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Introduction
Squamous cell carcinoma (SCC) accounts for >90% of
esophageal cancers worldwide[1]. Esophageal SCC
develops through a progressive sequence from mild to severe
dysplasia, carcinoma in situ and, finally, invasive
carcinoma[2,3]. This cancer shows marked variation in geographical
distribu-tion, with high frequencies in parts of China, Iran, South
Africa, Uruguay, France, Italy and Puerto
Rico[4]. The highest incidences are in the
Shansi, Henan and Hopei provinces in China, where the age-adjusted mortality rates are
151/100 000 for men and 115/100 000 for
women[5]. Risk factors for the disease include tobacco smoking, alcohol
consumption, ingestion of salt-pickled, salt-cured and moldy
foods, deficiencies in certain dietary vitamins and minerals
and, potentially, infection with human papilloma virus
(HPV)[4]. Research in China provides evidence
that N-nitroso compounds and their precursors are
etiological factors for esophageal SCC in the high-incidence
areas[6,7]. Nitrosamine carcinogens such as
N-nitrosomethylbenzylamine (NMBA) and
N-nitrosomethylamylamine (NMAA) have been identified in
the diets and gastric juices collected from subjects in
China[7]. In addition, contaminated foods often contain nitrates,
nitrites and secondary and tertiary amines, which act as
precursors for the formation of nitrosamine carcinogens in the
acidic conditions of the
stomach[8]. Thus, lifestyle changes,
especially the avoidance of tobacco and alcohol use and the
elimination of high salt and moldy foods, might be expected
to reduce the incidence and mortality of this disease.
Chemo-prevention may be another feasible approach and may have
special relevance in high-incidence areas of the
world[4].
Nitrosamine-induced tumorigenesis in the Fischer-344 rat
has proven to be a valuable animal model for studies of the
molecular biology and chemoprevention of esophageal
SCC[4]. Several nitrosamines, including NMBA and the
tobacco-specific nitrosamine, N-nitrosonornicotine (NNN),
induce tumorigenesis in the rat
esophagus[8]. NMBA is by far the
most potent inducer of tumors in the rat esophagus.
Its metabolic activation to DNA damaging species in the
esophagus, especially the formation of
O6-methylguanine (O6-MeGua) adducts in esophageal DNA, has previously
been described[4]. Repeat subcutaneous injection of NMBA
in F-344 rats results in esophageal tumor formation within
15_26 weeks (Figure 1). Several preneoplastic lesions
produced in NMBA-treated rat esophagus closely mimic lesions
observed in the human esophagus. These
lesions include simple hyperplasia, leukoplakia and epithelial
dysplasia (Figure 2). Squamous papilloma is the predominant tumor
type seen in the rat esophagus model. The incidence of SCC
is rather low because the rats often succumb to the
occlusive effects of large papillomas in their esophagi before
carcinomas can develop. In a typical tumor bioassay,
subcutaneous injections of NMBA at either 0.25 or 0.5 mg/kg
body weight three times per week for 5 weeks, or once per week for
15 weeks, result in a 100% tumor incidence by 26
weeks[9,10]. On average, these two concentrations of NMBA will
produce from 2_4 or 4_8 tumors per esophagus, respectively, at
26 weeks. In the past several years our laboratory and
others have used this model to develop surrogate end-point
biomarkers, identify novel targets for intervention and therapy
and evaluate putative chemoprevention agents against
esophageal SCC[11].
