ARTICLE pubs.acs.org/JAFC
Probiotics Prevent the Development of 1,2-Dimethylhydrazine (DMH)-Induced Colonic Tumorigenesis through Suppressed Colonic Mucosa Cellular Proliferation and Increased Stimulation of Macrophages Ning-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, Taiwan X Department of Emergency Medicine, Chi-Mei Medical Center, Liouying, Tainan, Taiwan ^ Department of Early Childhood Caring and Education, Chung Hwa University of Medical Technology, Tainan, Taiwan 4 Department of Emergency Medicine, Ditmanson Medical Foundation Chiayi Christian Hospital, Chiayi, Taiwan # Infectious Disease and Signaling Research Center, National Cheng Kung University, Medical College, Tainan, Taiwan O National 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 largely depend on the bacterial strain, and the precise mechanisms are not well understood. Therefore, we studied the effect of Bifidobacterium longum and/or Lactobacillus gasseri on the development of 1,2-dimethylhydrazine (DMH)-induced colonic precancerous lesions and tumors in mice while delineating the possible mechanisms involved. The results suggest that dietary consumption 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 tumor multiplicity and the size of the tumors. Administration of B. longum and L. gasseri suppressed the rate of colonic mucosa cellular proliferation in a manner correlating with the inhibition of tumor induction by DMH. In addition, the phagocytic activity of peritoneal macrophages was significantly increased in the DMH-treated mice that were fed various doses of B. longum, but not with L. gasseri or combined probiotics (B. longum + L. gasseri). We also found that L. gasseri significantly increased the proliferation of RAW264.7 macrophage cells through an increase in S phase DNA synthesis, which was related to the up-regulation of proliferating cell nuclear antigen (PCNA) and cyclin A. Taken together, these results demonstrate the in vivo chemopreventive efficacy and the immune 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-related mortality in many developed countries. The development of colorectal cancer involves various genetic and molecular changes in cell proliferation, cell survival, differentiation, metastasis, and tumor angiogenesis.1,2 Progression of this disease is commonly characterized by histologically distinct steps: colonic crypt hyperplasia, dysplasia, adenoma, adenocarcinoma, and distant metastasis.3 During this progression, the formation of aberrant crypt foci (ACF) in the early stage may represent a histological biomarker of colonic tumor development.4 Increases in the number and multiplicity of ACF are associated with an increased risk for the development of colorectal cancer.4,5 Despite the current understanding of the processes and mechanisms involved in colonic carcinogenesis, present therapies, including surgery, chemotherapy, radiotherapy, and molecular-targeted therapy, are still limited for advanced tumors. Thus, a growing amount of attention has been r 2011 American Chemical Society
focused on the investigation of the potential of dietary substances for both prevention and control of colon cancer through chemopreventive strategies.6 Epidemiological and experimental studies have demonstrated that dietary habits are associated with variations, either increases or decreases, in the risk of colon cancer.7,8 Intestinal bacteria play a significant role in the disease process, producing possible carcinogens and/or promoters of colon cancer.9 However, not all intestinal bacteria are deleterious; some bacteria are capable of competitively 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, 2011 Revised: November 2, 2011 Accepted: November 3, 2011 Published: November 03, 2011 13337
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Journal of Agricultural and Food Chemistry beneficial effect of certain lactic acid-producing enterobacterial food supplements, the so-called probiotics, in the prevention of colon cancer.11,12 Probiotics are live microorganisms that are used as dietary supplements with the aim of benefiting health by influencing the intestinal microbial balance.13 Among the major genera of colonic bacteria, bifidobacteria and lactobacilli are thought to have beneficial effects on the human host.14 In vivo animal studies in rats have shown that supplementation with Bifidobacterium longum reduces colon and liver carcinogenesis induced by 2-amino-3-methylimidazo[4,5-f]quinoline and colon cancer induced by azoxymethane.15,16 Dietary supplements of lactobacilli also increase the latency of induction of experimental colon cancer in rats,17 suggesting that lactobacilli and bifidobacteria may inhibit precancerous lesions and tumor development in animal models.18 The mode of action of probiotics is complex and not completely understood. Several mechanisms have been reported with respect to colon cancer prevention, such as modifying gut pH, antagonizing pathogens through the production of antimicrobial and antibacterial compounds, stimulating immunomodulatory cells, or competing with pathogens for available nutrients, receptors, and growth factors.19 21 Among these mechanisms, the immunomodulating and immunostimulating properties of probiotics have been well-documented.22 24 Increasing evidence from experimental and human studies suggests that probiotics modulate the host resistance against intestinal infections as well as a number of immune cell functions.25,26 The immunostimulatory effect of probiotics also depends on the degree of contact with lymphoid tissues while the bacteria are transiently colonizing the intestinal lumen.27,28 Animal studies demonstrated that gutassociated lymphoid tissue is stimulated by these surviving probiotics, resulting in enhanced production of cytokines and antibody.29 We have previously shown that dietary hydroxylated polymethoxyflavones or chitosan could strongly reduce the ACF and colorectal tumors in azoxymethane-treated mice.30,31 We also demonstrated that the molecular mechanisms involved in the chemoprevention of colonic tumorigenesis include antiproliferation, anti-inflammation, and antiangiogenesis.30,31 Here, we applied a similar model to examine the chemopreventive effects of probiotics in a DMH-induced colonic tumorigenesis model. In vivo antitumorigenic activities were evaluated using histopathology and immunohistochemistry for proliferating cell nuclear antigen (PCNA). Due to the importance of immunomodulating and immunostimulating properties of probiotics, we then tested the possibility that the antitumorigenic activities of probiotics might act through the enhancement of phagocytosis and proliferation of macrophages in vitro.
