Published OnlineFirst September 28, 2016; DOI: 10.1158/0008-5472.

CAN-15-3034

Cancer
Therapeutics, Targets, and Chemical Biology Research

Doxorubicin-Induced Systemic Inflammation Is

Driven by Upregulation of Toll-Like Receptor
TLR4 and Endotoxin Leakage
Lintao Wang1, Qian Chen1, Haixia Qi1, Chunming Wang2, Cheng Wang1,3, Junfeng Zhang1,
and Lei Dong1

Abstract
Doxorubicin is one of the most effective chemotherapeutic
agents used for cancer treatment, but it causes systemic inflam-
mation and serious multiorgan side effects in many patients. In
this study, we report that upregulation of the proinflammatory
Toll-like receptor TLR4 in macrophages by doxorubicin is an
important step in generating its toxic side effects. In patient serum,
doxorubicin treatment resulted in leakage of endotoxin and
inflammatory cytokines into circulation. In mice, doxorubicin
damaged the intestinal epithelium, which also resulted in leakage
of endotoxin from the gut flora into circulation. Concurrently,
doxorubicin increased TLR4 expression in macrophages both in
vitro and in vivo, which further enhanced the sensitivity of these
cells to endotoxin. Either depletion of gut microorganisms or
blockage of TLR4 signaling effectively decreased doxorubicin-
induced toxicity. Taken together, our findings suggest that doxo-
rubicin-triggered leakage of endotoxin into the circulation, in
tandem with enhanced TLR4 signaling, is a candidate mechanism
underlying doxorubicin-induced systemic inflammation. Our
study provides new insights for devising relevant strategies to
minimize the adverse effects of chemotherapeutic agents such as
doxorubicin, which may extend its clinical uses to eradicate
cancer cells. Cancer Res; 76(22); 1–12.
2016 AACR.

Introduction We postulated that the entry of endotoxin into the circulation

triggers TLR4-mediated systemic inflammation. Endotoxin is a
Increasing clinical evidence suggests that cancer therapeutic
major component of Gram-negative bacteria and a typical ligand
agents (CTA) can trigger systemic inflammation that leads to poor
of TLR4 (12). Under normal conditions, a large amount of
prognosis. For example, doxorubicin (DOX), one of the most
bacteria presenting endotoxin reside in the intestine and are
effective CTA to date, induces severe inflammatory responses in
strictly confined by the barrier of the intestinal mucosa. But this
various organs including the liver, kidney, intestine, and blood
barrier could be broken by doxorubicin, which is known to be
vessels (1–5), in addition to its known major adverse effect of
capable of disrupting the epithelium to induce oral ulcers, intes-
cardiac toxicity (6). The levels of proinflammatory cytokines, such
tinal inflammation, and hemorrhagic cystitis in cancer patients
as IL1 and TNFa, were significantly increased following doxorubicin
(13, 14). If doxorubicin caused damage in the intestinal mucosa,
administration; meanwhile, blocking their functions alleviated the
endotoxin could enter the circulation and stimulate systemic
tissue damage caused by doxorubicin (7–10). Further investigations
inflammation. Furthermore, doxorubicin was found to upregu-
uncovered the roles of the toll-like receptor (TLR) family in this
late the expression of TLR2 and 4 in cardiomyocytes (15). It is
process—in particular TLR4, as heart failure was completely unob-
possible that this molecule can directly elevate the level of TLR4 in
served in TLR4-knockout mice treated with doxorubicin (11).
macrophages, resulting in stronger inflammatory responses to
However, TLR4 is a receptor mainly responsible for innate immune
endotoxin and more severe damages to various organs.
response against bacterial infection; it remains unclear how it could
Therefore, we hypothesized that the disruption of the intestinal
be involved in these CTA-caused tissue damages.
mucosa barrier by doxorubicin, leading to the entry of endotoxin
into the circulation that elicits TLR4-mediated inflammation in
multiple organs, could serve as a new mechanism for the multi-
organ toxicity of doxorubicin in the body. To validate this, we
1
State Key Laboratory of Pharmaceutical Biotechnology, NJU compared the levels of endotoxin and inflammatory cytokines in
Advanced Institute for Life Sciences (NAILS), School of life sciences,
Nanjing University, Nanjing, China. 2State Key Laboratory of Quality serum samples of patients with or without doxorubicin treatment.
Research in Chinese Medicine, Institute of Chinese Medical Sciences, On the basis of the result, we tested the toxicity of doxorubicin in
University of Macau, Taipa, Macau SAR. 3Department of Clinical Lab- animal models in which gut microorganisms were depleted, and
oratory, Jinling Hospital, Nanjing University School of Medicine, Nanj-
ing, China. studied the correlation between TLR4 expression, doxorubicin
administration and global inflammatory responses both in vitro
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
and in vivo.
Corresponding Authors: Lei Dong, Nanjing University, 163 Xianlin Avenue,
Nanjing 210093, China. Phone/Fax: 85-25-89-68-1320; E-mail: Materials and Methods
leidong@nju.edu.cn; and Junfeng Zhang, jfzhang@nju.edu.cn
Reagents
doi: 10.1158/0008-5472.CAN-15-3034 Doxorubicin was purchased from Fenghua Biotech