Since the early 1990s, our laboratory has taken a
"food-based" approach to the prevention of gastrointestinal tract
cancers using freeze-dried
berries[12,13]. The rationale for the choice of berries and, in particular, black raspberries for
cancer prevention has been previously
described[13]. Black raspberries contain multiple agents that, by themselves, exhibit
chemopreventive effects in animals, including vitamins A, C,
E, and folic acid, calcium, selenium, β-sitosterol, ellagic and
ferulic acids, quercetin, and at least 5
anthocyanins[10,12]. The high level of anthocyanins is responsible for the dark color
and high-antioxidant potential of black
raspberries[13]. In an initial study, freeze-dried black raspberries (FBR),
administered at specific concentrations in a synthetic diet, produced
significant decreases in NMBA-induced esophageal tumors
in rats[10]. Specifically, 5% and 10% dietary FBR produced a
39% and 49% reduction, respectively, in esophageal tumor
multiplicity when administered in the diet 2 weeks before,
during and after treatment of rats with NMBA (see Figure 3A
for the complete carcinogenesis protocol). The berries were
found to reduce O6-MeGua adduct formation, preneoplastic
lesion development and cell proliferation rates in
NMBA-treated esophagus. In the same study, 5% and 10% FBR
produced 37% and 31% reductions in tumor multiplicity,
respectively, when administered in the diet immediately after
cessation of NMBA treatment and until the end of the
bioassay (see Figure 3B for the anti-promotion/progression
protocol). Using the anti-promotion/progression protocol,
Chen et al[14,15] have shown that 5% dietary FBR
downregul-ates the mRNA and protein expression levels of
cyclooxy-genase-2 (COX-2), inducible nitric oxide synthase (iNOS),
c-Jun (a component of activator protein-1 (AP-1), and
vascular endothelial growth factor (VEGF) in NMBA-treated
esophagus, and these reductions correlated with lower
levels of prostaglandin E2
(PGE2), nitrate/nitrite and microvessel
density in NMBA-treated esophagus, respec-tively. FBR,
therefore, can reduce the expression levels and activities of
genes associated with cellular proliferation, inflammation and
angiogenesis in the rat esophagus.
In contrast to previous studies that have examined black
raspberries for their anti-initiation and
anti-promotion/progression effects in the rat esophagus, the present study
evaluated FBR for their potential therapeutic effects on
esophageal carcinogenesis (see Figure 3C for the
therapeutic protocol). Specifically, 5%, 10% and 20% dietary FBR
were tested for their ability to reduce the incidence, number
and size of fully developed papillomas in NMBA-treated
esophagus. This study is important in that there is
considerable interest as to whether or not chemopreventive agents,
including natural foods, can be used in conjunction with
standard therapies (ie surgery, radiotherapy and
chemo-therapy) for the treatment of esophageal SCC and/or for the
prevention of its recurrence following treatment.
Materials and methods
Animals Male F-344 rats, 4 weeks of age, were purchased
from Harlan-Sprague Dawley (Indianapolis, IN, USA). The
animals were housed under standard conditions (20±2 ºC;
50%±10% relative humidity; 12-h light/dark cycles) and
maintained on a modified AIN-76A diet as previously described[10]. Food and water were provided
ad libitum and hygienic conditions were maintained by twice weekly cage
changes. Body weight and food consumption measurements
were recorded biweekly after administration of the berry diet
and for the duration of the study.
Chemicals NMBA, obtained from Ash Stevens (Detroit,
MI, USA), was greater than 98% pure as determined by high
performance liquid chromatography. Dimethyl sulfoxide
(DMSO) was purchased from the Sigma Chemical
Company (St Louis, MO, USA).
Diet preparation Black raspberries (Rubus
occidentalis) of the Bristol variety were supplied by the Stokes Raspberry
Farm (Wilmington, OH, USA). The berries were picked,
washed with water and frozen on the farm at -20 ºC within
2_4 h of the time of picking[12]. The berries were then shipped
frozen to Van Drunen Farms (Momence, IL, USA) where they
were freeze-dried. The FBR were packaged in double
polyethylene bags, placed in carton boxes and stored at -20 ºC.
The berries were then shipped frozen to the Parker Food
Science and Technology Building, Ohio State University,
and stored frozen until they were used in the experiments.
Berries were analyzed routinely for content of certain
vitamins, minerals, phenols, carotenoids and phytosterols
by Covance Laboratories (Madison, WI, USA) and for
anthocyanin and ellagic acid content in the laboratory of Dr
Steven Schwartz, Department of Food Science and
Techno-logy, Ohio State University. Data from these analyses
indicate that, with the exception of vitamin C, which degrades in
frozen berries, the components measured in FBR remain
relatively stable for at least 2 years when the berries are stored at
-20 °C. On a biweekly basis, FBR were mixed for 20 min in a
modified AIN-76A diet at concentrations of 5%, 10% and
20% using a Hobart mixer. The cornstarch in the diet was
reduced by 5%, 10%, and 20%, respectively, to provide a
similar caloric intake in the diets.
