Probiotics Prevent the Development of 1,2-Dimethylhydrazine (DMH)-Induced Colonic Tumorigenesis through Suppressed Colonic Mucosa Cellular Proliferation and Increased Stimulation of

Published: November 03, 2011

r 2011 American Chemical Society 13337 dx.doi.org/10.1021/jf203444d | J. Agric. Food Chem. 2011, 59, 13337–13345

ARTICLE

pubs.acs.org/JAFC

Probiotics Prevent the Development of 1,2-Dimethylhydrazine(DMH)-Induced Colonic Tumorigenesis through Suppressed ColonicMucosa Cellular Proliferation and Increased Stimulation ofMacrophagesNing-Ping Foo,†,X,^,4 Hui Ou Yang,† Hsueh-Huei Chiu,§ Hing-Yuen Chan,§ Chii-Cherng Liao,§

Chung-Keung Yu,*,‡,#,O and Ying-Jan Wang*,†

†Department of Environmental and Occupational Health, National Cheng Kung University, Medical College, Tainan, Taiwan§Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute, Hsinchu, Taiwan‡Department of Microbiology and Immunology, National Cheng Kung University, Medical College, Tainan, TaiwanXDepartment of Emergency Medicine, Chi-Mei Medical Center, Liouying, Tainan, Taiwan^Department of Early Childhood Caring and Education, Chung Hwa University of Medical Technology, Tainan, Taiwan4Department of Emergency Medicine, Ditmanson Medical Foundation Chiayi Christian Hospital, Chiayi, Taiwan#Infectious Disease and Signaling Research Center, National Cheng Kung University, Medical College, Tainan, TaiwanONational Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan

bS Supporting Information

ABSTRACT: Probiotics modulate immunity and inhibit colon carcinogenesis in experimental models, but these effects largelydepend on the bacterial strain, and the precise mechanisms are not well understood. Therefore, we studied the effect ofBifidobacterium longum and/or Lactobacillus gasseri on the development of 1,2-dimethylhydrazine (DMH)-induced colonicprecancerous lesions and tumors in mice while delineating the possible mechanisms involved. The results suggest that dietaryconsumption of probiotics (B. longum and L. gasseri) resulted in a significant inhibition of DMH-induced aberrant crypt foci (ACF)formation in male ICR mice. Long-term (24 weeks) dietary consumption of probiotics resulted in a reduction of colon tumormultiplicity and the size of the tumors. Administration of B. longum and L. gasseri suppressed the rate of colonic mucosa cellularproliferation in a manner correlating with the inhibition of tumor induction by DMH. In addition, the phagocytic activity ofperitoneal macrophages was significantly increased in the DMH-treated mice that were fed various doses of B. longum, but not withL. gasseri or combined probiotics (B. longum + L. gasseri). We also found that L. gasseri significantly increased the proliferation ofRAW264.7 macrophage cells through an increase in S phase DNA synthesis, which was related to the up-regulation of proliferatingcell nuclear antigen (PCNA) and cyclin A. Taken together, these results demonstrate the in vivo chemopreventive efficacy and theimmune stimulating mechanisms of dietary probiotics against DMH-induced colonic tumorigenesis.

KEYWORDS: probiotics, DMH, colon tumor, ACF, immunity

’ INTRODUCTION

Colorectal cancer is one of the major causes of cancer-relatedmortality in many developed countries. The development ofcolorectal cancer involves various genetic and molecular changesin cell proliferation, cell survival, differentiation, metastasis, andtumor angiogenesis.1,2 Progression of this disease is commonlycharacterized by histologically distinct steps: colonic crypt hy-perplasia, dysplasia, adenoma, adenocarcinoma, and distant meta-stasis.3 During this progression, the formation of aberrant cryptfoci (ACF) in the early stage may represent a histological bio-marker of colonic tumor development.4 Increases in the numberand multiplicity of ACF are associated with an increased risk forthe development of colorectal cancer.4,5 Despite the current un-derstanding of the processes andmechanisms involved in coloniccarcinogenesis, present therapies, including surgery, chemotherapy,radiotherapy, and molecular-targeted therapy, are still limited foradvanced tumors. Thus, a growing amount of attention has been

focused on the investigation of the potential of dietary substancesfor both prevention and control of colon cancer through chemo-preventive strategies.6

