Combined antioxidant (b-carotene, a-tocopherol and ascorbic acid) supplementation
increases the levels of lung retinoic acid and inhibits the activation of
mitogen-activated protein kinase in the ferret lung cancer model
Yuri Kim
1, Nalinee Chongviriyaphan
1,2, Chun Liu
1,
Robert M.Russell
1and Xiang-Dong Wang
1,1Nutrition and Cancer Biology Laboratory, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, USA
2Present address: Division of Nutrition, Department of Pediatrics, Faculty of Medicine at Ramathibodi Hospital, Mahidol
University, Rama 6 Road, Bangkok, Thailand 10400
To whom correspondence should be addressed at: The Nutrition and
Cancer Biology Laboratory, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA.
Tel:+1 617-556-3130; Fax: +1 617-556-3344;
Email: xiang-dong.wang@tufts.edu
Interactions among
b-carotene (BC), a-tocopherol (AT)
and ascorbic acid (AA) led to the hypothesis that using
a combination of these antioxidants could be more
beneficial than using a single antioxidant alone,
particu-larly against smoke-related lung cancer. In this
investiga-tion, we have conducted an animal study to determine
whether combined BC, AT and AA supplementation
(AOX)
protects
against
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis
in smoke-exposed (SM) ferrets. Ferrets were treated
for 6 months in the following four groups: (i) control,
(ii) SM
+ NNK, (iii) AOX and (iv) SM + NNK + AOX.
Results showed that the combined AOX supplementation
(i) prevented the SM
+ NNK-decreased lung
concentra-tions of retinoic acid (RA) and BC; (ii) inhibited the
SM
+ NNK-induced phosphorylation of Jun N-terminal
kinase
(JNK),
extracellular-signal-regulated
protein
kinase (ERK) and proliferating cellular nuclear antigen
proteins in the lungs of ferrets; and (iii) blocked the SM
+ NNK-induced up-regulation of total p53 and Bax
proteins, as well as phosphorylated p53 in the lungs of
ferrets. In addition, there were no lesions observed in
the lung tissue of ferrets in the control and/or the AOX
groups after 6 months of intervention, but combined
AOX supplementation resulted in a trend toward lower
incidence of both preneoplastic lung lesions and lung
tumor formation in SM
+ NNK + AOX group of ferrets,
as compared with the SM
+ NNK group alone. These
data indicate that combined AOX supplementation could
be a
useful
chemopreventive strategy
against lung
carcinogenesis through maintaining normal tissue levels
of RA and inhibiting the activation of mitogen-activated
protein
kinase
pathways,
cell
proliferation
and
phosphorylation of p53.
Introduction
Lung cancer has been the leading cause of cancer death in both
men and women in the United States for the past 10 years.
Cigarette smoking is the main risk factor for the development
of lung cancer and avoidance of tobacco products is the best
way to prevent tobacco-related cancers. However, the
addict-ive power of nicotine is strong and exposure to environmental
tobacco smoke (e.g. second-hand smoke) persists. Protection
by consuming a healthy diet may be an effective way to protect
against the harmful effects of tobacco smoke and reduce
lung cancer risk. Observational epidemiologic studies have
consistently demonstrated that individuals eating more fruits
and vegetables, which are rich in carotenoids and people
having higher serum
b-carotene (BC) levels have a lower
risk of cancer, particularly lung cancer (1). In contrast, two
intervention trials, the Alpha-Tocopherol, Beta-carotene
Can-cer Prevention Trial (ATBC) in Finland (2) and the
b-Carotene
and Retinol Efficacy Trial in the United States (3)
demon-strated an increased risk of lung cancer in heavy smokers
and asbestos workers if they consumed supplements containing
BC. A possible explanation of why high doses of BC in
smokers caused more lung cancer is that BC is susceptible
to oxidation in the free radical rich, antioxidant poor
environ-ment of the lungs of cigarette smokers (4,5). We found that
cigarette smoke enhanced the formation of oxidative excentric
cleavage products of BC in ferrets (6), which facilitate the
binding of benzo[a]pyrene metabolites to DNA (7) and induce
several cytochrome P450 (CYP) enzymes (such as CYP1A1/2
and CYP2A1, which activate tobacco-smoke procarcinogens)
in the lungs of ferrets (8). Furthermore, we have shown that
the induction of CYP enzymes in the lungs of ferrets exposed
to cigarette smoke and/or high dose of BC (30 mg/day)
enhances retinoic acid (RA) catabolism, which provides an
additional explanation for enhanced lung carcinogenesis (4,8).
Previous in vitro studies suggest that there are strong in vitro
interactions among BC,
a-tocopherol (AT) and ascorbic acid
(AA) in terms of mutual beneficial protection against oxidative
damage (9–11). AT and AA have the capability to regenerate
BC from its radical cation (9), thus preventing BC from being
further oxidized. In addition, BC can enhance AT antioxidant
efficiency by regenerating AT from its radical cation (10).
