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Cruciferous Vegetables and Cancer Prevention

Article in Nutrition and Cancer February 2001

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Genoveva Murillo Rajendra Mehta

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NUTRITION AND CANCER, 41(1&2), 1728
Copyright 2001, Lawrence Erlbaum Associates, Inc.
Cruciferous Vegetables and Cancer Prevention
Genoveva Murillo and Rajendra G. Mehta
Abstract: In recent years, cancer prevention by natural
products has received considerable attention. The potential
protective role of cruciferous vegetables and active compo-
nents present in these vegetables, such as isothiocyanates
and indole-3-carbinol, has been extensively studied in ex-
perimental in vitro and in vivo carcinogenesis models. Re-
sults have consistently shown that the chemopreventive
agents derived from this class of vegetables of the Cru-
ciferae family influence carcinogenesis during initiation and
promotion phases of cancer development. Similarly, reports
from epidemiological studies and clinical trials support this
notion. However, there is no comprehensive summary of all
these aspects of the association between cruciferous vegeta-
bles and cancer prevention. We have attempted to summa-
rize experimental carcinogenesis studies as well as clinical
trials and studies on the mechanism of action of selective
chemopreventive agents isolated and identified within these
natural products. Results clearly point toward a positive
correlation between cancer prevention of many target or-
gans and consumption of cruciferous vegetable or their
active constituents. Yet we are still far from complete under-
standing of the effects of combinations of chemopreventive
phytochemicals present in these cruciferous vegetables and
their overall mechanism(s) of action in providing protective
effects.
Introduction
For more than 25 years, the interdependence between nu-
trition and the development and progression of cancer has
been recognized. The key challenge has been to identify the
specific components responsible for contributing to this re-
lationship. In the United States, cancers of the lung, colon
and rectum, breast, and prostate account for almost half of
the total cancer incidence. In other countries, these types of
cancers occur with much less frequency (1). For example,
people in China, Japan, and Korea are 410 times less likely
to be diagnosed with colorectal or breast cancer than are
people in the United States (2). Studies have been employed
The authors are affiliated with the Department of Surgical Oncology, College of Medicine, University of Illinois, Chicago, IL 60612.
to elucidate the potential factors involved in geographical
variation among ethnic groups.
The existence of an etiologic relationship between dietary
practices and human cancer is evidenced by the data found
when the per capita consumption of specific foods (e.g., di-
etary fat) is compared with rates of cancer among different
countries. Similarly, ecological studies have concluded that
the differences in cancer rates are correlated with differences
in dietary patterns (13). The field of epidemiology has been
instrumental in highlighting the relationship between diet
and cancer incidence. For example, numerous reports have
supported the association between high fat intake and in-
creased incidence of colon cancer (37). Similarly, migrant
studies have been important in determining the association
between cancer rates and dietary habits (811). The relation-
ship between migration and cancer has been observed in
Polish and Japanese immigrants living in the United States
(8,9) and Australia (10) and Indian immigrants living in
London (11).
Cumulatively, epidemiological, etiologic, ecological, and
migration studies suggest that cancer rates are associated
with environmental factors. Because of these studies, nu-
merous investigations have attempted to identify dietary
agents that may inhibit the multistage process of carcino-
genesis. The field of nutrition and cancer in recent years re-
ceived a considerable stimulus with the finding of a number
of naturally occurring substances that have shown cancer-
preventive action in experimental models. Here we review
the role of cruciferous vegetables in cancer prevention.
Carcinogenesis and Chemoprevention
The process of carcinogenesis, in which normal cells be-
come malignant, is quite complex. To understand the activi-
ties of chemopreventive agents, a brief introduction to the
process of carcinogenesis may be useful. With skin car-
cinogenesis used as a model, it has been established that
there are three distinct stages in the development of cancer
(12,13). These stages are defined as initiation, promotion,
and progression. Initiation is irreversible damage of DNA
 Figure 1. Evolution stages of neoplasia. ACF, aberrant crypt foci.
caused by viral, chemical, or physical agents. When this
damage affects the function of genes that are involved in cell
differentiation, such as suppressor genes, protooncogenes,
or transcription factors, it may result in the development of
cancer (14). The possible sequence of events during the initi-
ation phase is shown in Fig. 1. Procarcinogens can be elimi-
nated from the body by nonenzymatic inactivation or can
form covalent DNA adducts, which in turn may lead to mu-
tation or transformation.
The promotion step is a process by which the initiated cell
is transformed into a preneoplastic or neoplastic cell (12).
This process is epigenetic and is affected by endogenous and
exogenous factors. The process of promotion is accompanied
by activation of protooncogenes or inactivation of suppressor
genes, which leads to defects in differentiation, growth con-
trol, and resistance to host immune response.
The third and final phase of carcinogenesis is known as
progression, where the malignant cells become increasingly
invasive and metastatic. This is probably due to increased
accumulation of genetic abnormalities, increased diversifi-
cation, and heterogeneity. For example, cumulative evi-
dence from molecular biology and cytogenetic studies
shows that most epithelial cancers contain multiple chromo-
somal deletions and rearrangements. In fact, although there
appears to be some unique target-organ specificity, there are
similarities in the malignant transformation for most epithe-
lial tissues. For example, increased heterozygosity by loci
mapping of chromosome 17 has been reported for lung,
breast, and colon, the most common targets for cancer,
18
whereas 3p deletions are associated with kidney and lung tu-
mors in humans. Thus knowledge of the mechanism of each
step of carcinogenesis can lead to development of novel ap-
proaches to cancer prevention.