Epidemiological and experimental studies have demonstratedthat dietary habits are associated with variations, either increasesor decreases, in the risk of colon cancer.7,8 Intestinal bacteria playa significant role in the disease process, producing possiblecarcinogens and/or promoters of colon cancer.9 However, notall intestinal bacteria are deleterious; some bacteria are capable ofcompetitively inhibiting carcinogen and mutagen formation,altering overall metabolism, or adsorbing and removing toxic/mutagenic metabolites.10 Of special interest in this regard is the

Received: August 26, 2011Revised: November 2, 2011Accepted: November 3, 2011

13338 dx.doi.org/10.1021/jf203444d |J. Agric. Food Chem. 2011, 59, 13337–13345

Journal of Agricultural and Food Chemistry ARTICLE

beneficial effect of certain lactic acid-producing enterobacterialfood supplements, the so-called probiotics, in the prevention ofcolon cancer.11,12 Probiotics are live microorganisms that areused as dietary supplements with the aim of benefiting health byinfluencing the intestinal microbial balance.13 Among the majorgenera of colonic bacteria, bifidobacteria and lactobacilli arethought to have beneficial effects on the human host.14 In vivoanimal studies in rats have shown that supplementation withBifidobacterium longum reduces colon and liver carcinogenesisinduced by 2-amino-3-methylimidazo[4,5-f]quinoline and coloncancer induced by azoxymethane.15,16 Dietary supplements oflactobacilli also increase the latency of induction of experimentalcolon cancer in rats,17 suggesting that lactobacilli and bifidobac-teria may inhibit precancerous lesions and tumor development inanimal models.18

The mode of action of probiotics is complex and not com-pletely understood. Several mechanisms have been reported withrespect to colon cancer prevention, such as modifying gut pH,antagonizing pathogens through the production of antimicrobialand antibacterial compounds, stimulating immunomodulatorycells, or competing with pathogens for available nutrients, receptors,and growth factors.19�21 Among these mechanisms, the immu-nomodulating and immunostimulating properties of probioticshave been well-documented.22�24 Increasing evidence fromexperimental and human studies suggests that probiotics mod-ulate the host resistance against intestinal infections as well as anumber of immune cell functions.25,26 The immunostimulatoryeffect of probiotics also depends on the degree of contact withlymphoid tissues while the bacteria are transiently colonizing theintestinal lumen.27,28 Animal studies demonstrated that gut-associated lymphoid tissue is stimulated by these survivingprobiotics, resulting in enhanced production of cytokines andantibody.29

We have previously shown that dietary hydroxylated poly-methoxyflavones or chitosan could strongly reduce the ACF andcolorectal tumors in azoxymethane-treated mice.30,31 We alsodemonstrated that the molecular mechanisms involved in thechemoprevention of colonic tumorigenesis include antiprolife-ration, anti-inflammation, and antiangiogenesis.30,31 Here, weapplied a similar model to examine the chemopreventive effectsof probiotics in a DMH-induced colonic tumorigenesis model. Invivo antitumorigenic activities were evaluated using histopathol-ogy and immunohistochemistry for proliferating cell nuclearantigen (PCNA). Due to the importance of immunomodulatingand immunostimulating properties of probiotics, we then testedthe possibility that the antitumorigenic activities of probioticsmight act through the enhancement of phagocytosis and pro-liferation of macrophages in vitro.

’MATERIALS AND METHODS

Materials. The probiotics (B. longum BCRC 910051 and Lactoba-cillus gasseri BCRC 910197) were provided by the Bioresource Collec-tion and Research Center, Food Industry Research and DevelopmentInstitute, Taiwan. They were supplied as a freeze-dried powder in sealedsachets, which contained either B. longum powder of approximately5 � 109 colony-forming units (cfu)/g or L. gasseri powder of approxi-mately 1 � 1011 cfu/g. The bacteria were kept at �20 �C until used.The probiotics were resuspended in saline and supplied to differentgroups of mice throughout the study as described below.Animals.Male Institute of Cancer Research (ICR) mice at 5 weeks

of age were purchased from the Laboratory Animal Center, National

Cheng Kung University (Tainan, Taiwan). After 1 week of acclimation,animals were randomly distributed into control and experimentalgroups. All animals were housed in a controlled atmosphere (25 �C at50% relative humidity) and with a 12 h light/12 h dark cycle. Animalshad free access to food (AIN-76 diet) and water ad libitum before theexperiment. Experiment groups included group 1, skimmilk (0.1668 g/mL)in drinking water; group 2, high dose of B. longum (HB, 1.3 � 108

