Antioxidant N-Acetylcysteine Attenuates Hepatocarcinogenesis by Inhibiting ROS/ER Stress in TLR2 Deficient Mouse
Hu Z-w (2013) Antioxidant N-Acetylcysteine Attenuates Hepatocarcinogenesis by Inhibiting ROS/ER Stress in TLR2 Deficient
Mouse. PLoS ONE 8(10): e74130. doi:10.1371/journal.pone.0074130
Antioxidant N-Acetylcysteine Attenuates Hepatocarcinogenesis by Inhibiting ROS/ER Stress in TLR2 Deficient Mouse
Heng Lin 0
Xiao-bo Liu 0
Jiao-jiao Yu 0
Fang Hua 0
Zhuo-wei Hu 0
Irina V. Lebedeva, Enzo Life Sciences, Inc., United States of America
0 Molecular Immunology and Pharmacology Group, State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (NO. BZ0150), Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences , Beijing , China
Hepatocellular carcinoma (HCC) remains one of the most deadly solid tumor malignancies worldwide. We recently find that the loss of toll-like receptor 2 (TLR2) activities promotes the diethylnitrosamine (DEN) induced hepatocellular carcinogenesis and tumor progression, which associates with an abundant accumulation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. This finding suggests that the ROS/ER stress plays a role in TLR2 modulated carcinogenesis of HCC. To investigate the mechanism of TLR2 activity defending against hepatocarcinogenesis, the TLR2-deficient mice were treated with or without antioxidant N-acetylcysteine (NAC) before DEN administration. We found that pretreatment of these animals with NAC attenuated carcinogenesis and progression of HCC in the TLR2-deficient mice, declined ROS/ER stress, and alleviated the unfold protein response and inflammatory response in TLR2-deficient liver tissue. Moreover, the NAC treatment significantly reduced the enhanced aggregation of p62 and Mallory-Denk bodies in the DEN-induced HCC liver tissue, suggesting that NAC treatment improves the suppressive autophagic flux in the TLR2-deficient liver. These findings indicate that TLR2 activity defends against hepatocarcinogenesis through diminishing the accumulation of ROS and alleviating ER stress and unfold protein response mediated inflammatory response in the liver.
Funding: This work was supported by grants from the National Natural Science Foundation of China (30973557), the Major Program of the National Natural
Science Foundation (81030056), the International Corporation Project supported by the Ministry of Science and Technology (2010DFB32900), and the Program for
Changjiang Scholars and Innovative Research Team in University (PCSIRT, No. IRT1007). Dr. Fang Hua is supported by a grant from the National Natural Science
Foundation of China (81101595). Dr. Heng Lin is supported by a grant from the Basic Research Program of the Institute of Materia Medica (2013CHX17). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Hepatocellular carcinoma (HCC) is one of the most commonly
diagnosed solid tumor malignancies and ranks as the third leading
cause of cancer-related death worldwide [1,2]. A verity of the risk
factors, including hepatitis virus infection, long-term excessive
alcohol intake, and nonalcoholic steatohepatitis elicit the
chronicinflammatory response to promote the initiation and progression
of HCC. Although the cellular transformation of HCC is known to
be a multistage process starting with increased cell density,
followed by atypia and frequent stromal invasion , the
underlying molecular mechanisms of hepatocarcinogenesis remain
to be completely elucidated [6,7].
Toll-like receptors (TLRs) are a family of pattern recognition
receptors (PRRs) that regulate the innate immune response .
They recognize distinct pathogen-associated molecular patterns
(PAMPs) [9,10], such as fractions of bacteria, virus, or fungi, and
damage-associated molecular patterns (DAMPs) released from the
damaged tissues. The activation of TLRs triggers immune
responses and builds up the first barrier against foreign invasion
or cellular malignance transformation. However, the activation of
TLRs may also lead to an uncontrolled and up-regulated innate
immune response, which in turn may result in inflammatory liver
disorders and HCC . TLRs are expressed in human liver,
where they become activated upon exposure to intestinal bacteria
which translocate via the portal vein . Almost all types of
liver cells, including hepatocytes , kupffer, stellate and
sinusoidal endothelial cells, express the functional TLR2 in the
liver . In primary hepatocytes, TLR2 ligands can activate the
nuclear factor kappa B (NF-kB) pathway . Human genetic
studies have revealed a significant association between TLR2
polymorphisms and HCC susceptibility [17,18]. TLR2/4
activation was implicated in the subsequent tumorigenesis in mouse
model of liver injury induced by
3,5-diethoxycarbonyl-1,4dihydrocollidine (DDC) . In contrast, mice deficient in
TLR4 showed a marked increase [20,21], while MyD88 deficient
mice showed a marked decrease in DEN-induced liver tumors
. Using the same well-established mouse HCC model, we find
recently that the TLR2 signaling plays a defense role against
HCC, through eliciting intracellular senescence and maintaining
autophagy flux in liver cells [22,23].
