World J Microbiol Biotechnol
DOI 10.1007/s11274-013-1499-6
REVIEW
A potential role of probiotics in colorectal cancer prevention:
review of possible mechanisms of action
Esther Swee Lan Chong
Received: 4 December 2012 / Accepted: 16 September 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract A number of investigations, mainly using
in vitro and animal models, have demonstrated a wide
range of possible mechanisms, by which probiotics may
play a role in colorectal cancer (CRC) prevention. In this
context, the most well studied probiotics are certain strains
from the genera of lactobacilli and bifidobacteria. The
reported anti-CRC mechanisms of probiotics encompass
intraluminal, systemic, and direct effects on intestinal
mucosa. Intraluminal effects detailed in this review include
competitive exclusion of pathogenic intestinal flora, alter-
ation of intestinal microflora enzyme activity, reduction of
carcinogenic secondary bile acids, binding of carcinogens
and mutagens, and increasing short chain fatty acids pro-
duction. Reduction of DNA damage and suppression of
aberrant crypt foci formation have been well demonstrated
as direct anti-CRC effects of probiotics on intestinal
mucosa. Existing evidence clearly support a multifaceted
immunomodulatory role of probiotics in CRC, particularly
its ability to modulate intestinal inflammation, a well
known risk factor for CRC. The effectiveness of probiotics
in CRC prevention is dependent on the strain of the
microorganism, while viability may not be a prerequisite
for certain probiotic anticancer mechanisms, as indicated
by several studies. Emerging data suggest synbiotic as a
more effective approach than either prebiotics or probiotics
alone. More in vivo especially human studies are warranted
E. S. L. Chong (&)
Institute of Food, Nutrition and Human Health, Massey
University, PO Box 11222, Palmerston North 4442,
New Zealand
e-mail: esther_swee@yahoo.com.sg
to further elucidate and confirm the potential role of pro-
biotics (viable and non-viable), prebiotics and synbiotics in
CRC chemoprevention.
Keywords Probiotics
Colorectal
Colon

Cancer
Mechanisms
Inflammation
Introduction
The term ‘probiotics’, meaning ‘for life’ in Greek, was first
used in 1965 to describe any substance or organism that
was beneficial in promoting microflora balance in the
intestinal system (Lilly and Stillwell 1965). More recent
definition describes ‘probiotics’ as preparation containing
adequate number of viable and defined microorganisms
that exert beneficial health effects in the host (de Vrese
et al. 2011). The majority of the probiotics available in the
market are lactic acid bacteria (LAB), members of the
genera of lactobacilli, bifidobacteria and streptococci. In
the early 1900s, a Russian microbiologist, Elie Metchnik-
off (1845–1916) first proposed the concept of ‘antibiosis’,
which depicted the inhibition of growth and activity of one
microorganism by another (Fric 2007). He hypothesized
that Lactobacillus. delbrueckii subsp. bulgaricus present in
fermented milk could slow the aging process by sup-
pressing proteolytic bacteria in the colon. In the same
period, Tissier also showed bifidobacteria as a useful
therapy for infant diarrhea (Gionchetti et al. 2000). E. coli
Nissle 1917, a non-LAB probiotic, was found to be
effective in treating infectious intestinal disorders (Verna
and Lucak 2010). There is good evidence to support the
efficacy of certain probiotic interventions in improving
acute infectious diarrhea, antibiotic-associated diarrhea,
irritable bowel syndrome, pouchitis, ulcerative colitis,
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necrotizing enterocolitis and Crohn disease (Sanders et al.
2013; Verna and Lucak 2010).
Intestinal microbiota, colorectal cancer risk factors
and probiotics
CRC ranks the third most common malignant disease
worldwide, with approximately 1 million new cases reg-
istered in 2008 (Bray et al. 2012). The prognosis for CRC
is poor with 5-year survival rate of 30–63 % (varying
among countries) and as low as 10 % for patients with
metastatic CRC (Geier et al. 2006; Gill and Rowland
2002). Established risk factors for CRC include diet and
obesity (Huxley et al. 2013; Uccello et al. 2012). There is a
strong body of epidemiological evidence that suggests a
causative role of red meat and alcohol consumption,
whereas diet rich in vegetables, fruits and dietary fiber
confers a protective effect (Boyle and Langman 2000;
Cross and Sinha 2004; Cross et al. 2010; Huxley et al.
2013; Potter 1996; World Cancer Research Fund/American
Institute for Cancer Research 2007). Meat-related com-
pounds, specifically heterocyclic aromatic amines (HCA),
polycyclic aromatic hydrocarbons (PAH), nitrites and
nitrates, are known mutagens involved in the aetiology of
CRC (Miller et al. 2013). Germ-free rodents developed
fewer spontaneous or induced tumours than conventional
rodents, suggesting a putative role of microbiota in intes-
tinal carcinogenesis (Tlaskalová-Hogenová et al. 2011;
Vannucci et al. 2008). Indeed, studies have revealed a
positive correlation between CRC and certain commensal
bacteria, including specific E. coli types, enterotoxigenic
Bacteroides fragilis and Streptococcus bovis. As an
example, B. fragilis was found enriched in the colon of
CRC patients versus healthy subjects. There are various
possible mechanisms by which B. fragilis may contribute
to colon carcinogenesis, including stimulation of cancer
cell proliferation and inducing inflammation via the TH17-
dependent pathway. B. fragilis has been implicated to
increase the genotoxicity of HCA, mutagens generated
during high-temperature cooking of meat (Culpepper and
Mai 2013). Conversely, an inverse association between
CRC and bifidobacteria as well as other butyrate-producing
bacteria was supported by existing evidence (Culpepper
and Mai 2013).
A study comparing composition of bacteria in the stool
of CRC patients and healthy individuals found a significant
dysbiosis. Specifically, there was a significant decrease in
core species bacteria, including bifidobacteria and Rumi-
nococcus bromii (Sobhani et al. 2013). Bifidobacteria was
also found reduced in the mucosa-adherent microbiota in
CRC patients, when compared to healthy individuals (Zhu
et al. 2013). Alteration of intestinal microbiome, including
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World J Microbiol Biotechnol
a decline in bifidobacteria, might be partly accountable for
the onset of CRC (Ohigashi et al. 2013). Firmicutes was
found to be abundant in the intestinal lumen in CRC
patients, and it was the most dominant bacteria found
adherent to precancerous adenomatous polyps (Sobhani
et al. 2013; Zhu et al. 2013). Diet can influence the com-
position of the microbiota. For instance, prebiotics such as
resistant starch, fructo-oligosaccharides and galacto-oligo-
saccharides, are non-digestible carbohydrates, which can
selectively promote the growth of bifidobacteria. Increase
in Ruminococcus bromii and a decrease in Firmicutes was
also observed with ingestion of prebiotic resistant starch
(Huxley et al. 2013). Therefore, dietary component such as
probiotics, prebiotics, or combination of both (synbiotics),
may protect against CRC partly by preventing intestinal
dysbiosis (changes in the normal microbiota). On the
contrary, obesity could increase the risk of CRC, possibly
by causing an imbalance of the intestinal microbiota.
Specifically, a decrease in bifidobacteria and an increase in
Firmicutes has been associated with obesity (Huxley et al.
2013). Age is another risk factor for CRC (Dunlop et al.
2013). A large cohort study, involving 35,292 adults aged
18–96 years, reported that bifidobacteria significantly
decreased, while E. coli and enterococci increased with age
(Enck et al. 2009). Therefore, alteration in the composition
of gut microbiota may be a part of the link between aging
and CRC.
Inflammation is increasingly recognised for its role in
colorectal tumour initiation and promotion (Grivennikov
2013). During the process of inflammation, reactive oxygen
species (ROS) and nitrogen intermediates are produced by
activated macrophages and neutrophil, causing DNA
damage and mutations of the colonic epithelial cells.
Inflammation causes disruption of intestinal barrier func-
tion, such as decrease in protective mucins and antimi-
crobial peptide production, which in turns increases
accessibility of the inflamed site to food-borned mutagens
and microorganisms with genotoxic capabilities present in
the lumen (Arthur et al. 2012; Grivennikov 2013). All these
events promote mutagenesis required for tumour initiation.
Inflammation also contributes to tumour promotion by
enhancing cancer cell proliferation and survival via various
possible pathways (e.g. Akt, NF-kB, Wnt/b-catenin)
(Grivennikov 2013).
Inflammatory bowel disease (IBD), such as ulcerative
colitis (UC) and Crohn’s disease (CD), is a well recognised
risk factor for CRC (Sobhani et al. 2013; Zhu et al. 2013).
Pathogenic invasion can give rise to acute colitis, however,
it appears that IBD is more associated with intestinal
dysbiosis (Azcárate-Peril et al. 2011; Grivennikov 2013).
Several microorganisms were over-represented in IBD,
including various bacteria from clostridia group, fusobac-
trium, mycobacterium, adherent invasive E. coli, Proteus
World J Microbiol Biotechnol
mirabilis, Klebsiella pneumonia and proteobacteria such as
Helicobacter spp (Grivennikov 2013; Zhu et al. 2013). For
instance, a 100-fold increase in E. coli was observed in an
experimental model of intestinal colitis (Arthur et al.
2012). Conversely, the abundance of bifidobacteria was
found to be reduced in patients with IBD (Fyderek et al.
2009).
