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Anti-inflammatory and anti-cancer activity of mulberry (Morus alba L.) root bark

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Background Root bark of mulberry (Morus alba L.) has been used in herbal medicine as anti-phlogistic, liver protective, kidney protective, hypotensive, diuretic, anti-cough and analgesic agent. However, the anti-cancer activity and the potential anti-cancer mechanisms of mulberry root bark have not been elucidated. We performed in vitro study to investigate whether mulberry root bark extract (MRBE) shows anti-inflammatory and anti-cancer activity. Methods In anti-inflammatory activity, NO was measured using the griess method. iNOS and proteins regulating NF-κB and ERK1/2 signaling were analyzed by Western blot. In anti-cancer activity, cell growth was measured by MTT assay. Cleaved PARP, ATF3 and cyclin D1 were analyzed by Western blot. Results In anti-inflammatory effect, MRBE blocked NO production via suppressing iNOS over-expression in LPS-stimulated RAW264.7 cells. In addition, MRBE inhibited NF-κB activation through p65 nuclear translocation via blocking IκB-α degradation and ERK1/2 activation via its hyper-phosphorylation. In anti-cancer activity, MRBE deos-dependently induced cell growth arrest and apoptosis in human colorectal cancer cells, SW480. MRBE treatment to SW480 cells activated ATF3 expression and down-regulated cyclin D1 level. We also observed that MRBE-induced ATF3 expression was dependent on ROS and GSK3β. Moreover, MRBE-induced cyclin D1 down-regulation was mediated from cyclin D1 proteasomal degradation, which was dependent on ROS. Conclusions These findings suggest that mulberry root bark exerts anti-inflammatory and anti-cancer activity.
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R E S E A R C H A R T I C L E Open Access
Anti-inflammatory and anti-cancer activity of
mulberry (Morus alba L.) root bark
Hyun Ji Eo
1
, Jae Ho Park
2
, Gwang Hun Park
1
, Man Hyo Lee
3
, Jeong Rak Lee
3
, Jin Suk Koo
1,4
and Jin Boo Jeong
1,4,5*
Abstract
Background: Root bark of mulberry (Morus alba L.) has been used in herbal medicine as anti-phlogistic, liver
protective, kidney protective, hypotensive, diuretic, anti-cough and analgesic agent. However, the anti-cancer activity
and the potential anti-cancer mechanisms of mulberry root bark have not been elucidated. We performed in vitro
study to investigate whether mulberry root bark extract (MRBE) shows anti-inflammatory and anti-cancer activity.
Methods: In anti-inflammatory activity, NO was measured using the griess method. iNOS and proteins regulating
NF-κB and ERK1/2 signaling were analyzed by Western blot. In anti-cancer activity, cell growth was measured by MTT
assay. Cleaved PARP, ATF3 and cyclin D1 were analyzed by Western blot.
Results: In anti-inflammatory effect, MRBE blocked NO production via suppressing iNOS over-expression in
LPS-stimulated RAW264.7 cells. In addition, MRBE inhibited NF-κB activation through p65 nuclear translocation
via blocking IκB-αdegradation and ERK1/2 activation via its hyper-phosphorylation. In anti-cancer activity, MRBE
deos-dependently induced cell growth arrest and apoptosis in human colorectal cancer cells, SW480. MRBE
treatment to SW480 cells activated ATF3 expression and down-regulated cyclin D1 level. We also observed that
MRBE-induced ATF3 expression was dependent on ROS and GSK3β. Moreover, MRBE-induced cyclin D1
down-regulation was mediated from cyclin D1 proteasomal degradation, which was dependent on ROS.
Conclusions: These findings suggest that mulberry root bark exerts anti-inflammatory and anti-cancer activity.
Keywords: Morus alba L. Mulberry root bark, Medicinal plant, Anti-inflammation, Anti-cancer
Background
Inflammation is an innate immune response by various
immune cells including macrophages for the protection
against the harmful stimuli such as virus and bacteria
[1]. As a consequence of excessive inflammatory response,
large amounts inflammatory mediators, such as nitric
oxide (NO) and prostaglandin E
2
(PGE
2
) are produced [2].
Inflammatory mediators-induced chronic inflammation is
considered to be a cause of numerous human diseases
including cancer, atherosclerosis, arthritis and septic shock
[3-5]. Among inflammatory mediators, NO is produced
by inducible nitric oxide synthase (iNOS) and results in
many disease processes such as carcinogenesis, obesity and
diabetes [6-8]. iNOS in macrophage is activated following
infection. Therefore, iNOS-mediated NO is a ubiquitous
mediator of a wide range of inflammatory conditions and
reflects degree of inflammation, thus providing a measure
of the inflammatory process [9].
It has been reported that development of cancer is asso-
ciated with inflammation [10,11]. Cancer is a major prob-
lem of public health in USA with the estimated 1.6 million
new cancer cases and 5.8 hundred thousand cancer deaths
occur in USA in 2013 [12]. Among inflammation-induced
cancers, colorectal cancer is the third leading cause of
cancer-related morbidity and mortality in USA [12].
Recently, cancer chemoprevention has received a great
attention and medicinal plants have been regarded as
effective anti-cancer sources [13].