cfu/mouse) and L. gasseri (L, 2.8 � 109 cfu/mouse); group 3, DMH +skimmilk (0.1668 g/mL); group 4, DMH+ low dose B. longum (LB, 1.3�107 cfu/mouse); group 5, DMH + high dose B. longum (HB, 1.3 � 108

cfu/mouse); group 6, DMH + L. gasseri (L, 2.8 � 109 cfu/mouse); andgroup 7, DMH + 1/2 high dose B. longum (1/2HB, 6.6 � 107 cfu/mouse) and 1/2 L. gasseri (1/2 L, 1.4� 109 cfu/mouse). DMH (AldrichChemicals, Milwaukee, WI; 20 mg/kg in saline, pH 7.0) was given to theanimals weekly for 10 weeks. All experimental animal care and treatmentfollowed the guidelines set by the Institutional Animal Care and UseCommittee.Experimental Procedure. The experimental protocol for this

study is shown in Figure 1A. Briefly, mice were randomly divided intoseven groups of 5�18 animals each. At 6 weeks of age, mice in groups3�7 were given DMH at a dose of 20 mg/kg via an intramuscularinjection once a week for 10 weeks, whereas groups 1 and 2 receivedsaline injections. Group 1 mice were fed standard AIN-76 diet andcomposition as described before,32 whereas group 2�7 mice were fedAIN-76 diet and received intragastric injections of different doses of theprobiotics or control, continued until the end of the study. All animalswere sacrificed using CO2 asphyxiation at 15 or 24 weeks for evaluationof aberrant crypts or tumors in colonic tissues. The entire colons wereexcised, cut longitudinally, rinsed with phosphate-buffered saline (PBS),and fixed flat between sheets of filter paper with 10% buffered formalinovernight for ACF and tumor number evaluation or immunohisto-chemistry.Determination of ACF and Tumors. The colons, fixed as

described above, were placed in Ringer’s solution containing 0.2%methylene blue for 20�30 min. After washing with PBS, the stainedcolons were placed luminal side up on a glass slide and kept moist withRinger’s solution. ACF, characterized as large, dark stained, elevatedlesions, were counted using a light microscope at a magnification of100� in rectal (2 cm), middle (2 cm), and cecal (1 cm) areas.33 Sampleswere examined blindly by two observers. Before being fixed in formalin,suspected macroscopic lesions were measured with a caliper, and theirdimensions were calculated by multiplying the two main diameters ofeach lesion. Colon tumors were divided into three types: microadeno-mas, adenomas, and macroadenomas. Adenomas were classified accord-ing to the guidelines of Morson et al.34

Measurement of Mitotic Index. The colon was embedded inparaffin and examined histologically after staining with hematoxylin andeosin. For morphometric assessment, cells of one longitudinal half of acomplete, well-orientated crypt were counted, representing a cryptcolumn. At least 20 crypt columns were assessed. At 1000� magnifica-tion, the numbers of mitotic figures per crypt column were counted.Only cells in definite metaphase or anaphase were regarded as mitoticfigures. Themitotic index (MI = number ofmitotic cells/total number ofcells � 100) was calculated for the crypt.35

Measurement of Cell Proliferation. To assess the proliferativeactivity and the distribution of proliferating cells in the colonic crypts, astandard immunohistochemical assay for the proliferating cell nuclearantigen (PCNA) was performed. Briefly, deparaffinized sections wererehydrated in a graded series of ethanol from 100 to 50% and then todistilled water. The sections were incubated with the primary antibodyto PCNA (PC10; Boehringer Mannheim, Mannheim, Germany) at a1:300 dilution overnight at 4 �C. A Level 2 Ultra Streptavidin detectionsystem (Signet Laboratories) was used with biotinylated goat anti-mouse IgG as the secondary antibody. The slides were counterstained



for 3 min with hematoxylin. In all cases, an independent observer,blinded to the experimental treatment, determined the quantification ofproliferation cells. A total of 10 crypts close to the rectal area werecalculated. The scoring for cell proliferation was the mean of the totalnumber of proliferating cells in each counted crypt column.Isolation of Mouse Peritoneal Macrophages and Phago-

