ARTHRITIS & RHEUMATISM
Vol. 50, No. 11, November 2004, pp 3504–3515
© 2004, American College of Rheumatology
Antiarthritic Effect of Bee Venom
Inhibition of Inflammation Mediator Generation by Suppression of NF-?B
Through Interaction With the p50 Subunit
Hye Ji Park,1Seong Ho Lee,1Dong Ju Son,1Ki Wan Oh,1Ki Hyun Kim,2Ho Sueb Song,2
Goon Joung Kim,2Goo Taeg Oh,3Do Young Yoon,4and Jin Tae Hong1
Objective. To investigate the molecular mecha-
nisms of the antiarthritic effects of bee venom (BV) and
melittin (a major component of BV) in a murine mac-
rophage cell line (Raw 264.7) and in synoviocytes ob-
tained from patients with rheumatoid arthritis.
Methods. We evaluated the antiarthritic effects of
BV in a rat model of carrageenan-induced acute edema
in the paw and in a rat model of chronic adjuvant-
induced arthritis. The inhibitory effects of BV and
melittin on inflammatory gene expression were mea-
sured by Western blotting, and the generation of pros-
taglandin E2(PGE2) and nitric oxide (NO) and the
intracellular calcium level were assayed. NF-?B DNA
binding and transcriptional activity were determined by
gel mobility shift assay or by luciferase assay. Direct
binding of BV and melittin to the p50 subunit of NF-?B
was determined with a surface plasmon resonance ana-
Results. BV (0.8 and 1.6 ?g/kg) reduced the
effects of carrageenan- and adjuvant-induced arthritis.
This reducing effect was consistent with the inhibitory
effects of BV (0.5, 1, and 5 ?g/ml) and melittin (5 and 10
?g/ml) on lipopolysaccharide (LPS; 1 ?g/ml)–induced
expression of cyclooxygenase 2, cytosolic phospholipase
A2, inducible NO synthase, generation of PGE2and NO,
and the intracellular calcium level. BV and melittin
prevented LPS-induced transcriptional and DNA bind-
ing activity of NF-?B via the inhibition of I?B release
and p50 translocation. BV (affinity [Kd] ? 4.6 ?
10?6M) and melittin (Kd? 1.2 ? 10?8M) bound directly
Conclusion. Target inactivation of NF-?B by di-
rectly binding to the p50 subunit is an important
mechanism of the antiarthritic effects of BV.
Rheumatoid arthritis (RA) is a chronic, destruc-
tive inflammatory disease characterized by the release of
numerous proinflammatory mediators such as prosta-
glandins (PGs) and nitric oxide (NO) (1,2). PGs have
been shown to increase cytosolic phospholipase A2
(cPLA2) and cyclooxygenase (COX) levels, and thereby
amplify their own production and increase the synthesis
of inflammatory cytokines (3,4). Elevated levels of PGs
were found in synovial cells treated with inflammation
mediators or in patients with RA (5,6). In activated
macrophages, large amounts of NO are generated by
inducible NO synthase (iNOS) (7). Many effectors of
NO production lead to the simultaneous release of other
inflammation mediators, such as prostaglandin E2
(PGE2) and prostaglandin I2, from the COX pathway
(6), and iNOS induction can be modified by cPLA2(8,9).
These synergistic effects may be one operational mech-
anism between iNOS and COX-2, as well as between
iNOS and cPLA2, by which physiologic or pathologic
responses are amplified in the inflammatory reaction
(10,11). Intracellular calcium also plays a pivotal role
because it is involved in the generation of the superoxide
Supported by a grant from the Basic Research Program of the
Korea Science and Engineering Foundation (R05-2002-000-00644-0),
and by the Brain Korea 21 Project in 2003.
1Hye Ji Park, BS, Seong Ho Lee, PhD, Dong Ju Son, MS, Ki
Wan Oh, PhD, Jin Tae Hong, PhD: Chungbuk National University,
Chungbuk, South Korea;2Ki Hyun Kim, MD, PhD, Ho Sueb Song,
PhD, Goon Joung Kim, MS: Kyungwon University, Gyeonggii, South
Korea;3Goo Taeg Oh, PhD: Ewha Women’s University, Seoul, South
science and Biotechnology, Taejon, South Korea.
Address correspondence and reprint requests to Jin Tae
Hong, MD, College of Pharmacy, Chungbuk National University, 48,
Gaesin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, South
Korea. E-mail: firstname.lastname@example.org.
Submitted for publication February 2, 2004; accepted in
revised form August 10, 2004.
4Do Young Yoon, PhD: Korea Research Institute of Bio-
anion and NO, the production of tumor necrosis factor
? (TNF?) (12), and the cPLA2-mediated liberation of
arachidonic acid (13,14). Therefore, elements that can
suppress the generation of these inflammation media-
tors and agents that reduce intracellular calcium levels
can potentially be used as therapies in the management
These representative inflammatory genes
(COX-2, cPLA2, iNOS, and TNF?) appear to be highly
regulated by a number of transcription factors, in par-
ticular NF-?B, which is expressed ubiquitously and can
be activated by a number of inflammatory and patho-
logic stimuli, including cytokines, oxidative stress, ultra-
violet light, and bacterial and viral products. Accord-
ingly, agents that modulate the activity of NF-?B have a
pronounced effect on iNOS, COX-2, and cPLA2expres-
sion in response to different inflammatory stimuli. Sev-
eral studies have demonstrated that agents inhibiting
NF-?B activation can be potential treatments for anti-
inflammatory diseases (15–17).