Sporn and co-workers (15,16) coined the term chemo-
prevention in 1976, and since then chemoprevention has be-
come a separate discipline of cancer research. Conceptually,
chemoprevention of cancer can be defined as an intervention
in the carcinogenic process by an agent either naturally de-
rived or synthetic in nature. An agent that blocks, arrests, or
reverses the progression of cancer can be termed a chemo-
preventive agent. In a broad sense, chemoprevention of cancer
is a mode of cancer control in which formation of the disease
can be prevented. In practice, this can best be achieved by the
dietary administration of chemical agents that can enhance
the physiological processes that protect the organism against
the growth of preneoplastic or cancer cell growth. Because
there exists a diversity of mechanisms for inhibition of carcino-
genesis, the likelihood that multiple approaches can be made
for identifying novel chemopreventive agents is also signifi-
cantly high. Investigations on the chemoprevention of cancer
focus on two categories of a population: the healthy popula-
tion and individuals who are at a higher risk of developing
cancer. Individuals in the general population certainly are ex-
posed to environmental carcinogens and are at risk of devel-
oping cancer; however, they are currently disease free. The
only attractive way of chemoprevention in healthy people is
by intelligent selection of foods (17). On the other hand, peo-
ple at high risk of developing cancer may reduce the risk by
Nutrition and Cancer 2001
selection of chemopreventive foods, as well as by use of
chemopreventive agents that may be purely synthetic.
Wattenberg and co-workers (18,19) classified chemopre-
ventive agents into two major categories: 1) blocking agents
or anti-initiators and 2) suppressing or antipromotional agents.
Some of the blocking agents in food include indoles, iso-
thiocyanates, and dithiolethiones in cruciferous vegetables,
terpenes from citrus fruits, organosulfurs from garlic, epi-
gallocatechin gallate from green tea and protease inhibitors
from beans, as well as some carotenes, coumarins, and cur-
cumins. On the other hand, antipromotional agents include
retinoids, vitamin D analogs, inhibitors of prostaglandin syn-
thesis, monoterpenes, selenium, inhibitors of polyamine
biosynthesis, and other agents selectively affecting the post-
initiation stage of carcinogenesis. Recent progress in the field
of chemoprevention has focused on the identification of mo-
lecular targets. On the basis of the mechanism of action of a
chemopreventive agent, more efficacious chemicals can be
synthesized. Thus the antioxidant properties of chemo-
preventive agents could serve as carcinogen detoxification
agents and be classified as anti-initiators. Similarly, gluta-
thione is a scavenger of carcinogens, and this reaction is cata-
lyzed by glutathione S-transferase. Therefore, glutathione S-
transferase or quinone reductase could inhibit carcinogen ac-
tion. The antipromotional effects of chemopreventive agents
involve a variety of molecular targets, affecting numerous sig-
nal transduction pathways. These molecular targets include, but
are not limited to, hormone receptors, cell cycle check-point
markers, transcription factors, mitogen-activated protein kin-
ases, second messengers such as adenosine 3,5-cyclic mono-
phosphate, rate-limiting enzymes including ornithine
decarboxylase, cyclooxygenases, cell junctions, and tumor
suppressor genes (e.g., p53). These targets are selective and
specific for chemopreventive agents and their action. In the
present review, we focus on the current status of cruciferous
vegetables in cancer chemoprevention.
Cruciferous Vegetables
Cruciferous vegetables are a group of vegetables named
for their cross-shaped flowers. Cruciferous vegetables in-
clude cabbage, broccoli, brussels sprouts, cauliflower, and
other members of the family Brassicaceae and genus Bras-
sica (Table 1). Several reports have demonstrated cruci-
ferous vegetables and/or their constituents to be potentially
cancer preventive (20). Isothiocyanates, degradation prod-
ucts of glucosinolates, which occur naturally in a variety of
cruciferous vegetables, are some of the most studied compo-
nents of cruciferous vegetables. More than 20 natural and
synthetic isothiocyanates have been studied for their ability
to prevent carcinogenesis (21). Volatile isothiocyanates are
released from thioglucosides in vegetables and contribute to
the distinctive odor and flavor of cruciferous vegetables.
Allyl isothiocyanate was identified nearly 50 years ago as a
compound particularly prevalent in cabbage. Phenethyl
isothiocyanates were found to be abundant in watercress
Vol. 41, Nos. 1&2
Table 1. Some Members of the Cruciferous Vegetable
Group
Arugula Horseradish
Beet greens Kale
Bok choy Kohlrabi
Broccoli Mustard greens
Broccoli sprouts Radishes
Brussels sprouts Rutabaga
Cabbage Swiss chard
Collard greens Turnips
Garden cress Turnip greens
(22). Our laboratory has extensively studied the chemo-
preventive effects of brassinin, an indole present in Chinese
cabbage, as a phytoalexin (23,24). Although a variety of
chemicals with chemopreventive potential are present in
these vegetables, their concentration depends on various fac-
tors, including rainfall, sun exposure, soil, and seed stock.
