-[2-ethanesulfonic acid])], pH 7.9, with
10 mM KCl, 0.1 mM EDTA (Ethylenediaminetetraacetic acid),
0.1 mM EGTA [Ethyleneglycol-bis-(β-amini ethyl ether N,N,
N,N-tetraacetic acid)], 1 mM DTT (DL-Dithiothreitol),
0.5 mM PMSF (phenylmethylsulfonyl fluoride), 5 μg/ml of
leupeptin, aprotinin, and pepstatin, incubated on ice for 15 min,
and then vortexed for 10 s with 0.6% Nonidet P-40. Nuclei
were separated from cytosol by centrifugation at 12,000 gfor
60 s. The supernatant was removed, and the pellet was sus-
pended in 50100 μlof20mMHEPES,pH7.9,with25%
glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,
0.5 mM PMSF, 10 μg/ml of leupeptin, aprotinin, and pepstatin.
The samples were incubated with rocking at 4 °C for 15 min,
and centrifuged for 5 min at 12,000 g. Protein concentration of
the supernatant was determined by Bicinchoninic Acid (BCA,
Pierce, USA) protein assay kit.
2.6. Western blot analysis
Macrophages were incubated with or without LPS in the
presence or absence of C. militaris butanol fraction (CMBF) or
cordycepin. Cells were harvested, washed twice with ice-cold
TrisHCl-buffered saline (TBS), and resuspended in Lysis
buffer 100 mM Tris, 5 mM EDTA, 50 mM NaCl, 50 mM β-
glycerophosphate, 50 mM NaF (Sodium fluoride), 0.5% NP-40,
1% sodium deoxycholate 0.1 mM sodium orthovanadate and 1%
PMSF. The cells were lysed by three cycles of freezing and
thawing in liquid nitrogen. The cytosolic fraction was obtained
from the supernatant after 12,000 gcentrifugation at 4 °C for
20 min. Samples (30 μg of protein) were separated on 10% SDS-
PAGE (Sodium dodecyl sulfate polyacrylamide gel electropho-
resis) and transferred to hybond-P PDVF membranes. The
membranes were blocked with 5% nonfat milk in TBS-Tween
(0.1%) (Junsei Chemical, JAPAN.) for 1 h and then incubated
with monoclonal antibody for mouse iNOS or polyclonal
antibody for COX-2 in TBS-Tween containing 1% nonfat milk
or BSA for 1 h. After washing three times with TBS-Tween, the
membrane was hybridized with secondary antibody conjugated
with horseradish peroxidase for 1 h and washed five times with
TBS-Tween. The membrane was incubated with enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech)
reagent for 2 min and exposed to X-ray film.
2.7. Reverse transcriptase-polymerase chain reaction analysis
Total RNA was prepared from RAW 264.7 cells using a Trizol
Reagent kit (Invitrogen, Korea). One microgram of total RNA
was converted to cDNA by treatment with 200 U of reverse
transcriptase and 500 ng of oligo-dT primer in 50 mM TrisHCl
(pH 8.3), 75 mM KCl, 3 mM MgCl
2
,10mMDTT,and1mM
dNTPs at 42 °C for 1 h. The reaction was stopped by heating at
70 °C for 15 min. Three microliters of the cDNA mixture was
used for enzymatic amplification. Polymerase chain reaction was
performed in 50 mM KCl, 10 mM TrisHCl (pH 8.3), 1.5 mM
MgCl
2
, 0.2 mM dNTPs, 2.5 U of Taq DNA polymerase, and
0.1 μM of each primer for iNOS and TNF-α. The amplification
was performed in a DNA thermal cycler under the following
condition: denaturation at 95 °C for 5 min for the first cycle; 95 °C
for 45 s annealing at 55 °C (iNOS) or 46 °C (TNF-α) for 30 s, and
extension at 72 °C for 45 s for 35 repetitive cycles. Final extension
was performed at 72 °C for 10 min. The PCR products were
electrophoresed on a 1.5% agarose gel and stained with ethidium
bromide. The primers were purchased from COSMO co, Ltd. The
primers used were 5-TCTTCGAAATCCCACCTGAC-3
(sense) and 5-CCATGATGGTCACATTCTGC-3(antisense)
for the iNOS, 5-ATGAGCACAGAAAGCATG-3(sense) and
5-TCACAGAGC AATGACTCC-3(antisense) for the TNF-α,
5-TCCTTCGTTGCCGGTCCACA-3(sense) and 5-CGTCTC
CGGAGTCCATCACA-3(antisense) for the β-Actin was used
as an internal control.
