doxorubicin-induced systemic inﬂammation is driven by ... · 6631. medchemexpress...

Therapeutics, Targets, and Chemical Biology

Doxorubicin-Induced Systemic Inflammation IsDriven by Upregulation of Toll-Like ReceptorTLR4 and Endotoxin LeakageLintao Wang1, Qian Chen1, Haixia Qi1, Chunming Wang2, Cheng Wang1,3, Junfeng Zhang1,and Lei Dong1

Abstract

Doxorubicin is one of the most effective chemotherapeuticagents used for cancer treatment, but it causes systemic inflam-mation and serious multiorgan side effects in many patients. Inthis study, we report that upregulation of the proinflammatoryToll-like receptor TLR4 in macrophages by doxorubicin is animportant step in generating its toxic side effects. In patient serum,doxorubicin treatment resulted in leakage of endotoxin andinflammatory cytokines into circulation. In mice, doxorubicindamaged the intestinal epithelium, which also resulted in leakageof 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 thesecells to endotoxin. Either depletion of gut microorganisms orblockage of TLR4 signaling effectively decreased doxorubicin-induced toxicity. Taken together, our findings suggest that doxo-rubicin-triggered leakage of endotoxin into the circulation, intandemwith enhanced TLR4 signaling, is a candidate mechanismunderlying doxorubicin-induced systemic inflammation. Ourstudy provides new insights for devising relevant strategies tominimize the adverse effects of chemotherapeutic agents such asdoxorubicin, which may extend its clinical uses to eradicatecancer cells. Cancer Res; 76(22); 6631–42. �2016 AACR.

IntroductionIncreasing clinical evidence suggests that cancer therapeutic

agents (CTA) can trigger systemic inflammation that leads to poorprognosis. For example, doxorubicin (DOX), one of the mosteffective CTA to date, induces severe inflammatory responses invarious organs including the liver, kidney, intestine, and bloodvessels (1–5), in addition to its known major adverse effect ofcardiac toxicity (6). The levels of proinflammatory cytokines, suchasIL1andTNFa,weresignificantly increasedfollowingdoxorubicinadministration; meanwhile, blocking their functions alleviated thetissuedamagecausedbydoxorubicin(7–10).Further investigationsuncovered the roles of the toll-like receptor (TLR) family in thisprocess—in particular TLR4, as heart failure was completely unob-served in TLR4-knockout mice treated with doxorubicin (11).However, TLR4 is a receptormainly responsible for innate immuneresponse against bacterial infection; it remains unclear how it couldbe involved in these CTA-caused tissue damages.

We postulated that the entry of endotoxin into the circulationtriggers TLR4-mediated systemic inflammation. Endotoxin is amajor component of Gram-negative bacteria and a typical ligandof TLR4 (12). Under normal conditions, a large amount ofbacteria presenting endotoxin reside in the intestine and arestrictly confined by the barrier of the intestinal mucosa. But thisbarrier could be broken by doxorubicin, which is known to becapable of disrupting the epithelium to induce oral ulcers, intes-tinal inflammation, and hemorrhagic cystitis in cancer patients(13, 14). If doxorubicin caused damage in the intestinal mucosa,endotoxin could enter the circulation and stimulate systemicinflammation. Furthermore, doxorubicin was found to upregu-late the expression of TLR2 and 4 in cardiomyocytes (15). It ispossible that thismolecule can directly elevate the level of TLR4 inmacrophages, resulting in stronger inflammatory responses toendotoxin and more severe damages to various organs.

Therefore, we hypothesized that the disruption of the intestinalmucosa barrier by doxorubicin, leading to the entry of endotoxininto the circulation that elicits TLR4-mediated inflammation inmultiple organs, could serve as a new mechanism for the multi-organ toxicity of doxorubicin in the body. To validate this, wecompared the levels of endotoxin and inflammatory cytokines inserum samples of patientswithorwithout doxorubicin treatment.On the basis of the result, we tested the toxicity of doxorubicin inanimal models in which gut microorganisms were depleted, andstudied the correlation between TLR4 expression, doxorubicinadministration and global inflammatory responses both in vitroand in vivo.

