Supported by a faculty research grant of Yonsei University College of Medicine for 2007 (6-20070224).
Article first published online: 22 MAR 2010
Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.
Inflammatory Bowel Diseases
Volume 16, Issue 9, pages 1514–1525, September 2010
How to Cite
Kim, S. W., Kim, H. M., Yang, K. M., Kim, S.-A., Kim, S.-K., An, M. J., Park, J. J., Lee, S. K., Kim, T. I., Kim, W. H. and Cheon, J. H. (2010), Bifidobacterium lactis inhibits NF-κB in intestinal epithelial cells and prevents acute colitis and colitis-associated colon cancer in mice. Inflamm Bowel Dis, 16: 1514–1525. doi: 10.1002/ibd.21262
Presented during UEGW 2008 in Vienna, Austria.
- Issue published online: 20 AUG 2010
- Article first published online: 22 MAR 2010
- Manuscript Accepted: 21 JAN 2010
- Manuscript Received: 14 JAN 2010
- Yonsei University College of Medicine for 2007. Grant Number: 6-20070224
- Bifidobacterium lactis;
- colitis-associated colon cancer;
- nuclear factor kappa B;
- inflammatory bowel disease
The aim of this study was to investigate the antiinflammatory effects of Bifidobacterium lactis on intestinal epithelial cells (IECs) and on experimental acute murine colitis and its tumor prevention effects on colitis-associated cancer (CAC) in mice.
Human HT-29 cells were stimulated with IL-1β, lipopolysaccharides, or tumor necrosis factor-α with and without B. lactis, and the effects of B. lactis on nuclear factor kappa B (NF-κB) signaling in IEC were examined. For in vivo study, dextran sulfate sodium (DSS)-treated mice were fed with and without B. lactis. Finally, we induced colonic tumors in mice by azoxymethane (AOM) and DSS and evaluated the effects of B. lactis on tumor growth.
B. lactis significantly suppressed NF-κB activation, including NF-κB-binding activity and NF-κB-dependent reporter gene expression in a dose-dependent manner, and suppressed IκB-α degradation, which correlated with the downregulation of NF-κB-dependent gene products. Moreover, B. lactis suppressed the development of acute colitis in mice. Compared with the DSS group, the severity of DSS-induced colitis as assessed by disease activity index, colon length, and histological score was reduced in the B. lactis-treated group. In the CAC model, the mean number and size of tumors in the B. lactis-treated group were significantly lower than those in the AOM group.
Our data demonstrate that B. lactis inhibits NF-κB and NF-κB-regulated genes in IEC and prevents acute colitis and CAC in mice. These results suggest that B. lactis could be a potential preventive agent for CAC as well as a therapeutic agent for inflammatory bowel disease. (Inflamm Bowel Dis 2010)
Inflammatory bowel disease (IBD) is a group of chronic and relapsing intestinal inflammatory disorders of unknown etiology. Currently, no drugs for the treatment of IBD have a nonrelapsing cure rate and few nontoxic therapeutic options are available to modulate intestinal inflammation. Furthermore, epidemiological studies have demonstrated an increased risk of colorectal cancer in patients with ulcerative colitis and Crohn's disease involving the colon.1, 2 In light of these facts, more effective and safe therapeutic and chemopreventive strategies for IBD are urgently needed.
The activation of the proinflammatory gene transcriptional program in intestinal epithelial cells (IECs) in response to challenges by bacterial products such as lipopolysaccharides (LPSs) or inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β), is associated with acute and chronic intestinal inflammation.3, 4 This signal ultimately converges on the nuclear factor kappa B (NF-κB) transcriptional system.5 Activation of NF-κB then upregulates the expression of various proinflammatory genes involved in intestinal inflammation.6 Because the NF-κB transcriptional system in IEC plays an essential role in the regulation of inflammation in patients with various intestinal disorders, targeting this signaling pathway may offer a therapeutic avenue for treating these diseases.7 Moreover, NF-κB is a multifunctional transcription factor that regulates the expression of a number of genes whose products are involved in tumorigenesis8, 9 and in regulating tumor development resulting from chronic inflammation.10 Persistent NF-κB activation has been suggested to contribute to cancer development. Furthermore, activation of NF-κB in response to chronic inflammation may be of particular relevance to gastrointestinal carcinogenesis, especially in colitis-associated cancer (CAC).11
A growing body of evidence suggests that certain probiotic bacteria can modulate intestinal inflammation, cell proliferation, and apoptosis, and intestinal epithelial homeostasis.12–14 Such properties may be useful for both immunomodulatory and cancer prevention strategies.15 Manipulation of intestinal bacterial flora has been used as an alternative health approach for disease prevention and treatment. Bifidobacterium lactis is a Gram-positive, anaerobic commensal-derived probiotic species.16 Interestingly, recent investigations have suggested that B. lactis has potent antiinflammatory and antiproliferative effects.17, 18 However, the precise mechanisms by which these probiotic bacteria exert their putative antiinflammatory and antitumorigenic influences are uncertain.