2.8. Statistical analysis
Data are presented as means ± S.E.M. of more than three
separate experiments performed in triplicate, except where
results of blots are shown, in which case a representative
experiment is depicted in figures. Comparisons between multiple
groups were performed with one-way ANOVA (Analysis of
Variance) with Bonferroni's test. Statistical significance was
considered significant when P< 0.05 and P< 0.01.
3. Results
3.1. Effects of C. militaris butanol fraction (CMBF) on NO
production and iNOS protein expression
Exposure of RAW 264.7 macrophages to LPS resulted in
nitrite production in time-dependent manner (data not shown).
RAW 264.7 cells (4 × 10
6
cells/well) were incubated for 16 h
with 1 μg/ml LPS. In activated macrophage, iNOS generates
large amounts of NO that causes acute or chronic inflammatory
disorders. To investigate whether C. militaris butanol fraction
can inhibit LPS-induced NO and iNOS expression, RAW 264.7
cells were pretreated for 30 min with various concentrations
of C. militaris butanol fraction and subsequently treated with
1μg/ml LPS. As shown in Fig. 1A, C. militaris butanol fraction
inhibited NO production of LPS-stimulated cells in a dose-
dependent manner. Western blot showed that C. militaris
butanol fraction decreased the level of iNOS protein in LPS-
stimulated cells in a dose-dependent manner (Fig. 1B).
194 H.G. Kim et al. / European Journal of Pharmacology 545 (2006) 192199
3.2. Identification of cordycepin from C. militaris butanol
fraction (CMBF)
To identify the major component of C. militaris butanol
fraction, the fraction was fractionated further (in the Materials
and methods section) and identified with HPLC using
acetonitrile/water as the mobile phase. Both cordycepin purified
from C. militaris and a commercial cordycepin (Sigma, USA)
were compared with HPLC (Fig. 2A). Also,
1
H NMR (Nuclear
Magnetic Resonance) was used to compare the purified
cordycepin and a commercial cordycepin (Fig. 2B). Results
from HPLC and
1
H NMR spectra showed that the cordycepin
purified from C. militaris was identical with a commercial
cordycepin. We confirmed that cordycepin purified from
C. militaris butanol fraction was an effective component of
anti-inflammation by HPLC and NMR spectra. Cordycepin
purchased from Sigma (USA) was used for the further tests.
3.3. Effect of cordycepin on LPS-induced NO production and
iNOS expression
Macrophage plays crucial roles in the initiation and
maintenance of inflammation. Stimulation of macrophages by
LPS results in nitric oxide (NO) production and the expression
of inducible nitric oxide synthase (iNOS). Cordycepin has been
known to be a component in C. militaris as an anti-tumor agent.
We first examined whether cordycepin inhibits the expression
of the NO and iNOS in LPS-induced macrophage. To assess the
inhibitory effects of cordycepin on LPS-induced NO produc-
tion, NO production of LPS-stimulated cells was measured in
the presence of cordycepin. As shown in Fig. 3A, cordycepin
inhibited NO production in a dose-dependent manner up to
30 μg/ml. To determine whether cordycepin exerts NO
inhibition of activated cells by blocking iNOS expression,
western blot analysis was carried out with whole cell lysates.