2016 American Association for Cancer Research. (purity>99%) and dissolved in saline. TAK-242 was provided by

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 Published OnlineFirst September 28, 2016; DOI: 10.1158/0008-5472.CAN-15-3034
Wang et al.
MedChem Express (purity>98%) and dissolved in a fat emulsion
(16). LPS (Lipopolysaccharides from Escherichia coli0111:B4)
was purchased from Sigma-Aldrich. Rapamycin was purchased
from Sangon Biotech (purity>98%) and dissolved in 0.9% sodi-
um chloride solution containing 1% DMSO at a concentration of
1 mg/mL. Other chemical reagents were purchased from Sangon
Biotech.
Commercial kits to determine the levels of creatine kinase
(CK), lactate dehydrogenase (LDH), blood urea nitrogen
(BUN), aspartate transaminase (AST), and alanine aminotrans-
ferase (ALT) were provided by Jiancheng Biotech. ELISA kits for
TNFa, IL1b, and IL6 were purchased from Sizhengbai Biotech.
Cell culture medium RPMI-1640 and FBS were purchased from
Gibco BRL and Hyclone, respectively.
Human serum samples
Serum samples were collected from healthy donors and
cancer patients with or without doxorubicin treatment. The
study was approved by the Nanjing University Human Research
Ethics Committee. All human samples were collected at Jinling
Hospital (Nanjing, China) after the informed consent was
obtained. We studied 10 healthy donors (Healthy donor; mean
age 53 years, SD ¼ 6), 10 cancer patients without doxorubicin
treatment (DOX patient; mean age 58 years, SD ¼ 7) and 14
cancer patients with doxorubicin treatment (DOXþ patient;
mean age 57 years, SD ¼ 10). Detailed information about the
clinical sample donors can be found in Supporting Information
(Supplementary Table S1).
Mouse peritoneal macrophages
Primary mouse peritoneal macrophages were used in all the
experiments. They were harvested and cultured according to a
published method (17). Purified macrophages (purity > 90%)
were incubated in 12-well plates (density of 1  106 per well) in
RPMI-1640 medium with 10% (v/v) FBS at 37 C in a humidified
atmosphere of 5% CO2.
To study the effect of gut microorganisms on proinflammatory
cytokine levels following doxorubicin administration, we treated
the mice with 20 mg/kg, i.p. doxorubicin. Macrophages were
harvested and cultured at days 0, 2, and 4 after doxorubicin
treatment. Serum-free medium was added for 12 hours before
it was collected for subsequent ELISA analyses.
To study the effect of doxorubicin on TLR4 expression in
peritoneal macrophages, we stimulated the cells with doxorubicin
(50 and 100 ng/mL, in PBS) for 12 or 24 hours, before the cells
were harvested for subsequent Western blotting analyses.
To verify whether doxorubicin administration increased cellu-
lar sensitivity to LPS, we treated the cells with doxorubicin (100
ng/mL) or/and LPS (100 ng/mL) for 24 hours before the cells were
harvested for subsequent ELISA analyses.
Animal treatments
C57BL/6N mice, ages 6 to 8 weeks, were provided by Vital River
Laboratories (Beijing, China). For the accuracy of experiments, all
of the control mice were littermates (18). The TLR4lps-del mice
(C57BL/10ScN background, The Jackson Laboratory Number:
003752), ages 6 to 8 weeks and carrying a spontaneous deletion
of TLR4, and littermates were purchased from the experimental
animal center of Nanjing University (Nanjing, China; refs.
19–22).
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Animal protocols were reviewed and approved by the Animal
Care and Use Committee of Nanjing University, and conformed
to the Guidelines for the Care and Use of Laboratory Animals
published by the National Institutes of Health. They were fed in a
specific pathogen-free (SPF) animal facility with controlled light
(12 hours light/dark), temperature and humidity, with food and
water available.
We established a depletion-of-gut-microorganisms (DGM)
mice model to study the source of endotoxin and its correlation
with doxorubicin's toxicity. Mice were initially given a treatment
of 1 g/L ampicillin, 0.5 g/L vancomycin, 1 g/L metronidazole,
1 g/L neomycin and 100 g/L glucose for 4 weeks. Mice were then
switched to a different antibiotic cocktail of 2 g/L streptomycin,
0.17 g/L gentamycin, 0.125 g/L ciprofloxacin, 1 g/L bacitracin, and
100 g/L glucose throughout the experiment. We confirmed intes-
tinal depletion by collecting feces, homogenizing them in PBS,
and serially diluting and plating the samples on trypticase soy agar
with 10% sheep blood (Thermo Fisher Scientific) in an anaerobic
chamber (37 C; 48 hours; refs. 23, 24).
To study the role of gut microorganisms in doxorubicin
induced multiorgan damages, mice were divided into four
groups: (i) Mice received drinking glucose solution without
the addition of microbiotic (WT group); (ii) DGM mice (DGM
group); (iii) WT group mice received 20 mg/kg doxorubicin,
intraperitoneal (i.p.) injection (WTþDOX group); and (iv)
DGM mice received 20 mg/kg doxorubicin, intraperitoneal
injection (DGMþDOX group). The animals were examined for
their body weights, mortality, and damages in different organs.
To verify whether endotoxin contributed to the toxicity of
doxorubicin, mice were divided into three groups: (i) DGM mice
received 20 mg/kg, i.p. doxorubicin administration (DGMþDOX
group); (ii) DGM mice received doxorubicin (20 mg/kg) and 2
mg/kg, i.p. LPS administration (DGMþDOXþ2mg/kg LPS