Because of the central role of NF-κB signaling in immune response and cancer development, we speculated that B. lactis mediates antiinflammatory and antiproliferative effects by modulating NF-κB signaling pathways in IEC. Therefore, we hypothesized that B. lactis could inhibit NF-κB in IEC and suppress in vivo acute and chronic intestinal inflammation and thereby suppress CAC. Thus, we aimed to investigate the effect of B. lactis on LPS- or cytokine-induced NF-κB signaling and proinflammatory and protumorigenic gene expression in HT-29 intestinal epithelial cancer cells. Moreover, we evaluated the antiinflammatory effects of B. lactis on acute colitis induced by dextran sodium sulfate (DSS) and the cancer prevention effects of B. lactis on CAC induced by azoxymethane (AOM) and DSS in a murine model.
MATERIALS AND METHODS
Bacterial Strains and Cell Lines
B. lactis (KCTC 5727, Korean collection for type cultures, Seoul, Korea) was obtained from Cell Biotech (Seoul, Korea). Lyophilized cultures (2 × 1010 colony-forming unit [CFU]/g) of B. lactis were dissolved with phosphate-buffered saline (PBS) to feed experimental animals in the acute colitis model. Mice were fed a diet including a 5 × 108 CFU/g diet of B. lactis in the CAC model. The B. lactis cells were suspended in deMan-Rogosa-Sharpe (MRS) broth (Difco Laboratories, Detroit, MI) and plated in MRS agar plates and cultured at 37°C under microaerobic conditions. Bacteria were counted using a plating technique and the cell counts in the bacterial suspension were estimated by optical density at an absorbance of 600 nm (UV-1601, Shimadzu, Kyoto, Japan). The B. lactis cells grown in MRS broth were collected by centrifugation (3000 rpm for 15 minutes), washed, and resuspended in 10 mL RPMI 1640, excluding antibiotics. The bacteria were added to the HT-29 cell culture wells at the appropriate dilution to reach a final concentration of 107, 108, or 109 CFU per mL of the incubation medium without antibiotics. The human colon cancer cell line HT-29 (KCLB 30038, Korean Cell Line Bank, Seoul, Korea) was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics.
DSS (36,000–50,000 Da; MP Biomedicals, Aurora, OH) was dissolved in distilled water at a concentration of 2% or 3.5% (w/v). A colonic carcinogen, AOM, was purchased from Sigma Chemical (St. Louis, MO).
Electrophoretic Mobility Shift Assay (EMSA)
To assess NF-κB activation, nuclear and cytoplasmic extracts were prepared. Briefly, harvested cells were lysed for 5 minutes in lysis buffer (5 mM KCl, 25 mM HEPES, 0.5 mM MgCl2, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 0.2% Triton X-100, complete protease inhibitor cocktail [Roche Diagnostics, Mannheim, Germany]), and then nuclei were pelleted by centrifugation at 6000 rpm for 15 minutes. Nuclei were lysed in a hypotonic buffer (350 mM NaCl, 10 mM HEPES, 25% glycerol, 0.1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, and protease inhibitors as above) for 30 minutes and debris was cleared by centrifugation at 13,000 rpm for 5 minutes. Bradford reagent (Bio-Rad Laboratories, Hercules, CA) was used to measure protein content. EMSA was performed using Lightshift chemiluminescent EMSA kit (Pierce, Rockford, IL) by following the manufacturer's protocol. Complementary NF-κB oligonucleotides 5′-AGT TGA GGG GAC TTT CCC AGG C-3′; 3′-GCC TGG GAA AGT CCC CTC AAC T-5′ were biotin-labeled separately using the Biotin end labeling kit (Pierce) and then annealed before use. Each binding reaction contained 1 × binding buffer (100 mM Tris, 500 mM KCl, 10 mM dithiothreitol, pH 7.5), and 2.5% glycerol, 5 mM MgCl2, 50 ng/μL poly (dIdC), 0.05% NP-40, 10 μg of nuclear extract, and 40 nM of biotin end-labeled target DNA. The contents were incubated at room temperature for 20 minutes. Complexes were separated on 4% nondenaturing polyacrylamide gel and were transferred to a nylon membrane. When the transfer was complete, DNA was crosslinked to the membrane at 120 mJ/cm2 using a UV crosslinker equipped with 254 nm bulbs. The biotin end-labeled DNA was detected using streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The membrane was exposed to X-ray film and developed using a Kodak film processor (Eastman Kodak, Rochester, NY).