Oxidative stress, which results from the generation of ROS by
environmental factors or mitochondrial dysfunction, associates
with liver tumorigenesis for either the direct causation of DNA
mutation or the causal link with the chronic inflammation [24,25].
Oxidative stress can be enforced by endoplasmic reticulum (ER)
stress, autophagic flux failure, or the accumulation of p62
aggregates . Meanwhile, cells undergoing high oxidative
stress can be cleared by the p38-MAPK-NFkB pathway mediated
apoptosis . It is possible that these signaling factors and
intracellular processes may be interrelated and/or triggered by a
common upstream factor that is causative of or responsive to
transformation of HCC.
In current study, the ROS and ER stress were found abundantly
accumulated in the DEN-induced HCC in TLR2-deficient mice.
Therefore, we investigated whether ROS is directly responsible for
the aggravated HCC because of TLR2 deficiency. We found that
the pretreatment of TLR2 deficient mice with antioxidant NAC
had rescued the development and progression of HCC in these
animals. Our studies indicate that, at least partially, by limiting
ROS accumulation and ER stress, TLR2 signaling plays a critical
role in defending against hepatocarcinogenesis.
DEN-induced liver cancer model
All procedures involving mice were carried out with prior
approval from the Animal Care and Use Committee of Institute of
Materia Medica, China. TLR2-deficient mice with B6
background and WT B6 mice (all males) were obtained from Jackson
Laboratory (Bar Harbor, ME, USA) and housed in pathogen-free
conditions. At 14 days old, a subset of WT mice were selected for
pre-treatment with anti-TLR2 antibody (to block TLR2 signaling),
IgG (negative control), and a subset of TLR22/2 mice were
selected for pre-treatment with NAC (antioxidant) or physiological
saline (n = 15 to 20 per group). At 15 days old, all of the
pretreated and untreated WT and untreated TLR22/2 mice were
injected with 25 mg/kg DEN (Sigma-Aldrich, St. Louis, MO,
USA) to induce HCC. Over the next six months, the
anti-TLR2and IgG-pretreated mice received weekly boosters (100 mg/kg).
The NAC-pretreated mice received every-other-day boosters
(100 mg/kg) for only three months and were left untreated for
the remaining three months. At the end of the six months, all mice
in the five groups were sacrificed to evaluate the development of
liver cancer. Tumor number and size were recorded. For this, the
whole liver was excised from each animal and washed in cold PBS.
An investigator who was blinded to the animals identity counted
the numbers of surface liver tumor nodules (.0.5 mm) for all liver
lobes. After counting, the livers were divided, with one portion
being stored in cold PBS and the other being sectioned and fixed
in 10% formalin for histological analysis using hematoxylin-eosin
(H&E) staining. To investigate the mechanism of carcinogenesis,
mice in WT, TLR22/2, physiological saline-pretreated TLR22/2,
NAC-pretreated TLR22/2 groups were sacrificed at the end of the
one months after DEN treatment for western blot analysis (n = 4 per
Cells, plasmid and transfection
Liver parenchymal and non-parenchymal cells were isolated
and plated as previously described . Parenchymal cells were
treated with or without 50 mM of H2O2 for 2 hrs and were
accessed for ROS related p62 aggregates measurement (n = 4 per
group). Mouse embryonic fibroblasts (MEFs) were primary
isolated and plated as previously described . MEFs were
treated with or without DEN (200 mg/ml) for 24 hr, and were
harvested for western blot measurement (n = 4 per group). TLR2
expressing plasmid pDUO-CD14/TLR2 was purchased from
InvivoGen (San Diego, CA). HepG2 cell line was obtained from
the ATCC (Rockville, MD, USA). To generate HepG2 cell
populations stably expressing TLR2, the control and the TLR2
expressing plasmids were both transfected into HepG2 cells with
Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to
the manufacturers instructions. After 24 hours of transfection,
stable transfectants were selected in medium containing 1 mg/ml
puromycin (InvivoGen, San Diego, CA). Single clones were
amplified by a dilution cloning technique.
Preparation of Triton X-100-insoluble aggregates and
Liver tissues were gently homogenated and lysed in RIPA lysis
buffer for 15 min on ice. The fraction of Triton X-100-soluble
proteins was collected by centrifugation (13000 rpm, 4uC,
15 min). The insoluble pellet was washed with RIPA buffer,
resolubilized by incubating with RIPA buffer containing 2 M urea,
and collected by centrifugation. After separation through
SDSGel, Triton X-100-soluble and insoluble p62 were detected by
immune blotting (n = 4 per group).
