Cancer-preventing attributes of probiotics an update

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International Journal of Food Sciences and Nutrition,
August 2010; 61(5): 473–496
Cancer-preventing attributes of probiotics: an update
MANOJ KUMAR
1
, ASHOK KUMAR
2
, RAVINDER NAGPAL
3
, DHEERAJ
MOHANIA
4
, PRADIP BEHARE
1
, VINOD VERMA
5
, PRAMOD KUMAR
1
,
DEV PODDAR
1
, P. K. AGGARWAL
1
, C. J. K. HENRY
6
, SHALINI JAIN
7
&
HARIOM YADAV
7
1
Dairy Microbiology Division, National Dairy Research Institute, Karnal (Haryana), India,
2
Department of Zoology, M.L.K. P.G. College, Balrampur (U.P.), India,
3
Department of
Biotechnology, Lovely Professional University, Phagwara, Punjab, India,
4
Animal Biochemistry
Division, National Dairy Research Institute, Karnal (Haryana), India,
5
AgResearch Ltd,
Ruakura Research Centre, Reproductive Technologies, East Street, Hamilton, New Zealand,
6
School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, UK, and
7
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, Maryland, USA
Abstract
Cancer is a serious global public health problem. Cancer incidence and mortality have been
steadily rising throughout the past century in most places of the world. There are several
epidemiological evidences that support a protective role of probiotics against cancer. Lactic acid
bacteria and their probioactive cellular substances exert many beneficial effects in the gastro-
intestinal tract, and also release various enzymes into the intestinal lumen and exert potential
synergistic (LAB) effects on digestion and alleviate symptoms of intestinal malabsorption.
Consumption of fermented dairy products with LAB may elicit anti-tumor effects. These effects
are attributed to the inhibition of mutagenic activity, the decrease in several enzymes implicated
in the generation of carcinogens, mutagens, or tumor-promoting agents, suppression of tumors,
and epidemiology correlating dietary regimes and cancer. Specific cellular components in lactic
acid bacteria seem to induce strong adjuvant effects including modulation of cell-mediated
immune responses, activation of the reticulo-endothelial system, augmentation of cytokine
pathways, and regulation of interleukins and tumor necrosis factors. Studies on the effect of
probiotic consumption on cancer appear promising, since recent in vitro and in vivo studies have
indicated that probiotic bacteria might reduce the risk, incidence and number of tumors of the
colon, liver and bladder. The protective effect against cancer development may be ascribed to
binding of mutagens by intestinal bacteria, may suppress the growth of bacteria that convert
procarcinogens into carcinogens, thereby reducing the amount of carcinogens in the intestine,
reduction of the enzymes b-glucuronidase and b-glucosidase and deconjugation of bile acids, or
merely by enhancing the immune system of the host. There are isolated reports citing that
administration of LAB results in increased activity of anti-oxidative enzymes or by modulating
circulatory oxidative stress that protects cells against carcinogen-induced damage. These
include glutathione-S-transferase, glutathione, glutathione reductase, glutathione peroxidase,
superoxide dismutase and catalase. However, there is no direct experimental evidence for
cancer suppression in human subjects as a result of the consumption of probiotic cultures in
Correspondence: Dr Hariom Yadav, National Institute of Diabetes and Digestive and Kidney Diseases, Diabetes Branch,
5W5872, CRC, Bld 10, Bethesda, MD 20892, USA. E-mail: yadavhariom@gmail.com
ISSN 0963-7486 print/ISSN 1465-3478 online 2010 Informa UK Ltd
DOI: 10.3109/09637480903455971
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fermented or unfermented dairy products, but there is a wealth of indirect evidence based largely
on laboratory studies.
Keywords: Probiotics, cancer, prebiotics, synbiotics, functional food
Introduction
Probiotics are defined as live microbial food ingredients (supplements) that confer
beneficial health effects to the host upon ingestion in adequate amounts (Salminen and
von Wright 1998). The concept of probiotics evolved at the turn of the twentieth
century from a hypothesis first proposed by Nobel Prize-winning Russian scientist Elie
Metchnikoff, who propounded that the long and healthy life of Bulgarian people
resulted from their consumption of fermented milk products (Bibel 1998). He believed
that, when consumed, the fermenting bacillus (Lactobacillus) positively influenced the
microbiota of the colon and, decreased their toxic activities. Several lactic acid bacteria
may help prevent initiation of cancer (Table I). It appears that LAB can reduce the
levels of colon enzymes that convert pro-carcinogens to carcinogens. Specifically, LAB
can reduce levels of the enzymes b-glucuronidase, nitro-reductase, and azo-reductase.
LAB may also be involved in the direct reduction of pro-carcinogens; for example, by
taking up nitrites and by reducing the levels of secondary bile salts (Fernandes and
Shahani 1990). Changes in enzyme activity in humans have been observed with
Lactobacillus acidophilus and Bifidobacterium bifidum (Marteau et al. 1990), and
Lactobacillus rhamnosus GG (LGG) (Goldin and Gorbach 1984). Animal studies
showed fewer tumors in those exposed to a carcinogen in the presence of LGG
compared with the animals exposed to the carcinogen without the benefit of LGG
(Goldin 1996). Human epidemiological reports have indicated that populations
ingesting fermented dairy products have a decreased risk of colon cancer (Kampman
et al. 1994). An anti-tumor effect has been reported by oral intake of LAB in in vitro
studies but, since colon carcinogenesis is a multistage process, the mechanisms if
validated remain to be elucidated (Hirayama and Rafter 1999). A preventive effect on
malignancy development could be mediated by different mechanisms involved in
detoxification of carcinogens.
In vitro studies have shown that, in general, live cells of probiotic bacteria possessed
higher anti-mutagenic activity. Intake of fermented milk by (L. acidophilus and
B. bifidum) influenced gut flora enzymes, like b-glucuronidase, b-glucosidase and
nitro-reductase (Wollowski et al. 2001). L. acidophilus and B. bifidum decreased the
activity of nitro-reductase and b-glucuronidase but increased that of b-glucosidase
(Marteau et al. 1990). This could be an advantage since b-glucosidase may release
flavonoids that have anti-mutagenic, anti-oxidative and immune-stimulatory effects
(Stoner and Mukhtar 1995). Most animal and human studies do indicate that feeding
certain LAB decreases fecal enzyme levels that may be involved in formation of
carcinogens. Also, beneficial effects can be attributed to immune-potentiating effects
by LAB strains. One specific effect was shown by heat-killed Lactobacillus plantarum
L-137, which restored the inhibited IL-12 production in DBA/2 mice with tumors
(Murosaki et al. 2000). The anti-tumor effects were found to be due to the activation of
macrophage by LC9018 (Okawa et al. 1993). Conjugated linoleic acid has been
highlighted recently because of its effect to reduce carcinogenesis, atherosclerosis
and body fats (Chin et al. 1992). Mycotoxins are well-known contaminants of grains
and other food raw materials in which Ochratoxin A is carcinogenic, genotoxic,
474 M. Kumar et al.
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immunosuppressive and nephrotoxic (Petzinger and Ziegler 2000). In some reports,
strains of Lactobacillus and Lactococcus were shown to be resistant to ochratoxin
A(5–10 microgram/disc), and just one strain of L. helveticus was sensitive (0.1
microgram/disc) (Piotrowska and Zakowska 2005). Furthermore, all strains tested
reduced the amount of ochratoxin A in the growth medium as measured after 120 h. L.
acidophilus CH-5, LGG and a couple of other strains reduced the initial level of
ochratoxin A by more than 50%. Intake of such LAB may potentially reduce carci-
nogenic/genotoxic effects of consumption of food contaminated by ochratoxin A.
