Scientific basis of biomarkers and benefits of functional foods for reduction of disease risk: Cancer

Abstract

One of the most promising areas for the development of functional foods lies in modification of the activity of the gastrointestinal tract by use of probiotics, prebiotics and synbiotics. While a myriad of healthful effects have been attributed to the probiotic lactic acid bacteria, perhaps the most controversial remains that of anticancer activity. However, it must be emphasised that, to date, there is no direct experimental evidence for cancer suppression in man as a result of consumption of lactic cultures in fermented or unfermented dairy products, although there is a wealth of indirect evidence, based largely on laboratory studies. Presently, there are a large number of biomarkers available for assessing colon cancer risk in dietary intervention studies, which are validated to varying degrees. These include colonic mucosal markers, faecal water markers and immunological markers. Overwhelming evidence from epidemiological, in vivo, in vitro and clinical trial data indicates that a plant-based diet can reduce the risk of chronic disease, particularly cancer. It is now clear that there are components in a plant-based diet other than traditional nutrients that can reduce cancer risk. More than a dozen classes of these biologically active plant chemicals, now known as 'phytochemicals', have been identified. Although the vast number of naturally occurring health-enhancing substances appear to be of plant origin, there are a number of physiologically active components in animal products (such as the probiotics referred to above) that deserve attention for their potential role in cancer prevention.

Full text

Scientific basis of biomarkers and benefits of functional
foods for reduction of disease risk: cancer
Joseph J. Rafter*
Department of Medical Nutrition, Karolinska Institutet, NOVUM, S-141 86 Huddinge, Sweden
One of the most promising areas for the development of functional foods lies in modification of
the activity of the gastrointestinal tract by use of probiotics, prebiotics and synbiotics. While a
myriad of healthful effects have been attributed to the probiotic lactic acid bacteria, perhaps the
most controversial remains that of anticancer activity. However, it must be emphasised that, to
date, there is no direct experimental evidence for cancer suppression in man as a result of con-
sumption of lactic cultures in fermented or unfermented dairy products, although there is a
wealth of indirect evidence, based largely on laboratory studies. Presently, there are a large
number of biomarkers available for assessing colon cancer risk in dietary intervention studies,
which are validated to varying degrees. These include colonic mucosal markers, faecal water
markers and immunological markers. Overwhelming evidence from epidemiological, in vivo,
in vitro and clinical trial data indicates that a plant-based diet can reduce the risk of chronic
disease, particularly cancer. It is now clear that there are components in a plant-based diet
other than traditional nutrients that can reduce cancer risk. More than a dozen classes of
these biologically active plant chemicals, now known as ‘phytochemicals’, have been identified.
Although the vast number of naturally occurring health-enhancing substances appear to be of
plant origin, there are a number of physiologically active components in animal products
(such as the probiotics referred to above) that deserve attention for their potential role in
cancer prevention.
Probiotics: Cancer: Biomarkers: Phytochemicals: Animal products
Introduction
In recent years, there has been a growing interest in the
concept of ‘functional foods’ by both the food industry
and the consumer. Functional foods are defined as food-
stuffs that improve overall health and/or reduce the risk
of disease. Thus, with the consumer becoming more inter-
ested in foods which benefit health and the food industry
beginning to understand the market potential of functional
foods, solid research activity in the area has been initiated
world-wide. In addition, when one considers the costs to
society of diseases such as obesity, cardiovascular disease,
diabetes, food allergies, osteoporosis and cancer, all of
which may be influenced by diet, the potential of well-
characterised functional foods becomes even more evident.
Although a large number of naturally occurring health-
enhancing substances are of plant origin, there are a
number of physiologically active components in animal
products that deserve attention for their potential role in
disease prevention.