Bee venom (BV) contains a variety of different
peptides, including melittin (a major component of BV),
apamin, adolapin, and mast cell degranulating peptide
(18,19). A few studies have shown that BV therapy
attenuates RA in humans (18) and in experimental
animals (20), and that BV has an antiinflammatory
effect (20,21). However, systematic experiments demon-
strating the molecular mechanisms of the antiarthritic
effects of BV have not been reported. To gain better
insight into these mechanisms, we first reevaluated the
antiarthritic effects of BV in rat models of arthritis, and
then investigated the antiarthritic mechanisms of BV
and melittin in a murine macrophage cell line (Raw
264.7) and in synoviocytes obtained from RA patients.
We focused on targeted disruption of NF-?B by BV or
melittin as a possible mechanism of antiinflammatory
MATERIALS AND METHODS
Chemicals. Rabbit polyclonal antibodies to cPLA2
(dilution 1:500), goat polyclonal antibody to COX-2 (1:500),
and TNF? (1:500), p50 (1:500), p65 (1:500), I?B? (1:500),
phospho-I?B? (1:200), mouse polyclonal antibody to iNOS
(1:500), and all of the secondary antibodies used in Western
blot analysis were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). T4 polynucleotide kinase was obtained from
peroxidase–labeled donkey anti-rabbit secondary antibody,
and ECL detection reagent were obtained from Amersham
Pharmacia Biotech (Piscataway, NJ). Reagents for sodium
dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis
were purchased from Bio-Rad (Hercules, CA). Lipopolysac-
charide (LPS), Griess reagent, anti–?-actin monoclonal anti-
body, MTT, and melittin were purchased from Sigma (St.
Louis, MO). All other reagents were purchased from Sigma
unless otherwise stated. BV was purchased from You-Miel BV
(Hwasoon, South Korea). The composition of the BV was as
follows: 45–50% melittin, 2.5–3% apamin, 2–3%. MCD pep-
tide, 12% PLA2, 1% lyso-PLA, 1–1.5% histidine, 4–5% 6pp
lipids, 0.5% secarpin, 0.1% tertiapin, 0.1% procamine, 1.5–2%
hyaluronidase, 2–3% amine, 4–5% carbohydrate, and 19–27%
other, including protease inhibitor, glucosidase, invertase, acid
phosphomonoesterase, dopamine, norepinephrine, and un-
known amino acids, with ?99.5% purity.
Adjuvant-induced arthritis (AIA). Male Sprague-
Dawley rats (Samtako, Osan, Kyungki, Korea) were main-
tained in accordance with guidelines for the care and use of
laboratory animals of the National Institute of Toxicological
Research of the Korea Food and Drug Administration. To
determine the approximate dose of BV, the antiinflammatory
activity of BV was first assessed with the carrageenan paw
edema test according to the methods of Sugishita et al (22). BV
(0.4, 0.8, 1.6, and 3.2 ?g/kg) or vehicle (saline) was adminis-
tered directly into the plantar surface of the right hind paw 30
minutes before injection of carrageenan (0.05 ml; 3% [weight/
volume] in saline) into the subplantar area of the right hind
paw. Volumes of the injected and contralateral paws were
measured with a Letica water plethysmometer (Panlab, Bar-
celona, Spain) 1, 3, and 5 hours after the induction of edema.
The maximum (3.2 ?g/kg) dose of BV did not cause a toxic
response, and greatly reduced paw edema (40% reduction
compared with the contralateral paws). We therefore used 0.8
or 1.6 ?g/kg of BV in the animal model.
AIA was elicited in Sprague-Dawley rats by injecting
0.1 ml of Mycobacterium butyricum (10 mg/ml) in mineral oil
into the base of the tail. Paw volumes were measured with a
plethysmometer at the beginning of the experiment. Animals
with edema values 1.1 ml greater than normal paws were then
randomized into treatment groups. BV (0.8 or 1.6 ?g/kg) or
vehicle (saline) was directly administered into the plantar
surface of the right hind paw from 1 day after AIA induction
to day 14. The magnitude of the inflammatory response was
evaluated by measuring the volume of both hind paws. On day
15, rats were anesthetized and placed on a radiographic box at
a distance of 90 cm from the x-ray source (BLD-150RK;
FOMA Bohemia, Hradec Kra ´love ´, Czech Republic), and the
arthritic hind paws were exposed to the x-ray beam for 0.01
seconds at 40 kW. Paws were oriented horizontally relative to
the detector. Radiographs were scored by an investigator
(HJP) who was blinded to the treatment information, using the
following scale: 0 ? no bone damage, 1 ? tissue swelling and
edema, 2 ? joint erosion, and 3 ? bone erosion and osteo-
Cell culture. Raw 264.7, a mouse macrophage-like cell
line, and THP-1, a human monocytic cell line, were obtained
from American Type Culture Collection (Manassas, VA).