Luciferase Reporter Gene Expression Assay
To examine reporter gene expression, cells were transiently transfected with the pNF-κB Luc plasmid and control pRenilla Luc plasmid (Invitrogen, Carlsbad, CA) for 24 hours. Transfected cells were treated with B. lactis at 108 to 109 CFU per mL of the incubation medium without antibiotics for 16 hours and stimulated with TNF-α (10 μM). They were analyzed for NF-κB reporter gene and control vector activity using lysis buffer and reagents from Promega (Madison, WI).
Western Blot Analysis
Cells were harvested on ice by washing twice with cold PBS, scraping, and resuspending. Protein concentration was determined using the Bradford assay with bovine serum albumin as a reference. Samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were prepared by mixing aliquots of the protein with NuPAGE sample buffer (Invitrogen) and heated at 70°C for 10 minutes. Protein samples were run on NuPAGE 4%–12% gradient Bis-Tris gels at 150V for 1 hour with MES SDS running buffer (Invitrogen). For Western blot analysis, gels were electrotransferred to a polyvinylidene difluoride membrane (Invitrogen) using the Xcell Surelock electrophoresis and transfer apparatus (Invitrogen). Blots were blocked with 5% (w/v) skim milk in Tris-buffered saline solution containing 0.1% Tween 20 (Pierce) and incubated overnight at 4°C with antibodies against p65 NF-κB, active NF-κB, IκB-α, phospho-IκB-α (p-IκB-α), cyclooxygenase-2 (COX-2), Histone H1 (Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (Sigma). Blots were additionally incubated with secondary antibodies conjugated with horseradish peroxidase for 1 hour at room temperature and, finally, revealed with the Enhanced Chemiluminescence Western blot detection reagent (Amersham Biosciences, Freiburg, Germany).
Quantitative Real-time Reverse-transcription Polymerase Chain Reaction (RT-PCR)
B. lactis-pretreated cells were stimulated with TNF-α for 4–8 hours. Total RNA was extracted using TRIzol Reagent (Invitrogen) and 1 μg of RNA was reverse-transcribed using SuperScript First-Strand Synthesis kit (Invitrogen) according to the manufacturer-recommended protocol. The cDNAs were mixed in triplicate using SYBR Green master mix (Applied Biosystems, Foster City, CA) and pairs of primers (4 pmol of each primer). PCR was done using primers for COX-2 (5′-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3′ and 5′-AGA TCA TCT CTG CCT GAG TAT CTT-3′), matrix metallopeptidase 9 (MMP-9; 5′-TGA CAG CGA CAA GAA GTG-3′ and 5′-CAG TGA AGC GGT ACA TAG G-3′), vascular endothelial growth factor-A (VEGF-A; 5′-GCT GCT CTA CCT CCA CCA TGC-3′ and 5′-GTT AAC TTC CGC GTT TGC TC-3′), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5′-TGA TGA CAT CAA GAA GGT GG-3′ and 5′-TTT CTT ACT CCT TGG AGG CC-3′). Samples were amplified in a 7500 real-time PCR System (Applied Biosystems) for 40–45 cycles using the following PCR variables: 95°C for 30 seconds, 60–62°C for 1 minute, and 72°C for 1 minute. Finally, quantitative analysis was performed using the relative standard curve method and the results were reported as the relative expression or fold change as compared to the calibrator after normalization of the transcript level to the endogenous control, GAPDH.
Animal Model and Assessment
Six-week-old male C57BL/6 mice (Orient, Seongnam, Korea) were maintained on a 12:12-hour light:dark cycle under specific pathogen-free conditions. The mice had access to a standard diet and water until they reached the desired age (9 weeks). All experiments using animals were reviewed and approved by the Institutional Animal Care and Use Committee of Yonsei University Severance Hospital, Seoul, Korea.
In the acute colitis model, mice were given 3.5% DSS in drinking water for 7 days. B. lactis was administered daily by oral gavage until Day 6 in the low-dose group (2 × 109 CFU/day) and the high-dose group (2 × 1010 CFU/day). DSS-treated groups received 0.9% normal saline in a comparable volume by the same route. Normal control mice received filtered water alone. Mice were sacrificed on Day 7 and clinical parameters and pathology were evaluated.