cytosis Assays. Peritoneal macrophages were collected from theperitoneal fluid as previously described.36 Briefly, mice were euthanizedusing CO2 anesthesia followed by cervical dislocation. The peritonealcavities were lavaged with 5 mL of cold Hank’s balanced salt solution(HBSS; Gibco) to collect peritoneal cells. The cells were washed twice inHBSS and resuspended in a 5% BSA solution at 2 � 105 cells/mL.Assessment of the phagocytic ability of peritoneal macrophages usingflow cytometry was based on the methods of Wan et al., with minormodifications.37 Briefly, 10 μL of fluorescein isothiocyanate-labeledEscherichia coli and 500 μL of peritoneal macrophages (2 � 105 cells/mL)were mixed and incubated at 37 �C for 2 h. Samples were then centri-fuged at 1500 rpm for 10 min, and the pellet was resuspended in 1 mL ofHBSS. The level of phagocytic activity was determined using a FACS-Calibur flow cytometer (Becton Dickinson Instruments).Measurement of IgA Antibody. An ELISA-based assay to

quantify mouse IgA in the intestinal fluid samples was performed usinga Mouse IgA ELISA Quantification Set (catalog no. E90-103, BethylLaboratories, Inc., Montgomery, TX).Cell Line and Cell Culture. RAW264.7 cells, derived frommurine

macrophages, were obtained from the American Type Culture Collec-tion (Rockville, MD). RAW264.7 cells were cultured in RPMI-1640supplemented with 10% endotoxin-free, heat-inactivated fetal calf serum(GIBCO, Grand Island, NY), 100 units/mL penicillin, and 100 μg/mLstreptomycin in a humidified incubator (37 �C, 5% CO2). Theprobiotics were added at the indicated doses. For control specimens,the same volume of H2O was added instead of the probiotics. Cellviability was estimated using the trypan blue exclusion assay as previouslydescribed.38

Cell Cycle Analysis. Cells were plated in 10 cm dishes. After serumstarvation with 0.04% FCS for 24 h to render them quiescent and tosynchronize their cell cycle activities, the cells were returned to mediawith 10% FCS with test material or control. RAW264.7 cells wereharvested at various time points. BrdU (10 μg/10 mL) was added 1 hbefore cell collection. Cells were then washed once with 1� PBS, fixedwith 75% alcohol, stored at 4 �C for 1 h, and centrifuged at 500g and10 �C for 10 min, at which point pellets were collected. After denatura-tion of the DNA with 1 mL of 2 N HCl/Triton X-100, pellets werecollected after centrifugation at 500g for 10 min. Cells then wereneutralized with 1 mL of 0.1 M Na2B4O7 3 10H2O (pH 8.5). Aftercentrifugation at 500g for 10 min, pellets were treated with 1 mL of 0.5%Tween 20/1% BSA/PBS to adjust cell concentration. Next, 20 μL ofanti-BrdU was added, and cells (1 � 106) were incubated at roomtemperature for 30 min and washed once with 1 mL of Tween 20/1%BSA/PBS; the pellets were collected using centrifugation at 500g for10 min. Flow cytometry analyses were performed after the addition ofpropidium iodide.Western Blot Analysis. Treated and untreated cells were rinsed

three times with ice-cold PBS pelleted at 800g for 5 min and lysed in500 μL of freshly prepared extraction buffer (10 mM Tris-HCl, pH 7,140 mM sodium chloride, 3 mM magnesium chloride, 0.5% (v/v)Nonidet P-40 (NP-40), 2 mM phenylmethanesulfonyl fluoride, 1% (w/v)aprotinin, and 5 mM dithiothreitol (DTT)), for 20 min on ice. Theextracts were centrifuged for 30 min at 10000g. Proteins were loaded at50 μg/lane on 12% (w/v) SDS�polyacrylamide gel (SDS-PAGE),blotted, and probed using specific antibodies, including PCNA, P27,cyclin A, cyclin B, cyclin E, cdk-2, and cdc-2 (Transduction Laboratories,Lexington, KY) and then detected using a chemiluminescence (ECL)detection system (Amersham Life Science, Arlington Heights, IL).

The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was used as the control for equal protein loading.Statistics. Data are expressed as the mean ( SD. Statistical signifi-

cancewas determinedusing either Student’s t test for comparison betweenmeans or one-way analysis of variance with a post hoc Dunnett’s test.Differences were considered to be significant when p < 0.05.

