Promotion of intestinal carcinogenesis by Streptococcus bovis
Stéphan Ellmerich1,
Marie Schöller1,
Benoît Duranton,
Francine Gossé,
Michel Galluser,
Jean-Paul Klein1 and
Francis Raul2
Université Louis Pasteur, Institut National de la Santé et de la Recherche Médicale, Contrat Jeune Formation 95-09, Institut de Recherche contre les Cancers de l'Appareil Digestif, 1 place de l'hôpital, BP 426, 67091 Strasbourg cedex, France and
1 Institut National de la Santé et de la recherche Médicale, Unité 392, Illkirch-Graffenstaden, France
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Abstract
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The involvement of Streptococcus bovis, an member of the human gut flora, in colorectal neoplastic diseases is an object of controversy. The aim of this study was to determine the effects of S.bovis and of antigens extracted from the bacterial cell wall on early preneoplastic changes in the intestinal tract. Adult rats received i.p. injections of azoxymethane (15 mg/kg body weight) once per week for 2 weeks. Fifteen days (week 4) after the last injection of the carcinogen, the rats received, by gavage twice per week during 5 weeks, either S.bovis (1010 bacteria) or wall-extracted antigens (100 µg). One week after the last gavage (week 10), we found that administration of either S.bovis or of antigens from this bacterium promoted the progression of preneoplastic lesions through the increased formation of hyperproliferative aberrant colonic crypts, enhanced the expression of proliferation markers and increased the production of IL-8 in the colonic mucosa. Our study suggests that S.bovis acts as a promoter of early preneoplastic lesions in the colon of rats. The fact that bacterial wall proteins are more potent inducers of neoplastic transformation than the intact bacteria may have important implications in colon cancer prevention.
Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; BHI, brainheart infusion broth; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; WEA, wall-extracted antigens.
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Introduction
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Environmental factors and hereditary factors are important in the etiology of colon cancer (1). The involvement of the intestinal microflora in the pathogenesis of colon cancer has been hypothesized (2). In a recent paper, Beebe and Koneman (3) reviewed the evidence in favor of the idea that the recovery of certain bacterial species from blood culture indicates the presence of an underlying, occult, or undiagnosed neoplasm. For example, Streptococcus bovis, a member of the gastrointestinal tract flora of humans, became diagnostically important, since it was shown that bacteremia and endocarditis due to these bacteria are often associated with colorectal neoplastic diseases (4,5). Increased faecal carriage of S.bovis in patients with colorectal cancer was also reported (6).
It has been suggested that alteration in local conditions and disruption of capillary channels at the site of neoplasm allowed S.bovis to proliferate and gain entry into the blood stream, whereas other studies indicated genito-urinary or dental diseases as possible sources of S.bovis entry (7). Local actions of cytokines or of chemical mediators able to promote vasodilatation and the enhancement of capillary permeability, may support bacterial entry at the tumour site, and increase bacterial adherence to various cells (8). It has also been speculated that S.bovis produces a carcinogen that induces intestinal cancer (6). In contrast, other reports have shown that there seems to be no clear association between S.bovis and human colorectal carcinoma (9,10).
In view of the potential involvement of S.bovis in intestinal carcinogenesis it was the aim of this study to determine the effects of S.bovis and of antigens extracted from the bacterial cell wall on preneoplastic histopathological changes in the intestinal tract of rats pre-treated with the carcinogen azoxymethane (AOM). We studied the changes in mucosal polyamines and the expression of proliferating cell nuclear antigen (PCNA), two indicators of a hyperproliferative state in the colonic mucosa (11,12), as well as the production of pre-inflammatory cytokines (IL-8) in the intestinal mucosa.
We present experimental evidence that both S.bovis and antigens from this bacterium promote the progression of early preneoplastic lesions in the colonic mucosa of AOM pre-treated rats.
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Materials and methods
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Bacterial strain and cell wall antigens
The bacterial strain used in this study was S.bovis NCTC 8133. Bacteria were grown anaerobically in brainheart infusion broth (BHI) (Difco Laboratories, USA) supplemented with 1% glucose.