’ MATERIALS AND METHODS Materials. The probiotics (B. longum BCRC 910051 and Lactobacillus gasseri BCRC 910197) were provided by the Bioresource Collection and Research Center, Food Industry Research and Development Institute, Taiwan. They were supplied as a freeze-dried powder in sealed sachets, which contained either B. longum powder of approximately 5 109 colony-forming units (cfu)/g or L. gasseri powder of approximately 1 1011 cfu/g. The bacteria were kept at 20 C until used. The probiotics were resuspended in saline and supplied to different groups 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
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Cheng Kung University (Tainan, Taiwan). After 1 week of acclimation, animals were randomly distributed into control and experimental groups. All animals were housed in a controlled atmosphere (25 C at 50% relative humidity) and with a 12 h light/12 h dark cycle. Animals had free access to food (AIN-76 diet) and water ad libitum before the experiment. Experiment groups included group 1, skim milk (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 + skim milk (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); and group 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 (Aldrich Chemicals, Milwaukee, WI; 20 mg/kg in saline, pH 7.0) was given to the animals weekly for 10 weeks. All experimental animal care and treatment followed the guidelines set by the Institutional Animal Care and Use Committee. Experimental Procedure. The experimental protocol for this study is shown in Figure 1A. Briefly, mice were randomly divided into seven groups of 5 18 animals each. At 6 weeks of age, mice in groups 3 7 were given DMH at a dose of 20 mg/kg via an intramuscular injection once a week for 10 weeks, whereas groups 1 and 2 received saline injections. Group 1 mice were fed standard AIN-76 diet and composition as described before,32 whereas group 2 7 mice were fed AIN-76 diet and received intragastric injections of different doses of the probiotics or control, continued until the end of the study. All animals were sacrificed using CO2 asphyxiation at 15 or 24 weeks for evaluation of aberrant crypts or tumors in colonic tissues. The entire colons were excised, cut longitudinally, rinsed with phosphate-buffered saline (PBS), and fixed flat between sheets of filter paper with 10% buffered formalin overnight for ACF and tumor number evaluation or immunohistochemistry. 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 stained colons were placed luminal side up on a glass slide and kept moist with Ringer’s solution. ACF, characterized as large, dark stained, elevated lesions, were counted using a light microscope at a magnification of 100 in rectal (2 cm), middle (2 cm), and cecal (1 cm) areas.33 Samples were examined blindly by two observers. Before being fixed in formalin, suspected macroscopic lesions were measured with a caliper, and their dimensions were calculated by multiplying the two main diameters of each lesion. Colon tumors were divided into three types: microadenomas, adenomas, and macroadenomas. Adenomas were classified according to the guidelines of Morson et al.34 Measurement of Mitotic Index. The colon was embedded in paraffin and examined histologically after staining with hematoxylin and eosin. For morphometric assessment, cells of one longitudinal half of a complete, well-orientated crypt were counted, representing a crypt column. At least 20 crypt columns were assessed. At 1000 magnification, the numbers of mitotic figures per crypt column were counted. Only cells in definite metaphase or anaphase were regarded as mitotic figures. The mitotic index (MI = number of mitotic cells/total number of cells 100) was calculated for the crypt.35 Measurement of Cell Proliferation. To assess the proliferative activity and the distribution of proliferating cells in the colonic crypts, a standard immunohistochemical assay for the proliferating cell nuclear antigen (PCNA) was performed. Briefly, deparaffinized sections were rehydrated in a graded series of ethanol from 100 to 50% and then to distilled water. The sections were incubated with the primary antibody to PCNA (PC10; Boehringer Mannheim, Mannheim, Germany) at a 1:300 dilution overnight at 4 C. A Level 2 Ultra Streptavidin detection system (Signet Laboratories) was used with biotinylated goat antimouse IgG as the secondary antibody. The slides were counterstained 13338
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Journal of Agricultural and Food Chemistry for 3 min with hematoxylin. In all cases, an independent observer, blinded to the experimental treatment, determined the quantification of proliferation cells. A total of 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 each counted crypt column.