The iNOS proteins were hardly detectable in resting macro-
phages RAW 264.7, while large amount of iNOS protein was
induced upon exposure to LPS alone (Fig. 3B). Treatment of
cordycepin to the cells decreased LPS-induced synthesis of
iNOS protein in a dose-dependent manner. To characterize
whether cordycepin decreases NO production via suppression
Fig. 1. Cordyceps militaris butanol fraction (CMBF) inhibits the production of
NO and expression of iNOS. RAW 264.7 cells (4 × 10
6
cells/well) were
incubated for 16 h with 1 μg/ml LPS in the presence or absence of various
concentrations of CMBF. Unstimulated cells were incubated under the same
conditions but in the absence of LPS. (A) RAW 264.7 cells were pretreated with
the indicated concentrations of cordycepin for 30 min before incubation with
LPS (1 μg/ml) for 16 h. The culture supernatants were subsequently isolated and
analyzed for nitrite levels. (B) Macrophages were stimulated with LPS in the
absence or presence of CMBF for 16 h, and the nuclear-free lysate was
immunoblotted with iNOS antibody. Data are expressed as mean ± S.E.M.
(n= 3). Asterisks indicate a significant difference from LPS alone (P< 0.05,
⁎⁎P<0.01). Significance was determined by Student's t-test (< 0.001).
Fig. 2. Identification of cordycepin. (A) Chromatogram of cordycepin (220 nm) by HPLC using acetonitrile/water as the mobile phase (described in Materials and
methods). (B) Expansion of the cordycepin (sigma) and purified cordycepin in the 300 MHz proton NMR (Buker Avancr 300) in dimethyl sulfoxide (DMSO).
195H.G. Kim et al. / European Journal of Pharmacology 545 (2006) 192199
of iNOS mRNA levels, the levels of iNOS mRNA in cells
treated with LPS in the presence of various concentrations of
cordycepin were determined (Fig. 3C). RT-PCR analysis of
LPS-activated macrophages treated with cordycepin showed
suppression of iNOS mRNA expression. We found that
cordycepin decreased the levels of iNOS protein and mRNA
in LPS-stimulated cells in a concentration-dependent manner.
To find whether cordycepin has cytotoxicity on macrophage
cells, RAW 264.7 cells were pretreated for 30 min with various
concentrations (up to 30 μg/ml) of cordycepin and were
incubated for 16 h. No significant cytotoxicity of cordycepin in
RAW 264.7 cells was found (data not shown).
3.4. Effect of cordycepin on COX-2 expression and production
of TNF-α
Because the level of COX-2, a pro-inflammatory enzyme, is
important to address the extent of inflammation, the effect of
cordycepin on the inhibition of COX-2 expression was
investigated. RAW 264.7 cells were incubated with LPS
(1 μg/ml) in the presence or absence of various concentrations
of cordycepin. Cordycepin suppressed the level of COX-2
expression increased in RAW 264.7 cells by treatment of LPS in
a dose-dependent manner (Fig. 4A). Also, the level of pro-
inflammatory cytokines is important in inflammation, and the
effect of cordycepin on the inhibition of TNF-α, a pro-
inflammatory cytokine, was investigated. The mRNA levels of
TNF-αinduced by LPS were significantly decreased in a dose-
dependent manner by treatment with cordycepin as shown in
Fig. 4B. These data indicated that pro-inflammatory enzyme,
COX-2 and pro-inflammatory cytokine, TNF-αwere sup-
pressed by treatment of cordycepin.
3.5. Effect of cordycepin on phosphorylation of IκBα
NF-κB is a transcription factor that modulated the expression
of variety of genes involved in immune and inflammatory
responses, including iNOS, COX-2 and TNF-α.Inan
unstimulated cell, NF-κB resides in the cytoplasm as an inactive
NF-κBIκB complex. Under the stimulus effects, IκB becomes
phosphorylated and is subsequently degraded, allowing NF-κB
to translocate into the nucleus (Makarov, 2000). IκBαcan be
phosphorylated at its Ser-32 and -36 residues by IκB kinase
(IKK) complex, which marks for ubiquitin-dependent IκBα
degradation. The translocation of NF-κB to the nucleus is
preceded by the phosphorylation of IκB. After a 2 h activation of
macrophages by LPS, the serine phosphorylated IκBαprotein
was increased as detected by Ser32-phosph-specific IκBα
protein. Cordycepin inhibited LPS-mediated IκBαphosphory-
lation in a dose-dependent manner (Fig. 5A). To investigate
whether cordycepin could affect nuclear translocation of NF-κB,
western immunoblot analysis for NF-κB p65 was carried out
with nuclear extracts of LPS-stimulated macrophages RAW
Fig. 3. Effect of cordycepin on LPS-induced NO production(A), iNOS protein
(B) and iNOS mRNA expression (C). (A) RAW 264.7 cells (4 ×10
6
cells/well)
were pretreated with the indicated concentrations of cordycepin for 30 min
before incubation with LPS (1 μg/ml) for 16 h. The culture supernatants
were subsequently isolated and analyzed for nitrite levels. Data are expressed as
mean ± S.E.M (n= 3). Asterisks indicate a significant difference from LPS alone
(P<0.03, ⁎⁎P< 0.01). Significance was determined by Student's t-test
(<0.001). (B) The cells were lysed, and the lysates were analyzed by
immunoblotting used anti-iNOS. The blot was stripped of the bound antibody
and reprobed with anti-βactin to confirm equal loading. (C) Macrophage cells
were pretreated with the indicated concentrations of cordycepin for 30 min before
incubation with LPS (1 μg/ml) for 8 h. Total RNA was prepared and RT-PCR
analysis was performed as described in Materials and methods.