To test the physiological relevance of B. lactis-mediated blockade of CAC in vivo, we used an AOM/DSS-induced colon cancer model. The mice were divided into three groups: a control group, an AOM group in which vehicle were fed, and an AOM + BL (B. lactis) group in which mice were fed a 5 × 108 CFU/g diet of B. lactis (n = 5 each group). Mice in the AOM and AOM + BL groups were injected intraperitoneally with AOM (10 mg/kg). After 1 week they were fed 2% DSS in the drinking water over 5 days, followed by 16 days of regular water. This cycle was repeated three times. On the ninth experimental week all the mice were sacrificed and their colons were resected.
In both the acute colitis and the CAC model the mice were examined daily for behavior, water/food consumption, body weight, stool consistency, and the presence of gross blood in the stool or at the anus. A previously validated clinical disease activity index (DAI) that ranged from 0 to 4 was calculated based on the following parameters: stool consistency (0, normal; 2, loose; 4, diarrhea), gross bleeding (0, absence; 2, blood tinged; 4, presence), and weight loss (0, none; 1, 1%–5%; 2, 5%–10%; 3, 10%–20%; 4, >20%).19 The calculated DAI = (weight loss + stool consistency + gross bleeding)/3. The severity of colitis was evaluated by an independent observer who was blinded to the treatment.
Histology and Immunohistochemistry
Postmortem, the entire colon was removed from the cecum to the anus and opened longitudinally. Subsequently, samples of colonic tissue were either fixed in 10% buffered formalin, embedded in paraffin, and stained hematoxylin-eosin. Histological examination was performed on three samples of the distal colon for each animal. All histological quantification was performed in a blinded fashion using a scoring system described previously.20 Briefly, three parameters were measured: severity of inflammation (0, none; 1, slight; 2, moderate; 3, severe), extent of injury (0, none; 1, mucosal; 2, mucosal and submucosal; 3, transmural), and crypt damage (0, none; 1, basal one-third damaged; 2, basal two-thirds damaged; 3, only surface epithelium intact; 4, entire crypt and epithelium lost). The score of each parameter was multiplied by a factor that reflected the percentage of tissue involvement (31, 0%–25%; 32, 26%–50%; 33, 51%–75%; 34, 76%–100%), and all numbers were summed. The combined histopathological scores ranged from 0 to 40. Colon length as an indirect marker of inflammation was also measured. For the precise evaluation of CAC in colon tissue the presence and tumor load was determined in the whole colon by a blinded pathologist. Tumor counts were performed and tumor sizes were calculated. Paraffin blocks were sectioned and stained for COX-2, IL-6, and IκB-α (Santa Cruz Biotechnology) using the Dako REALTM Envision Kit (DakoCytomation, Carpinteria, CA) and the slides were counterstained with hematoxylin–eosin. Images were obtained using a microscope (Olympus BX41; Olympus Optical, Tokyo, Japan).
Isolation of Primary Mouse IEC
Colons were cut longitudinally, washed three times in calcium/magnesium-free HBSS (Invitrogen), cut into pieces 0.5 cm long, and incubated at room temperature in 40 mL of calcium/magnesium-free HBSS containing 10 mM DTT for 30 minutes, then incubated for 60 minutes in 1 mM EDTA in CMF-HBBS at 4°C. Epithelial cells were detached as intact crypts by 10 vigorous shakes of the vessel. The supernatant was filtered on cell strainer (BD Biosciences, San Jose, CA), centrifuged for 5 minutes at 400 g, and the IEC pellets were lysed for subsequent Western blot analysis.
Experimental results are expressed as mean values ± standard error of the mean (SEM). Statistical Package for the Social Sciences (SPSS/PC+ 11.0, Chicago, IL) was used for all analyses. Significance was determined using the Mann–Whitney U-test or Student's t-test and were accepted when P-values were less than 0.05.
B. lactis Inhibits IL-1β or TNF-α-mediated NF-κB Activation in IEC
First, we evaluated the molecular effects of B. lactis in a colorectal cancer cell line, HT-29, which possess a range of endogenous NF-κB activity. We performed EMSA to determine whether B. lactis decreases the DNA binding activity of NF-κB. B. lactis inhibited IL-1β or TNF-α-dependent NF-κB activation in a dose-dependent manner (Fig. 1A; Supporting Fig. 1A). Furthermore, TNF-α activated NF-κB in a time-dependent manner in B. lactis-untreated cells, which was significantly inhibited in B. lactis-treated cells (Fig. 1B; Supporting Fig. 1B).
To evaluate the effects of B. lactis on NF-κB gene expression, an NF-κB-dependent luciferase (Luc) reporter gene assay was performed because DNA binding may not correlate with effects on gene expression. The results of luciferase reporter assay showed that B. lactis suppressed TNF-α-mediated NF-κB activation in a dose-dependent manner (Fig. 1C).
