lactobacillus salivarius ren inhibits rat oral cancer induced by 4 … · research article...

Research Article

Lactobacillus Salivarius REN Inhibits Rat Oral CancerInduced by 4-Nitroquioline 1-Oxide

Ming Zhang1, Fang Wang1, Lu Jiang1, Ruihai Liu3, Lian Zhang2, Xingen Lei4, Jiyou Li2, Jingli Jiang1,Huiyuan Guo1, Bing Fang1, Liang Zhao1, and Fazheng Ren1

AbstractDespite significant advances in cancer therapy, cancer-related mobility and mortality are still rising.

Alternative strategies such as cancer prevention thus become essential. Probiotics represent an emerging

option for cancer prevention, but studies are limited to colon cancers. The efficiency of probiotics in the

prevention of other cancers and the correlative mechanism remains to be explored. A novel probiotics

Lactobacillus salivarius REN (L. salivarius REN) was isolated from centenarians at Bama of China, which

showed highly potent antigenotoxicity in an initial assay. 4-nitroquioline 1-oxide (4NQO)-induced oral

cancermodel was introduced to study the anticancer activity of L. salivariusREN in vivo. The results indicated

that oral administration of probiotic L. salivarius REN or its secretions could effectively suppress 4NQO-

induced oral carcinogenesis in the initial and postinitial stage, and the inhibition was in a dose-dependent

manner. A significant decrease of neoplasm incidence (65%–0%)was detected in rats fedwith the high dose

of L. salivarius REN [5 � 1010 CFU/kg body weight (bw)/d]. In vivo evidences indicated that the probiotics

inhibited 4NQO-induced oral cancer by protecting DNA against oxidative damage and downregulating

COX-2 expression. L. salivarius REN treatment significantly decreased the expression of proliferating cell

nuclear antigen (PCNA) and induced apoptosis in a dose-dependent manner. Our findings suggest that

probiotics may act as potential agents for oral cancer prevention. This is the first report showing the

inhibitory effect of the probiotics on oral carcinogenesis. Cancer Prev Res; 6(7); 686–94. �2013 AACR.

IntroductionCancer is the leading cause of death worldwide. Despite

great progress in the recent years, the overall survive rate ofcancer is still low, especially for late-stage diseases (1).Cancer prevention remains an attractive strategy since theintroduction of cancer chemoprevention by Sporn (2) andWattenberg and colleagues (3). Among the various chemo-preventing agents, COX-2– selective inhibitors were themost promising candidates due to their efficiency in cancerprevention and low toxicity (4–6). However, prolongadministration of some COX-2–selective inhibitors wasfound to be associated with a significant increase in cardio-vascular events (7, 8), which resulted in the withdrawn ofVioxx from the market, followed by Bextra (valdecoxib).

These facts indicate that cancer prevention with pharma-cologic agents is associatedwithpractical challenges: benefitdoes not always overweigh risk, especially when long-termadministration is required for subjects with unknown riskfactors. New candidates are heavily investigated to improvethe benefit-risk ratio of cancer prevention agents.

The microbial genome project and related studies haverevealed that the gut microbiome may play a far moreimportant role in human health (9–12). Probiotics is agroup of health beneficial strains, which have been used forcenturies inhumanhistory all over theworld. Recent studiessuggested that probioticsmay represent an emerging optionfor cancer prevention and treatment. Different from che-mopreventive agents, probiotics have a well-establishedhistory in safety and may act via multiple mechanisms andpathways. Several groups have reported the potential ofusing probiotics in cancer prevention, but studies are main-ly focused on colon tumors in vitro and in vivo (13–15).Some general mechanisms have been proposed, such asantimicrobial effects against carcinogen-producing micro-organisms, antigenotoxic activities against internal andexternal carcinogens, and activation of gut mucosalimmune system (16–18). These results indicate that pro-biotics are effective agent for colon cancer inhibition. How-ever, the possibility of preventing other cancers and thecorrelative mechanisms remain to be explored.