Fig. 4. Effect of cordycepin on LPS-induced COX-2 protein expression, and
TNF-αmRNA expression. (A) RAW 264.7 cells (4 × 10
6
cells/well) were
pretreated with the indicated concentrations of cordycepin for 30 min before
incubation with LPS (1 μg/ml) for 16 h. The cells were lysed, and the lysates
were analyzed by immunoblotting used anti-COX-2. The blot was stripped of
the bound antibody and reprobed with anti-βactin to confirm equal loading. (B)
Raw cells were pretreated with cordycepin for 30 min and then treated with LPS
in the presence of cordycepin for 8 h. Total RNAwas isolated and used for TNF-
αor actin RT-PCR. Total RNA was prepared and RT-PCR analysis was
performed as described in Materials and methods.
196 H.G. Kim et al. / European Journal of Pharmacology 545 (2006) 192199
264.7. Amount of NF-κB p65 in the nucleus was markedly
increased upon exposure to LPS alone, but cordycepin inhibited
LPS-mediated nuclear translocation of NF-κB p65 (Fig. 5A). To
determine whether cordycepin was associated with the suppres-
sion of NF-κB activation, the NF-κB inhibitor, curcumin was
used. Curcumin (25 uM) and cordycepin (20 μg/ml) showed the
inhibition of IκBαphosphorylation in LPS-induced macrophage
cell (Fig. 5B). We then examined whether the PI3K inhibitor
(LY294002) could affect the activation-induced proteolysis of
IκBαinhibitor protein, which traps NF-κB dimmer in the
cytosol. When cells were pretreated for 30 min with 10 μM
LY294002, LPS-induced IκB phosphorylation was inhibited,
indicating that the LPS-induced proteolysis was partially
prevented by LY294002 (Fig. 5C). LPS-induced IκB phosphor-
ylation was both inhibited by LY294002 and cordycepin,
indicating the involvement of PI3-kinase pathway.
3.6. Inhibition of Akt and p38 kinase phosphorylation in
response to cordycepin in RAW 264.7 cells
The expression of iNOS is regulated by pathways that
involved Akt and MAPKs in macrophages. The Akt signal
molecule is known to regulate NF-κB activation via IKK
activation. Activation of IKK is mediated by phosphorylation
through various upstream kinases such as NF-κB-inducing kinase,
NK-κB-activating kinase, and Akt, which are involved in cellular
signaling in response to pro-inflammatory stimuli (Islam et al.,
2004; Giri et al., 2004). Therefore, the effects of cordycepin on
LPS-induced Akt and p38 phosphorylation were examined. To
investigate whether Akt pathway was involved in the regulation of
macrophage inflammation, we examined the phosphorylation of
Akt after stimulation of RAW 264.7 with LPS. Phosphorylation of
Akt was inhibited by cordycepin in a dose-dependent manner,
while nonphosphorylated Akt remained the same (Fig. 6A).
