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THE “LITTLE” BUT POWERFUL PROBIOTICS AGAINST COLON CANCER
Dokos Charalampos and Mironidou-Tzouveleki Maria
Laboratory of Pharmacology, Medical School, Aristotle University of Thessaloniki,
Greece
Keywords: Colon Cancer (colon Ca), probiotics, diet, nutrition, anti-mutagenic agents.
Correspondence author
Dr. Maria Mironidou-Tzouveleki
Anaesthesiologist
Associate Professor of Pharmacology
Komninon 4, 57010, Filyro, Thessaloniki, Greece
[email protected]
Tel: 00302310678385, Fax: 00302310678385.
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Summary
Colon cancer (Colon Ca) is a type of cancer that has great morbidity and
mortality rate in western countries. Although the molecular and genetical basis of
colorectal cancer is well established, environmental factors are still under research. Diet
seems to be a major contributor to colon Ca, associated with the balance of colonic
microflora. Probiotics, live microorganisms usually in dairy fragment cultures, are in the
scope of current research as general anti-mutagenic agents in gastrointestinal diseases
including colon Ca. Laboratory animal studies have shown that two species:
Lactobacillus and Bifidobacteria have anti-mutagenic properties in colorectal cancer.
However no direct evidence from dietary intervention or epidemiological studies exists
that correlates diet and nutrition as contributors to colon Ca. Nevertheless research is
still going on to throw light on the precise mechanisms that probiotics act as antimutagenic agents in colon Ca.
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Introduction
Colorectal cancer is a serious form of cancer, which attacks both men and women.
Surprisingly it is the second most serious form of cancer in men, after lung cancer and
women, following breast cancer. Over the past 20-30 years colorectal cancer has shown
a little decrease in general population based on epidemiological studies. These studies
imply that diet seems to be an important factor of increasing the risk of colorectal
cancer.1- 4
On the other hand human gut is a natural habitat of a powerful bacterial
community colonized by 300-500 different bacteria species.5,6 In experimental models,
colonic microflora is a contributor to gastrointestinal diseases and also colon cancer
(CCA).1,6-8 Laboratory animal studies also support that probiotics, prebiotics (nondigestible carbohydrates that stimulate lactic acid bacteria in gut) and synbiotics (agents
that combine probiotics and prebiotics bacteria) have a positive effect on CCA.1,4,5,7
Although reports in current literature indicate that human diet is a contributor to CCA,
there is no direct evidence for cancer suppression in humans. CCA’s molecular identity
is well defined and described but epidemiological, animal and human dietary
intervention studies must be accomplished because little is known about the potential
mechanisms through which probiotic bacteria may inhibit CCA.
Probiotics
Human species live in synbiosis and the majority of the bacteria is found in colon.4
Colonic microflora starts immediately after birth (sterile infant colon) and within few
days is well established. Naturally the type of delivery and diet might affect the
rhythm of colonization and type of colonic microflora. After birth these bacteria are
vulnerable to environmental factors. These factors vary from country to country
according to economic and social status.4,5 Examples of great interest are low-birthweight premature infants delivered by caesarian section and antibiotic administration to
infants which can dramatically change the colonization type of microflora.9
The term probiotic was derived from the Greek word “pro-biotikos” meaning “life”.