Dulbecco’s modified Eagle’s medium (DMEM), penicillin,
streptomycin, and fetal bovine serum (FBS) were purchased
from Gibco Life Technologies (Rockville, MD). Raw 264.7
cells were grown in DMEM with 10% FBS, 100 units/ml
penicillin, and 100 ?g/ml streptomycin at 37°C in 5% CO2
humidified air. THP-1 cells were grown in RPMI 1640 with
L-glutamine and 25 mM HEPES buffered saline (Gibco Life
BEE VENOM INTERACTION WITH p50 SUBUNIT OF NF-?B3505
Technologies) supplemented with 10% FBS, 100 units/ml
penicillin, and 100 ?g/ml streptomycin at 37°C in 5% CO2
Synoviocyte culture. Synovial tissues were obtained,
with consent, from 9 RA patients who were undergoing total
knee replacement or arthroscopic synovectomy. From these
samples, 3 cell lines were studied. To avoid the effects of
steroids on the synovial cells, the patients were instructed to
cease use of steroid medication for 2 months before the
surgery. All patients met the American College of Rheuma-
tology (formerly, the American Rheumatism Association) 1987
revised criteria for RA (23). Synovial tissues were minced and
treated for 4 hours with 4 mg/ml collagenase 1 (Worthington,
Freehold, NJ) in DMEM at 37°C in 5% CO2. Dissociated cells
were plated in DMEM supplemented with 10% FBS, penicillin
(100 units/ml), and streptomycin (100 ?g/ml). Cells were used
between the third and fifth passages.
Determination of NO. Cells were grown in 24-well
plates and then incubated for 72 hours with or without LPS (1
?g/ml) in the absence or presence of various concentrations of
BV (0.5–5 ?g/ml) or melittin (5 or 10 ?g/ml). The nitrite
accumulation in the supernatant was assessed by Griess reac-
tion (24). Each 100 ?l of culture supernatant was mixed with
an equal volume of Griess reagent and incubated at room
temperature for 10 minutes. The absorbance at 550 nm was
measured in an automated microplate reader, and a series of
known concentrations of sodium nitrite was used as a standard.
Determination of PGE2. Cells were cultured for 24
hours, and then they were treated with LPS (1 ?g/ml) in the
absence or presence of various concentrations of BV (0.5–5
?g/ml) or melittin (5 or 10 ?g/ml) in the presence of tritiated
arachidonic acid (0.4 ?Ci/ml; Perkin Elmer, Boston, MA) for
48 hours. Briefly, PGs were extracted and separated by thin-
layer chromatography on a silica gel G plate using develop-
ment solution (ethyl acetate/isooctane/acetic acid/water [110:
corresponding to each PG was scraped off, and the radioactiv-
ity was determined with a liquid scintillation counter (Perkin
Elmer). Similarly, the liberation of arachidonic acid was deter-
mined in the medium after extraction and separation using
petroleum ether/diethyl ether/acetic acid (40:40:1 [v/v/v]) as the
development solution (25).
Measurement of intracellular calcium. Intracellular
calcium was measured by fluorescence ratio imaging of the
calcium indicator dye Fura 2 according to the methods of
Grynkiewicz et al (26). Briefly, the cells (1.3 ? 106cells/ml)
were loaded with 5 ?M Fura 2 at 37°C for 30 minutes and
imaged with a Delta Scan system (Photon Technology, Prince-
ton, NJ). Average calcium concentration data were expressed
as a ratio of the fluorescence emissions obtained using 2
different excitation wavelengths (340 and 380 nm).
Western blot analysis. Cells were homogenized with
lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.02% NaN3,
0.2% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 ?l/ml
aprotinin, 1% igapel 630 [Sigma], 10 mM NaF, 0.5 mM EDTA,
0.1 mM EGTA, and 0.5% sodium deoxycholate) and centri-
fuged at 23,000g for 1 hour. Equal amounts of proteins (80 ?g)
were separated on SDS–12% polyacrylamide gels and then
transferred to a nitrocellulose membrane (Hybond ECL; Am-
ersham Pharmacia Biotech). Blots were blocked for 2 hours at
room temperature with 5% (w/v) nonfat dried milk in Tris
buffered saline (10 mM Tris, pH 8.0, 150 mM NaCl) containing
0.05% Tween 20. The membrane was incubated for 5 hours at
room temperature with specific antibodies: goat polyclonal
COX-2, TNF?, and p50 antibody (1:1,000), and mouse mono-
clonal cPLA2and iNOS antibodies (1:500) (Santa Cruz Bio-
technology). The blot was then incubated with the correspond-
ing conjugated anti-rabbit IgG–horseradish peroxidase (Santa
Cruz Biotechnology). Immunoreactive proteins were identified
with the ECL Western blot detection system. The protein
bands were scanned by densitometry using MyImage (SLB,
Seoul, South Korea) and quantified with Labworks 4.0 soft-
ware (UVP, Upland, California).
Preparation of nuclear extracts and electrophoretic
mobility shift assay (EMSA). Gel shift assays were performed
according to the manufacturer’s recommendations (Promega).
