Mulberry leaf extract inhibit hepatocellular carcinoma cell proliferation via depressing IL-6 and TNF-α derived from adipocyte

Abstract

Epidemiological studies have revealed that obesity and being overweight are associated with increased cancer risk. Adipose tissue is regarded as an endocrine organ that secretes proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which are related to the progression of hepatocellular carcinoma (HCC). In this study, adipocytes from 3T3-L1 cells were induced and stained with Oil Red O, which revealed marked intracellular lipid accumulation. Adding 15% conditioned medium (CM) from adipogenic -differentiated 3T3-L1 cells, which contained adipocyte-derived factors, to a culture medium of HepG2 cells was discovered to promote cell proliferation by a factor of up to 1.3 compared with the control. Mulberry leaf extract (MLE), with major components including chlorogenic acid and neochlorogenic acid, was revealed to inhibit CM-promoted HepG2 cell proliferation. The inhibitory effect of MLE on the proliferation of the signal network was evaluated. Expression of the CM-activated IκB/NFκB, STAT3, and Akt/mTOR pathways were reduced when MLE was administered. Although adipocyte-derived factors are complex, administrating anti-TNF-α and anti-IL-6 revealed that MLE blocks signal activation promoted by TNF-α and IL-6. Taken together, these results demonstrated that MLE targets the proliferation signal pathway of the inflammatory response of adipocytes in HCC and could be to prevent obesity-mediated liver cancer.

Full text

Original Article
Mulberry leaf extract inhibit hepatocellular
carcinoma cell proliferation via depressing IL-6 and
TNF-aderived from adipocyte
Chun-Hua Chang
a
, Yu-Tzu Chang
a
, Tsui-Hwa Tseng
c,d,**
,
Chau-Jong Wang
a,b,*
a
Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan
b
Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan
c
Department of Medical Applied Chemistry, Chung Shan Medical University, Taichung, Taiwan
d
Department of Medical Education, Chung Shan Medical University Hospital, Taichung, Taiwan
article info
Article history:
Received 25 September 2017
Received in revised form
13 December 2017
Accepted 18 December 2017
Available online 1 February 2018
Keywords:
Hepatocellular carcinoma (HCC)
Obesity
Proinflammatory cytokines
Mulberry leaf extract (MLE)
Proliferation
abstract
Epidemiological studies have revealed that obesity and being overweight are associated
with increased cancer risk. Adipose tissue is regarded as an endocrine organ that secretes
proinflammatory cytokines such as tumor necrosis factor-a(TNF-a) and interleukin-6 (IL-
6), which are related to the progression of hepatocellular carcinoma (HCC). In this study,
adipocytes from 3T3-L1 cells were induced and stained with Oil Red O, which revealed
marked intracellular lipid accumulation. Adding 15% conditioned medium (CM) from adi-
pogenic -differentiated 3T3-L1 cells, which contained adipocyte-derived factors, to a cul-
ture medium of HepG2 cells was discovered to promote cell proliferation by a factor of up
to 1.3 compared with the control. Mulberry leaf extract (MLE), with major components
including chlorogenic acid and neochlorogenic acid, was revealed to inhibit CM-promoted
HepG2 cell proliferation. The inhibitory effect of MLE on the proliferation of the signal
network was evaluated. Expression of the CM-activated IkB/NFkB, STAT3, and Akt/mTOR
pathways were reduced when MLE was administered. Although adipocyte-derived factors
are complex, administrating anti-TNF-aand anti-IL-6 revealed that MLE blocks signal
activation promoted by TNF-aand IL-6. Taken together, these results demonstrated that
MLE targets the proliferation signal pathway of the inflammatory response of adipocytes in
HCC and could be to prevent obesity-mediated liver cancer.
Copyright ©2018, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan
LLC. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
*Corresponding author. Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan.