B. lactis Inhibits p65 NF-κB Translocation by Blocking IκBα Degradation
Since the degradation of IκB-α results in nuclear translocation of p65 NF-κB, the effects on TNF-α- or LPS-induced nuclear translocation and the state of IκB-α were examined using Western blot of nuclear and cytosolic extracts. Translocation of NF-κB was abolished by B. lactis in a time-dependent manner (Fig. 2A; Supporting Fig. 2). Because nuclear translocation of NF-κB is preceded by proteolytic degradation of IκB-α by phosphorylation level, phosphorylation of IκB-α in the cytoplasm was analyzed. TNF-α-or LPS-induced IκB-α degradation occurred in control cells not treated with B. lactis, but B. lactis pretreatment stabilized IκB-α (Fig. 2; Supporting Fig. 2).
B. lactis Suppresses NF-κB-dependent Tumorigenic Genes
COX-2 contributes to carcinogenesis by promoting cell proliferation. TNF-α can induce the expression of genes in IEC involved in tumor metastasis, including MMP, VEGF, and COXs that are regulated by NF-κB. We investigated whether B. lactis can modulate TNF-α-induced expression of COX-2, VEGF, and MMP-9 in vitro by real-time RT-PCR and Western blot, respectively. B. lactis strongly inhibited transcriptional activation of these genes in HT-29 cells challenged with TNF-α (Fig. 3). Similarly, protein levels of COX-2, MMP-9, and VEGF decreased in a time-dependent fashion as determined by Western blot analysis (Fig. 3; Supporting Fig. 3). These overall patterns of B. lactis responsiveness appear to correlate best with the level of inactivated NF-κB and demonstrate that B. lactis suppresses NF-κB activation and the expression of the related tumorigenic genes.
B. lactis Prevents the Development of Acute Colitis in Mice
To test the physiological relevance of B. lactis-mediated blockade of NF-κB activation in vivo we used a DSS-induced acute murine colitis model. The mice were divided into four groups: Control, DSS, DSS + LBL (mice were supplemented with B. lactis in a low dose, 2 × 109 CFU/day), and DSS + HBL (a high dose of B. lactis, 2 × 1010 CFU/day). There were no significant differences in the starting body weights between control and experimental groups in the acute preventive model. Administration of a high dose of B. lactis produced a significant recovery of body weight induced by DSS-induced colitis (Fig. 4A).
On gross examination, B. lactis-treated groups (DSS + LBL and DSS + HBL) had lower levels of clinical DAI than DSS groups (Fig. 4B). Blinded histological injury scoring was quantified in the distal colon. In the DSS group the histological severity of colitis assessed by the overall score was significantly higher than that in controls (Fig. 4C). DSS induced complete destruction of epithelial architecture with a loss of crypts and epithelial integrity, submucosal edema, and intense infiltration of inflammatory cells including neutrophils and lymphocytes in all layers (Fig. 5; Supporting Fig. 4A). Treatment of DSS-fed mice with B. lactis led to a significant attenuation of experimental colitis, with a reduced total score compared with the DSS group (Fig. 4D). These observations correlate well with clinical and macroscopic findings in a dose-dependent manner.
Because our data from the in vitro study suggested that B. lactis could exert its antiinflammatory effects by blocking NF-κB in IEC, we investigated this signaling in the DSS-colitis model to reconfirm it in vivo. The expression of IκB, the active form of NF-κB, and IL-6 in colonic mucosa was examined by immunohistochemistry staining. In mice with DSS-induced colitis, IκB-α was strongly negative in both epithelial cells and submucosal inflammatory cells, but treatment with B. lactis markedly increased the degree of IκB-α (Fig. 5I–L; Supporting Fig. 4A,B) staining and decreased the active form NF-κB (Fig. 5M–P; Supporting Fig. 4C) and IL-6 (Fig. 5Q–T; Supporting Fig. 4D) in the colonic tissue. Moreover, the expression of IκB, phosphorylated IκB, and IL-6 in colonic tissue extracts and isolated IEC from colonic tissues were examined by Western blot analysis (Supporting Fig. 4E). Administration of B. lactis significantly increased the expression of IκB but decreased the phosphorylated IκB and IL-6 expression in both colonic tissues (Fig. 6A–C) and isolated IEC (Fig. 6D).