Root bark of mulberry (Morus alba L.) has been used in
herbal medicine as anti-phlogistic, liver protective, kidney
* Correspondence: jjb0403@anu.ac.kr
Equal contributors
1
Department of Bioresource Sciences, Andong National University, Andong
760749, South Korea
4
Insititute of Agricultural Science and Technology, Andong National
University, Andong 760380, South Korea
Full list of author information is available at the end of the article
© 2014 Eo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Eo et al. BMC Complementary and Alternative Medicine 2014, 14:200
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protective, hypotensive, diuretic, anti-cough and analgesic
agent [14]. However, the anti-cancer activity and the
potential anti-cancer mechanisms of mulberry root bark
have not been elucidated.
Activating transcription factor 3 (ATF3) is a member
of the ATF/CREB subfamily of the basic-region leucine
zipper (bZIP) family. In human colorectal cancer, ATF3
expression was suppressed compared to normal adjacent
tissue [15]. Up-regulation of ATF3 expression can induce
apoptosis in colorectal cancer cells. In addition, ATF3
induces p53 activation [16,17] and inhibits Ras-mediated
carcinogenesis and cyclin D1 expression [18]. Cyclin D1
regulates cell cycle transition from G1 to S phase by form-
ing cyclin-dependent kinase (CDK) 4 and 6 [19]. Cyclin
D1 overexpression was observed in 68.3% of human
colorectal cancer [20]. Thus, up-regulation of ATF3
and down-regulation of cyclin D1 are major molecular
targets for treatment of colorectal cancer.
In light of the therapeutic potential of mulberry root
bark in inflammation-induced colorectal cancer, this study
was performed to elucidate the biological mechanism
by which mulberry root bark inhibits an inflammatory
response in LPS-stimulated RAW264.7 cells and induces
the inhibition of cell growth and apoptosis in human
colorectal cancer cells. Here, for the first time, we re-
ported that mulberry root bark extracts attenuated NO
production by suppressing iNOS expression via regulating
the activations of NF-κB and ERK1/2. In addition, it
induced cell growth arrest and apoptosis by activating
ATF3 expression and cyclin D1 proteasomal degradation
in colorectal cancer cells, SW480.
Methods
Materials
Cell culture media, Dulbeccos Modified Eagle medium
(DMEM) was purchased from Gibco Inc. (NY, USA). LPS
(Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-
2.5-diphenyltetrazolium bromide (MTT) were purchased
from SigmaAldrich (St. Louis, MO, USA). SB203580,
PD98059 were purchased from Calbiochem (San Diego,
CA). SB216763 and N-Acetyl Cysteine (NAC) were pur-
chased from SigmaAldrich. Antibodies against iNOS,
ATF3 and cyclin D1 were purchased from Santa Cruz
Biotechnology, Inc (Santa Cruz, CA, USA). Other anti-
bodies against IκB-a, p65, ERK1/2, phospho-ERK1/2
(Thr202/Tyr204) and b-actin were purchased from Cell
Signaling (Bervely, MA, USA). All chemicals were pur-
chased from Sigma-Aldrich, unless otherwise specified.
Sample preparation
The plant sample, Mulberry (Morus alba L. voucher num-
ber: PARK1002(ANH)) root bark, was kindly provided by
the Bonghwa Alpine Medicinal Plant Experiment Station,
Korea. One kilogram of mulberry root bark was extracted
with 1000 ml of 80% methanol with shaking for 24 h.
After 24 h, the methanol-soluble fraction was filtered and
concentrated to approximately 20 ml volume using a vac-
uum evaporator and then fractioned with petroleum ether
and ethyl acetate in a separating funnel. The ethyl acetate
fraction was separated from the mixture, evaporated by
a vacuum evaporator, and prepared aseptically and kept
in a refrigerator until use.
Cell culture and treatment
Mouse macrophage cell line, RAW264.7 and human
colorectal cancer cell line, SW480 were purchased
from Korean Cell Line Bank (Seoul, Korea) and grown
in DMEM supplemented with 10% fetal bovine serum
(FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.
These cells were maintained at 37°C under a humidified at-
mosphere of 5% CO
2
. Mulberry root bark extracts (MRBE)
were dissolved in dimethyl sulfoxide (DMSO) and then
treated to cells. DMSO was used as a vehicle and the
final DMSO concentration was not exceeded 0.1% (v/v).
Measurement of nitric oxide (NO) production
Inhibitory effect of mulberry root bark extracts on the
production of NO in LPS-stimulated RAW264.7 cells
was evaluated using literature [21]. Briefly, RAW264.7
cells were plated in 12-well plate for overnight. Cells were
pre-treated with mulberry root bark extracts at the indi-
cated concentrations for 2 h and then co-treat with LPS
(1 μg/ml) for the additional 18 h. After 18 h, 200 μlofthe
media was mixed with equal amount of Griess reagent
(1% sulfanilamide and 0.1% N-1-(naphthyl) ethylenediamine-
diHCl in 2.5% H
3
PO
4
). The mixture was incubated for
the additional 5 min at the room temperature and the
absorbance was measured at 540 nm.