Briefly, 1 ? 106cells/ml were washed twice with 1? phosphate
buffered saline (PBS) followed by the addition of 1 ml of PBS,
and the cells were scraped into a cold Eppendorf tube. Cells
were centrifuged at 15,000g for 1 minute, and the resulting
supernatant was removed. Solution A (50 mM HEPES, pH 7.4,
10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol
[DTT], 0.1 ?g/ml phenylmethylsulfonyl fluoride, 1 ?g/ml pep-
statin A, 1 ?g/ml leupeptin, 10 ?g/ml soybean trypsin inhibitor,
10 ?g/ml aprotinin, and 0.5% Nonidet P40) was added to the
pellet in a 2:1 ratio (v/v) and allowed to incubate on ice for 10
minutes. Solution C (solution A ? 10% glycerol and 400 mM
KCl) was added to the pellet in a 2:1 ratio (v/v) and vortexed
on ice for 20 minutes. The cells were centrifuged at 15,000g for
7 minutes, and the resulting nuclear extract supernatant was
collected in a chilled Eppendorf tube. Consensus oligonucleo-
tides were end-labeled using T4 polynucleotide kinase and
?32P-ATP for 10 minutes at 37°C. Gel shift reactions were
assembled and allowed to incubate at room temperature for 10
minutes, followed by the addition of 1 ?l (50,000–200,000
counts per minute) of32P-labeled oligonucleotide and another
20 minutes of incubation at room temperature. Subsequently,
1 ?l of gel-loading buffer was added to each reaction and
placed on 4% nondenaturing gels and electrophoresed until
the dye was three-fourths of the way down the gel. The gel was
dried at 80°C for 1 hour and exposed to film overnight at 70°C.
The relative density of the protein bands was scanned by
densitometry using MyImage (SLB) and quantified with Lab-
works 4.0 software (UVP).
Transfection and assay of luciferase activity. Raw
264.7 or THP-1 cells were transfected with pNF-?B-Luc plas-
mid (5? NF-?B; Stratagene, La Jolla, CA) using a mixture of
plasmid and Lipofectamine Plus in Opti-MEM, according to
the manufacturer’s specifications (Invitrogen, Carlsbad, CA).
The control pCMV? (Clontech, Palo Alto, CA) was cotrans-
fected to monitor the transfection efficiency. After 24 hours,
the cells were then cotreated with BV (or melittin) and LPS.
Luciferase activity was measured using a luciferase assay kit
(Promega) and WinGlow software, according to the manufac-
turer’s instructions (Berthold, Wildbad, Germany).
Surface plasmon resonance (SPR) analysis. BIAcore
2000 and a CM5 sensor chip were both supplied by BIAcore
(Uppsala, Sweden). HEPES buffered saline (pH 7.4, contain-
3506 PARK ET AL
ing 10 mM HEPES, 1 mM EDTA, 0.001% Tween 20, and
0.15M NaCl) was used as the constant flow buffer unless
otherwise stated. All buffers and solutions used during BIA-
core analysis were created using ultrapure water, then de-
gassed and sterile-filtered. Activated CM-dextran matrix car-
riedoutby mixing ethyl-N-(dimethylaminopropyl)
carbodiimide and N-hydroxysuccimide was surfaced on the
sensor chip. Recombinant p50 protein or melittin was then
layered onto the CM-dextran sensor chip, followed by blockage
of the chip using 1M ethanolamine, pH 8.5. Serial dilutions of
BV, melittin, or p50 antibody were prepared using HEPES
buffered saline, and then flowed sequentially with increasing
concentrations. The regeneration of protein interaction was
tested by changing the pH of the solution and was found to
occur optimally at pH 12. The BIAcore 2000 system continu-
ously monitored the change in mass at the sensor surface, and
the kinetics of protein interaction was analyzed with BIAevalu-
ation 3.0 software (BIAcore).
Immunocytochemistry. To determine whether melittin
could be taken up into the cells, cells (1 ? 105cm2) were
cultured on a chamber slide (Lab-Tak II chamber slider
system; Nalge Nunc International, Naperville, IL) and then
treated with melittin labeled with superior Alexa Fluor 488 dye
(Molecular Probes, Eugene, OR). Cells were incubated for 24
hours at 37°C and were then fixed in 4% paraformaldehyde.
The membrane was permeabilized by exposure for 5 minutes
to 0.2% Triton X-100 in PBS, and the cells were placed in
blocking serum (5% horse or goat serum in PBS). Immuno-
fluorescence images were acquired using a confocal laser
scanning microscope (dual-wavelength scan, MRC1024; Bio-
Rad) with a 60? oil immersion objective.
Statistical analysis. Data were analyzed using one-way
analysis of variance followed by Tukey’s test as a post hoc test.
P values less than 0.05 were considered significant.
Effects of BV on AIA. Consistent with previous
studies of rat models of chronic AIA, BV (0.8 and 1.6
?g/kg) significantly reduced hind paw edema (Figure
1A). Consistent with the inhibitory effects on the paw
swelling volume, the clinical score was also reduced in
BV-treated animals (Figure 1B). Radiographic examina-
tion of the hind paws revealed bone matrix resorption,
soft tissue swelling, and osteophyte formation at the
joint margin of rats 15 days after adjuvant injection, all
of which were markedly reduced by BV treatment (data
not shown). BV treatment did not affect body weight
progression and produced no behavioral alteration (data
not shown), suggesting that BV itself did not cause any
Inhibitory effects of BV and melittin on LPS-
induced PGE2and NO generation. The maximum pro-
duction of NO and PGE2was seen 12 or 24 hours after
stimulation in a time course study (data not shown). We
therefore determined NO production at 12 hours and
PGE2at 24 hours after stimulation. The concentration
of nitrite (?M) in cell supernatant after treatment with
LPS (1 ?g/ml) alone or with various doses of BV (0.5–5
?g/ml) was determined using Griess reagent. Significant,
dose-dependent inhibition of LPS-induced PGE2was
induced by treatment with BV both in Raw 264.7 cells
(Figure 2A, part a) and in synoviocytes (Figure 2A, part
b). We also observed that BV significantly inhibited
LPS-induced production of NO in a dose-dependent
manner in both Raw 264.7 cells (Figure 2B, part a) and
synoviocytes (Figure 2B, part b). The inhibitory effect of
melittin on the production of PGE2and NO was similar
or slightly greater compared with BV in both Raw 264.7
Figure 1. Effects of bee venom (BV) on adjuvant-induced arthritis in
rats. BV was administered daily into the plantar surface of the right
hind paw for 14 days, beginning 1 day after the injection of adjuvant.