Materials and MethodsReagents

Doxorubicin was purchased from Fenghua Biotech(purity>99%) and dissolved in saline. TAK-242 was provided by

1State Key Laboratory of Pharmaceutical Biotechnology, NJUAdvanced Institute for Life Sciences (NAILS), School of life sciences,Nanjing University, Nanjing, China. 2State Key Laboratory of QualityResearch in Chinese Medicine, Institute of Chinese Medical Sciences,University of Macau, Taipa, Macau SAR. 3Department of Clinical Lab-oratory, Jinling Hospital, Nanjing University School of Medicine, Nanj-ing, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Lei Dong, Nanjing University, 163 Xianlin Avenue,Nanjing 210093, China. Phone/Fax: 85-25-89-68-1320; E-mail:[email protected]; and Junfeng Zhang, [email protected]

doi: 10.1158/0008-5472.CAN-15-3034

�2016 American Association for Cancer Research.

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MedChem Express (purity>98%) and dissolved in a fat emulsion(16). LPS (Lipopolysaccharides from Escherichia coli0111:B4)was purchased from Sigma-Aldrich. Rapamycin was purchasedfrom Sangon Biotech (purity>98%) and dissolved in 0.9% sodi-um chloride solution containing 1%DMSO at a concentration of1 mg/mL. Other chemical reagents were purchased from SangonBiotech.

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 forTNFa, IL1b, and IL6 were purchased from Sizhengbai Biotech.Cell culture medium RPMI-1640 and FBS were purchased fromGibco BRL and Hyclone, respectively.

Human serum samplesSerum samples were collected from healthy donors and

cancer patients with or without doxorubicin treatment. Thestudy was approved by the Nanjing University Human ResearchEthics Committee. All human samples were collected at JinlingHospital (Nanjing, China) after the informed consent wasobtained. We studied 10 healthy donors (Healthy donor; meanage 53 years, SD ¼ 6), 10 cancer patients without doxorubicintreatment (DOX� patient; mean age 58 years, SD ¼ 7) and 14cancer patients with doxorubicin treatment (DOXþ patient;mean age 57 years, SD ¼ 10). Detailed information about theclinical sample donors can be found in Supporting Information(Supplementary Table S1).

Mouse peritoneal macrophagesPrimary mouse peritoneal macrophages were used in all the

experiments. They were harvested and cultured according to apublished method (17). Purified macrophages (purity > 90%)were incubated in 12-well plates (density of 1 � 106 per well) inRPMI-1640 medium with 10% (v/v) FBS at 37�C in a humidifiedatmosphere of 5% CO2.

To study the effect of gut microorganisms on proinflammatorycytokine levels following doxorubicin administration, we treatedthe mice with 20 mg/kg, i.p. doxorubicin. Macrophages wereharvested and cultured at days 0, 2, and 4 after doxorubicintreatment. Serum-free medium was added for 12 hours beforeit was collected for subsequent ELISA analyses.

To study the effect of doxorubicin on TLR4 expression inperitonealmacrophages, we stimulated the cellswith doxorubicin(50 and 100 ng/mL, in PBS) for 12 or 24 hours, before the cellswere harvested for subsequent Western blotting analyses.

To verify whether doxorubicin administration increased cellu-lar sensitivity to LPS, we treated the cells with doxorubicin (100ng/mL) or/and LPS (100ng/mL) for 24hours before the cells wereharvested for subsequent ELISA analyses.

Animal treatmentsC57BL/6Nmice, ages 6 to 8weeks, were provided by Vital River

Laboratories (Beijing, China). For the accuracy of experiments, allof 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 deletionof TLR4, and littermates were purchased from the experimentalanimal center of Nanjing University (Nanjing, China; refs.19–22).

Animal protocols were reviewed and approved by the AnimalCare and Use Committee of Nanjing University, and conformedto the Guidelines for the Care and Use of Laboratory Animalspublished by the National Institutes of Health. They were fed in aspecific pathogen-free (SPF) animal facility with controlled light(12 hours light/dark), temperature and humidity, with food andwater available.

We established a depletion-of-gut-microorganisms (DGM)mice model to study the source of endotoxin and its correlationwith doxorubicin's toxicity. Mice were initially given a treatmentof 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 thenswitched to a different antibiotic cocktail of 2 g/L streptomycin,0.17 g/L gentamycin, 0.125 g/L ciprofloxacin, 1 g/L bacitracin, and100 g/L glucose throughout the experiment. We confirmed intes-tinal depletion by collecting feces, homogenizing them in PBS,and serially diluting andplating the samples on trypticase soy agarwith 10% sheep blood (Thermo Fisher Scientific) in an anaerobicchamber (37�C; 48 hours; refs. 23, 24).