Certain microorganisms are responsible for inducing and
maintaining the inflammatory disease. For examples,
microorganisms including B. fragilis, Citrobacter roden-
tium, adherent E. coli and Clostridium difficile have been
shown to disrupt intestinal barrier, and subsequently
inducing IBD and CRC in some cases (Grivennikov 2013;
Zhu et al. 2013). A recent study demonstrated monocol-
onization with either mouse commensal adherent-invasive
E. coli NC101 or human commensal Enterococcus faecalis
OG1RF caused severe colitis in azoxymethane (AOM)-
treated IL10-/- mice (Arthur et al. 2012). Both treatments
resulted in similar levels of colitis and profile of cytokines
involved in inflammation, including IL-6, IL-17, IL-18, IL-
23, IL-1b, IFN-c and TNF-a. Interesting, while majority of
the mice treated with E. coli NC101 developed invasive
mucinous carcinoma, mice treated with E. faecalis rarely
develop tumours. The authors detected the presence of
polyketide synthases (pks) in E. coli NC101, but not E.
faecalis. Furthermore, pks? E. coli was detected more fre-
quently in CRC patients and IBD patients than controls (67
vs. 40 vs. 21 %). Their further investigations confirmed pks
alone could promote DNA damage in vitro and promote
tumorigenesis in mice. Pathogenic island pks is involved in
sysnthesis of genotoxin named colibactin, which has been
associated with CRC (Brotherton and Balskus 2013). The
findings of Arthur et al. (2012) indicate an important role of
pks? bacteria in chronic inflammation, as well as the
development and invasiveness of CRC. Taken together,
existing evidence suggest intestinal dysbiosis can induce
inflammatory diseases and vice verse. Moreover, inflam-
mation-associated expansion of microorganisms with
genotoxic capabilities such as pks? bacteria and Bacteroi-
des fragilis, can promote colorectal tumorigenesis.
Collectively, changes in intestinal microbiota composi-
tion are at least part of the link between the CRC and
several key risk factors discussed, including diet, obesity,
aging, and inflammation. Therefore, dietary intake probi-
otics may represent a logical strategy to restore the normal
microbiome thus reduce the risk of CRC. Pala et al. (2011)
reported significant CRC risk reduction attributed to
yoghurt consumption among 45,241 volunteers recruited
from the European Prospective Investigation into Cancer
and Nutrition (EPIC) cohort. There are several strengths of
this study. In particular, given the involvement of five
EPIC centres which varied considerably in terms of food
intake, a broad range of yoghurt intake was covered, which
allowed sufficient power to detect an association between
CRC and yogurt consumption. The food questionnaire
asked specifically about yogurt consumption alone, not
combined with other dairy products. Other strengths
claimed by the authors include more precise definition of
yogurt exposure, and less variation in probiotic content of
yohurt, compared to other similar studies. One main
weakness of this study, however, was that healthy behav-
iours or lifestyle was found to be associated with yogurt
consumption, thus could not be completely ruled out as a
possible confounding factor, though the results had been
adjusted for other CRC risk factors (Pala et al. 2011). A
randomized dietary intervention trial, involving 398 CRC
patients, showed daily consumption of L. casei (1 g after
each meal), was effective in preventing the development of
colorectal tumours (Ishikawa et al. 2005). Currently, there
is a lack of human data which directly link probiotics
consumption and the risk of CRC. Nevertheless, a number
of in vitro and in vivo mechanistic studies have provided
evidence that probiotics may prevent the risk of CRC. A
broad and complex range of possible mechanisms have
been reported, as depicted in Fig. 1 and reviewed in further
details below.
Potential mechanisms of probiotic action in CRC
chemoprevention
Intraluminal effects
Adhesion of probiotics and competitive exclusion
of pathogenic intestinal flora
Probiotics may reduce the risk of CRC by competitive
exclusion of pathogenic bacteria involved in carcinogene-
sis. Animal studies have shown that ingestion of lactoba-
cilli and bifidobacteria are able to increase LAB and reduce
the faecal putrefactive microorganisms (e.g. coliforms) that
have been implicated with synthesis of putative carcino-
gens in the colon (Gaudier et al. 2005; O’Mahony et al.
2001; Rafter 2003). This effect is linked to a decrease in
tumorigenesis in the IL-10 knockout mice model
(O’Mahony et al. 2001). A fermented milk, containing B.
lactis, L. lactis, S. thermophilus, and L. bulgaricus, was
found to improve inflammation in a mouse model of colitis
(Veiga et al. 2010). Administration of the fermented milk
resulted in an increase in butyrate-producing bacteria, and
a concomitant decrease of enterobacteriaceae strains
Klebsiella pneumoniae and Proteus mirabilis, which were
capable of inducing colonic inflammation (Veiga et al.
2010). A human study showed that Lactobacillus aci-
dophilus administered through fermented milk decreased
faecal putrefactive flora with a concomitant increase in
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Fig. 1 Mechanisms of action of probiotics in CRC prevention. There
are various possible mechanisms of action by which probiotics may
play a chemopreventive role in CRC including: competitive exclusion
of pathogenic intestinal flora, alteration of intestinal microflora
enzyme activity, reduction of carcinogenic secondary bile acids,
binding of carcinogens and mutagens (e.g. heterocyclic amines),
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World J Microbiol Biotechnol
reduction of DNA damage, suppressing the formation of aberrant
crypt foci, improving barrier function, increasing short chain fatty
acids production, improving folate status that in turn may modulate
DNA methylation in colonocytes and modulation of immune response
(e.g. controlled inflammation and humoral response) (keys: ? |
blocking action)
World J Microbiol Biotechnol
commensal lactobacilli (Ayebo et al. 1980). Similarly, a
randomized, double-blind and placebo controlled trial
(n = 26 healthy adults) demonstrated three-week con-
sumption of a probiotics strain Lactobacillus plantarum
through fermented rose-hip drink (0.7 g probiotics/100 ml)
significantly increased the faecal bacfidobacteria and lac-
tobacilli; a decrease in pathogenic sulphite-reducing clos-
tridia was evident after 1 week of probiotics treatment
(Johansson et al. 1998). Perioperative administration of B.
longum and L. johnsonii was found to be effective to
reduce pathogens enterococci and enterobacteriaceae in
CRC patients (Gianotti et al. 2010). In the Simulator of the
Human Intestinal Microbial Ecosystem (SHIME) model, L.
acidophilus or L. casei was shown to increase LAB with a
concomitant decrease of fecal coliforms and clostridia
(Chaikham et al. 2012).
Competitive exclusion of pathogenic microflora by
probiotics could involve two types of competitions occur-
ring among the intestinal microbiota, i.e. competition for
nutrients and competition for adhesion at the intestinal
mucosa. There are limiting nutrients available at the distal
part of the colon. It has been suggested that probiotics
compete for nutrients and grow at the expense of other
intestinal microflora (Fooks and Gibson 2002). Adhesion is
a key prerequisite for colonization of microorganisms in
the intestinal cavity (Tareb et al. 2013). in vitro studies
demonstrated that L. acidophilus, L. casei and L. plantar-
ium were highly adhesive to the intestinal mucosa, thus
obstructing or excluding the adhesion of enteropathogens
such as Salmonella typhimurium and E. coli (Bernet et al.
1994; Dhanani and Bagchi 2013; Hudault et al. 1997). L.
johnsonii and L.casei Shirota were shown to reduce the
adhesion of Salmonella typhimurium in an intestinal
mucosa model (Tuomola et al. 1999). L. acidophilus was
inhibitory to growth and adhesion of pathogen Campylo-
bacter jejuni to Caco-2 cells, by a competitive exclusion
mechanism (Campana et al. 2012).
Two probiotic strains, L. rhamnosus CNCM-I-3698 and
L. farciminis CNCM-I-3699 were found to be highly
adherent to various intestinal matrix models, and elicit a
high exclusion potential against the binding of pathogens,
especially Campylobacter jejuni (Tareb et al. 2013). In
addition, these probiotics displayed non-species specific
coaggregation with pathogens, including Salmonella,
E. coli, Listeria monocytogens and Campylobacter. This
mechanism reduces the distance between the probiotics
and pathogens, thus enhancing the efficiency of antimi-
crobial lactic acid and bacteriocins produced by probiotics
to act against the pathogens (Tareb et al. 2013). Inter-
estingly, this study found that both heat-treated L.
rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-
3699 were more effective than their viable forms in terms
of adhesion to intestinal mucosa and exclusion of
pathogens. The authors hypothesized that compounds such
as exopolysaccharides, produced by cells as protective
barrier against heat, may in fact increase the adhesive
potential of heat-killed probiotics. Nonetheless, as
reviewed by Tareb et al. (2013), some studies have shown
drastic decrease in adhesive ability of probiotics due to
heating. Further investigations are warranted to confirm
the effects of heat treatment on the adhesion potential of
probiotics in the intestinal mucosa.
Alteration of intestinal microflora enzyme activity
The chemopreventive role of probiotics for CRC is also
supported by their ability to reduce intestinal microflora
enzyme activity. The human body detoxifies foreign
compounds and drugs via hepatic synthesis of glucuronides
prior to their entering into the intestines. A bacterial
enzyme, b-glucuronidase, with broad substrate specificity
can hydrolyse a number of glucuronides, causing the
release of carcinogens into the colon, including PAH (e.g.
benzo[a]pyrene), an important risk determinant for CRC
(Rafter 2003). A manipulation of this enzyme has been
shown to modulate colonic carcinogenesis in rat model
(Takada et al. 1982; Verma and Shukla 2013a, b). Similar
to b-glucuronidase, another bacterial enzyme named b-
glucosidase, breaks down plant gycosides cycasin in the
colon, generating aglycones, the majority of which are
carcinogens (Laqueur et al. 1983; McIntosh et al. 2013;
Salminen et al. 1998). Nitroreductase reduces the N-nitro
compounds (e.g. nitrobenzenes, nitropyrenes) to amines,
which are usually carcinogenic and mutagenic; nitrate
reductase participates in the generation of highly toxic and
carcinogenic nitrite (Facchini and Griffiths 1981; Hamer
et al. 2012; Salminen et al. 1998). Biochemical analysis of
faecal samples has shown that nitroreductase activity is
significantly higher in colon cancer patients than healthy
subjects (Nakamura et al. 2002). Azoreductase has also
attracted attention because of its involvement in converting
the azo compounds contained in some drugs and food
colouring agents to carcinogenic aromatic amines (Hamer
et al. 2012; Nakamura et al. 2002). Commensal bacteria
clostridia, E. coli and B. fragilis have exhibited high
activities of these procarcinogenic enzymes (i.e. b-glucu-
ronidase, b-glucosidase, nitroreductase and azoreductase)
(Cole et al. 1985; Nakamura et al. 2002; Verma and Shukla
2013b). Studies have also demonstrated clostridia, B. fra-
gilis and E. coli to be over-presented in colon cancer
patients (Culpepper and Mai 2013; Drasar et al. 1976;
Legakis et al. 1981). Taken together, modulation of bac-
terial enzyme activities represents an important chemo-
preventive approach for CRC.