Animal bioassay Rats were randomized into 6 groups
and placed on a AIN-76A diet (Table 1). They were then
treated as follows: Group 1 was injected subcutaneously
with 0.2 mL of a solution containing 20% DMSO in water
(the solvent for NMBA) once per week for 15 weeks. Group
2 was fed a diet containing 20% FBR only. Groups 3_6 were
injected subcutaneously with NMBA (0.5 mg/kg body weight) once per week for 15 weeks. To monitor for
papilloma development, at the beginning of week 19, 5 rats from
the NMBA control group (Group 3) were killed and their
tumors were counted. The animals had an average of 5_6
papillomas per esophagus, all of which exceeded 1 mm in
diameter. Immediately, therefore, animals in Groups 4_6 were
placed on diets containing 5%, 10% and 20% FBR,
respectively (Figure 3C). The rats remained on the berry diets until
26 weeks when animals in all groups were killed. At
necropsy, the esophagus of each animal was opened longitudinally
and the surface tumors were mapped, counted and measured.
Lesions greater than 0.5 mm in diameter were considered to
be tumors. Assuming a spheroid shape, a volume estimate
for each papilloma was calculated[16]. Each esophagus was
fixed on laminated cards in 10% neutral buffered formalin
(NBF) to be processed for histopathology. In addition,
sections of the liver, colon, kidney and spleen were harvested
and fixed in NBF to evaluate possible toxicity of FBR.
Immunohistochemical analysis of cell
proliferation At the end of the study, the entire esophagus from 5 rats per
group was stained for the cell proliferation marker,
proliferating cell nuclear antigen (PCNA), as previously
described[10]. In brief, following antigen retrieval and treatment with 3%
H2O2, tissue sections were incubated with the primary
antibody, monoclonal mouse anti-PCNA (BioGenex, San
Ramon, CA, USA) for 30 min. Rat-absorbed link secondary
antibody (biotinylated anti-immunogobulin) followed for 30
min, and streptavidin-horseradish peroxidase for another 30
min. Slides were then incubated in 3,3'-diaminobenzidene
for 3.5 min to permit biomarker visualization. The slides were
counterstained with hematoxylin, dehydrated and cover
slipped with Permount (Fisher Scientific, Pittsburgh, PA,
USA). Positive (colon) and negative controls (mouse
antiserum) were included in each run. PCNA-stained slides
were viewed at 200× with a Nikon bright-field microscope
mounted with a high-resolution spot camera. The camera
was interfaced with a computer containing a matrix frame
grabber board and image analysis software (Simple PCI
Imaging Systems; Complix, Cranberry Township, PA, USA).
The basal layer of the esophagus was scanned and a
minimum of 10 fields (1500_2000 cells) was quantified to
determine the mean labeling index (LI). The LI was computed by
dividing the positive nuclear-stained area by the total nuclear
area; the LI was expressed as a percentage.
Statistical analysis All statistical procedures were
carried out using the NCSS 97 statistical software package (NCSS
Statistical Software, Kaysville, UT, USA). Body weight, food
consumption, tumor multiplicity and tumor size data were
analyzed for statistical significance (P<0.05) using
anova followed by Newman-Keuls' multiple comparisons and
Kruskal-Wallis tests. Differences in tumor incidence were
determined using Fisher's exact probability test and
c2 and Kruskal-Wallis tests.
For survival analysis, the means, medians and
standard deviations of tumor numbers and average volumes were
computed for each group defined by treatment
(Vehicle, 20% BRB, NMBA, NMBA + 5% BRB, NMBA + 10% BRB, NMBA
+20% BRB). The groups were compared using a one-way
anova. If the residuals were non-normal or had unequal
variances, a non-parametric Kruskal-Wallis test was carried
out. A significant group effect was followed by multiple
comparisons. Tukey's method was used for all pairwise
comparisons. The overall alpha was set at 0.05. The mean
survival time, standard error and median survival time were
calculated for each group using the Kaplan-Meier
method[17]. If the overall test of equality of survival curves was
signifi-cant, then follow-up multiple comparisons were carried out
using log rank tests.
Results
There were no differences in mean body weight or food
consumption for rats in all groups prior to being fed berry
diets. From weeks 23 to 26, however, there were significant
reductions in body weight in rats treated with NMBA only
or with NMBA and all three dietary concentrations of berries
compared with Group 1 and 2 controls (data not shown).
These reductions in body weight resulted from increases in
the number and size of tumors throughout the esophagus,
leading to a reduced passage of food.
There were no significant differences in esophageal
tumor incidences, numbers or volumes in NMBA-treated rats
fed 5%, 10% or 20% FBR (Groups 4_6) compared with rats
treated with NMBA only (Group 3) (Table 2).
Histopathological examination of sections of the liver, colon, stomach,
kidney and spleen from animals fed 20% FBR indicated that
the berries did not produce toxic effects in any of these
organs.