Aflatoxin B
1
(AFB1), a mycotoxin produced by the common fungi Aspergillus flavus
and Aspergillus parasiticus, is an established human hepato-carcinogen (Vamio et al.
1993). Various strains of LAB isolated from either dairy products or healthy human
microbiota showed that two probiotic L. rhamnosus strains, LGG and LC705, were
found to be highly efficient in binding a range of mycotoxins, including AFB1. Earlier,
a single viable bacterium was found to bind >10
7
AFB1 molecules (Haskard et al.
2000).
Protective and prevention of cancer occurrence
Liver cancer is the sixth most commonly diagnosed cancer in the world, and the third
most common cause of death from cancer, according to Cancer Research UK. Despite
Table I. Potential probiotic bacteria having cancer-preventing properties.
Probiotic bacteria References
B. polyfermenticus Jeong et al. (2003), Park et al. (2004, 2007)
B. longum ATCC 15708 Challa et al. (1997), Vanderhoof (2001), Biasco et al. (1991), Pool-Zobel
et al. (1996), Rowland et al. (1998), Reddy and Rivenson (1993), Singh
et al. (1997), Reddy (1998), Haskard et al. (2000), Lin and Chang (2000)
Lactobacillus GG Goldin et al. (1996), Drisko et al. (2003), Femia et al. (2002), Caro et al.
(2005), Ahotupa et al. (1996), Fang and Polk (2002), Lam et al. (2007)
L. casei (LC9018) Aso et al. (1995), Lidbeck et al. (2000), Okawa et al. (1993)
L. casei Shirota Matsumoto et al. (2009)
L. acidophilus Lidbeck et al. (1992), Biffiet al. (1997),Vanderhoof (2001), Biasco et al.
(1991), Pool-Zobel et al. (1996), Arimochi et al. (1997), Goldin et al.
(1980), Haskard et al. (2000)
L. gasseri Vanderhoof (2001), Pool-Zobel et al. (1996)
L. confusus, Vanderhoof (2001), Pool-Zobel et al. (1996)
S. thermophilus Vanderhoof (2001), Pool-Zobel et al. (1996)
B. breve ATCC 15700 Vanderhoof (2001), Pool-Zobel et al. (1996), Onoue et al.
(1997), Chalova et al. (2008)
L. delbrueckeii Rafter (1995)
B. infantis Biffiet al. (1997)
B. lactis Femia et al. (2002), Sekine et al. (1995)
L. plantarum L-137,
L. plantarum JCM 1551,
L. plantarum KLAB21,
L. plantarum 2592
Murosaki et al. (2000), Ando et al. (2004), Rhee and Park,
(2001), Kruszewska et al. (2002), Khanafari et al. (2007)
P. pentosaceus 16:1 Kruszewska et al. (2002)
L. paracasei F19 Kruszewska et al. (2002)
L. reuteri Iyer et al. (2008)
B. adoleascentis ATCC 15703 Chalova et al. (2008)
VSL#3 Otte et al. (2009)
Cancer-preventing attributes of probiotics 475
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these figures, the cancer remains relatively rare, with 18,500 new cases in the USA
every year, and about 3,000 in the UK. The highest incidences of the disease are in east
and southeast Asia, particularly China, and for this reason the current researchers
looked at the effects of probiotic supplements on markers for the disease. The biggest
risk factor for the disease is said to be chronic hepatitis B virus infection, but
consumption of foods (contaminated with aflatoxins) are also established causes of
liver cancer.
AFB1 is classified as a group I carcinogen in humans by International Agency for
Research on Cancer. However, the exact mechanisms of AFB1 hepatocarcinogenesis
have not been fully elucidated. But some studies have suggested that oncogenes are
critical molecular targets for AFB1, and AFB1 causes characteristic genetic changes in
the p53 tumor suppressor gene and ras proto-oncogenes. The hepatic carcinogen
AFB1 is metabolized in the liver by at least four different P450s, all of which exhibit
large inter-individual differences in the expression levels. These differences could affect
the individual risk of hepatocellular carcinoma. Some researchers provided additional
evidence that reactive oxygen species (ROS) and oxidative DNA damage may be
involved in AFB1-induced p53 and ras mutations. AFB1–glutathione (GSH) conju-
gation is the major pathway for the detoxification of aflatoxin metabolites (Figure 1).
This reaction is catalyzed by glutathione-S-transferase (GST) and plays a major role in
modulation of AFB1 adduct formation to nuclear DNA. Changes recorded in hepatic
GST activity during development of rats can alter the balance between AFB1–GSH
conjugation and AFB1–DNA adduct formation. Some probiotic bacterial strains
successfully bind mycotoxins; L. rhamnosus binds away AFB1 in vivo, thus reducing
bio-absorption of the toxin from the gut, while L. acidophilus and Bifidobacterium longum
neutralize AFB1 and AFM1 by a similar binding mechanism (Haskard et al. 2000).
Epidemiological studies in Africa and Asia (where high levels of exposure occur) link
AFB1 exposure and hepato-cellular carcinomas (Bulatao-Jayme et al. 1982; Bosch and
Munoz 1988), and the risk is synergistic with hepatitis viral infections. The mechanism
of action for AFB1 mutagenicity begins with metabolic activation by CYP3A4,
CYP3A5 and/or CYP1A2 (Garner et al. 1972; Reynolds et al. 1987) that forms an
exo-8,9-epoxide and subsequent adduct formation and DNA damage (Essigmann et al.
Aspergillus spp.
Aflatoxin B1
Aflatoxin-exo8,9-epoxide
CYP 1A2
CYP 3A4
DNA
Alatoxin-N7-Guanine
Genotoxicity
Aflatoxin-S-G
AFB1-mercapturate
Aflatoxin-dihydrodiol
Aflatoxin dialdehyde
Aflatoxin-Albumin
Inhibition-Probiotic
by binding mechanism
Detoxification by antioxidants
produced by probiotics
Figure 1. Mechanism of action of probiotics during liver cancer progression induced by AFB1.
476 M. Kumar et al.
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1977; Iyer et al. 1994). This damage has been shown in vitro to cause guanine
nucleotide substitutions (Lilleberg et al. 1992) specifically to codon 249 of the p53
gene (Aguilar et al. 1993). Mutations in specific oncogenes and tumor-suppressor
genes (e.g. K-ras, p53) are involved in tumor development in the colon and liver, as
they occur at high frequencies in human colon and liver cancers (Erdman et al. 1997).