Western blot analysis
Liver tissues (n = 4 per group) were gently homogenated and
lysed in RIPA lysis buffer for 15 min on ice. Total protein was
collected by centrifugation (13000 rpm, 4uC, 15 min) and
quantified using a bicinchoninic acid kit. Equal amounts of
protein from each mouse liver were loaded onto a SDS-Gel,
resolved by electrophoresis, and transferred to a PVDF
membrane. Non-specific binding was blocked by incubating with 5%
nonfat milk solution. 8-ohdG, p62, Bip, phospho-eIF2a, eIF2a,
CHOP, IRE1a, phospho-JNK1/2, JNK1/2, and cleaved
caspase3 were detected by incubation with the corresponding primary
antibodies, including anti-8-ohdG (Santa Cruz Biotechnology,
CA, USA), anti-p62 (Sigma-Aldrich, St. Louis, MO, USA),
antiBip, anti-phospho-eIF2a, anti-eIF2a, anti-CHOP, anti-IRE1a,
anti-phospho-JNK1/2, anti-JNK1/2, and anti-cleaved caspase-3
(Cell Signaling Technology, MA, USA), followed by incubation
with the appropriate enzyme-conjugated secondary antibodies
(Invitrogen, CA, USA). The immunoreactive bands were
visualized with chemiluminescence using the ECL Western Blotting
Substrate (Pierce) and following the manufacturers protocol.
The molecular probe, 29,79-Dichlorofluorescein Diacetate
(H2DCF-DA, Sigma-Aldrich, St. Louis, MO, USA) was used to
detect ROS. Liver cells were in situ released by enzymatic
digestion with collagenase, and were loaded with H2DCF-DA
(10 mM in phenol-free medium) for 30 min (n = 4 per group). Cells
were then rinsed with PBS and measured by flow cytometer
(Becton-Dickinson Biosciences) using the accompanying CellQuest
software. For each measurement, .10,000 events were counted.
Frozen liver sections (n = 4 per group) were incubated with
5 mM Dihydroethidium (DHE, Sigma-Aldrich, St. Louis, MO) for
30 min (37uC). After washes with PBS, the ROS level in the tissue
was measured by confocal microscopy (Leica).
ROS related P62 aggregates assay
Liver parenchymal and non-parenchymal cells were primary
isolated and were loaded with 5 mM DHE for 30 min (37uC, n = 4
per group). Cells were rinsed with PBS containing 0.1% saponin,
blocked with 1% BSA, and incubated with antibodies against p62
for 30 min (25uC). Cells were then washed with PBS, incubated
with donkey anti-rabbit IgG labeled with Alexa Fluor 488
(Invitrogen, CA, USA) at 4uC for 30 min, and measured by flow
cytometer (Becton-Dickinson Biosciences) using the accompanying
CellQuest software. For each measurement, .10,000 events were
SD. Between-group differences were analyzed using the two-tailed
Students t-test. A p-value of ,0.05 was considered statistically
Immunohistochemistry and immunofluorescence
A portion of the unprocessed liver tissues were fixed in 4%
paraformaldehyde, dehydrated, embedded in paraffin, and sliced
into 4 mm thick sections. After dewaxing in xylene and rehydrating
in a graded alcohol series, the sections were processed for antigen
retrieval by heating in citrate buffer, pH 6.0, for 30 min at 95uC.
Immunodetection of TLR2 and p62 was carried out in a
humidified chamber, as follows (n = 4 per group). After three
washes in PBS for 5 min each, the sections were covered with 3%
bovine serum albumin (BSA) and incubated at 37uC for 30 min.
Then, primary antibody replaced the BSA and the slides were
incubated at 4uC overnight. After three washes in PBS for 5 min
each, the sections were incubated with goat anti-rabbit IgG
labeled with horseradish peroxidase-streptavidin complex
(Invitrogen, CA, USA) at 37uC for 30 min. After three washes in PBS for
5 min each, the immunoreactive proteins were detected by
staining with DAB (Abcam) according to the manufacturers
instructions. Eight magnifications (206) fields per liver were
assessed for the level of p62. The average immunostaining
intensities are analyzed.
Primary isolated liver parenchymal and non-parenchymal cells
were planted on coverslips for 4 hr (n = 4 per group). These slides
were fixed in 4% paraformaldehyde. After 3 washes in PBS for
5 min each, the specimens were covered with 3% BSA at 37uC for
30 min, and then were incubated with primary antibodies at 4uC
overnight. These specimens were rinsed in PBS and incubated
with Alexa Fluor labeled second antibodies (Invitrogen, CA, USA)
at 37uC for 30 min. After three washes in PBS for 5 min each, the
slides were covered with DAPI, and detected by confocal
Single-cell suspensions were prepared from unprocessed normal
liver tissues (n = 4 per group), as follows. The liver was minced
with scissors into pieces of 1 mm and placed in 10 ml of ice-cold
dissociation medium. After draining through a plastic tea sieve, the
pieces of tissue were re-suspended in 50 ml of cold, fresh
dissociation medium and kept on ice until further processing
when the suspensions were placed in an atmosphere of 95% 02 and
5% C02 with continuous shaking at 37uC. After 20 min, the
nondissociated pieces of tissue were collected by passing the suspension
through a tea sieve and then re-incubated in 50 ml of fresh
dissociation medium. This process was repeated three times. Cells
from each filtrate were collected by centrifugation (706g, 30uC,
10 min), then washed in 10 ml of incubation medium,
recentrifuged, and resuspended in 50 ml of incubation medium to
give the working dilution. The percentage of apoptotic cells were
assessed by staining with Annexin V-APC and propidium iodide
and detection by flow cytometer. For each measurement, .10,000
events were counted.