Fax: þ866 4 23248167
** Corresponding author. Chung Shan Medical University, Number 110, Section 1, Jiankuo North Road, Taichung 402, Taiwan.
E-mail addresses: tht@csmu.edu.tw (T.-H. Tseng), wcj@csmu.edu.tw (C.-J. Wang).
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.jfda-online.com
journal of food and drug analysis 26 (2018) 1024e1032
https://doi.org/10.1016/j.jfda.2017.12.007
1021-9498/Copyright ©2018, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Hepatocellular carcinoma (HCC) is a highly fatal disease.
Common risk factors for the development of HCC include viral
infections caused by the hepatitis B or hepatitis C viruses,
excess alcohol intake, and some dietary carcinogens, aflatoxin
in particular. In addition, growing evidence shows that obesity
increases the risk of developing HCC [1,2]. Several patho-
physiological mechanisms linking obesity to cancer have been
suggested as related to the systemic and local effects of dys-
regulated adiposity [3]. Obese adipose tissue mainly releases
proinflammatory cytokines consist of TNF-a, IL-6, leptin,
resistin, angiotensin II, and plasminogen activator inhibitor 1
to induce inflammatory response. mRNA expression studies
indicates that adipocytes can produce TNF-a, IL-1band IL-6
[4e6]. TNF-aand IL-6 activate transcription factors NF-kB
and STAT3, respectively, which can enhance cell growth and
prevent apoptosis [7]. Additionally, adipose tissue functions as
a key endocrine organ, releasing multiple bioactive sub-
stances that may be involved in a complex network of survival
signal pathways such as PI3K/Akt/mTOR, which is believed to
be critical during HCC development [8]. Therefore, cytokine
blockers or natural diets that block signal pathways activated
by adipocyte-derived factors may decrease the risk of devel-
oping obesity-promoted HCC.
Accumulated evidence suggests that a high intake of plant
foods is associated with a lower risk of developing a chronic
disease and cancer. Plant polyphenols, which have manifold
biological roles, have received substantial attention [9e12].
They generally present in fruits, vegetables, and tea. Mulberry
leaves (Morus alba L.), which are commonly used to feed silk-
worms, are known to be rich in polyphenols such as quercetin
and caffeic acid [13] and are used as a traditional medicine to
treat several metabolic diseases including dyslipidemia, dia-
betes, fatty liver disease, and hypertension [14e16]. Previous
research has revealed that mulberry leaf extract (MLE) can
effectively inhibit the proliferation and migration of vascular
smooth muscle cells, improve vascular endothelial function,
and reduce atheroma burden [17,18]. Ann et al. discovered
that mulberry leaves have beneficial effects on obesity-related
fatty liver disease through their regulation of hepatic lipid
metabolism, fibrosis, and the antioxidant defense system [19].
Because of the etiological link between obesity and liver can-
cer, plant polyphenolsdwith their antioxidant and anti-
inflammatory propertiesdhave drawn increasing interest for
their possible role in chemoprevention. However, little is
known about the effect of plant polyphenols on the progres-
sion of adipocyte-induced HCC progression. Hence, the pre-
sent study investigated the inhibitory effect of MLE on
adipocyte-derived factor-induced HepG2 cell proliferation
and attempted to verify the molecular mechanism involved.
2. Materials and methods
2.1. Chemicals and reagents
Dulbecco's modified Eagle medium: nutrient mixture F-12
(DMEM/F-12), minimum essential media, fetal bovine serum
(FBS), L-glutamine, and penicillin-streptomycin were pur-
chased from Gibco BRL (Grand Island, NY, USA). In addition, 3-
(4,5-dimethylthiazol-zyl)-2,5- diphenyltetrazolium bromide
(MTT), antimouse IgG peroxidase conjugate, antirabbit IgG
peroxidase conjugate antibodies, and b-actin antibodies were
purchased from SigmaeAldrich (St. Louis, MO, USA). Anti-p38,
NF-kB (p65), STAT3, IkB, mTOR, and GSK3bantibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Antiphosphorylated p38 (Thr 180/Tyr 182), phosphory-
lated STAT3 (Ser 727), phosphorylated mTOR (Ser 2448),
phosphorylated IkB (Ser 32/36), and phosphorylated GSK3b
(Ser21/9) antibodies were obtained from Cell Signaling Tech-
nology (Beverly, MA, USA). IL-6 and TNF-aantibodies were
purchased from Abcam Biotechnology (Abcam, Cambridge,
MA, USA).
