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© 2006 American Society for Nutrition J. Nutr. 136:1483-1487, June 2006

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Bioproduction of Conjugated Linoleic Acid by Probiotic Bacteria Occurs In Vitro and In Vivo in Mice1,2

Julia B. Ewaschuk, John W. Walker, Hugo Diaz and Karen L. Madsen3

Division of Gastroenterology, University of Alberta, Edmonton, Alberta, Canada

3 To whom correspondence should be addressed. E-mail: karen.madsen{at}ualberta.ca.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Probiotics have been shown to reduce the incidence of colon cancer in animal models. The mechanisms responsible for this activity are poorly defined. Conjugated linoleic acids (CLA) are a group of isomers of linoleic acid (LA) possessing anti-inflammatory and anticarcinogenic properties, which can be produced from LA by certain bacterial strains. In this study, the ability of probiotic bacteria to exert anticarcinogenic effects through the production of CLA was assessed. Incubation of probiotic bacteria (VSL3, Lactobacillus acidophilus, L. bulgaricus, L. casei, L. plantarum, Bifidobacterium breve, B. infantis, B. longum, and Streptococcus thermophilus) in the presence of LA yielded CLA production as measured by gas chromatography. Conditioned medium, containing probiotic-produced CLA, reduced viability and induced apoptosis of HT-29 and Caco-2 cells, as assessed by MTT assay and DNA laddering, respectively. Western blotting demonstrated an increased expression of PPAR{gamma} in cells treated with conditioned medium compared with LA alone. Incubation of murine feces with LA after administering VSL3 yielded 100-fold more CLA than feces collected prior to VSL3 feeding. This study supports a role for supplemental probiotics as a strategy both for attenuating inflammation and for preventing colon cancer.

KEY WORDS: • lactobacilli • bifidobacteria • colon cancer

The colonic microflora plays a critical role in human health and disease and has been implicated in the pathogenesis of colorectal carcinoma. Colon cancer is the second leading cause of cancer mortality in industrialized countries (1) and environmental factors appear to have a prominent role in its development (2,3). Studies of animal models of colon cancer provide evidence that the colonic microflora is involved in the etiology of carcinogenesis (4). Furthermore, specific strains of bacteria have been implicated in the pathogenesis of colon cancer, including Streptococcus bovis (5), Bacteroides (6), and Clostridia (7). Conversely, some probiotic strains of bacteria have demonstrated protective effects against tumor production (8–11). Lactobacillus acidophilus and Bifidobacterium longum have been shown to reduce incidence of colonic tumors and aberrant crypt foci, respectively, in animal models (12,13). A cocktail of probiotic strains was recently demonstrated to increase the colonic apoptotic index in normal rats (14). Several mechanisms for these protective actions have been defined, including binding of potential mutagens (15) and reduced activity of enzymes involved in carcinogen formation (16).

Conjugated linoleic acid (CLA)4 is a term defining a group of positional (e.g., 7:9, 9:11, 10:12, and 11:13) and geometric (i.e., cis or trans) isomers of linoleic acid (C18, cis-9:cis-12) that have been shown to exert numerous health benefits, including antiatherogenic, antidiabetic, anti-inflammatory and anticarcinogenic properties (17). In vitro, CLA inhibits the growth of HT-29 and Caco-2 colon cancer cells (18). CLA-treated SW480 colonic tumor cells possess increased caspase-3 and caspase-9 activities and reduced Bcl-2 expression compared with controls (19). CLA has been shown to reduce the incidence of colonic, skin, mammary, and prostate carcinogenesis in animal models (20). Rats supplemented with CLA showed reduced incidence of colonic tumors and increased apoptotic indices in response to the administering of 1,2-dimethylhydrazine (21), and numerous mechanisms for this action have been defined. CLA is a ligand for the peroxisome PPAR{gamma}, and growth of colon cancer cells is repressed in a dose-dependent fashion by CLA exposure (22). Furthermore, growth inhibition was abrogated by the repression of PPAR{gamma}. Activation of PPAR{gamma} by CLA, and the subsequent transcription of PPAR{gamma}-responsive genes, has been demonstrated (23). In addition, the improvement of dextran sodium sulfate colitis upon administering CLA has been attributed to PPAR{gamma} activation (23). CLA also reduced the mRNA ratio of Bax/Bcl-2 in the colonic mucosa of rats (24), thereby decreasing cellular proliferation and inducing apoptosis of the colonic mucosa. Expression of ErbB3, a protein implicated in the development of colon cancer, is also reduced in HT-29 cells when exposed to CLA (25).