One such example of a functional food, which has been
the focus of increasing research activity in recent years, are
probiotics: live microbial feed supplements that benefi-
cially affect the host animal by improving its intestinal
microbial balance (Fuller, 1989). Probiotics usually refer
to highly selected lactic acid bacteria, e.g. Lactobacillus
spp., Bifidobacterium spp. and Streptococcus spp., with
defined gut survival properties and associated biological
activities, that can be ingested in fermented milk products
or as a supplement. The list of healthful effects attributed
to probiotic bacteria is extensive and includes alleviation
of lactose intolerance symptoms, serum cholesterol
reduction, anticancer effects, alleviating constipation and
relieving vaginitis, to name but a few. The vast majority
of studies on the anticancer effects deal with colorectal
cancer (Hirayama & Rafter, 2000), although there are
some on breast and bladder cancer.
There is also a large amount of evidence, from epi-
demiological, in vivo, in vitro and clinical trial data,
indicating that a plant-based diet can reduce the risk of
chronic disease, particularly cancer. In 1992, a review of
200 epidemiological studies showed that cancer risk in
* Corresponding author: Dr J. J. Rafter, fax + 46 8 711 66 59, email joseph.rafter@mednut.ki.se
Abbreviations: COX-2, cyclo-oxygenase-2; SYNCAN, Synbiotics and Cancer Prevention in Humans.
British Journal of Nutrition (2002), 88, Suppl. 2, S219–S224 DOI: 10.1079/BJN2002686
q International Life Sciences Institute 2002

people consuming diets high in fruits and vegetables was
only one-half that in those consuming few of these foods
(Block et al. 1992). It is now clear that there are com-
ponents in a plant-based diet other than traditional nutrients
that can reduce cancer risk. More than a dozen classes of
these biologically active plant chemicals, now known as
‘phytochemicals’, have been identified. Several examples
are given below.
Functional foods from animal sources
Dairy products
Epidemiological studies regarding the effect of milk and
dairy products on colorectal cancer incidence provide
conflicting results. Some studies report a negative associ-
ation between consumption of dairy products and disease
incidence, whereas others report no or even a positive
association. One reason for this discrepancy in the epi-
demiological observations may lie in the fact that some
dairy products, such as cheese and cream, contain large
amounts of animal fat, which is considered a risk factor
by some for colorectal cancer. In line with this assumption,
it appeared that the positive correlation between dairy
products and colorectal cancer found in one study was
due to cheese consumption (Iscovich et al. 1992). In
addition, an international correlation study observed a
negative correlation between consumption of dairy pro-
ducts and colorectal cancer only after adjustment for
animal fat intake (McKeown-Eyssen & Bright-See,
1984). Most of the studies that reported no association
did not adjust for fat intake. Hence, epidemiological
studies provide weak evidence that consumption of milk
and dairy products could protect against colorectal cancer.
The main hypothesis to explain this effect has been the
high Ca content of dairy products. Thus, many previous
studies have focused on Ca and consequently examined
the effects of dietary Ca supplements (Lapre et al. 1993)
or Ca-deprived milk (Govers et al. 1996) on risk markers
for the disease, in controlled dietary intervention studies.
While the results of these studies generally do show a
protective effect of Ca on the biomarkers, this is not
always the case. However, recently, a great deal of
research has been dealing with other components in dairy
products, particularly fermented dairy products, i.e. the
probiotic bacteria.
Many studies confirm the involvement of the endo-
genous microflora in the onset of colon cancer. This
makes it reasonable to think that changing the intestinal
microflora could influence tumour development. Thus,
considerable attention has focused on dietary components
that can influence the gut microflora as a strategy for
colon cancer prevention. In particular, evidence has been
accumulating from animal studies for a protective role
for probiotics, prebiotics (non-digestible carbohydrates
that stimulate lactic acid bacteria numbers in the gut) and
synbiotics (combinations of pro- and prebiotics).
Several studies have indicated that addition of probiotic
cultures (e.g. Lactobacillus rhamnosus GG, Lactobacillus
salivarius or Bifidobacterium longum ) to the diet of rats
treated with colon carcinogens reduces colon tumour
incidence and multiplicity (Hirayama & Rafter, 2000).