2.2. Preparation of MLE
Mulberry leaves were collected in Dadu Township, central
Taiwan. Fresh mulberry leaves (200 g) were harvested and
immediately dried at 50 C. The dried leaves were then heated
at 90 C in 3 L of deionized water for 1 h. After filtration, the
residue was removed and the aqueous extract was lyophilized
(80 C, 12 h) to obtain MLE. The dried powder was stored at
80 C and MLE was filtrated using a 0.22-mm filter prior to use
in the following experiments.
2.3. Cell culture
Murine 3T3-L1 fibroblast cells (BCRC 60159) and HepG2 cells
(BCRC 60025) were purchased from Bioresource Collection
and Research Center. The 3T3-L1 cells were grown in DMEM
(Invitrogen Inc.) supplemented with 10% FBS, 4 mM L-
glutamine, and 1% penicillin-streptomycin. HepG2 cells are
a human hepatoblastoma cell line. Cells were maintained in
a minimum essential medium (Invitrogen Inc.) supple-
mented with 10% FBS, 2 mM L-glutamine, 1% penicillin-
streptomycin, 0.1 mM nonessential amino acids, and
1.0 mM sodium pyruvate (Invitrogen Inc.), and then incu-
bated at 37 C in a humidified atmosphere of 5% CO
2
and
95% air.
2.4. Adipocyte formation assay and preparation of CM
The 3T3-L1 cells (preadipocytes) were seeded at a density of
10
6
/mL in six-well plates and grown to confluence. Cell dif-
ferentiation into adipocytes was induced using DMEM sup-
plemented with 10% FBS, 4.5 mM rosiglitazone (RSG), 1 mg/mL
insulin, 0.25 mM dexamethasone (DEX), and 0.5 mM iso-
butylmethylxanthine (IBMX) for 4 days, with the medium
replaced every 2 days. On day 5, the medium was replaced
with a medium containing 10% FBS supplemented with 1 mg/
mL insulin for 4 additional days, with the medium again
replaced every 2 days. On day 9, the medium was replaced
with a medium containing 10% FBS for 4 days, with the me-
dium replaced every 2 days. At this point, preadipocytes were
induced to become adipocytes and lipid droplets became
clearly visible. On day 13, the medium was replaced with a
serum-free medium. The observed induction of adipogenic
differentiation from preadipocytes is summarized in Table 1.
journal of food and drug analysis 26 (2018) 1024e1032 1025

The medium was collected as a conditioned medium (CM) and
stored at 80 C until use.
2.5. Oil Red O staining
Adipocyte differentiation was detected by staining lipids with
Oil Red O (SigmaeAldrich). Oil Red O stock solution (5 mg/mL)
was prepared in isopropanol and stored at 4 C prior to use.
Fresh Oil Red O working solutions were prepared by mixing
the stock solution with distilled water (6:4), followed by in-
cubation for 10 min and filtration through a 0.22-mm filter.
Cells were washed twice with phosphate buffered saline (PBS)
and fixed with 4% formaldehyde for 1 h at room temperature.
Subsequently, the fixative solution was removed and the cells
were washed twice with PBS. The Oil Red O working solution
was added and covered the cells for 30 min. The cells were
then washed with distilled water, dried, and examined using
an inverted optical microscope.