’RESULTS

Treatmentwith Probiotics Inhibited DMH-Induced ColonicACF and Tumor Formation.During the experiment (Figure 1A),all mice were monitored to investigate whether treatment withprobiotics caused any adverse effects. No significant alteration ofwater consumption or bodyweight gain was observed in any of thegroups ofmice given the probiotics during the experimental period(supplementary data, Figure S1). Toxic effects due to the DMHtreatment were not observed during the initiation of treatment(data not shown).The efficacy of probiotic (B. longum and L. gasseri) adminis-

tration on inhibiting DMH-induced ACF formation was deter-mined. Colonic ACF were identified and analyzed under a lightmicroscope after methylene blue staining. Table 1 summarizesthe incidence, the number of ACF per mouse (multiplicity), andthe distribution of ACF in the colon after 15 weeks of treatment.All mice developed ACF in the colon after DMH treatment. Themean number of ACFper colon in theDMHalone group (group 3)was 20.7( 5.6, whereasmice treated withDMH and fed differentdoses of the probiotics showed a significantly lower number ofACF. Roughly, a 25�30% reduction of ACF was found in theprobiotic administration groups (groups 4�7). The reducedACF occurred in the middle to cecal part of the colon. However,the incidence of ACF in the probiotic administration groups wassimilar to that in the DMH-treated group, with a 100% incidence.Moreover, we did not find a dose-dependent or synergistic effectof B. longum and L. gasseri in the inhibition of DMH-inducedACF formation.We further evaluated the anticolonic tumorigenesis activity of

long-term treatment of probiotics. Mice were fed B. longum orL. gasseri for 24 weeks, the colonic tissues were collected, andtumors in the colon were divided into three types: microadeno-mas, adenomas, and macroadenomas (Figure 1B), classifiedaccording to the guidelines of Morson et al.34 As shown inTable 2, the mean number of tumors in the DMH-treated groupwas 11.8 ( 2.5, whereas the numbers of tumors were decreasedin both B. longum (7.1( 1.7) and L. gasseri (7.9( 2.3) groups.Furthermore, we found a significant reduction in the numberof microadenomas and adenomas in the B. longum group(35 and 43%, respectively) and in the L. gasseri group(37 and 31%, respectively), as compared with the DMH-alonegroup. The incidence of colonic tumors was not significantlydifferent between the B. longum and L. gasseri groups. Inaddition, there was no difference in the tumor size betweenthe two groups.Probiotics Inhibited DMH-Induced Colonic Mucosa Cell

Proliferation. Due to the observation that the treatment withprobiotics inhibited DMH-induced colonic ACF and tumorformation, the markers of colonic mucosa cell proliferation werefurther examined to explore the mechanisms of probiotic inhibi-tion of colon tumorigenesis. As shown in Figure 2, the mitoticindex was significantly increased in the DMH-treated group ascompared with the control groups fed a normal or probiotic diet.Mice treated with DMH and fed different doses of B. longum



showed a significantly lower mitotic index compared with theDMH-treated group. However, the efficacies of L. gasseri and the1/2 (B. longum + L. gasseri) groups were less than that of theB. longum group, with there being no significant difference betweenthese groups. In addition, we also examined the PCNA labeling ofcells (a marker for cell proliferation) in the colonic crypts of micetreated with DMH and/or probiotics. The results shown inFigure 3 indicate that the number of proliferating cells/cryptswas significantly increased in the DMH-treated group as com-pared with the control groups. Mice treated with DMH and fed

both B. longum and L. gasseri showed a significantly lower numberof proliferating cells compared with the DMH-treated group.Phagocytic Function of Peritoneal Macrophages and the

IgA Antibody in theMurine Colon.The phagocytic activities ofthe peritoneal macrophages are shown in Figure 4. Peritonealmacrophages from the DMH-treated mice fed different doses ofB. longum for 15 weeks exhibited significantly greater phagocyticactivity than cells from control mice. However, the phagocyticactivities of macrophages from the DMH-treated mice fedL. gasseri and the 1/2 (B. longum + L. gasseri) were lower than thatof theB. longum group. There was no significant difference betweenthose groups and the control group. To examine whether changesin phagocytic activity observed in the DMH-treated mice fed B.longum were related to mucosal antibody response, IgA wasquantified using enzyme immunoassays. As shown in Figure 4C,the mucosa IgA levels tended to be higher in the DMH-treatedmice fed different doses of B. longum, but were not significantlydifferent from those in the control mice.Effects of Probiotics on the Incorporation of BrdU andCell