Isolation of Mouse Peritoneal Macrophages and Phagocytosis Assays. Peritoneal macrophages were collected from the peritoneal fluid as previously described.36 Briefly, mice were euthanized using CO2 anesthesia followed by cervical dislocation. The peritoneal cavities were lavaged with 5 mL of cold Hank’s balanced salt solution (HBSS; Gibco) to collect peritoneal cells. The cells were washed twice in HBSS and resuspended in a 5% BSA solution at 2 105 cells/mL. Assessment of the phagocytic ability of peritoneal macrophages using flow cytometry was based on the methods of Wan et al., with minor modifications.37 Briefly, 10 μL of fluorescein isothiocyanate-labeled Escherichia coli and 500 μL of peritoneal macrophages (2 105 cells/mL) were mixed and incubated at 37 C for 2 h. Samples were then centrifuged at 1500 rpm for 10 min, and the pellet was resuspended in 1 mL of HBSS. The level of phagocytic activity was determined using a FACSCalibur flow cytometer (Becton Dickinson Instruments). Measurement of IgA Antibody. An ELISA-based assay to quantify mouse IgA in the intestinal fluid samples was performed using a Mouse IgA ELISA Quantification Set (catalog no. E90-103, Bethyl Laboratories, Inc., Montgomery, TX). Cell Line and Cell Culture. RAW264.7 cells, derived from murine macrophages, were obtained from the American Type Culture Collection (Rockville, MD). RAW264.7 cells were cultured in RPMI-1640 supplemented with 10% endotoxin-free, heat-inactivated fetal calf serum (GIBCO, Grand Island, NY), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator (37 C, 5% CO2). The probiotics were added at the indicated doses. For control specimens, the same volume of H2O was added instead of the probiotics. Cell viability was estimated using the trypan blue exclusion assay as previously described.38 Cell Cycle Analysis. Cells were plated in 10 cm dishes. After serum starvation with 0.04% FCS for 24 h to render them quiescent and to synchronize their cell cycle activities, the cells were returned to media with 10% FCS with test material or control. RAW264.7 cells were harvested at various time points. BrdU (10 μg/10 mL) was added 1 h before cell collection. Cells were then washed once with 1 PBS, fixed with 75% alcohol, stored at 4 C for 1 h, and centrifuged at 500g and 10 C for 10 min, at which point pellets were collected. After denaturation of the DNA with 1 mL of 2 N HCl/Triton X-100, pellets were collected after centrifugation at 500g for 10 min. Cells then were neutralized with 1 mL of 0.1 M Na2B4O7 3 10H2O (pH 8.5). After centrifugation 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 of anti-BrdU was added, and cells (1 106) were incubated at room temperature for 30 min and washed once with 1 mL of Tween 20/1% BSA/PBS; the pellets were collected using centrifugation at 500g for 10 min. Flow cytometry analyses were performed after the addition of propidium iodide. Western Blot Analysis. Treated and untreated cells were rinsed three times with ice-cold PBS pelleted at 800g for 5 min and lysed in 500 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. The extracts were centrifuged for 30 min at 10000g. Proteins were loaded at 50 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).
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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 significance was determined using either Student’s t test for comparison between means or one-way analysis of variance with a post hoc Dunnett’s test. Differences were considered to be significant when p < 0.05.