MAPKs have important functions as mediators of cellular
responses to extracellular signals. MAPKs important to macro-
phage cells include p38, c-jun N-terminal kinase (JNK), and
extracellular signal-regulated kinase (ERK) (Chan and Riches,
2001; Kim et al., 2004). Specifically, p38 plays a important role in
LPS-induced NO production and iNOS induction (Chen and
Wang, 1999). Cordycepin also decreased the phosphorylation level
of p38 protein in LPS-stimulated cells in a concentration-
dependent manner (Fig. 6B). These findings indicate that
cordycepin is effective on the inhibition of Akt and p38
phosphorylation in LPS-induced macrophage cells.
4. Discussion
The present study was undertaken to elucidate the pharmaco-
logical and biological effects of cordycepin from C. militaris on
Fig. 5. Effect of cordycepin on LPS-mediated phosphorylation of IκBαand
nuclear translocation of NF-κB p65. (A) RAW 264.7 cells (4× 10
6
cells/well)
were pretreated with cordycepin for 30 min and stimulated with LPS for 30 min
and 2 h. Cytosolic and nuclear extracts of the cells were subjected to western
immunoblot analysis with anti-phospho-IκBα(Ser-32/36), antibody (Cell
Signaling #9241). NF-κB p65(Cell Signaling #3037). Nuclear extracts of the
cells were subjected to Western immunoblot analysis with anti-NF-κB p65
antibody. (B) Inhibition of LPS-induced IκB phosphorylation by curcumin (NF-
κB inhibitor) and cordycepin. Macrophage were incubated with LPS without or
with 25 uM curcumin and 20 uM cordycepin for 30 min. Cytosolic fractions
were prepared and analyzed for IκB protein by western blot. This experiment
was repeated three times with similar results. (C) Macrophages RAW 264.7
(4 × 10
6
cells/well) were pretreated with LY294002 (PI3K inhibitor: Promega,
#v1201) and cordycepin for 30 min and stimulated with LPS (1 μg/ml) for
indicated periods (30 min). Cytoplasmic extracts of the cells were subjected to
western immunoblot analysis with anti-phospho-IκBα(Ser-32/36) antibody
(Cell Signaling #9241) to measure IκBαphosphorylation. This experiment was
repeated three times with similar results.
Fig. 6. Effect of cordycepin on Akt and p38 phosphorylation in RAW 264.7
macrophage cells. (A) Effect of cordycepin on Akt phosphorylation (Cell
Signaling #9271) and Akt (Cell Signaling #9272). Macrophages (4 × 10
6
cells/
well) were treated with or without lipopolysaccharide (1 μg/ml), or with
lipopolysaccharide plus different concentrations of cordycepin. Control values
were obtained in the case of lipopolysaccharide and cordycepin. (B) Effect of
cordycepin on p38 phosphorylation (BD SCIENCE 612280). Cordycepin was
pretreated with 30 min before stimulation. Western blot analysis was performed
using 50 μg of total cellular protein. Cell lysates were prepared to perform
electrophoresis and immunoblotting as described in the Materials and methods
section by employing a specific anti-phospho-Akt and p38 antibody or anti-Akt
and p38 antibody. Cells were harvested 30 min after the LPS treatment. Results
are representative of four independent experiments.
197H.G. Kim et al. / European Journal of Pharmacology 545 (2006) 192199
the inhibition of inflammatory mediators in macrophage. We
showed that cordycepin suppresses the production of NO, iNOS
(induce nitric oxide synthase) and COX-2 in LPS-stimulated
RAW 264.7 cells. The molecular mechanism by which cordycepin
inhibits the expression of these inflammatory mediators appear to
involve the inhibition of NF-kB, Akt and p38 activation. These
findings suggest that cordycepin may prevent inflammation by
suppressing NF-κB-mediated inflammatory genes.