The expression levels of these genes can be measured and used to demonstrate the
effect of various dietary components on tumor formation in colonic mucosa.
Addition of the probiotic B. longum to the diet of rats was shown to exert a strong
anti-tumor activity on colonic mucosa by reducing the expression level of ras-p21
expression and cell proliferation (Singh et al. 1997; Reddy 1998). LGG administration
determined the upregulation and downregulation of 334 and 92 genes, respectively,
shown by microarray analysis, and further real-time polymerase chain reaction con-
firmed the reliability of the analysis—it mainly affects the expression of genes involved
in immune response and inflammation (transforming growth factor-beta and TNF
family members, cytokines, nitric oxide synthase 1, defensin alpha 1), apoptosis, cell
growth and cell differentiation (cyclins and caspases, oncogenes), cell–cell signaling
(intracellular adhesion molecules), cell adhesion (cadherins), signal transcription and
transduction (Caro et al. 2005). Colorectal cancer represents a major public health
problem accounting for over 1 million cases and about 0.5 million deaths worldwide
(Chau and Cunningham 2006). Survival from colon cancer at 5 years has been found to
vary demographically and estimated to be 65% in North America, 54% in Western
Europe, 34% in Eastern Europe and 30% in India (Parkin et al. 2005). Dietary
interventions and natural bioactive supplements have now been extensively studied to
reduce the risks of colon cancer as a cause of prevention instead of cure. Postulated
mechanisms include reduction in the activity of several cancer-causing bacteria,
des-mutagenic and anti-carcinogenic properties (Collins and Gibson 1999; Chau
and Cunningham 2006). Probiotics have been found by several researchers to decrease
fecal concentrations of enzymes and secondary bile salts, and to reduce absorption of
harmful mutagens that may contribute to colon carcinogenesis (Rafter 1995). Other
studies suggest that normal intestinal flora can influence carcinogenesis by producing
enzymes (glycosidase, B-glucuronidase, azoreductase, and nitroreductase) that
transform precarcinogens into active carcinogens (Goldin et al. 1980; Goldin
1990; Marteau et al. 1990; Ling et al. 1994; Pedrosa et al. 1995). Certain probiotics
may protect the host from this activity. L. acidophilus and Lactobacillus casei supple-
mentation in humans helped to decrease levels of these enzymes, as shown by fecal
specimens (Hayatsu and Hayatsu 1993; Lee and Salminen 1995; Lidbeck et al. 1992).
In animal studies, the aforementioned bacterial enzymes have been suppressed with
the administration of LGG (Drisko et al. 2003). Other LAB have shown similar
results; however, the relationship between enzyme activity and cancer risk needs
further investigation.
Several mechanisms have been proposed as to how LAB may inhibit colon cancer,
including enhancing the host’s immune response, altering the metabolic activity of the
intestinal microbiota, binding and degrading carcinogens, producing antimutagenic
compounds, and altering the physiochemical conditions in the colon (Hirayama and
Rafter 2000) (Figure 2). During carcinogenesis in the colon, aberrant crypts can be
recognized as early neoplastic lesions in both rodents and humans (Bird 1995).
A number of natural chemopreventive agents or medicinal plants that inhibit the
development of aberrant crypt foci (ACF) have been demonstrated to prevent colon
Cancer-preventing attributes of probiotics 477
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cancer in rodents (Finley et al. 2000; Ohishi et al. 2002), thereby suggesting that ACF
assays in the rodent colon can be employed as good biomarkers in colon carcinogen-
esis. Colon carcinogenesis is also known to be a pathological consequence of persistent
oxidative stress, resulting in DNA damage and mutations in cancer-associated genes,
cycle arrest or cell death, and mutations and regeneration in which the cellular
overproduction of reactive oxygen molecules and reactive nitrogen species have
been implicated (Payne et al. 1999; Bartsch and Nair 2002; Korenaga et al.
2002; Nair et al. 2002) In an in vitro experiment, Bacillus polyfermenticus exerted
antioxidative, antigenotoxic, and anticarcinogenic effects (Jeong et al. 2003; Park et al.
2004). Consumption of large quantities of dairy products such as yoghurt and
fermented milk containing Lactobacillus or Bifidobacterium may be related to a lower
incidence of colon cancer (Shahani and Ayebo 1980). Oral administration of LAB has
been shown to effectively reduce DNA damage, induced by chemical carcinogens, in
gastric and colonic mucosa in rats. By the comet assay, L. acidophilus,Lactobacillus
gasseri,Lactobacillus confusus,Streptococcus thermophilus,Bifidobacterium breve and B.
longum were antigenotoxic toward N¢-nitro-N-nitrosoguanidine (MNNG) (Pool-Zobel
et al. 1996). These bacteria were also protective toward 1,2-dimethylhydrazine
(DMH)-induced genotoxicity. Metabolically active L. acidophilus cells, as well as an
acetone extract of the culture, prevented MNNG-induced DNA damage, while heat-
treated L. acidophilus was not antigenotoxic. Goldin et al. (1996) showed that a specific
strain of L. casei subsp. rhamnosus designated GG can interfere with the initiation or
early promotional stages of DMH-induced intestinal tumor genesis and that this
effect is most pronounced for animals fed a high-fat diet. Feeding of L. acidophilus
and B. longum suppressed the formation of ACF and tumor incidence, induced by
azoxymethane (AOM) (Kulkarni and Reddy 1994; Arimochi et al. 1997; Singh et al.
1997; Challa et al. 1997; Rowland et al. 1998) or DMH (Abdelali et al.
Steps leading to cancer
DMH (Procarcinogen)
Carcinogen (Azoxymethanemethyldiazoniumion)
Reactions with cellular targets (colonocytes)
Carcinogen-DNA adduct
Neoplastic manifestation
Cancer (Adenocarcinoma)
Probiotics by
binding mechanism
Probiotics by
antioxidants
Inhibition and detoxification
Figure 2. Mechanism of action of probiotics during colon cancer progression.