We have shown that the in vitro addition of both AT and
AA into the incubation mixture of BC with smoke-exposed
ferret lung tissue can inhibit the production of excentric
cleavage metabolites of BC, but increase the levels of retinal
and RA (12). Although pre-treatment of human lung cells with
both vitamin E and BC has been shown to provide protection
against DNA strand breaks induced by tobacco-specific
nitrosamines (11), the combination of BC (20 mg/day) and
vitamin E (50 mg/day) were not found to be protective against
smoke-related lung cancer in the ATBC study. However,
vitamin C, which facilitates both recycling and stability of
AT and BC as well as converting BC into RA (12–14), was
Abbreviations:AA, ascorbic acid; AT,a-tocopherol; BC, b-carotene; ERK,
extracellular-signal-regulated protein kinase; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PCNA, proliferating cellular nuclear antigen; RA, retinoic acid.
not used in the ATBC study and would be expected to be low
in this population of heavy smokers. In a follow-up study
from the first National Health and Nutrition Examination
Survey, the subjects in the highest quartiles of carotenoid,
vitamin E and vitamin C consumption had a 68% lower risk
of lung cancer than those in the lowest quartiles of all three
nutrients (15). A recent prospective cohort study exploring
the question of whether combinations of dietary antioxidants
affect lung cancer morbidity and mortality in male smokers has
shown that men with the highest antioxidant index scores had
a significantly lower (16%) risk of lung cancer than men with
the lowest scores (16). Although it is possible that using a
combination of these antioxidants would be advantageous in
antioxidant protection against smoke-related lung cancer, more
evidence supporting the hypothesis and exploration of the
possible mechanism(s) involved are needed.
Jun N-terminal kinase (JNK) and
extracellular-signal-regulated protein kinase (ERK) belong to mitogen-activated
protein kinase (MAPK) family and are activated by
phos-phorylation in response to many extracellular stimuli and
environmental stress factors (e.g. smoke exposure) and may
play an important role in carcinogenesis (17–19). JNK
was shown to phosphorylate c-Jun on sites serine-63 and
serine-73 (20) and increase AP-1 transcription activity (21)
and, eventually, mediate cell proliferation and apoptosis.
ERK induced c-Jun through phosphorylation and activation
of AP-1 component ATF1 at serine-63 (22). In addition,
JNK and ERK mediate phosphorylation of the p53 tumor
suppressor (19,23). Phosphorylation of p53 plays a role in
cell accumulation in G
1phase of the cell cycle and signals
oxidative stress in cells. Phospho-p53 appears to be expressed
in cells undergoing apoptosis. Serine-15 in human p53 is
sensitive to oxidative stress-induced phosphorylation (24).
Previously, we have shown that high dose of BC
supplementa-tion promotes the smoke-induced phosphorylasupplementa-tion of JNK
and p53 (25). However, it is not known whether BC combined
with AT and AA supplementation can affect the MAPK
signaling pathway or phosphorylation of p53 and its
downstream gene Bax.
The ferret (Mustela putorius furo) has been shown to be
an excellent model for studying carotenoid absorption
and metabolism (6,26–30). Recently, we have performed a
6-month in vivo study in ferrets exposed to both tobacco
smoke and a carcinogen
[4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK] found in cigarette smoke (31).
Based on the close similarity of the animal’s lung tumor
pathology to humans, we have demonstrated the ferret to be
a good model for lung carcinogenesis. In the present report,
we conducted a ferret study to determine whether combined
BC, AT and AA supplementation protects against altered
BC metabolism, activation of the MAP kinase pathway and
chemical carcinogen-induced carcinogenesis in the lungs
of smoke-exposed ferrets.
Materials and methods
Animals, diet and study group
Adult male ferrets (1.2–1.4 kg) from Marshall Farms (North Rose, NY) were housed in an American Association of Accreditation of Laboratory Animal Care-accredited animal facility at the Human Nutrition Research Center on Aging at Tufts University (HNRCA), fed a semipurified ferret diet (Research Diets, New Brunswick, NJ) and provided water ad libitum. Accord-ing to our previous study, the average daily food intake of the ferret, with an average body weight of 1.3 kg, is 80 g/day. In 80 g of this diet, a ferret gets
a negligible amount of BC, 0.43 mg of retinyl palmitate, 2 mg AT and no AA. All animals were quarantined for a minimum of 1 week to ascertain their health status before the experiment began. During the experimental period, ferret body weights were recorded weekly. After the experimental period, all ferrets were terminally exsanguinated under deep isoflurane anesthesia. Tissues were collected and stored at80C until analyzed. All experimental procedures carried out on the ferrets were reviewed and approved by the Animal Care and Use Committee of the HNRCA.
Short-term (6-week) study. Thirty-six ferrets were randomly assigned to four groups for a 6-week experiment as follows: (i) control (neither vitamin
supplemented nor exposed to cigarette smoke), n¼ 9; (ii) smoke exposed
(SM) without any supplementation, n¼ 9; (iii) smoke exposed plus low dose BC (LBC, 0.48 mg/kg body wt/day), AT (22 mg/kg body wt/day) and AA (3 mg/kg body wt/day) supplementation, n¼ 9; and (iv) smoke exposed plus high dose BC (HBC, 2.4 mg/kg body wt/day), AT (22 mg/kg body wt/day) and
AA (3 mg/kg body wt/day) supplementation, n¼ 9.