The production of NO, PGE
2
, IL-1β, and TNF-αis an
important part of immune response to many inflammatory
stimuli. However, excessive production of these mediators is
seen in many acute and chronic human diseases, including septic
shock, hemorrhagic shock, multiple sclerosis, rheumatoid
arthritis, ulcerative colitis, and atherosclerosis (Tilg et al.,
1992; Lind, 2003; Bertolini et al., 2001). Thus, the suppression
of these mediators may be an effective therapeutic strategy for
preventing inflammatory reaction and diseases. Furthermore,
nonsteroidal anti-inflammatory drugs inhibited the production
of NO and iNOS by the suppression of NF-κB activation (Chen
et al., 1998). Several natural antioxidant polyphenol com-
pounds, such as Quercetin, resveratrol, sesquiterpene lactone,
and theaflavin have been shown to directly inhibit the expression
of NF-κB-dependent cytokines, iNOS, and COX-2 genes (Lin
et al., 1999; Steffen et al., 1998). The suppressive effects of these
antioxidant compounds on the production of these inflammatory
mediators are associated with their antioxidant activities. It has
been shown that Cordyceps sp. possessed a strong antioxidant
activity (Won and Park, 2005; Li et al., 2001). The antioxidant
NF-κB inhibitors, such as N-acetyl-cysteine (NAC) and ethyl
pyruvate (EP), inhibit the production of inflammatory mediators
through suppression of their gene expression and also prevent
inflammation (Song et al., 2004). We demonstrated that
cordycepin decreased IκB phosphorylation in RAW 264.7
cells stimulated with LPS. This inhibitory effect could be
associated with the down-regulation of iNOS and COX-2
expression. Inhibition of LPS-induced Akt activation led to the
down-regulation of NF-κB activation, resulting in decreased
iNOS expression and NO production. NF-κB-activating cyto-
kine such as TNF-αwas also down-regulated by the inhibition of
Akt activation, leading to a decrease NO production. LPS-
mediated phosphorylation of Akt was blocked by cordycepin.
Events necessary for the activation of NF-κB (such as IκB
phosphorylation and nuclear translocation) were suppressed by
cordycepin. These results place Akt upstream of NF-κB
activation in the sequence of signaling events, whereby
activation of Akt up-regulates iNOS promoter activity, leading
to transcription and translation of iNOS and increased NO
production. The activation of NF-κB requires phosphorylation
of IκB, which then targets IκB for ubiquitination and
degradation (Karl et al., 2002). Inhibition of Akt, which was
demonstrated as diminished Akt phosphorylation by cordyce-
pin, caused decreased phosphorylation of IκB and attenuated the
degradation of IκB in LPS-induced macrophage cells.
Our results demonstrated that cordycepin inhibits the
phosphorylation of Akt in LPS-stimulated macrophages.
Therefore, the PI3KAkt pathway modulates the expression
of inflammatory agent in LPS-induced macrophage.
The mitogen-activated protein (MAP) kinase plays a critical
role in the regulation of cell growth and differentiation, and in
the control of cellular responses to cytokines and stresses.
MAP kinases are involved in the signaling pathway for LPS-
induced iNOS expression. LPS activates all three types of
MAPKs p38, JNK, and ERK in mouse macrophages
(Chen and Wang, 1999). The exact signaling pathways among
the three types of MAPKs are still unclear; however, there is a
cross-talk and signal convergence among the MAPKs. Many
of the upstream kinases and downstream substrates are the
same for each of the major cascades (Jordan et al., 2000).
Activation of p38 by LPS resulted in the stimulation of NF-
κB-specific DNA-protein binding and the subsequent expres-
sion of inducible form of NO synthase and NO release in
RAW 264.7 macrophage (Chen and Wang, 1999). Cordycepin
also decreased the phosphorylation level of p38 protein in
LPS-stimulated cells in a concentration-dependent manner.
Our results demonstrated that cordycepin inhibits the
production of NO production in LPS-stimulated macrophages.
This anti-inflammatory effect occurs by the down-regulation of
iNOS, COX-2 expression and TNF-αgene expression via the
suppression of NF-κB activation, Akt and p38 phosphorylation.
Thus, cordycepin might be relevant for clinical use for inflam-
matory diseases.
Acknowledgments
This work was supported by a grant from the Regional
Innovation which was conducted by the Ministry of Commerce
Industry and Energy, and BioGreen 21 Program, Rural
Development Administration, Republic of Korea.