group); and (iii) DGM mice received doxorubicin (20 mg/kg)
and LPS (5 mg/kg) administration (DGMþDOXþ5mg/kg LPS
group). The animals were examined for their body weights,
mortality, and damages in different organs.
To study the influence of doxorubicin treatment on TLR4
expression in peritoneal macrophages, the mice were treated with
doxorubicin (20 mg/kg, i.p.) or an equal volume of PBS for 2 days,
before their hearts, kidneys, livers and intestines were harvested
for TLR4 analysis.
To assess whether doxorubicin treatment would increase the
toxicity of LPS in vivo, mice were divided into four groups: (i)
Mice treated with saline at the equal volume as used in other
experimental groups (Sham group); (ii) WT mice received 20 mg/
kg, i.p. doxorubicin administration (DOX group); (iii) WT mice
received 5 mg/kg, i.p. LPS administration (LPS group); (iv) WT mice
received both doxorubicin (20 mg/kg) and LPS (5 mg/kg) admin-
istration (DOXþLPS group). The animals were examined for their
body weights, mortality and damages in different organs.
To study the role of TLR4 in the process of doxorubicin-caused
intestinal impairment and systemic inflammation, TAK-242 (10
mg/kg body weight, i.v. injection) was used to inhibit the signal
transduction of TLR4. The TLR4lps-del mice were further used to
confirm the function of TLR4 in the generation of doxorubicin's
toxicity. Having been challenged by doxorubicin or doxorubicin
combined with LPS, the animals were examined for damage in
their tissues and the cytokines in the serum.
To study the role of mTOR signaling in the side effect of
doxorubicin, mice were divided into two groups: (i) Mice treated
Cancer Research
 Published OnlineFirst September 28, 2016; DOI: 10.1158/0008-5472.CAN-15-3034
with 2 mg/kg, i.p. Rapamycin per day resolved in 0.9% sodium
chloride solution containing 1% DMSO for 30 days before they
were injected with doxorubicin (20 mg/kg; RapamycinþDOX
group). (ii) Mice treated with 0.9% sodium chloride solution
containing 1% DMSO (i.p.) per day at the equal volume as
the RapamicinþDOX group for 30 days before receiving doxo-
rubicin (20 mg/kg, DOX group). The animals were examined
for their body weights, mortality, and damage in different
organs.
Serum marker of multiple organ damages and cytokine analysis
The serum and cell supernatant were collected and stored at
80 C until analysis, in which the levels of CK, LDH, BUN, AST,
and ALT were assessed with corresponding kits. The levels of the
inflammatory cytokines (TNFa, IL1b, and IL6) were determined
by ELISA kits.
Histological and immunofluorescence analysis
The cardiac, renal, hepatic, and intestinal tissue samples were
fixed in Bouin's buffer, embedded in paraffin and sectioned. For
histologic analyses, the slices were counterstained with hematox-
ylin and eosin (H&E). For the histologic quantitative analyses of
gut tissues, inflammatory foci, crypt depth, and altered villi were
scored for each section. For immunofluorescence (IF) analysis, the
slices were washed with PBS and blocked with 5% BSA for 1 hour,
followed by incubation in primary (4 C; overnight) and second-
ary antibodies (room temperature; 45 minutes).
Western blotting analysis
Proteins extracted from cell lysate or tissue homogenate were
separated with SDS-PAGE and transferred onto polyvinylidine
difluoride (PVDF) membranes (Biotrace). The membranes were
blocked by 5% skimmed milk (room temperature; 1 hour) with
gentle shaking, blotted with primary antibodies (4 C; overnight)
and secondary antibodies (room temperature; 1 hour), conse-
quently, with proper rinse. The bands were visualized with Lumi-
GLO Reagent (CST).
Statistical analysis
Data are presented as mean  SEM. The differences between
groups were analyzed using the Student t test when only two
groups were compared or one-way ANOVA when more than two
groups were compared. A P value of 0.05 was considered
significant.
Results
Endotoxin from gut microbiota causes inflammation and tissue
damage following doxorubicin administration
To study the role of endotoxin in doxorubicin-induced inflam-
mation and tissue damages, we measured the levels of both
endotoxin and TNFa in different human serum samples. Their
levels in patients who received doxorubicin treatment (endotoxin,
342.39 U/L; TNFa, 73.44 pg/mL) were significantly higher than
those in patients without doxorubicin treatment (endotoxin,
127.5 U/L; TNFa, 9.7 pg/mL) and healthy donors (endotoxin,
200.10 U/L; TNFa, 13.54 pg/mL; Fig. 1A). We assumed that the
increased endotoxin came from gut microbiota, a main reservoir
of bacteria in the body. To validate this, we examined whether
doxorubicin would trigger less severe inflammation in DGM mice,
in which gut microorganisms were experimentally removed.
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Gut Flora Promoted Doxotubicin-Induced Inflammation
Indeed, we detected remarkably lower levels of endotoxin and
TNFa in the serum of DGMþDOX mice than in the WTþDOX
group (endotoxin: 56.26 vs. 121.53 U/L; TNFa: 65.34 vs. 189.65
pg/mL; Fig. 1B). Also, lower levels of inflammatory cytokines were
detected in the supernatant of the macrophages isolated from the
abdominal cavity of DGMþDOX mice than those from WTþDOX
mice (TNFa: 7.68 vs. 57.26 pg/mL; IL1b: 83.03 vs. 177 pg/mL; IL6:
162.67 vs. 752.58 pg/mL; Fig. 1C). Further inspection of the gut