Several strains of bacteria that are considered to have probiotic effects (i.e., lactobacilli and bifidobacteria) are capable of converting linoleic acid to CLA. However, CLA production has not been described as a mechanism by which probiotics exert anticarcinogenic effects. In this study, we investigate the ability of probiotic compounds currently in clinical use to produce CLA in vitro and ex vivo, and examine their effects on cell viability and apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Materials. Unless otherwise stated, all chemicals and reagents were purchased from Sigma.

    Assessment of CLA conjugation by probiotic strains. Lyophilized probiotic strains (0.01 g VSL3 [L. casei, L. plantarum, L. acidophilus, L. delbrueckii subsp bulgaricus, B. infantis, B. breve, B. longum, S. salivarius subsp. thermophilus]; VSL Pharmaceuticals) were incubated overnight at 37°C in 10 mL cell culture medium (RPMI 1640) or 10 mL Mann-Rogosa-Sharpe (MRS) broth in the presence or absence of 0.5 g/L of linoleic acid (LA) suspended in 0.05% Tween-80. Samples were centrifuged at 3000 x g for 15 min. Lipids were immediately extracted from the resultant supernatant as previously described (26). Briefly, 24 mL of 2:1 chloroform:methanol solution and 8 mL of 0.88% NaCl were mixed with 2 mL of medium. Ten mL of the lower layer were dried under nitrogen at 40°C and resuspended in hexane. Fatty acid methyl esters were produced by incubating samples with 40 µL methyl acetate and 80 µL sodium methoxide for 15 min at 50°C. Methylated fatty acids were subjected to gas chromatography on a Varian 3600 GC (Varian) using a SP2560 column (Supelco). Integration and quantitation were performed using Class-VP Chromatography Data System (version 4.2, Shimadzu Scientific Instruments).

    Cell culture studies. HT-29 and Caco-2 cells were obtained from American Type Culture Collection and cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS; Cansera) and DMEM-F12 with 5% FCS, respectively. Cells were grown to 80% confluence in 6-well plates for Western blotting and 96-well plates for viability assays. For the preparation of conditioned medium (CM), 0.01 g probiotic bacteria were incubated overnight with 0.5 g/L LA in RPMI 1640. Live bacteria were removed by filtration through a 0.2 µm syringe filter and CM was placed on HT-29 or Caco-2 monolayers. Serial 100-fold dilutions of CM were plated on brain-heart infusion medium to ensure the absence of viable bacteria.

    Western blotting. For Western blot analysis, HT-29 cells were lysed in Mono-Q buffer, and 50 µg of protein subjected to electrophoresis on 10% SDS-polyacrylamide gels as previously described (27). Anti-PPAR{gamma} (Upstate) was used to detect PPAR{gamma}, using an enhanced chemiluminescence light-detecting kit (Amersham). To confirm equal loading of protein, Western blots were stained with Ponceau S.

    Cell viability assay. Cells grown in 96-well plates were incubated for 24 h with CM (0.01 g probiotic bacteria incubated overnight with 0.5 g/L LA in RPMI 1640). The medium was aspirated, cells washed 3 times with PBS, and then incubated with 0.5 g/L (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in dimethyl sulfoxide (DMSO) for 4 h. DMSO was added to each well, incubated for 5 min, and read on a Beckman DU-640 plate reader (Beckman Coulter) at 570 nm. Background was read at 650 nm and subtracted.