A fraction of wall-extracted antigens (WEA) was prepared from 18 h culture. Bacteria were centrifuged (5000 g, 20 min, 4°C), the pellet was washed three times with phosphate-buffered saline (PBS) and was then shaken for 1 h at 4°C with 0.5 M phosphate buffer pH 6 in the presence of glass beads as has been described (13). The suspension was centrifuged (5000 g, 20 min, 4°C), the supernatant was extensively dialysed against water, lyophilized and resuspended in PBS. This extract was kept frozen at 80°C until use.
Animals and treatments
The experiments were conducted according to the National Research Council Guide for use and care of laboratory animals with the authorization (no. 00573) of the French Ministry of Agriculture.
Male Wistar rats (n = 18) weighing 230245 g, were housed under standardized conditions (22°C; 60% relative humidity; 12 h light/12 h dark cycle, 20 air changes/h) and fed a standard diet with free access to drinking water. All animals received i.p. injections of 15 mg AOM/kg body weight once each week for 2 weeks.
Two weeks after the last injection with AOM (week 4), the rats were randomly divided into three groups which received controlled isocaloric diets (234 kcal/kg/day) during the whole experimental period. The diet (UAR A05, Villemoisson/Orge, France) contained 13.5% protein as casein and fish protein, 62% carbohydrates as wheat starch, 3% lipids as soya and fish oil, 6% salt mixture and 1% vitamin mixture.
The rats received either 1 ml of S.bovis suspension (1010 bacteria, group I) or S.bovis WEA (100 µg, group II) in BHI twice per week during 5 weeks by gavage. The control AOM-treated rats received 1 ml of BHI. One week after the last gavage (week 10) the animals were killed.
The body weights of the animals of the three groups showed no significant differences: 460 ± 24 g (control group), 481 ± 19 g (group I) and 489 ± 30 g (group II).
The entire colon was collected for histological and biochemical analyses.
Cytokine assays
Intestinal mucosal samples were homogenized in 500 µl of PBS containing EDTA (10 mM), phenylmethylsulfonyl fluoride (2 mM) and gentamicin (40 µg/ml) and the suspension was centrifuged (10 000 g, 15 min, 4°C). IL-8 was measured in the supernatant using the heterologous two-site sandwich ELISA kit according to the manufacturer's instructions. The enzyme immunoassay kit for IL-8 was from Diaclone (Besancion, France).
Assessment of aberrant crypts and tumors in the colon
The determination of aberrant hyperproliferative crypts and tumors was performed on a segment of 5 cm in length corresponding to the distal part of the colon. The segment was washed with physiological saline, cut open, pinned out flat and fixed in 10% buffered formalin. The colon was stained with 0.2% methylene blue for 5 min, rinsed in KrebsRinger buffer, placed onto a glass slide and examined microscopically using a low power objective (x5) for assessment of the number of aberrant crypts (14,15) and of the presence of tumors. All counts were made by two blinded observers (F.G. and M.S.). The criteria for the identification of aberrant crypts were: (i) an increased size; (ii) a thicker epithelial cell lining; and (iii) an increased pericryptal zone relative to normal crypts.
PCNA immunostaining
Formalin-fixed, paraffin-embedded tissue sections (5 µm) were cut and taken onto silanized slides. After removal of the paraffin and rehydration, endogenous peroxidase was blocked by 0.3% hydrogen peroxide in methanol for 20 min. Immunohistochemical staining was performed using a biotinylated monoclonal anti-PCNA antibody followed by the biotinstreptavidin immunoperoxidase method according to the manufacturer's instructions (Zymed Laboratories Inc., San Francisco, CA). The nuclei were counterstained with hematoxylin.
Only U-shaped longitudinally cut crypts with open lumina along the crypt axis, and the base of the crypt touching the muscularis mucosae were evaluated by the two blinded observers. The proliferation index indicates the ratio of the number of immunoreactive nuclei/crypt to the total number of nuclei/crypt.