478 M. Kumar et al.
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1995; Gallaher et al. 1996). In addition, it has been reported that colonization of
bacteria with an ability to produce genotoxic compounds and high b-glucuronidase
activity enhanced progression of ACF induced by DMH in rats, and that the additional
colonization of B. breve reduced the number of ACF with four or more crypts/focus and
crypt multiplicity that are reliable predictors of malignancy (Onoue et al. 1997). AOM-
induced colon tumor development was also suppressed with a decrease in colonic
mucosal cell proliferation and tumor ornithine decarboylase and ras-p21 activities
(Hirayama and Rafter 2000). There was a report on the anti-tumorigenic activity of
the prebiotic inulin, enriched with oligofructose, in combination with the probiotics
L. rhamnosus and Bifidobacterium lactis in the AOM-induced colon carcinogenesis rat
model (Femia et al. 2002). Reddy and Rivenson (1993) reported that lyophilized
cultures of B. longum administered in the diet to rats inhibited liver, colon and
mammary tumors, induced by the food mutagen 2-amino-3-methyl-3H-imidazo
(4,5-f) quinoline (IQ). Goldin and Gorbach (1980) showed that dietary supplements
of L. acidophilus not only suppressed the incidence of DMH-induced colon carcino-
genesis but also increased the latency period in rats. Feeding of fermented milk
increased the survival rate of rats with chemically induced colon cancer (Shackelford
et al. 1983). B. polyfermenticus was also determined to protect against the DNA damage
induced by MNNG, a direct-acting carcinogen with alkylating properties in CHO-K1
cells and in human lymphocytes, and was also determined to inhibit the growth of
Caco-2 human colon cancer cells in a dose-dependent manner, as shown by the results
of an MTT assay. These in vitro results of B. polyfermenticus were confirmed in the
present in vivo animal study. Our current results indicate clearly that the dietary feeding
of B. polyfermenticus (3.1 x 10
8
cfu/day) effectively suppresses the occurrence of colonic
ACF induced by DMH when administered 1 week prior to treatment with the
carcinogen (Park et al. 2007). Using AOM-induced aberrant crypt foci in rats,
Reddy et al. (1997) found that a stimulated growth of bifidobacteria in the colon
could lead to the inhibition of colon carcinogenesis. Reduction in GST activity is
associated with an increased risk for Colorectal Cancer (CRC) (Szarka et al. 1995).
The use of lactulose separately or in conjunction with B. longum has been demonstrated
to significantly increase GST levels in rat colon (Challa et al. 1997). These results led to
the suggestion that probiotics could suppress colon cancer.
There are 57 cytochrome P450s encoded in the human genome, mainly catalyzing
the metabolism of steroids, bile acids, eicosanoids, drugs and xenobiotic chemicals.
However, some of the P450s are also active carcinogens. Past epidemiological research
has shown increased risk of colon cancer in individuals with high P450-1A2 activity.
The metabolic activation of food-borne heterocyclic amines to colon carcinogens in
humans is hypothesized to occur via N-oxidation followed by O-acetylation to form the
N-acetoxy arylamine that binds to DNA to give carcinogen-DNA adducts. These steps
are catalyzed by hepatic cytochrome P450-1A2 and N-acetyltransferase-2 (NAT-2),
respectively (Lang et al. 1994) (Table II). It has been postulated that probiotics such as
Bifidobacterium could lower the risks of colon cancer, by producing metabolites that
could affect the mixed-function of P450s and subsequently affect the conversion of
azoxymethane from proximate to ultimate carcinogen (Campbell and Hayes 1976).
GST is an important family of phase II detoxification enzymes, which play a crucial role
in protecting the colon mucosa from dietary carcinogens.
Burns and Rowland (2000) suggested that increasing the amount of LAB in the
colon decreases the ability of microbiota to produce carcinogens. A randomized,
Cancer-preventing attributes of probiotics 479
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Table II. Examples of carcinogens, their biomarkers and potential probiotics that reduce their effects.
Target
organ
Biomarkers
Carcinogen Probiotics References Genetic susceptibility Phenotypic biomarker
Activating enzyme with
genetic
polymorphisms
Detoxifying enzyme
with genetic
polymorphisms
Adduct Gene
mutation
AFB1 Liver L. rhamnosus,
L. acidophilus,
B. longum
Haskard et al. (2000) CYP3A4
a
, CYP3A5,
CYP1A2
Glutathione
transferases,
glucuronyl
transferases
Adducts in DNA,
albumin, hemogloblin
and urinary metabolites
p
53
mutation
at codon 249
AGG to AGT
IQ Colon,
breast
B. longum,
S. thermophilus,
B. animalis
Reddy and Rivenson
(1993), Tavan et al. (2002)
CYP1A2, NAT1
b
GST, GPx, NAT2 N-(deoxyguanosin-
8-yl)-2-amino-3-
methylimidazo-[4,5-f]
quinoline (dG-C8-IQ)
k-ras
MEIQ Colon,
breast
S. thermophilus,
B. animalis
Reddy and Rivenson
(1993), Tavan et al. (2002)
CYP1A2, NAT1
b
GST, GPx, NAT2, glucuronyltransferase C-8 guanine
adducts
Kras
PhIP Colon,
breast
S. thermophilus,
B. animalis
Reddy and Rivenson
(1993), Tavan et al. (2002)
CYP1A2, NAT1
b
GST, GPx, NAT2 DNA adduct and
adducts in albumin
C-8 guanine adducts
P53, K-ras
Trp-P-2 Liver,
colon
B. longum Reddy and Rivenson (1993) CYP1A2, CYP1A1, and
CYP2B
GST DNA adduct Kras
DMH Colon L. acidophilus,
L. casei sub sp.
rhamnosus,
B. breve
Goldin et al. (1980,
1996), Onoue et al. (1997)
CYP1A1, CYP2E1 GST, glucuronyl
transferases
O6-Methylguanine
(O6-MeGua) adduct
Kras, c-myc
AOM Colon L. acidophilus,
B. longum
Arimochi et al. (1997),
Kulkarni et al. (1994),
Challa et al. (1997),
Rowland et al. (1998)
CYP1A1, CYP2E1 GST, glucuronyl
transferases
O6-Methylguanine
(O6-MeGua) adduct
Kras, c-myc
MNNG Colon L. acidophilus,
L. gasseri,
L. confusus,S.
Pool et al. (1996),
Park et al. (2007),
Caldini et al. (2005)
CYP1A2, CYP1A3 GST N7-Methyl
deoxyguanosine
Kras
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Table II (Continued)
Target
organ
Biomarkers
Carcinogen Probiotics References Genetic susceptibility Phenotypic biomarker
thermophilus,
B. breve,
B. longum,
B. polyfermenticus
3¢-monophosphate
(N7-MedGp)
DMBA Mammary
cancer,
liver
cancer
L. bulgaricus
191 R
Nadathur et al. (1995) CYP1A1 and CYP1B1 GST DNA adduct K-ras
4NQO Tongue
cancer
L. bulgaricus
191 R, B. longum
(ATCC 15708),
L. acidophilus
(ATCC 4356)
Nadathur et al. (1995), Lin
and Chang, (2000)
CYP1A1 GST, GPx DNA adduct H-RAS
DMAB Colon,
prostate,
pancreas,
urinary
bladder
cancer
L. bulgaricus
191 R
Nadathur et al. (1995) CYP1A2, NAT1
b
GST DNA adduct K-ras
MC Lung
cancer,
liver
cancer,
mammary
cancer
L. casei strain
shirota
Takagi et al. (1999) CYP1A1, CYP1A2 GST-a, UDP,
glucuronyl
transferase, quinone
oxido-reductase I
(NQO1), aldehyde
dehydrogenase
(ALDH)
DNA adduct K-ras
BaP Lung
cancer
B. adoleascentis
ATCC 15703,
B. breve
Chalova et al. (2008) CYP1A1, CYP1B1 GST, glucuronyl
transferase
Benzo[a]pyrene diol
epoxide
Kras, p53
a
CYP, cytochrome P450.
b
NAT, N-acetyltransferase.