epithelium sections indicated that doxorubicin treatment caused
focal accumulation of inflammatory cells in the lamina propria
and interstitial edema, as well as shortening of small intestinal
villis in WT mice; however, these impairments were much less
severe in the DGM group (Fig. 1D and E).
The above findings suggested that endotoxin from gut micro-
biota mediated doxorubicin-triggered inflammation—but were
these endotoxin responsible for doxorubicin's toxicity to various
tissues? To answer this question, we examined whether removal of
gut microbiota—the source of endotoxin—could abolish the
multiorgan damages caused by doxorubicin. We found no differ-
ence between the DGMþDOX mice and the untreated controls. All
animals in the DGMþDOX group survived through the 8-day
period without significant body loss, in sharp contrast with the
WTþDOX mice that suffered a consistent body weight loss until all
of them died between days 6 and 8 (Fig. 2A). Similarly, the
DGMþDOX mice had significantly lower levels of CK (691.84 vs.
1,677.14 U/L), LDH (1,481.63 vs. 2,058.5 U/L), BUN (16.56 vs.
23.89 mmol/L) and ALT (29.57 vs. 69.56 U/L) than WTþDOX
mice, indicating that removal of gut microorganisms could effec-
tively prevent the toxicity of doxorubicin to the cardiac (CK and
LDH), renal (BUN) and hepatic (ALT) tissues, respectively (Fig.
2B). Furthermore, we observed that, compared with the
DGMþDOX group, the WTþDOX group showed serious disor-
ganization of myofibrillar arrays and intense infiltration with
neutrophil granulocytes in cardiac tissue samples, and cytoplasmic
vacuolization in the renal and hepatic tissue samples (Fig. 2C). To
verify whether endotoxin is a direct cause of those side effects of
doxorubicin, the DGM mice were treated with different levels of
LPS (2 and 5 mg/kg) following doxorubicin administration. Fol-
lowing doxorubicin and LPS administration, endotoxin in the
serum of the DGMþDOXþ2 mg/kg LPS and DGMþDOXþ5
mg/kg LPS groups showed higher level than in the DGMþDOX
group (Supplementary Fig. S1). We detected remarkably higher
levels of proinflammatory factors (TNFa and IL1b) in the serum of
DGMþDOXþ2mg/kg LPS and DGMþDOXþ5mg/kg LPS groups
than in the DGMþDOX group (TNFa: 77.76 and 167.57 vs. 42.19
pg/mL; IL1b: 215.71 and 309.92 vs. 113.49 pg/mL; Fig. 2D and
Supplementary Fig. S2). We also observed lower survival rates and
body weights in DGMþDOXþ2 mg/kg LPS and DGMþDOXþ5
mg/kg LPS groups than in the DGMþDOX group (Fig. 2D and
Supplementary Fig. S3). The DGMþDOXþ2 mg/kg LPS mice and
DGMþDOXþ5 mg/kg LPS mice had significantly higher levels of
CK (2,303.16 and 3,399.11 vs. 691.84 U/L), LDH (2,257.68 and
3,037.83 vs. 1,481.63 U/L), BUN (19.62 and 26.02 vs. 15.89
mmol/L), and ALT (58.43 and 89.59 vs. 29.57 U/L) than
DGMþDOX mice. These findings indicated that endotoxin in
circulation could be a direct cause of doxorubicin to the cardiac
(CK and LDH), renal (BUN), and hepatic (ALT) tissues, respec-
tively (Supplementary Fig. S4A). Moreover, compared with the
DGMþDOX group, damages in heart, kidney, liver, and intestine
were more severe in the DGMþDOXþ2 mg/kg LPS mice and
DGMþDOXþ5 mg/kg LPS mice (Supplementary Fig. S4B). These
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Wang et al.
data confirmed that removal of gut microbiota was effective in
abolishing the damage of doxorubicin to multiple tissues/organs.
Collectively, we demonstrated that the entry of endotoxin from the
gut to the circulation, as a consequence of doxorubicin-caused
intestinal damage, could be an important mechanism for doxo-
rubicin-induced systemic inflammation and multiorgan toxicity.
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Figure 1.
Endotoxin and inflammatory factor
levels are upregulated following DOX
administration. A, serum concentrations
of endotoxin (ET) and TNFa of healthy
donors (healthy donor; n ¼ 10), patients
without DOX treatment (DOX patient;
n ¼ 10) and patients with DOX treatment
(DOXþ patient; n ¼ 14).  , P < 0.05 versus
healthy donor/DOX patient and DOXþ
patient. B, serum levels of endotoxin
and TNFa of WT, DGM, WTþDOX,
and DGMþDOX groups at the indicated
time points (n ¼ 10).  , P < 0.05
versus WTþDOX/DGMþDOX.
C, concentrations of TNFa, IL1b, and
IL6 in macrophage supernatant of WT,
DGM, WTþDOX, and DGMþDOX mice
groups at the indicated time points
(n ¼ 10).  , P < 0.05 versus WTþDOX/
DGMþDOX. D, representative histologic
images of H&E staining of the intestines
of WT, WTþDOX, and DGMþDOX. E, the
numbers of inflammatory foci depicted
per millimeter (E, left, indicated with
yellow arrowhead in D), crypt depth
reflecting edema (E, middle, indicated
with red arrowhead in D), and the
reduced length of villi (E, right,
indicated with black arrowhead in D)
were measured in five ilea on 100 villi
per ileum from WT, WTþDOX and
DGMþDOX (n ¼ 10).  , P < 0.05
versus WT/WTþDOX and
WTþDOX/DGMþDOX.
Elevated level of TLR4 increases endotoxin toxicity following
doxorubicin administration
As TLR4 is involved in doxorubicin-induced cardiopathy (11)
and an important receptor of endotoxin (25), we assessed wheth-
er its expression was altered in response to doxorubicin admin-
istration. We observed that the doxorubicin treatment elevated
Cancer Research
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Gut Flora Promoted Doxotubicin-Induced Inflammation