A, Hind paw volume and B, clinical score were determined as
described in Materials and Methods. Values are the mean and SEM
from 10 animals per group. ? ? P ? 0.05 compared with adjuvant-
induced arthritis control group.
BEE VENOM INTERACTION WITH p50 SUBUNIT OF NF-?B 3507
cells and synoviocytes. However, BV or melittin alone
had no cytotoxic effect and did not affect the basal
production of PGE2and NO (data not shown).
Inhibitory effects of BV and melittin on LPS-
enhanced intracellular calcium levels. Experiments
were carried out to investigate the effect of BV and
melittin on intracellular calcium mobilization in Raw
264.7 cells. BV or melittin alone did not significantly
change calcium mobilization in Raw 264.7 cells (data not
shown). The response to LPS was rapid, and the intra-
cellular calcium level was significantly increased. The
elevated intracellular calcium level was reduced in a
dose-dependent manner by treatment with BV in Raw
264.7 cells (Figure 2C). The reduction of intracellular
calcium with melittin was similar to that with BV.
Interestingly, calcium response to LPS and sodium ni-
troprusside was not found in synoviocytes (data not
shown). The insensitivity of synoviocytes was also found
after treatment with thapsigargin, a well-known agent
that releases calcium by activation of Ca2?-ATPase. We
therefore could not determine the change in intracell-
ular calcium level after treatment with BV and melittin
Inhibitory effects of BV and melittin on LPS-
induced COX-2, cPLA2, and iNOS expression. To verify
whether the inhibition of PGE2and NO generation is
due to decreased protein expression, the effects of BV
and melittin on COX-2, cPLA2, and iNOS protein
expression were determined by Western blotting. The
maximum expression of COX-2, cPLA2, and iNOS was
Figure 2. Effects of bee venom (BV) and melittin on lipopolysaccharide (LPS)–induced prostaglandin E2(PGE2) and nitric oxide
(NO) generation and the intracellular calcium level in Raw 264.7 cells and synoviocytes. A, Inhibition of LPS-induced PGE2
production by BV and melittin in Raw 264.7 cells (a) and in synoviocytes (b). B, Inhibition of nitrite release by BV and melittin.
Raw 264.7 cells (a) and synoviocytes (b) were treated with LPS with or without 0.5–5 ?g/ml BV or 5 or 10 ?g/ml melittin at 37°C
for 24 hours. The amount of NO or PGE2in medium was measured as described in Materials and Methods. Values are the mean
and SEM of 3 independent experiments performed in triplicate. ? ? P ? 0.05 compared with LPS alone. C, Raw 264.7 cells were
loaded with 5 ?M Fura 2 for 30 minutes, then 0.5–5 ?g/ml BV (a) or 5 or 10 ?g/ml melittin (b) was added in the absence or
presence of LPS. [Ca2?]iwas monitored by fluorescence ratio imaging, using the ratio of excitation at 340 and 380 nm (F340/380).
The traces are from a single experiment, and are representative of 3 separate cell preparations.
3508PARK ET AL
seen 24 hours after LPS treatment in Raw 264.7 cells
but 6 hours after treatment in synoviocytes (results
not shown). Therefore, expression patterns were de-
termined at those designated times with the same con-
centration of BV and melittin that caused a reduction of
PGE2and NO generation. LPS-induced COX-2, cPLA2,
and iNOS protein expression in Raw 264.7 cells (Figure
3A) and in synoviocytes (Figure 3B) was inhibited by BV
and melittin in a dose-dependent manner, consistent
with reducing effects on the generation of PGE2and
Inhibition of NF-?B–dependent luciferase activ-
ity. Transcriptional regulation involving the activation of
NF-?B has been implicated in the LPS-induced expres-
sion of COX-2, cPLA2, and iNOS (7). To determine the
role of BV in NF-?B–dependent gene transcription, we
conducted a transient transfection assay with a fusion
gene containing SV40 promoter, 5 repeats of the con-
sensus NF-?B binding sequence, and the luciferase
reporter gene. Raw 264.7 and THP-1 cells were trans-
fected with this promoter-reporter gene construct, and
transcriptional activities were measured after stimulat-
ing the cells with LPS with or without BV or melittin. As
shown in Figure 4, cotreatment of the transfected cells
with BV significantly inhibited the luciferase activity
induced by the different agents in Raw 264.7 cells
(Figure 4A) and in synoviocytes (Figure 4B). LPS in-
duced a 7–8-fold increase in luciferase activity; however,
in the presence of BV and melittin, the increased
luciferase activity was markedly reduced in Raw 264.7
cells and THP-1 cells. Luciferase activity induced by
TNF? or TNF? with LPS (8–11-fold increase) was also
reduced by BV or melittin. Compared with the effect of
BV (5 ?g/ml), the inhibitory effect of melittin (10 ?g/ml)
on luciferase activity was greater after stimulation with
LPS, TNF?, or TNF? with LPS.