Additionally, it has been reported that normal food prepara-
tion methods can substantially inactivate isothiocyanate ac-
tivity (25). Consequently, it is difficult to correlate vegetable
consumption and intake of protective compounds. For this
reason, attention has focused on using isolated compounds
(26) and/or a special seed stock yielding sprouts rich in se-
lective chemopreventive agents, such as isothiocyanates, to
ensure adequate levels of the active compound.
Epidemiological Investigations of Cruciferous
Vegetables and Cancer
The medicinal value of cruciferous vegetables has been
reported since ancient times (27). In more recent years, at-
tention has focused on the anticancer effects of cruciferous
vegetables. The epidemiological data have been recently re-
viewed by Verhoeven et al. (28). Of the seven prospective
studies, five (71%) reported an inverse association between
the consumption of one or more Brassica vegetables and
cancer risk. Similarly, Verhoeven et al. examined evidence
from case-control studies. Consumption of cabbage, broc-
coli, cauliflower, or brussels sprouts was inversely associ-
ated with risk of cancer in 37 (70%) studies, whereas 11
(20%) studies reported no association and 7 (13%) showed a
positive association between cruciferous vegetables and risk
of cancer. Cumulatively, these studies provide evidence sup-
porting an anticancer effect of cruciferous vegetables.
More recently, Zhang et al. (29) studied the association
between non-Hodgkins lymphoma and fruit and vegetable
intake. The association was examined among 88,410 women
who took part in the Nurses Health Cohort Study. Partici-
pants who provided dietary information in 1980 were fol-
lowed for up to 14 yr. During this period, 199 cases of
non-Hodgkins lymphoma were reported. This study found
that higher intake of cruciferous vegetables was associated
with a relative risk (RR) of 0.67 for women who consumed
five or more servings per week compared with those con-
suming less than two servings per week.
19
 Similarly, increased consumption of cruciferous vegeta-
bles has been reported to be associated with a decreased risk
of developing prostate cancer. Cohen et al. (30) examined
the association of fruit and vegetable intake and prostate
cancer risk among newly diagnosed men residing in the Se-
attle, WA, area. Cases consisted of men 65 yr old who had
been newly diagnosed with prostate cancer. Controls were
men residing in the same area and of similar age. The results
of the study showed that, for cruciferous vegetable intake,
adjusted for total vegetable intake and other covariates, the
odds ratio for comparison of three or more servings per week
with less than one serving per week was 0.59 [95% confi-
dence interval (CI) = 0.390.90], with a two-sided P trend =
0.02. These results suggest that high consumption of vegeta-
bles, particularly cruciferous vegetables, is associated with a
reduced risk of prostate cancer.
The potential benefits of cruciferous vegetables in fight-
ing colon cancer have been summarized in a recent meta-
analysis. This study reviewed 20 epidemiological studies in
the literature in which the effects of cruciferous vegetable
intake on risk for developing colon cancer were examined.
By controlling for the effects of overall vegetable intake, it
was concluded that cruciferous vegetables themselves con-
fer a separate protective benefit against colon cancer. On the
basis of the findings from the meta-analytic study, the au-
thors concluded that, with each 10 g of cruciferous vegeta-
bles consumed per day, one could expect an 8% decrease in
risk for colorectal cancer (31).
The link between cruciferous vegetables and gene inter-
actions in humans has recently been investigated. London et
al. (32) examined the effects of a common deletion polymor-
phism of glutathione S-transferases M1 and T1 (enzymes
responsible for conjugating isothiocyanates, leading to
more rapid elimination) on the risk for lung cancer in a
population of 18,244 men aged 4564 yr residing in Shang-
hai, China. Data were obtained from annual contacts with
the surviving members of the cohort and biannual reviews
of cancer reports from the Shanghai cancer registry and
death certificates. Total isothiocyanate concentrations in
the urine were measured before diagnosis. Blood samples
were collected to determine glutathione S-transferase M1
and T1 status of 232 incident cases of lung cancer and 710
matched controls selected from the cohort. This study
found that individuals with detectable levels of isothio-
cyanates in the urine had a decreased risk of lung cancer
(smoking-adjusted RR = 0.65, 95% CI = 0.430.97). How-
ever, the greatest protection against lung cancer was ob-
served among individuals with homozygous deletion of
glutathione S-transferases M1 (RR = 0.36, 95% CI = 0.20
0.63) and particularly with deletion of glutathione S-trans-
ferase M1 and T1 (RR = 0.28, 95% CI = 0.130.57). This
study supports the use of isothiocyanates to reduce lung can-
cer risk in humans. Furthermore, this study shows that the
effect of isothiocyanates is strongest in people without the
gene coding for glutathione S-transferase, in whom isothio-
cyanate metabolism is predicted to be slower.
20
Cruciferous Vegetables and Experimental
Carcinogenesis
Some of the earliest work on cancer chemoprevention by
components of cruciferous vegetables was reported by Wat-
tenberg and co-workers (3335). Treatment with benzyl
isothiocyanate 2 h before the carcinogen inhibited 7,12-
dimethylbenz[a]anthracene (DMBA)-induced mammary
carcinogenesis. Bresnick et al. (36) fed cabbage to rats after
the N-methyl-N-nitrosourea (MNU)-induced initiation event
was completed to evaluate its effects on the promotion phase
of mammary carcinogenesis. Results indicated that 5% and
10% dried cabbage in the diet reduced the tumor multiplicity
in these animals. However, cabbage was ineffective in the
group of rats consuming a high-fat diet. Since then, several
groups have studied the effects of synthetic aromatic isothio-
cyanates to show inhibition of carcinogen-induced mam-
mary, forestomach, and lung carcinogenesis. Chung and
colleagues (37) showed that aromatic isothiocyanates are
powerful inhibitors of tobacco-specific nitrosamine-induced
lung carcinogenesis in rodent models. The experiments to
evaluate the structure-activity relationship of isothiocyanate
against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-in-
duced lung carcinogenesis have shown that the longer the
alkyl chain (up to 6 carbons), the more potent the inhibition.