Figure 2.
DOX toxicity to multiple organs is lenient
when gut microorganisms were deleted.
A, body weights and survival rates in the
WT, DGM, WTþDOX, and DGMþDOX
groups (n ¼ 10). B, serum levels of CK,
LDH, BUN, AST, and ALT in WT, DGM,
WTþDOX and DGMþDOX groups
(n ¼ 10).  , P < 0.05 versus WTþDOX/
DGMþDOX. C, representative histologic
images of H&E staining of the heart,
kidney and liver of WT, DGM, WTþDOX,
and DGMþDOX. D, serum levels of
TNFa in DGMþDOX, DGMþDOXþ
2mg/kg LPS and DGMþDOXþ5mg/kg
LPS groups (n ¼ 10).  , P < 0.05 versus
DGMþDOX/DGMþDOXþ2 mg/kg LPS
or DGMþDOXþ5 mg/kg LPS, and
survival rates in DGMþDOX,
DGMþDOXþ2 mg/kg LPS, and
DGMþDOXþ5 mg/kg LPS groups.

the level of TLR4 expression in the ex vivo cultured peritoneal this drug resulted in upregulated expression of TLR4 in the cardiac,
macrophages, and that doxorubicin in a higher dose (100 vs. 50 renal, hepatic, and intestinal tissues, as evidenced by both Western
ng/mL) exerted a stronger augmentation effect (Fig. 3A). A similar blotting (Fig. 3B) and immunofluorescent staining (Fig. 3C).
effect of doxorubicin was found in mice: The administration of Interestingly, treatment of macrophages with a combination of

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Wang et al.
doxorubicin and LPS stimulated remarkably higher levels of
inflammatory cytokines (TNFa, IL1b, and IL6), compared with
using LPS alone (Fig. 3D). This finding not only further indicated
that doxorubicin could directly upregulate the expression of TLR4
and thereby increase the cellular sensitivity to LPS, but also
inspired us to assess whether doxorubicin treatment could ampli-
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fy the toxicity of endotoxin/LPS at a global level. Indeed, we
detected significantly higher serum levels of TNFa and IL1b in
mice receiving combined treatment of doxorubicin and LPS than
those treated with LPS alone (TNFa: 188.6 vs. 58.64 pg/mL; IL1b:
309.64 vs. 207.18 pg/mL; Fig. 4A), and observed lower body
weights and survival rates in the former group (Fig. 4B). In
Figure 3.
TLR4 expression level is upregulated
following DOX administration. A,
representative Western blotting and
quantitative results showing the protein
levels of TLR4 in peritoneal macrophages
in response to PBS or DOX.  , P < 0.05
versus DOX/PBS and 50 ng/mL DOX/
100 ng/mL DOX. Left, representative
Western blotting. Right, bar graphs
illustrating quantitative results. B,
representative Western blotting and
quantitative results showing the protein
levels of TLR4 in the cardiac, renal, hepatic,
and intestinal tissues of the mice following
PBS or DOX administration.  , P < 0.05
versus DOX/PBS. Left, representative
Western blotting. Right, bar graphs
illustrating quantitative results (n ¼ 10).
C, immunofluorescent staining for TLR4 on
heart, kidney, liver, and intestine slices of
wild-type mice treated with PBS/DOX.
Red, TLR4; blue, DAPI. D, levels of
TNFa, IL1b, and IL6 in macrophage
supernatant in the PBS, DOX, LPS, and
DOXþLPS groups (n ¼ 10).  , P < 0.05
versus LPS/DOXþLPS.
Cancer Research
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Gut Flora Promoted Doxotubicin-Induced Inflammation