Figure 3. Effects of BV and melittin on LPS-induced inflammatory gene expression in Raw 264.7 cells and synoviocytes. A, Raw
264.7 cells were treated with 0.5–5 ?g/ml BV or 5 or 10 ?g/ml melittin in the presence of LPS at 37°C for 24 hours. B, Synoviocytes
were treated with 0.5–5 ?g/ml BV or 5 or 10 ?g/ml melittin in the presence of LPS at 37°C for 6 hours. Equal amounts of total
proteins (80 ?g/lane) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and expression of
cyclooxygenase 2 (COX-2), cytosolic phospholipase A2(cPLA2), inducible nitric oxide synthase (iNOS), and ?-actin (as an internal
control) was detected by Western blotting using specific antibodies. Quantification of band intensities from 3 independent
experimental results was determined by densitometry. Values are the mean and SEM from 3 experiments performed in triplicate.
? ? P ? 0.05 compared with the LPS-treated group. See Figure 2 for other definitions.
BEE VENOM INTERACTION WITH p50 SUBUNIT OF NF-?B 3509
Inhibition of NF-?B activation. NF-?B DNA
binding activity was then measured by EMSA in nuclear
extracts of Raw 264.7 cells and synoviocytes treated with
LPS alone, or in combination with BV or melittin for 1
hour, which was the time to induce NF-?B activation
maximally (results not shown). BV inhibited the LPS-
induced specific NF-?B DNA binding activity in a
dose-dependent manner both in Raw 264.7 cells and in
synoviocytes (Figure 5A, parts a and b). Melittin also
significantly reduced NF-?B activation by LPS. The
specificity of DNA binding was evidenced either by
supershift assay using antibodies for the p65 and p50
components of NF-?B (Figure 5A, part c) or by compe-
tition assay with the addition of excessive unlabeled/cold
oligonucleotides (Figure 5A, part d).
Inhibition of LPS-induced release of I?B and
nuclear translocation of the p50 subunit of NF-?B.
Release of the inhibitory I?B subunit and nuclear trans-
location of the p65 or p50 subunit are involved in
activation of NF-?B. The kinetics of I?B release was
studied by Western blot analysis. As shown in Figure 5B,
LPS-induced I?B release was markedly inhibited by BV
and melittin both in Raw 264.7 cells (Figure 5B, part a)
and in synoviocytes (Figure 5B, part b). Next, to study
the translocation of subunits of NF-?B into the nucleus
during NF-?B activation, we determined the appearance
of the p50 and p65 subunits of NF-?B in the nuclear
extracts. BV and melittin treatment dose-dependently
resulted in delayed and reduced nuclear translocation of
the p50 subunit, but had no effect on p65 translocation.
The inhibitory effect of melittin on the release of I?B
and nuclear translocation of the p50 subunit by melittin
was similar to that of BV. To further examine the
mechanism of I?B release, phosphorylation of I?B was
investigated. LPS-induced phosphorylation of I?B in
both cells was reduced by treatment with BV or melittin.
Figure 4. Effects of BV and melittin on LPS-induced NF-?B–dependent luciferase activity in Raw 264.7 cells and THP-1 cells. Raw
264.7 cells (A) and THP-1 cells (B) were transfected with pNF-?B-Luc plasmid (5? NF-?B) and then activated with LPS or tumor
necrosis factor ? (TNF?) in the absence or presence of 0.5–5 ?g/ml BV or 5 or 10 ?g/ml melittin for 2 hours, and then the luciferase
activity was determined. Values are the mean and SEM of 3 independent experiments performed in triplicate. The level of induction
was calculated relative to the luciferase activity in unstimulated transfected cells. ? ? P ? 0.05 compared with the LPS-, TNF?-, or
LPS ? TNF?–treated group. RLU ? relative luciferase units; ?-gal ? ?-galactosidase (see Figure 2 for other definitions).
3510 PARK ET AL
Moreover, the inhibitory effect of BV or melittin on I?B
phosphorylation in Raw 264.7 cells (Figure 5C, part a)
and in synoviocytes (Figure 5C, part b) was partially
reversed by the phosphatase inhibitor H-7 in a dose-
BV and melittin interaction with the p50 subunit
of NF-?B. To determine whether p50 may be a target for
the inhibitory effect of BV and/or melittin on NF-?B
activity, we studied the interaction between p50 and BV
or melittin in a cell-free system. Protein p50 was incu-
bated with the NF-?B DNA binding element in the
absence or presence of BV or melittin, and then EMSA
was performed to determine the ability of p50 to bind to
the NF-?B binding element. An inhibitory effect of BV
and melittin on the ability of p50 to bind to the NF-?B
binding element was observed (Figure 6A).