2.6. Cell viability
The HepG2 cells were seeded at a density of 5 10
4
/mL in 24-
well plates and treated with CMs of various concentrations
(0%e25%) for 24 or 48 h. After incubation, their cytotoxicity
was determined using an MTT assay. In brief, the medium was
changed and MTT solution (5 mg/mL) was added to each well
and incubated for 4 h at 37 C. After washing with PBS, 1 mL
isopropanol was added to the purple blue formazan, and the
absorbance was measured spectrophotometrically at 563 nm.
2.7. WST assay
Cell proliferation was measured using the WST-1 cell prolif-
eration reagent kit (Roche Applied Science, Mannheim, Ger-
many). HepG2 cells were plated in 24-well plates at a density
of 5 10
4
cells per well. After treatment with 15% CM and
MLEs of various concentrations, 10 mL/well of WST-1 was
added to the medium, followed by incubation at 37 Cina
humidified atmosphere of 5% CO
2
for 0.5 h. Cellular viability
was determined by measuring the cells’absorbance using an
ELISA reader at 440 nm.
2.8. BrdU incorporation assay
The BrdU Cell Proliferation ELISA colorimetric assay kit (Roche
Applied Science, IN, USA) was used to quantitate cell prolif-
eration according to the manufacturer's protocol. HepG2 cells
were seeded at a density of 10
4
in 96-well plates and incubated
overnight. The cells were then treated with adipocyte-CM and
combined with MLE in various concentrations (1e4 mg/mL) for
48 h. BrdU solution (10 mM) was added to each well and left for
24 h at 37 C. Cellular proliferation was determined by
measuring the cells'absorbance using an ELISA reader at
450 nm.
2.9. Nuclear extraction
Nuclear and cytoplasmic fractionations were performed using
NE-PER Nuclear and Cytoplasmic Extraction Reagents ac-
cording to the manufacturer's instructions (Thermo Scienti-
fic). Briefly, 2 10
6
HepG2 cells were induced with an
adipocyte-CM and treated with MLE of different concentra-
tions (1e4 mg/mL) for 48 h. After centrifugation (500 g for
5 min), the supernatant was removed. Then, 200 mL of CER I
buffer was added to the cell pellets, which were then vigor-
ously shaken for 15 s. The cell mixtures were incubated on ice
for 10 min, and then added to 11 mL of CER II buffer. The cell
mixtures were again incubated on ice for 1 min and shaken
vigorously for 5 s. The cell suspension was subsequently
centrifuged at 16,000 g for 5 min. The supernatants (cyto-
plasmic extract) were kept on ice. The cell pellets were
resuspended using 100 mL of nuclear extraction reagent buffer
(NERbuffer) on ice for 40 min with 15 s of vigorous shaking
every 10 min. Following centrifugation at the maximum speed
Table 1 eInduction of adipogenic differentiation from preadipocytes (3T3-L1). Adipose differentiation of 3T3-L1
preadipocytes was induced as described in Materials and Methods.
journal of food and drug analysis 26 (2018) 1024e10321026

of 16,000 g for 10 min, the supernatants (nuclear extracts) thus
obtained was transferred to a new Eppendorf tube and stored
on ice or at 80 C until use.
2.10. Western blot analysis
210
6
cells per well were seeded in 10-cm dishes in the
presence of 0.5e4.0 mg/mL MLE and 15% CM. Total protein
extracts were prepared in radioimmunoprecipitation assay
buffer (RIPA buffer) (50 mM Tris-HCl, 1 mM EDTA, 150 mM
NaCl, 1% NP-40) containing protease inhibitors. Protein con-
centration was measured using a Bradford protein assay kit
(Bio-Rad, USA). Cell lysate with an equal amount of protein
(50 mL) was loaded onto 8%e10% SDS-PAGE and transferred
onto a nitrocellulose membrane (PALL, USA). The membranes
were blocked in 5% milk in Tris-buffered saline (TBS) plus 0.5%
Tween for 1 h at room temperature and then incubated with
specific primary antibodies at 4 C overnight, after which the
secondary antibody was conjugated to horseradish peroxi-
dase for 1 h. The immunocomplex was revealed using
enhanced chemiluminescence with a chemiluminescence
detection kit (Millipore, USA) and was exposed using a Fuji-
Film LAS-4000 mini (Tokyo, Japan). Protein quantity was
determined using FujiFilm Multi Gauge 2.2.