addition, the animals receiving combined treatment had remark- animals treated with LPS alone (Fig. 4C). The maximum number
ably elevated levels of CK (4,143.26 vs. 1,957.75 U/L), LDH of cytoplasmic vacuolizations and intense infiltrations with neu-
(3,051.66 vs. 1,721.16 U/L), BUN (20.86 vs. 16.00 mmol/L), trophil granulocytes was also found in various tissue samples
AST (178.41 vs. 33.07 U/L), and ALT (98.4 vs. 44.35 U/L) than the from the DOXþLPS group (Fig. 4D).

Figure 4.
TLR4 upregulation is involved in DOX-caused toxicity. A, serum levels of TNFa and IL1b in Sham, DOX, LPS, and DOXþLPS groups (n ¼ 10).  , P < 0.05 versus
LPS/DOXþLPS. B, body weights and survival rates in the Sham, DOX, LPS, and DOXþLPS groups (n ¼ 10).  , P < 0.05 versus LPS/DOXþLPS. C, serum levels of CK,
LDH, BUN, AST, and ALT in Sham, DOX, LPS, and DOXþLPS groups (n ¼ 10).  , P < 0.05 versus LPS/DOXþLPS. D, representative histologic images of H&E
staining of the heart, kidney, liver of Sham, DOX, LPS, and DOXþLPS.

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Wang et al.
TLR4 is involved in doxorubicin-induced intestinal impairment
and multiorgan damages
To validate the essential role of TLR4 in the whole process of
doxorubicin-caused intestinal impairment, systemic inflamma-
tion and multiorgan damages, we suppressed or completely
abolished its function in the animal models, by injecting TAK-
242 or using TLR4lps-del mice, respectively. Histologic observation
revealed that the damages to the intestinal tissues were clearly
reduced in TAK-242-treated and TLR4lps-del mice administrated
with doxorubicin, in comparison with those in the ShamþDOX
group (Fig. 5A). Meanwhile, the serum levels of endotoxin and
inflammatory cytokines were significantly lower in TAK-242-
treated and TLR4lps-del mice receiving doxorubicin treatment than
in the ShamþDOX animals (endotoxin: 97.99 and 88.90 vs.
121.53 U/L; TNFa: 79.74 and 38.77 vs. 254.57 pg/ml; IL1b:
127.51 and 115.45 vs. 222.05 pg/mL; Fig. 5B and C), suggesting
that suppressing or abolishing TLR4 functions could relieve the
intestinal damages and endotoxin leakage into the circulation
caused by doxorubicin administration.
The role of TLR4 in doxorubicin-induced multiorgan tox-
icity was subsequently confirmed. We observed higher body
weights and lower death rates in TAK-242þDOXþLPS and
TLR4lps-delþDOXþLPS groups than in the ShamþDOXþLPS
group (Fig. 6A). In addition, TAK-242þDOXþLPS and
TLR4lps-delþDOXþLPS groups had remarkably decreased levels
of CK (1,604.23 and 1,405.33 vs. 3,579.97 U/L), LDH (1,186.47
and 930.33 vs. 2,284.7 U/L), BUN (11.99 and 7.88 vs. 19.26
mmol/L), and ALT (66.43 and 39.82 vs. 89.24 U/L) than
the animals in the ShamþDOXþLPS group (Fig. 6B). Further-
more, compared with the ShamþDOXþLPS group, the TAK-
242þDOXþLPS and TLR4lps-delþDOXþLPS groups showed slight
disorganizations of myofibrillar arrays and intense infiltrations
with neutrophil granulocytes in cardiac tissue samples, and the
lower number of cytoplasmic vacuolizations in the renal and
hepatic tissue samples (Fig. 6C).
Discussion
The severe adverse effects of doxorubicin on multiple organs,
notably including its cardiac toxicity leading to life-threatening
heart failure, have substantially restricted its extended clinical use
(6). However, their underlying mechanism remains elusive. Here,
we report for the first time that the entry of endotoxins (ET) to the
circulation from gut microbiota, due to the disruption of gut
epithelium by doxorubicin, substantially contributes to doxoru-
bicin-caused systemic inflammation and multiorgan damages.
This finding provides a new answer to what causes systemic
inflammation in the cancer patients receiving doxorubicin treat-
ment. In fact, evidences developed both in clinically and in
laboratory have long demonstrated that doxorubicin could cause
devastating systemic inflammation in vivo, including hepatitis,
nephritis, phlebitis, and mucositis throughout the digestive
tract, and increase inflammatory cytokines levels in the circulation
(1–5). Anti-inflammation treatments not only suppressed doxo-
rubicin-caused myocardial inflammation but also prevented
cardiaomyopathy (7–9). Previous studies suggested that the sys-