TUNEL (terminal deoxynucleotidyl transferasemediated
dUTP nick-end labeling) staining was performed with a kit
(Roche, USA) following the manufacturers instructions (n = 4 per
group). Eight magnifications (206) fields per liver were assessed for
the level of p62. The percentage of TUNEL-positive cells is
All statistical analyses were carried out using the SPSS version
11.5 software (Chicago, IL, USA). Data are expressed as mean 6
DEN causes more liver tumors in TLR2-deficient mice and
mice treated with anti-TLR2 antibody
DEN treatment induced liver tumors in all of the WT and
TLR22/2 mice within six month of injection. We first examined
the expression of TLR2 in liver tissues after HCC occurred.
Immunohistochemical analysis showed that the highest expression
level of TLR2 was found in normal liver tissue adjacent HCC;
TLR2 expressed in the central zone of HCC was higher than that
in the HCC peripheral region (Figure 1A). Immune blotting
analysis verified that the expression level of TLR2 was lower in the
HCC peripheral region than that in HCC adjacent area
(Figure 1B). These findings suggested the roles of TLR2 in the
development of the DEN-induced HCC. Indeed, a significantly
increased number and larger volume of liver tumors had been
observed in TLR22/2 mice in compared to WT counterparts at 6
months after DEN injection (Figure 1C, D). Blockade of TLR2
signaling by the pretreatment of WT mice with an anti-TLR2
antibody also increased the number, volume and size of
DENinduced HCCs (Figure 1C, D). These results suggest that the
TLR29s activity can protect against DEN-induced HCC
Enhanced DEN-induced HCC by TLR2-deficiency is
associated with ROS accumulation and ER stress
To test whether the increased tumor number in TLR22/2 mice
was associated with the oxidative stress and/or ER stress, we
measured the expression and distribution profiles of oxidative
stress and ER stress markers. We found that more ROS
accumulated in the liver tissues from TLR22/2 mice than WT
mice at one month after DEN injection (Figure 2A and 2B). Also,
the expression level of 8-hydroxyguanosine (8-ohdG), a biomarker
of oxidative stress-damaged products of DNA, increased
remarkably in the TLR22/2 mice (Figure 2C).
Because intracellular accumulation of ROS may either result
from the accumulation of p62, a cargo receptor of selective
autophagy, or result in the aggregation of p62 in cells [27,28], we
examined the protein level of p62 in the liver tissues obtained from
the different groups of mice. The expression of p62 was
significantly higher in the livers from the DEN-treated TLR22/2
mice than that from the DEN-treated WT mice (Figure 2D). Western
blotting revealed that the DEN-injured liver from the TLR22/2
mice showed an enhanced unfold protein response (UPR)
(Figure 3A): the ER stress chaperone protein, Bip, was
downregulated and ER stress sensors such as phospho-eIF2a, IRE1a, and
CHOP were up-regulated in TLR22/2 mice, indicating an increase
in ER stress in the livers. In contrast to the results of increased ROS
accumulation and ER stress, less apoptosis was induced in the
DENinjured TLR22/2 livers than their WT counterparts livers
(Figure 3B to D).
Our previous work indicates that the liver-infiltrating
macrophages reduce in TLR2 deficient mice in response to DEN insult
. Thus, TLR2 regulation of DEN-induced cancerogenesis is a
liver cell specific effect through a cytochrome P450 2E1 dependent
ROS-generating manner in liver . Hepatocytes are more
susceptible to DEN-induced damage than other types of liver cells.
Thus, we evaluated the expression of TLR2 and F4/80 (a marker
of marophages) in parenchymal and non-parenchymal cells by
Immunofluorescence-staining analysis. We found that not only
kupffer cells but also hepatocytes were TLR2 positive (Figure
S1A). Indeed, more ROS production and p62 aggregates were
detected in parenchymal than non-parenchymal cells (Figure S1B);
and treatment of these cells with H2O2 caused more ROS and p62
accumulation in TLR22/2 parenchymal cells than WT
parenchymal cells (Figure S1C). We also found a decrease in the
expression of phospho-eIF2a, CHOP, IRE1a, and phospho-JNK
in HepG2 cells stably overexpressing TLR2 (Figure S2C). We next
examined if TL2 could modulate stresses in other cell types. An
increase in the expression of phospho-eIF2a, CHOP, and
phospho-JNK was found in primary isolated MEFs from
TLR22/2 mice in a manner independent of DEN treatment
(Figure S2A). Thus, this modulation seems not limit to liver cells or
Antioxidant reagent NAC attenuates DEN-induced HCC
in TLR2-deficient mice
Because TLR2 deficiency aggravated HCC associated with
oxidative and ER stress, we tested whether an antioxidant agent
had a protective role in the DEN-induced HCC in TLR22/2
mice. Indeed, NAC treatment led to significantly less numbers,
smaller volume and sizes of HCC in the DEN-injured liver from
TLR22/2 mice when compared to the untreated TLR22/2 mice
(Figure 4AE). These observations indicate that the
anti-hepatocarcinogenesis effect of TLR2 signaling in response to DEN insult,
at least partially, relies on the role of TLR2s activity in the
attenuation of ROS accumulation and oxidant stress.