Cycle Regulatory Genes of RAW264.7 Cells. Due to theimportance of immunomodulating and immunostimulating pro-perties of probiotics, we examined their effects on the proliferationof macrophage in vitro. As shown in Figure 5A, L. gasseri at a doseof 106 cfu/mL significantly increased proliferation of RAW264.7macrophage cells after 24 h of treatment. However, there was nosignificant difference between the macrophage cells treated withB. longum and the control group. As L. gasseri increased thegrowth of macrophage cells more significantly than did B. longum,we chose L. gasseri for the following experiments.To confirm that L. gasseri could increase the S phase of macro-

phage cells, BrdU cell cycle analysis was conducted. As shown inFigure 5B, treatment of macrophage cells with L. gasseri at doses

Figure 1. (A) Experimental treatment protocol in DMH-induced colon carcinogenesis. (B) ACF, characterized as large, dark stained, elevated lesions,were counted using a light microscope at a magnification of 100�. Suspected macroscopic lesions were measured with a caliper, and their dimensionswere calculated by multiplying the two main diameters of each lesion. Tumors in the colon were divided into three types, thatis, microadenomas,adenomas, and macroadenomas. (C) Representative H&E stained colon adenoma (100�magnification). Histopathological examination of the tumorswas performed. The results showed adenoma but not adenocarcinoma in all tumors.

Table 1. Effects of Each Group on the Formation of AberrantCrypt Foci (ACF) in Colons Induced by 1,2-Dimethylhydra-zine (DMH)a

distribution of ACF

in the colon (%)

group

incidence

(%)

no. of

ACF rectal middle cecal

control (n = 5) 5 (0) 0 0 0 0

B. + L. (n = 5) 5 (0) 0 0 0 0

DMH (n = 8) 8 (100) 20.7 ( 5.6 # 25 50 25

DMH + L. B. (n = 9) 9 (100) 14.5 ( 3.3 * 34 44 22

DMH + H. B. (n = 9) 9 (100) 15.3 ( 4.8 * 30 45 25

DMH + L. (n = 8) 8 (100) 14.3 ( 3.2 * 27 50 23

DMH + B. + L. (n = 9) 9 (100) 15.8 ( 3.4 * 30 46 24aData are presented as the mean ( SD. One-way ANOVA. #,p < 0.05,the group was significantly different from control and probiotics treatedgroups, respectively. *, p < 0.05, the group was significantly differentfrom DMH-treated group.



of 106 cfu/mL significantly increased BrdU incorporation at 18 hafter treatment, as compared with control (control versusL. gasseri, 49.2 versus 63.8%, respectively). This result suggeststhat the L. gasseri-induced proliferative effect on macrophage cellsoccurs through an increase of S phase DNA synthesis. Thus, wenext examined how L. gasseri regulated the expression of cell cycleassociated genes by Western blot analysis. Figure 5C shows thattreatment of macrophage cells with 106 cfu/mL of L. gasserisignificantly increased the expression of PCNA and cyclin A at 12and 18 h. However, the expressions of cyclin B, cyclin E, Cdc-2,and Cdk-2 were not changed over time. These findings suggestedthat up-regulation of PCNA and cyclin Amight be involved in theL. gasseri-induced increase of S phase DNA synthesis in macro-phage cells.

’DISCUSSION

In the present study, we demonstrate that dietary consump-tion of probiotics (B. longum and L. gasseri) resulted in significantinhibition of DMH-induced ACF formation in male ICR mice.Long-term (24 weeks) dietary consumption of probiotics alsoresulted in a reduction of colon tumor multiplicity and the size of

the tumors without any noticeable adverse effects, indicatinglong-term safety and chemopreventive efficacy of dietary pro-biotics. These findings strongly suggest the chemopreventivepotential of dietary administration of the probiotics againstcolonic tumorigenesis. Although the mechanism of inhibitionof colon cancer by probiotics is not clear, we believe that thiseffect may proceed through diverse mechanisms, including alter-ation of physiological conditions such as the pH of the colonicmicroenvironment, as well as the immune response of the host.39

The probiotics and their metabolites may affect the mixedfunction oxygenases, especially the cytochrome P450 familymembers, which are believed to be involved in the conversionof DMH from proximate to ultimate carcinogen.40,41 In addition,protective effects could be achieved by probiotics interacting with(adsorbing, binding, and/or catabolizing) the initiating carcinogenin the intestinal lumen, thereby reducing potency and/or availabilityin the lumen.42

Our current study also analyzed the modifying effects ofprobiotics on colonic mucosal cell proliferation with MI andPCNA labeling to determine if the modulation of these cellularand biochemical events, relevant to colon carcinogenesis, couldbe effectively used to monitor the inhibition of colon cancer.