Long-term (6-month) study. Forty-four male ferrets were randomly assigned to four groups as follows: (i) control (neither vitamin supplemented
nor exposed to cigarette smoke), n ¼ 9; (ii) smoke-exposed plus
NNK-treated (SM + NNK), n ¼ 12; (iii) combined antioxidant supplemented
[AOX, BC (0.85 mg/kg body wt/day), AT (22 mg/kg body wt/day) and AA
(3 mg/kg body wt/day)], n¼ 9; and (iv) SM + NNK treated plus combined
AOX supplemented, n¼ 14.
Cigarette smoke exposure and NNK treatment
We conducted a short-term (6-week) study using the ferret model exposed to smoke only and a long-term (6-month) study using the ferret model exposed to both smoke and NNK treatment, as described previously (31). In brief, we exposed ferrets to smoke from non-filtered cigarettes (Standard Research Cigarettes, Type 2R4F, Tobacco-Health Research Institute, University of Kentucky, Lexington, KY) (6). Ferrets were exposed to cigarette smoke twice (30 min each time) per day throughout the experimental period. We showed that this amount of smoke exposure in the ferret is similar to that found in humans smoking one pack of cigarettes per day. For the carcinogen treatment, 50 mg NNK/kg body weight was given by i.p. injection at 4-week intervals for a total of 4 doses over 4 months. This dose was based on the report that mink receiving a single of dose (150 mg) of tobacco-specific N-nitrosamine, N0-nitrosonornicotine (NNN) did not show any toxic effects (32). Control groups were given a sham injection of normal saline. The sham-exposed ferrets were housed in a separate room and went through the exact same procedures as the smoke-exposed animals, except that they received neither smoke nor carcinogen exposure.
BC, AT and AA supplementation
Natural all-trans BC (Cognis, Cincinnati, OH) and all-rac-a-tocopherol acetate (Roche Vitamins, NJ) were dissolved in 1 ml of corn oil and fed orally (not gavage) to the supplemented ferrets every morning. Ferrets like to eat corn oil and lick it spontaneously. AA (Roche Vitamins) was freshly prepared by dissolving it in distilled water and then fed orally to the supplemented ferrets every morning at a dose of 3 mg AA/kg body wt/day, which is equivalent to 210 mg AA/day in a 70 kg man. Ferrets in the control group
and SM+ NNK group were fed the basal diet plus 1 ml corn oil without
antioxidants. In the short-term study, we used BC at low (0.48 mg BC/kg body wt/day) and high (2.4 mg BC/kg body wt/day) doses, which had been previously shown to be protective and harmful, respectively, in smoke-exposed ferrets (33). Based on our previous study (26), the total absorption of intact BC by ferrets is5 times lower than that in the human. The LBC at 0.48 mg BC/kg body wt/day in the ferret is therefore equivalent to 6 mg BC/day in a 70 kg man. The HBC at 2.4 mg BC/kg body wt/day in the ferret is equivalent to 30 mg BC/day in a 70 kg man. Based on the result from the short-term study, 0.85 mg BC/kg body wt/day in ferrets (equivalent to 12 mg BC/day in a 70 kg man) was chosen for the long-term study in order to maintain the lung RA concentrations at a normal level. From these short-term data, the final sample size of ferrets chosen in the long-term study was one that would allow a 80% chance of detecting statistically significant differences among the groups at a 0.05 level of significance, assuming a 50% reduction of both the RA level and the incidence of lung squamous metaplasia in the
lungs of the SM+ BC + AT + AA group as compared with the SM exposed
alone group.
Tissue extraction and HPLC analysis
BC (33), AT (30,34), AA (35) and retinoids including retinyl palmitate, retinol and RA in lung tissue homogenates were measured by HPLC and UV detection, as described previously with some modifications. A total of 0.2 g of lung tissue (wet weight) in a mixture of normal saline and methanol (2:1, v/v) was homogenized on ice for 30 s, followed by addition of 100ml
of internal standard (retinyl acetate). After adding 5 ml of chloroform:methanol (2:1, v/v), the mixture was vortexed and centrifuged for 10 min at 800 g at 4C. Four milliliter of hexane was added after the lower layer had been collected.
The chloroform and hexane layers were evaporated under N2 and a 50ml
aliquot of the extract reconstituted with ethanol was injected into a gradient reverse-phase HPLC system, consisting of a Waters 2695 separation module, a Waters 2996 photodiode array detector (Waters Corporate, Milford, MA)
and a 0.46 · 8.3 cm Pecosphere-3 C18 cartridge column (Perkin-Elmer
Analytical Instruments, Shelton, CT). The HPLC mobile phase consisted of acetonitrile:tetrahydrofuran:water (50:20:30, v/v/v, 1% ammonium acetate in water) as solvent A and acetonitrile:tetrahydrofuran:water (50:44:6, v/v/v, 1% ammonium acetate in water) as solvent B at flow rate of 1 ml/min. Individual compounds were identified by coelution with standards and were quantified by determining peak areas in the HPLC chromatograms calibrated against known amount of standards. Vitamin levels were corrected for extraction loss based on the recovery of the internal standard. All procedures were carried out under red light.