B. lactis Suppresses the Development of Colitis-associated Colon Cancer in Mice
Next we examined the tumor prevention effects of B. lactis in a murine CAC model. Body weights were measured daily during the experimental period. Weight gain in DSS-treated mice was much lower than in control mice. Rapid weight reduction started 19 days after the second cycle of 2% DSS, and this reduction was sustained in both the AOM and the AOM + BL groups. However, B. lactis attenuated the DSS-induced weight loss (Fig. 7A). The mice that received AOM had markedly shorter colons compared to control mice, whereas the colons of mice that received AOM + BL were markedly longer than the colons of mice in the AOM group (Fig. 7B). Although partial destruction of epithelial architecture, submucosal edema, predominant lymphocytic infiltration, and several lymphoid follicles were observed in the AOM group, the maintenance of crypt architecture and minimal infiltration of inflammatory cells were observed in the AOM + BL group (Fig. 8A–C).
On magnified gross examination, no tumors were found in mice treated with saline. All mice treated with AOM plus DSS developed tumors. These tumors were located in the middle to distal colon and were primarily broad-based adenomas with high-grade dysplasia and varying degrees of inflammatory cell infiltration. We observed a dramatic decrease in tumor incidence in B. lactis-treated mice (Fig. 7C). The mean number of tumors was 4.5 in the B. lactis group, which was markedly lower than that of the AOM group (10.9). When tumors were classified into three groups on the basis of size (below 1 mm, more than 1 mm, and not more than 3 mm, or above 3 mm), none of the tumors observed in the B. lactis group were found to be more than 3 mm in diameter and most tumors were less than 1 mm. In contrast, the number of tumors between 1 mm and 3 mm was significantly higher in the AOM (n = 7.6) group compared with the B. lactis (n = 1.3) group (P = 0.014).
In the AOM group, IκB-α was weakly positive in both destroyed epithelial cells and submucosal inflammatory cells. Treatment with B. lactis markedly attenuated the degree of IκB-α degradation in the colonic tissue. There was a significant increase of IκB-α in the AOM + BL group compared with the AOM group (Fig. 8D–F; Supporting Fig. 5A). As inhibition of COX-2 is an accepted chemopreventive strategy, we tested the effect of COX-2 inhibition in our model. Although strong COX-2 expression was seen in both epithelial cells and submucosal inflammatory cells in the AOM group, reduced immunoreactivity for COX-2 was observed in epithelial cells and in the basal layer in B. lactis-treated mice (Fig. 8G–I; Supporting Fig. 5B).
The present study was undertaken to investigate the potential antiinflammatory and cancer preventive effects of B. lactis on intestinal inflammation and cancer and to understand the mechanisms involved. Recently, a few studies have demonstrated the antiproliferative and proapoptotic effects of Bifidobacterium on various cancer cell lines.12, 16, 21 However, the precise mechanism of their antiinflammatory and antitumorigenic influences remains unclear, although a speculative mechanism implicates the immunomodulating properties of probiotic bacteria.22
Because NF-κB is considered a key player in inflammatory processes and cell proliferation, it provides a mechanistic link between inflammation and cancer.23 We hypothesized that B. lactis might modulate NF-κB activation in IEC, through which it could also inhibit acute colitis and CAC in mice. To test these hypotheses, we first investigated the impact of B. lactis on cytokine signaling and elucidated its mode of action using IEC. Our results demonstrate that B. lactis strongly suppresses TNF-α- and IL-1β-induced NF-κB signaling in IEC, which highlights a possible mechanism for its antiinflammatory action in the intestine. We used EMSA and a nuclear assay for the NF-κB p65 in HT-29 cells to demonstrate that B. lactis inhibits NF-κB DNA binding activity and translocation of NF-κB into the nucleus by suppression of IκB-α degradation. Moreover, NF-κB-dependent reporter gene activation by TNF-α could be inhibited by preincubation with B. lactis in a dose-dependent manner. Next, to clarify whether B. lactis would modulate the inflammation in the intestines of animals as well as IEC, we investigated the effects of B. lactis in a murine DSS-induced colitis model that is a commonly used model for the inflammatory component of IBD. In our study, B. lactis appeared to prevent DSS-induced colitis. Furthermore, it was clearly demonstrated by immunohistochemistry and Western blot that B. lactis is associated with the inhibitory modulation of IκB-α degradation. This is in line with the results of the in vitro study that demonstrated a B. lactis-mediated NF-κB inhibition in IEC. These findings indicate the novel protective effects of B. lactis against inflammation in the colon, suggesting its potential clinical value in the treatment of IBD.