2.11. Statistical analysis
Data reported are means ±standard deviations of three in-
dependent experiments analyzed using analysis of variance.
Significant differences were defined as P<0.05.
3. Results and discussion
3.1. Induction of adipogenic differentiation from
preadipocyte 3T3-L1
Mulberry leaves have recently been discovered to have a
beneficial effect on obesity-induced hepatic lipogenesis and
fibrosis [19]. However, the effect of MLE on obesity-mediated
hepatic cancer cell proliferation remains unknown. To
address this deficiency in the literature, the effect of MLE on
adipocyte-mediated hepatic cancer cell proliferation and its
signaling pathway was investigated in this study. Cancer-
associated adipocytes were previously proposed to interact
reciprocally with cancer cells and affect cancer progression
[20]. To clarify the chemopreventive potential of MLE
regarding obesity-associated HCC in vitro, adipocyte from 3T3-
L1 preadipocytes was prepared according to the process pre-
sented in Table 1. Postconfluent 3T3-L1 preadipocytes were
discovered to have a fibroblast-like shape (Fig. 1, left) when
treated with differentiation medium containing a mixture of
RSG, DEX, IBMX, and insulin. On day 13, round adipocytes
were observed after adipogenic differentiation (Fig. 1, middle).
In addition, cells with intracellular lipid accumulation were
markedly stained by Oil Red O, suggesting that 3T3-L1 pre-
adipocytes differentiate into mature adipocytes (Fig. 1, right).
3.2. Promoting cell proliferation of HepG2 using CM
from adipocytes
Fig. 2A illustrated the effect of CM from adipocytes on the
viability of HepG2 cells. Cell viability was highest when
treatment with 15% CM for 24 h and 48 h was performed;
therefore, 15% CM was used in the subsequent experiments.
3.3. Inhibiting CM-induced proliferation in HepG2 cells
using MLE and its components
Previous studies have reported that natural polyphenols have
anticancer potential [21], so whether MLE inhibits CM-induced
HepG2 proliferation was investigated. Treatment with CM for
24 or 48 h was demonstrated to significantly promote HepG2
proliferation, whereas MLE treatment decreased this effect at
both treatment durations (Fig. 2B). When treatment with MLE
was administered at 2 and 4 mg/mL, the absorbance was lower
than that of the control group, implying that MLE not only
inhibited CM-promoted HepG2 proliferation but also blocked
the endogenous proliferation effect of HepG2 cells. In our
previous report, the composition of MLE were identified using
high-performance liquid chromatography and liquid chro-
matographyemass spectrometry. Eight phenolic compounds
were identified: neochlorogenic acid (35.5%), crypto-
chlorogenic (31.7%), chlorogenic (23.8%), rutin (9.2%), iso-
quercitrin (5.6%), astragalin acid (5.3%), nicotiflorin (3.5%), and
protocatechuic acid (1.3%) [22] The effect of major phenolic
acids on CM-induced proliferation was also investigated. CGA
and nCGA were discovered to significantly inhibit CM-
promoted proliferation and endogenous-mediated prolifera-
tion in HepG2 cells (Fig. 2C).