Table 2. Effects of Probiotics on the Formation of Colon Tumors in Mice Induced by 1,2-Dimethylhydrazine (DMH)*

classification of tumors

group incidence (%) no. of tumors/colon microadenoma adenoma macroadenoma

DMH (n = 8) 8 (100) 11.8 ( 2.5 5.7 ( 1.5 4.9 ( 1.6 1.3 ( 1.7

DMH + H. B. (n = 9) 9 (100) 7.1 ( 1.7 * 3.7 ( 1.1 * 2.8 ( 1.3 * 0.6 ( 0.9

DMH + L. (n = 8) 8 (100) 7.9 ( 2.3 * 3.6 ( 1.9 * 3.4 ( 1.9 * 0.8 ( 1.5*Data are presented as the mean ( SD. t tests. *, p < 0.05, the group was significantly different from DMH treated group.

Figure 2. Measurement of mitotic index. (A) The colon was embedded in paraffin and examined histologically after staining with hematoxylin andeosin. For morphometric assessment, cells of one longitudinal half of a complete, well-orientated crypt were counted, representing a crypt column. a,control; b, DMH alone; c, DMH + LB.; d, DMH + HB.; e, DMH + L.; f, DMH + (B. + L.). (B) At least 20 crypt columns were assessed. At 1000�magnification, the numbers of mitotic figures per crypt column were counted. Mitotic index (MI = number of mitotic cells/total number of cells� 100)was calculated for the crypt. Data are expressed as the mean( SE of five to nine samples. #, p < 0.05, compared with control and the probiotic groups. /,p < 0.05, compared with the DMH alone group. DMH combined with the B + L group treated with a half dose of HB. and L., respectively.



We found that the administration of B. longum and L. gasseri sup-pressed the rate of cellular proliferation in a manner correlatingwith inhibition of tumor induction by DMH. Hyperproliferationof colonic epithelial cells is induced by administration of bileacids, fatty acids, and certain carcinogens.43�45 Enhanced labeling

indices in patients with neoplastic lesions and in carcinogen-treated experimental animals have been observed in all sites ofthe colon and the uninvolved colonic mucosa. Lipkin46 andTerpstra et al.47 have suggested that the patterns and rates ofmucosal cell proliferation may be acceptable measures of colon

Figure 3. Measurement of cell proliferation. (A) To assess the proliferative activity and distribution of proliferating cells in the colonic crypts, standardimmunohistochemical for PCNAwas performed. a, control; b, DMH alone; c, DMH+LB.; d, DMH+HB.; e, DMH+ L.; f, DMH+ (B. + L.). (B) A totalof 10 crypts close to the rectal area were calculated. The scoring for cell proliferation was the mean of the total number of proliferating cells in eachcounted crypt column. Data are expressed as the mean( SE of five to nine samples. #, p < 0.05, compared with the control and the probiotic groups. /,p < 0.05, compared with the DMH alone treated group.

Figure 4. Measurement of phagocytic activity and IgA antibody. (A, B) The level of phagocytic activity of peritoneal macrophages was determined usinga FACSCalibur flow cytometer. a, control; b, probiotics alone; c, DMH alone; d, DMH + LB.; e, DMH +HB.; f, DMH + L.; g, DMH + (B. + L.). (C) AnELISA-based assay to quantify mouse IgA in intestinal fluid samples was performed using theMouse IgA ELISAQuantification Set. Data are expressed asthe mean ( SE of five to nine samples. #, p < 0.05, compared with the DMH alone treated group.



cancer risk and that modulation of cellular proliferation by agentsknown to prevent cancer formation might therefore serve as anintermediate end-point in cancer prevention trials. In animals,probiotic ingestion was shown to prevent carcinogen-inducedpreneoplastic lesions and colon tumors.48�50 The results of thepresent study, showing a significant inhibition of DMH-inducedcell proliferation that correlates with suppression of DMH-induced colon tumor multiplicity and tumor volume by dietaryB. longum and L. gasseri, are in line with these observations. Pre-vious studies have suggested that variations in apoptosis in thecolon mucosa may be correlated with carcinogenesis,51 higherapoptosis being associated with a lower risk of developing cancer.Surprisingly, apoptosis was not increased in the probiotic groupswhen compared with the control group (data not shown).Therefore, in the present study apoptosis in the normal mucosaand carcinogenesis were not correlated.