To study the role of gut microorganisms in doxorubicininduced multiorgan damages, mice were divided into fourgroups: (i) Mice received drinking glucose solution withoutthe addition of microbiotic (WT group); (ii) DGM mice (DGMgroup); (iii) WT group mice received 20 mg/kg doxorubicin,intraperitoneal (i.p.) injection (WTþDOX group); and (iv)DGM mice received 20 mg/kg doxorubicin, intraperitonealinjection (DGMþDOX group). The animals were examined fortheir body weights, mortality, and damages in different organs.

To verify whether endotoxin contributed to the toxicity ofdoxorubicin, mice were divided into three groups: (i) DGMmicereceived 20mg/kg, i.p. doxorubicin administration (DGMþDOXgroup); (ii) DGM mice received doxorubicin (20 mg/kg) and 2mg/kg, i.p. LPS administration (DGMþDOXþ2mg/kg LPSgroup); and (iii) DGM mice received doxorubicin (20 mg/kg)and LPS (5 mg/kg) administration (DGMþDOXþ5mg/kg LPSgroup). The animals were examined for their body weights,mortality, and damages in different organs.

To study the influence of doxorubicin treatment on TLR4expression in peritoneal macrophages, themice were treated withdoxorubicin (20mg/kg, i.p.) or an equal volumeof PBS for 2days,before their hearts, kidneys, livers and intestines were harvestedfor TLR4 analysis.

To assess whether doxorubicin treatment would increase thetoxicity of LPS in vivo, mice were divided into four groups: (i)Mice treated with saline at the equal volume as used in otherexperimental groups (Sham group); (ii) WT mice received 20 mg/kg, i.p. doxorubicin administration (DOX group); (iii) WT micereceived5mg/kg, i.p. LPSadministration (LPSgroup); (iv)WTmicereceived both doxorubicin (20 mg/kg) and LPS (5 mg/kg) admin-istration (DOXþLPS group). The animals were examined for theirbody weights, mortality and damages in different organs.

To study the role of TLR4 in the process of doxorubicin-causedintestinal impairment and systemic inflammation, TAK-242 (10mg/kg body weight, i.v. injection) was used to inhibit the signaltransduction of TLR4. The TLR4lps-del mice were further used toconfirm the function of TLR4 in the generation of doxorubicin'stoxicity. Having been challenged by doxorubicin or doxorubicincombined with LPS, the animals were examined for damage intheir tissues and the cytokines in the serum.

To study the role of mTOR signaling in the side effect ofdoxorubicin, mice were divided into two groups: (i) Mice treated

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with 2 mg/kg, i.p. Rapamycin per day resolved in 0.9% sodiumchloride solution containing 1% DMSO for 30 days before theywere injected with doxorubicin (20 mg/kg; RapamycinþDOXgroup). (ii) Mice treated with 0.9% sodium chloride solutioncontaining 1% DMSO (i.p.) per day at the equal volume asthe RapamicinþDOX group for 30 days before receiving doxo-rubicin (20 mg/kg, DOX group). The animals were examinedfor their body weights, mortality, and damage in differentorgans.

Serummarker ofmultiple organ damages and cytokine analysisThe 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 theinflammatory cytokines (TNFa, IL1b, and IL6) were determinedby ELISA kits.

Histological and immunofluorescence analysisThe cardiac, renal, hepatic, and intestinal tissue samples were

fixed in Bouin's buffer, embedded in paraffin and sectioned. Forhistologic analyses, the slices were counterstained with hematox-ylin and eosin (H&E). For the histologic quantitative analyses ofgut tissues, inflammatory foci, crypt depth, and altered villi werescored for each section. For immunofluorescence (IF) analysis, theslices 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 analysisProteins extracted from cell lysate or tissue homogenate were

separated with SDS-PAGE and transferred onto polyvinylidinedifluoride (PVDF) membranes (Biotrace). The membranes wereblocked by 5% skimmed milk (room temperature; 1 hour) withgentle 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 analysisData are presented as mean � SEM. The differences between

groups were analyzed using the Student t test when only twogroups were compared or one-way ANOVA when more than twogroups were compared. A P value of �0.05 was consideredsignificant.