Because NAC treatment was able to reduce the DEN-induced
HCC development in TLR22/2 mice, we tested whether this
agent inhibited the accompanying increase in ER stress factors.
We found that the treatment of mice with NAC reduced the levels
of insoluble p62 aggregates, but had little effect on p62 in the
soluble fraction in liver (Figure 5A). The livers from NAC-treated
TLR22/2 mice also showed a significantly reduced number of
Mallory-Denk bodies, which are cytoplasmic protein aggregates in
hepatocytes (Figure 5B). Consistent with these findings, NAC
treatment was also shown to significantly up-regulate Bip and
down-regulate phospho-eIF2a, phospho-JNK1/2, total eIF2a,
JNK1/2, and IRE1a(Figure 5C). Intriguingly, NAC treatment
had no detectable effect on the ER stress sensor CHOP
(Figure 5C). Collectively, these results suggest that TLR2 activity
prevents the DEN-induced HCC by diminishing oxidative stress
and ER stress, which induces autophagic flux and attenuates
UPR-mediated inflammation in response to DEN liver injury
HCC has been characterized as a chronic inflammation-driven
cancer and the studies of animal models of chemically-induced
HCC have revealed the crucial roles of inflammatory signaling in
disease onset and severity . Hepatic immunity is predominantly
Figure 2. TLR2-deficiency results in increases in ROS accumulation and oxidative DNA damage in DEN-injured liver tissues. (A)
Representative images of DHE-detected ROS (left panel, red) in the livers of WT and TLR22/2 mice at one month post-DEN injection (n = 4 per group).
Scale bar, 75 mm. (B) DCF fluorescence-detected ROS in livers of WT and TLR22/2 mice (n = 4 per group). (C) Western blot detection of
8hydroxyguanosine adduct crossed proteins in livers of WT and TLR22/2 mice at one month post-DEN injection. Data are presented as mean 6 SEM
(n = 4 per group). (D) Representative images of p62 immunostaining in livers of WT and TLR22/2 mice at one month post-DEN injection (left panel).
Scale bars, 100 mm. The p62 intensities are presented as mean 6 SD (n = 4 per group, right panel).
innate . The pattern recognition receptors, especially TLRs, play
a central role in maintaining such balance between homeostasis and
inflammatory state in liver . We recently find that knockout
TLR2 enhances DEN-induced hepatocarcinogenesis because the
liver immune network fails to respond to DEN challenge in TLR2
deficient livers, resulting in a less infiltration of macrophages and
release of immune factors. This immune-network is necessary for
maintaining the functional autophagy flux, cellular senescence, and
cellular death undergoing cellular stress [22,23]. In this study, we
further prove that ROS/ER stress is directly responsible for the
aggravation of liver carcinogenesis in TLR2 deficient mice. The
treatment of animals with the antioxidant agent NAC can attenuate
ROS/ER stress to prevent UPR-induced inflammation and p62
aggregation in DEN-treated TLR22/2 liver. Thus, NAC treatment
interrupts the positive feedback of the ROS/ER stress-p62
aggregation-UPR-induced inflammation and reverses the TLR2
deficiency increased susceptibility of HCC development and
In hepatocytes, DEN is metabolized by cytochrome P450 2E1
via a ROS-generating reaction. DEN administration thus causes a
large amount of ROS production in WT hepatocytes, which is not
only responsible for liver injury and necrosis of hepatocytes, but
also induces DNA damage and genomic instability .
Consequently, DAMPs are released from these injured and necrotic
hepatocytes and activate PRRs on macrophages and liver cells.
The activated macrophages intake debris of necrotic hepatocytes
and secrete immune factors that further activate autophagy and
senescence response in hepatocytes . The activated immune
network in turn supports the cell death and survival signaling
pathways of hepatocytes to counter against cellular stresses.
Therefore, the production of ROS plays a critical role in
maintaining homeostasis in response to DEN insult to protect
against tumorigenesis and progression.
ER stress is triggered when unfolded proteins accumulate in the
ER and is one of the endogenous sources of ROS accumulation
. P62 is a cargo receptor of selective autophagy and can deliver
ubiquitin decorated proteins into the autophagy-lysosome
degradation pathway . Once cells are undergoing ER stress or
failure of autophagic flux, p62 and its bonded proteins would
became aggregation. P62 aggregation is another source of ROS
production and accumulation . In turn, ROS is a powerful
trigger of ER stress and p62 aggregates [26,28]. Thus, a positive
feedback loop is established among the ROS production, ER stress
and p62 aggregation in DEN-injured liver. For this reason, once
the accumulated ROS is reduced by the treatment of NAC, the
ER stress and p62 aggregates are decreased and HCC
development is diminished in the TLR22/2 liver.