The precise mechanisms by which lactic acid bacteria
may inhibit colon cancer are presently unknown. However,
such mechanisms might include: enhancing the host’s
immune response; binding and degrading potential
carcinogens; quantitative and/or qualitative alterations in
the intestinal microflora incriminated in producing putative
carcinogen(s) and promoters (e.g. bile acid-degrading
bacteria); producing anti-tumourigenic or anti-mutagenic
compounds in the colon; alteration of the metabolic
activities of intestinal microflora; alteration of physico-
chemical conditions in the colon; and effects on the
physiology of the host.
A wide range of studies have also been conducted in
rodent models, including transgenic mice, on the cancer-
modulating effect of dietary prebiotics such as fructo-
oligosaccharides, inulins and lactulose. These have
consistently demonstrated a reduction in the endpoints
measured — colonocyte DNA damage, aberrant crypt
foci in the colon (believed to be pre-neoplastic lesions),
number of tumours per animal, size and growth rate of
the tumours, mean survival time, increase in life span
(Pierre, 1997). In all animal experiments where mixtures
of probiotics (Bifidobacterium spp., Lactobacillus casei,
L. rhamnosus ) and prebiotics (lactulose, long-chain
inulin, short-chain fructo-oligosaccharides) were tested,
the effect of the mixture on the measured biomarkers
was greater than the sum of the two separately, suggesting
a combinational advantage of synbiotics.
However, to date, there is no definite evidence that
probiotics actually reduce the risk of human colon
cancer. The epidemiological studies are inconsistent
(Hirayama & Rafter, 2000). Consumption of large quan-
tities of dairy products such as yoghurt and fermented
milk containing Lactobacillus or Bifidobacterium may be
related to a lower incidence of colon cancer. An epidemio-
logical study performed in Finland demonstrated that,
despite the high fat intake, colon cancer incidence was
lower than in other countries because of the high consump-
tion of milk, yoghurt and other dairy products (Intestinal
Microbiology Group, 1977). In two population-based
case–control studies of colon cancer, an inverse associ-
ation was observed for yoghurt (Peters et al. 1992) and cul-
tured milk (Young & Wolf, 1988) consumption, adjusted
for potential confounding variables. It can also be men-
tioned that an inverse relationship has been demonstrated
between the frequency of consumption of yoghurt and
other fermented milk products and breast cancer in
women (van’t Veer et al. 1989). On the other hand, two
companion American prospective studies, the 1980–1988
follow-up of the Nurses’ Health Study and the 1986–
1990 follow-up of the Health Professionals’ Follow-up
Study, did not provide evidence that intake of dairy pro-
ducts is associated with a decreased risk of colon cancer
(Kampman et al. 1994a). In a cohort study in The Nether-
lands, it was shown that the intake of fermented dairy pro-
ducts was not significantly associated with colorectal
cancer risk in an elderly population with a relatively
wide variation in dairy product consumption, although a
weak non-significant inverse association with colon
cancer was observed (Kampman et al. 1994b).
J. J. RafterS220

In addition, there are some studies examining the effect
of probiotics on the biomarkers for colon cancer risk (see
below) in healthy volunteers and patients. Consumption
of lactic acid bacteria by volunteers has been shown to
reduce the mutagenicity of urine and faeces associated
with the ingestion of carcinogens in cooked meat (Lidbeck
et al. 1992). Mucosal cell proliferative activity in upper
colonic crypts of patients with colon adenomas (believed
to be a risk factor for tumour development) decreased
significantly after the administration of Lactobacillus
acidophilus and Bifidobacterium bifidus cultures (Biasco
et al. 1991).
Beef
An anticarcinogenic fatty acid known as conjugated
linoleic acid was first isolated from grilled beef in 1987
(Ha et al. 1987). Nine different isomers of conjugated
linoleic acid have been reported as occurring naturally in
food. Conjugated linoleic acid is unique in that it is
found in highest concentrations in fat from ruminant
animals (e.g. beef, dairy, lamb). In recent years, conjugated
linoleic acid has been shown to be effective in suppressing
forestomach tumours in mice, aberrant colonic crypt foci in
rats and mammary tumours in rats (Ip & Scimeca, 1997).