’ RESULTS Treatment with Probiotics Inhibited DMH-Induced Colonic ACF and Tumor Formation. During the experiment (Figure 1A),
all mice were monitored to investigate whether treatment with probiotics caused any adverse effects. No significant alteration of water consumption or body weight gain was observed in any of the groups of mice given the probiotics during the experimental period (supplementary data, Figure S1). Toxic effects due to the DMH treatment were not observed during the initiation of treatment (data not shown). The efficacy of probiotic (B. longum and L. gasseri) administration on inhibiting DMH-induced ACF formation was determined. Colonic ACF were identified and analyzed under a light microscope after methylene blue staining. Table 1 summarizes the incidence, the number of ACF per mouse (multiplicity), and the distribution of ACF in the colon after 15 weeks of treatment. All mice developed ACF in the colon after DMH treatment. The mean number of ACF per colon in the DMH alone group (group 3) was 20.7 ( 5.6, whereas mice treated with DMH and fed different doses of the probiotics showed a significantly lower number of ACF. Roughly, a 25 30% reduction of ACF was found in the probiotic administration groups (groups 4 7). The reduced ACF occurred in the middle to cecal part of the colon. However, the incidence of ACF in the probiotic administration groups was similar to that in the DMH-treated group, with a 100% incidence. Moreover, we did not find a dose-dependent or synergistic effect of B. longum and L. gasseri in the inhibition of DMH-induced ACF formation. We further evaluated the anticolonic tumorigenesis activity of long-term treatment of probiotics. Mice were fed B. longum or L. gasseri for 24 weeks, the colonic tissues were collected, and tumors in the colon were divided into three types: microadenomas, adenomas, and macroadenomas (Figure 1B), classified according to the guidelines of Morson et al.34 As shown in Table 2, the mean number of tumors in the DMH-treated group was 11.8 ( 2.5, whereas the numbers of tumors were decreased in both B. longum (7.1 ( 1.7) and L. gasseri (7.9 ( 2.3) groups. Furthermore, we found a significant reduction in the number of 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-alone group. The incidence of colonic tumors was not significantly different between the B. longum and L. gasseri groups. In addition, there was no difference in the tumor size between the two groups. Probiotics Inhibited DMH-Induced Colonic Mucosa Cell Proliferation. Due to the observation that the treatment with probiotics inhibited DMH-induced colonic ACF and tumor formation, the markers of colonic mucosa cell proliferation were further examined to explore the mechanisms of probiotic inhibition of colon tumorigenesis. As shown in Figure 2, the mitotic index was significantly increased in the DMH-treated group as compared with the control groups fed a normal or probiotic diet. Mice treated with DMH and fed different doses of B. longum 13339
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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 dimensions were 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 tumors was performed. The results showed adenoma but not adenocarcinoma in all tumors.
Table 1. Effects of Each Group on the Formation of Aberrant Crypt Foci (ACF) in Colons Induced by 1,2-Dimethylhydrazine (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) DMH + L. (n = 8)
9 (100) 15.3 ( 4.8 * 8 (100) 14.3 ( 3.2 *
30 27
45 50
25 23
DMH + B. + L. (n = 9) 9 (100) 15.8 ( 3.4 *
30
46
24
Data are presented as the mean ( SD. One-way ANOVA. #,p < 0.05, the group was significantly different from control and probiotics treated groups, respectively. *, p < 0.05, the group was significantly different from DMH-treated group. a
showed a significantly lower mitotic index compared with the DMH-treated group. However, the efficacies of L. gasseri and the 1/2 (B. longum + L. gasseri) groups were less than that of the B. longum group, with there being no significant difference between these groups. In addition, we also examined the PCNA labeling of cells (a marker for cell proliferation) in the colonic crypts of mice treated with DMH and/or probiotics. The results shown in Figure 3 indicate that the number of proliferating cells/crypts was significantly increased in the DMH-treated group as compared with the control groups. Mice treated with DMH and fed
both B. longum and L. gasseri showed a significantly lower number of proliferating cells compared with the DMH-treated group. Phagocytic Function of Peritoneal Macrophages and the IgA Antibody in the Murine Colon. The phagocytic activities of the peritoneal macrophages are shown in Figure 4. Peritoneal macrophages from the DMH-treated mice fed different doses of B. longum for 15 weeks exhibited significantly greater phagocytic activity than cells from control mice. However, the phagocytic activities of macrophages from the DMH-treated mice fed L. gasseri and the 1/2 (B. longum + L. gasseri) were lower than that of the B. longum group. There was no significant difference between those groups and the control group. To examine whether changes in phagocytic activity observed in the DMH-treated mice fed B. longum were related to mucosal antibody response, IgA was quantified using enzyme immunoassays. As shown in Figure 4C, the mucosa IgA levels tended to be higher in the DMH-treated mice fed different doses of B. longum, but were not significantly different from those in the control mice. Effects of Probiotics on the Incorporation of BrdU and Cell Cycle Regulatory Genes of RAW264.7 Cells. Due to the importance of immunomodulating and immunostimulating properties of probiotics, we examined their effects on the proliferation of macrophage in vitro. As shown in Figure 5A, L. gasseri at a dose of 106 cfu/mL significantly increased proliferation of RAW264.7 macrophage cells after 24 h of treatment. However, there was no significant difference between the macrophage cells treated with B. longum and the control group. As L. gasseri increased the growth 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 macrophage cells, BrdU cell cycle analysis was conducted. As shown in Figure 5B, treatment of macrophage cells with L. gasseri at doses 13340
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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
11.8 ( 2.5
adenoma
macroadenoma
DMH (n = 8)
8 (100)
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 and eosin. 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.