It was shown that 6-phenylhexyl isothiocyanate reduced
lung cancer multiplicity by 85% in animal experiments (38).
Some data on inhibition of carcinogenesis by cruciferous
vegetables are summarized in Table 2. Given the large
amount of data available, only a sample of the types of stud-
ies will be discussed; several reviews have previously dis-
cussed some of these data (21,3942).
Indole-3-carbinol has been the most studied component
of cruciferous vegetables. It has been demonstrated to have
chemopreventive activity in several different animal models
of carcinogenesis, including mammary gland (43,44), colon
(45,46), tongue (47), lung (48), forestomach (35), endo-
metrium (48), cervix (49), skin (50), bone marrow (51), and
bladder (52). Grubbs et al. (43) showed that indole-3-car-
binol, at 50 or 100 mg/day, was highly effective against
DMBA- or MNU-induced rat mammary tumors when ad-
ministered during the initiation or promotion phase of car-
cinogenesis. These results showed that indole-3-carbinol
can prevent mammary carcinogenesis by direct- and indi-
rect-acting carcinogens. Various animal models have been
employed to investigate the effectiveness of indole-3-car-
binol against colon cancer (45,46). Guo et al. (45) examined
the protective effects of indole-3-carbinol against the devel-
opment of colonic aberrant crypts in rats. Animals were
given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) at Weeks 3 and 4 of a 16-wk period. Indole-3-carbi-
nol was provided in the diet (0.1%) at initiation, at postini-
tiation, or continuously throughout the study. The results
show that indole-3-carbinol was a potent inhibitor of PhIP-
induced aberrant crypt foci during all three stages of car-
cinogenesis (initiation, postinitiation, and with continuous
Nutrition and Cancer 2001
Table 2. Compounds in Cruciferous Vegetables Effective Against Cancer Modelsa
Treatment Type of Cancer Model(s) Effect Ref.

Cabbage supplements Mammary MNU, rat Inhibition 36

20% Brussels sprouts Mammary DMBA, rat Inhibition 89
Isothiocyanate Lung NNK, mouse Inhibition 90
Mammary DMBA, rat Inhibition 19,91
Colorectal MAM, rat Inhibition 92
Liver NDEA, rat Inhibition 93
Esophagus NBMA, rat Inhibition 94
a-Naphthylisothiocyanate Liver 3-Me-DAB, rat Inhibition 95
Liver Ethionine, rat Inhibition 96
Bladder BHBN, rat Inhibition 97
Phenethyl derivatives Mammary DMBA, rat Inhibition 91
Lung B[a]P, mouse Inhibition 98
Lung NNK, rat Inhibition 37
Lung BOP, hamster Inhibition 99
Forestomach B[a]P, mouse Inhibition/no effect 98
Skin B[a]P, mouse No effect 98
Nasal cavity NNK, rat No effect 90
Liver NNK, rat No effect 90
Esophagus NBMA, rat Inhibition 100
PEITC-NAC Lung NNK, mouse Inhibition 38
Esophageal NMBA, rat Inhibition 101
Colon, ACF AOM, rat Inhibition 102
Forestomach DMBA, mouse Inhibition 91
Indole-3-carbinol Laryngeal HPV-induced, mouse Inhibition 103
Mammary DMBA, rat Inhibition 43
Mammary MNU, rat Inhibition 43
Mammary MNU, rat Enhancement 61
Mammary Spontaneous Inhibition 44
Colon PhIP, rat Inhibition 45
Colon AOM, rat Inhibition 46
Colon DMH, rat Enhancement 57
Colon DMH, mice Enhancement 58
Tongue NQO, rat Inhibition 47
Forestomach B[a]P, mouse Inhibition 35
Endometrium Rats, spontaneous Inhibition 48
Thyroid DMD, rat Enhancement 59
Cervical-vaginal Transgenic mice Inhibition 48
Skin DMBA-TPA, mouse Inhibition 49
Bone marrow Cyclophosphamide-induced micronuclei Inhibition 50
formation, mouse
Liver AFB1, rainbow trout Enhancement 5254
Liver DMD, rat Enhancement 59
Liver DEN, newborn/young rats Enhancement 55
Pancreas BOP, hamsters Enhancement 58
Bladder NNK, rat Inhibition 51
S-methyl-methanethiosulfonate Colorectal AOM, rat Inhibition 63
Sulforaphane Colon, ACF AOM, rats Inhibition 75
Brassinin Mammary DMBA, mouse mammary organ culture Inhibition 22
Skin DMBA/TPA, mouse Inhibition 23

a: Abbreviations are as follows: AFB1, aflatoxin B1; ACF, aberrant crypt foci; AOM, azoxymethane; B[a]P, benzo[a]pyrene; BHBN, N-butyl-N-(4-
hydroxybutyl)nitrosamine; BOP, N-nitroso(2-oxopropyl)amine; DEN, diethylnitrosamine; DMBA, 7,12-dimethylbenz[a]anthracene; DMD,
diethylnitrosamine (DEN) + N-methyl-N-nitrosourea (MNU) + dihydroxy-di-N-propyl-nitrosamine (DHPN); DMH, 1,2-dimethylhydrazine; HPV, human
papilloma virus; MAM, methylazoximethanol acetate; 3-Me-DAB, 3-methyl-4-dimethylaminoazobenzene; NDEA, N-nitrosodiethylamine; NBMA, N-
nitrosobenzylmethylamine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NQO, 4-nitroquinoline-1-oxide; PEITC-NAC, phenethyl
isothiocyanate-N-acetylcysteine; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; TPA, 12-O-tetradecanoylphorbol-13-acetate.