Optimally functioning innate and acquired immune systemsare essential for host defense against invading pathogens andspontaneously developing cancers.52 The results of our studiesdemonstrate that the phagocytic activity of peritoneal macro-phages was significantly increased in the DMH-treated mice feddifferent doses of B. longum but not L. gasseri or 1/2 (B. longum +L. gasseri). Enhanced phagocytic activity of peritoneal macro-phages from animals given dietary probiotics has previously been

demonstrated.53 It has also been shown that the level of enhance-ment depends on the strain, dose, and viability of the probioticsused.54 Augmentation of specific serum and/or mucosal anti-body responses has been reported, showing that different strainsof the probiotics vary greatly in their ability to enhance humoralimmunity.55 The mucosa IgA levels examined in the currentstudy tended to be higher in the DMH-treated mice fed differentdoses of B. longum, but were not significantly different from thosein the control mice. The precise mechanisms of probiotic stimula-tion of the immune system are not fully understood. It is possiblethat when present in large numbers, probiotics or their productsare able to gain access to the gut-associated lymphoid tissue orsystemic immune system.56 In addition, the intestinal epitheliumcontains a variety of immunoregulatory cells, and thus the probioticsand their productsmay exert their influence through these cells.57

Furthermore, the interaction between the probiotics or theirproducts and immunocompetent cells (such as macrophages)results in the secretion of cytokines that are known to have amultitude of effects on the functioning of the immune system.54

In our current studies, we also found that L. gasseri, at doses of106 cfu/mL, significantly increased proliferation of RAW264.7macrophage cells through increased S phase DNA synthesis.Although relatively little information is known for S phase control inthe mechanisms of cell cycle regulation, it has been demonstrated

Figure 5. (A) Effects of probiotics on the proliferation of RAW264.7 cells. Viable cells were determined using a trypan blue exclusion assay. Data are themean ( SE of three independent experiments. #, p < 0.05, versus controls. (B) Effects of probiotics on BrdU incorporation in RAW264.7 cells.Percentage of cells with BrdU incorporation was quantitated using a FACSCalibur flow cytometer. Data are the mean ( SE of three independentexperiments. #, p < 0.05, versus controls. (C) Time-dependent effect of probiotics on the expression of S phase regulatory proteins in RAW264 cells. Thecells were harvested, and the protein extracts were separated using SDS-PAGE. After electrophoresis, the proteins were transferred and probed with theproper dilution of the specific antibodies. GAPDH served as an internal control.



that PCNA, a key molecule involved in DNA replication machi-nery, has been associated with S phase regulation.58,59 The CDKwere recognized as key regulators of cell cycle progression throughtheir association with regulatory subunits called cyclins.60,61 Ourcurrent results demonstrating that L. gasseri treatment increasedexpression of PCNA as well as cyclin A suggested that these werethe key molecules involved in the probiotic-induced DNA syn-thesis in macrophage cells. These findings also suggested thatprobiotics could directly regulate immune cell growth, whichmight be one of the mechanisms involved in our immune stimu-lation findings in mice.

In summary, our study has supported earlier observations thatcertain probiotic bacteria are capable of diminishing colon tumordevelopment in a carcinogen-induced colonic tumorigenesis model.Inhibition of colon carcinogenesis is associated with the modula-tion of colonic cell proliferation, leading to suppressed colonicACF and tumor formation. Immunomodulating and immun-ostimulating properties, such as enhanced phagocytic activity ofperitoneal macrophages and increased proliferation of macro-phage cells, could play partial roles in the chemopreventiveefficacy of dietary probiotics in DMH-induced colonic tumor-igenesis. More probiotic studies are needed to examine furtherother possible mechanisms for their potential benefit.

’ASSOCIATED CONTENT

bS Supporting Information. Changes in (A) body weightgain and (B) water consumption in mice of experimental groupsthroughout the experimental period (Figure S1). This material isavailable free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*(Y.-J.W.) Postal address: Department of Environmental andOccupational Health, National Cheng Kung University MedicalCollege, 138 Sheng-Li Road, Tainan 70428, Taiwan. Phone: 886-6-235-3535, ext. 5804. Fax: 886-6-2752484. E-mail: [email protected]. (C.-K.Y.) Postal address: Department ofMicrobiology and Immunology, National ChengKungUniversity,Medical College, 138 Sheng-Li Road, Tainan, Taiwan 70101.E-mail: [email protected].

Funding SourcesWe appreciate the commission of this study by the Food IndustryResearch and Development Institute and the financial support(93-EC-17-A-17-R7-0525) of the Ministry of Economic Affairs,Republic of China, and the Chi Mei Medical Center, Liouying(CMNCKU9913).

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