Fig. 1 eThe differentiation of adipocytes was evaluated using Oil Red O staining. Mouse preadipocytes, 3T3-L1 cell line
showed a fibroblast-like shape (left panel). Preadipocytes were induced into adipogenic differentiation by differentiation-
induced media (middle panel). Adipose differentiation of 3T3-L1 preadipocytes evaluated by Oil-red-O staining of lipid
droplets (right panel). The adipogenic-differentiated cell showed positive staining for Oil-red-O. Red-colored expression
indicated lipid droplets in differentiated adipocyte (arrowhead).
journal of food and drug analysis 26 (2018) 1024e1032 1027

3.4. Deactivation of CM-induced NF-kB, STAT3, and
mTOR using MLE
Adipocytes have been proposed to induce a low-grade in-
flammatory response that in turn increases TNF-aand IL-6
expression. One previous study reported that TNF-aand
IL-6 can stimulate the proliferation and progression of HCC
[20].TNF-aand IL-6 activate NF-kB and STAT3, respectively.
The effect of MLE on the phosphorylation of IkBandp38
MAPK, which are upstream of NF-kB(Fig. 3A), was evaluated.
The results revealed that CM increased the expression of p-
IkB but not that of p-p38 and enhanced the nuclear trans-
location of NF-kB p65. MLE treatment decreased the
expression of p-IkB and nuclear translocation of NF-kB p65.
In addition, CM increased the expression of pSTAT3, which
was reversed by the MLE treatment (Fig. 3B). Therefore, MLE
inhibited the NF-kB and STAT3 signaling pathways, which
are involved in HCC proliferation. Adipocyte-derived factors
are complex and may activate other signaling networks
such as that of mTOR, which is frequently upregulated in
HCC and is associated with cell proliferation. The effect of
CM on the mTOR pathway was thus examined. CM was
revealed to increase the phosphorylation of Akt and mTOR,
which was reduced by the administration of MLE (Fig. 3C).
These results demonstrated that MLE can block the PI3K/
Akt/mTOR signaling pathway.
3.5. Effect of anti-IL-6 and anti-TNF-aon CM-induced
signal activation
To determine whether CM promotes signal activation through
the TNF-aand IL-6 mediated signaling pathways, the phos-
phorylation of p-STAT, p-IkB, p-Akt, and p-mTOR was per-
formed using the administration of anti-IL-6 and anti-TNF-a.
The results indicated that anti-IL-6 blocked the CM-activated
STAT pathway whereas anti-TNF-ablocked the CM-
activated IkB/NF-kB pathway (Fig. 4A and B). Furthermore,
administration of anti-IL-6 and anti-TNF-aalso blocked the
CM-activated Akt/mTOR signaling pathway (Fig. 4C). Finally,
treatment with MLE, anti-IL-6, or anti-TNF-awere all
demonstrated using BrdU incorporation analysis to inhibit
CM-induced HepG2 proliferation (Fig. 5). Thus, MLE blocks
signal activations promoted by TNF-aand IL-6, resulting in the
antiproliferation of HepG2 cells.
Fig. 2 eInhibition of adipocyte-conditioned medium (CM)-induced cell proliferation by MLE in HepG2 cells. (A) 5 £10
4
HepG2
cells were treated with CM of different concentrations (0%e25%) in serum-free culture medium for 24 and 48 h. Cell viability
was analyzed using the MTT assay as described in the text (B) 5 £10
4
HepG2 cells per well were treated with adipocyte-
conditioned medium alone and in combination with MLE of various concentrations (1, 2 and 4 mg/mL) for 24 and 48 h (C)
5£10
4
HepG2 cells per well were induced with adipocyte-conditioned medium and treated with nCGA (0.1 mg/mL) and CGA
(0.1 mg/mL) for 24 and 48 h. Cell proliferation was evaluated using the WST assay. The result represents an average of four
independent experiments ±SD. The result represents an average of three independent experiments ±SD.
#
p<0.05
compared with the control.*p<0.05, **p<0.005 versus CM group.
journal of food and drug analysis 26 (2018) 1024e10321028

4. Discussion
Epidemiological studies have demonstrated that obesity and
being overweight are associated with increased risk of cancers
such as HCC. In obese patients, lipid accumulation in the liver
increases the demand on the endoplasmic reticulum, thus
provoking oxidative stress, causing the production of reactive
oxygen species, and activating inflammatory pathways.