feeding of indole-3-carbinol). However, the inhibition of
large aberrant crypt foci (4 crypt/focus) was only signifi-
cantly different from the control group when indole-3-carbi-
nol was fed during the initiation phase. These results support
the theory that indole-3-carbinol has a protective role against
PhIP-induced colon carcinogenesis, with the greatest protec-
tion being obtained during the initiation phase.
A transgenic mouse model was used to examine whether
the administration of physiological doses of indole-3-carbi-
nol would prevent cervical-vaginal cancer in mice express-

Vol. 41, Nos. 1&2 21

ing the transgenes for human papilloma virus type 16 (49).
At 45 wk of age, virgin mice were implanted subcutane-
ously with 17b-estradiol (0.125 mg or 0.250 mg per 60-day
release) and placed on an AIN-76A diet or an AIN-76A diet
enriched with 2,000 ppm indole-3-carbinol. At Week 24,
surviving animals were euthanized. Results showed that 19
of the 25 control transgenic mice developed cervical-vaginal
cancer within 6 mo, and the remainder of the mice formed
dysplasia. In the treatment group, only 5% (2 of 24) of the
animals developed cancer, and the rest of the mice exhibited
dysplasia or hyperplasia. These findings suggest that indole-
3-carbinol is a good chemopreventive candidate against the
development of cervical-vaginal cancer associated with hu-
man papilloma virus.
Brassinin, an indole-based phytoalexin found naturally in
Chinese cabbage, has also been shown to have potential
chemopreventive properties in several animal models. In our
laboratories, brassinin was shown to significantly induce
quinone reductase activity in mammary glands in organ cul-
ture and to inhibit skin and mammary carcinogenesis (23,24).
Although indole-3-carbinol has shown great promise in
many cancer models, it has also been reported to exhibit ad-
verse promoting effects in a number of animal models. For
example, exposure to indole-3-carbinol during the postini-
tiation (promotion) stage was shown to strongly enhance
aflatoxin B1-induced liver tumorigenesis in rainbow trout
(5355), diethylnitrosamine-induced liver tumorigenesis in
newborn or young rats (56), 1,2-dimethylhydrazine-induced
colon tumorigenesis in rats (57) and mice (58), N-nitrobis(2-
oxopropyl)amine-induced pancreas tumorigenesis in ham-
sters (59), liver and thyroid gland tumorigenesis induced by
diethylnitrosamine + MNU + dihydroxy-di-N-propylnitro-
samine (60), and MNU-induced mammary carcinogenesis in
rats (61). Although several of the earlier studies (53,55) have
been criticized for using relatively high dietary doses, other
studies (62) have used relatively low dietary doses and still
observed significant promotional effects by indole-3-carbi-
nol when administered during the promotional phase of car-
cinogenesis. Thus clinical trials with the aim of investigating
the chemopreventive effects of indole-3-carbinol should be
approached with caution, until its mechanism(s) of action in
enhanced tumor promotion and the relation to human cancer
risk are better understood.
Cruciferous Vegetables and Clinical Studies
The experimental basis for the use of isothiocyanates in
clinical studies was first introduced by Chung et al. (22). In
this early work, volunteers were fed a breakfast containing 56
g of watercress, a cruciferous vegetable rich in gluconas-
turtiin. Urine samples were collected over a 24-h period and
analyzed for metabolites. Results derived from high-perform-
ance liquid chromatography analysis showed the presence of
a compound coeluting with a synthetic N-acetyl conjugate of
phenethyl isothiocyanate (PEITC-NAC). These investigations
22
have been followed by several other studies that have exam-
ined the fate of isothiocyanates and glucosinolates in humans
(63,64). Shapiro et al. (65) investigated the levels of urinary
dithiocarbamates to quantify the amounts of cruciferous vege-
tables consumed by healthy volunteers. Subjects were admitted
to the testing facilities and placed on a control diet that ex-
cluded all foods containing the compounds of interest. The test
vegetable was administered at 7 AM, and breakfast was with-
held until 10 AM. Results of this study showed that, in non-
smokers, urinary dithiocarbamates were detected only after
consumption of cruciferous vegetables and condiments rich in
isothiocyanates and/or glucosinolates. Dosing studies re-
vealed that eating horseradish containing graded doses of
isothiocyanates led to a rapid excretion of proportionate
amounts of urinary dithiocarbamates. Similarly, ingestion of
broccoli in which myrosinase was heat inactivated also led to
proportionate but lower levels of urinary dithiocarbamates.
Thus these studies highlight the importance of myrosinase in
the conversion of glucosinolates.