Isolation of cytosol and nuclear fraction
Nuclear and cytosolic fractions were prepared follow-
ing the manufacturers protocols of nuclear extract kit
(Active Motif, Carlsbad, CA, USA). Briefly, RAW264.7
cells were washed with ice-cold PBS containing phos-
phatase inhibitors and harvested with 1xhypotonic buf-
fer for 15 min at 4°C. After adding detergent, the cells
were centrifuged at 15,000 rpm for30 min and then the
supernatants were collected as cytoplasmic fraction.
Nuclear fractions were collected by suspending nuclear
pellet with lysis buffer and centrifugation.
MTT assay
The 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazo-
lium bromide (MTT) assay was used to measure cell
proliferation. Briefly, SW480 cells were seeded onto 96-well
culture plate at a density of 50,000 cells per well. The cells
were treated with MRBE for 24 h. Then, 50 μlofMTT
solution (1 mg/ml) was added to each well. The resulting
Eo et al. BMC Complementary and Alternative Medicine 2014, 14:200 Page 2 of 9
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crystals were dissolved in DMSO. The formation of forma-
zan was measured by reading absorbance at a wavelength
of 570 nm.
SDS-PAGE and Western blot
Cells were washed with 1 × phosphate-buffered saline
(PBS), and lysed in radioimmunoprecipitation assay (RIPA)
buffer (Boston Bio Products, Ashland, MA, USA) supple-
mented with protease inhibitor cocktail (Sigma Aldrich)
and phosphatase inhibitor cocktail (Sigma Aldrich),
and centrifuged at 12,000 × g for 10 min at 4°C. Protein
concentration was determined by the bicinchoninic acid
(BCA) protein assay (Pierce, Rockford, IL, USA) using bo-
vine serum albumin (BSA) as the standard. The proteins
were separated on SDS-PAGE and transferred to PVDF
membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
The membranes were blocked for non-specific binding with
5% nonfat dry milk in Tris-buffered saline containing 0.05%
Tween 20 (TBS-T) for 1 h at room temperature and then
incubated with specific primary antibodies in 5% nonfat dry
milk at 4°C overnight. After three washes with TBS-T,
the blots were incubated with horse radish peroxidase
(HRP)-conjugated immunoglobulin G (IgG) for 1 h at room
temperature and chemiluminescence was detected with
ECL Western blotting substrate (Amersham Biosciences)
andvisualizedinPolaroidfilm.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was prepared using a RNeasy Mini Kit
(Qiagen, Valencia, CA, USA) and total RNA (1 μg) was
revese-transcribed using a Verso cDNA Kit (Thermo
Scientific, Pittsburgh, PA, USA) according to the manufac-
turers protocol for cDNA synthesis. PCR was carried out
using PCR Master Mix Kit (Promega, Madison, WI, USA)
with primers for human ATF3, human cyclin D1 and
human GAPDH as follows: human ATF3: 5-gtttgaggatttt
gctaacctgac-3, and reverse 5-agctgcaatcttatttctttctcgt-3;
human cyclin D1: forward 5-aactacctggaccgcttcct-3and
reverse 5-ccacttgagcttgttcacca-3; huaman GAPDH:
forward 5-acccagaagactgtggatgg-3and reverse 5-
ttctagacggcaggtcaggt-3.
Statistical analysis
Statistical analysis was performed with the Studentsun-
paired t-test, with statistical significance set at *, P < 0.05.
Results
The effect of MRBE on NO production and iNOS
expression in LPS-stimulated RAW264.7 cells
Macrophages play an important role in inflammatory re-
sponse by producing inflammatory mediators such as NO,
PGE
2
and TNF-α[22]. So, we used the mouse macrophage
cell line RAW264.7 cells for evaluating anti-inflammatory
effect of MRBE. iNOS-mediated NO is associated with
cytotoxicity and tissue damage and involved in several
processes such as chronic inflammation and immuno-
regulation [23]. To determine if MRBE could reduce NO
generation by LPS, RAW264.7 cells were pretreated with
MRBE for 2 h and then co-treated with LPS (1 μg/ml) for
the additional 18 h. As shown in Figure 1A, treatment
of LPS without MRBE induced NO overproduction in
LPS-stimulated RAW264.7 cells, while pretreatment of
MRBE suppressed LPS-mediated NO overproduction.
Since NO production is regulated by iNOS expression,
the effect of MRBE on iNOS expression was evaluated by
Western blot. As shown in Figure 1B, LPS overexpression
was detected in the cells stimulated LPS alone. However,
MRBE inhibited iNOS expression in LPS-stimulated
RAW264.7 cells. From these results, MRBE-induced
decrease of NO production may result from the inhibition
of LPS-induced iNOS overexpression in RAW264.7 cells.
Inhibitory effect of MRBE on LPS-induced NF-κB and
ERK1/2 activation in RAW264.7 cells
To elucidate the effect of MRBE on NF-κB activation,
we performed a Western blot for IκB-αdegradation in
LPS-stimulated RAW264.7 cells. As shown in Figure 2A,
LPS induced IκB-αdegradation at 15 min after the stimu-
lation. However, pretreatment of MRBE attenuated IκB-α
degradation in a dose-dependent manner. p65 nuclear
translocation resulted from IκB-αdegradation are essential
in NF-κB activation. Thus we examined whether MRBE
inhibits p65 nuclear translocation. As shown in Figure 2B,
LPS markedly increased an amount of p65 in the nucleus
of RAW264.7 cells. However, pretreatment of MRBE dose-
dependently inhibited LPS-induced p65 nuclear transloca-
tion in RAW264.7 cells. There is a growing evidence that
NF-κB activation is modulated by ERK1/2 activation [24].