Lactobacillus salivarius (L. salivarius) REN was a novelstrain isolated from fecal samples of healthy centenarians

Authors'Affiliations: 1KeyLaboratory of Functional Dairy, College of FoodScience and Nutritional Engineering, China Agricultural University; Depart-ment of 2Food Science and 3Peking University Cancer Hospital, School ofOncology, Peking University, Beijing, China; and Department of 4AnimalScience, Cornell University, Ithaca, New York

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

M. Zhang and F. Wang contributed equally to this work.

Corresponding Author: Fazheng Ren, China Agricultural University, P.O.Box 303,No. 17 TsinghuaEast Road,Beijing 100083,China. Phone: 86-10-62736344; Fax: 86-10-62736344; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-12-0427

�2013 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 6(7) July 2013686

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Published OnlineFirst May 8, 2013; DOI: 10.1158/1940-6207.CAPR-12-0427

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living in Bama (China), one of the 5 longevity villages in theworld. In aprevious study, itwas found thatL. salivariusRENcould decrease the 4-nitroquioline 1-oxide (4NQO)-induced genotoxicity in vitro (19). To further verify thesepotential anticarcinogensis effect in vivo, inhibitory activityof L. salivarius REN on 4NQO-induced oral carcinogenesiswas investigated in male F344 rats. In addition, the effect ofL. salivarius REN on DNA oxidative damage, expression ofCOX-2, and cell proliferation and apoptosis activity intongue tissue were assessed.

Materials and MethodsMicrobial strains and culture conditionsL. salivariusRENwas isolated from fecal samples collected

from centenarians who lived at Bama, Guanxi Province,China. This is one of the 5 longevity regions in the world.L. salivariusRENwas identified on the basis of the sequencesof 16S ribosomal RNA gene and the sugar fermentationpattern, using the API50 CH kit (BioM�erieux) and a com-puter-aided identification program (version 4.0, Bio-M�erieux). Three commercial strains, Lactobacillus rhamno-sus GG [LGG; American Type Culture Collection (ATCC)53103], Bifidobacterium animalis subsp. Lactis Bb12 (Bb12;ATCC 27536), and Lactobacillus casei Shirota (LCS; ATCC53103) were provided by Chr. Hansen Company andYakult Central Institute, respectively. All strains were cul-tured in Man–Rogosa–Sharpe (MRS) liquid medium at37�C until late log phase.

Preparation of microbial cells and its secretionsThe strain was cultured for 12 hours in MRS liquid

medium, harvested by centrifugation (4,000 g, 10minutes),washed twice, and adjusted to appropriate concentrations

in sterile saline (0.9% w/v) for 4NQOmetabolism test andoral administration.

The strain collected was then resuspended in deionizedwater to make a final concentration. This mixture wasincubated for 1 hour at room temperature, and the watersoluble secretion was harvested by centrifugation (4,000 g,10minutes, at room temperature). The concentration of thesecretion was defined as the concentration of the strain indeionized water for oral administration.

HPLC analysis of 4NQO metabolism in vitroThe microbial cells were adjusted to appropriate concen-

trations (1� 109 CFU/mL) in sterile saline (0.9%w/v), and4NQO was added at a final concentration of 20 ppm.Coincubation was protracted under anaerobic conditionsfor different period of time and with agitation (200 rpm,37�C). The supernatant was recovered by centrifugation,filtered using a 0.45 mmmembrane, and analyzed by high-performance liquid chromatography (HPLC) as describedin Wang and colleagues (19).

Animals, diets, and experimental procedureMale F344 rats, 4 weeks old (76 � 17 g, Vital River Lab

Animal Technology Co. Ltd.) were housed 5 in eachplastic cage, in an air-conditioned room with 12-hourlight/dark cycle. The temperature and relative humiditywere kept at 25 � 2�C and 50 � 10%. After 2 weeksquarantine, the rats were randomized into 12 groups. Allrats were fed with basal diet (Rodent Chow Product,KeAoXie Li feeds Co. Ltd.) and allowed free access todeionized water, with or without 4NQO (Sigma-Aldrich).A total of 170 rats were divided into 12 groups as shownin Fig. 1. From week 2, rats in groups 1 to 9 were given 20

Figure 1. Experimental protocol.