temic inflammation was stimulated by tissue damage or cell
apoptosis due to the toxicity of this drug (26). However, inflam-
matory responses triggered by tissue damage should be transient
and self-restricted. Instead, doxorubicin caused chronic and much
more destructive inflammation that usually includes the activa-
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tion of TNF signaling pathway, generation of massive ROS and
persistent expression of multiple proinflammatory cytokines—all
of which belong to typical characteristics of inflammatory
responses against endotoxin (27, 28). In addition, the central
role of TLR4 in mediating this pathologic process—as demon-
strated by both our and other studies—further suggests that
endotoxin is the most likely antigen to trigger tissue inflamma-
tion, because the principal function of this receptor is to recognize
the component of invaded bacteria, notably including endotoxin
(17, 29).
The main source of endotoxin in the body is the Gram-negative
bacteria residing in the intestinal tract (30, 31). Healthy epithe-
lium confines the endotoxin in the intestine and prevents them
from entering the circulation. The epithelial cells are constantly
under self-renewal, as a mechanism to resist the disturbance from
food intake and digestion process, which nevertheless makes
themselves more vulnerable to the chemotherapeutic agents than
other normal cells. This is why patients receiving doxorubicin
treatment always suffer from peptic ulcer and even gastrointesti-
nal hemorrhage (32). Damages in the intestinal epithelium will
inevitably lead to the leakage of endotoxin into the circulation. In
our study, we observed severe damages in the intestinal epithe-
lium of doxorubicin-treated mice, including the loss of epithelial
cells, the shrink of the villus and large areas of inflammatory
infiltration. Before causing systemic inflammation, the leakage of
endotoxin first triggers inflammatory responses in the intestine,
which in turn exaggerates the tissue damages. This dynamic
pathologic process was evidenced by the result that removal of
gut microbiota prevented the damage caused by doxorubicin
administration. It is very likely that both the initial damage to
the epithelium and the subsequent inflammation collaboratively
promoted the leakage of endotoxin to the circulation.
Circulating endotoxin is closely related to the heart failure.
Clinical investigations discovered that the circulating endotoxin
was elevated in HF patients and the monocytes from the patients
were hypersensitive to LPS (33, 34). Recent studies suggested that
the circulating endotoxin originated from gut microflora, and that
the systemic inflammatory responses stimulated by the circulating
endotoxin substantially contributed to the pathology of heart
failure (35). These findings were consistent with our study that
elimination of intestinal microflora dramatically alleviated the
inflammation response in multiple organs and decreased the mor-
tality rate in the doxorubicin-treated animals. Besides endotoxin,
other microbial products, such as nucleic acids or polysaccharides,
may also contribute to the inflammation via TLR2/9–mediated
pathways, which were previously reported to be involved in the
generation of doxorubicin's toxicity (36, 37).
Because the mTOR signaling pathway is involved in LPS-medi-
ated inflammation and tissue damages (38, 39), we wondered
whether systemic inflammation would be lenient if mTOR sig-
naling was suppressed. The mice were treated with doxorubicin
and Rapamycin. We detected higher body weights and survival
rates in the DOXþRapamycin group than in the doxorubicin
group (Supplementary Fig. S5). The levels of CK, LDH, BUN, and
ALT were lower in the DOXþRapamycin group than in the
doxorubicin group (Supplementary Fig. S6A). Moreover, lenient
damages in cardiac, renal and hepatic tissue samples were
observed in the DOXþRapamycin group than the doxorubicin
group (Supplementary Fig. S6B). These findings indicated that
mTOR signaling pathway could play a role in mediating the side
effects of doxorubicin. It has been widely accepted that the
Cancer Research
 Published OnlineFirst September 28, 2016; DOI: 10.1158/0008-5472.CAN-15-3034