of 106 cfu/mL significantly increased BrdU incorporation at 18 h after treatment, as compared with control (control versus L. gasseri, 49.2 versus 63.8%, respectively). This result suggests that the L. gasseri-induced proliferative effect on macrophage cells occurs through an increase of S phase DNA synthesis. Thus, we next examined how L. gasseri regulated the expression of cell cycle associated genes by Western blot analysis. Figure 5C shows that treatment of macrophage cells with 106 cfu/mL of L. gasseri significantly increased the expression of PCNA and cyclin A at 12 and 18 h. However, the expressions of cyclin B, cyclin E, Cdc-2, and Cdk-2 were not changed over time. These findings suggested that up-regulation of PCNA and cyclin A might be involved in the L. gasseri-induced increase of S phase DNA synthesis in macrophage cells.
’ DISCUSSION In the present study, we demonstrate that dietary consumption of probiotics (B. longum and L. gasseri) resulted in significant inhibition of DMH-induced ACF formation in male ICR mice. Long-term (24 weeks) dietary consumption of probiotics also resulted in a reduction of colon tumor multiplicity and the size of
the tumors without any noticeable adverse effects, indicating long-term safety and chemopreventive efficacy of dietary probiotics. These findings strongly suggest the chemopreventive potential of dietary administration of the probiotics against colonic tumorigenesis. Although the mechanism of inhibition of colon cancer by probiotics is not clear, we believe that this effect may proceed through diverse mechanisms, including alteration of physiological conditions such as the pH of the colonic microenvironment, as well as the immune response of the host.39 The probiotics and their metabolites may affect the mixed function oxygenases, especially the cytochrome P450 family members, which are believed to be involved in the conversion of 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 carcinogen in the intestinal lumen, thereby reducing potency and/or availability in the lumen.42 Our current study also analyzed the modifying effects of probiotics on colonic mucosal cell proliferation with MI and PCNA labeling to determine if the modulation of these cellular and biochemical events, relevant to colon carcinogenesis, could be effectively used to monitor the inhibition of colon cancer. 13341
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Figure 3. Measurement of cell proliferation. (A) To assess the proliferative activity and distribution of proliferating cells in the colonic crypts, standard immunohistochemical for PCNA was performed. a, control; b, DMH alone; c, DMH + LB.; d, DMH + HB.; e, DMH + L.; f, DMH + (B. + L.). (B) A total of 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 each counted 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 using a 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) An ELISA-based assay to quantify mouse IgA in intestinal fluid samples was performed using the Mouse IgA ELISA Quantification Set. Data are expressed as the mean ( SE of five to nine samples. #, p < 0.05, compared with the DMH alone treated group.
We found that the administration of B. longum and L. gasseri suppressed the rate of cellular proliferation in a manner correlating with inhibition of tumor induction by DMH. Hyperproliferation of colonic epithelial cells is induced by administration of bile acids, fatty acids, and certain carcinogens.43 45 Enhanced labeling
indices in patients with neoplastic lesions and in carcinogentreated experimental animals have been observed in all sites of the colon and the uninvolved colonic mucosa. Lipkin46 and Terpstra et al.47 have suggested that the patterns and rates of mucosal cell proliferation may be acceptable measures of colon 13342
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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 the mean ( 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 independent experiments. #, p < 0.05, versus controls. (C) Time-dependent effect of probiotics on the expression of S phase regulatory proteins in RAW264 cells. The cells were harvested, and the protein extracts were separated using SDS-PAGE. After electrophoresis, the proteins were transferred and probed with the proper dilution of the specific antibodies. GAPDH served as an internal control.