Thus, we evaluated the effects of MRBE on phosphoryl-
ation of ERK1/2 in LPS-stimulated RAW264.7 cells using
Western blot to further investigate whether inhibition of
NF-κB activation by MRBE was associated with modula-
tion of ERK1/2. As shown in Figure 2C, Increase of phos-
phorylation of ERK1/2 was observed in LPS-stimulated
RAW264.7 cells without MRBE. However, MRBE attenu-
ated LPS-induced ERK1/2 phosphorylation. Overall, these
results suggest that MRBE may inhibit the inflammatory
response by ERK1/2-mediated NF-κBactivationinLPS-
stimulated RAW264.7 cells.
Effect of MRBE on cell viability and apoptosis in human
colorectal cancer cell line, SW480
There are a number of evidences indicating that MRBE
shows anticancer activity in human leukemia cells [25,26].
To observe whether MRBE affects viability and apoptosis
in human colorectal cancer cell line, SW480, we carried
out MTT assay for cell viability and Western blot for
apoptosis. As shown in Figure 3A, treatment of MRBE
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dose-dependently reduced the viability of SW480 cells by
43%, 71% and 83% at 6.25 μg/ml, 12.5 μg/ml and 25 μg/ml,
respectively. Apoptosis was also induced by MRBE treat-
ment (Figure 3B).
Effect of MRBE on the levels of ATF3 and cyclin D1 in
protein and mRNA in SW480 cells
There is growing evidence that activating transcription
factor 3 (ATF3) is linked to cell growth arrest and apoptosis
in colorectal cancer. To investigate whether MRBE activates
ATF3 expression in human colorectal cancer cells, SW480
cells were treated with MRBE at the indicated concentra-
tions for 24 h. As shown in Figure 4A and 4C, MRBE
induced ATF3 expression in the levels of both protein and
mRNA. Time-course experiment showed that induction of
ATF3 by MRBE occurred after 1 h stimulation (Figure 4D).
We also evaluated whether MRBE regulates cyclin D1
level in SW480 cells since cyclin D1 is associated with
cell growth arrest and apoptosis. As shown in Figure 4B,
MRBE decreased the protein level of cyclin D1 in a
dose-dependent manner. However, decrease in mRNA
level of cyclin D1 by MRBE treatment was not observed
(Figure 4C). In time-course experiment for cyclin D1
(Figure 4D), MRBE significantly reduced cyclin D1 protein
level after 1 h stimulation.
GSK3βand ROS-dependent ATF3 activation of MRBE in
SW480 cells
To elucidate the molecular mechanism for MRBE-induced
ATF3 expression, we evaluated several signaling pathways
affected by MRBE. SW480 cells were pretreated with kinase
inhibitors such as PD98059 (ERK1/2 inhibitor), SB203580
(p38 inhibitor) and SB216763 (GSK-3βinhibitor), and NAC
(ROS scavenger) for 2 h prior to incubation with 25 μg/ml
of MRBE. As shown in Figure 5A, MRBE-induced ATF3 ex-
pression was observed in the cells pretreated with PD98059
and SB203580. However, pretreatments of SB216763
and NAC diminished MRBE-induced ATF3 expression
Figure 1 Effect of MRBE on NO production (A) and iNOS (B) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-treated with
MRBE at the indicated concentrations for 2 h and then co-treated with LPS (1 μg/ml) for the additional 18 h. After treatment, NO production was
measured using the media and Griess reagent and cell lysates were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with iNOS
antibody for Western blot. iNOS protein was visualized using ECL detection. Actin was used as internal control. DMSO was used as a vehicle.
Values given are the mean ± SD (n = 3). *p < 0.05 compared to LPS treatment without MRBE.
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(Figure 5B). Collectively, these results suggest the pathways
of GSK-3βand ROS may be involved in MRBE-induced
ATF 3 express ion.
ROS-dependent cyclin D1 proteasomal degradation by
MRBE in SW480 cells
We found that MRBE dose-dependently attenuated cyclin
D1 protein level whiles it did not affect cyclin D1 mRNA
level (Figure 4B and 4C). Thus, we asked whether cyclin
D1 protein stability was affected by MRBE treatment.
Figure 5C shows that MG-132, a well-known proteasome
inhibitor, completely blocked MRBE-induced cyclin D1
down-regulation. This result suggests that MRBE-induced
cyclin D1 down-regulation may result from proteasomal
degradation. SW480 cells were pretreated with kinase
inhibitors such as PD98059, SB203580, SB216763 and
NAC for 2 h, and then co-treated with 25 μg/ml of
MRBE for the additional 1 h to elucidate the molecular
mechanism for cyclin D1 proteasomal degradation affected
by MRBE. As shown in Figure 5D and 5E, MRBE-induced
cyclin D1 down-regulation was observed in the cells
pretreated with PD98059, SB203580 and SB216763.