Lactobacilli and bifidobacteria have much lower enzyme
activities associated with carcinogenesis compared with
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clostridia, bacteroides and eubacteria (Burns and Rowland
2000). Furthermore, animal studies have demonstrated that
probiotics preparations consisting LAB are able to reduce the
activity of above mentioned procarcinogenic enzymes
(Chang et al. 2012; Goldin and Gorbach 1984; Kulkarni and
Reddy 1994; Lee et al. 2009a; Rowland et al. 1998; Verma
and Shukla 2013b). For instance, L. GG or L. acidophilus
treatment led to a significant reduction in b-glucuronidase
activity, while the administration of L. casei or L. plantum
decreased the nitroreductase activity in 1,2-dimethylhydra-
zine dihydrochloride (DMH)-treated rats (Verma and Shukla
2013b). This study observed a reduction of b-glucosidase
activity with B. bifidum treatment. Another study using the
DMH model showed L. acidophilus KFRI342 inhibited b-
glucosidase and b-glucuronidase activity (Chang et al.
2012). B. longum was also found to lower these two enzyme
activities in healthy rats (Lee et al. 2009a).
Several small clinical trials showed similar inhibitory
effects of LAB on these carcinogenic enzymes (Bouhnik
et al. 1996; Lee et al. 2013; Ling et al. 1992, 1994; Marteau
et al. 1990). For instance, a recent study showed consump-
tion of probiotic yoghurt, containing L. GG, resulted in a
decrease in b-glucuronidase activity in patients with irritable
bowel syndrome (Lee et al. 2013). A randomised placebo-
controlled study conducted by Bouhnik et al. (1996) dem-
onstrated that bifidobacterium species reduced the b-glucu-
ronidase activity in human intestine (n = 12). A larger
randomized placebo-controlled study involving 64 female
subjects reported the effects of Lactobacillus GG in reducing
faecal enzyme activities of b-glucuronidase and nitrore-
ductase (Ling et al. 1994). Lactobacillus acidophilus and L.
casei Shirota were also shown to reduce colonic bacterial
enzyme activity in humans (De Preter et al. 2008; Goldin
et al. 1980; Spanhaak et al. 1998). However, some lactoba-
cillus strains such as L. rhamnosus DR20 and L. plantarum
failed to reduce bacterial b-glucuronidase activity in human
studies (Goossens et al. 2003; Tannock et al. 2000). Thus, the
ability of probiotics lactobacilli to reduce the enzyme
activities of intestinal bacteria appears to be strain-specific.
It is plausible that probiotics modulate the bacterial
enzyme activity in the colon by a combination of mecha-
nisms including: (1) competitive exclusion of pathogenic
microflora; (2) ability of LAB in synthesizing antibacterial
substance (e.g. bacteriocin) that inhibit the growth of other
microorganism; and (3) ability to generate acids (e.g. lactic
acids) that lower the pH of the colon, which leads to
changes in bacterial enzyme activities in the colonic lumen
(Commane et al. 2005).
Reduction of carcinogenic secondary bile acids
As shown in epidemiological studies, high-fat diets have
been associated with increased risk of CRC (Boyle and
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World J Microbiol Biotechnol
Langman 2000; Ou et al. 2012). This correlation can be
partially explained with respect to bile acid metabolism.
Increased dietary fat intake leads to higher hepatic syn-
thesis of primary bile acids, cholic acid (CA) and cheno-
deoxycholic acid (CDCA), which are needed for lipid
digestion and absorption in the small intestine. Most of
these primary bile acids are reabsorbed in the ileum and
circulated back to the liver via the portal vein (i.e. enter-
ohepatic circulation). However, a proportion escapes the
enterohepatic circulation (20 %) and enters the colonic
lumen (Nagengast et al. 1995). In the colon, CA and CDCA
are metabolized by anaerobic microorganisms (e.g. Clos-
tridium leptum, Clostridium bifermentans, Eubacterium
lentum) into the secondary bile acids deoxycholic acid
(DCA) and lithocholic acid (LCA) (Pavlović et al. 2012;
Takahashi and Morotomi 1994). This microbial biotrans-
formation involves 7a-dehydroxylation catalyzed by a
bacterial enzyme, 7a-dehydroxylase. Both DCA and LCA
are highly genotoxic and mutagenic (Nagengast et al. 1995;
Wilpart and Roberfroid 1986). DCA has also been impli-
cated with mucosal proliferation, predisposing the host to
an increased risk of CRC. Secondary bile acids induce
mucosal proliferation via various possible mechanisms
including: (1) stimulation of protein kinase C, which plays
an important role in tumourigenesis and growth factors
activities; and (2) exerting cytotoxic effects on epithelial
cells, which leads to compensatory cell renewal (Nagengast
et al. 1995). Furthermore, both DCA and LCA might
stimulate the biosynthesis of mutagenic fecapentaenes
present in feces (Ou et al. 2012). A study found a strong
correlation between constipation and colon cancer (Prom-
thet et al. 2010). It has been hypothesized that constipation
led to longer transits time in the colon, thus extending the
exposure of intestinal mucosa to mutagens in the lumen,
including secondary bile acids and fecapentaenes (Prom-
thet et al. 2010).
Indeed, epidemiological studies have observed that
populations with a high risk of CRC and high animal fat
consumption excrete more secondary bile acids (Ou et al.
2012, 2013; Reddy et al. 1983). The recent studies of Ou
et al. (2012, 2013) showed that African Americans, who
consumed a high-fat diet, had higher concentrations of
secondary bile acid (DCA and LCA), as compared to native
Africans who consumed a low-fat diet. Furthermore, the
butyrate-producing bacteria were significantly less abun-
dant in the African Americans than the native Africans.
Studies also showed that patients with colorectal adenomas
had elevated serum DCA level (Bayerdörffer et al. 1993,
1995; Pavlović et al. 2012). Collectively, existing data
suggested a pathogenic role of secondary bile acids in the
development of CRC.
Common probiotics strains were found to be lack of 7a-
dehydroxylase activity, thus unable to metabolize or
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convert CA into secondary bile acids (Kurdi et al. 2003;
Pavlović et al. 2012; Takahashi and Morotomi 1994).
Furthermore, probiotics could reduce the formation of
secondary bile acids by physically binding to primary bile
acids, meaning less substrate for 7a- dehydroxylation (De
Boever et al. 2000). An in vitro study a possible mecha-
nism, involving the binding of CA to an exocellular
polysaccharides (EPS) of probiotics, that adhered to the
cells as capsular EPS (Pigeon et al. 2002). These authors
hypothesized that the presence of EPS would facilitate the
removal of bile acids from the body. Indeed, a recent
animal study showed administration of L. plantarum
KCTC3928 led to a significant increase in fecal bile acid
excretion by 45 % (Jeun et al. 2010). An in vitro investi-
gation by Kurdi et al. (2003) provide a different mecha-
nism by which probiotics could reduce the production of
carcinogenic secondary bile acid. They demonstrated that
many species of bifidobacteria and lactobacilli were able to
entrap CA, the most abundant primary bile acids in the GI
tract (30–40 %) (Kurdi et al. 2000, 2003). Entrapment of
CA resulted in less substrate for bacterial 7a-dehydroxy-
lation process, thus less secondary bile acids formation.
They hypothesized that CA accumulation in these probio-
tics cells was driven by two possible mechanisms: (1) CA
diffused through the bacterial membrane and dissociated in
the bacterial cytoplasm via the transmembrane proton
gradient force, and (2) higher cytoplasmic or intracellular
pH of the bacteria (i.e. DpH) led to further diffusion and
dissociation of CA. The negatively charged cholate anion
was entrapped in the bacterial cytoplasm due to their
polarity.
Collectively, these in vitro and animal findings indicated
that probiotics could possibly reduce secondary bile pro-
duction in the human body. Indeed, a recent clinical study
revealed probiotic Clostridium butyricum Miyairi (3.0 g/
day) administration for 4 weeks significantly reduce serum
DCA level in patients with pancolitis (Sato et al. 2012).
Nevertheless, more clinical studies are required to dem-
onstrate the effects of probiotics on bile acid profile in
humans and its implications in CRC.
Binding of carcinogens and mutagens
Apart from high-fat diets, diets that are high in meat have
also been associated with a higher risk of CRC, mainly
attributed to some meat-related carcinogens and mutagens.
These compounds include N-nitroso compounds, HCA
such as 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and
3-amino-1-methyl-5H-pyrido[4,3-b]indole (TrpP2), as well
as polycyclic aromatic hydrocarbons such as benzo(a)pyr-
ene (BaP) found abundant in meat cooked at high
temperature (Cross and Sinha 2004; Cross et al. 2010;
Geier et al. 2006; Zhao et al. 2013).
A recent study investigated the binding ability of 15
lactobacilli strains to BaP in vitro (Zhao et al. 2013). L.
plantarum CICC 22135 and L. pentosus CiCC 23163 were
shown to exhibit the highest binding ability. Cell viability
was not a prerequisite for this binding effect, since both
heat-killed and viable forms of these probiotics displayed
similar binding efficiency. Their mechanistic experiment
demonstrated that the binding of these probiotics to BaP
was resulted from a physical adsorption of BaP to pepti-
doglycans, a cell wall component (Zhao et al. 2013).
Another in vitro study showed probiotics significantly
decreased b-glucuronidase activity stimulated by PhIP
treatment (Nowak et al. 2012). The authors regarded
binding of probiotics to PhIP as one of the possible
mechanisms underlying their findings.
Probiotics LAB were previously shown to bind HCA
carcinogens including IQ and TrpP2; this action correlated
significantly with a decrease in mutagenicity (Orrhage
et al. 1994). An animal study demonstrated that various
lactobacillus strains were highly effective in preventing
HCA-induced DNA damage partly because of their ability
to adhere to HCA directly (Zsivkovits et al. 2003). Others
also showed that the absorption of TrpP1 and TrpP2 in GI
tract and various tissues were reduced by dietary LAB,
plausibly by their adhesion to these mutagens in the
intestine (Orrhage et al. 2002; Zhang and Ohta 1993). In
humans, consumption of L. casei was shown to reduce
urinary mutagens associated with fried ground beef, sug-
gesting the ability of this probiotic microorganism to bind
and degrade these mutagens (Hayatsu and Hayatsu 1993).