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controlled study by Aso and Akazan (1992) demonstrated that the recurrence of
bladder tumors was delayed with daily intake of L. casei. They performed another study
that was larger (125 patients) and placebo-controlled, and found that L. casei reduced
the recurrence of tumors in all patients except those with more than one recurrent
tumor (Aso et al. 1995). In an animal model with DMH-induced colon cancer, it was
shown that LGG significantly reduced the incidence of colon tumors (Goldin et al.
1996). LAB administered to animals have been shown to prevent carcinogen-induced
preneoplastic tumors and lesions (Wollowski et al. 2001) and also reduced the growth
and viability of the HT-29 human colon cancer line (Hirayama and Rafter 2000). L.
gasseri,L. confusus,S. thermophilus,B. breve,B. longum, and L. acidophilus strains
showed an antigenotoxic effect after MNNG administration (Vanderhoof 2001), and
L. acidophilus,L. confusus,S. thermophilus,L. delbrueckeii,L. gasseri,B. longum, and B.
breve inhibited DNA damage from DMH (Rafter 1995).
Supplementation of the chow with 0.5% lyophilized B. longum caused pronounced
inhibition of the incidence of 3-amino-1-methyl-5H-pyrido[4,3-6]indole (Trp-P-2)-
induced liver and colon tumors (i.e. 80% and 100% reduction in tumor frequencies,
respectively) (Reddy and Rivenson 1993); however, the concentration of tryptophan
pyrolysates in meats are much lower than those of other heterocyclic amines (HCAs)
(Felton et al. 2000). Fermented milks prepared by S. thermophilus or Bifidobacterium
animalis inhibited the induction of preneoplastic lesions (ACF in the colon) and
DNA migration caused by a HCA mixture containing IQ, 2-amino-3,4-dimethyl-
3H-imidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]
pyridine (PhIP) (ratio 1:1:1) (Tavan et al. 2002). Anti-mutagenicity of an acetone
extract of a L. bulgaricus 191R fermented yoghurt observed a significant dose-depen-
dent anti-mutagenic activity against several mutagens including MNNG,
4-nitroquinoline-N-oxide, 3,2-dimethyl-4-aminobiphenyl, 9,10-dimethyl-1,2-benz[a]
anthracene and Trp-P2. This study suggests that a metabolite of the LAB may be
responsible for their anti-carcinogenic properties (Nadathur et al. 1995). Bifidobacter-
ium infantis and L. acidophilus bacteria strains inhibited the growth of the MCF7 breast
cancer cell line (Biffiet al. 1997). Studies have suggested that the host’s immune
response may be stimulated by B. infantis, leading to tumor suppression or regression
(Hirayama and Rafter 2000). The metabolic activity of the intestinal microbiota may
also be altered with administration of LAB (Goldin and Gorbach 1984). The alteration
of the physicochemical conditions in the colon may influence colon cancer (Modler
et al. 1990), and suggest that reducing the intestinal pH may prevent the growth of
putrefactive bacteria. In a 3-month study, L. acidophilus and B. bifidum were admin-
istered to patients with colonic adenomas. The result was a decrease in fecal pH and
cell proliferative activity in the upper colon (Biasco et al. 1991). The mechanisms of the
links of probiotics to anti-tumor activity are not completely clear, but offer useful
potential material for future cancer studies.
Possible modes of actions of probiotics: as evidenced by various probiotic
strains
Reduction of reactive oxygen species load
Oxidative stress results when the balance between the production of ROS overrides the
antioxidant capability of the target cell; oxidative damage from the interaction of
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reactive oxygen with critical cellular macromolecules may occur. ROS may interact
with and modify cellular protein, lipid, and DNA, which results in altered target cell
function. The accumulation of oxidative damage has been implicated in both acute and
chronic cell injury including possible participation in the formation of cancer. Oxidative
stress occurs in a cell or tissue when the concentration of ROS generated exceeds the
antioxidant capability of that cell (Sies 1991). ROS can be produced both endoge-
nously and exogenously (Figure 3). Endogenous oxidative stress can be the result of
normal cellular metabolism and oxidative phosphorylation. The metabolism of sub-
stances by the P450 enzyme system generates oxygen free radicals through normal or
futile cycling mechanisms (Parke and Ioannides. 1990). Exogenous sources of ROS
can also impact on the overall oxidative status of a cell. Drugs, hormones, and other
xenobiotic chemicals can produce ROS by either direct or indirect mechanisms (Trush
and Kensler 1991; Halliwell 1996). Alternatively, oxidative stress can also occur when
there is a decrease in the antioxidant capacity of a cell. Non-enzymatic antioxidant
levels (vitamin E, vitamin C, GSH, etc.) and enzymatic antioxidant levels (superoxide
dismutase, GSH peroxidase, and catalase) in the cell can be decreased through
modification in gene expression, decreased in their uptake in the diet, or can be
overloaded in ROS production, which creates a net increase in the amount of oxygen
free radicals present in the cell (Vuillaume 1987; Barber and Harris 1994). Several
human chronic disease states, including cancer, have been associated with oxidative
stress produced through either an increased free radical generation and/or a decreased
antioxidant level in the target cells and tissues (Trush and Kensler 1991; Rice-Evans and
Burdon 1993). Also it is well known that LGG and LGG-fermented milk were potent
scavengers of superoxide anion and inhibitors of lipid peroxidation reactions in vitro
(Ahotupa et al. 1996). Glutathione peroxidase (GPx) and glutathione-S-transferase
Endogenous source of
ROS
mitochondria
cytochrome P450
peroxisomes
inflammatary cells
Exogenous source of
ROS
radiation
ozone
hyperoxia
xenobiotics
Protein damage
(DNA repair
proteins,
Caspases)
DNA damage and
mutation
8-oxo-dG
nitrosamines
8-nitroguanine
S-nitrosothiol
DNA-strand breako
Lipid
peroxidation
4HNE
MDA
Arachidonic
acid cascade
Eicosanoids
Cell
proliferation
Probiotic
bacteria
Source
Enzymatic
(SOD, CAT, GSH
Peroxidase)
Nonenzymatic
(Vit E, GSH, Vit C)
Cell membrane
Figure 3. Effect of probiotics on ROS production that are produced by endogenous and exogenous sources.
Cancer-preventing attributes of probiotics 483
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(GST) are detoxification/biotransformation enzymes that are involved in the detoxifi-
cation of toxic substances such as xenobiotics, carcinogens, free radicals and peroxides
by conjugating these substances with GSH (Hayes and Pulford 1995). Since the ultimate
carcinogenic form of DMH and AFB1 is a toxic electrophile (methyldiazonium ion and
carbonium ion) and exo-8,9-epoxide, GPx and GST take considerable significance in
promoting carcinogen detoxification (Beckett and Hayes, 1993). ROS are produced
during passage of nutrients through the gastrointestinal tract. The natural production of
host antioxidants decreases rostrally. It is well known that oxidative damage forms part in
the pathogenesis of cancer, cirrhosis, atherosclerosis and other chronic diseases.