However, NAC attenuated cyclin D1 down-regulation
by MRBE. These findings suggest that MRBE induced
ROS-dependent cyclin D1 proteasomal degradation.
Discussion
Although controlled inflammatory response is beneficial
to defend and protect the body from harmful factors such
as physical damage, precursor chemicals and microbial
invasion, inflammation can induce adverse effects such as
cancer, atherosclerosis, arthritis and septic shock on the
body if the regulation of inflammation is dysfunctional
[27]. Since inflammation is associated with many inflamma-
tory mediators such and pathways that lead to a wide range
of changes in pathology, it is difficult to treat inflammation
[27]. Non-steroidal anti-inflammatory drugs (NSAIDs) have
long been used for the treatment of inflammation. How-
ever, recently, because of side effects of NSAIDs, herbal
medicinal plants have received a great attention and a large
number of mechanistic studies have been reported.
Nitric oxide (NO) plays an important role in inflam-
mation pathogenesis [28]. Although NO has an anti-
inflammatory effect under controlled inflammatory
response, it induces chronic inflammation due to over-
production under abnormal regulation [28]. In addition,
Figure 2 Effect of MRBE on IκB-αdegradation (A), p65 nuclear translocation (B) and ERK1/2 phosphorylation (C) in LPS-stimulated
RAW264.7 cells. RAW264.7 cells were pre-treated with MRBE at the indicated concentrations for 2 h and then co-treated with (1 μg/ml) for
15 min (for Western blot of IκB-αand ERK1/2 phosphorylation) or 30 min (for Western blot of p65). DMSO was used as a vehicle. Cell lysate were
resolved by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies against IκB-α, p-ERK1/2, total ERK1/2 and p65. The proteins
were then visualized using ECL detection. Actin was used as an internal control.
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Figure 4 Effects of MRBE on ATF3 and cyclin D1 expression in mRNA and protein level in SW480 cells. (A, B) SW480 cells were treated
with MRBE at the indicated concentrations for 24 h. Cell lysates were subjected to SDSPAGE and the Western blot was performed using
antibodies against cyclin D1, ATF3 and actin. For RT-PCR analysis of ATF3 and cyclin D1 gene expression (C), total RNA was prepared after MRBE
treatment for 24 h. (D) SW480 cells were treated with MRBE (25 μg/ml) for the indicated times. Cell lysates were subjected to SDSPAGE and the
Western blot was performed using antibodies against cyclin D1, ATF3 and actin. Actin and GAPDH were used as internal control for Western blot
and RT-PCR, respectively. DMSO was used as a vehicle.
Figure 3 Effect of MRBE on cell growth (A) and apoptosis (B) in SW480 cells. SW480 cells were treated with MRBE at the indicated
concentration for 24 h. Cell growth was measured sung MTT solution and expressed as absorbance (A
570
). *P < 0.05 compared to cell without
MRBE treatment. Apoptosis by MRBE was evaluated with Western blot against cleaved PARP. Actin was used as an internal control. DMSO was
used as a vehicle.
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NO is involved in inflammation-induced human diseases
such as cancer, rheumatoid arthritis, diabetes, septic shock
and cardiovascular diseases [29,30]. Therefore, NO is a
key target for managing inflammatory diseases. NO is
synthesized by nitric oxide synthases (NOSs) from the
amino acid l-arginine [31,32]. Among three types NOS
isoforms including neuronal NOS (nNOS), endothelial
NOS (eNOS) and inducible NOS (iNOS), only iNOS is
overexpressed in LPS-stimulated macrophage and sub-
sequently generates NO [33]. Mulberry root bark ex-
tracts (MRBE) attenuated the over-production of NO
by suppressing LPS-induced iNOS over-expression in
RAW264.7 cells. These results suggest that MRBE may
exert an anti-inflammatory effect.
There are growing evidences that several agents inhibit-
ing NO production have been shown to suppress NF-κB
activation [34-37]. NF-κB regulates expression of various
genes associated with apoptosis, proliferation, cancer pro-
gression and inflammation [38]. In inflammatory response
under the external stimuli such as LPS, Activated IκB-α
kinase (IKK) phosphorylates IκB-α.PhosphorylatedIκB-α
is subsequently ubiquitinated and degraded by the 26S
proteasome, which thereby releases NF-κBfromthe
cytoplasmic NF-κB-IκBαcomplex and results in NF-κB
nuclear translocation. Translocated NF-κB activates the
expressions of target genes associated with inflammation
such as iNOS. Thus, NF-κB is the key transcription factor
inducing inflammatory response and a promising target
for anti-inflammation [39,40]. In present study, we dem-
onstrated that MRBE inhibits the nuclear translocation of
NF-κB p65 via blocking LPS-induced IκB-αdegradation
in RAW264.7 cells.
In addition, some agents inhibiting NO production and
NF-κB activation also suppressed the activation of mitogen-
activated protein kinases (MAPKs) [37]. The activation
of JNK, p38 and ERK1/2 protein regulates iNOS expression
and modulates NF-κB activity [41]. We found that MRBE
inhibits phosphorylation of ERK1/2 induced by LPS in
RAW264.7 cells. These results suggest that inhibition
of NF-κB and ERK pathway is a potential mechanism
by which MRBE exerts anti-inflammatory activity.