Overall, these studies have demonstrated that probiotics
can decrease the bioavailability of the HCA mutagens,
preventing the interactions between these mutagens and
colonic cells through the binding mechanism.
Several in vitro studies demonstrated certain lactobacilli
strains (e.g. L. rhamnosus GG, L. rhamnosus LC-705, L.
rhamnosus TISTR 541) could also bind to aflatoxin B1, B2,
G1, and G2 carcinogens produced by Aspergillus fungi and
commonly found in moldy foods, especially nuts (Cenci
et al. 2008; Elsanhoty et al. 2013; Haskard et al. 2001;
Hernandez-Mendoza et al. 2009; Kankaanpää et al. 2000).
Noteworthily, heat treatment had been found to enhance
the binding capacity of tested probiotics to aflatoxin B1, B2,
G1, and G2 (Elsanhoty et al. 2013). It is now clear that
microorganism cell wall elements, particularly peptido-
glycans and polysaccharides, play a key role as the binding
site for mutagens, including aflatoxin, HCA and ben-
zo(a)pyrene (Cenci et al. 2008; Pizzolitto et al. 2012;
Sreekumar and Hosono 1998a, b; Zhang and Ohta 1991;
Zhao et al. 2013).
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This binding mechanism between carcinogens and pro-
biotics is dependent on strains, mutagens and pH (i.e. can
be reversed under neutral or alkaline pH conditions). Dif-
ferent probiotics strains were shown to vary in their ability
to bind a particular mutagen (Elsanhoty et al. 2013; Her-
nandez-Mendoza et al. 2009; Zhao et al. 2013). Type of
mutagen is another determinant. For instance, Bifidobac-
terium longum and L. acidophilus were found bound poorly
to AFB1, but effectively to HCA mutagens IQ and PhIP
in vitro (Bolognani et al. 1997). Nonetheless, these authors
did not observe any reduction in absorption or tissue dis-
tribution of these mutagens and their hepatic genotoxicity
activity in an animal model. Noteworthily, the degree of
binding varied according to pH conditions (e.g. greatest at
pH 5 for B. longum). Therefore, this lack of effect could be
explained by the marked variations in pH along the GI
tract. Specifically, binding of carcinogens by probiotics
might not take place or might be reversible in certain
regions of the GI system, where the pH is neutral or
alkaline (e.g. ileum, certain part of colon) (Bolognani et al.
1997). Another animal study, however, had demonstrated
that probiotic L. casei Shirota treatment was effective to
reduce blood serum aflatoxin B1 (AFB1) and hepatotoxic
effects of aflatoxin in AFB1-treated rats (Nasrabadi et al.
2013). A recent human study suggested binding of probi-
otics to HCA could have partially contributed to the
reduced fecal mutagenicity (Shaughnessy et al. 2011).
Overall, while in vitro evidence appears to be convincing,
more research is warranted to confirm the role and capacity
of probiotic in binding carcinogens in vivo, strain differ-
ences, and the clinical implications for CRC.
Chemopreventive role of short chain fatty acids
One of the key roles of colonic microflora is to recover
energy from the carbohydrates that are not digested in the
upper GI tract, via fermentation in the colon. Fermentable
carbohydrates include dietary fibers, resistant starch, non-
starch polysaccharides (e.g. cellulose, pectins), non-
digestible oligosaccharides (e.g. inulin, oligofructose) and
lactulose (Challa et al. 1997; Salminen et al. 1998; Wol-
lowski et al. 2001). The colonic fermentation process
produces 1- to 6-carbon SCFA as the chief metabolites,
namely acetate, propionate and butyrate in the ratio of
3:1:1 (Hijova and Chmelarova 2007). Studies had shown a
population with low incidence of colon colorectal polyps
and colon cancer (e.g. native Africans) had high fecal
SCFA concentrations (Ou et al. 2012, 2013; Segal et al.
1995). The total faecal SCFA concentration was signifi-
cantly inversely related to total faecal pH (Segal et al.
1995). The findings of this study agreed well with an earlier
study by Walker et al. (1986), which investigated the
relation between faecal pH and prevalence of colon cancer
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among four South African populations. The White popu-
lation, who had a significantly higher mean faecal pH
value, tended to have a higher prevalence of colon cancer
than the other groups of populations with lower mean fecal
pH value (i.e. blacks, Indians, Coloureds) (Walker et al.
1986).
It had been shown that probiotic bacteria can up-regulate
the SCFA production and could involve several mecha-
nisms. Probiotics such as Lactobacillus plantarum and
Butyrivibrio fibrisolvens were shown to produce SCFA
directly during the fermentation in vivo (Johansson et al.
1998; Ohkawara et al. 2005). In the SHIME model,
encapsulated L. acidophilus LA5 or L. casei 01 along with
a pressurized longan juice product significantly increase
lactobacilli and bifidobacteria, which led to an enhanced
the fermentative capacity of the ecosystem, as indicated by
a marked increase in production of SCFA, including pro-
pionate, acetate and butyrate (Chaikham et al. 2012).
Administration of milk containing probiotics, B. lactis, L.
lactis, S. thermophilus, and L. bulgaricus, significantly
increased the production of these three SCFA, while
causing a decrease in lactic acid in a mouse colitis model
(Veiga et al. 2010). Concurrently, this study observed an
increase in endogenous bacteria Anaerostipes caccae and
Eubacterium hallii. Indeed, these two bacteria had been
previously reported as lactic-acid consuming as well as
butyrate-producing bacteria (Duncan et al. 2002; Duncan
et al. 2004). These microflora utilized lactic acid such as
those produced by probiotics LAB and convert it into
SCFA, a mechanism that could partly explain the finding of
Veiga et al. (2010) on SCFA levels. Several strains of
lactobacilli, bifidobacteria, and Entercoccus faecalis were
shown to enhance the SCFA production by pig caecal
bacteria (Sakata et al. 2003). The authors suggested that
probiotics accelerate the break down of carbohydrates that
are resistant to indigenous microflora, thereby increasing
the SCFA production in vivo.
The anticarcinogenic effects of SCFA, especially buty-
ric acid, were well demonstrated in relation to CRC
(Commane et al. 2005; Salminen et al. 1998). As shown by
the study by Ohkawara et al. (2005), the reduction in ACF
observed in experimental animals was partially associated
with high level of butyrate acids produced by Butyrivibrio
fibrisolvens. Lankaputhra and Shah (1998) conducted an
in vitro study to investigate the antimutagenic capacity of
SCFA against eight mutagens. It was shown that butyric
acid exerted inhibitory effect on all these mutagen. Fur-
thermore, butyrate exhibited the highest antimutagenic
effect compared to other organic acids produced by lacto-
bacilli and bifidobacteria (e.g. acetate acid, lactic and
pyruvic acids) (Lankaputhra and Shah 1998). The mecha-
nisms of chemopreventive action of butyrate are multi-
faceted as discussed below.
World J Microbiol Biotechnol
About 70–90 % of the butyrate produced by colonic
fermentaion are metabolised by colon cells (Hijova and
Chmelarova 2007). Indeed, butyrate is the principal sub-
strate for oxidative metabolism in the colonocytes, i.e. the
major energy source for the cells, especially in the distal
colon (Salminen et al. 1998; Sengupta et al. 2006). Rat distal
colon cells treated with butyrate were found to be more
resistant to DNA and cell damage caused by oxidative stress
(induced by hydrogen peroxide), compared to cells treated
with acetate and propionate (Abrahamse et al. 1999). This
observation could be explained by the fact that butyrate is
the most preferred fuel, among various SCFA, for colono-
cytes (Hijova and Chmelarova 2007). Thus, the colon cells
treated with butyrate had higher energy level than those
treated with other SCFA to fight against oxidative stress.
Butyrate had been shown to stimulate the production of
glutathione S-transferase (GST), an important Phase II
detoxifying enzyme. Specifically, GST is involved in the
detoxification of compounds that can cause oxidative
damage (e.g. hydrogen peroxide). GST also catalyses the
conjugation between electrophilic mutagen and sulfhydryl
compounds (e.g. reduced glutathione and cysteine), a
process that produces remarkably stable thioether com-
pounds (i.e. mutagen becomes inactive or non-mutagenic)
(Challa et al. 1997; Jakoby 1978; Lo et al. 2002). An
in vitro experiment demonstrated that butyrate treatment
resulted in higher GST expression and activity, which in
turn reduced the genotoxicity induced by 4-hydroxy-2-
nonenal in human colon cancer cells lines HT29 and HT29
clone 19A (Ebert et al. 2001). Induction of GST could be
one of the key mechanisms underlying the antimutagenic
action of butyrate, which might partly explain the chemo-
preventive role of probiotics in CRC. As demonstrated by
Challa et al. (1997), administration of Bifidobacterium
longum and lactulose inhibited the ACF formation induced
by AOM in rats. Concurrently, there was an increase in
GST activity and decrease in cecal pH, which correlated
with a lower number of ACF observed in this treatment
group than the controls. The authors did not measure the
SCFA profile in these experimental animals. Nonetheless,
it is possible that their findings (i.e. on ACF, GST and cecal
pH) were closely related to an enhanced SCFA production,
particularly butyrate, attributed to the probiotics treatment.
The role of butyrate in cell cycle modulation, colon cell
differentiation and apoptosis was well demonstrated by
in vitro studies (Boffa et al. 1992; Di Luccia et al. 2013;
Hague et al. 1995; Matsuki et al. 2013; Salminen et al.