Determination of polyamines
Colonic mucosal samples were homogenized in 10 parts (w/v) of 0.2 M perchloric acid, and the homogenates were centrifuged at 3000 g for 10 min after standing for 16 h at 2°C. The supernatants were diluted with 0.2 M perchloric acid and 200 µl aliquots were applied on a reversed-phase column for separation. The polyamines (putrescine, spermidine and spermine) were determined by separation of their ion pairs formed with n-octanesulfonic acid, reaction of the column effluent with o-phthalaldehyde/2-mercaptoethanol reagent and monitoring of fluorescence intensity (16).
Statistics
Data are reported as means ± SE. Statistical differences between groups were evaluated by one-way ANOVA and specific differences were identified using the StudentNewmanKeuls multiple comparison test.
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Results
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Production of IL-8 in colonic mucosal samples
IL-8 in mucosal samples of groups I and II was compared with the IL-8 level in animals injected with AOM only. As shown in Figure 1
, small amounts of IL-8 were detected in the mucosa of control rats, the level of IL-8 was ~4- and 3-fold higher in the mucosa of rats receiving S.bovis and S.bovis WEA fraction, respectively.

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Fig. 1. IL-8 content in the colonic mucosa of AOM-pre-treated rats. Values are means ± SE of six animals/group. Open column, AOM-treated controls; hatched column, rats treated with AOM + S.bovis; dotted column, rats treated with AOM + S.bovis WEA. Columns not sharing a common superscript differ significantly (P < 0.05).
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Formation of aberrant crypt foci (ACF) and adenomas
As shown in Table I
, all rats injected with AOM developed numerous abnormal and hyperplasic colonic crypts, regardless of treatment. However, the administration of either S.bovis or S.bovis WEA resulted in a 1.8-fold increase of the number of aberrant colonic crypts, when compared with control rats treated with AOM. In the group receiving bacterial WEA, colonic adenomas were present in 50% of the rats. In the two other groups the colon remained tumor free.
Effect of treatments on colonic cell kinetics
Since AOM promotes hyperproliferative changes in the colon, we examined PCNA expression in histological sections of the colonic crypts. As shown in Figure 2
, the proliferation index was increased 2-fold in rats receiving either S.bovis or bacterial WEA when compared with rats treated with AOM only, but no significant changes were observed between rats treated with S.bovis or S.bovis WEA. An increased number of crypt cells engaged in proliferation was observed in rats treated with the bacteria or with the bacterial antigen when compared with the AOM-treated controls.

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Fig. 2. PCNA labelling in colonic crypts of AOM-pre-treated rats. The proliferation index for a given crypt was defined as the percentage of immunoreactive crypt cells of the total number of crypt cells. At least six well-oriented and well stained crypts were used to obtain a reliable proliferation index for each animal. Values are means ± SE of six animals/group. Open column, AOM-treated controls; hatched column, rats treated with AOM + S.bovis; dotted column, rats treated with AOM + S.bovis WEA. Columns not sharing a common superscript differ significantly (P < 0.05).
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Polyamine content in the mucosa of the colon
The rats treated with AOM and either the bacterial suspension or bacterial WEA, showed a significant increase in the mucosal content of the polyamines, putrescine, spermidine and spermine, as compared with controls (Figure 3
). The highest amounts of putrescine and spermine were measured in rats receiving S.bovis WEA fraction.

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Fig. 3. Polyamine content of the colonic mucosa of AOM-pre-treated rats. Values are means ± SE of six animals/group. Open column, AOM-treated controls; hatched column, rats treated with AOM + S.bovis; dotted column, rats treated with AOM + S.bovis WEA. Columns not sharing a common superscript differ significantly (P < 0.05).
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Discussion
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The chemical carcinogen AOM induces colonic adenomas and adenocarcinomas, and to a lesser extent small intestinal tumors (17). Administration of AOM causes a continuum of morphological changes ranging from normal intestinal epithelium to carcinoma, which are biologically and histologically quite similar to those seen in humans (17). Because of the potential progression of early changes to malignancy, the study of premalignant hyperproliferative lesions and of aberrant crypts is crucial for the understanding of the pathogenesis of colon cancer. In this regard, the identification of environmental factors that are able to enhance the malignant process is of high importance.