ResultsEndotoxin fromgutmicrobiota causes inflammation and tissuedamage following doxorubicin administration

To study the role of endotoxin in doxorubicin-induced inflam-mation and tissue damages, we measured the levels of bothendotoxin and TNFa in different human serum samples. Theirlevels inpatientswho receiveddoxorubicin treatment (endotoxin,342.39 U/L; TNFa, 73.44 pg/mL) were significantly higher thanthose 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 theincreased endotoxin came from gut microbiota, a main reservoirof bacteria in the body. To validate this, we examined whetherdoxorubicinwould trigger less severe inflammation inDGMmice,in which gut microorganisms were experimentally removed.

Indeed, we detected remarkably lower levels of endotoxin andTNFa in the serum of DGMþDOX mice than in the WTþDOXgroup (endotoxin: 56.26 vs. 121.53 U/L; TNFa: 65.34 vs. 189.65pg/mL; Fig. 1B). Also, lower levels of inflammatory cytokinesweredetected in the supernatant of the macrophages isolated from theabdominal cavity ofDGMþDOXmice than those fromWTþDOXmice (TNFa: 7.68 vs. 57.26pg/mL; IL1b: 83.03 vs. 177pg/mL; IL6:162.67 vs. 752.58 pg/mL; Fig. 1C). Further inspection of the gutepithelium sections indicated that doxorubicin treatment causedfocal accumulation of inflammatory cells in the lamina propriaand interstitial edema, as well as shortening of small intestinalvillis in WT mice; however, these impairments were much lesssevere in the DGM group (Fig. 1D and E).

The above findings suggested that endotoxin from gut micro-biota mediated doxorubicin-triggered inflammation—but werethese endotoxin responsible for doxorubicin's toxicity to varioustissues? To answer this question, we examinedwhether removal ofgut microbiota—the source of endotoxin—could abolish themultiorgan damages caused by doxorubicin. We found no differ-ence between theDGMþDOXmice and the untreated controls. Allanimals in the DGMþDOX group survived through the 8-dayperiod without significant body loss, in sharp contrast with theWTþDOXmice that suffered a consistent bodyweight loss until allof them died between days 6 and 8 (Fig. 2A). Similarly, theDGMþDOX 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 WTþDOXmice, indicating that removal of gut microorganisms could effec-tively prevent the toxicity of doxorubicin to the cardiac (CK andLDH), renal (BUN) and hepatic (ALT) tissues, respectively (Fig.2B). Furthermore, we observed that, compared with theDGMþDOX group, the WTþDOX group showed serious disor-ganization of myofibrillar arrays and intense infiltration withneutrophil granulocytes in cardiac tissue samples, and cytoplasmicvacuolization in the renal and hepatic tissue samples (Fig. 2C). Toverify whether endotoxin is a direct cause of those side effects ofdoxorubicin, the DGM mice were treated with different levels ofLPS (2 and 5 mg/kg) following doxorubicin administration. Fol-lowing doxorubicin and LPS administration, endotoxin in theserum of the DGMþDOXþ2 mg/kg LPS and DGMþDOXþ5mg/kg LPS groups showed higher level than in the DGMþDOXgroup (Supplementary Fig. S1). We detected remarkably higherlevels of proinflammatory factors (TNFa and IL1b) in the serumofDGMþDOXþ2mg/kg LPS and DGMþDOXþ5mg/kg LPS groupsthan in theDGMþDOX group (TNFa: 77.76 and 167.57 vs. 42.19pg/mL; IL1b: 215.71 and 309.92 vs. 113.49 pg/mL; Fig. 2D andSupplementary Fig. S2). We also observed lower survival rates andbody weights in DGMþDOXþ2 mg/kg LPS and DGMþDOXþ5mg/kg LPS groups than in the DGMþDOX group (Fig. 2D andSupplementary Fig. S3). The DGMþDOXþ2 mg/kg LPS mice andDGMþDOXþ5 mg/kg LPS mice had significantly higher levels ofCK (2,303.16 and 3,399.11 vs. 691.84 U/L), LDH (2,257.68 and3,037.83 vs. 1,481.63 U/L), BUN (19.62 and 26.02 vs. 15.89mmol/L), and ALT (58.43 and 89.59 vs. 29.57 U/L) thanDGMþDOX mice. These findings indicated that endotoxin incirculation 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 theDGMþDOX group, damages in heart, kidney, liver, and intestinewere more severe in the DGMþDOXþ2 mg/kg LPS mice andDGMþDOXþ5 mg/kg LPS mice (Supplementary Fig. S4B). These