1998; Young et al. 2005). In relation to CRC, several
studies showed the effect of butyrate in activating p21,
which in turn could impede the cell cycle at G1 phase,
allowing genomic repair to take place (Archer et al. 1998;
Di Luccia et al. 2013; Siavoshian et al. 2000). A study
demonstrated the effect of butyrate in modulating some
twenty-three genes in HT29 colon cancer cell, including a
gene called tob-1. A high level of tob-1 was shown to cause
growth arrest of these colon cancer cells, plausibly at the
G1/S phase of cell cycle (Della Ragione et al. 2001). A
recent in vitro study suggested probiotics suppressed can-
cer cell proliferation, by downregulating cyclin D1 and
cyclin E1 gene expression, and SCFA might be the active
factor for this effect (Matsuki et al. 2013). The authors also
observed an upregulation of gene encoding for alkaline
phosphatase, a key biomarker for intestinal epithelial cell
differentiation. Moreover, butyrate, as an inhibitor of his-
tone deacetylase, could increase histone acetylation that
played a regulatory role in transcriptional activity in
eukaryotic cells (Struhl 1998).
An animal study showed that increased luminal butyrate
facilitated the rate of apoptosis in the distal colon, espe-
cially at the base and proliferative zone of the crypt of the
colon of AOM-treated rats (Clarke et al. 2012). Both viable
and heat-killed formed of L. GG and L. paracasei were
shown to inhibit cell proliferation, as well as induce
apoptosis in CRC (DLD-1) and gastric cancer (HGC-27)
cell lines (Orlando et al. 2012). In another in vitro study,
combined treatment of probiotics (L. casei, L. acidophilus)
and 5-fluorouracil (5-FU) was more effective than 5-FU
alone in inducing apoptosis of CRC cells (LS513) (Baldwin
et al. 2010). Several studies showed the effects of SCFA,
particularly butyrate, in initiating apoptosis in colon cancer
cells lines, via the histone hyperacetylation-mediated
pathway that improve mitochondrial function (Hague et al.
1995; Heerdt et al. 1997; Medina et al. 1997). In addition,
butyrate could induce apoptosis via activating GPR109A
receptor, a tumour suppressor which was found silence in
CRC (Thangaraju et al. 2009). The apoptotic process
involved downregulation of Bcl-2, Bcl-xL and cyclin D1.
Furthermore, this study also demonstrated the activation of
GPR109A by butyrate led to suppression of nuclear factor-
kB (NF-kB) activation, which had been linked to inflam-
matory conditions and inflammation-induced CRC (Than-
garaju et al. 2009). Propionibacteria, which can produce
propionate and acetate, have also been shown to stimulate
apoptosis of various human CRC cells in vitro (HT29,
Caco2) (Jan et al. 2002).
Taken together, there is a reasonable body of evidence
to support that the production of SCFA particularly buty-
rate is one of the principal mechanisms of the anticarci-
nogenic action of probiotics in the colon.
Direct effects on intestinal mucosa
Blocking the initiation phase: reduction of DNA damage
Studies also showed that probiotics could prevent CRC by
inhibiting DNA damage. In a recent clinical study,
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genotoxicity of fecal water from atopic patients, measured
using comet assay, was found be to higher than healthy
subjects, indicating a higher risk for CRC (Roessler et al.
2012). This study reported administration of a probiotic
drink, containing L. paracasei Lpc-37, L. acidophilus 74-2,
and B. lactis DGCC 420, led to an increase in lactobacilli,
as well as a significant reduction in genotoxic activity of
fecal water in the patients. A dietary intervention trial
demonstrated daily consumption of 300 g probiotic yogurt
(containing L. acidophilus 145 and B. longum 913) for
6 weeks resulted in reduced feacal water-induced geno-
toxicity in human colon cancer cells HT29clone19A, i.e.
less DNA strand break-inducing agents in faeces (Ober-
reuther-Moschner et al. 2004). A study investigated the
effects of a combined treatment, comprised of three test
agents: crucifera, chlorophyllin, and a yogurt drink con-
taining L. casei, L. bulgaricus and S. thermophilus, in
healthy subjects (Shaughnessy et al. 2011). These subjects
were also fed a diet containing meat cooked at high tem-
perature (200 °C). The combined treatment significantly
reduced the HCA-induced DNA damage in the colorectal
cells (isolated from rectal biopsies of the subjects), and
probiotics might have played a part in this protective effect
(Burns and Rowland 2004; Kumar et al. 2010; Pool-Zobel
et al. 1993a, b, 1996; Renner and Münzner 1991; Roessler
et al. 2012; Shaughnessy et al. 2011).
Burns and Rowland (2004) showed that B. Bb12 and L.
plantarum significantly reduced the faecal water-induced
DNA damage in HT29 human colon tumor cells, whereas
Streptococcus thermophilus did not confer this antigeno-
toxicity effect. Pool-Zobel et al. (1996) demonstrated that
many species of LAB, particularly lactobacilli and bifido-
bacteria, gave protective effects against DNA damage
induced by carcinogen DMH in the colon mucosa of rats.
In their previous animal study, Pool-Zobel et al. (1993a)
demonstrated the effect of viable L. casei cells in reducing
the genotoxicity induced by mutagen N-methyl-N-nitro-N-
nitrosoguanidine (MNNG) in the colon of rats. Consis-
tently, a probiotic curd prepared with lactobacilli exhibited
protective effects against DMH-induced DNA damage in
rats (Kumar et al. 2010).
L.casei was also shown to be effective in decreasing
DNA damage induced by nitrosated beef extract in the
Salmonella typhimurium Ames assay (Renner and Münzner
1991). A study investigated the antimutagenic potential of
several bifidobacteria strains against benzo[a]pyrene
(B[a]P), measured as the degree of reduction of B[a]P-
induced mutation, through a modified S. typhimurium
Ames Test (Lo et al. 2002). B[a]P is a highly carcinogenic
and mutagenic PAH compound that results from incom-
plete combustion of organic materials, and is found in food
cooked at high temperature (e.g. charbroiled food) and the
atmosphere (e.g. automotive exhaust fumes, tobacco
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smoke). It was demonstrated that B. lactis Bb-12 and B.
longum CCRC 14634 exhibited the significant antimuta-
genicity activity against B[a]P. The authors suggested that
the mechanisms of this antimutagenic action of bifidobac-
teria were plausibly: (1) binding of bifidobacteria and the
mutagen; (2) inactivation of P450-mediated monooxygen-
ase system, which was accountable for conversion of B[a]P
into active and mutagenic B[a]P-7,8-dihydrodiol 9,10-
epoxide compounds (e.g. these compounds can form DNA
adducts with guanine) (Lo et al. 2002).
Taken together, the antigenotoxicity effect of probio-
tics is reasonably well demonstrated in vitro and in vivo,
though more studies involving human subjects are war-
ranted. This mechanism has a significant implication in
preventing CRC, because genetic changes (i.e. DNA
damage, oncogenes and tumor suppressor gene muta-
tions) are critically involved in the initial phase and
development of CRC (Grivennikov 2013; Vogelstein
et al. 1988).
Inhibition of the promotion phase of CRC
Probiotics may also reduce the risk of CRC by suppressing
the promotion phase of CRC by two key mechanisms: (1)
preventing formation of aberrant crypt foci (ACF) and (2)
improving colonic barrier functions, as discussed below.
Aberrant crypt foci are recognised as precursors of
colorectal adenomas (Chang et al. 2012; Takayama et al.
1998, 2011). ACF are characterised by hyperproliferation
and lack of cell differentiation (Culpepper and Mai 2013).
CRC patients were found to have more ACF compared to
patients with non-malignant lesions, and the majority of the
ACF had K-ras mutation, one of the key genetic events in
colonic carcinogenesis (Fig. 2) (Chung 2000; Commane
et al. 2005; Culpepper and Mai 2013; Jiang et al. 2013;
Takayama et al. 1998). K-ras mutations were linked to
increased expression and activity of DNA methyltransfer-
ase, cyclin D1 and gastrin, all of which were involved in
aetiology of CRC (Chung 2000). An assessment found that
as the number of ACF increased, the risk of a patient
having colonic advanced neoplasms increased (Seike et al.
2006).
The DMH-induced colon cancer model had been com-
monly used to investigate the effects of probiotics in ACF
formation and positive results were consistently reported
by several studies. For instance, L. acidophilus KFRI342,
isolated from kimchi (a tradiotinal Korean food) significant
reduced the number of DMH-induced ACF (Chang et al.
2012). Another recent study reported suppressive effect of
L. GG and L. acidophilus against ACF formation in the
DMH model (Verma and Shukla 2013b). An earlier
investigation observed a reduction in the incidence of
DMH-induced tumors in the colon of F344 rats, attributed
World J Microbiol Biotechnol
Fig. 2 Colorectal carcinogenesis. Genetic events occurring during colorectal cancer carcinogenesis and potential chemopreventive approaches
to the supplementation of L. GG (Goldin et al. 1996). The
authors concluded that L. GG administration inhibited
initiation or early promotional phase of CRC tumorigene-
sis. Administration of a probiotic bacteria, Bacillus poly-
fermenticus, significantly reduced the number of DMH-
induced ACF in F344 rats, when compared to the controls
(DMH-treated, no probiotics supplementation) (Park et al.
2007). Furthermore, the probiotics-treated group showed
significantly less DMH-induced DNA damage, less blood
lipid peroxidation, and increased Total Radical Trapping
Antioxidant Potential (TRAP) by 9.3 % versus the con-
trols. These findings suggest that Bacillus polyfermenticus
is able to suppress both the initiation and promotion phase
of carcinogenesis (i.e. less preneoplastic lesions). A study
investigated the chemopreventive potential of Butyrivibrio
fibrisolvens for CRC using Jcl:ICR mice model treated
with DMH (Ohkawara et al. 2005). The investigators
reported the administration of Butyrivibrio fibrisolvens for
4 weeks (109 cfu/dose, 3 times/week) significantly reduced
the incidence of ACF. Concomitantly, they also observed
several phenomenon: (1) a reduced b-glucuronidase
activity, (2) a higher rate butyrate acid production, and (3)
enhanced immune response, i.e. increased natural killer
(NK) and natural killer T (NKT) cells, all of which may
partially explain the effect of Butyrivibrio fibrisolvens in
inhibiting the ACF formation.