Indeed probiotics are living organisms with the aim of benefiting health by balancing
intestinal microflora. These valuable microorganisms were first characterized and
described in modern times by Fuller10 as “live microbial feed supplements which
beneficially affect the host animal by improving its microbial balance”. From the past
until nowadays the key of success and admiration for probiotics in general population
seems to be the huge effects of probiotics on gastrointestinal tract. In the past yoghurt,
ferment milk products and cheese were favorite dairy products among consumers. A
yoghurt per se, which is by the way a probiotic food product, was a favorite habit
years ago. It is very important to remember that the Russian Nobel prizewinner Elie
Metchnikoff observed a high life expectancy in Bulgarian shepherds who ate large
amounts of ferment-milk products.3 In fact today experts still debate whether yoghurt
starter cultures such as Lactobacillus bulgaricus and Streptococcus thermophilus should
be considered as probiotics. Notice that the yoghurt starter cultures are sensitive to high
pH conditions in the digestive tract and do not have a high colonization type in colon
tract. Benefits of these starter cultures, which are still discussed, are the improvement
of lactose digestion and reinforcement of immune system.5,11,12
Lactose maldigestion is a frequent disease that occurs in adults and in persons with
bowel resection or enteritis. Persons with lactose maldigestion have better digestion and
tolerance of lactose contained in yoghurt than in milk. Yoghurt bacteria deliver their
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lactase enzyme and digest lactose. It is also believed that this intestinal delivery of
lactose is much slower than that of milk.10,12
As a result a microorganism so as to be defined as an effective probiotic agent
must have the following properties: (a) adhere to cells, (b) exclude or reduce
pathogenic adherence, (c) persist and multiply, (d) produce acids, hyperoxide and
bacteriocins (defined as local antibiotics) antagonist to pathogenic growth, (e) be safe,
noninvase, noncarcinogenic, (f) coaggregate to form normal balanced flora.13
Although these characteristics have been proposed as to be named a specific strain
“probiotic”, it seems they are not enough for public safety. FAO (Food and Agriculture
Organization) and WHO (World Health Organization) have strict guidelines about
candidate bacteria strains to be used as probiotics. Except for the above properties,
functional and safety assessments need to be accomplished. Functional tests include in
vitro and animal studies. Safety assessments include a minimum of series of tests
including antibiotic resistance pattern, metabolic activities, toxin production, hemolytic
activity and three phases of Clinical Studies each described by certain guidelines.12
Above any matter of public safety various studies indicate that probiotics have a
wide range of general biological effects on human organism such as: (a) in vitro and
in vivo enhance human immunity system reinforcement9-11,13-15 (b) reduce incidence or
duration of specific diarrhea illnesses (e.g acute viral diarrhea)9,10,13,15 (c) contribute to
the prevention and treatment of gastrointestinal conditions (e.g H. Pylori infection) and
inflammatory bowel diseases (e.g Cronh’s disease)5,9,11,14,15 (d) improve asthmatic and
food allergy status of children8 and (e) cholesterol lowering levels.9,13,16
Probiotics and Colon cancer (CCA)
a) Laboratory animal studies
Several animal models have been proposed for CCA research associated with
probiotics, prebiotics and synbiotics. Probiotics are the most supportive evidence of the
theory supposing that diet contributes to at least 75% of cases with CCA. Prebiotics are
the “new entrance” that gains field in research but modern trends implicate that
synbiotics are more effective than either pro- or prebiotics.1,7
The main two genera of probiotics that are widely used in cancer research are:
Bifidobacterium and Lactobacillus species.1,13,14 These Gram-positive bacteria that are
extensively used and named as probiotics have all the above mentioned characteristics
so as to be probiotics according to FAO and WHO guidelines.9,11
Rodent model studies support the anti-carcinogenic properties for probiotics in
CCA.1 These probiotics strains have the ability to modify colonic bacteria activities17
that take part in initation of CCA through a production of carcinogens, cocarninogens