Activation of TLR2 stimulates several signaling pathways in
response to the DEN-induced ROS accumulation. The ASK1/
p38 MAPK/NF-kB signaling pathway is a major sensor for
cellular ROS and drives these higher mutant risk cells into
apoptotic cell death . Each of the ERK, JNK, and Akt
pathways has been reported involving in the positive or negative
regulation of proliferation, apoptosis, and autophagy in response
to ROS. For instance, The UPR-activated JNK pathway links the
ER-stress to inflammation in much broader cell types in
responding to cellular stress . The activation of autophagy
under ER-stress is necessary for cell survival in a JNK dependent
manner . Therefore, JNK is an important downstream
molecule of UPR and plays a dominant pro-survival role through
an UPR-JNK-autophagy pathway . Indeed, DEN treatment
causes an attenuation of the ASK1/p38 MAPK/NF-kB and
PI3K/Akt signaling [22,23] but an increase in the activity of JNK
Figure 3. TLR2 deficiency enhances ER stress in DEN-injured liver tissues. (A) Western blot detection of Bip, phospho-eIF2a, eIF2a, CHOP,
IRE1a, phospho-JNK1/2 and JNK1/2 in livers of WT and TLR22/2 mice at one month post sham or DEN treatment. Data are presented as mean 6 SD
(n = 4 per group, right panel). (B) Apoptosis quantified by Annexin V staining is presented as mean 6 SD (n = 4 per group). (C) Representative images
of TUNEL staining (green) with liver sections from WT and TLR22/2 mice at one month post DEN treatment. The cell nuclei were counterstained with
DAPI (blue). Scale bar, 75 mm. Eight magnified (206) fields per liver were counted for the percentage of TUNEL-positive cells. Data are presented as
mean 6 SD (n = 4 per group, right panel). (D) Western blot detection of cleaved caspase-3 in livers of WT and TLR22/2 mice at one month post DEN
treatment. Data are presented as mean 6 SD (n = 4 per group, bottom panel).
Figure 4. Antioxidant NAC ameliorates TLR22/2-aggravated HCC progression. (A) Representative images of gross liver specimens from
TLR22/2 mice treated with or without NAC at six months post-DEN injection. Scale bars, 1 cm. (B) NAC treatment decreased the number of surface
tumor nodules in TLR22/2 mice. Each dot represents the tumor nodules from each mouse. Scale bars indicate the mean 6 SD (n = 20 per group). (C)
NAC treatment decreased the volume of surface tumors. Data are presented as mean 6 SD (n = 20 per group). (D) Representative H&E stained liver
sections from TLR22/2 mice treated with or without NAC. Scale bar, 500 mm. (E) The calculated tumor area (percentage of liver) is presented as mean
6 SD (n = 20 per group).
pathways (Figure 3A) in livers from TLR2-deficient mice,
suggesting that HCC cells containing higher ROS and DNA
damages have more chance to survive in TLR22/2 livers.
Molecular therapeutic strategies against cancer by targeting the
components of the innate immune system rely on a precise
understanding of the tumor stage and pathogenic mechanisms
. TLR agonists have been shown to enhance immune
responses against tumors in both animal models and humans.
Indeed, the anti-tumor activities of Imiquimod, a ligand for TLR7,
and CpG DNA, a ligand for TLR9, have been demonstrated in
several clinical trials [39,40]. TLR2 agonist MALP-2, a 2 kDa
synthetic lipopeptide with macrophage-stimulatory activity, is
currently in use as a cancer immunotherapy [41,42]. Based on the
evidence presented herein, which shows that TLR2 prevents HCC
development, it is possible that MALP-2 may be a useful activator
of TLR2 for suppressing HCC development.
In conclusion, a molecular mechanism underlying the
TLR2mediated anti-tumor effects involves, at least partially, the
prevention of ROS accumulation and ER stress onset. More
importantly, treatment with the antioxidant reagent, NAC, can
eliminate ROS accumulation, alleviate the ER stress, interrupt the
positive feedback of p62 aggregation and ROS production in the
DEN-injured liver tissue, and reduce the burden of liver tumors.
Subsequent studies are needed to uncover the precise components
and signal pathway and their dynamic interactions that regulate
the signal from TLR2 activation to suppression of oxidant and ER
stressors in HCC.
Figure S1 Expression of TLR2 associates with ROS
accumulation and p62 aggregates in parenchymal cells.
(A) Representative images of WT primary parenchymal (left
panel), non-parenchymal (middle panel), and TLR22/2
nonparenchymal cells (right panel) stained with TLR2 and F4/80.