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Lactobacillus Salivarius REN Inhibits Oral Cancer

www.aacrjournals.org Cancer Prev Res; 6(7) July 2013 687




ppm 4NQO in drinking water for 8 weeks. Groups 2 to 7were administered with L. salivarius REN at 5 � 106, 5 �108, or 5 � 1010 colony-forming units (CFU)/kg bodyweight (bw) once per day, started either from week 1 orweek 9, until the end of experiment. Group 8 and 9 wereadministrated with secretion of L. salivarius REN (2 � 108

CFU/mL drinking water) w/o 4NQO (20 ppm). All of theanimals were sacrificed under ether anesthesia 32 weeksafter the commencement of the experiment. Tongues werecut approximately into 2 halves; one portion was used for8-OHdG assays and the other for histopathology andimmunohistochemistry assays. For histologic examina-tion, tissue and gross lesions were fixed in 10% bufferedformalin, embedded in paraffin blocks, and the histologicsections were stained with hematoxylin and eosin. Epi-thelial lesions (hyperplasia, dysplasia, and neoplasia) inthe oral cavity were diagnosed according to the criteriadescribed by Banoczy and Csiba and Kramer and collea-gues (20, 21). The diagnosis was randomized and singleblinded.

8-OHdG levels of tongue epithelium tissue8-OHdG concentration was determined using a Rat 8-

OHdG ELISA kit (Cosmo Bio), according to the manufac-turer’s instruction. ELISA was carried out in triplicate and ina blinded fashion.

ImmunohistochemistryThe paraffin sections were deparaffinized, rehydrated,

and subsequently incubated with a polyclonal primaryantibody (COX-2, Cell Signaling Technology; PCNA,NOVUS) at room temperature for 1 hour. The secondaryantibody was incubated for 15 minutes at room temper-ature, followed by incubation with strepavidin-POD(Dako) for 15 minutes. Antibody binding was visualizedusing AEC solution (Dako). The tissues were then coun-terstained by hematoxylin solution. Slides were subse-quently reviewed in a blinded fashion. The overall COX-2staining intensity was scored as follows (22, 23): �, nostaining; �, weakly positive (weaker than the immuno-positivity of macrophages) over less than 10% of the area;þ, weakly positive over more than 10% of the area; andþþ, strongly positive (equal or more than the immuno-positivity of macrophages) over more than 10% of thearea. For proliferating cell nuclear antigen (PCNA) immu-noreactivity, the index was determined by counting thepercentage of positive cells in the total cell counts indefined fields.

Terminal deoxynucleotidyl transferase–mediateddUTP nick end labeling (TUNEL) assay

Terminal deoxynucleotidyl transferase–mediated dUTPnick end labeling (TUNEL) assay was conducted with thecommercially DeadEnd Colorimetric TUNEL System (Pro-mega) according to the manufacturer’s instructions. Theapoptosis indexwas determined by counting the percentageof TUNEL-positive cells in the total cell counts in definedfields.

Statistical analysisStatistical analysis on the incidence of lesions and immu-

noreactivity COX-2were conducted using Fisher exact prob-ability test. The data of 8-OHdG levels and positive cellsratio in immunohistochemistry were analyzed using theStudent t test. The results were considered statistically sig-nificant if the P value was less than 0.001, 0.01, or 0.05.

ResultsL.salivariusREN-induced 4NQOdecomposition in vitro

The metabolism profiles of 4NQO by L. salivarius RENwere analyzed by HPLC, and the results were shown in Fig.2A. In the cases treatedwithother probiotics (LGG, Bb12, orLCS), only a single new peak (peak 2; Fig. 2A, b) wasobserved on HPLC in coordination with the disappearanceof the 4NQO peak (peak 4), regardless of time and strainconcentration. This newly formed peak was fairly stable,and no obvious decrease was observed during the experi-mental courses for all the other strains tested. This peak waslater confirmed to be 4-hydroxy aminoquinoline-l-oxide(HAQO; found Mþ1 ¼ 177.0, peak 1; Fig. 2B). However,when 4NQO (peak 3) was coincubated with L. salivariusREN, 2 new compounds (peaks 1 and 4) appeared (Fig. 2A,c–h), and the relative height of each peak is changing withtime. If incubation timewas long enough (12 hours), all theother peaks would disappear, leaving peak 1 as the onlyproduct (Fig. 2A, h). Liquid chromatography-mass spec-trometry (LC-MS) analysis of 2 new compounds was 4-aminoquinoline (4AQ; expected Mþ1 146.08) and 4-ami-noquinoline-1-oxide (4AQO; expected Mþ1 161.07).4HAQOwas the proximate carcinogen, whereas 4AQO and4AQ were either less or nontoxic compounds comparedwith 4NQO.