    DNA laddering. HT-29 cells were seeded in 60 mm plastic dishes and allowed to reach 80% confluency. After 96 h, under increasing concentrations of CM (0, 1, 10, 20, 50, and 100%), cells were collected in lysis buffer (1 mol/L NaCl, 0.2 mol/L Tris pH 8.0, 0.1 mol/L EDTA, and 5 µL proteinase K) and incubated for 45 min at 55°C. A total of 400 µL of 5 mol/L NaCl were added and the sample was centrifuged several times (10,000 x g; 10 min). DNA was ethanol precipitated at –20°C, overnight. After centrifugation, the DNA was washed with 70% ethanol and dried. The pellet was resuspended in Tris-EDTA buffer with RNase A. DNA was electrophoresed through an ethidium bromide–containing agarose gel (1.8%).

    Animal in vivo studies. To determine whether probiotics maintained the capacity to conjugate linoleic acid to CLA in vivo, adult (14 wk) 129/SvEv mice (Taconic; n = 4) were fed 30 µL of probiotic (0.03 g VSL3 in 10 mL water) for 3 d. Mice had free access to standard laboratory chow (LabDiet, PMI; Table 1) and water. Two fecal pellets were collected from each mouse prior to and after receiving VSL3. The pellets were incubated in 10 mL anaerobic thioglycolate medium and 10 mL MRS broth containing 0.5 g/L linoleic acid at 37°C overnight. Lipids were extracted and subjected to gas chromatography for CLA, as described above. All experiments were performed according to the Institutional Guidelines for the Care and Use of Laboratory Animals in Research and with the permission of the University of Alberta Health Sciences Animal Policy and Welfare Committee.


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  		TABLE 1 Composition of laboratory diet

 
    Statistical analyses. Data are expressed as means ± SEM. Analyses were performed using the statistical software SPSS (version 13.0, SPSS). Differences between means were evaluated using 1-way ANOVA and differences among treatment means were assessed using Tukey's test, or Tamhane's T2 test when variances were unequal. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    CLA production by probiotics. All bacterial strains in the VSL3 probiotic compound converted LA to CLA to varying degrees (Fig. 1). The combination of all strains (VSL3) did not increase CLA production compared with individual strains. The highest rates of conversion were by L. bulgaricus and S. thermophilus and the lowest by L. acidophilus (Fig. 1). Conversion to the cis-9, trans-11 isomer and the trans-10, cis-12 isomer was similar by each strain; all strains produced both isomers. No CLA was detected in medium incubated with probiotics in the absence of LA (data not shown). Production of CLA was similar in MRS broth (data not shown).


Figure 1
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  		FIGURE 1  All strains of VSL3 were capable of producing both cis-9, trans-11 and trans-10, cis-12 isomers of CLA from LA. The combination of all strains (VSL3) did not increase CLA production compared with individual strains. Values are means ± SEM, n = 2. Means without a common letter differ, P < 0.05.

 
    Effects of CLA on cell viability. CM containing CLA induced a significant decrease in cellular viability compared with controls (either LA or probiotic alone, Fig. 2A). Furthermore, at 1, 10, and 20%, CM did not affect cell viability, but at 50 and 100% cell viability was significantly reduced (Fig. 2B). Conditioned medium (20–100%) from L. bulgaricus induced DNA laddering (Fig. 3).


Figure 2
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  		FIGURE 2  Growth of HT-29 colonic carcinoma cells was inhibited by CM from each strain of VSL (La, L. acidophilus; Lb, L. bulgaricus; Lc, L. casei; Lp, L. plantarum; Bb, B. breve; Bi, B. infantis; Bl, B. longum; St, S. thermophilus) (A) in a dose dependent manner (B). Values are means ± SEM, n = 3. Values without a common letter differ, P < 0.05. Black bars are control or probiotic without LA, white bars are control or probiotics with LA.