cancer risk and that modulation of cellular proliferation by agents known to prevent cancer formation might therefore serve as an intermediate end-point in cancer prevention trials. In animals, probiotic ingestion was shown to prevent carcinogen-induced preneoplastic lesions and colon tumors.48 50 The results of the present study, showing a significant inhibition of DMH-induced cell proliferation that correlates with suppression of DMHinduced colon tumor multiplicity and tumor volume by dietary B. longum and L. gasseri, are in line with these observations. Previous studies have suggested that variations in apoptosis in the colon mucosa may be correlated with carcinogenesis,51 higher apoptosis being associated with a lower risk of developing cancer. Surprisingly, apoptosis was not increased in the probiotic groups when compared with the control group (data not shown). Therefore, in the present study apoptosis in the normal mucosa and carcinogenesis were not correlated. Optimally functioning innate and acquired immune systems are essential for host defense against invading pathogens and spontaneously developing cancers.52 The results of our studies demonstrate that the phagocytic activity of peritoneal macrophages was significantly increased in the DMH-treated mice fed different doses of B. longum but not L. gasseri or 1/2 (B. longum + L. gasseri). Enhanced phagocytic activity of peritoneal macrophages from animals given dietary probiotics has previously been
demonstrated.53 It has also been shown that the level of enhancement depends on the strain, dose, and viability of the probiotics used.54 Augmentation of specific serum and/or mucosal antibody responses has been reported, showing that different strains of the probiotics vary greatly in their ability to enhance humoral immunity.55 The mucosa IgA levels examined in the current study tended to be higher in the DMH-treated mice fed different doses of B. longum, but were not significantly different from those in the control mice. The precise mechanisms of probiotic stimulation of the immune system are not fully understood. It is possible that when present in large numbers, probiotics or their products are able to gain access to the gut-associated lymphoid tissue or systemic immune system.56 In addition, the intestinal epithelium contains a variety of immunoregulatory cells, and thus the probiotics and their products may exert their influence through these cells.57 Furthermore, the interaction between the probiotics or their products and immunocompetent cells (such as macrophages) results in the secretion of cytokines that are known to have a multitude of effects on the functioning of the immune system.54 In our current studies, we also found that L. gasseri, at doses of 106 cfu/mL, significantly increased proliferation of RAW264.7 macrophage cells through increased S phase DNA synthesis. Although relatively little information is known for S phase control in the mechanisms of cell cycle regulation, it has been demonstrated 13343
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Journal of Agricultural and Food Chemistry that PCNA, a key molecule involved in DNA replication machinery, has been associated with S phase regulation.58,59 The CDK were recognized as key regulators of cell cycle progression through their association with regulatory subunits called cyclins.60,61 Our current results demonstrating that L. gasseri treatment increased expression of PCNA as well as cyclin A suggested that these were the key molecules involved in the probiotic-induced DNA synthesis in macrophage cells. These findings also suggested that probiotics could directly regulate immune cell growth, which might be one of the mechanisms involved in our immune stimulation findings in mice. In summary, our study has supported earlier observations that certain probiotic bacteria are capable of diminishing colon tumor development in a carcinogen-induced colonic tumorigenesis model. Inhibition of colon carcinogenesis is associated with the modulation of colonic cell proliferation, leading to suppressed colonic ACF and tumor formation. Immunomodulating and immunostimulating properties, such as enhanced phagocytic activity of peritoneal macrophages and increased proliferation of macrophage cells, could play partial roles in the chemopreventive efficacy of dietary probiotics in DMH-induced colonic tumorigenesis. More probiotic studies are needed to examine further other possible mechanisms for their potential benefit.
’ ASSOCIATED CONTENT
bS
Supporting Information. Changes in (A) body weight gain and (B) water consumption in mice of experimental groups throughout the experimental period (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*(Y.-J.W.) Postal address: Department of Environmental and Occupational Health, National Cheng Kung University Medical College, 138 Sheng-Li Road, Tainan 70428, Taiwan. Phone: 8866-235-3535, ext. 5804. Fax: 886-6-2752484. E-mail: yjwang@ mail.ncku.edu.tw. (C.-K.Y.) Postal address: Department of Microbiology and Immunology, National Cheng Kung University, Medical College, 138 Sheng-Li Road, Tainan, Taiwan 70101. E-mail:
[email protected]. Funding Sources
We appreciate the commission of this study by the Food Industry Research 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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