The connection between inflammation and the develop-
ment of colorectal cancer is well-established [42]. Thus, we
evaluated whether MRBE possesses anti-cancer activity and
elucidated its potential mechanisms in human colorectal
cancer cells, SW480.
ATF3 has been known as a stress-responsive product
[43]. There are growing evidences that the ATF3 expres-
sion was repressed in normal cells but might be rapidly
induced by various pathological stimuli [44,45]. Moreover,
ATF3 expression plays an important role in apoptosis
induced by a variety of anti-cancer compounds such as
berberine [46], conjugated linoleic acid [47], curcumin
[48] and 3,3-diindolylmethane [49] in human colorectal
cancer cells, which indicates that ATF3 could function as
a pro-apoptotic mediator. We found that MRBE induced
cell growth arrest and apoptosis, and activated ATF3
expression in the levels of mRNA and protein in SW480.
We also found that MRBE-activated ATF3 expression was
reduced by the treatments SB216763 (GSK3βinhibitor)
Figure 5 ROS/GSK3β-dependent ATF3 expression and ROS-dependent cyclin D1 proteasomal degradation by MRBE. (A, B) SW480 cells
were pre-treated with PD98059 (20 μM), SB203580 (20 μM), SB216763 (20 μM) or NAC (20 mM) for 2 h and then co-treated with MRBE (25 μg/ml)
for 1 h. Cell lysates were subjected to SDSPAGE and the Western blot was performed using antibodies against ATF3 and actin. (C, D, E) SW480
cells were pre-treated with MG132 (5 and 10 μM), PD98059 (20 μM), SB203580 (20 μM), SB216763 (20 μM) or NAC (20 mM) for 2 h and then
co-treated with MRBE (25 μg/ml) for 1 h. Cell lysates were subjected to SDSPAGE and the Western blot was performed using antibodies against
cyclin D1 and actin. Actin was used as an internal control and DMSO was used as a vehicle.
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and NAC (ROS scavenger), which indicates that MRBE-
induced ATF3 expression could be dependent on GSK3β
and ROS. Indeed, there is a report that ROS induced
ATF3-mediated apoptosis in human colorectal cancer cells
[50].However,theeffectofGSK3βon ATF3 expression has
not been elucidated. Thus, it is necessary that MRBE-
induced ATF3 is affected by GSK3βdirectly or indirectly.
These results suggest that MRBE could induce apoptosis
through GSK3βand ROS-dependent ATF3 activation.
Moreover, we observed that MRBE treatment down-
regulated cyclin D1 protein level but not mRNA in
SW480 cells. Thus, we hypothesized that MRBE-induced
decrease in cyclin D1 level may be mediated from its
proteasomal degradation, and found that pretreatment of
MG132 (proteasome inhibitor) suppresses MRBE-induced
cyclin D1 down-regulation. There are some kinases re-
ported to degrade cyclin D1 such as p38 [51], ERK1/2 [52],
GSK3β[53] and ROS [54]. From using kinase inhibitor,
we conclude that MRBE-induced cyclin D1 degradation
requires ROS; however, p38, ERK1/2 and GSK3βare not
involved in MRBE-induced cyclin D1 degradation. Because
cyclin D1 is a well-known cell cycle regulatory protein,
MRBE-induced cell growth arrest could be mediated
from cyclin D1 proteasomal degradation.
Mulberry root bark has been reported to have various
active components such as mulberroside A, oxyresveratrol,
mulberrofuran G, kuwanon C, kuwanon G, kuwanon H
and morusin. Among these active components, kuwanon
C and kuwanon G possess an anti-inflammatory effect
[55,56]. In anti-cancer activity, morusin induce apoptosis
and suppress NF-kB in human colorectal cancer cells [57].
From these studies, anti-inflammatory and anti-cancer
activity by MRBE may be contributed by kuwanon C,
kuwanon G or morusin. But, we do not exclude the pos-
sibility that other active components could also mediate
an anti-inflammatory and anti-cancer activity.
Conclusions
Taken together, our report is the first to show that MRBE
exerts anti-inflammatory and anti-cancer activity. Anti-
inflammatory effect of MRBE is mediated from inhibiting
NF-κB and ERK1/2 activation. Anti-cancer activity of MRBE
is associated with ROS-dependent cyclin D1 proteasomal
degradation and ROS/ GSK3β-dependent ATF3 expression.
Abbreviations
MRBE: Mulberry root bark extract; NO: Nitric oxide; iNOS: Inducible nitric
oxide synthease; NF-κB: Nuclear factor-kappaB; ERK1/2: Extracellular
signal-related kinase 1/2; ATF3: Activating transcription factor 3; ROS: Reactive
oxygen species.
Competing interest
The authors declare that they have no conflict interest.
Authorscontributions
JBJ directed and HJE designed the study. HJE, JHP, GHP, MHL, JRL and JSK
performed the experiments. HJE and JHP drafted manuscript. JHP, GHP, MHL,
JRL, JSK and JBJ corrected the manuscript. All authors read and approved
the final manuscript.
Acknowledgement
This study was supported by the BK21 PLUS program of Ministry of Education
and by Bio-industry Technology Development Program (112144-02-2-SB010),
Ministry of Agriculture, Food and Rural Affairs.