Histopathology and immunohistochemistry
The histopathology examination and procedures used for the lung have been described elsewhere (31,33). The WHO International Association for the Study of Lung Cancer classification of lung and pleural tumors (36,37) and the WHO Histological Classification of Tumors of the Respiratory System for domestic animals (37) were used as guidelines for histopathological diagnoses. The right upper lobe of each ferret lung was inflated and fixed by an intratracheal instillation of 10% formalin. The samples were then embed-ded in paraffin. The lung sections, 5mm in thickness, were made using an AO microtome and stained with hematoxylin and eosin. Lung lesions were independently examined and diagnosed by a pathologist and by two invest-igators who were blinded to the treatment groups. If any premalignant or malignant lesions were observed (histological examination plus confirmation by immunohistochemistry) in a ferret, the ferret was considered as ‘positive’. Otherwise, the animal was considered ‘negative’. Localization of phospho-p53 (serine-15, Cell signaling, Beverly, MA) in a lung sections from a tumor
bearing animals in the SM+ NNK group was analyzed by using
immunohis-tochemistry, as described previously (33). Western blot analysis
Western blot analysis of protein levels was carried out for JNK, ERK, Bax, phospho-JNK and phospho-ERK. Briefly, lung tissues were incubated in
extraction buffer (25 mM HEPES, 300 mM NaCl, 1.5 MgCl2, 0.2 mM
EDTA, 0.05% Triton X-100, and 20 mMb-glycerophosphate and a mixture
of protease inhibitors) with agitation at 4C for 30 min. The mixture was then centrifuged and supernatants were collected. Western blot analysis for p53, phospho-p53 at serine-15 and proliferating cellular nuclear antigen (PCNA) were carried out using nuclear protein extracts from the lungs of ferrets. The lung tissues were homogenized gently with ice-cold buffer A [10 mM Tris–HCl (pH 7.5), 10% glycerol, 10 mM KCl, 10 mM monothioglycerol with a mixture of protease inhibitors] and nuclei were collected by centrifu-gation for 30 min at 3200 g. Nuclear pellets were solubilized in buffer B [10 mM Tris–HCl (pH 7.5), 10% glycerol, 600 mM KCl, 1 mM DTT, 10 mM monothioglycerol with a mixture of protease inhibitors]. The extracts were centrifuged for 30 min at 100 000 g; the resulting supernatants are referred to as the nuclear extract. SDS–PAGE and western blot analysis were carried out according to the standard protocols using antibodies against total JNK, phospho-JNK, PCNA (Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies against phospho-p53 (serine-15), total ERK, phospho-ERK, Bax were purchased from Cell Signaling and antibody against p53 was purchased from Calbiochem (San Diego, CA).
Statistical analysis
All results are expressed as means ± SEM. Data were analyzed by ANOVA followed by Tukey’s honest significant difference test, unless otherwise indicated. Differences were considered significant at P< 0.05.
Results
Lung squamous metaplasia and lung concentration of RA in
ferrets after 6 weeks of treatment
There were no significant differences in ferret body weight
among the treatment groups at the beginning, during,
or after the 6-week experiment (data not shown). The
results of the pathological examination for the effect of
com-bined antioxidant supplementation on smoke-induced lung
squamous metaplasia in ferrets after 6 weeks of treatment
are presented in Table I. There was one animal in the control
group having lung squamous metaplasia, probably due to
spon-taneous occurrence. Although there were no significant
differ-ences in the incidence of lung squamous metaplasia among the
control, the SM alone and the SM
+ HBC + AT + AA groups,
the number of ferrets with squamous metaplasia in the SM
+
LBC
+ AT + AA group was significantly lower than that in the
SM group (Table I). Lung RA concentrations were
signific-antly lower in the SM group than in the control group, as we
reported before (33). Although LBC in the presence of AT and
AA did restore the lung RA concentration to that of the control
group, the lung RA concentration was significantly higher in
the SM
+ HBC + AT + AA group as compared with the control
group (Table I).
Concentration of antioxidants (BC, AT and AA) and retinoids
(RA, retinol and retinyl palmitate) in lung tissue of ferrets
after 6 months of treatment
The AOX supplemented groups showed significantly higher
lung BC after 6 months, as compared to non-supplemented
groups (Table II). Interestingly, in the presence of AT and
AA, the lung BC concentration in the SM
+ NNK + AOX
was maintained at the level equal to that of the AOX alone
group. AT levels in lung tissues of ferrets were significantly
higher in AOX group than in the control group (2.2-fold
difference) (Table II). The concentration of AT in lung tissues
was not affected by SM exposure plus NNK treatment in the
non-supplemented group, whereas AT concentration was
significantly higher in SM
+ NNK + AOX group, compared
with the AOX alone group. There were significantly lower
lung levels of both reduced AA (28% lower) and total AA
(38% lower) among smoke-exposed plus NNK-treated ferrets,
as compared with control animals with or without AOX
supplementation. However, the level of either reduced
or total AA lung concentrations was not affected by AOX
supplementation.