Traditionally, bacterial infections have not been considered as a major cause of cancer. However, bacteria have been linked to cancer by two mechanisms: chronic inflammation and production of carcinogenic metabolites (18). It has been reported that only 20% of germ-free rats were shown to develop chemically induced colon tumors as compared with 93% of their counterparts hosting normal intestinal flora (19). More recently, it was suggested that some intestinal bacteria might behave as promoters and some as anti-promoters in 1,2-dimethylhydrazine-induced colonic ACF in gnotobiotic and normal rats (20). Helicobacter pylori has been the first bacterium that has been identified as a definite cause of cancer in humans by the International Agency for Research on Cancer (18). Although numerous studies have focused on H.pylori, little is known about the flora associated with the colonic mucosa. In this regard, Swidsinski et al. (21) quantified bacteria in colonic biopsy specimens of normal and cancer patients by polymerase chain reaction. They found that the colonic mucosa of patients with colorectal carcinoma but not normal colonic mucosa was colonized by intracellular Escherichia coli. In H.pylori infections, the gastric levels of IL-8 strongly correlate with inflammation and the degree of gastritis (22). Monolayers of human colon epithelial cells exposed to invasive strains of bacteria have been shown to express pre-inflammatory cytokines including IL-8 (23). It was also reported that colonic cancer cells exposed in vitro to Clostridium difficile toxin A showed induced IL-8 production (24). In the present study, we show that S.bovis as well as cell wall antigens from this bacteria are able to increase the production of IL-8 in the colonic mucosa of rats, suggesting direct interactions between S.bovis and the colonic mucosal cells. These results are in agreement with those obtained in in vitro experiments in our laboratory showing that binding of S.bovis WEA to various cell lines including human colonic cancer cells (Caco-2) stimulates the production of IL-8 by those cells (25). Production of inflammatory cytokines such as TNF-
, IL-1ß and IL-6, and the chemokine IL-8 contribute to the normal defence mechanisms of the host (26) leading to the formation of nitric oxide and free radicals such as superoxide, peroxynitrites, hydroxyl radicals as well as alkylperoxy radicals (27). Owing to their potent mutagenicity all these molecular species can contribute to the neoplastic processes by modifying cellular DNA. On the other hand, the production in the colonic mucosa of angiogenic factors like IL-8 triggered by S.bovis antigens may also favor the progression of colon carcinogenesis (28,29).
The observation that normal rats treated with S.bovis did not develop hyperplastic colonic crypts (data not shown) indicates that the bacteria exert their pathological activity in the colonic mucosa only when preneoplastic lesions are established. The WEA from S.bovis exhibited a higher effect on the promotion of preneoplastic lesions in the colonic mucosa than the living bacteria. This was obvious from the appearance of colonic adenoma in 50% of the rats, by a higher number of aberrant colonic crypts and by the increase in the proliferation markers, namely the polyamine content and the expression of PCNA.
Preliminary studies realized in our laboratory with another oral Streptococcus species (Streptococcus gordonii) have shown that under identical experimental conditions, the number of preneoplastic lesions (ACF) in the colon was similar in S.gordonii-treated rats (22 ± 2) and in controls treated by AOM only. Our study suggests that S.bovis and related cell wall protein act as promoters of carcinogenesis in a chemically induced animal model. Such a promoting effect of S.bovis on intestinal carcinogenesis requires confirmation using another experimental model, for example transgenic mice, which are genetically predisposed to develop intestinal tumors, to determine whether such effects are also observed in non-chemically induced colon cancer models.
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Acknowledgments
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This work was supported by a grant (no. 9342) from Association pour la Recherche sur le Cancer (ARC).
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Notes
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2 To whom correspondence should be addressed Email: francis.raul{at}ircad.u-strasbg.fr 
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Received August 23, 1999;
revised November 1, 1999;
accepted December 16, 1999.