or procarcinogens.5 Oral administration in rats of these lactic acid bacteria effectively
reduces DNA damage induced by chemical carcinogens in colonic mucosa1 such as Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) (Fig.1). Generally N-nitroso compounds are
a group of genotoxic substances best known as initiators and promoters of CCA.5,17
The anti-mutagenic activities of Lactobacillus and Bifidobacteria species have been
shown experimentally both in killed and alive bacterial cells. According to these data,
live Lactobacillus and Bifidobacteria microorganisms showed a higher anti-mutagenic
activity than killed bacterial cells of these species.13,14 Also it has been reported that
oral administration in rats of the carcinogen 1,2-dimethyl hydrazine (DMH) in
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combination with lactic acid bacteria (e.g bacteria from Lactobacillus species) or
yoghurt, significantly have prevented DNA damage in cells of gastrointestinal tract
(Fig.2).3
Another mutagen that has been reported in CCA rats is IQ (2-amino-3-methylo-3Hinidizo(4,5-f)quinoline). IQ is a heterocycle aromatic amine produced by food pyrolisis
(Fig. 3). Food mutagen IQ has a multitarget organospecificity with specific cancer
induction in Zymbal gland, skin, colon, oral cavity and mammary gland of rodents.18 It
has been proved that lyophilized B. longum strains in male and female rats inhibited
the formation of CCA tumors induced by IQ.1,18 Notice that IQ is not directly
mutagenic but is metabolized by gut bacteria to active mutagen metabolite 7-OH IQ.19
Modern trends in laboratory animal studies imply the insertion of preneoplastic
lesions such as the aberrant crypt foci (ACF) (Fig.4).1,20 ACF is a putative neoplastic
lesion from adenomas and carcinomas that may develop in the colon of rodent
models.13,20 It has been identified as a preneoplastic lesion visible in the colon of
rodents as early as two weeks after carcinogen administration (e.g AOM, DMH). Also
ACF has been identified in humans with easy quantify methods and because of its
distinct preneoplastic nature. Normally few dysplastic ACF both in human subjects and
rodents will be developed into cancers.17,20 Studies indicate that probiotics (L.
acidophilus and B. longum) inhibit the formation of ACF induced by azoxymethane
(AOM) (Fig.5).1,16 A decrease is also observed in β-glucoronidase activity 13 and
heterocycle amines concentration.5,13
As mentioned above CCA is a well-defined type of cancer in both rodents and
humans, with a series of precise histological changes in colonic tract with parallel
mutations, activations and deletions of oncogenes and tumor suppression genes (Fig.6).
At the same time members of colon microbial community promote CCA by producing
specific enzymes such as β-glycosidase, β-glucuronidase, azoreductase, nitroreducatse.
These enzymes are transformed into active carcinogens resulting in CCA.4,11 The
question that now rises is when and how probiotics act in CCA process.
In conclusion environmental factors such as diet have a major role in development
of CCA in rodent models. However at this point it must be noticed that current
literature indicated the differences between human and rodent caecum and colon. This
could be a point of debate among researchers about the usefulness of those experiments
using rodent models with CCA. Nevertheless laboratory rat models provide a supportive
evidence for a laboratory based phenomenon and imply the association between
nutritional and diet habits with CCA.17
b) Human-epidemiological studies
Rodent model studies have proved the correlation between CCA and diet.
However few epidemiological studies associate human diet with CCA. High
consumption of dairy products containing Lactobacillus or/and Bifidobacterium may be
related to lower risk of CCA.1 Epidemiological studies have related cancers in systemic
organs, such as colon and stomach to inappropriate diet routine.19 Nevertheless more
human epidemiological studies are needed so as to make definite conclusions about the
beneficial effects of probiotics on the prevention or treatment of CCA.
From the few epidemiological studies one that is of great interest has to do with
the ingestion of L. acidophilus or best known as L. casei. Scientists have associated the
activity of this probiotic bacterium with the significantly reduced levels of precarcinogen enzymes such as β-glucoronidase.5,11 Faecal and urine samples of healthy
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volunteers showed low mutagenic activity after consumption of fried meat and L.