Yamazaki et al. (2000) studied the short term and long-
term effects of administration of live L. casei Shirota on
ACF and colon cancer induced by azoxymethane (AOM) in
rats. The study demonstrated that 8 and 12 weeks admin-
istrations of the probiotic significantly lowered the number
of large colonic ACF (C4 aberrant crypts per focus)
compared to the non-probiotic treated group. The long-
term experiment revealed less incidence of colon cancers in
the probiotic treated group 25 weeks after the AOM
injection. These findings indicate short- and long-term
treatments of L. casei Shirota suppress carcinogen-induced
tumorigenesis in vivo (Yamazaki et al. 2000). A recent
study showed that beetroot juice, fermented with L. brevis
0944 and L. paracasei 0920, significantly suppressed the
N-Nitroso-N-methylurea (MNU)-induced ACF in rat colon
(Klewicka et al. 2012).
There are a few data suggesting that probiotics can
protect against the loss of colonic barrier function (i.e.
increased mucosal permeability), another characteristic
physiological event occurring during promotion phase of
CRC (Commane et al. 2005; Salminen et al. 2004). The
colonic barrier function refers to the blocking of luminal
contents including chemicals, bacteria, bacterial metabo-
lites and toxins from permeating through the epithelial
cell lining. Certain lactobacillus strains, such as L. brevis,
L. plantarum, L. GG and L. reuteri have exhibited a
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stabilizing effect on mucosal barrier in animal and human
studies (Garcia-lafuente et al. 2001; Gotteland et al. 2001;
Kennedy et al. 2000; Mao et al. 1996). For instance, Mao
et al. (1996) showed that ingestion of L. plantarum or L.
reuteri ameliorate the impaired epithelial barrier function
due to treatment of methotrexate (a drug used in chemo-
therapy) in rats. The plausible mechanisms underlying the
protective effect of probiotics in colonic barrier function
include: (1) production of antioxidants, which protects the
epithelial barrier structure against damage caused by
reactive oxygen species (Gotteland et al. 2001), and (2)
maintenance of intestinal mucus layer and tight junction
structure, which will be further discussed in this review,
under a separate section titled ‘Maintenance or enhance-
ment of intestinal barrier function by probiotics’.
Systemic effects
Immunomodulatory functions of probiotics
The immune system of mice raised in germ-free environ-
ment does not develop completely. Compared to normally
colonized mice, germ-free mice showed various immuno-
logical defects, including fewer Peyer’s patches, dendritic
cells (DC), geminal centres and T cells, as well as thinner
lamina propria (Dongarrà et al. 2013; Hardy et al. 2013;
Round and Mazmanian 2009). Intestinal epithelial cells of
germ-free mice showed reduced levels of ATP, IL-25 and
expression of Toll-like receptor 9 (TLR9) (Azcárate-Peril
et al. 2011). Specific commensals such as species of Bifi-
dobacterium, Bacteroides, and Lactobacillus had been
shown to drive or restore the development of gut-associ-
ated lymphoid tissue (GALT) and epithelial immune
functions (Azcárate-Peril et al. 2011; Hardy et al. 2013).
These data suggest a pivotal role of microbiota in the
development and functions of the gut immune system.
Probiotics exhibit multifaceted mechanisms in modulating
the immune system, which can be potentially beneficial in
CRC prevention, as discussed further below.
Maintenance or enhancement of intestinal barrier function
by probiotics It has been shown that the mucus layer in
inflamed mucosa of IBD patients are thinner as compared
to those of normal subjects (Fyderek et al. 2009). The GI
tract is protected by a mucus layer consisting of large
mucin glycoproteins, which functions as a physical barrier
to exclude luminal pathogens, viruses, parasites and toxins
(Kim and Khan 2013). Among the various mucins secreted
by intestinal goblet cells, the predominant one is mucin 2
(MUC2). Animal studies using MUC2-deficient mice
observed spontaneous inflammation of colon, colitis and
development of CRC (Sheng et al. 2012). Defects in
assembly and processing of the MUC2 complex result in
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thinner mucus barrier and chronic inflammation in the
distal colon of mice with mutations in gene encoding for
MUC2 (Sheng et al. 2012). Treatment of probiotics (i.e. L.
GG or a mixture of Streptococcus thermophilus, L. aci-
dophilus, and B. lactis) was effective to promote the res-
toration of colonic tissue, which could be associated with
an increased MUC2 gene expression, in rats with trinitro-
benzenesulfonic acid (TNBS)-induced colitis (Amit-Ro-
mach et al. 2010). Daily administration of a probiotic
mixture (four species of lactobacilli, three species of bifi-
dobacteria, and Streptococcus salivarius subsp. Thermofi-
lus) for 1 week upregulated colonic MUC2 gene
expression and increased luminal mucin content by 60 %
in normal Wistar rats (Caballero-Franco et al. 2007). Cell
culture experiments using colonic epithelial cells had
demonstrated the ability of probiotics (e.g. L. plantarum, L.
GG) to increase mucin gene expression (MUC2 and
MUC3), extracellular mucin secretion and inhibition of
enteropathogenic adherence in vitro (Caballero-Franco
et al. 2007; Mack et al. 2003). These data suggest a pro-
tective role of probiotic role in maintaining the mucus layer
integrity, which is essential for an effective intestinal bar-
rier function.
Tight junction (TJ) functions as another physical barrier
against the invasion of pathogens and toxins into the
intestinal tissues. TJ holds epithelial cells together and are
responsible to control permeability of the paracellular
pathway. Aberrant distribution and downregulation of TJ
proteins leads to increased epithelial permeability thus
increased infiltration of proinflammatory stimuli into the
lamina propria, which subsequently triggers inflammation
and exacerbates mucosal barrier dysfunction (Karczewski
et al. 2010). Altered epithelial permeability has been
implicated in IBD, celiac disease and the early stages of
CRC development (Karczewski et al. 2010; Mennigen
et al. 2009). Some probiotic strains have been shown to
stabilize epithelial TJ structure in vitro and in vivo
(Anderson et al. 2010; Karczewski et al. 2010; Mennigen
et al. 2009). In an animal model of colitis, a probiotic
mixture, consisting of species of Lactobaccillus, Bifido-
bacterium, and Streptococcus, enhanced the maintenance
of TJ structural protein expression (occludin, claudin
family, and zonula occludens), and prevented undesirable
changes to epithelial permeability (Mennigen et al. 2009).
A probiotic strain L. plantarum MB452 was shown to
enhance Caco-2 TJ integrity, possibly associated with a
parallel upregulation of TJ-related genes expression,
including those encoding occludin and scaffold protein
zonula occludens (Anderson et al. 2010). A randomized
crossover human study demonstrated that administration of
L. plantarum significantly increased the levels of occludin
and zonula occludens in the TJ structure (Karczewski et al.
2010). The modulatory effect of L. plantarum on epithelial
World J Microbiol Biotechnol
integrity observed in this study could be mediated via the
Toll-like receptor 2 (TLR2) in the gut epithelium (Kar-
czewski et al. 2010).
Recognition of probiotics by immune system: toll-like
receptors and anti-CRC responses Probiotics exert its
immunomodulatory activities via binding to pattern rec-
ognition receptors (PRRs), which are present on the surface
of immune cells such as dendritic cells, macrophages,
monocytes and intestinal epithelial cells (Bron et al. 2012;
Sivieri et al. 2013). PPRs, including Toll-like receptors
(TLRs), C-type lectin receptors (CLRs) and NOD-like
receptors (NLRs), recognise specific microorganism-asso-
ciated molecular patterns derived from bacteria including
probiotics (Bron et al. 2012). Studies have suggested pro-
biotics express various ligands recognised by TLRs, CLRs
and NLRs (Shida and Nanno 2008). Nonetheless, among
various PPRs, TLRs such as TLR2 and TLR9 are most
investigated and important for immunomodulatory actions
of probiotics (Bron et al. 2012; Dongarrà et al. 2013;
Plantinga et al. 2011).
In a mouse model of colitis-induced CRC, TLR2 defi-
ciency led to dysregulated cell proliferation, reduced
apoptosis in the crypt, and early formation of aberrant crypt
foci and colonic tumours (Lowe et al. 2010). Compared to
wild type mice, TLR-2 deficient mice exhibited increased
inflammation and higher serum levels of inflammatory
cytokines IL-6 and IL-17A. Therefore, TLR2 signalling
could be crucial in preventing pro-tumour inflammatory
skewing and plays a protective role in colitis-associated
CRC development and progression (Lowe et al. 2010). It
had been shown that TLR2 deficiency in mice led to
colonic mucosal DNA methylation changes, for instance, at
the DNase-hypersensitivity regions and CTCF-binding
sites, which have been implicated in CRC development
(Kellermayer et al. 2011). Furthermore, alterations of gene
expression involved in immune processes, such as Ifit2 and
Anpep, in the absence of TLR2, suggest an important role
of TLR2 signalling in regulating immune system and CRC
development (Kellermayer et al. 2011). Logically, probi-
otics may modulate the risk of CRC in part through a
TLR2-dependent pathway. TLR2 recognises peptidogly-
can, lipoproteins, lipoteichoic acids and teichoic acids,
which are the components of Gram-positive bacteria, such
as lactobaccilli and bifidobacteria (Kaji et al. 2010; Sivieri
et al. 2013). Indeed, peptidoglycan from lactobacilli
inhibited proinflammatory cytokine IL-12 production by
macrophages via TLR2 (Shida et al. 2009). Combination of
L. plantarum and L. casei synergistically induced IL-10
production in macrophages through a TLR2-dependent
pathway (Kaji et al. 2010).
IL-10 downregulates proinflammatory cytokines and
promotes the development of regulatory T cells (Tregs)
(Feleszko and Jaworska 2013; Kaji et al. 2010). Tregs are
involved in regulation of T cell-mediated immune
responses, maintenance of immune tolerance and preven-
tion of inflammatory disorders (Bron et al. 2012; Feleszko
and Jaworska 2013). In relation to IBD, probiotics have
been shown to activate Tregs, which in turn lead to several
actions that reduce intestinal inflammation: (1) upregula-
tion of T-helper 2 (Th2) cytokines (e.g. IL-10); (2)
downregulation of Th1 type proinflammatory cytokines
(e.g. IL-12) and (3) suppression of proinflammatory
responses mediated by IFN-c (Clarke and Mullin 2008).