B. longum (ATCC 15708) and L. acidophilus (ATCC 4356) showed antioxidative activity,
inhibiting linoleic acid peroxidation by 28–48% and also showed the ability to scavenge
the a-diphenyl-b-picrylhydrazyl free radical, scavenging 21–52%. The intact cells of
these two intestinal bacteria demonstrated a high inhibitory effect on the cytotoxicity of
4-nitroquinoline-N-oxide (4NQO). Cytotoxicity of 4NQO was reduced by L. acidophilus
by approximately one-half, and by almost 90% by B. longum. Nevertheless, no inhibition
of cytoxicity was observed for intracellular cell-free extracts of 10
9
cells of B. longum and
L. acidophilus (Lin and Chang 2000). Pediococcus pentosaceus 16:1 and L. plantarum 2592
produced antioxidants after 18 h growth corresponding to 100 microgram vitamin C,
and L. paracasei F19 slightly less but another L. paracasei did not exert antioxidative
activity, again emphasizing that these characteristics are strain dependent (Kruszewska
et al. 2002). In a recent study, obligatory homofermentative lactobacilli produced high
antioxidant activity whereas this was highly strain dependent among facultatively and
obligate heterofermentative lactobacilli (Annuk et al. 2003).
Binding/adsorption of carcinogens
Several carcinogens such as heterocyclic amines and AFB1 are reportedly adsorbed or
bound in vitro by LAB and other intestinal bacteria. A concomitant decrease in
mutagenicity is often reported. (Orrhage 1994; Bolognani et al. 1997). In-depth
investigations have also showed that cultured milk possessed des-mutagenicity and
this activity increased with increasing numbers of viable cells, indicating that probiotics
could play an important role in the inhibition of mutagenicity (Usman 1998). Thya-
garaja and Hosono (1993) found that probiotic isolated from ‘idly’, a traditional cereal
pulse product of India, could exert des-mutagenicity on various spice mutagens,
heterocyclic amines and aflatoxins. Subsequent studies on the des-mutagenicity
properties of probiotics suggested that the des-mutagenic substances may reside in
the cellular envelope of the bacterial cell wall (Singh et al. 1997). A cell wall preparation
of B. infantis was found to inhibit tumor activity in mouse peritoneal cells (Sekine et al.
1995), while a cell wall preparation of heat-killed L. casei (LC9018) was found to
induce immunity against tumor induction in a randomized, controlled and compar-
ative study involving 223 patients with stage III cervical cancer. Also there are some
potential probiotics, Bifidobacterium adoleascentis ATCC 15703 and B. breve ATCC
15700, that exhibited higher antimutagenicity against benzo[a]pyrene (BaP) and
sodium azide (Chalova et al. 2008). Strains of B. lactis were shown to express anti-
mutagenic properties, probably linked to cell wall constituents. The anti-mutagenic
effect occurred even after acid and bile treatment, mimicking the gastrointestinal
transport; interestingly, enhanced in the presence of whole milk (Lo et al. 2004).
One mechanism for this effect could be binding of mutagens, as heterocyclic aromatic
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amines were shown to be bound to the cell wall of certain bacteria such as B. longum
and other LAB, and thereby be detoxified (Orrhage et al. 1994). However, the anti-
mutagenic effect of L. plantarum KLAB21 was mediated by three glycoproteins that are
secreted extracellularly (Rhee and Park 2001).
Antigenotoxic activity of several Lactobacillus species against 4-nitroquinoline-
1-oxide was shown in in vitro tests, whereas only one L. acidophilus strain inhibited
MNNG. All strains also showed anti-mutagenic properties and were viable after the
tests (Caldini et al. 2005). Also, mutagens were suggested to be bound to the cell wall
of probiotics.
Effect on production of bacterial enzymes and metabolites
Oral administration of some LAB/fermented milks to animals and humans leads to a
decrease in certain metabolites and enzymes purported to be involved in synthesis or
activation of carcinogens, genotoxins and tumor promoters such as b-glucuronidase,
b-glucosidase, nitrate reductase and ammonia. Such changes in enzyme activities
concomitantly suppressed tumors. This would appear to be due to the low specific
activity of these enzymes in LAB (Saito and Rowland 1992). Such changes in enzyme
activity or metabolite concentration have been suggested to be responsible for the
decreased level of pre-neoplastic lesions or tumors seen in carcinogen-treated rats given
prebiotics and probiotics (Reddy and Rivenson 1993; Rowland et al. 1998).
Stimulation of host enzymes involved in carcinogen inactivation
There are isolated reports citing that administration of LAB results in increased activity
of antioxidative enzymes or processes that protect cells against carcinogen-induced
damage. These include GST, GSH, GSH reductase, and GSH peroxidase, which
consequently reduce the apoptotic rate and the necrobiotic changes in liver. Such
mechanisms of protection could be effective against a wide range of dietary carcinogens
possibly influencing several cancer sites. Lowering of hepatic GSH levels and enhanced
lipid peroxidation was studied in animals dosed with AFB1 and other carcinogens. It
may be useful to employ hepatic lipid peroxidation as a biochemical marker during
AFB1-initiated hepato-carcinogenesis (Corrales et al. 1992; Aguilar-Delfinetal.
1996; Gasso et al. 1996). Many food-borne carcinogens such as heterocyclic amines
and polycyclic aromatic hydrocarbons are known to be conjugated to GSH, which
appears to result in inactivation. The enzyme involved, GST, is found in the liver and in
other tissues including the gut. A study of the effect of B. longum and lactulose on AOM-
induced ACF in the colon showed that the activity of GSH in the colonic mucosa was
inversely related to the ACF numbers (Challa et al. 1997). Such a mechanism of
protection would be effective against a wide range of dietary carcinogens that the various
LAB can inhibit genotoxicity of dietary carcinogens. The degree of inhibition was
strongly species dependent. For example, Pool-Zobel et al. (1993) demonstrated that
L. casei and L. lactis inhibited the mutagenic activity of nitrosated beef by over 85%,
whereas L. confusus and Lactobacillus sake had no effect. Pre-treatment of mice with
L. casei 1 h before the administration of genotoxins can lead to a moderate reduction
(P<0.05) in the genotoxicity of pro-carbazine and urethane (Abraham and Paul 2001).
Using the technique of single-cell microgel electrophoresis (Comet assay), Pool-Zobel
et al. (1993) investigated the ability of range of species of LAB to inhibit DNA damage in
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the colon mucosa of rats treated with the carcinogens MNNG or DMH. All the strains of
lactobacilli and bifidobacteria tested—L.acidophilus (isolated from a yoghurt sample), L.
gasseri,L. confusus,B. breve and B. longum—prevented MNNG-induced DNA damage
when given at a dose of 10
10
cells/kg body weight 8 h before feeding the carcinogen. In
most cases the DNA damage was reduced to a level similar to that in untreated rats. S.
thermophilus was not as effective as the other LAB strains. The comet assay has also been
used to evaluate the effect of a prebiotic, lactulose on DNA damage in the colonic
mucosa. Rats that were fed a diet containing 3% lactulose and given DMH exhibited less
DNA damage in colon cells than similarly treated animals fed a sucrose diet. In the latter
animals, the percentage of cells with severe DNA damage comprised 33% of the total,
compared with only 12.6% in the lactulose-fed rats (Rowland et al. 1998).