RA concentrations in lung tissue were significantly lower
(90%) in the SM
+ NNK group as compared with the control
group. However, the RA concentration in the lungs of ferrets
of the AOX group was higher (2 fold difference) than that of
the control group. The RA concentration was restored to
Table I. The effect of 6 weeks of smoke-exposure (SM) with or without
antioxidants (BC, AT and AA) supplementation on lung squamous metaplasia and lung concentration of RA in ferrets
Number of ferrets having lung squamous metaplasia/the total number in each group
Lung concentration of RA (pmol/g) Control 1/9 (11%)a,b 24.17 ± 3.89a SM 4/9 (44%)a 10.52 ± 0.83b SM+ LBC + AT + AA 0/9 (0%)b 14.55 ± 1.51b SM+ HBC + AT + AA 1/9 (11%)a,b 48.01 ± 13.34c
Number of ferrets with squamous metaplasia/the total number of ferrets in each group (%). SM, smoke-exposed; LBC, low dose (equivalent
daily intake of 6 mg BC in a 70 kg man) ofb-carotene supplemented;
HBC, high dose (equivalent daily intake of 30 mg BC/day in a 70 kg
man) ofb-carotene supplemented; AT, a-tocopherol dose (100 mg
AT/day) supplemented; AA, ascorbic acid (210 mg AA/day); RA, retinoic acid.
a,b,cFor a given column, data not sharing a common superscript letter are statistically significantly different at P< 0.05 (Fisher’s Exact test for the incidence of lung squamous metaplasia).
control levels in the SM
+ NNK groups by BC supplementation
in the presence of AT and AA (Table II). We observed
no significant differences in the concentrations of lung retinol
and/or retinyl palmitate among the four groups (Table II).
Phosphorylated JNK, phosphorylated ERK and PCNA protein
expression in the lung tissue of ferrets
We observed no difference in protein levels of total JNK
among four treatment groups (Figure 1A). As compared
with the control group, phospho-JNK protein levels in the
smoke-exposed plus NNK-treated group were higher (58%).
This up-regulation of JNK phosphorylation in SM
+ NNK
group was significantly inhibited by AOX treatment.
Further-more, ERK phosphorylation was significantly higher in SM
+
NNK treatment (2.2-fold difference), as compared with
the control or the AOX group (Figure 1B); whereas, AOX
supplementation prevented the SM+NNK-induced ERK
phos-phorylation. The levels of total ERK were not different among
four groups. Since MAPK phosphorylation mediates cell
proliferation, PCNA protein expression in lung tissue was
examined to evaluate a potentially higher cell proliferation
with SM
+ NNK treatment. Compared with the control
group or the AOX treated group, PCNA expression was
significantly higher (36%) in the SM
+ NNK group
(Figure 1C). This up-regulated PCNA expression in the
SM
+ NNK group was inhibited by AOX supplementation
(Figure 1C).
Levels of p53, phosphorylated p53 (serine-15) and Bax in the
lung tissue of ferrets
There was higher (50–63%) total p53 protein level in the
group exposed to SM
+ NNK (Figure 2A) compared with
the control group, the AOX group and the SM
+ NNK +
AOX group. No differences in total p53 protein levels were
observed among the control groups, the AOX group and the
SM
+ NNK + AOX group. Because p53 phosphorylation is
mediated by MAPKs such as JNK and ERK, we examined
p53 phosphorylation. As compared with the control and the
AOX groups, p53 protein phosphorylation at serine-15 was
higher in the groups exposed to SM
+ NNK (2-fold
differ-ence) (Figure 2B). This SM
+ NNK-induced phosphorylation
of p53 was blocked by AOX supplementation. No difference
in phopho-p53 protein levels were observed between the
AOX group, the SM
+ NNK + AOX group and the control
group. Further analysis of p53 phosphorylation using the
antibody against phosphorylated p53 showed significantly
higher
phospho-p53
expression
in
the
tumor
region
(Figure 3A and C), as compared with non-tumor regions
(Figure 3A and B). These data indicate that higher p53
phosphorylation in the tumor region may explain the higher
levels of p53 phosphorylation in SM
+ NNK group, as
com-pared with the control group. Since phosphorylation of
p53 stabilizes and activates p53, we examined Bax, a major
downstream gene of p53. Bax protein expression was
signi-ficantly higher (70%) in the SM
+ NNK group, compared
with the control group (Figure 2C). AOX supplementation
prevented this induction of Bax by SM
+ NNK treatment.
No difference in Bax protein expression levels were observed
between the SM
+ NNK + AOX group, the control group or
the AOX treated group.
Lung preneoplastic and neoplastic lesions
The treatment of ferrets with combined cigarette smoke and
NNK resulted in both the formation of preneoplastic lesions
and neoplastic lesions (Figure 4). The preneoplastic lesions
included both squamous dysplasia (Figure 4A) and atypical
adenomatous hyperplasia (Figure 4B), which are precancerous
lesions for squamous cell carcinoma and adenocarcinoma,
respectively. Neoplastic lesions, including squamous cell
carcinomas (Figure 4C) and adenocarcinomas (Figure 4D),
were also detected in the SM
+ NNK exposed groups.
These preneoplastic and neoplastic lesions were not detected
in the control group or the AOX group. Preneoplastic lesions
including squamous metaplasia, squamous dysplasia and
atypical adenomatous hyperplasia were observed in the lung
tissues of 10 of 12 (83%) SM
+ NNK ferrets, but only 7 of 14
(50%) ferrets treated with SM
+ NNK but supplemented with
AOX (Table III), which constituted a trend (33% decrease, P
¼
0.11) toward lower incidence of preneoplastic lesions in the
SM
+ NNK + AOX group, compared with the SM + NNK
group. Neoplastic lesions including squamous cell carcinoma,
adenocarcinoma
and
adenosquamous
carcinoma
were
observed in the lung tissues of 6 of 12 (50%) ferrets treated
with SM
+ NNK, but only 2 out of 14 (14%) ferrets treated with
SM
+ NNK + AOX (Table III), which showed a trend (36%
decrease, P
¼ 0.09) toward lower incidence of neoplastic
lesions in the AOX supplemented group, compared with the
SM
+ NNK group. However, it is important to note that neither
preneoplastic lesions nor neoplastic lesions were observed in
the lung tissue of ferrets in the control or AOX alone groups
after 6 months of intervention (Table III). In addition, there
were no significant differences in ferret body weight among the
treatment groups at the beginning, during or after the 6-month
experiment (data not shown).