Given that myrosinase is deactivated with heat and that we
commonly consume cruciferous vegetables in cooked form,
the need to determine whether dietary glucosinolates could be
converted to isothiocyanates after being cooked became ap-
parent. To quantify the uptake of isothiocyanates, Getahun
and Chung (25) used a urinary marker based on the cyclo-
condensation product formed by the reaction of isothio-
cyanates and their conjugates with 1,2-benzenedithiol. Nine
volunteers consumed a total of 350 g of cooked watercress in
which myrosinase activity was completely deactivated. The
amount of glucosinolates ingested by each subject was esti-
mated to be 475 mol. Twenty-four-hour urinary samples
from the volunteers showed that total urinary excretion of
conjugated isothiocyanate was 1.27.3% of the total amount
ingested. In a second experiment, subjects who consumed 150
g of uncooked watercress excreted isothiocyanates in the
range of 17.277.7% of the total ingested isothiocyanates.
These results demonstrate that even when myrosinase is inac-
tivated, some conversion of glucosinolates to isothiocyanates
takes place in humans after ingestion of cooked watercress.
However, the extent of conversion is considerably less than
the amount of uncooked vegetables ingested. Thus normal
food preparation (cooking) can substantially inactivate the
isothiocyanate-related activity. Consequently, to ensure ade-
quate levels of the potential chemopreventive compounds, the
use of supplements may be required to adequately study the
true effects of these compounds.
Rosen et al. (66) recently investigated the use of indole-3-
carbinol for the treatment of recurrent respiratory papilloma-
tosis, a rare disease characterized by benign papillomatous
growths of the aerodigestive mucosa (phase I trial). In this
study, 18 patients were treated with oral indole-3-carbinol for
18 mo. Outcome measures included a change in papilloma
growth rate and the need for surgery after the treatment. Ces-
sation of papilloma growth was found in 33% of the study pa-
tients, and they did not require surgery. Six patients had
reduced papilloma growth rate, whereas six patients showed
no clinical response to indole-3-carbinol. These results sug-
Nutrition and Cancer 2001
gest that indole-3-carbinol may be beneficial for the treatment
of recurrent respiratory papillomatosis (66).
Bell et al. (67) recently conducted a study to examine the
use of indole-3-carbinol for the treatment of cervical intra-
epithelial neoplasia (CIN). Thirty patients with biopsy-
proven CIN II-III were randomized, and placebo or indole-
3-carbinol was administered orally at 200 or 400 mg/day for
12 wk. All patients underwent colposcopy at the initial visit
as well as their visits at Weeks 4, 8, and 12. At the end of the
study, none (0 of 10) of the patients in the placebo group had
a complete regression of CIN, whereas four of the eight pa-
tients in the 200 mg/day arm and four of the nine patients in
the 400 mg/day arm had complete regression on the basis of
their biopsy at Week 12. The results show an RR of 0.50
(95% CI = 0.250.99, P = 0.023) for the 200 mg/day group
and 0.55 (95% CI = 0.310.99, P = 0.032) for the 400 mg/
day group. These data show a statistically significant regres-
sion of CIN in patients receiving indole-3-carbinol orally
compared with the placebo group.
In addition to naturally derived chemopreventive agents
from cruciferous vegetables, attention has also been focused
on synthetic chemicals exhibiting properties similar to in-
doles and thiols present in natural products. One such
example is oltipraz [5-(2 pyranzinyl)-4-methyl-1,2-dithiole-
3-thione], an agent previously used in humans as an anti-
schistosomal agent. Animal studies have demonstrated that
oltipraz is a potent inducer of phase II detoxification en-
zymes and inhibitor of colon cancer (68) and development
of mammary lesions in organ culture (69). Human studies
have examined the use of oltipraz among individuals at high
risk for hepatocellular carcinoma (70) and patients with
resected colon polyps (71). Wang et al. (70), in a random-
ized, placebo-controlled, double-blind phase II chemopre-
ventive study, examined oltiprazs ability to modulate phase
I and phase II enzymes. In the study, 234 healthy adults were
randomly assigned to receive 125 mg of oltipraz daily, 500
mg of oltipraz weekly, or a placebo. Phase I and phase II me-
tabolites of aflatoxin B1 were identified and quantified by
immunoaffinity chromatography and liquid chromatography
coupled with mass spectrometry or fluorescence detection.
Results showed that 1 mo of supplementation with oltipraz
at 500 mg/wk led to a 51% decrease in median levels of
phase I metabolite aflatoxin M1 excreted in urine compared
with administration of placebo (P = 0.03) but had no effect
on phase II metabolites (P = 0.871). By contrast, daily ad-
ministration of 125 mg of oltipraz resulted in a 2.6-fold in-
crease in the phase II metabolite aflatoxin-mercapturic acid
(P = 0.17) but had no effect on excreted aflatoxin M1 levels
(P = 0.682). This study shows it is feasible to induce phase I
and phase II enzymes with oltipraz. Further studies of dose/
schedule and biological end points are necessary.