Scale bar, 10 mm. (B) Liver parenchymal cells produced more
ROS and p62 aggregates. Primary parenchymal cells (PC, red, left
panel) and non-parenchymal cells (NPC, black, middle panel) was
detected by flow cytometer, and measured for ROS and p62
aggregates (right panel). (C) H2O2 induced more ROS and p62
aggregates in TLR22/2 parenchymal cells. Primary isolated WT
and TLR22/2 parenchymal cells was treated with 50 mM H2O2
for 2 hrs, and was detected by flow cytometer (PC, blue, left
panel). WT (red) and TLR22/2 (black) parenchymal cells was
Figure S2 Expression of TLR2 associates negatively
with ER stress in MEFs or HepG2 cells. (A) Expression of
Bip, phospho-eIF2a, eIF2a, CHOP, IRE1a, phospho-JNK1/2
and JNK1/2 was detected with Western blotting in MEFs treated
with or without DEN (200 mg/ml for 24 h). Data are
representative immune blots (left panel) and mean 6 SEM (n = 4 per
group, right panel). (B) The stable overexpression of TLR2 was
identified in the clone 1 of HepG2 cells. (C) Expression of Bip,
phospho-eIF2a, eIF2a, CHOP, IRE1a, phospho-JNK1/2 and
JNK1/2 was detected with Western blotting in HepG2 cells
overexpressing TLR2 or control vector. Data are representative blots
(left panel) and mean 6 SEM (n = 4 per group, right panel).
Conceived and designed the experiments: HL ZH. Performed the
experiments: HL XL JY. Analyzed the data: HL. Contributed reagents/
materials/analysis tools: HL FH. Wrote the paper: HL ZH.
1. Parkin DM , Bray F , Ferlay J , Pisani P ( 2005 ) Global cancer statistics . CA Cancer J Clin 55 : 74 - 108 .
2. Siegel R , Ward E , Brawley O , Jemal A ( 2011 ) Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths . CA Cancer J Clin 61 : 212 - 236 .
3. Takayama T , Makuuchi M , Hirohashi S , Sakamoto M , Okazaki N , et al. ( 1990 ) Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma . Lancet 336 : 1150 - 1153 .
4. Sakamoto M , Hirohashi S , Shimosato Y ( 1991 ) Early stages of multistep hepatocarcinogenesis: adenomatous hyperplasia and early hepatocellular carcinoma . Hum Pathol 22 : 172 - 178 .
5. Kudo M ( 2009 ) Multistep human hepatocarcinogenesis: correlation of imaging with pathology . J Gastroenterol 44 : 112 - 118 .
6. Forner A , Llovet JM , Bruix J ( 2012 ) Hepatocellular carcinoma . Lancet 379 : 1245 - 1255 .
7. Sia D , Villanueva A ( 2011 ) Signaling pathways in hepatocellular carcinoma . Oncology 81 : 18 - 23 .
8. O9Neill LA ( 2008 ) The interleukin-1 receptor/Toll-like receptor superfamily: 10 years of progress . Immunol Rev 226 : 10 - 18 .
9. Li X , Jiang S , Tapping RI ( 2010 ) Toll-like receptor signaling in cell proliferation and survival . Cytokine 49 : 1 - 9 .
10. Kimbro K , Parker S ( 2009 ) Recent patents in Toll-like receptor pathways and relevance to cancer . Recent Pat Anticancer Drug Discov 4 : 189 - 195 .
11. Seki E , Brenner DA ( 2008 ) Toll-like receptors and adaptor molecules in liver disease: update . Hepatology 48 : 322 - 335 .
12. Schwabe RF , Seki E , Brenner DA ( 2006 ) Toll-like receptor signaling in the liver . Gastroenterology 130 : 1886 - 1900 .
13. Matsumura T , Degawa T , Takii T , Hayashi H , Okamoto T , et al. ( 2003 ) TRAF6-NF-kappaB pathway is essential for interleukin-1-induced TLR2 expression and its functional response to TLR2 ligand in murine hepatocytes . Immunology 109 : 127 - 136 .
14. Liu S , Gallo DJ , Green AM , Williams DL , Gong X , et al. ( 2002 ) Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide . Infect Immun 70 : 3433 - 3442 .
15. Szabo G , Dolganiuc A , Mandrekar P ( 2006 ) Pattern recognition receptors: a contemporary view on liver diseases . Hepatology 44 : 287 - 298 .
16. Gao B , Jeong WI , Tian Z ( 2008 ) Liver: An organ with predominant innate immunity . Hepatology 47 : 729 - 736 .
17. Junjie X , Songyao J , Minmin S , Yanyan S , Baiyong S , et al. ( 2012 ) The association between Toll-like receptor 2 single-nucleotide polymorphisms and hepatocellular carcinoma susceptibility . BMC Cancer 12 : 57 .
18. Nischalke HD , Coenen M , Berger C , Aldenhoff K , Muller T , et al. ( 2012 ) The toll-like receptor 2 (TLR2) -196 to -174 del/ins polymorphism affects viral loads and susceptibility to hepatocellular carcinoma in chronic hepatitis C . Int J Cancer 130 : 1470 - 1475 .
19. Bardag-Gorce F , Oliva J , Lin A , Li J , French BA , et al. ( 2010 ) SAMe prevents the up regulation of toll-like receptor signaling in Mallory-Denk body forming hepatocytes . Exp Mol Pathol 88 : 376 - 379 .