Table II.Lung concentrations of BC, AT, AA and retinoids (RA, ROH, RP) in the four groups of ferrets after 6 months of treatment with smoke, NNK and
antioxidants (BC, AT, AA) in various combinations BC (pmol/g) AT (nmol/g) Reduced AA (mmol/g) Total AA (mmol/g) RA (pmol/g) ROH (nmol/g) RP (nmol/g) Control NDa 28.8 ± 5.7a 0.65 ± 0.08a 1.40 ± 0.26a 23.71 ± 3.54a 1.17 ± 0.23a 12.9 ± 2.7a SM+ NNK NDa 33.1 ± 6.9a 0.47 ± 0.07b 0.87 ± 0.09b 2.37 ± 0.23b 0.92 ± 0.20a 18.8 ± 3.5a AOX 8542 ± 1979b 64.5 ± 18.6b 0.73 ± 0.10a 1.20 ± 0.10a 42.27 ± 2.50c 1.15 ± 0.21a 9.9 ± 1.1a SM+ NNK + AOX 7834 ± 1546b 114.5 ± 24.0c 0.50 ± 0.09b 0.87 ± 0.14b 29.33 ± 2.24a 1.32 ± 0.31a 21.5 ± 5.7a
SM+ NNK, smoke-exposed plus NNK-treated; AOX, antioxidant supplemented (BC, AT, AA); SM + NNK + AOX, smoke-exposed plus NNK-treated
plus antioxidant supplemented; RA, retinoic acid; ROH, retinol; RP, retinyl palmitate.
a,b,cFor a given column, data not sharing a common superscript letter are statistically significantly different at P< 0.05. Values are expressed as means ±
Fig. 1.Expressions of total JNK, phospho-JNK, phospho-ERK, total ERK and PCNA protein levels were measured by western blot in lung tissue from four groups of ferrets after 6 months of treatment [Ctrl, control
(n¼ 9); SM + NNK, smoke-exposed plus NNK-treated (n ¼ 10); AOX,
antioxidant supplemented (BC, AT, AA) (n¼ 9); SM + NNK + AOX,
smoke-exposed plus NNK-treated plus antioxidants (BC, AT, AA) supplemented (n¼ 12)]. (A) Upper portions of each panel are
representative western blots in the same order as in the bar graph. The bar graph shows the intensity of the protein signal of phospho-JNK/total JNK, which was expressed by the relative values means ± SEM. Asterisk indicates that this value is significantly different from all others (P< 0.05). Fisher’s Least Significant Difference test was performed. The sizes were 46 kDa for JNK and phospho-JNK. (B) Upper portions of each panel are representative western blots in the same order as in the bar graph. The bar graph shows the intensity of the protein signal of phospho-ERK/total ERK, which was expressed by the relative values means ± SEM. Asterisk indicates that this value is significantly different from all others (P< 0.01). The sizes were 42 and 44 kDa for phospho-ERK and total ERK. (C) Upper portions of each panel are representative western blots in the same order as in the bar graph. The bar graph shows the intensity of the protein signal of PCNA was expressed by the relative values means ± SEM. Asterisk indicates that this value is significantly different from others (P< 0.05). The size was 34 kDa for PCNA.
Fig. 2.Expressions of total p53, phospho-p53 and Bax protein levels were measured by western blot in lung tissue from four groups of ferrets after 6 months of treatment [Ctrl, control (n¼ 9): SM + NNK,
smoke-exposed plus NNK-treated (n¼ 10); AOX, antioxidant supplemented
(BC, AT, AA) (n¼ 9); SM + NNK + AOX, smoke-exposed plus
NNK-treated plus antioxidants (BC, AT, AA) supplemented (n¼ 12)]. (A)
Upper portions of each panel are representative western blots in the same order as in the bar graph. The bar graph shows the intensity of the protein signal of total p53 was expressed by the relative values means ± SEM. Asterisk indicates that this value is significantly different from all others (P< 0.05). The size was 53 kDa for total p53. (B) Upper portions of each panel are representative western blots in the same order as in the bar graph. The bar graph shows the intensity of the protein signal of phospho-p53 at serine 15, which was expressed by the relative values means ± SEM. Asterisk indicates that this value is significantly different from all others (P< 0.01). (C) Upper portions of each panel are representative western blots in the same order as in the bar graph. The bar graph shows the intensity of the protein signal of Bax, which was expressed by the relative values means ± SEM. Asterisk indicates that this value is significantly different from others (P< 0.05). The size was 23 kDa for Bax.