A Kaplan-Meier survival curve of animals in Groups 3_6
(NMBA-treated rats with and without berry diets) is shown
in Figure 4. Included in the survival data were rats that died
during the course of the experiment from week 19 through to
week 26, as well as those killed at the end of the bioassay
(week 26). Overall, there were no significant differences in
survival between rats in Groups 3_6 (p=0.146). The effects
of FBR on the PCNA LI in the esophagus of rats in all groups
are shown in Table 3. As indicated, the PCNA LI in the
esophagus of rats fed 20% FBR (Group 2) was similar to that
in vehicle controls (Group 1). NMBA treatment (Group 3)
resulted in a >2-fold increase in the PCNA LI relative to
vehicle or berry controls (Groups 1 and 2). The esophagi of
NMBA-treated animals fed 5%, 10% or 20% (Groups 4_6)
had somewhat higher PCNA LI than the NMBA controls
(Group 3); however, these differences were not significant.
Discussion
We have reported on the ability of FBR to prevent the
development of NMBA-induced papillomas in the rat
esophagus when the berries were fed at 5% and 10% of the diet
either before, during or after treatment of rats with the
carcinogen (Figure 3A) or shortly after carcinogen treatment
until the end of the bioassay (Figure
3B)[10,12]. When administered in the diet before, during and after treatment of the
rats with NMBA, the berries were shown to influence the
metabolism of NMBA leading to a reduced formation of
O6-MeGua adducts in esophageal
DNA[10]. Subsequently, treatment of rats with dietary FBR was shown to inhibit the
metabolism of NMBA in esophageal explant cultures and in
liver microsomes in vitro, and to stimulate the activity of
glutathione-S-transferase in the liver, all of which could be
responsible for the reduced rate of adduct formation
in vivo[18]. In this same protocol (Figure 3A), dietary berries were found
to reduce the growth rate of preneoplastic cells in
NMBA-treated esophagi almost to the level seen in untreated
esophagi[10]. Subsequently, FBR were shown to
downregul-ate the mRNA and protein expression levels of COX-2, iNOS,
c-Jun and VEGF in pre-neoplastic esophagus when
administered in the diet shortly after treatment of the rats with NMBA
(Figure 3B)[14, 15]. These changes in gene expression were
associated with reduced levels of PGE2, nitrate/nitrite and
microvessel density in pre-neoplastic esophageal epithelium,
indicating that berries influence the expression of genes
involved in cell proliferation, inflammation and angiogenesis.
In vitro studies involving the induction of luciferase reporter
genes with benzo(a)pyrene-7,8-diol-9,10-epoxide (B[a]PDE)
in mouse epidermal JB-6 clone 41 cells have shown that
extracts of FBR inhibit signal transduction pathways
leading to activation of AP-1 and NF-κB, resulting in
downregula-tion of VEGF and COX-2
expression[19_21]. These extracts were also shown to selectively inhibit the proliferation of
oral cavity cancer cells in vitro and to stimulate their
apopto-sis[22].
In the present study, FBR were evaluated for their
potential therapeutic effects against NBMA-induced rat
esophageal papillomas. FBR were added to the diet at week 19 of
the standard bioassay, when the esophagi contained an
average of 5_6 papillomas each (Figure 3C). The results of
this study were disappointing in that the berries did not
reduce the incidence, number or size of papillomas when
given in the diet for a period of 7 weeks. In addition,
although there was a trend toward enhanced survival in
NMBA-treated animals fed berries compared with NMBA
controls, the results were not significant. The reasons for
the lack of response of already developed papillomas to berry
treatment are unknown, however, it is possible that the
active compounds in berries, some of which appear to be the
anthocyanins[23], are not well absorbed into the papillomas.
This might be because of the relatively thick layer of kertain
on the papilloma surface. It is also possible that the
conversion of dysplastic lesions to papillomas is accompanied by
genetic events that render the papillomas resistant to the
inhibitory effects of berry compounds. Finally, the
treatment period (7 weeks) may have been too short for the FBR
to be effective. Unfortunately, the treatment period could
not be extended because of the progressive loss of
NMBA-treated animals, beginning at 22 weeks of the bioassay. This
loss resulted from the inability of rats with developed
papillomas to swallow food and to respiratory failure in rats with
large papillomas that press against the trachea and restrict
the passage of air.
Acknowledgements
We thank Dr Amy FERKETICH and Dr Li-shu WANG for
statistical analysis of the data, and Mr Ronald NINES for
assistance with the animal bioassay.
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