casei.1,5,11,17 Probably the mutagenic compounds were taken up or degraded by the
probiotic bacteria.15 L. casei Shirota (present in “Yakult” preparation) reduced also the
recurrence rate of superficial bladder cancer. It has been reported to exhibit antitumor,
immunostimulatory and antimicrobial activities. “Yakult” is a Japanese milk-based drink
consumed by approximately 26 million people every day.9,11,21
Epidemiological studies are too difficult to be accomplished because human
intervention studies lack well-validated biomarkers of CCA so risk biomarkers are under
development.4 The SYNCAN project, funded by European Union (www.syncan.be),
involves eight research centers in Europe so as to study the biochemical markers of
CCA including colonic mucosal markers, faecal water markers and immunological
markers. This 12 week randomized, double-blinded placebo controlled trial uses food
supplement containing Lactobacillus GG, Bifidobacterium Bd12 and Raftilose Synergyl
in adenoma patients of CCA (Fig.7).1,4
The few epidemiological studies that exist are contradictory. Some studies could
not find association between ferment-milk products consumption and risk of CCA.
Others show a significant but lower incidence of CCA with probiotics strains of
ferment-milk products.1,3,12
So not all studies show a correlation between probiotics and microflora
activities. Different probiotics may or may not induce significant changes in human
colonic microflora.17,22 It is obvious that there is a problem both in accurate counting of
the colonic microflora and a quantitative assessment for research purpose. Molecular
biology’s methods such as fluorescent label and 16S RNA oligonucleotide probes
designed as primers for polymerase chain reaction (PCR) have already been used for
this purpose.17,23,24 Indeed in near future, genomics and proteomics will investigate the
effect of pro- and prebiotics on gene expression and post-transciptional modifications.
Data from molecular biology’s research will acquire further process so as to identify
human groups responsive to pro- and prebiotics intervention.
Using sequence analysis of clone libraries from amplified ribosomal DNA we
can identify certain bacteria species suitable for colonic microflora.23,24 These new
techniques of molecular biology like gene engineering may aid in designing and
inserting new generations of probiotics in human colon. These new recombinant
probiotics will be humanized and they are under development. They will produce
predetermined therapeutic proteins for general gastrointestinal disease including CCA in
humans (e.g subolin).13
Conclusions
A lot has already been done in the area of laboratory animal studies. From
these rodent studies there is indirect evidence that specific probiotics strains contribute
to inhibition of CCA caused by carcinogenic compounds. Unfortunately few
epidemiological studies give hope so as to associate probiotics and prevention of CCA
in general population. However there are unanswered questions about how probiotics
inhibit CCA development: is it an alteration of the metabolic activities of intestinal
microflora? an alteration of physicochemical conditions in colon? a binding and
degradation of potential carcinogens or production of antimutagenic compounds?
Anyway work has to be done so as to have a definite and well trusted answer for the
correlation between probiotics and risk of human CCA with the aid of molecular
biology’s techniques.
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Taking account of the results in current literature on both human and laboratory
animal studies, the theory that states that specific probiotics strains may inhibit the
development of human CCA seems to gain more and more researchers trust;
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Figures
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12
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DIET INTERVENTION
12 Weeks
20 Patients/Group
PATIENTS WITH
HIGH RISK
FOR COLON CANCER
(POLYPECTOMISED)
CONTROL DIET
Blood, faeces and
biopsy samples
to BIOMARKER
PATIENTS WITH
HISTORY
OF COLON CANCER
(DUKES B)
NETWORK
SYNBIOTIC DIET
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Legends
Fig. 1: Chemical structure of N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).
Fig. 2: Chemical structure of 1,2-dimethyl hydrazine (DMH).
Fig. 3: Chemical structure of 2-amino-3-methylo-3H-inidizo(4,5-f)quinoline (IQ).
Fig. 4: Histological sample
www.researchcanceruk.org).
of
aberrant
crypt
foci
(ACF)
(adapted
from
Fig. 5: Chemical structure of azoxymethane (AOM).
Fig. 6: Sequential genetic changes that are associated with dysplasia and malignant
neoplasia of the colon (adapted in Expert Review in Molecular Medicine ©1999
Cambridge University Press).
Fig. 7: Simplified diagram of SYNCAN project.
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