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    This study was carried out to examine the antimutagenic and anticancer effects of cordycepin isolated from the butanol fraction of Cordyceps militaris. The cordycepin itself did not show any mutagenic effect against the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), benzo (alpha)pyrene (B(alpha)P) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1) using the Ames test. The cordycepin (3'-deoxyadenosine, 100 mug/plate) showed inhibitory effects of approximately 85.0%, 52.5% and 32.7% on the mutagenesis induced by 4NQO, B(alpha)P and Trp-P-1 against strain TA98 respectively. Inhibitory effects of 70.2%, 73.1%, 81.7% and 67.7% were also observed on the mutagenesis induced by MNNG, 4NQO, B(alpha)P and Trp-P-1 against strain TA 100 respectively. The treatment of 100 mug/well cordycepin showed the strongest of 90.0% against A549 cell line (human lung carcinoma) among other cancer cell line, whereas the least cytotoxicity only 11.3% was abserved on human normal cell 293.
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    Mice injected with lipopolysaccharide (LPS) develop lethal septic shock, accompanied by elevated serum NOx, interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and TNF-receptor levels. Elevated NO levels are thought to play a central role in tissue damage observed during septic shock. In vitro data indicate that IFN-gamma and TNF-alpha play an important role in LPS-induced NO release. Further, interleukin 10 (IL-10) has been shown to inhibit the release of pro-inflammatory cytokines such as IFN-gamma and TNF-alpha. Therefore, in the present study, we investigated the role of IFN-gamma, TNF-alpha, and IL-10 in LPS-induced NO release. To this end, mice were pretreated with anti-IFN-gamma, anti-TNF-alpha, anti-IL-10 mAbs or combinations of these 2 h before LPS-challenge. The results indicate that IFN-gamma, TNF-alpha as well as IL-10 are involved in the regulation of LPS-induced NO release. Blocking either IFN-gamma or TNF-alpha has no effect on LPS-induced NO release, however, blocking both IFN-gamma and TNF-alpha nearly completely prevents NO release after LPS challenge, suggesting that the presence of either TNF-alpha or IFN-gamma is essential for induction of NO release after LPS challenge. Further, the results obtained with anti-IL-10 treatment suggest the presence of an IL-10 inducible factor which together with IFN-gamma and TNF-alpha regulates LPS-induced NO release.
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    Full-text available
    Endotoxins of Gram-negative microbes fulfill as components of the outer membrane a vital function for bacterial viability and, if set free, induce in mammalians potent pathophysiological effects. Chemically, they are lipopolysaccharides (LPS) consisting of an O-specific chain, a core oligosaccharide, and a lipid component, termed lipid A. The latter determines the endotoxic activities and, together with the core constituent Kdo, essential functions for bacteria. The primary structure of lipid A of various bacterial origin has been elucidated and lipid A of Escherichia coli has been chemically synthesized. The biological analysis of synthetic lipid A partial structures proved that the expression of endotoxic activity depends on a unique primary structure and a peculiar endotoxic conformation. The biological lipid A effects are mediated by macrophage-derived bioactive peptides such as tumor necrosis factor alpha (TNF). Macrophages possess LPS receptors, and the lipid A regions involved in specific binding and cell activation have been characterized. Synthetic lipid A partial structures compete the specific binding of LPS or lipid A and antagonistically inhibit the production of LPS-induced TNF. LPS toxicity, in general, and the ability of LPS to induce TNF are also suppressed by a recently developed monoclonal antibody (IgG2a), which is directed against an epitope located in the core oligosaccharide. At present we determine molecular and submolecular details of the specificity of the interaction of lipid A with responsive host cells with the ultimate aim to provide pharmacological or immunological therapeutics that reduce or abolish the fatal inflammatory consequences of endotoxicosis.