20. Wang Z , Yan J , Lin H , Hua F , Wang X , et al. ( 2013 ) Toll-like receptor 4 activity protects against hepatocellular tumorigenesis and progression by regulating expression of DNA repair protein Ku70 in mice . Hepatology 57 : 1869 - 1881 .
21. Wang Z , Lin H , Hua F , Hu ZW ( 2013 ) Repairing DNA damage by XRCC6/ KU70 reverses TLR4-deficiency-worsened HCC development via restoring senescence and autophagic flux . Autophagy 9 : 925 - 927 .
22. Lin H , Yan J , Wang ZY , Hua F , Yu JJ , et al. ( 2013 ) Loss of Immunity-supported senescence enhances susceptibility to hepatocellular carcinogenesis and progression in TLR2-deficient mouse . Hepatology 57 : 171 - 82 .
23. Lin H , Hua F , Hu ZW ( 2012 ) Autophagic flux, supported by toll-like receptor 2 activity, defends against the carcinogenesis of hepatocellular carcinoma . Autophagy 8 : 1859 - 1861 .
24. Marra M , Sordelli IM , Lombardi A , Lamberti M , Tarantino L , et al. ( 2011 ) Molecular targets and oxidative stress biomarkers in hepatocellular carcinoma: an overview . J Transl Med 9 : 171 .
25. Malhi H , Kaufman RJ ( 2011 ) Endoplasmic reticulum stress in liver disease . J Hepatol 54 : 795 - 809 .
26. Zhang K , Kaufman RJ ( 2008 ) From endoplasmic-reticulum stress to the inflammatory response . Nature 454 : 455 - 462 .
27. Mathew R , Karp CM , Beaudoin B , Vuong N , Chen G , et al. ( 2009 ) Autophagy suppresses tumorigenesis through elimination of p62 . Cell 137 : 1062 - 1075 .
28. Moscat J , Diaz-Meco MT ( 2009 ) p62 at the crossroads of autophagy , apoptosis, and cancer. Cell 137 : 1001 - 1004 .
29. Dolado I , Swat A , Ajenjo N , De Vita G , Cuadrado A , et al. ( 2007 ) p38alpha MAP kinase as a sensor of reactive oxygen species in tumorigenesis . Cancer Cell 11 : 191 - 205 .
30. Smedsrd B , Pertoft H ( 1985 ) Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence . J Leukoc Biol 38 : 213 - 230 .
31. Conner DA ( 2001 ) Mouse embryo fibroblast (MEF) feeder cell preparation . Curr Protoc Mol Biol Chapter 23: Unit 23 .2.
32. Sakurai T , He G , Matsuzawa A , Yu GY , Maeda S , et al. ( 2008 ) Hepatocyte necrosis induced by oxidative stress and IL-1 alpha release mediate carcinogeninduced compensatory proliferation and liver tumorigenesis . Cancer Cell 14 : 156 - 165 .
33. Maeda S , Kamata H , Luo JL , Leffert H , Karin M , et al. ( 2005 ) IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis . Cell 121 : 977 - 990 .
34. Gao B , Jeong WI , Tian Z ( 2008 ) Liver: An Organ with Predominant Innate Immunity . Hepatology 47 : 729 - 736 .
35. Johansen T , Lamark T ( 2011 ) Selective autophagy mediated by autophagic adapter proteins . Autophagy 7 : 279 - 296 .
36. Ogata M , Hino S , Saito A , Morikawa K , Kondo S , et al. ( 2006 ) Autophagy is activated for cell survival after endoplasmic reticulum stress . Mol Cell Biol 26 : 9220 - 9231 .
37. Clarke R , Cook KL , Hu R , Facey CO , Tavassoly I , et al. ( 2012 ) Endoplasmic reticulum stress, the unfolded protein response, autophagy, and the integrated regulation of breast cancer cell fate . Cancer Res 72 : 1321 - 1331 .
38. Yan J , Wang ZY , Yang HZ , Liu HZ , Mi S , et al. ( 2011 ) Timing is critical for an effective anti-metastatic immunotherapy: the decisive role of IFNc/STAT1- mediated activation of autophagy . PLoS One 6 : e24705 .
39. Hemmi H , Kaisho T , Takeuchi O , Sato S , Sanjo H , et al. ( 2002 ) Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway . Nat Immunol 3 : 196 - 200 .
40. Hemmi H , Takeuchi O , Kawai T , Kaisho T , Sato S , et al. ( 2000 ) A Toll-like receptor recognizes bacterial DNA . Nature 408 : 740 - 745 .
41. Schneider C , Schmidt T , Ziske C , Tiemann K , Lee KM , et al. ( 2004 ) Tumour suppression induced by the macrophage activating lipopeptide MALP-2 in an ultrasound guided pancreatic carcinoma mouse model . Gut 53 : 355 - 361 .
42. Shingu K , Kruschinski C , Luhrmann A , Grote K , Tschernig T , et al. ( 2003 ) Intratracheal macrophage-activating lipopeptide-2 reduces metastasis in the rat lung . Am J Respir Cell Mol Biol 28 : 316 - 321 .