 

Figure 3
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  		FIGURE 3  At concentrations >20%, probiotic-produced CLA results in apoptosis, as demonstrated by DNA laddering, of HT-29 cells. Representative of duplicate assays.

 
    Effect of CM on PPAR{gamma} expression. CLA has been shown to activate PPAR{gamma} (23). Thus, we examined the effect of CM on PPAR{gamma} expression. Cells incubated under CM and CLA demonstrated enhanced expression of PPAR{gamma} compared with cells incubated with LA alone and probiotic alone (Fig. 4).


Figure 4
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  		FIGURE 4  Probiotic-produced CLA induces an upregulation of PPAR{gamma} in HT-29 cells. Lb, Lactobacillus bulgaricus; NT, no treatment. Representative of duplicate assays.

 
    Ex vivo CLA production. Following the administering of VSL3, fecal pellets from 129/SvEv mice had a 100-fold increase in CLA-producing capacity under anaerobic conditions (3.60 ± 0.16) compared with pellets collected prior to administering the probiotic (0.21 ± 0.09). No differences in CLA-production were seen due to the administering of probiotic upon incubation of feces in aerobic medium (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
In this study, we investigated a previously unreported mechanism of probiotic action: the production of CLA. We demonstrated that probiotic strains in VSL3 have the capacity to convert LA to CLA, inducing the upregulation of PPAR{gamma}, a reduction in cancer cell viability, and the induction of apoptosis. This LA conjugating capacity is maintained in vivo.

The ability of microbes to convert LA to CLA has been known for nearly 40 y (28) and has been investigated mainly in ruminants. CLA was originally identified as a potential anticarcinogen in grilled beef (29). The microbial production of vaccenic acid (trans-11, 18:1), which is subsequently converted to CLA by eukaryotic {Delta}-9 desaturase, yields a major source of dietary CLA in beef and in dairy products (30,31). Several studies have investigated the bioproduction of CLA by various lactobacilli and bifidobacteria (32–35), but the action of this CLA has not been investigated. Because substantial evidence exists demonstrating the capacity of CLA to inhibit carcinogenesis (36,37), the possibility that probiotics may be working through this action warranted investigation.

Although there is no direct evidence for the prevention or treatment of colorectal malignancies in humans using probiotics, numerous in vivo animal studies have been carried out. These have demonstrated that probiotics are capable of reducing the incidence of colonic tumor formation and aberrant crypt formation, thereby suppressing bacterial enzyme activities and reducing DNA damage (12,13,38–41). In addition, a synbiotic combination of prebiotics and probiotics has been demonstrated to facilitate an apoptotic response to a genotoxic carcinogen in the large intestine of rats (42). Mechanistic studies indicate that probiotics may exert anticarcinogenic properties by altering colonic metabolism, degrading carcinogens, producing antimutagenic compounds, and enhancing host immune responses (43). Conversely, Yan and Polk (44) demonstrated, in vitro, that probiotics prevent cytokine-induced apoptosis of colonic epithelial cells. In our study, we also observed an increase in cellular viability in response to a probiotic conditioned medium (Fig. 2). However, conditioned medium containing CLA resulted in the opposite effect and reduced cellular viability in a dose-dependent manner (Fig. 3). This reduction was not attributed merely to the presence of LA, because LA-containing medium did not significantly reduce cellular viability.