Probiotics administration induced regulatory dentritic cells,
which in turn promoted the induction of CD4?Foxp3?
Tregs in vivo (Kwon et al. 2010). The increase in
CD4?Foxp3? Tregs at the inflamed site ameliorated the
disease progression in mice with IBD. The same probiotics
administration also led to significant decrease in proin-
flammatory cytokines IL-17, IFN-c and TNF-a in mice
with TNBS-induced colitis (Kwon et al. 2010).
Inhibitory effect of probiotics on TLR4 and COX-2
expression Cyclooxygenase-2 (COX 2) has been impli-
cated in inflammatory diseases and CRC development
(Otte et al. 2008). The administration of L.plantarum Lp91
significantly reduced COX-2 and TNF-a, while upregu-
lating IL-10 levels and MUC2 gene expression in the colon
tissues of mice with TNBS-induced colitis (Duary et al.
2012). Treatment of B. lactis or L. casei also downregu-
lated colonic COX-2 expression in colitis rat model (Peran
et al. 2007). E. coli Nissle 1917, probiotic mixture VSL#3
and media conditioned by these probiotics had been shown
to decrease COX-2 expression and prostaglandin E2
secretion induced by gastrin and TNF-a in intestinal epi-
thelial cells (Colo320 or SW480) (Otte et al. 2008). A
treatment involving probiotic mixture VSL#3 led to
reduction in COX-2, NF-kB, TNF-a, IL-6 and inducible
nitric oxide synthase, while increasing IL-10 expression in
colonic tissue of rats with dextran sulfate sodium-induced
colitis (Dai et al. 2013). TLR4 is required for the induction
of COX-2 and has been implicated in the development of
colitis associated CRC (Abreu 2010; Santaolalla et al.
2012). Overexpression of TLR4, which has observed in
colons of IBD patients and experimental colitis models,
upregulates NF-kB activation, which in turns induces
COX-2 expression (Lee et al. 2009b). The study of Lee
et al. (2009b) demonstrated that L. suntoryeus HY7801 had
potent inhibitory effect on NF-kB activation in vitro, and
downregulated the expression of TLR4 and COX-2 in
TNBS-induced colitic mice. Several probiotic strains were
found to inhibit TLR4-linked NF-kB activation, which
could be one of the mechanisms by which probiotics
downregulate colonic COX-2 expression in inflammatory
disorders (Lee et al. 2009b).
123

Probiotics enhance innate immune functions Mammalian
defensins, which are part of the innate immune system in
the GI, have pharmaceutical potential given its antimicro-
bial, antiviral and immunomodulatory activities. In relation
to cancer, defensins has been implicated to exert cytotoxic
activity on tumour cells involving membrane lysis and
DNA damage, as well as anti-angiogenic effects (Droin
et al. 2009). Defensins have potential to overcome the
evasion of tumour cells on immunosurveillance. For
instance, murine b-defensin 2 has been shown to promote
dentritic cell maturation, which in turns triggers type 1
polarized immune responses, such as production of proin-
flammatory cytokines IL-12, IL-1a, IL-1b and IL-6 (Bi-
ragyn et al. 2002). Beta-defensins are expressed in the
mucosa and epithelial cells (Droin et al. 2009; Paolillo
et al. 2009). Treatment of human colon cancer cells Caco-2
with probiotics L. plantarum significantly upregulates
human b-defensin 2 (HDB-2) mRNA expression and HBD-
2 secretion in a dose-dependent manner, through the
induction of TLR2 (Paolillo et al. 2009). A probiotic
mixture Symbioflor 2, containing several E. coli genotypes,
has been shown to increase HDB-2 synthesis in human, and
a dose-dependent induction of HDB-2 in Caco-2 cells
(Möndel et al. 2008). Similar in vitro findings have been
reported for probiotic mixture VSL#3 and Lactobacili
(Schlee et al. 2008).
Immunoglobulin A (IgA) at mucosal surfaces is a major
component of specific immunity against the invasion of
pathogenic microorganisms (Feleszko and Jaworska 2013).
Antibody production, particularly IgA, is markedly reduced
in the intestine of germ-free mice (Round and Mazmanian
2009). Indeed, TLR ligation by microbiota on intestinal
epithelial cells was shown to promote the production of
IgA by B cells into the lumen (Abreu 2010). Animal
studies have revealed certain probiotics strains such as
lactobacilli elicit antigen-specific IgA responses at the
mucosal surfaces. Probiotics administration in human was
shown to increase to both serum and fecal IgA levels
(Feleszko and Jaworska 2013). IgA has been implicated to
have anti-inflammatory function and direct cytotoxic effect
on tumour cells (Schwartz-Albiez et al. 2009). A study
showed administration of yogurt containing probiotics was
effective to suppress the development of carcinoma in the
large intestine of mice treated with carcinogen 1,2-
dimethylhydrazine (Perdigon et al. 1995). The anticarci-
nogenic action of probiotics observed in this study was
associated with upregulation of IgA, T-lymphocyte and
colonic macrophages activities. Furthermore, both animal
and human studies have demonstrated increases of natural
killer (NK) cells and its activity in response to probiotics
administration (Sivieri et al. 2013). For instance, a clinical
trial revealed three-week intake of L. casei strain Shirota
enhanced the cytotoxicity activity of natural killer (NK)
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World J Microbiol Biotechnol
cells (Nagao et al. 2000). Dietary supplementation of
Bifidobacterium lactis led to increases in T-lymphocyte
and NK cells in the systemic circulation, as well as
upregulation of NK cell cytotoxicity activity (Gill et al.
2001). Consumption of a probiotic drink, containing L.
casei Shirota, was also effective in enhancing NK cell
activity in healthy elderly subjects (Dong et al. 2013).
Furthermore, L. casei Shirota was shown to stimulate
the production of inflammatory cytokines such as tumour
necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and
interferon-c (IFN-c), which in turn suppressed the devel-
opment of tumour and prolonged the survival in mice
treated with a carcinogen (Matsuzaki 1998). IFN-c is
involved in activation of NK cells and macrophages;
therefore it plays a critical role in cancer prevention
(Meydani and Ha 2000). Human and animals constantly
produce IFN-c as a defence against neoplasms (Bocci
1981). Lactobacillus rhamnosus E509 was shown to be
effective in stimulating IFN-c in human peripheral blood
mononuclear cells (Miettinen et al. 1998). In a study
involving 24 human subjects, daily intake of 450 g of
yogurt for 4 months led to an increased IFN-c production
in T-lymphocytes (Halpern et al. 1991). It has been sug-
gested that aging can result in decline in physiological
interferon response, which increases the tendency of
tumour development during the aging process (Bocci
1981). In this regard, a study showed that lactobacilli were
able to restore the IFN-c status in aged mice (Muscettola
et al. 1994). Excessive inflammatory response is not
desirable because of its association with CRC development
(Collins et al. 2006; Grivennikov 2013; Munkholm 2003).
Interestingly, probiotics are able to induce and control
Tregs activity as discussed above, which may promote a
balance of cytokine production (i.e. a balancing act
between proinflammatory and anti-inflammatory immune
response).
In summary, there is a strong body of evidence which
support modulation of immune responses as one of the key
anti-CRC mechanisms of probiotics. In particular, probio-
tics have been well demonstrated to regulate inflammatory
processes, enhance intestinal barrier functions and innate
immune responses, all of which are critical to prevent
colorectal tumourigenesis.
Folate production and DNA methylation
Probiotics not only contribute to increased SCFA produc-
tion, but also play a role in the synthesis of B vitamins such
as vitamin B12 and folate in humans (Rossi et al. 2011).
Epideomiologic evidence suggests that an adequate folate
intake and status is important in CRC prevention (Gio-
vannuci 2002; World Cancer Research Fund/American
Institute for Cancer Research 2007). Folate can protect
World J Microbiol Biotechnol
against CRC via different mechanisms particularly the
DNA synthesis and methylation which plays an important
role in CRC carcinogenesis (Chung 2000; World Cancer
Research Fund/American Institute for Cancer Research
2007). Chronic deficiency of folate can reduce the
S-adenosylmethionine (SAM): S-adenosylhomocysteine
(SAH) ratio, thus decreasing the normal cellular DNA
methylation capacity (McCabe and Caudill 2005). Aberrant
DNA methylation (i.e. hypomethylation or hypermethyla-
tion) leads to abnormal gene expression and cell differen-
tiation (Kellermayer et al. 2011). It has been suggested that
DNA hypomethylation occurs during the early phase of
CRC carcinogenesis (Fig. 2) (Commane et al. 2005; Cravo
et al. 1994).
A considerable number of studies have shown that folate
intake or status regulates DNA methylation in humans
(McCabe and Caudill 2005). In relation to CRC, a group
showed that the DNA methylation in rectal mucosa was
significantly improved in CRC patients as a result of folate
treatment (Cravo et al. 1994, 1998). A randomized dietary
intervention by Mohammad et al. (2006) demonstrated that
L. acidophilus supplementation (1012 CFU/day) in yogurt
for 42 days significantly increased the plasma levels of
folate and vitamin B12 (cobalamin) in children. The pro-
biotic supplementation also resulted in reduced plasma
total homocysteine and urinary methymalonic levels (i.e.
improved folate and vitamin B12 status) (Mohammad et al.
2006). An in vitro study identified three strains of bifido-
bacteria that were able to produce high amount of folate in
medium, namely B. adolescentis MB 227, B. adolescentis
MB 239 and B. pseudocatenulatum MB 116 (Pompei et al.