Functional foods from plant sources
Soyabeans
Several classes of anticarcinogens have been identified in
soyabeans, including protease inhibitors, phytosterols,
saponins, phenolic acids, phytic acid and isoflavones. Of
these, isoflavones (genistein and daidzein) are particularly
noteworthy because soyabeans are the only significant
dietary source of these compounds. Isoflavones are
heterocyclic phenols structurally similar to the oestrogenic
steroids. Because they are weak oestrogens, isoflavones
may act as anti-oestrogens by competing with the naturally
occurring endogenous oestrogens. This may explain why
populations that consume significant amounts of soya
have reduced risk of oestrogen-dependent cancer. How-
ever, more epidemiological data and clinical intervention
trials are needed to investigate the role of soya in reducing
cancer risk.
Flaxseed
There has been an increasing interest in fibre-associated
compounds known as lignans. The two primary mam-
malian lignans, enterodiol and its oxidation product,
enterolactone, are formed in the intestinal tract by bacterial
action on plant lignan precursors. Flaxseed is one of the
richest sources of mammalian lignan precursors. Because
enterodiol and enterolactone are structurally similar to
both naturally occurring and synthetic oestrogens, and
have been shown to possess weakly oestrogenic and
anti-oestrogenic activities, they may also play a role in
the prevention of oestrogen-dependent cancers. In rodents,
flaxseed has been shown to decrease tumours of the colon,
mammary gland and lung. Phipps et al. (1993) demon-
strated that the ingestion of 10 g of flaxseed per day elicited
several hormonal changes associated with reduced breast
cancer risk. However, as is the case with soya, epidemio-
logical data are required to support the hypothesis that
enterodiol and enterolactone have anticarcinogenic
properties in man.
Tomatoes
Tomatoes have received much attention in recent years
because of interest in lycopene, the primary carotenoid in
this fruit, and its potential role in cancer risk reduction
(Weisburger, 1998). In a prospective cohort study of
more than 47 000 men, those who consumed tomato
products ten or more times per week had less than one-
half the risk of developing advanced prostate cancer
(Giovannucci et al. 1995). Interestingly, lycopene is the
most abundant carotenoid in the prostate gland. Other
cancers whose risk have been inversely associated with
serum or tissue levels of lycopene include breast, digestive
tract, cervix, bladder and skin (Clinton, 1998). Proposed
mechanisms by which lycopene could influence cancer
risk are related to its antioxidant function. Lycopene is
the most efficient quencher of singlet oxygen in biological
systems (Di Mascio et al. 1989).
Garlic
Garlic (Allium sativum ) is probably the herb most widely
quoted in the literature for medicinal purposes. The intact
garlic bulb contains an odourless amino acid, which is
converted enzymatically by allinase into allicin when the
garlic cloves are crushed (Block, 1992). Allicin then
decomposes spontaneously to form numerous sulfur-
containing compounds, some of which have been investi-
gated for their chemopreventive activity. Garlic com-
ponents have been shown to inhibit tumourigenesis in
several experimental models. However, additional reports
have shown garlic to be ineffective. Inconclusive results
are likely to be due to differences in the type of garlic
compounds or preparations used by various investigators.
Several epidemiological studies show that garlic may be
effective in reducing human cancer risk (Dorant et al.
1993). However, it should be mentioned that not all such
studies have shown garlic to be protective. A review of
twenty epidemiological studies (Ernst, 1997) suggested
that allium vegetables, including onions, may confer a
protective effect on cancers of the gastrointestinal tract.
Tea
Much attention has focused on the polyphenolic constitu-
ents of tea, particularly green tea. Polyphenols comprise
up to 30 % of the total dry weight of fresh tea leaves.