Oxidative stress can induce DNA damage, which leads to
genomic instability. The enhanced production of proin-
flammatory cytokines such as TNF-aand IL-6, which can lead
to hepatic inflammation, promotes abnormalities in liver cells
[23]. Researchers have reported a number of biologically active
Fig. 3 eEffects of adipocyte-conditioned medium (CM) induced cell proliferation by MLE on protein levels of TNF-a/IL-6
signaling-related proteins in HepG2 cells. HepG2 cells were induced with adipocyte-conditioned medium and treated with
MLE of various concentrations (1e4 mg/mL) for 48 h. Whole cell extracts (50 mg/lane) were separated on 8e10% SDS PAGE
followed by Western blot analyses. Each target protein band was detected with respective antibody. b-actin and C23 were
used as the cytosolic and nuclear protein loading control, respectively. Quantification of phosph-IkB and NFkB(A), phosph-
Stat3/Stat3(B) and phosph-Akt/Akt and phosph-mTOR/mTOR(C) protein level. The graph represents mean values of three
independent experiments, and error bars represent means ±SD of the experiments.
#
p<0.05 compared with the
control.*p<0.05 versus CM group. C, control; CM, adipocyte-conditioned medium.
journal of food and drug analysis 26 (2018) 1024e1032 1029

compounds in MLE that have effective anti-inflammatory,
antioxidant, and hepatoprotective activities [24,25]. This
study discovered that administration of a medium from
cultured adipocytes increased the proliferation of HepG2
(Fig. 2), which was associated with activating the IL-6 and
TNF-asignaling pathways (Fig. 4). The results also demon-
strated that MLE treatment decreased CM-promoted HepG2
proliferation by blocking proliferation signaling pathways
including the STAT3, IkB/NFkB, and Akt/mTOR pathways.
Cell survival signaling plays a critical role in the patho-
genesis of cancer. Most studies on IL-6 signaling in hepatic
epithelia have focused on proliferation and the characteriza-
tion of the pathways involved [26]. Numerous different path-
ways are known to be activated by IL-6, such as the STAT3,
p38/MAPK, and PI3K/AKT pathways. The CM was revealed
herein to activate the STAT3 and Akt/mTOR pathways, but
this activation was reduced by the administration of MLE to
HepG2 cells (Fig. 3). TNF-a, crucial to cancer-related inflam-
mation, can activate p38/MAPK and I kappa B kinase (IKK) [27]
IKK activation is associated with the activation of NFkB, a
transcription factor. The role of NFkB in cancer cells appears
to involve the regulation of cell proliferation, control of
apoptosis, and stimulation of invasion/metastasis. The pre-
sent study additionally revealed that Akt/mTOR activation in
HepG2 is also mediated by TNF-a. The CM-activated IKK and
NFkB, but this activation was reduced by the administration of
MLE to HepG2 cells (Fig. 3). Adipose tissue is considered to be
not simply a reservoir of stored energy but also an active
secretory organ that releases inflammatory cytokines, adipo-
kines, and growth factors, leading to an increased risk of HCC
development [28]. Whether these signal activations are regu-
lated by other components released by adipocytes merits
further investigation. The carcinogenesis of HCC is a multi-
factor and complex process involving chromosomal aberra-
tions, epigenetic alterations, and the activation of complex
signaling pathways. A previous study reported that mTOR is
frequently upregulated in HCC and that growth factors such
as insulin-like growth factor II are secreted from HepG2 [29].
HepG2 may activate endogenous proliferation signaling
pathways in a serum-free culture (control). Comparing Figs. 3
and 4 reveals that MLE inhibited not only CM-activated pro-
liferation signaling but also endogenous proliferation
signaling, thus demonstrating its anti-HCC role of targeting
proliferation signaling pathways.