General observations of animalThe body weights of all 4NQO treatment groups, with or

without probiotics, were significantly lower than the con-trol group (except group 8; Supplementary Table S1). Incomparison of group 10 with 12, the oral administration ofL. salivarius REN or its secretions showed no significant signof any indexes tested, which indicates that this species is safefor the model animals.

Inhibition effect of L. salivariusRENon the incidence oftongue tumors and preneoplastic lesions

Neoplasms lesions were found in the tongues of alleffected rats but no metastasis was observed (Table 1).Thirteen out of 20 rats in the 4NQO-treated group (group1)were histologically diagnosed with either papilloma orsquamous cell carcinoma, accounting for 65% in total,whereas a significant decrease was observed in all the othergroups treated with 4NQO and L. salivarius REN (groups 2–7). The effects were in a dose-dependant manner, and thebest effectwas observed in group4,when thehighest dosageof L. salivariusREN (1010 cfu/kg bw)was given to the rats forthe whole experimental period. No incidence of neoplast-ismwas observed in this group, offering 100%protection tothe rats exposed to 4NQO. Reducing the dosages resulted in

Zhang et al.

Cancer Prev Res; 6(7) July 2013 Cancer Prevention Research688




decreased effects but the effect was still significantly differ-ent from group 1, even at the lowest dosage (106 cfu/kg bw,groups 2 and 5, P < 0.05). When L. salivarius RENwas givenafter the exposure to 4NQO (groups 5–7), the efficacy wasslightly decreased when compared with groups 2 to 4, butno significant difference was detected (P > 0.05). Ratstreated with the secretions of L. salivarius REN were alsowell protected from the 4NQO-induced lesions as shown ingroups 8 and 9. These results indicated that at least a part ofthe activity was in the microbial secretion.In addition to neoplasms, a number of hyperplasias

and dysplasias that are considered to be preneoplasticlesions for oral cancer were observed in groups 1 to 9 butnone in groups 10 to 12 (Table 1). Tongue squamous cellhyperplasia were classified into 2 categories of hyperpla-sia (simple and papillary) and 3 types of dysplasia (mild,moderate, and severe) according to the degree of cellularatypism. The incidences of total dysplasia in groups 2 to 9were significantly lower than that of group 1 (100%, P <0.05). Different from the results of neoplasm and dys-plasia, a relatively higher percentage of hyperplasia wasobserved in all the treated groups. However, the incidence

of hyperplasia in groups 4 and 8 was lower than group 1(100%, P < 0.05). These findings suggested that L. sali-varius REN was a powerful agent for oral neoplasmsprevention.

L. salivarius REN protected against the DNA oxidativedamage in vivo

Theoxidative damage in tonguemucosawasmeasuredby8-OHdG assay and shown in Fig. 2C. A high level of 8-OHdGwas observed in the 4NQO-treated group, indicatingthat DNA oxidative damage was involved in the initiationand progression of 4NQO-induced carcinogenesis. In mostof the L. salivarius REN and its secretions treatment groups(7 out of 8), the levels of 8-OHdG were significantlydecreased when compared with the control group (P <0.05 or P < 0.01). When L. salivarius REN was given for thewhole experimental period (group 2–4), the decrease ofthe 8-OHdG levels was more remarkable, whereas it hadless effects when given after 4NQO exposure (group 5–7,P < 0.05). In addition, the levels of 8-OHdG in group 4was not significantly different from the normal tissue(group 12), which suggested that long-term and high-

Figure 2. L.salivarius RENprotected against the oxidativedamage in vitro and in vivo. A, HPLCprofiles of 4NQO (20 ppm)coincubated with L.salivarius REN indifferent time. a, 4NQO alone for 3hours. b, 4-NQO coincubated withBb12, LGG, or LCS (1 � 109 cfu/mL)for 3 hours. c–h, 4-NQOcoincubatedwith L.salivarius REN (1 � 109

cfu/mL) for 1 to 12 hours. B, LC-MSof 4NQO treated with L.salivariusREN after 9 hours (g): Peak 1, AQ,expected MW145.07; Peak 2,HAQO, expected MW 176.06; Peak3, AQO, expected MW160.06. C,8-OHdG levels of tongue epitheliumtissue (mean � SD, n ¼ 10).�, P < 0.05 in a t test comparing with4NQO group; ��, P < 0.01 in a t testcomparing with 4NQO group.