Discussion
To our knowledge, this is the first report of an in vivo study
showing a beneficial effect of a combination of antioxidant
vitamins (BC, AT and AA) against lung damage induced by
tobacco smoke and NNK treatment in ferrets. The present
study shows that the ferret is a good model for studying
lung cancer chemoprevention with antioxidants and for
study-ing the molecular mechanism of carcinogenesis in the early
stages of smoke-related lung cancer.
Previously, we demonstrated the level of BC in the lung
tissue of smoke-exposed animals receiving low-dose BC
(0.48 mg/kg body wt/day) or high-dose BC (2.4 mg/kg
30 mg body wt/day) was significantly lowered with smoke
exposure (33). However, in the present study, the concentration
of BC was not lower with SM
+ NNK (Table II) when the
animal was fed with BC (0.85 mg/kg body wt/day) in the
presence of AT and AA, indicating that combined AT and
AA can prevent smoke-enhanced BC degradation. This is
in agreement with our previous demonstration that the
forma-tion of excentric cleavage metabolites of BC are higher in the
lungs of smoke-exposed ferrets than non-smoke-exposed
ferrets (6) and in vitro tissue incubation results showing that
this enhancement of excentric cleavage of BC can be blocked
by AT and AA (12).
We selected an AT dose of 22 mg/kg body wt, equivalent
to a daily intake of 100 mg AT in a 70 kg man based on a pilot
study revealing that the bioavailability of AT in the ferret
is
6% of that in a human. This dose has been shown in clinical
trials to improve immune responsiveness (40.2–536 mg AT
equivalents) (38) and to protect against LDL oxidation (17.4–
804 mg AT equivalents) without any toxic effect (39,40). For
the AA dosage, 3 mg/kg body wt/day in ferret (equivalent
to 210 mg AA in a 70 kg man) was chosen, since this dose
has been shown to afford smokers an equivalent protection
from AA hypovitaminosis as nonsmokers (41,42).
The lung AT concentration was significantly higher in the
AOX supplemented groups (Table II). Unexpectedly, however,
lung AT concentration was not lower in SM
+ NNK group
than in the control group. There is a lack of information about
the lung tissue concentration of AT in smokers and conflicting
information on the effects of smoke on plasma AT. Whereas
AT concentration was lowered by smoke in most in vitro
incubation studies (1,43,44), the plasma AT concentrations
of smokers either did not differ (44) or were lower than
those found in nonsmokers (45). However, we found that
AT concentration in lung tissue was higher with SM
+
NNK
+ AOX, compared with that of the control with AOX
supplementation. The mechanism for this is currently
unknown.
The concentration of lung AA was lower in the SM
+ NNK
group compared with the control groups in this study, in
concordance with previous human studies [(e.g. smokers
have significantly lower plasma levels of vitamin C compared
with nonsmokers) (46)]. This implies that the dose of smoke
exposure in this animal study was sufficient to produce a
smoking environment that mimics the human condition.
Inter-estingly, even with AA supplementation (3 mg/kg body wt,
which is equivalent to 210 mg/day in humans), lung levels of
AA in the smoke-exposed ferrets were not restored to control
levels (Table II). Therefore, it is possible that the function of
BC and AT in the lung tissue of smokers in the ATBC study
(2), and in a recent trial of head and neck cancer patients using
combined BC
+ AT to prevent second primary cancers (47),
was diminished by lack of sufficient AA to recycle both
antioxidants. The levels of reduced AA and total AA were
not different after AA supplementation in lung tissues of
ferrets, which is probably due to saturation of tissue AA,
since unlike humans, ferrets synthesize their own AA (48,49).
Cigarette smoke exposure is a strong risk factor for
lung cancer since it promotes genomic instability and the
development of neoplasia by modulating molecular pathways
involved in cell differentiation, cell proliferation and
apop-tosis. It has been reported that components of smoke or
smoke exposure itself increase MAPK, including JNK
(50–52) and ERK phosphorylation (52,53), in cell models.
In the present study, we demonstrated that phosphorylation
Fig. 3. Localization of phospho-p53 in the squamous carcinoma region in
the SM+ NNK group after 6 months of SM + NNK treatment. (A)
Non-tumor region and tumor region (· 100). (B) Non-tumor region (higher magnification, 400·) from (A); (C), Tumor-region (higher magnification, 400·) from (A).
of JNK and ERK was higher in the SM
+ NNK-treated ferrets,
as compared with the control group and this increased
phos-phorylation
was
inhibited
by
AOX
supplementation
(Figure 1A and B). It has been reported that activation and
phosphorylation of JNK at serine-63 and serine-73 residues
can result in activation of AP-1 (54,55), which may explain
increased cell proliferation (Figure 1C) and tumorigenesis
induced by smoke-exposure and NNK treatment. The levels
of total JNK and total ERK proteins were not different across
treatment groups, indicating that SM
+ NNK treatment and
AOX affect JNK and ERK activities by phosphorylation of
JNK and ERK, rather than by affecting protein amount.
Importantly, we have shown that lower levels of RA in
the lungs of the SM
+ NNK group were completely restored
to normal levels by AOX supplementation in our studies
(Tables I and II). Interestingly, we have previously shown
that high dose BC (2.4 mg/kg body wt/day) supplementation
decreased RA concentrations in the lungs of smoke-exposed
ferrets in vivo (6) and that the production of RA from BC
in the lung tissue of smoke exposed ferrets was substantially
increased by adding AT and AA to in vitro incubations (12).