Gut Flora Promoted Doxotubicin-Induced Inflammation

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data confirmed that removal of gut microbiota was effective inabolishing the damage of doxorubicin to multiple tissues/organs.Collectively, wedemonstrated that the entry of endotoxin from thegut to the circulation, as a consequence of doxorubicin-causedintestinal damage, could be an important mechanism for doxo-rubicin-induced systemic inflammation and multiorgan toxicity.

Elevated level of TLR4 increases endotoxin toxicity followingdoxorubicin 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

Figure 1.

Endotoxin and inflammatory factorlevels are upregulated following DOXadministration. A, serum concentrationsof endotoxin (ET) and TNFa of healthydonors (healthy donor; n ¼ 10), patientswithout DOX treatment (DOX� patient;n¼ 10) and patients with DOX treatment(DOXþ patient; n¼ 14). � , P < 0.05 versushealthy donor/DOX� patient and DOXþ

patient. B, serum levels of endotoxinand TNFa of WT, DGM, WTþDOX,and DGMþDOX groups at the indicatedtime points (n ¼ 10). � , P < 0.05versus WTþDOX/DGMþDOX.C, concentrations of TNFa, IL1b, andIL6 in macrophage supernatant of WT,DGM, WTþDOX, and DGMþDOX micegroups at the indicated time points(n ¼ 10). � , P < 0.05 versus WTþDOX/DGMþDOX. D, representative histologicimages of H&E staining of the intestinesofWT, WTþDOX, and DGMþDOX. E, thenumbers of inflammatory foci depictedper millimeter (E, left, indicated withyellow arrowhead in D), crypt depthreflecting edema (E, middle, indicatedwith red arrowhead in D), and thereduced length of villi (E, right,indicated with black arrowhead in D)were measured in five ilea on 100 villiper ileum from WT, WTþDOX andDGMþDOX (n ¼ 10). � , P < 0.05versus WT/WTþDOX andWTþDOX/DGMþDOX.

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the level of TLR4 expression in the ex vivo cultured peritonealmacrophages, and that doxorubicin in a higher dose (100 vs. 50ng/mL) exerted a stronger augmentation effect (Fig. 3A). A similareffect of doxorubicin was found in mice: The administration of

this drug resulted inupregulated expressionof TLR4 in the cardiac,renal, hepatic, and intestinal tissues, as evidencedbybothWesternblotting (Fig. 3B) and immunofluorescent staining (Fig. 3C).Interestingly, treatment of macrophages with a combination of

Figure 2.

DOX toxicity tomultiple organs is lenientwhen gut microorganisms were deleted.A, body weights and survival rates in theWT, DGM, WTþDOX, and DGMþDOXgroups (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 histologicimages of H&E staining of the heart,kidney and liver of WT, DGM, WTþDOX,and DGMþDOX. D, serum levels ofTNFa in DGMþDOX, DGMþDOXþ2mg/kg LPS and DGMþDOXþ5mg/kgLPS groups (n ¼ 10). � , P < 0.05 versusDGMþDOX/DGMþDOXþ2 mg/kg LPSor DGMþDOXþ5 mg/kg LPS, andsurvival rates in DGMþDOX,DGMþDOXþ2 mg/kg LPS, andDGMþDOXþ5 mg/kg LPS groups.






doxorubicin and LPS stimulated remarkably higher levels ofinflammatory cytokines (TNFa, IL1b, and IL6), compared withusing LPS alone (Fig. 3D). This finding not only further indicatedthat doxorubicin could directly upregulate the expression of TLR4and thereby increase the cellular sensitivity to LPS, but alsoinspired us to assess whether doxorubicin treatment could ampli-

fy the toxicity of endotoxin/LPS at a global level. Indeed, wedetected significantly higher serum levels of TNFa and IL1b inmice receiving combined treatment of doxorubicin and LPS thanthose 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 bodyweights and survival rates in the former group (Fig. 4B). In

Figure 3.