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dosage administration of L. salivarius REN could effectivelyprevent DNA from oxidative damage in vivo.

L. salivarius REN downregulated the expression ofCOX-2 in vivo

Statistical results for COX-2 immunohistochemical stain-ing of rat tongue mucosa are summarized in Table 2.Representative immunohistochemical expression of COX-2 in different groupswere shown in Fig. 3. In normal tonguetissues, slight immunoreactivity for COX-2 was observed intongue squamous epithelium. In group 1 (4NQO treatment

alone), COX-2 expression was prominent in tongue neo-plasms. Oral administration of L. salivarius REN (group 3and 7) significantly decreased COX-2 immunoreactivity inthe tongue neoplasms when compared with group 1 (P <0.05). In addition, oral administration of L. salivarius RENsignificantly increased the incidence of preneoplasticlesions with weak (group 4, 6, 7) or negative (group 2, 4,8) immunoreactivity of COX-2 (P < 0.05 or P < 0.01). Theseresults indicated that the immunoreactivity of COX-2 wasdownregulated by administration of L. salivarius REN inboth neoplastic and preneoplastic tissues.

Table 1. Incidence of tongue neoplasms and preneoplastic lesions of rats during or after 4-NQO exposure

Neoplasms Dysplasia HyperplasiaGroupNo. Treatment

No. ofrats(final) Papilloma SCC Total Mild Moderate Severe Total Simple Papillary Total

1 4NQO Alone 20 1 (5%) 12 (60%) 13 (65%) 2 (10%) 1 (5%) 17 (85%) 20 (100%) 20 (100%) 12 (60%) 20 (100%)2 4NQOþ106cfu/kg REN 15 0 (0%) 5 (33%) 5 (33%)c 4 (27%) 1 (7%) 5 (33%)b 10 (67%)b 14 (93%) 7 (47%) 14 (93%)3 4NQOþ108 cfu/kg REN 15 1 (7%) 0 (0%)a 1 (7%)b 2 (13%) 1 (7%) 1 (7%)a 4 (27%)a 13 (87%) 7 (47%) 13 (87%)4 4NQOþ1010 cfu/kg REN 15 0 (0%) 0 (0%)a 0 (0%)a 2 (13%) 1 (7%) 0 (0%)a 3 (20%)a 9 (60%)b 9 (60%) 9 (60%)b

5 4NQO!106 cfu/kg REN 15 0 (0%) 4 (27%) 4 (27%)c 1 (7%) 1 (7%) 4 (27%)a 6 (40%)a 13 (87%) 10 (67%) 13 (87%)6 4NQO!108 cfu/kg REN 15 0 (0%) 3 (20%)c 3 (20%)c 1 (7%) 1 (7%) 3 (20%)a 5 (33%)a 12 (80%) 11 (73%) 14 (93%)7 4NQO!1010 cfu/kg REN 15 0 (0%) 1 (7%)b 1 (7%)b 2 (13%) 1 (7%) 1 (7%)a 4 (27%)a 11 (73%)c 7 (47%) 12 (80%)8 4NQOþSecretions 15 0 (0%) 2 (13%)b 2 (13%)b 2 (13%) 2 (13%) 2 (13%)c 6 (40%)a 11 (73%)c 6 (40%) 11 (73%)c

9 4NQO!Secretions 15 0 (0%) 2 (13%)b 2 (13%)b 5 (33%) 2 (13%) 2 (13%)a 9 (60%)b 12 (80%) 7 (47%) 12 (80%)10 108 cfu/kg REN 10 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)11 Secretions 10 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)12 No treatment 10 0 (0%) 0 (0%) 0 (0%) 0 0%) 0 (0%) 0 (0%) 0 (0%) 0( 0%) 0 (0%) 0 (0%)

aSignificantly different from group 1 by Fisher exact probability test (P < 0.001).bSignificantly different from group 1 by Fisher exact probability test (P < 0.01).cSignificantly different from group 1 by Fisher exact probability test (P < 0.05).