A previous study by Kamlage et al. (45) concluded that intestinal micro-organisms do not supply rats with systemic CLA, insofar as CLA did not accumulate in tissues upon administration of LA conjugating microbes to germ-free rats. However, those investigators found, as we did, that LA-conjugation activity of feces was increased after administering microbes known to produce CLA. In contrast, Chin et al. (46) reported that conventionally raised, linoleic acid–fed mice had a 5- to 10-fold increase of CLA content in body tissues (liver, lung, kidney, skeletal, muscle, and adipose) compared with germ-free mice. Bioformation of CLA in the proximal small intestine is less likely to occur due to the reduced microbial population relative to the ileum and colon. However, probiotic-produced CLA has the potential to be produced and absorbed by epithelial cells in the distal ileum, and also to interact with colonocytes, exerting local beneficial effects. Indirect evidence of colonic uptake of CLA is also provided by studies showing CLA to activate the nuclear receptor, PPAR{gamma}, in colonocytes (23), because such activation requires CLA uptake into the cell (47). The amount of linoleic acid available for CLA production in the colon would vary and is dependant upon the amount ingested and the efficacy of absorption in the small intestine. However, studies have shown that humans generally excrete ~20 mg of linoleic acid/d (48), suggesting that substrate is available for microbial production of CLA. Although additional LA could be made available to the colon for increased CLA production via increased dietary intake, high levels of n-6 fatty acids have been shown to promote chemically induced carcinogenesis (49). Thus, increasing LA intake is not recommended at this time (49).

There is a possibility that coprophagy by mice may introduce CLA to the small intestine, and thus making it systemically available. Coprophagy could also result in probiotics being reingested in the feces and reintroduced to the colon, thus increasing the nominal dosage.

In addition to the two isomers of CLA we reported, cis-9, trans-11 and trans-10, cis-12, there are numerous other isomers that may have been present in the CM that we did not measure, such as trans-9, trans-11 (32). There is some evidence to suggest that different isomers of CLA exert differential effects. The current study investigated only two isomers: cis-9, trans-11 and trans-10, cis-12. Combined, these isomers appear to inhibit the growth of colon cancer cells. Other CLA isomers may have been present that were not detected by the gas chromatographic methodology used in this study. Although detrimental effects of the trans-10, cis-12 isomer on eicosanoid production (50) and carcinogenesis (51) have been reported, these pertain to the administering of synthetic CLA that is subsequently absorbed and accumulates systemically. In vivo, CLA in the colon is likely unabsorbed and is thus unlikely to have similar deleterious effects.

CLA has numerous additional reported benefits, including altering body composition, improving lipid profiles, modifying both the innate and adaptive immune responses, and improving insulin resistance [reviewed in (52)]. Animal studies demonstrate that the anticarcinogenic effects of CLA are observable at dosages of 0.5–1% (w:w) of the total diet (24,53,54). Whether probiotic-produced CLA has any of these effects is unknown. However, altering colonic inflammation by locally produced CLA may have implications in the mechanisms by which probiotics ameliorate inflammatory bowel disease, and an investigation of the effects of probiotic-produced CLA on markers of inflammation is warranted.


   ACKNOWLEDGMENTS
 
The authors thank Francis Cheung, Naomi Beswick, John Kennelly, and Jody Backer for technical assistance.


   FOOTNOTES
 
1 Presented in abstract form at the American Gastroenterological Association Annual Meeting, Chicago, IL. (2005). [Ewaschuk JB, Walker JB, Madsen KL. Probiotics exert anti-proliferative effects on colonic tumor cells via production of conjugated linoleic acid (abstract). Digestive Disease Week 2005 Program, p. 844]. Back

2 Support for this study was provided by the Canadian Institutes for Health Research, the Alberta Heritage Foundation for Medical Research, and the Crohn's and Colitis Foundation of Canada. Julia Ewaschuk is supported by a joint Canadian Association of Gastroenterology/Canadian Institute for Health Research/Astra Zeneca Fellowship and the Alberta Heritage Foundation for Medical Research Incentive Award. Back

4 Abbreviations used: CLA, conjugated linoleic acid; CM, conditioned medium; LA, linoleic acid; MRS, Mann-Rogosa-Sharpe; VSL3, lyophilized probiotic bacteria (L. acidophilus, L. bulgaricus, L. casei, L. plantarum, B. breve, B. infantis, B. longum, and S. thermophilus). Back

Manuscript received 22 November 2005. Initial review completed 28 January 2006. Revision accepted 1 March 2006.


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