TLR4 expression level is upregulatedfollowing DOX administration. A,representative Western blotting andquantitative results showing the proteinlevels of TLR4 in peritoneal macrophagesin response to PBS or DOX. � , P < 0.05versus DOX/PBS and 50 ng/mL DOX/100 ng/mL DOX. Left, representativeWestern blotting. Right, bar graphsillustrating quantitative results. B,representative Western blotting andquantitative results showing the proteinlevels of TLR4 in the cardiac, renal, hepatic,and intestinal tissues of the mice followingPBS or DOX administration. � , P < 0.05versus DOX/PBS. Left, representativeWestern blotting. Right, bar graphsillustrating quantitative results (n ¼ 10).C, immunofluorescent staining for TLR4 onheart, kidney, liver, and intestine slices ofwild-type mice treated with PBS/DOX.Red, TLR4; blue, DAPI. D, levels ofTNFa, IL1b, and IL6 in macrophagesupernatant in the PBS, DOX, LPS, andDOXþLPS groups (n ¼ 10). � , P < 0.05versus LPS/DOXþLPS.

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addition, the animals receiving combined treatment had remark-ably elevated levels of CK (4,143.26 vs. 1,957.75 U/L), LDH(3,051.66 vs. 1,721.16 U/L), BUN (20.86 vs. 16.00 mmol/L),AST (178.41 vs. 33.07U/L), andALT (98.4 vs. 44.35U/L) than the

animals treated with LPS alone (Fig. 4C). The maximum numberof cytoplasmic vacuolizations and intense infiltrations with neu-trophil granulocytes was also found in various tissue samplesfrom 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 versusLPS/DOXþLPS. B, body weights and survival rates in the Sham, DOX, LPS, and DOXþLPS groups (n¼ 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&Estaining of the heart, kidney, liver of Sham, DOX, LPS, and DOXþLPS.






TLR4 is involved indoxorubicin-induced intestinal impairmentand multiorgan damages

To validate the essential role of TLR4 in the whole process ofdoxorubicin-caused intestinal impairment, systemic inflamma-tion and multiorgan damages, we suppressed or completelyabolished its function in the animal models, by injecting TAK-242 or using TLR4lps-del mice, respectively. Histologic observationrevealed that the damages to the intestinal tissues were clearlyreduced in TAK-242-treated and TLR4lps-del mice administratedwith doxorubicin, in comparison with those in the ShamþDOXgroup (Fig. 5A). Meanwhile, the serum levels of endotoxin andinflammatory cytokines were significantly lower in TAK-242-treated and TLR4lps-del mice receiving doxorubicin treatment thanin 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), suggestingthat suppressing or abolishing TLR4 functions could relieve theintestinal damages and endotoxin leakage into the circulationcaused by doxorubicin administration.

The role of TLR4 in doxorubicin-induced multiorgan tox-icity was subsequently confirmed. We observed higher bodyweights and lower death rates in TAK-242þDOXþLPS andTLR4lps-delþDOXþLPS groups than in the ShamþDOXþLPSgroup (Fig. 6A). In addition, TAK-242þDOXþLPS andTLR4lps-delþDOXþLPS groups had remarkably decreased levelsof CK (1,604.23 and 1,405.33 vs. 3,579.97 U/L), LDH (1,186.47and 930.33 vs. 2,284.7 U/L), BUN (11.99 and 7.88 vs. 19.26mmol/L), and ALT (66.43 and 39.82 vs. 89.24 U/L) thanthe animals in the ShamþDOXþLPS group (Fig. 6B). Further-more, compared with the ShamþDOXþLPS group, the TAK-242þDOXþLPS and TLR4lps-delþDOXþLPS groups showed slightdisorganizations of myofibrillar arrays and intense infiltrationswith neutrophil granulocytes in cardiac tissue samples, and thelower number of cytoplasmic vacuolizations in the renal andhepatic tissue samples (Fig. 6C).