Table 2. COX-2 immunohistochemistry staining of tongue lesions

Preneoplastic lesions Neoplasms

No. of lesions with COX-2antibody staining (%)

No. of lesions with COX-2antibody staining (%)

GroupNo. Treatment

No. of lesionsexamined � � þ þþ

No. of lesionsexamined � � þ þþ

1 4NQO Alone 21 0 (0) 6 (29) 9 (43) 6 (29) 21 0 (0) 1 (5) 4 (19) 16 (76)2 4NQOþ106 cfu/kg REN 24 6 (25)a 9 (38) 6 (25) 3 (13) 13 0 (0) 1 (8) 4 (31) 8 (62)3 4NQOþ108 cfu/kg REN 30 2 (7) 10 (33) 11 (37) 7 (23) 3 0 (0) 0 (0) 3 (100)a 0 (0)a

4 4NQOþ1010 cfu/kg REN 18 4 (22)a 12 (67)a 0 (0)b 2 (11) 0 0 0 0 05 4NQO!106 cfu/kg REN 21 4 (19) 7 (33) 6 (29) 4 (19) 12 0 (0) 2 (16) 4 (33) 6 (50)6 4NQO!108 cfu/kg REN 22 4 (18) 16 (73)b 2 (9)a 0 (0)b 8 0 (0) 2 (25) 2 (25) 4 (50)7 4NQO!1010 cfu/kg REN 19 3 (16) 14 (74)b 2 (11)a 0 (0)a 3 0 (0) 1 (33) 2 (67) 0 (0)a

8 4NQOþSecretions 22 8 (36)b 10 (45) 4 (18) 0 (0)b 5 0 (0) 0 (0) 2 (40) 3 (60)9 4NQO!Secretions 23 4 (17) 8 (35) 11 (48) 0 (0)b 5 0 (0) 1 (20) 2 (40) 2 (40)

NOTE: �, negative; �, weakly positive; þ, moderate positive; þþ, strong positive.aSignificantly different from group 1 by Fisher exact probability test (P < 0.001).bSignificantly different from group 1 by Fisher exact probability test (P < 0.01).

Zhang et al.





L. salivarius REN modulated the proliferation andapoptosis activities in vivoRepresentative immunohistochemical expression of

PCNA and TUNEL in different groups were shown in Fig.3. Results for PCNA immunohistochemical staining of ratnonlesional tongue squamous epithelium were summa-rized in Table 3. A high level of PCNA expression(31.8%) was observed in the 4NQO-treated group. How-ever, in most of the L. salivarius REN and its secretionstreatment groups (7 out of 8), the levels of PCNA expressionwere significantly decreased when compared with the con-trol group (P < 0.05 or P < 0.01). The results indicated thatthe proliferative activity of tongue squamous epitheliumcells was significantly inhibited by L. salivarius REN.Results for TUNEL staining of rat lesional tongue squa-

mous epithelium were also summarized in Table 3. Ingroup 1 (4NQO treatment alone), apoptosis index had nosignificant difference when compared with the controlgroup (P > 0.05). In most of the L. salivarius REN and itssecretion treatment groups (7 out of 8), the levels of apo-ptosis index were significantly decreased when comparedwith the control group (P < 0.05 or P < 0.01). However, nosignificant induction of apoptosis was observed in L. sali-varius REN treatment alone, which implied that L. salivariusREN had no negative effect on normal cells.

DiscussionThe results in the current study indicated that oral admin-

istration of probiotic L. salivariusRENor its secretions couldeffectively suppress 4NQO-induced oral carcinogenesis andthe inhibition was in a dose-dependent manner. Interest-ingly, no SCC or papilloma was detected in rats fed withhigh dose of L. salivarius REN (group 4). The strain coulddecompose 4NQO in vitro, resulted in less toxic com-

pounds. The strain protectedDNAagainst oxidative damageinduced by 4NQO in vivo, significantly downregulatedCOX-2/PCNA expression, and induced apoptosis in adose-dependent manner. These results clearly suggestedthat L. salivarius REN could act as a very good candidatefor cancer prevention agent.

Oral carcinogenesis is a multistage, slow progress. Theinitiation, proliferation, and development of tumorsinvolved many mechanism and pathways. Mutagenicity byDNA oxidative damage, overexpression of COX-2 and pros-taglandin, resistance to apoptosis, continuous proliferationmay all contribute to oral carcinogenesis. The interventionof these multistage processes by modulating different intra-cellular signaling pathways and mechanisms provides amolecular basis for chemoprevention with probiotics.