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    The nucleoside analogue cordycepin (3'-deoxyadenosine), when protected against ADA deamination, is specifically cytotoxic for TdT-positive leukemia cells. Cordycepin-treated, ADA-inhibited, TdT-positive cells undergo the classic changes associated with drug-induced apoptosis: reduction in cell volume, chromatin clumping, membrane blebbing, and 180-bp multimer DNA laddering on agarose gels. In common with the apoptosis seen in normal TdT-positive thymocytes, following exposure to various agents, apoptosis induced by cordycepin in TdT-positive leukemia cells was associated with increased protein kinase A (PK-A) activity. Unlike thymocyte apoptosis however, no elevation in cAMP levels was seen preceding the rise in PK-A activity. Ex vivo we show that cordycepin monophosphate can activate PK-A as efficiently as cAMP. On this basis we speculate that cordycepin monophosphate in TdT-positive cells may be able to activate PK-A in place of cAMP, and that PK-A may phosphorylate TdT, augmenting its activity as an endonuclease. In cell-free experiments, the activity of recombinant TdT as an endonuclease digesting supercoiled plasmid DNA into linear fragments was dramatically increased following phosphorylation of TdT by PK-A. A role for TdT as an apoptotic endonuclease in TdT-positive leukemia cells following cordycepin exposure is now the subject of on-going work.
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    Full-text available
    Extracts from certain Mexican Indian medicinal plants used in traditional indigenous medicine for the treatment of inflammations contain sequiterpene lactones (SLs), which specifically inhibit the transcription factor NF-κB (Bork, P. M., Schmitz, M. L., Kuhnt, M., Escher, C., and Heinrich, M. (1997) FEBS Lett. 402, 85–90). Here we show that SLs prevented the activation of NF-κB by different stimuli such as phorbol esters, tumor necrosis factor-α, ligation of the T-cell receptor, and hydrogen peroxide in various cell types. Treatment of cells with SLs prevented the induced degradation of IκB-α and IκB-β by all these stimuli, suggesting that they interfere with a rather common step in the activation of NF-κB. SLs did neither interfere with DNA binding activity of activated NF-κB nor with the activity of the protein tyrosine kinases p59fyn and p60src. Micromolar amounts of SLs prevented the induced expression of the NF-κB target gene intracellular adhesion molecule 1. Inhibition of NF-κB by SLs resulted in an enhanced cell killing of murine fibroblast cells by tumor necrosis factor-α. SLs lacking an exomethylene group in conjugation with the lactone function displayed no inhibitory activity on NF-κB. The analysis of the cellular redox state by fluorescence-activated cell sorter showed that the SLs had no direct or indirect anti-oxidant properties.
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    Full-text available
    The antifungal activity of the nucleoside analog 3'-deoxyadenosine (cordycepin) was studied in a murine model of invasive candidiasis. When protected from deamination by either deoxycoformycin or coformycin, both of which are adenosine deaminase inhibitors, cordycepin exhibited potent antifungal efficacy, as demonstrated by prolongation of survival and a decrease in CFU in the kidneys of mice treated with cordycepin plus an adenosine deaminase inhibitor. The antifungal effect was seen with three different Candida isolates: Candida albicans 64, a relatively fluconazole-resistant clinical isolate of C. albicans (MIC, 16 micrograms/ml), and the fluconazole-resistant Candida krusei. Cordycepin and related compounds may provide another avenue for the discovery of clinically useful antifungal drugs.
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    Pulmonary fibrosis can complicate diverse pulmonary and systemic pathologies. In many cases the underlying cause remains unidentified. Mortality from the disease is increasing steadily in the UK and USA. The clinical features are well-described, but patients frequently present at an advanced stage, and current treatments have not improved the poor prognosis. There is a compelling need to identify the fibrotic process earlier and to develop new therapeutic agents. Increased collagen deposition is central to the pathology and interest over the last decade has focused on the role of cytokines in this process. These polypeptide mediators are believed to be released from both circulating inflammatory and resident lung cells in response to endothelial and epithelial injury. Key cytokines currently implicated in the fibrotic process are transforming growth factor-beta, tumour necrosis factor-alpha and endothelin-1. This article outlines the evidence implicating these mediators in the pathogenesis of pulmonary fibrosis and also considers the possible role of cytokines with antifibrotic effects, such as interferon-gamma. The "balance" of positively and negatively regulating cytokines is discussed, and the potential for interaction with other factors including viruses, hormones and altered antioxidant status is also considered. Finally, potential novel therapeutic approaches are discussed, together with suggestions for future studies and clinical trials. As the outcomes of different avenues of research over the last ten years are brought together, it is clear that there is now a hitherto unrivalled opportunity to begin to tackle the treatment of this devastating disease.