Enhancing the host’s immune response
In animal and human studies with probiotic treatments (L. casei,L. acidophilus,or
B. bifidus) shown to influence several aspects of immune function, enhancing secretory
IgA production was observed in which L. casei is most effective in stimulating secretory
IgA (Perdigon et al. 1991) and increasing the systemic immune response in malnour-
ished animals (Perdigon 1992). There are several other examples of probiotics (L. casei,
LGG, and other strains) that can affect cytokine production and promote a non-
specific immune response by enhancing phagocytosis of pathogens (Erickson and
Hubbard 2000). Another study showed that mice fed LAB had higher splenocyte
proliferation in response to mitogens for T cells and B cells (De Simone et al.
1993; Erickson and Hubbard 2000). LAB shows anti-tumor activity against sarcoma
180, a transplantable mouse tumor (Yokokura et al. 1981). One explanation for tumor
suppression by LAB may be mediated through an immune response of the host. Sekine
et al. (1985) suggested that B. infantis stimulates the host-mediated response, leading to
tumor suppression or regression. In addition, there are studies to suggest that LAB play
an important role and function in the host’s immunoprotective system by increasing
specific and non-specific mechanisms to have an anti-tumor effect (Kato et al.
1983; De Simone et al. 1993; Schiffrin et al. 1995). L. casei Shirota (LcS) has been
shown to have potent anti-tumor and anti-metastatic effects on transplantable tumor cells
and to suppress chemically induced carcinogenesis in rodents. Also, intra-pleural
administration of LcS into tumor-bearing mice has been shown to induce the production
of several cytokines, such as IFN-g, IL-1 and TNF-a, in the thoracic cavity of mice,
resulting in the inhibition of tumor growth and increased survival (Matsuzaki 1998). An
additional study has indicated that oral administration of BLP, a preparation of viable
L. casei YIT 9018, potentiated systemic immune responses that modified T-cell func-
tions in tumor-bearing mice (Kato et al. 1994). It has also been demonstrated that
B. longum and B. animalis promote the induction of inflammatory cytokines (IL-6,
TNF-a) in mouse peritoneal cells (Sekine et al. 1994). Suppression of colon tumor
growth by yoghurt in DMH-treated mice was associated with suppression of the
inflammatory immune response (Wollowski et al. 2001). Many case studies have shown
that consumption of fermented milk resulted in reduction of risk of breast, liver and
colon cancer (Aso and Akazan 1992).
Among them, LcS showed especially high potency. LcS is a strain of LAB that has
been selected for its specific biological activity in humans. This strain is not directly
cytotoxic to tumor cells in vitro; it has been postulated that its anti-tumor action may be
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mediated by augmentation of the host’s immune system (Kato et al. 1981) and also
exhibited marked anti-tumor activity against human malignant cancer cells in clinical
trials (Aso and Akazan 1992; Aso et al. 1995). This idea stimulated further studies of
the anti-tumor and immunoregulatory actions of LcS in various experimental models
(Matsuzaki et al. 1985, 1988; Miake et al. 1985), and it has been demonstrated that oral
administration of LcS has beneficial effects in both humans and animals. It has also
been reported that LcS induced recovery of host immune responses that were decreased
by treatment with carcinogens and augmented the natural killer activity and T-cell
functions of host immune cells. After LcS is ingested by the host, it is incorporated
into M cells in Peyer’s patches and digested to form active components. In Peyer’s
patches, macrophages or dendritic cells that phagocytosed LcS gained the ability to
produce several cytokines, especially TNF. The components of LcS digested in Peyer’s
patches were then recognized through Toll-like receptor 2 in antigen-presenting cells,
resulting in the production of several cytokines that elicited varied responses in host
immune cells (Takeshi et al. 2007). LcS exerted a strong inhibitory effect on carcino-
genesis in mice through the regulation of host immune cells (Takagi et al. 1999) in a
3-methylcholanthrene (MC)-induced carcinogenesis model (Noguchi et al. 1996) that
induced various tumors such as colon, liver, lung, uterine cervix, and mammary gland
cancer models (Rao and Hussain 1988; Baral and Maity 1992). Oral feeding of mice with
LcS inhibits MC-induced tumorigenesis by modulating the host immune responses that
are disrupted during MC carcinogenesis. A possible mechanism of the prevention of the
carcinogenesis is the activation of natural killer (NK) cells. NK cells are large granular
lymphocytes derived from bone marrow, and these cells display non-MHC-restricted
cytotoxicity against a variety of tumors. It is well recognized that NK cells act as cytolytic
effector cells of the innate immune system. Oral feeding of LcS to MC-treated mice
rendered their NK cells tumoricidal in terms of both quality and quantity, resulting in the
suppression of tumor incidence (Takagi et al. 2001). Other possible mechanism of action
in which effector cells that may respond to LcS are dendritic cells. Dendritic cells are
thought to be one of the most important types of cells involved in the presentation of
several antigens and in the production of cytokines (Banchereau and Steinman
1998; Rescigno et al. 1999). Recent studies showed Lactobacillus strain-specific activity
in prevention of murine tumorigenesis and in induction of IL-12 release by bone
marrow-derived dendritic cells in vitro (Takagi et al. 2008). Recently, Matsumoto
et al. (2009) described the role of the LAB strain LcS for prevention of colon cancer
by targeting immune system. They reported that L. casei produces polysaccharide, which
suppresses colon carcinogenesis by suppressing synthesis of IL-6 and signal transducer
and activator of transcription 3 in a mouse model.
These findings suggest that treatment with probiotics has the potential to ameliorate
or prevent tumorigenesis through modulation of the host’s immune system, specifically
cellular immune responses. Elucidating the precise mechanism of cancer prevention by
probiotic bacteria may help identify a novel and effective molecular target for cancer
prevention.
Production of conjugated linoleic acid from castor oil
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
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anti-atherogenic, anti-diabetic, anti-inflammatory and anticarcinogenic properties
(Maggiora et al. 2004). In vitro, CLA inhibits the growth of HT-29 and Caco-2 colon
cancer cells (Lampen et al. 2005). CLA-treated SW480 colonic tumor cells possess
increased caspase-3 and caspase-9 activities and reduced Bcl-2 expression compared
with controls (Miller et al. 2002). CLA has been shown to reduce the incidence of
colonic, skin, mammary, and prostate carcinogenesis in animal models (Belury 2002).
Rats supplemented with CLA showed reduced incidence of colonic tumors and
increased apoptotic indices in response to the administering of DMH (Kim and
Park 2003). Some probiotic strains of bacteria have demonstrated protective effects
against tumor production (Sekine et al. 1995; Biffiet al. 1997; O’Mahony et al.