DiscussionThe severe adverse effects of doxorubicin on multiple organs,

notably including its cardiac toxicity leading to life-threateningheart failure, have substantially restricted its extended clinical use(6). However, their underlyingmechanism remains elusive. Here,we report for the first time that the entry of endotoxins (ET) to thecirculation from gut microbiota, due to the disruption of gutepithelium by doxorubicin, substantially contributes to doxoru-bicin-caused systemic inflammation and multiorgan damages.

This finding provides a new answer to what causes systemicinflammation in the cancer patients receiving doxorubicin treat-ment. In fact, evidences developed both in clinically and inlaboratory have long demonstrated that doxorubicin could causedevastating systemic inflammation in vivo, including hepatitis,nephritis, phlebitis, and mucositis throughout the digestivetract, and increase inflammatory cytokines levels in the circulation(1–5). Anti-inflammation treatments not only suppressed doxo-rubicin-caused myocardial inflammation but also preventedcardiaomyopathy (7–9). Previous studies suggested that the sys-temic inflammation was stimulated by tissue damage or cellapoptosis due to the toxicity of this drug (26). However, inflam-matory responses triggered by tissue damage should be transientand self-restricted. Instead, doxorubicin caused chronic andmuchmore destructive inflammation that usually includes the activa-

tion of TNF signaling pathway, generation of massive ROS andpersistent expression ofmultiple proinflammatory cytokines—allof which belong to typical characteristics of inflammatoryresponses against endotoxin (27, 28). In addition, the centralrole of TLR4 in mediating this pathologic process—as demon-strated by both our and other studies—further suggests thatendotoxin is the most likely antigen to trigger tissue inflamma-tion, because the principal function of this receptor is to recognizethe component of invaded bacteria, notably including endotoxin(17, 29).

Themain source of endotoxin in the body is theGram-negativebacteria residing in the intestinal tract (30, 31). Healthy epithe-lium confines the endotoxin in the intestine and prevents themfrom entering the circulation. The epithelial cells are constantlyunder self-renewal, as amechanism to resist the disturbance fromfood intake and digestion process, which nevertheless makesthemselves more vulnerable to the chemotherapeutic agents thanother normal cells. This is why patients receiving doxorubicintreatment always suffer from peptic ulcer and even gastrointesti-nal hemorrhage (32). Damages in the intestinal epithelium willinevitably lead to the leakage of endotoxin into the circulation. Inour study, we observed severe damages in the intestinal epithe-lium of doxorubicin-treated mice, including the loss of epithelialcells, the shrink of the villus and large areas of inflammatoryinfiltration. Before causing systemic inflammation, the leakage ofendotoxin first triggers inflammatory responses in the intestine,which in turn exaggerates the tissue damages. This dynamicpathologic process was evidenced by the result that removal ofgut microbiota prevented the damage caused by doxorubicinadministration. It is very likely that both the initial damage tothe epithelium and the subsequent inflammation collaborativelypromoted the leakage of endotoxin to the circulation.

Circulating endotoxin is closely related to the heart failure.Clinical investigations discovered that the circulating endotoxinwas elevated in HF patients and the monocytes from the patientswere hypersensitive to LPS (33, 34). Recent studies suggested thatthe circulating endotoxin originated from gutmicroflora, and thatthe systemic inflammatory responses stimulatedby the circulatingendotoxin substantially contributed to the pathology of heartfailure (35). These findings were consistent with our study thatelimination of intestinal microflora dramatically alleviated theinflammation 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–mediatedpathways, which were previously reported to be involved in thegeneration of doxorubicin's toxicity (36, 37).

Because the mTOR signaling pathway is involved in LPS-medi-ated inflammation and tissue damages (38, 39), we wonderedwhether systemic inflammation would be lenient if mTOR sig-naling was suppressed. The mice were treated with doxorubicinand Rapamycin. We detected higher body weights and survivalrates in the DOXþRapamycin group than in the doxorubicingroup (Supplementary Fig. S5). The levels of CK, LDH, BUN, andALT were lower in the DOXþRapamycin group than in thedoxorubicin group (Supplementary Fig. S6A). Moreover, lenientdamages in cardiac, renal and hepatic tissue samples wereobserved in the DOXþRapamycin group than the doxorubicingroup (Supplementary Fig. S6B). These findings indicated thatmTOR signaling pathway could play a role in mediating the sideeffects of doxorubicin. It has been widely accepted that the