Figure 5. Effects of BV and melittin on LPS-induced NF-?B activation, release of I?B, and nuclear translocation of the p50 subunit
of NF-?B in Raw 264.7 cells and synoviocytes. A, Activation of NF-?B was determined by electrophoretic mobility shift assay (EMSA),
as described in Materials and Methods. Nuclear extracts from Raw 264.7 cells (a) and synoviocytes (b) treated either with LPS alone
or with BV (0.5–5 ?g/ml) or melittin (5 or 10 ?g/ml) were incubated in binding reactions of32P-labeled oligonucleotide containing the
?B sequence. NF-?B DNA binding activity was determined by EMSA. Each panel is representative of 3 similar experiments performed
in triplicate. For supershift assays, nuclear extracts from cells treated with LPS were incubated for 1 hour before EMSA with specific
antibodies against the p50 and p65 NF-?B isoforms (c). Asterisk indicates supershift assay using p50 antibody. For competition assays,
nuclear extracts from cells treated with LPS were incubated for 1 hour before EMSA with unlabeled NF-?B oligonucleotide or labeled
stimulating protein 1 and activator protein 1 (d). B, Raw 264.7 cells (a) were treated with 0.5–5 ?g/ml BV or 5 or 10 ?g/ml melittin
in the presence of LPS at 37°C for 24 hours. Nuclear, cytosolic, and total proteins (50 ?g) extracted 24 hours after treatment were used
to determine the expression of p50, p65, I?B, and ?-actin (as an internal control). Synoviocytes (b) were treated with 0.5–5 ?g/ml BV
or 5 or 10 ?g/ml melittin in the presence of LPS at 37°C for 6 hours. Equal amounts of total proteins (80 ?g/lane) were subjected to
10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and expression of p50, p65, I?B, and ?-actin proteins was detected
by Western blotting using specific antibodies. C, Levels of p-I?B protein in cytosolic lysates of Raw 264.7 cells (a) and synoviocytes
(b) treated with 0.5–5 ?g/ml BV or 5 or 10 ?g/ml melittin in the presence of LPS with or without phosphate inhibitor (H-7).
Immunoblots were performed with a p-I?B–specific antibody. Each panel is representative of 3 similar experiments performed in
duplicate. See Figure 2 for other definitions.
BEE VENOM INTERACTION WITH p50 SUBUNIT OF NF-?B 3511
Radiographic analysis has defined the presence
of cysteine residues in the structure of p50 of the DNA
binding domain, and some investigations have shown
that target disruption of cysteine residues of the p50
subunit can inhibit NF-?B activation (27). To explore
this possibility, p50 and NF-?B DNA binding element
mixtures were incubated with BV or melittin in the
presence of DTT, and then DNA binding activity was
measured by EMSA. DTT reversed the inhibitory effect
of BV or melittin on p50 DNA binding ability in a
dose-dependent manner (results not shown). To further
investigate direct binding of BV or melittin with p50,
surface plasmon resonance analysis was then performed.
The analysis clearly demonstrated that BV or melittin
bound to the p50 subunit of NF-?B immobilized onto
the surface of a sensor chip, and increasing doses of BV
and melittin clearly showed increased binding activity
with p50 (Figures 6B and C). The binding affinity (Kd) of
BV and melittin to p50 was 4.6 ? 10?6M and 1.2 ?
10?8M, respectively. The binding affinity (Kd) of p50 to
anti-p50 antibody was 2.4 ? 10?10M under the condi-
Next, the interaction between melittin immobi-
lized onto a sensor chip and immunoprecipitated p50
extracted from a nuclear fraction of cells treated with
either LPS alone or LPS with BV or melittin was
studied. The interaction between melittin and immuno-
precipitated p50 extracted from a nuclear fraction of
Figure 6. Uptake of melittin into cells and interaction between bee venom (BV) or melittin and p50. A, Recombinant human p50 was
incubated with the NF-?B DNA binding element in the absence or presence of BV or melittin, and binding capacity was determined
by electrophoretic mobility shift assay. B–E, Representative surface plasmon resonance binding kinetic traces from 2 separate
experiments with similar results. B and C, Full kinetic data set for the ability of BV (B) and melittin (C) to bind to immobilized p50.
D and E, Representative kinetic traces for the interaction between melittin immobilized onto a surface chip and p50 extracted from
nuclear fraction with each treatment of Raw 264.7 cells (D) or synovial joint (E). F, Uptake of melittin into the membrane and nucleus
of Raw 264.7 cells (original magnification ? 360). RU ? resonance units; Kd? binding affinity; negative ? cells treated with unlabeled
melittin; positive ? cells treated with melittin labeled with Alexa Fluor 488.
3512PARK ET AL
cells treated with LPS with BV or melittin was reduced
much more than the interaction with immunoprecipi-
tated p50 extracted from the nuclear fraction of cells
treated with LPS alone. This inhibitory effect was dose-
dependent (Figure 6D). A similar reduced interaction
was also found in the interaction between melittin and
p50 in a nuclear fraction extracted from synovial joints
treated with the combination of LPS and BV (Figure
6E). We also monitored the uptake of melittin into cells.
We labeled melittin with Alexa Fluor 488 using a
protein-labeling kit, and incubated Raw 264.7 cells were
treated with the conjugated melittin with Alexa Fluor.
The uptake of the labeled melittin into the cells was
viewed under a confocal laser-scanning microscope. As
seen in Figure 6F, BV was taken up into the membrane
and nucleus of the cells.
To gain a better understanding of the molecular
mechanisms of the antiinflammatory effects of BV on
RA, we reevaluated the inhibitory effect of BV in a rat
model of acute and chronic RA. Consistent with other
findings (20–23,28), the antiarthritic effect of BV was
also found in the present study. Treatment with BV
resulted in a great reduction in tissue swelling and
osteophyte formation in the model of chronic arthritis as
well as edema formation in the model of acute arthritis
in this study. These effects were observed in our study
with a much lower dose of BV (?g/kg) than the doses
used in other studies (mg/kg) (20). Different sources,
methods of extract or purification of BV and composi-
tion, different animal models, or other unknown factors
may cause this discrepancy. Although further study is
needed for determination of an effective dose, our data
show that the antiarthritic effects of BV are related to its
The antiinflammatory properties of BV and the
molecular action of this compound were demonstrated.