Our results are consistent with the results of several other studies. It was recently reported that some strains of bifidobacteria are effective in inhibiting LPS-induced activation of IL-8 production and the NF-κB activation pathway.16, 24 However, only NF-κB promoter transcriptional activity was measured in this study. For in vivo study it was reported in one study that B. lactis showed intestinal antiinflammatory activity in a TNBS model of rat colitis without the demonstration of its action mechanisms.25 Our finding is further supported by the recent observation that Bifidobacterium inhibits DSS-induced colitis in mice with a similar dose of bacteria to ours.26 On the contrary, commensal bacteria, including Lactobacillus spp., have been shown to influence the regulatory pathways of the mammalian intestinal epithelium by directly modulating the ubiquitin–proteasome system.12 Moreover, a previous report suggested that B. lactis can transiently activate NF-κB and proinflammatory gene expression both in vitro and in vivo.27Bifidobacterium animalis was also shown to cause duodenitis and colitis in a susceptible host.28 Taken together, these findings suggest that all Bifidobacterium strains are not protective in the intestine and different probiotics may mediate different signaling pathways. Further studies are necessary to elucidate the exact roles of each strain of Bifidobacterium in NF-κB signaling. Moreover, recent evidence indicated that IEC-derived NF-κB signaling might be essential to protect these cells against acute injury such as DSS or radiation.29–31 Further research is necessary to draw a concrete conclusion concerning this controversial impact of NF-κB blockage on acute intestinal damage.
In addition to its key role in inflammation, NF-κB activates the transcription of numerous genes capable of suppressing apoptosis, suggesting a pivotal role in inflammation-related carcinogenesis.9 In particular, a recent investigation demonstrated that IKKβ is related to inflammation and tumorigenesis in murine models of colitis-associated cancer.10 The anti-NF-κB action of sulfasalazine, which is known to prevent colon cancer in patients with IBD, is mediated by the direct inhibition of IKKα and IKKβ.32 Based on these findings, B. lactis may have similar effects on CAC and IBD. Encouraged by the antiinflammatory effects of B. lactis on intestines in mice, we further sought to investigate the effect of B. lactis on CAC in a murine model. B. lactis significantly reduced the number of colon tumors in an inflammation-related colon cancer model. Although the antiinflammatory activity of probiotics has been reported previously, to our knowledge, this is the first report on a specific inhibitory effect of bifidobacteria on colitis-induced carcinogenesis in epithelial cells through NF-κB modulation, suggesting a role for bifidobacteria in down-modulation of inflammation and tumorigenesis. This effect of B. lactis which suppressed IκB-α degradation in both acute colitis and colonic cancer suggests a mechanistic effect comparable to sulfasalazine. Our findings correspond to those of some previous reports. It was reported that B. lactis treatment reduced colonic TNF-α production and COX-2 expression.33 Moreover, it has been reported that dietary intake of Bifidobacterium culture significantly inhibited the development of AOM-induced aberrant crypt foci and blocked the induction of colon and liver tumors by 2-amino-3-methyl-imidazo [4, 5-f] quinolone, a food mutagen.17
In this study, B. lactis inhibited the expression of COX-2, an NF-κB-dependent mediator, in colitis-related cancer. COX-2 is an enzyme that catalyzes the production of prostaglandin E2 (PGE2) from arachidonic acid, and has been linked to proliferation and metastasis of tumor cells.34 VEGF, a key molecule involved in angiogenesis, and MMP, a class of enzymes involved in tissue remodeling, are known as positive regulators of intestinal tumorigenesis,35 which is also regulated by NF-κB and COX-2. COX-2, VEGF, and MMP-9, all of which have NF-κB binding sites in their promoters, are also induced by TNF-α. mRNA or protein induction of these genes was inhibited by B. lactis in our study. Similar to our findings, a COX-2 inhibitor inhibited both colon carcinogenesis and colitis in a murine colitis model.36, 37 The COX-2 level has been shown to be increased in IBD38 and in colon cancer.39 However, in other studies B. lactis upregulated COX-1 but downregulated COX-2 in Caco-2 cells40 and one such probiotic, Lactobacillus rhamnosus GG, prevented cytokine-induced apoptosis in two different IEC models41 and induced COX-2 expression in T84 colon epithelial cells.42 Moreover, COX-1 and -2 has been shown to have a crucial role in the defense of the intestinal mucosa.43 Therefore, the complex, interactive mechanisms of COX-2 in intestinal inflammation or cancer remain to be further determined.
In conclusion, the results of this study indicate that B. lactis blocks cytokine-induced NF-κB signaling and proneoplastic gene expression in IEC. Moreover, B. lactis prevents acute colitis and decreases AOM-induced carcinogenesis in a murine model of CAC by the inhibition of IκB-α degradation. These results provide clues to understanding the molecular mechanisms by which B. lactis mediates its antiinflammatory and anticarcinogenic effects both in vitro and in vivo. B. lactis has great potential as a therapeutic agent for disorders of chronic intestinal inflammation and as an agent for cancer prevention.