Author details
1
Department of Bioresource Sciences, Andong National University, Andong
760749, South Korea.
2
Department of Medicinal Plant Science, Jungwon
University, Goesan 367805, South Korea.
3
Gyeongbuk Institute for
Bio-industry, Andong 760380, South Korea.
4
Insititute of Agricultural Science
and Technology, Andong National University, Andong 760380, South Korea.
5
Department of Medicinal Plant Resources, Andong National University,
Andong 760380, South Korea.
Received: 12 March 2014 Accepted: 23 June 2014
Published: 25 June 2014
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doi:10.1186/1472-6882-14-200
Cite this article as: Eo et al.:Anti-inflammatory and anti-cancer activity
of mulberry (Morus alba L.) root bark. BMC Complementary and Alternative
Medicine 2014 14:200.
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In current study, we have investigated the anti-oral cancer effects of developed functional beverage, which integrates fucoidan complex (FC) with natural/organic ingredients including vegetable juice (V), mulberry (M) and wheatgrass (W), designated as VMW-FC cocktail. FC has been recognized as functional food with nutraceutical characteristics, other cocktail components are known to individually contain luxuriant bioactive factors including sulfated polysaccharides and polyphenols etc., exerting various curative impacts. Our results demonstrated that VMW-FC significantly inhibited oral cancer proliferation in a dose- and time-dependent manner. It also retarded cell cycle regulators (CDKs and cyclins) and upregulated cyclin-dependent kinase inhibitor p21. Additionally, VMW-FC suppressed cell migration along with inhibited EMT markers. Further, VMW-FC significantly attenuated xenograft tumor growth in NOD-SCID mice without further adverse effects. Conclusively, VMW-FC strongly inhibits oral cancer through inducing apoptosis, G2/M cell cycle arrest and reducing migration-related EMT markers, which may be primarily attributed to sulfated polysaccharides in beverage cocktail.
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Purpose: This study aims to define the role of adiponectin (APN) in preventing goblet cell apoptosis and in differentiation of epithelial cells to goblet cell lineage resulting in greater mucus production and hence greater protection from chronic inflammation-induced colon cancer (CICC). Methods: Six- to eight-week-old male APNKO and C57BL/6 (WT) mice were randomly distributed to three treatment groups: DSS, DMH, DSS + DMH and control. Chronic inflammation was induced in DSS and DSS + DMH group by administrating 2 % DSS in drinking water for 5 days followed by 5 days of normal drinking water and this constitutes one DSS cycle. Three cycles of DSS were administered to induce chronic inflammation. Cancer was induced in both APNKO and WT mice in DMH and DSS + DMH groups by intraperitoneal injections of DMH (20 mg/kg body weight) once for DSS + DMH group and once per week for 12 weeks for DMH group. On day 129, the colon tissue was dissected for mucus thickness measurements and for genomic studies. HT29-C1.16E and Ls174T cells were used for several genomic and siRNA studies. Results: APNKO mice have more tumors and tumor area in DSS + DMH group than WT mice. APN deficiency downregulated goblet to epithelial cell ratio and enhanced the colonic mucosal erosion with reduced mucus thickness. APN increases Muc2 production with no affect on Muc1 production. APN abated goblet cell apoptosis, while APN deficiency reduced epithelial to goblet cell differentiation. Conclusion: APN may be involved in reducing the severity of CICC by preventing goblet cell apoptosis and increasing epithelial to goblet cell differentiation.
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Each year the American Cancer Society estimates the numbers of new cancer cases and deaths that will occur in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival. Incidence data were collected by the National Cancer Institute (Surveillance, Epidemiology, and End Results [SEER] Program), the Centers for Disease Control and Prevention (National Program of Cancer Registries), and the North American Association of Central Cancer Registries. Mortality data were collected by the National Center for Health Statistics. A total of 1,658,370 new cancer cases and 589,430 cancer deaths are projected to occur in the United States in 2015. During the most recent 5 years for which there are data (2007-2011), delay-adjusted cancer incidence rates (13 oldest SEER registries) declined by 1.8% per year in men and were stable in women, while cancer death rates nationwide decreased by 1.8% per year in men and by 1.4% per year in women. The overall cancer death rate decreased from 215.1 (per 100,000 population) in 1991 to 168.7 in 2011, a total relative decline of 22%. However, the magnitude of the decline varied by state, and was generally lowest in the South (15%) and highest in the Northeast (20%). For example, there were declines of 25% to 30% in Maryland, New Jersey, Massachusetts, New York, and Delaware, which collectively averted 29,000 cancer deaths in 2011 as a result of this progress. Further gains can be accelerated by applying existing cancer control knowledge across all segments of the population. CA Cancer J Clin 2015;000:000000. V C 2015 American Cancer Society.