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unwanted toxicity of doxorubicin largely comes from its stimu-lation of intracellular production of ROS that damages cellmembrane and induces drastic apoptosis (40, 41). Nevertheless,ROS is also likely to contribute to the systemic inflammationduring doxorubicin treatment, as there were studies that demon-strated that ROS could significantly increase the level of TLRs, suchas 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 beenhanced by doxorubicin treatments in vivo or in vitro (44–46).Moreover, the upregulation of TLRs (including TLR4) seems acommon phenomenon in the inflammation process as there isevidence that LPS, oxidative LDL and CpG could all increase the

expression of some TLRs. Taken together, the upregulation ofTLR4 might be due to the inflammation reactions triggered bydoxorubicin and the release of some key inflammatorymediators(ROS, IL6, GM-CSF).

Our findings may provide insights for understanding theimmunotoxicology of other cancer chemotherapeutic agents,because systemic inflammation is commonly found in the cancerpatients undertaking chemotherapy, not only limited to anthra-cyclines (47). The side effects of chemotherapeutics were mostlyattributed to their toxicity to cells in normal organs and tissues(48, 49). The inflammatory reactions following the chemotherapywere always considered as a consequence, but not the reason, ofsuch cytotoxicity (50). Nevertheless, our present finding that theproduct of gut microflora leaking from the intestinal tract

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,andTLR4lps-delþDOX.B,serumconcentrationsofendotoxin inSham,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.






promoted systemic inflammation during the treatment withchemotherapeutics suggested that inflammation could directlyderive from chemotherapy and might play a significant role ininducing 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 thatthe gut microflora was the source of some key inflammatorymediators, such as endotoxin, the application of antibiotics totemporarily eliminate the bacteria in gutmay be a convenient andeffective way to reduce the chemotherapy related physiological

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 ratesin the Sham, TAK-242, TLR4lps-del, ShamþDOXþLPS, TAK-242þDOXþLPS, and TLR4lps-delþDOXþLPS groups (n¼ 10). B, serum levels of CK, LDH, BUN, and ALT inSham, 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.

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toxicity. Accordingly, several other strategies, inhibition of TLRsignal pathway and suppression of the mTOR signaling pathwayand restoration of intestine integrity, can be investigated aspotential approaches to prevent or alleviate adverse effects ofdoxorubicin and other chemotherapeutic agents.

In conclusion, our present study demonstrates that doxorubi-cin-caused disruption of the intestinal epithelium, which enablesthe leakage of endotoxin from gut microflora into circulation, isan 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 inboth the intestine and other tissues, which further amplified theglobal immunotoxicity of endotoxin. Either depletion of gutmicroflora or inhibition of TLR signaling proved effective inabolishing or alleviating DOX's toxicity in the animal models.This can be a general mechanism for further understanding andcontrol of systemic toxicity caused by a broader class of cancerchemotherapeutic agents.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Wang, C. Wang, J. Zhang, L. DongDevelopment of methodology: L. Wang, L. Dong

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Wang, Q. Chen, H. Qi, C. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Wang, H. Qi, C. Wang, L. DongWriting, review, and/or revision of the manuscript: L. Wang, Q. Chen,C. Wang, L. DongAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Wang, C. Wang, L. DongStudy supervision: C. Wang, J. Zhang, L. Dong

Grant SupportJ. 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 theProgram for New Century Excellent Talents in University (NCET-13-0272x);all authors received the support of the National Natural Science Foundationof China (31271013, 31170751, 31200695, 31400671, 51173076,91129712 and 81102489) and Nanjing University State Key Laboratory ofPharmaceutical Biotechnology Open Grant (02ZZYJ-201307). C. Wangacknowledges the funding supports from Macau Science and TechnologyFund (FDCT 048/2013/A2) and University of Macau (MYRG2015-00160-ICMS-QRCM).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 6, 2015; revised August 10, 2016; accepted September 6,2016; published OnlineFirst September 28, 2016.

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2016;76:6631-6642. Published OnlineFirst September 28, 2016.Cancer Res Lintao Wang, Qian Chen, Haixia Qi, et al. Upregulation of Toll-Like Receptor TLR4 and Endotoxin LeakageDoxorubicin-Induced Systemic Inflammation Is Driven by

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