- 17Anti-inflammatory activity of probiotic Bifidobacterium: enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells. World J Gastroenterol. 2008; 14: 2511–2516., , , et al.
- 25A comparative study of the preventative effects exerted by three probiotics, Bifidobacterium lactis, Lactobacillus casei and Lactobacillus acidophilus, in the TNBS model of rat colitis. J Appl Microbiol. 2007; 103: 836–844., , , et al.
Additional supporting information may be found in the online version of this article.
|IBD_21262_sm_suppfig1.tif||97K||Supporting Figure 1. B. lactis inhibits NF-κB activation in HT-29 cells (quantitative analysis of data described in the main article using densitometry). (A) Cells were preincubated with B. lactis for 16 h, followed by an incubation with 10 μM of IL-1β or TNF-α for 1 h and the nuclear extracts were analyzed for NF-κB activation by EMSA, as described in Materials and Methods. (B) Cells were preincubated with B. lactis, treated with TNF-α (10 μM) for the indicated times, and then subjected to EMSA. Quantification of the each band intensity was measured by densitometry (GS-800; Biorad, Hercules, CA), analysed using Quantity One software (Bio-rad) and represented as relative folds. Data are expressed as mean ± SEM (n ≥ 4). * P < 0.05 compared with control; ** P < 0.05 compared with IL-1β or TNF-α treated cells; †P < 0.05 compared with BL untreated cells. BL, Bifidobacterium lactis.|
|IBD_21262_sm_suppfig2.tif||124K||Supporting Figure 2. B. lactis inhibits TNF-α-induced p65 NF-κB translocation and IκBα degradation in HT-29 cells (quantitative analysis of data described in the main article using densitometry). (A) Cells were preincubated with B. lactis for 16 h, followed by an incubation with TNF-α (10 μM) for 1 h. Nuclear (NE) and cytoplasmic (CE) extracts were analyzed for p65 localization and IκB-α phosphorylation/degradation by Western blotting. Histone H1 is used as control. (B) Cells were preincubated with B. lactis for 16 h, followed by an incubation with LPS (10 μg/ml) for 1 h. Quantification of the each band intensity was measured by densitometry, analysed using Quantity One and represented as relative folds. Data are expressed as mean ± SEM (n = 5). * P < 0.05 compared with BL untreated cells. BL, Bifidobacterium lactis.|
|IBD_21262_sm_suppfig3.tif||89K||Supporting Figure 3. B. lactis suppresses TNF-α-induced NF-κB-dependent expression of tumorigenic genes (quantitative analysis of data described in the main article using densitometry). (A) Cells were preincubated with B. lactis for 16 h, followed by an incubation with TNF-α (10 μM) for the indicated times. Whole-cell extracts were prepared. The protein level analyzed by Western blotting. (B) VEGF and MMP-9 protein level were analyzed by Western blotting. Quantification of the each band intensity was measured by densitometry, analyzed using Quantity One program and represented as relative fold. Data are expressed as mean ± SEM (n ≥ 3). * P < 0.05 compared with BL untreated cells. BL, Bifidobacterium lactis.|
|IBD_21262_sm_suppfig4.tif||863K||Supporting Figure 4. Histopathology in the acute colitis model (quantitative analysis of data described in the main article using densitometry). (A) Representative immunohistochemistric statining for IκB-α using isotype antibody (rabbit) and control antibodies of colonic samples taken from mice in the acute colitis model and IgG negative controls (magnification: ×200). (B-D) Relative changes of IκB-α (B), NF-κB (C), and IL-6 (D) protein expression in immunohistochemistry of colonic samples taken from mice in the acute colitis model were determined using densitometry (Metamorph; Universal Imaging, West Chester, PA). (E) Isolated intestinal epithelial cells from the mice colonic tissues for Western blotting. Data are expressed as mean ± SEM (n = 5). * P < 0.05 compared with DSS group. LBL, low-dose B. lactis group (2 × 109 CFU/day); HBL, high-dose B. lactis group (2 × 1010 CFU/day); BL, Bifidobacterium lactis.|
|IBD_21262_sm_suppfig5.tif||77K||Supporting Figure 5. Histopathology in the CAC model (quantitative analysis of data described in the main article using densitometry). Relative changes in IκB-α (A) and COX-2 (B) protein expression were determined using densitometry (Metamorph). Data are expressed as mean ± SEM (n ≥ 3). * P < 0.05 compared with AOM group. AOM, azoxymethane; BL, Bifidobacterium lactis.|
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