Mechanisms of Action
Most reports on the antimutagenic activity or chemo-
preventive activity of cruciferous vegetables have addressed
its anti-initiation properties. One principal mechanism by
Vol. 41, Nos. 1&2
which an anti-initiation agent can exert its effects is inhibi-
tion of carcinogen activation. Almost all the carcinogens
present in foods require metabolic activation (72). These
carcinogens include aflatoxin, nitrosamine, polycyclic hy-
drocarbons, heterocyclic amines, and hydrazines. In addi-
tion, it has been reported that extracts of cauliflower and
cabbage interfere with the production of mutagens by nitro-
sation (73). There is considerable agreement that the active
agents include ascorbic acid, cysteine, or other compounds
acting as reducing agents. Osawa and co-workers (74)
showed that ascorbic acid is responsible for the chemical re-
duction of the 1,2-dinitro-2-methyl pyrrole, the mutagenic
nitrosation product of ascorbic acid, and the nonmutagenic
compound 1-nitro-2-methyl-4-amino pyrrole. On the other
hand, Stohs and co-workers (75) identified four specific
compounds isolated from Savoy chieftain cabbage that dem-
onstrated antimutagenic activity induced by MNU and 2-
aminoanthracene. These compounds, b-sitosterol, pheophy-
tin, nonacosane, and nonacosanone, are notable, especially
because they are likely to be present in most plants. These
compounds were shown to present different activity profiles
against MNU and 2-aminoanthracene; therefore, it was ar-
gued that these compounds were achieving their antimuta-
genicity through more than one biological mechanism.
Another mechanism that has been proposed by Munzner
(76) related to the antimutagenic activity of many vegeta-
bles, including cabbage, brussels sprouts, and kohlrabi, was
the stimulation of the S-9 mix normally used to metabolize,
and sometimes activate, mutagens. This observation serves
to bridge the antimutagenic potential discussed above and
the large body of data that makes it clear that, in animals,
there is a strong stimulation of many of the native detoxifica-
tion systems by extracts of various Brassica species.
The earliest work on the induction of these enzyme sys-
tems was actually an attempt by Wattenberg and Loub (35)
to explain variations in baseline levels of aryl hydrocarbon
hydroxylase in different rat colonies. The variation was ulti-
mately ascribed to the presence of alfalfa as an occasional
component in rat chow. This observation was followed by
an examination of the ability of many foods to stimulate
this enzyme. Loub et al. (77) demonstrated that many com-
pounds present in members of the Brassicaceae family (e.g.,
indole-3-carbinol, 3,3-diindolylmethane, and indole-3-ace-
tonitrile) were also active in this regard. These compounds
significantly induced hydrocarbon hydroxylase activity in
the livers of rats consuming augmented basal chow (77).
In subsequent reports, the authors demonstrated that th\e
ability of intestinal enzymes to detoxify many xenobiotic
compounds, including the indoles noted above, correlated to
brussels sprouts or cabbage consumption in rats and humans.
The enzyme systems involved included a number of mixed-
function oxidases such as phenacetin O-dealkylase, 7-ethoxy-
coumarin O-dealkylase, hexobarbital hydroxylase, and
benzo[a]pyrene hydroxylase. A direct correlation was later
established by McDanell and co-workers (78) between the in-
duction of these activities and the concentration of these com-
pounds. These later studies also demonstrated that the various
23
active compounds differed in their ability to stimulate en-
zymes in different organs of the body. They noted, for in-
stance, that the ascorbic acid conjugate of indole-3-carbinol is
the most active compound in stimulating the mixed-function
oxidase population of the gut, whereas indole-3-carbinol was
the strongest inducer of liver enzymes.
Tanaka and colleagues (47) reported the ability of in-
dole-3-carbinol to inhibit tongue carcinogenesis induced
with 4-nitroquinoline-1-oxide. Meanwhile, Bradfield and
Bjeldanes (79) not only confirmed the earlier results of Loub
et al. (77) but also demonstrated that the enzyme glutathione
S-transferase was also strongly induced by Brussels sprouts.
This enzyme, unlike those discussed earlier, is not a P-450-
type enzyme but represents, rather, a phase II detoxification
system that acts to conjugate and clear toxicants from the sys-
tem. The significance of this difference cannot be overstated.
For most P-450-type enzymes, their ability to detoxify many
mutagens must always be balanced by their ability to activate
other mutagens. In the case of glutathione S-transferase, there
are no such drawbacks; rather, an increase in this enzyme
alone directly resulted in an 87% reduction in the binding
of aflatoxin to hepatic DNA in vivo (80). A wide spectrum
of compounds, including the glucosinolates, such as sini-
grin and progoitrin, and their derivatives, such as allyl-
isothiocyanate, goitrin, indole-3-carbinol, and indole-aceto-
nitrile, induce glutathione S-transferase. In other systems, it
is induced even more strongly by xanthotoxin and some
flavonoids.
In recent years, Talalay and co-workers (81) employed in-
duction of quinone reductase [(NAD9P)H:(quinone accep-
tor)oxidoreductase] as a phase II detoxification biomarker.
Using enzyme induction as a parameter, they identified
Figure 2. Proposed mechanisms of action of cruciferous vegetables.
24
erucin, a sulfide analog of sulforaphane in broccoli. Erucin
also induced quinone reductase activity in murine hepatoma
cells in culture. However, it was relatively less potent than
sulforaphane. Mehta et al. (24) synthesized several analogs of
brassinin (a phytoalexin present in Chinese cabbage) and
evaluated their ability to induce quinone reductase activity.