Catechins are the predominant and most significant of all
tea polyphenols (Graham, 1992). The four major green
tea catechins are epigallocatechin-3-gallate, epigallo-
catechin, epicatechin-3-gallate and epicatechin. Much of
the work on the health effects of tea has focused on its
cancer chemopreventive effects. Research results from labora-
tory animals tend to support a cancer chemopreventive
Reduction of disease risk: cancer S221

effect of tea components (Dreosti et al. 1997). However, the
epidemiological studies are still somewhat inconclusive. It
has been suggested that benefits from tea consumption are
restricted to high intakes in high-risk populations (Kohl-
meier et al. 1997). The consumption of five or more
cups of green tea per day was shown to be associated
with decreased recurrence of stage I and stage II breast
cancer in Japanese woman (Nakachi et al. 1998).
Broccoli and other cruciferous vegetables
Epidemiological evidence has also associated the frequent
consumption of cruciferous vegetables with decreased
cancer risk. In a review of eighty-seven case–control
studies, Verhoeven et al. (1996) demonstrated an inverse
association between consumption of total brassica vege-
tables and cancer risk. The percentages of case – control
studies showing an inverse association between consump-
tion of cabbage, broccoli, cauliflower and Brussels sprouts
and cancer risk were 70, 56, 67 and 29 %, respectively. The
anticarcinogenic properties of cruciferous vegetables have
been attributed to their relatively high content of gluco-
sinolates (Verhoeven et al. 1997). Glucosinolates are a
group of glycosides stored within cell vacuoles of all
cruciferous vegetables. Myrosinase, an enzyme found in
plant cells, converts these compounds to a variety of
hydrolysis products, including isothiocyanates and indoles.
While a wide variety of naturally occurring and synthetic
isothiocyanates have been shown to prevent cancer in
animals (Hecht, 1995), attention has been focused on a
particular isothiocyanate isolated from broccoli, i.e.
sulforaphane. Sulforaphane has been shown to be a good
inducer of a particular phase II enzyme, quinone reductase.
Indole-3-carbinol has received attention for its cancer
chemopreventive properties, particularly of the mammary
gland. In addition to the induction of phase I and phase
II detoxification reactions, indole-3-carbinol may reduce
cancer risk by modulating oestrogen metabolism.
Citrus fruits
Several epidemiological studies have shown that citrus
fruits are protective against a variety of human cancers.
Although oranges, lemons, limes and grapefruits are a
principal source of such important nutrients as vitamin
C, folate and fibre, Elegbede et al. (1993) have suggested
that another component is responsible for the anticancer
activity. Citrus fruits are particularly high in a class of
phytochemicals known as the limonoids (Hasegawa &
Miyake, 1996). In recent years, evidence has been
accumulating in support of the cancer-preventative
effect of limonene (Gould, 1997). Crowell (1997)
showed this compound to be effective against a variety
of both spontaneous and chemically induced rodent
tumours.
However, mindful of the importance of the overall
dietary pattern in cancer risk reduction, one must question
the clinical implications of a single phytochemical in
isolation.
Biomarkers available for assessing diet-related
changes in colon cancer risk
Since colon cancer has been a major target for the cancer
chemopreventive effects of probiotics and functional food
components, I shall limit my examples of ‘biomarkers’ to
this cancer form. Presently, there are a large number of
biomarkers available for assessing colon cancer risk in
dietary intervention studies, which are validated to varying
degrees. These include colonic mucosal markers, faecal
water markers and immunological markers.
Colon mucosa biomarkers
1. Adhesion of Gram-negative bacteria
2. Modulation of cyclo-oxygenase-2 (COX-2)
3. Proliferation
4. K-ras
5. Genetic instability
6. Apoptosis
7. DNA-repair integrity
8. Metastasis markers
9. Microsatellite instability
10. Oxidative DNA damage
11. Gene-specific damage
Faecal water markers
In recent years, there has been considerable interest in the
role of the aqueous phase of human faeces (faecal water) in
studies examining the mechanisms underlying the dietary
aetiology of colon cancer. The motivation is that com-
ponents of this faecal fraction are more likely to be able
to exert untoward effects on the cells of the colonic epi-
thelium than components bound to food residues and the
bacterial mass.