2001; Saikali et al. 2004). L. acidophilus and B. longum have been shown to reduce
incidence of colonic tumors and aberrant crypt foci, respectively, in animal models
(Rowland et al. 1996; McIntosh et al. 1999). A cocktail of probiotic strains was recently
demonstrated to increase the colonic apoptotic index in normal rats (Linsalata et al.
2005). Several strains of bacteria that are considered to have probiotic effects
(i.e. lactobacilli and bifidobacteria) are capable of converting linoleic acid to CLA.
In this study, we investigated a previously unreported mechanism of probiotic action:
the production of CLA. We demonstrated that probiotic strains in VSL3 (L. casei,
L. plantarum,L. acidophilus,L. delbrueckii subsp. bulgaricus,B. infantis,B. breve,
B. longum,S. salivarius subsp. thermophilus) have the capacity to convert linoleic
acid to CLA, inducing the upregulation of PPARg, a reduction in cancer cell viability,
and the induction of apoptosis. This linoleic acid-conjugating capacity is maintained
in vivo (Julia et al. 2006). One LAB strain, L. plantarum JCM 1551, was shown to
efficiently produce conjugated linoleic acid from castor oil in the presence of lipase,
which is an interesting ‘side effect’of anti-tumor activities of LAB (Ando et al. 2004).
By regulation of apoptosis
Apoptosis is a genetically regulated active process abolishing cell populations in both
physiological and pathological processes. Impaired apoptosis, either due to expression
of oncogenes or mutations of tumor suppressor genes, leads to an uncontrolled
accumulation of malignant cells, and eventually to cancer.
Caspase-3 is one of the cysteine proteases that play a major role in the execution of
apoptosis (Nicholson 1999). A number of genetic and biochemical studies suggest that
caspase activation is essential for the occurrence of the apoptotic phenotype of cell
death (Janicke et al. 1998) A variety of caspase substrates are involved in the regulation
of DNA structure, repair and replication. Caspase-3 substrate cleavage has been
observed under oxidative stress in different pathological conditions (Nicholson and
Thornberry 1997). Aflatoxins are one of the most dangerous mycotoxins known, owing
to their high toxicity to both animals and human. AFB1, a metabolite of Aspergillus
flavus, is a potent hepatotoxic and hepatocarcinogenic mycotoxin. One of manifesta-
tions of AFB1-induced toxicity is oxidative stress (Souza et al. 1999). Recently, it is
accepted that oxidative stress is an apoptosis inducer (Chandra et al 2000). Many
agents that induce apoptosis are either oxidants or stimulators of cellular oxidative
metabolism. Conversely, many inhibitors of apoptosis have antioxidant activities or
enhance cellular antioxidant defenses (Freeman and Grapo 1982). The GSH redox
cycle is an important component of the antioxidant machinery in cells. In normal cells,
a primary defense against oxidative damage is provided by antioxidants such as GSH
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and the onset of apoptosis is associated with a fall in intracellular GSH in numerous
cellular systems (Ratan et al. 1994).
There are isolated reports citing that administration of LAB results in increased
activity of antioxidative enzymes or process that protect cells against carcinogen-
induced damage. These include GST, GSH, GSH reductase, and GSH peroxidase,
which consequently reduce the apoptotic rate and the necrobiotic changes in
liver. Such mechanisms of protection could be effective against a wide range of
dietary carcinogens possibly influencing several cancer sites. Some probiotics
(e.g. LGG) prevent cytokine-induced apoptosis in intestinal epithelial cell models.
Also it is well known that LGG and LGG-fermented milk are potent scavengers of
superoxide anion and inhibitors of lipid peroxidation reactions in vitro (Ahotupa et al.
1996).
Culture of LGG with either mouse or human colon cells activates the anti-apoptotic
Akt/protein kinase B. This model probiotic also inhibits activation of the pro-apoptotic
p38/mitogen-activated protein kinase by TNF, IL-1aor IFN-g. Furthermore, products
recovered from LGG culture broth supernatant showed concentration-dependent
activation of Akt and inhibition of cytokine-induced apoptosis. These observations
suggest a novel mechanism of communication between probiotics and colon epithelia
that increases survival of intestinal cells normally found in an environment of
pro-apoptotic cytokines (Fang and Polk 2002). LGG enhanced gastric ulcer healing
via attenuation of the cell apoptosis to cell proliferation ratio and an increase in
angiogenesis. Regulators of these processes such as ornithine decarboxylase, B-cell
lymphoma 2 (Bcl-2), vascular endothelial growth factor and epidermal growth factor
receptor are likely to be involved in the healing action of LGG for gastric ulcer
(Lam et al. 2007). B. longum is a probiotic, known for its beneficial effects to the
human gut and even for its immune-modulatory and anti-tumor activities. Recently,
many studies have indicated an intimate relation between probiotic bacteria and the
gut mucosa and its influence on human cellular homeostasis. Bacteria were incubated
with Caco-2 cells to investigate apoptotic deletion. Co-cultures of Caco-2 cells with
adherent strains (B12 and B18) of B. longum induced DNA fragmentation, indicating
that it can induce apoptotic deletion of Caco-2 cells. Thus it helps in restoring the
ecology of damaged colon tissues. Lactobacillus reuteri secretes factors that potentiate
apoptosis in myeloid leukaemia-derived cells induced by TNF, as indicated by
intracellular esterase activity, terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate nick end-labeling assays and poly(ADP-ribose) polymer-
ase cleavage. L. reuteri downregulated nuclear factor-kB-dependent gene products
those mediate cell proliferation (Cox-2, cyclin D
1
) and cell survival (Bcl-2, Bcl-xL).
Moreover Otte et al. (2009) demonstrated that a probiotic combination call VSL#3
significantly suppressed the COX-2 expression in a cancer cell line (Colo320 and
SW480 intestinal epithelial cells). L. reuteri suppressed TNF-induced nuclear
factor-kB activation, including nuclear factor-kB-dependent reporter gene
expression, in a dose-dependent and time-dependent manner. L. reuteri may
regulate cell proliferation by promoting apoptosis of activated immune cells via
inhibition of IkBaubiquitination and enhancing pro-apoptotic mitogen-activated
protein kinase signaling. An improved understanding of L. reuteri-mediated effects
on apoptotic signaling pathways may facilitate development of future probiotic-
based regimens for prevention of colorectal cancer and inflammatory bowel disease
(Iyer et al. 2008).
Cancer-preventing attributes of probiotics 489
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Conclusions
Although there is no direct evidence for the prevention or treatment of cancer
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 tumor formation and aberrant crypt formation, thereby suppressing bac-
terial enzyme activities and reducing DNA damage. Mechanistic studies indicate that
probiotics may exert anticarcinogenic properties by altering colonic metabolism, degrad-
ing carcinogens, producing antimutagenic compounds, and enhancing host immune
responses and by preventing cytokine-induced apoptosis of colonic epithelial cells.
Therefore LAB offer potential as chemo-protective agents and thus further research
is clearly needed to quantify the beneficial effects for prevention of human cancer.
Declaration of interest: The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
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