There is growing evidence suggesting that, aside from the abil-
ities of cruciferous vegetables to inhibit metabolic activation of
phase I enzymes and to induce detoxification of carcinogens
via phase II enzymes, several other mechanisms may play piv-
otal roles in the cancer-preventive properties of these com-
pounds. Figure 2 summarizes the mechanisms by which
cruciferous vegetables potentially inhibit tumorigenesis.
In estrogen-dependent cancers, such as breast and endo-
metrial cancer, it was first demonstrated that cruciferous
vegetables elicit a protective effect via their effects on the
estrogen metabolism pathway. Specifically, cruciferous veg-
etables have been shown to shift production of the estrogen
metabolite 16a-hydroxyestrone to the less potent estrogen
metabolite 2-hydroxyestrone. This change in estrogen me-
tabolite production has been shown to be protective against
estrogen-dependent cancers, because, much like estradiol,
16a-hydroxyestrone has been reported to increase breast
cancer proliferation in vitro (82,83) and to promote mam-
mary gland tumors in murine models (83). Unlike 16a-
hydroxyestrone, 2-hydroxyestrone has a low affinity for the
estrogen receptors and is rapidly methylated by catechol-O-
methyl transferase (84), making it less available to the estro-
gen receptors and, thus, less tumorigenic.
More recently, Meng et al. (85) reported that compounds
in cruciferous vegetables are able to modulate estrogen re-
ceptor (ER) transcription activity. In this study, a reporter
Nutrition and Cancer 2001
gene driven by the ER was used to study the effects of
indole-3-carbinol on ER-a signaling in human tumor cells.
The results showed that indole-3-carbinol (10125 mol/l)
significantly repressed the 17b-estradiol-activated ER-a
signaling in a dose-dependent manner. Furthermore, indole-
3-carbinol downregulated expression of the estrogen-
responsive genes pS2 and cathepsin-D and upregulated
BRCA1. Thus these studies suggest that compounds in cru-
ciferous vegetable may not only affect estrogen metabolism
but also function at the transcriptional level.
Meng et al. (86) studied the effects of indole-3-carbinol
on cell migration and invasion behavior in ER-positive
MCF-7 and ER-negative MDA-MB-468 human breast can-
cer cell lines. They found that indole-3-carbinol (50 or 100
M) elicited a significant inhibition in in vitro cell adhesion,
migration, and invasion as well as lung metastasis in vivo.
Indole-3-carbinol also suppressed the 17b-estradiol-stimu-
lated migration and invasion in estrogen-responsive MCF-7
cells. These findings indicate that the anti-invasion and anti-
migration activities of indole-3-carbinol occur via estrogen-
independent and estrogen-dependent pathways. Moreover,
indole-3-carbinol was found to cause a significant dose-
dependent increase in E-cadherin, three major catenins (a,
b, and g), and BRCA1 expression. These findings demon-
strate that indole-3-carbinol can activate the function of
invasion-suppressor molecules associated with the suppres-
sion of invasion and migration in breast cancer cells.
Various reports have suggested that the chemopreventive
effects of cruciferous vegetables may be associated with their
ability to induce cell cycle arrest in cancerous cells (87,88).
Telang and colleagues (87) examined the effects of treating
chemically transformed breast epithelial (184-B5/BP-
reduction mammoplasty-derived cells) and MDA-MB231
breast epithelial cancer cells with indole-3-carbinol. They
found that 50 M indole-3-carbinol resulted in a 137210%
increase in Q/P ratio (Q = G0, P = S + M) and a twofold in-
crease in cellular apoptosis. Similarly, isothiocyanates have
been shown to induce apoptosis in several cancerous cell
lines. Chiao et al. (88) investigated the effects of PEITC-NAC
on tumor cell growth of human prostate cancer cell lines
LNCaP (androgen-dependent) and DU-145 (androgen-
independent) cells. This study found that PEITC-NAC ar-
rested cells in the S and G2/M phases of the cell cycle, thus
blocking cells from entering replicating phases. Moreover,
HeLa cervical cancer cells treated with apoptosis-inducing
concentrations of isothiocyanates stimulated an induction of
caspase-3/CPP32-like activity, a caspase involved in apo-
ptosis (100). This study suggests that isothiocyanates may in-
duce apoptosis through a caspase-3-dependent mechanism.
Thus cruciferous vegetables have multiple modes of actions.
As a class, these compounds can mediate their actions as anti-
initiators and antipromoters. It is quite remarkable that so
many biological activities have been demonstrated for plants
as commonly consumed as these plants.
In this review, we have summarized the cancer-preven-
tive potential of many members of the Brassicaceae family.
Although the correlation of cruciferous vegetables and pre-
Vol. 41, Nos. 1&2
vention of carcinogenesis is strong, it must always be
stressed that, to understand the relevance of these reports on
the human condition, many further studies need to be done
to specifically address questions of the stability, bioavail-
ability, transport, and metabolism. The possible additive or
even synergistic effects of these compounds are unknown.
The additional effects of normal food preparation proce-
dures present another factor that is yet largely unexplored
with respect to the cancer-preventive properties of phyto-
chemicals present in these vegetables.
Acknowledgments and Notes
This work was supported by National Cancer Institute Program Proj-
ect P01 CA-48112. Address correspondence to Dr. Rajendra Mehta, Dept.
of Surgical Oncology (MC/820), University of Illinois College of Medi-
cine, Clinical Science Bldg., 840 S. Wood St., Chicago, IL 60612-7322.
Submitted 26 April 2001; accepted in final form 13 September 2001.
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