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Nuclear Factor kappa B (NF-kappaB) is an inducible transcription factor that tightly regulates the expression of a large cohort of genes. As a key component of the cellular machinery NF-kappaB is involved in a wide range of biological processes including innate and adaptive immunity, inflammation, cellular stress responses, cell adhesion, apoptosis and proliferation. Appropriate regulation of NF-kappaB is critical for the proper function and survival of the cell. Aberrant NF-kappaB activity has now been implicated in the pathogenesis of several diseases ranging from Inflammatory Bowel Disease to autoimmune conditions such as Rheumatoid Arthritis. Systems governing NF-kappaB activity are complex and there is an increased understanding of the importance of nuclear events in regulating NF-kappaB's activities as a transcription factor. A number of novel nuclear regulators of NF-kappaB such as IkappaB-zeta and PDLIM2 have now been identified, adding another layer to the mechanics of NF-kappaB regulation. Further insight into the functions of these molecules raises the prospect for better understanding and rational design of therapeutics for several important diseases.
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In the past ten years several research fields have converged to show that the tiny molecule nitric oxide (NO), a reactive gas, functions both as a signalling molecule in endothelial and nerve cells and as a killer molecule by activated immune cells—and it can be used as a new medicine by inhalation. This article reviews the biology of this remarkable molecule and discusses the implications for clinical medicine.
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Atopic dermatitis is a chronic, inflammatory disease of the skin with increased transepidermal water loss. Both an abnormal inflammatory response and a defective skin barrier are known to be involved in the pathogenesis of atopic dermatitis. Protease activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is activated by both trypsin and a specific agonist peptide, SLIGKV-NH2. PAR2 is expressed in suprabasal layers of the epidermis and regulates inflammatory responses and barrier homeostasis. In this study, we show that nordihydroguaiaretic acid (NDGA) inhibits the PAR2-mediated signal pathway and plays a role in skin barrier recovery in atopic dermatitis. Specifically, NDGA reduces the mobilization of intracellular Ca(2+) in HaCaT keratinocytes by down-regulating inflammatory mediators, such as interleukin-8, thymus and activation-regulated chemokine and intercellular cell adhesion molecule-1 in HaCaT keratinocytes. Also, NDGA decreases the protein expression of involucrin, a differentiation maker of keratinocyte, in both HaCaT keratinocytes and normal human epidermal keratinocytes. We examined NDGA-recovered skin barrier in atopic dermatitis by using an oxazolone-induced atopic dermatitis model in hairless mice. Topical application of NDGA produced an increase in transepidermal water loss recovery and a decrease in serum IgE level, without weight loss. Accordingly, we suggest that NDGA acts as a PAR2 antagonist and may be a possible therapeutic agent for atopic dermatitis.
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Recent studies demonstrate that tolfenamic acid (TA) induces apoptosis and suppresses the development and progression of several types of cancers. However, the underlying mechanisms are complex and remain to be fully elucidated. NF-κB plays a critical role in inflammation, cancer development, and progression. Although non-steroidal anti-inflammatory drugs (NSAIDs) modulate NF-κB signaling pathway in different ways, the link between NF-κB and TA-induced apoptosis of colorectal cancer cells has yet to be thoroughly investigated. In the present study, we examined the effects of TA on the NF-κB pathway and apoptosis. TA activated NF-κB transcriptional activity and binding affinity of NF-κB to DNA. TA-induced NF-κB activation was mediated by an increased phosphorylation and proteosomal degradation of IκB-α and subsequent p65 nuclear translocation. We also observed that TA stabilized p65 and increased nuclear accumulation via an increase of p65 phosphorylation at Ser276 residue, which was mediated by p38 MAPK and ERK. The knockout of p53 blocked TA-induced transcriptional activation of NF-κB, but not the p65 nuclear accumulation. TA increased transcriptional activity of p53 and the binding affinity of p53 with p65, which are mediated by p38 MAPK and ERK-stimulated Ser276 phosphorylation. TA-induced apoptosis was ameliorated by the knockout of p65 and p53 and the point mutation of p65 at Ser276 residue. We demonstrate a novel molecular mechanism by which TA induced the NF-κB and apoptosis in human colorectal cancer cells.
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Each year, the American Cancer Society estimates the numbers of new cancer cases and deaths expected in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival based on incidence data from the National Cancer Institute, the Centers for Disease Control and Prevention, and the North American Association of Central Cancer Registries and mortality data from the National Center for Health Statistics. A total of 1,660,290 new cancer cases and 580,350 cancer deaths are projected to occur in the United States in 2013. During the most recent 5 years for which there are data (2005-2009), delay-adjusted cancer incidence rates declined slightly in men (by 0.6% per year) and were stable in women, while cancer death rates decreased by 1.8% per year in men and by 1.5% per year in women. Overall, cancer death rates have declined 20% from their peak in 1991 (215.1 per 100,000 population) to 2009 (173.1 per 100,000 population). Death rates continue to decline for all 4 major cancer sites (lung, colorectum, breast, and prostate). Over the past 10 years of data (2000-2009), the largest annual declines in death rates were for chronic myeloid leukemia (8.4%), cancers of the stomach (3.1%) and colorectum (3.0%), and non-Hodgkin lymphoma (3.0%). The reduction in overall cancer death rates since 1990 in men and 1991 in women translates to the avoidance of approximately 1.18 million deaths from cancer, with 152,900 of these deaths averted in 2009 alone. Further progress can be accelerated by applying existing cancer control knowledge across all segments of the population, with an emphasis on those groups in the lowest socioeconomic bracket and other underserved populations. CA Cancer J Clin 2013;. © 2013 American Cancer Society.