4NQO-induced oral cancer model is the best amongstthe available experimental systems for sequential studies oforal carcinogenesis (24). The morphology and the molec-ular events showed that 4NQO-induced tumors resemblehuman squamous cell carcinoma of the head and neckinduced by tobacco or other oral carcinogens (25). Thecarcinogenic action of 4NQO is thought to be initiated byenzyme reduction in vivo to give 4HAQO. The latter is aproximate carcinogen, which forms adduct with DNA tocause oxidative damage andmutation, which is similar withthe carcinogenic progress caused by tobacco and smoking(25). Our assay revealed that L. salivarius REN and itssecretion could effectively reduce the toxicity of 4NQO viasequential decomposition of 4HAQO to give less or no toxicproduct(s) 4AQO and 4AQ (Fig. 2A). This set a very solidbase for further investigation to evaluate the potential of L.salivarius REN as cancer prevention agents. Therefore, wechose 4NQO model to further investigate these effects invivo.

Figure 3. Immunohistochemistry of COX-2, PCNA, and TUNEL staining in tongue lesions induced by administration of 4-NQO. a–d, showed representativeimmunohistochemistry of COX-2 in preneoplastic lesions with different immunopositivity. a, normal tongue epithelium, COX-2 expression wasnegative, �. b, hyperplasia lesion, COX-2 was expressed weakly, �. c, dysplasia lesion, COX-2 expression was moderately positive, þ. d, SCC, COX-2expression was strongly positive, þþ. e and f, showed representative immunohistochemistry of PCNA. e, normal tongue epithelium, PCNA expressionwas weakly positive. f, hyperplasia lesion, PCNA expression was positive. g and h, showed representative immunohistochemistry of TUNEL. g, hyperplasialesion, TUNEL expression was positive. h, normal tongue epithelium, TUNEL expression was negative.






Oxidative DNA damage is a preliminary step during rattongue carcinogenesis induced by 4NQO (26). Kohda andcolleagues concluded that the 8-OHdG,whichwas regardedas amajormarker ofDNAoxidative damage (27, 28), canbeattributed to 4HAQO-DNA adducts in 4NQO animal mod-el. High level of 8-OHdG reflects the increased oxidativedamage of DNA (28). Several reports have suggested thatprobiotics could be efficacious in reduction ofDNAdamagecaused by carcinogenic chemical agents in vitro and in vivo(29, 30). However, this mechanism had not been proveneffective in cancer prevention. In the current study, L.salivarius REN or its secretions could dose dependentlydecrease the levels of 8-OHdG. The reduction of 8-OHdGlevels was extremely obvious in group 4, whichwas fedwithhighdose of L. salivariusREN(1010 cfu/kg bw) togetherwith4NQO exposure. These results are in concordant with the4NQO metabolism profile observed in vitro (Fig. 2A).

However, it must be pointed out that a significantneoplasm inhibitory effect was also observed significantlyin groups treated with L. salivarius REN after 4NQOadiminstration, where 4NQO metabolism by L. salivariusREN was not feasible. Hence, there must be other pre-vention mechanisms involved, in addition to 4NQOdecomposition.

Increased COX-2 expression has been associated withinflammation and the development of cancers (31–34).Some COX-2–specific inhibitors can prevent these condi-tions, at least in part, via inhibition of the expression ofCOX-2 (22, 23, 35). These findings suggested that inhibi-tion of COX-2 may be another mechanism to suppress thedevelopment of 4NQO-induced oral carcinogenesis. Sev-eral species of probiotics were also proven to reduce theinflammation response by downregulating the COX-2expression in vivo (36, 37). However, this mechanism had

not been proven effective in cancer prevention. In thecurrent study, oral administration of REN or its secretionssignificantly reduced the overexpression of COX-2 in4NQO-treated rats (Table 3).

This result is very encouraging and yet conflicting. Asmentioned previously, prolong usage of COX-2 inhibitorshave been reported to be associated with cardiovascularside effects (7, 8, 38). These side effects have been attrib-uted to the prevented production of the provasodilatorprostacyclins in the endothelial and smooth muscle cellsas a consequence of COX-2 inhibition (39). This impliesthat certain amount of COX-2 is expressed in these tissuesand may play important physiologic functions (40). Howcould REN function via the same mechanism as COX-2inhibitors but without the side effects, which has beenproven by the strains’ origin? A possible explanationcould be that probiotics would downregulate the over-expressed COX-2 rather than inhibit it. As part of the gutmicrobial composition, probiotics may benefit from aprolong supply of moderate level of COX-2–inhibitingactivity, but this fine-tuning mechanism remains to beexplored.