Fig. 4 eInhibition of adipocyte-conditioned medium (CM)-induced signal activation by anti-IL-6 and anti-TNF-ain HepG2
cells (AeC) HepG2 cells were induced with adipocyte-conditioned medium and treated with anti-IL-6 (10 mg/mL) or anti-TNF-
a(1 ng/mL) for 48 h. Whole cell extracts (50 mg/lane) were separated on 10% SDS PAGE followed by Western blot analyses.
Each target protein band was detected with respective antibody. b-actin was used as an internal control. Quantification of
the pSTAT3, NF-kB p65, pIkB, pAkt and pmTOR protein level were put on right site. (D) HepG2 cells were treated with or
without adipocyte-conditioned medium and the HepG2 cells were presence MLE 4 mg/ml with anti-IL-6 or anti-TNF-afor 0,
24 and 48hours. Proliferation was measured by the WST assay. All the experiments were performed thrice in triplicates.
#
p<0.05 versus control group and * p<0.05 versus CM group.
journal of food and drug analysis 26 (2018) 1024e10321030

In recent years, the biological properties of plant poly-
phenols with chemopreventive potential have attracted
increasing interest. CGA and its isomer nCGA have been
associated with anticarcinogenic, anti-inflammatory, and
antioxidant activities and have been demonstrated to
reduce the risk of cardiovascular disease and type 2 diabetes
[22,30]. CGA and nCGA can suppress breast cancer cell
viability and growth while having no effect on MCF-10A
normal breast epithelial cells. In Caco-2 human colon can-
cer, both CGA and nCGA also showed similar effects [22,31].
In our previous report, 0.5% of MLE exhibited a significant
effect to decrease the obesity induced NAFLD, inflammation
and oxidative stress. The polyphenol composition analysis
revealed that chlorogenic and its isomers account for
approximately 0.47 mg when mice were fed with 10 g diet
containing 0.5% MLE. We speculated that administration
2.35 mg/day of chlorogenic and its isomers could prevent
NAFLD in human (500 g diet/day). Thus, we hypothesized
that chlorogenic and its isomers should be effective in
preventing obesity-induced liver cancer at similar concen-
trations CGA was also shown to suppress carbon
tetrachloride-induced NF-kB activation and decrease the
levels of TNF-a, IL-6, and IL-1bin rat serum [32]. The
composition of MLE except for nCGA and CGA includes
other polyphenols. Studies showed that isoquercitrin sup-
press colon cancer proliferation and inflammation [34].
Rutin, a polyphenolic bioflavonoid, has anti-tumor effect on
lung cancer cells. When cells were treated with rutin caused
a significant reduction in lipid peroxidation and LDH activ-
ity; restored antioxidant enzyme activity and modulated the
expression of inflammatory [35]. Additional studies revealed
that astragalin acid inhibited TNF-ainduced NF-kB activity
and suppressed tumor growth and induced cancer cell
apoptosis in vivo [36]. Obesity, an abnormal or excessive fat
accumulation in adipose tissue, is considered a chronic in-
flammatory disease and increases the risk of developing
HCC. Chronic overexpression of inflammatory mediators in
cell microenvironments enhances tumor promotion and
progression. This paper is the first report on polyphenol-
containing MLE inhibiting adipocyte-derived factor-
enhanced hepatoma cell proliferation through the blocking
of inflammatory mediator-activated signaling pathways.
In conclusion, the results presented herein demonstrated
that MLE targets the proliferation signal pathways of the in-
flammatory response of adipocytes in HCC and has potential
for chemoprevention in obesity-mediated liver cancer.
Conflicts of interest
The authors do not have any possible conflicts of interest.
Acknowledgements
This work was supported by Ministry of Science and Tech-
nology Grant (MOST 104-2632-B-040-002), Taiwan.
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