1. Cytotoxicity
2. COX-2 induction
3. Caspase induction
4. Calprotectin levels
5. Activator protein-1 activation
6. Bile acid levels
7. Effects on metastasis
8. Genotoxicity
9. Effects on cell metabolism
10. Gene induction
Immunological and inflammatory response markers
In the colon:
1. Suppression of COX-2 induction by pro-inflammatory
cytokines
In blood:
1. Natural killer cells
2. Lymphocyte proliferation
3. Cytokines (interleukin-2, interleukin-b, tumour necro-
sis factor)
Thus, in designing a dietary intervention study/clinical trial
J. J. RafterS222

to study anticancer effects (colon cancer) of functional
foods, it is recommended to use as many of the above
‘state-of-the-art’ biomarkers as is feasible.
Examples of studies addressing changes in
biomarker response to a functional food
Shift from dairy product-rich to dairy product-free diet
Cytotoxicity of faecal water is now an accepted risk marker
for colon cancer and several studies have correlated
toxicity of this faecal fraction with a higher colonic cell
proliferation and increased colon cancer risk (Lapre
´
&
Van der Meer, 1992). Genotoxicity per se in human
faeces is also generally accepted as a risk marker for
colon cancer. Recently, Pool-Zobel et al. (1996) have
demonstrated, by employing the single cell gel electrophor-
esis (COMET) assay, that colon carcinogens induced gen-
otoxicity in the colon of rats and that this genotoxicity was
altered by dietary manipulations.
To determine whether the cytotoxicity and genotoxicity
of the aqueous phase of human stools (faecal water) were
affected by a change in dairy product intake, eighteen
healthy male and female volunteers were randomly divided
into two groups (Glinghammar et al. 1997). In a cross-over
design, the volunteers shifted from their normal dairy
product-rich to a dairy product-free diet. Nutritional
analysis of the food consumed, during the study period,
showed a significant decrease in energy intake from
9000 kJ/d to 7866 kJ/d (P ¼ 0.004), due to decreased
intakes of protein and fat. Carbohydrate and fibre intakes
remained unchanged during the intervention. Ca intake
decreased significantly from 1488 mg/d to 372 mg/d
(P , 0.001), with phosphate and vitamin D displaying
similar significant decreases (P , 0.001). Cytotoxicity of
faecal water, analysed by the HT-29 cytotoxicity assay,
indicated a significant decrease (P ¼ 0.025) in cell survival
from 34 % to 20 % when dairy products were excluded
from the participants’ diet. This effect is most likely due
to the decreased intake of dairy Ca and possibly phosphate.
The COMET assay, used to analyse genotoxicity of faecal
waters, indicated no differences brought about by the diet-
ary intervention. These findings indicate that a shift from a
dairy product-rich to a dairy product-free diet results in a
significant effect (P ¼ 0.025) on an accepted risk marker
for colon cancer, and may suggest that the mechanism by
which dairy products are protective is at the level of
tumour promotion rather than initiation.
Clinical trial to examine the effect of a synbiotic
preparation on colon cancer risk biomarkers
in adenoma patients
Another example of such a study is the ongoing Symbiotics
and Cancer Prevention in Humans (SYNCAN) project,
funded by the EU, and involving eight research centres
in Europe. It involves a twelve-week, randomised,
double-blind placebo-controlled trial of a food supplement
containing L. rhamnosus GG, Bifidobacterium Bb-12 and
Raftilose Synergyl in adenoma patients. In this study, all
of the colon cancer risk biomarkers, listed above, will be
measured. In parallel, a long-term tumourigenesis study
in rats, using the same synbiotic combination and assaying
for the same biomarkers, is being carried out. It is hoped
that the results of this study will provide much needed
information on the cancer-protective effects of synbiotics
in man and on the underlying mechanisms.
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