Cell proliferation and apoptosis play important roles inmultistage carcinogenesis. It has been found that someprobiotics could also retard the growth of tumor volumeand enhance the apoptosis of tumor cells, but studies weremainly focused on colon tumors (41–43). Our results(Table 3) showed a significant antiproliferation and proa-poptotic effect that correlates with suppression of 4NQO-induced oral cancer incidence by dietary L. salivarius RENare in line with these observations. No significant inductionof apoptosis was observed in L. salivarius REN treatmentalone, which implied that L. salivarius REN had no negativeeffect on normal cells.

Table 3. PCNA, TUNEL-positive cell ratio (%) in the tongue squamous epithelium

Nonlesional area Lesional area

Group No. Treatment PCNA TUNEL

1 4NQO 31.84 � 5.08d 5.82 � 3.282 4NQOþ5 � 106cfu/kg REN 26.25 � 6.38 9.60 � 3.763 4NQOþ5 � 108 cfu/kg REN 19.53 � 9.35b 14.42 � 3.03b,c

4 4NQOþ5 � 1010 cfu/kg REN 18.91 � 6.89b 12.22 � 2.58a,d

5 4NQO!5 � 106 cfu/kg REN 31.96 � 2.68d 9.75 � 1.33a,c

6 4NQO!5 � 108 cfu/kg REN 18.69 � 4.72b 14.76 � 1.68b,d

7 4NQO!5 � 1010 cfu/kg REN 22.53 � 3.54b 14.64 � 2.84b,d

8 4NQOþSecretions 23.65 � 2.23b,c 16.56 � 3.26b,d

9 4NQO!Secretions 19.62 � 4.36b 13.43 � 2.95b,d

10 5 � 108 cfu/kg REN 19.92 � 1.74b 7.05 � 3.7011 Secretions 21.14 � 2.57b 7.71 � 4.0212 Control 19.83 � 2.39b 5.48 � 3.22

aSignificantly different from group 1 by Student t test (P < 0.05).bSignificantly different from group 1 by Student t test (P < 0.01).cSignificantly different from group 12 by Student t test (P < 0.05).dSignificantly different from group 12 by Student t test (P < 0.01).

Zhang et al.





In conclusion, our results indicate that L. salivarius RENand its secretions have significant inhibitory effects on oralcarcinogenesis induced by 4NQO, and such effects may berelated to the metabolism of 4NQO, prevention of DNAfromoxidedamage, suppressionof cell proliferation, induc-tion of cell apoptosis, and/or downregulation of COX-2expression. This is the first report showing the inhibitoryeffect of the probiotics on oral carcinogenesis, to theauthors’ knowledge. Take into account the safety profile ofprobiotics in general, L. salivarius REN could act as a verygood candidate for preventive agent against oral cancer,although clinical investigations are needed.

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

Authors' ContributionsConception and design: M. Zhang, F. Wang, L. Jiang, R.H. Liu, H. Guo,F. RenDevelopment of methodology: M. Zhang, F. Wang, J. Li, F. Ren

Acquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Zhang, J. Jiang, L. ZhaoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):M. Zhang, F. Wang, L. Jiang, R.H. Liu, X.Lei, J. LiWriting, review, and/or revision of the manuscript: M. Zhang, L. Jiang,R.H. Liu, X. Lei, J. Li, F. RenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Zhang, R.H. Liu, L. Zhang,J. Jiang, B. Fang, L. Zhao, F. RenStudy supervision: L. Jiang, R.H. Liu, L. Zhang, F. Ren

Grant SupportThis work was supported by the Ministry of Science and Technology of

China (2011AA100903, 2012BAD28B08), Beijing Excellent PHD ThesisProgram (YB20101001902), and Beijing Science and Technology Project(D101105046010001).

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 October 24, 2012; revised March 6, 2013; accepted April 22,2013; published OnlineFirst May 8, 2013.

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Zhang et al.





2013;6:686-694. Published OnlineFirst May 8, 2013.Cancer Prev Res Ming Zhang, Fang Wang, Lu Jiang, et al. 4-Nitroquioline 1-Oxide

REN Inhibits Rat Oral Cancer Induced byLactobacillus Salivarius

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