Gut Flora Promoted Doxotubicin-Induced Inflammation

Figure 5.
DOX-caused intestinal damage relieves when TLR4 was suppressed or abolished. A, representative histologic images of H&E staining of the intestine of Sham,
TAK-242, TLR4lps-del, ShamþDOX, TAK-242þDOX, and TLR4lps-delþDOX. B, serum concentrations of endotoxin in Sham, TAK-242, TLR4lps-del, ShamþDOX, TAK-242þDOX,
and TLR4lps-delþDOX groups (n ¼ 10).  , P < 0.05 versus ShamþDOX/TAK-242þDOX or TLR4lps-delþDOX. C, serum concentrations of TNFa and IL1b in Sham,
TAK-242, TLR4lps-del, ShamþDOX, TAK-242þDOX, and TLR4lps-delþDOX groups (n ¼ 10).  , P < 0.05 versus ShamþDOX/TAK-242þDOX or TLR4lps-delþDOX.

unwanted toxicity of doxorubicin largely comes from its stimu-
lation of intracellular production of ROS that damages cell
membrane and induces drastic apoptosis (40, 41). Nevertheless,
ROS is also likely to contribute to the systemic inflammation
during doxorubicin treatment, as there were studies that demon-
strated that ROS could significantly increase the level of TLRs, such
as TLR9 and TLR2 (42). Besides ROS, other inflammatory med-
iators, such as IL6, GM-CSF and IFNg were also found to upre-
gulate the expression of TLR-4 in different published studies (43).
Intriguingly, the expression of IL6, GM-CSF, and IFNg could all be
enhanced by doxorubicin treatments in vivo or in vitro (44–46).
Moreover, the upregulation of TLRs (including TLR4) seems a
common phenomenon in the inflammation process as there is
evidence that LPS, oxidative LDL and CpG could all increase the
expression of some TLRs. Taken together, the upregulation of
TLR4 might be due to the inflammation reactions triggered by
doxorubicin and the release of some key inflammatory mediators
(ROS, IL6, GM-CSF).
Our findings may provide insights for understanding the
immunotoxicology of other cancer chemotherapeutic agents,
because systemic inflammation is commonly found in the cancer
patients undertaking chemotherapy, not only limited to anthra-
cyclines (47). The side effects of chemotherapeutics were mostly
attributed to their toxicity to cells in normal organs and tissues
(48, 49). The inflammatory reactions following the chemotherapy
were always considered as a consequence, but not the reason, of
such cytotoxicity (50). Nevertheless, our present finding that the
product of gut microflora leaking from the intestinal tract

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 Published OnlineFirst September 28, 2016; DOI: 10.1158/0008-5472.CAN-15-3034

Wang et al.

Figure 6.
Toxicity of multiple organs after combined administration of DOX and LPS is lenient when TLR4 was suppressed or abolished. A, body weights and survival rates
in the Sham, TAK-242, TLR4lps-del, ShamþDOXþLPS, TAK-242þDOXþLPS, and TLR4lps-delþDOXþLPS groups (n ¼ 10). B, serum levels of CK, LDH, BUN, and ALT in
Sham, TAK-242, TLR4lps-del, ShamþDOXþLPS, TAK-242þDOXþLPS, and TLR4lps-delþDOXþLPS groups (n ¼ 10).  , P < 0.05 versus ShamþDOXþLPS/TAK-
242þDOXþLPS or TLR4lps-delþDOXþLPS. C, Representative histologic images of H&E staining of the heart, kidney, and liver of Sham, TAK-242, TLR4lps-del,
ShamþDOXþLPS, TAK-242þDOXþLPS, and TLR4lps-delþDOXþLPS groups.

promoted systemic inflammation during the treatment with
chemotherapeutics suggested that inflammation could directly
derive from chemotherapy and might play a significant role in
inducing systemic toxicity. As such, we could develop new strat-
egies aimed at controlling inflammation for the treatment of
chemotherapy-related adverse effects. As our study indicated that
the gut microflora was the source of some key inflammatory
mediators, such as endotoxin, the application of antibiotics to
temporarily eliminate the bacteria in gut may be a convenient and
effective way to reduce the chemotherapy related physiological

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 Published OnlineFirst September 28, 2016; DOI: 10.1158/0008-5472.CAN-15-3034
toxicity. Accordingly, several other strategies, inhibition of TLR
signal pathway and suppression of the mTOR signaling pathway
and restoration of intestine integrity, can be investigated as
potential approaches to prevent or alleviate adverse effects of
doxorubicin and other chemotherapeutic agents.
In conclusion, our present study demonstrates that doxorubi-
cin-caused disruption of the intestinal epithelium, which enables
the leakage of endotoxin from gut microflora into circulation, is
an important cause for the common adverse effects of doxorubi-
cin, including systemic inflammation and multiorgan damages.
The administration of doxorubicin not only impaired the epithe-
lial barrier, but also directly upregulated the expression of TLR4 in
both the intestine and other tissues, which further amplified the
global immunotoxicity of endotoxin. Either depletion of gut
microflora or inhibition of TLR signaling proved effective in
abolishing or alleviating DOX's toxicity in the animal models.
This can be a general mechanism for further understanding and
control of systemic toxicity caused by a broader class of cancer
chemotherapeutic agents.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: L. Wang, C. Wang, J. Zhang, L. Dong
Development of methodology: L. Wang, L. Dong
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Gut Flora Promoted Doxotubicin-Induced Inflammation
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): L. Wang, Q. Chen, H. Qi, C. Wang
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computational analysis): L. Wang, H. Qi, C. Wang, L. Dong
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Grant Support
J. Zhang was supported by the National Basic Research Program of China
(2012CB517603) and the National High Technology Research and Devel-
opment Program of China (2014AA020707); L. Dong was supported by the
Program for New Century Excellent Talents in University (NCET-13-0272x);
all authors received the support of the National Natural Science Foundation
of China (31271013, 31170751, 31200695, 31400671, 51173076,
91129712 and 81102489) and Nanjing University State Key Laboratory of
Pharmaceutical Biotechnology Open Grant (02ZZYJ-201307). C. Wang
acknowledges the funding supports from Macau Science and Technology
Fund (FDCT 048/2013/A2) and University of Macau (MYRG2015-00160-
ICMS-QRCM).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
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Cancer Research
 Published OnlineFirst September 28, 2016; DOI: 10.1158/0008-5472.CAN-15-3034

Doxorubicin-Induced Systemic Inflammation Is Driven by

Upregulation of Toll-Like Receptor TLR4 and Endotoxin
Leakage
Lintao Wang, Qian Chen, Haixia Qi, et al.

Cancer Res Published OnlineFirst September 28, 2016.

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