2007a). Furthermore, their biosynthesis of folate was not
influenced by pH, carbohydrate and exogeneous folate. The
authors postulated that these bifidobacteria strains, partic-
ularly B. adolescentis MB 239, could work as an excellent
folate-producing probiotics in the colon environment. The
investigators confirmed these in vitro findings in a sub-
sequent animal study (Pompei et al. 2007b). They showed
that supplementation of probiotic mixture consisting of
these three bifidobacteria strains significantly improved the
folate status in Wistar rats. Some researchers also explore
the potential of using probiotics to increase the folate
content of food. For instance, the use of a combination of
yogurt starter culture, Streptococcus thermophillus, and a
probiotic strain, Bifidobacterium animalis, was able to
achieve a substantial production of folate during the fer-
mentation process of milk, resulting in more than six-fold
increase in folate content in the end product (Crittenden
et al. 2003). A recent study showed a combination of three
probiotic strains, L. bulgaricus CRL 871, S. thermophilus
CRL 803, and S. thermophilus CRL 415 could be used to
achieve a yogurt product with high folate content (Laiño
et al. 2013).
Taken together, probiotics appear to be an interesting
way to maintain adequate folate status, thereby maintaining
a healthy DNA methylation that is essential for CRC pre-
vention. Nonetheless, there is a lack of studies demon-
strating the relation between folate biosynthesis of
probiotics, DNA methylation and risk of CRC.
Prebiotics, synbiotics and CRC chemoprevention Data on
the effects of prebiotics in relation to CRC have accumu-
lated. A novel resistant starch (stearic-acid complexed
high-amylose cornstarch) treatment was shown to
decreased the number of ACF and mucin depleted foci in
AOM-treated rats (Zhao 2012). This study also highlighted
different cooking methods could give rise to different anti-
cancer activity. Specifically, bread-baked starch diet was
found to exert a greater inhibitory effect on ACF devel-
opment than water-boiled starch diet. Treatment with pre-
biotic, inulin or lactulose, led to decrease in ACF and
microbial enzyme activity of b-glucuronidase and b-glu-
cosidase in DMH-treated rats. Short-chain fructooligosac-
charides (SC-FOS) administration increased intestinal
lactobacilli and bifidobacteria counts in colitis rats, with a
parallel increase in anti-inflammatory activity, as indicated
by a decrease in inflammatory myeloperoxidase activity,
leukotriene B4 production and iNOS expression (Lara-
Villoslada et al. 2006). This study also showed fermenta-
tion of SC-FOS occurred in the ileum and cecum, sug-
gesting that distal colonic fermentation was not a
prerequisite for the anti-inflammatory effects of these
prebiotics. Inulin and lactulose reduced the genotoxicity
induced by fecal water or 4-nitroquinoline-1-oxide (4-
NQO) in HT-29 human colon adenocarcinoma cells
(Adebola et al. 2013). Since 4-NQO is known to generate
free radicals as well as DNA adducts, this study suggests
future study to determine which form of DNA damage may
be impeded by inulin and lactulose. Unlike inulin-type
linear fructans, less is known about the bioactivity of
branched fructans in relation to CRC. A branched fructans
derived from Agave angustifolia plant, was recently shown
to increase bifidobacteria population, SCFA production and
decrease ammonia concentrations, in the SHIME model
(Allsopp et al. 2013). Furthermore, the resulting fermen-
tation supernatant was found to enhance the intestinal
barrier function, measured as transepithelial electrical
resistance in a Caco-2 cell monolayer.
There is a growing body of evidence which suggest the
advantage of combining both probiotics and prebiotics,
namely, synbiotics. Combination of resistant starch and
probiotic B. lactis significantly enhanced the acute apop-
totic response to genotoxic carcinogen in the distal colon in
the AOM rat model (Le Leu et al. 2005). Either of the two
components alone, however, was found to be ineffective in
promoting this pro-apoptotic response. In their subsequent
123

investigation, the same synbiotic combination was further
proved to be more effective than resistant starch or B. lactis
alone, in reducing neoplasm incidence and multiplicity of
colonic tumours in AOM-treated rats (Le Leu et al. 2010).
A clinical trial has reported synbiotic intervention of
resistant starch and B. lactis significant increase fecal
Lachnospiraceae spp. (Worthley et al. 2009). Recently,
Lachnospiraceae was identified as abundant and stable part
of the core microbiota in adult colon, and under-repre-
sentation of Lachnospiraceae had been correlated with IBD
(Sekelja et al. 2011). Furthermore, Lachnospiraceae was
associated with a protective effect against the development
of intestinal adenomas in a mouse colon cancer model (Mai
et al. 2007). Collectively, Lachnospiraceae could play a
role in the anti-CRC activity of synbiotic observed by Le
Leu et al. (2010). Another synbiotic intervention consisting
of oligofructose-enriched inulin, L. rhamnosus GG and B.
lactis, was shown to elicit effects on several intermediate
biomarkers for CRC in polypectomized and colon cancer
patients (Rafter et al. 2007). These effects include an
increase in bifidobacteria and lactobacilli, suppression of
colonic cell proliferation and IL-2 secretion, as well as an
improved epithelial barrier function and a reduced expo-
sure to genotoxins. A synbiotic consisting of B. lactis, ol-
igofructose and inulin, significantly enhanced apoptosis,
and decreased the development of ACF in a dimethylhy-
drazine (DMH)-induced CRC model (Dias et al. 2010). A
combination of LAB administration was found to decrease
b-glucosidase, b-glucuronidase and tryptophanase activi-
ties, as well as lower serum cholesterol in rats (Lee et al.
2011).
In vitro studies also have demonstrated the advantage of
a synbiotic treatment than either prebiotic or probiotic
alone. A synbiotic preparation, consisting of B. breve, L.
lactis and oligoalternan, was more effective than various
strains of LAB alone in inhibiting cancerous cell HT-29
proliferation. This effect could be attributed to an upreg-
ulation of intestinal alkaline phosphatase, a biomarker of
colonocyte differentiation (Grimoud et al. 2010). Com-
pared to a dietary fiber source wheat aleurone alone,
addition of probiotics LGG and B. lactis enhanced butyrate
production in a fermentation model, as well as an increase
in cell cycle arrest at G0/G1 in HT-29 cells (Borowicki
et al. 2011). A group investigated the effects of eight
synbiotics, involving combinations of LAB with carbohy-
drates, such as isomaltulose, cellobiose, raffinose and
maltotriose, which were previously shown to stimulate
growth of LAB under laboratory conditions (van Zanten
et al. 2012). All of the synbiotics increased acetic and
butyrate acid production in a colonic model system. A
decrease in the modified ratio of Bacteroidetes/Firmicutes
was observed, which was correlated with the increase in
SCFA production. This study showed the synbiotics tested
123
World J Microbiol Biotechnol
can affect the microbial composition in a way which might
have implications in human health.
Safety of probiotics use The long history of use of pro-
biotic bacteria, especially lactobacilli and bifidobacteria in
foods (e.g. fermented dairy products) and their ubiquitous
presence in the human GI tract, provides reasonable evi-
dence regarding the safety of dietary LAB (Salminen et al.
1998; Snydman 2008). Nevertheless, given the mecha-
nisms of actions of probiotics as discussed above, there are
potential theoretical risks of probiotics use. These risks
could include excessive immune stimulation, adverse
metabolic activities, infection (endocarditis and bactere-
mia), transmigration of pathogenic bacteria, and transfer of
antibiotic-resistance from probiotics to pathogenic bacteria
(e.g. Tetracycline resistance in Lactobacillus plantarum;
vancomycin resistance in many lactobacilli strains)
(Snydman 2008).
One of the key safety concerns is that LAB has been
associated with cases of infection, i.e. infective endocar-
ditis and bacteremia (Boyle et al. 2006). Nonetheless, two
Finnish studies demonstrated that there was no significant
increase in lactobacilli or probiotics related infection cases
between 1990 and 2000, even though there had been a
significant increase in Lactobacillus GG consumption over
that decade (Salminen et al. 2002; Saxelin et al. 1996). The
LAB, lactobacilli and bifidobacteria which are the most
commonly used probiotics have been issued the ‘generally
regarded as safe’ (GRAS) status (Snydman 2008). A
review reported that dietary intake of LAB was well-tol-
erated and demonstrated to be safe in 143 human clinical
studies conducted between 1961 and 1998, involving a
total of 7526 subjects (Naidu et al. 1999). In relation to the
safety of probiotics use in patient receiving nutritional
support, Whelan and Myers (2010) conducted a systematic
review of case reports, randomized controlled trials, and
nonrandomized trials, covering a total of 53 trials and 4131
patients received probiotics. This extensive review found
that probiotics had either no or positive effects in relation
to safety outcomes (e.g. mortality, infection) (Whelan and
Myers 2010). Consistently, other reviews also concluded
that clinical evidence available to date supports the safety
of consumption of LAB probiotics (Borriello et al. 2003;
Charalampopoulos et al. 2009; Saavedra 2001; Snydman
2008). Nevertheless, it is noteworthy that not all probiotics
have GRAS status. Some members of enterococci, strep-
tococci and bacilli, which are also used as probiotic
organisms, are not generally regarded as safe since they
contain opportunistic pathogens, especially enterococci
(Boyle et al. 2006; Salminen et al. 1998; Snydman 2008).
In summary, there is a reasonable body of evidence to
support the use of probiotics. However, individuals with
immune deficiency should use probiotics with caution
World J Microbiol Biotechnol
because of the risk of infection such as endocarditis,
pneumoniae and sepsis (Boyle et al. 2006; Floch 2013;
Kuitunen 2011; Vankerckhoven et al. 2008; Young and
Huffman 2003). Microorganisms with known virulence
genes should not be developed for probiotics use (Van-
kerckhoven et al. 2008). Readers are referred to recom-
mendations of the EU-PROJECT workshop for more
details on biosafety assessment of microorganism for
human consumption, including taxonomy and typing,
in vitro assessment of virulence and in vivo investigations
(Vankerckhoven et al. 2008).
Conclusions
In conclusion, there is a convincing body of evidence
suggesting various potential mechanisms of action of pro-
biotics in CRC prevention. It is clear that the chemopre-
ventive effects of probiotics are dependent on the strain of
the microorganism. Emerging data suggest viability may
not be a prerequisite for probiotics to exert its anti-CRC
activity. Synbiotics is likely to be more effective than
either prebiotics or probiotics alone, as indicated by the
growing body of data. More in vivo, especially human
studies are warranted to further elucidate and confirm the
potential chemopreventive role of probiotics (viable and
non-viable), prebiotics and synbiotics in CRC.
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