In this study, 0.5–5 ?g/ml noncytotoxic BV inhibited
LPS-induced PGE2and NO production in Raw 264.7
cells. The inhibitory action of BV on the generation of
inflammation mediators was also effective in synovio-
cytes from RA patients. The inhibitory effect of BV was
consistent with that of a well-known COX-2 inhibitor,
indomethacin (10 mM) (data not shown). Thus, the
inhibitory effects on BV on the inflammatory reaction in
AIA may be due, at least in part, to the inhibition of
COX-2 expression. In inflammatory arthritis, there has
also been evidence indicating that the affected tissues
produce large amounts of NO that could act as a
proinflammatory agent to cause tissue injury (29,30). In
this regard, the reducing effect of BV on NO may also
contribute to the antiarthritic effect of BV. NO may also
influence the actions of TNF?, which is significantly
involved in the influx of inflammatory cells, erosion of
joint cartilage, and bone destruction in inflammatory
arthritis (31,32). BV exhibits important inhibitory effects
on the generation of inflammation mediators, including
TNF?, and their enzyme expression (data not shown).
We also found that BV has an inhibitory effect on
LPS-induced reactive oxygen species (ROS) generation
(data not shown) as well as calcium release. Thus, the
antiarthritic effects of BV may be related to the multiple
inhibitory effects on the generation of inflammation
mediators such as PGE2, NO, and TNF? as well as on
the release of ROS and intracellular calcium (33).
NF-?B is one of the most important regulators of
expression of proinflammatory genes such as COX-2,
cPLA2, iNOS, and TNF? (34,35). In the present study,
we have shown that BV inhibits DNA binding and
transcriptional activity of NF-?B in a dose-dependent
manner in both Raw 264.7 cells and synoviocytes. This
effect was also found in THP-1 cells, suggesting that the
inhibitory effect of BV may be not cell specific. The
promoter region of the murine gene encoding iNOS and
COX-2 contains NF-?B binding sites (5,36). The inhib-
itory effect was consistent with the reduction of the
release of I?B detected in the cytosol by suppression of
phosphorylation of I?B, and the decrease of transloca-
tion of the p50 subunit of NF-?B but not p65. The
specific phosphatase inhibitor H-7 reversed this inhibi-
tory effect of BV or melittin.
These results suggest that BV or melittin inhib-
ited the DNA binding activity of NF-?B through inhibi-
tion of I?B phosphorylation, thereby inhibiting p50
translocation, resulting in reduction of inflammatory
gene expression. Our data showing the inhibitory effect
of BV or melittin on LPS-induced ROS generation
support two possible perspectives. Cellular redox equili-
brium has been shown to be a critical factor in NF-?B
activation through disruption of the pathways that in-
volve either p50 homodimer–DNA binding on the one
hand, or phosphorylation of I?B on the other (37,38).
The direct interaction between BV and melittin
and p50 was evidenced by SPR analysis. The binding
affinity of BV (Kd? 4.6 ? 10?6M) or melittin (Kd?
1.2 ? 10?8M) to p50 is ?50–500 times weaker than the
binding affinity of p50 to anti-p50 (Kd? 2.4 ? 10?10M).
However, the binding affinity is comparable with or
much stronger than the binding affinity of p50 to DNA
(Kd? 3–5.4?8M) (37,39,40). It was also found that p50
BEE VENOM INTERACTION WITH p50 SUBUNIT OF NF-?B 3513
extracted from cells treated with LPS alone bound to
melittin that had been immobilized onto the surface of
the sensor chip with much greater affinity than did p50
extracted from cells treated with the combination of LPS
and BV or melittin. Moreover, inhibitory effects of BV
and melittin on the ability of p50 to bind to the NF-?B
DNA element in a cell-free system were also found. We
found that melittin was taken up into the cells and
translocated into the nucleus, where p50 is translocated
when NF-?B is activated. Therefore, it is possible that
BV or melittin directly binds to p50 in either the cytosol
or the nucleus, thereby inhibiting NF-?B activation.
The radiographic structure of p50 has shown the
presence of cysteine residues, which are critical for
optimal DNA–protein interactions (38,41), and cysteine
residues of p50 have been considered to be a target for
redox regulation (42). Further evidence shows that DTT
reversed the ability of p50 to bind to the NF-?B DNA
element as well as the direct binding of melittin. These
results show that BV or melittin may modify a sulfhydryl
group of p50 protein (43), thereby hindering p50 affinity
to the NF-?B DNA binding element.
An experiment to identify the binding site of p50
with melittin is currently being conducted. There have
been intensive searches for molecules that inhibit the
activation of NF-?B though various modes of action
(15,34,41,44). The present study demonstrates that the
inhibition of NF-?B activation by BV, through the direct
ability of BV and/or melittin to bind to p50, may be an
important molecular mechanism in the suppressive ef-
fect of BV on inflammatory reactions. The potency of
BV (or melittin) in the inhibition of the inflammatory
response may be of great benefit in degenerative and
inflammatory diseases such as RA. The extent of the
inhibitory effects of melittin in most parameters deter-
mined in the present study is similar to or greater than
those of BV itself, suggesting that melittin may be a
major causative component in the pharmacologic effects
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