This present in vivo study confirms our in vitro observations (8)
and indicates that combined AT and AA may enhance central
cleavage of BC, thereby increasing RA production from BC,
or inhibit excentric cleavage of BC, thereby decreasing of RA
catabolism. RA is the ligand for retinoid receptors (RARs and
RXRs) and appears to have the potential for chemopreventive
protection against cancer (56). It has been shown that RA
regulates the growth and differentiation of bronchial epithelial
cells in vitro and suppresses lung carcinogenesis in animal
studies (57). Recently, we demonstrate that 9-cis RA
supple-mentation in the A/J mouse model provides protection against
lung carcinogenesis and this effect may be mediated in
part by 9-cis RA induction of RAR-b (58). Although the
effi-cacy and complex biological functions of retinoids in human
lung cancer prevention need more investigation (59), our study
indicates that the restoration of normal levels of RA by AOX
supplementation in smoke-exposed lung tissue may contribute
to the protective effects of AOX against lung carcinogenesis. It
has been reported that RA can inhibit phosphorylation
of MAPKs, such as JNK and ERK, by upregulation of MAP
kinase-phosphatase-1 which dephosphorylates MAPK (60–
62), thereby preventing abnormal cell proliferation. This
sug-gests that the mechanism behind the inhibitory effect of AOX
supplementation against the phosphorylation of JNK and ERK
could be due to increased lung RA levels in SM
+ NNK + AOX
group. This is supported by our recent observation that the
induction of MAP kinase-phosphatase-1 was associated with
the concentration of RA in the lungs of ferrets (25). This was
also supported by the fact that PCNA was significantly higher
in the lungs of SM
+ NNK when the levels of RA were lower as
Fig. 4.Representative preneoplastic and neoplastic lesions seen in the lungs of ferrets after 6 months of SM+ NNK treatment. (A) Squamous dysplasia, HE (400·). (B) Atypical adenomatous hyperplasia, HE (100·). (C) Squamous cell carcinoma, HE (200·). (D) Adenocarcinoma, HE (200·).
Table III. The incidence of preneoplastic lesion and neoplastic lesions in the four groups of ferrets after 6 months of treatment
Preneoplastic lesion1,3 Neoplastic lesion2,3
Control 0/9 (0%)a 0/9 (0%)a AOX 0/9 (0%)a 0/9 (0%)a SM+ NNK 10/12 (83%)b P¼ 0.11 6/12 (50%) b P¼ 0.09 SM+ NNK + AOX 7/14 (50%)b 2/14 (14%)a,b
SM+ NNK, smoke-exposed plus NNK-treated; AOX, antioxidant
supplemented (b-carotene, a-tocopherol and ascorbic acid);
SM+ NNK + AOX, smoke-exposed plus NNK-treated plus
antioxidant supplemented.
1Preneoplastic lesion, number of ferrets with preneoplastic lesions including squamous metaplasia, squamous dysplasia and atypical adenomatous hyperplasia/the total number of ferrets in each group (%). 2
Neoplastic lesion, number of ferrets with neoplastic lesions including squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma/the total number of ferrets in each group (%). 3
Data not sharing a common superscript letters for a given value indicate that the incidence of lung squamous metaplasia statistically significantly different from each other (p< 0.05, Fisher’s Exact test).
compared with the control group or the AOX supplemented
group (Figure 1C). Furthermore, this increase in PCNA
labeling was prevented when the lower levels of lung RA in
the SM
+ NNK group were restored to normal in the SM +
NNK
+ AOX group. In addition, our study suggests that the
inhibition of JNK activation by the combined antioxidants may
help to ‘rescue’ the functions of RAR because it has been
recently reported that activation of JNK contributes to RAR
dysfunction by phosphorylating RAR-alpha and inducing
degradation through the ubiquitin-proteasomal pathway (63).
This hypothesis warrants further investigation.
Phosphorylation of JNK and ERK mediate phosphorylation
of p53, an important tumor suppressor that plays a critical
role in the cell-injury response to various stressors (19,20).
Overexpression of p53 protein is associated with cigarette
consumption (64). There is a dose–response relationship
between the quantity of tobacco consumed and the frequency
of p53 gene mutation in lung cancer patients (65). p53
phosphorylation at serine-15 facilitates both the accumulation
and functional activation of p53 (66,67). Solhaug et al. (52)
have shown that B[a]P-metabolites, B[a]P-7,8-DHD and
BPDE-1 induced accumulation and phosphorylation of p53
at serine-15 in Hepa1c1c7 cells. In the present study, we
have shown combined AOX (BC, AT and AA) inhibited the
phosphorylation of p53 induced by SM
+ NNK treatment
(Figure 2B). These findings are consistent with the lower
expression of phospho-JNK and phospho-ERK in the SM
+
NNK
+ AOX groups as compared with the SM + NNK group.
In addition, total p53 protein level was also increased in
parallel with phospho-p53 in SM
+ NNK-treated groups versus
all other groups, suggesting that the accumulation and
func-tional activation of p53 by its phosphorylation are mediated by
JNK and ERK.
p53 phosphorylation at serine-15 was upregulated in the
SM
+ NNK groups compared with the control group
(Figure 2B). Post-translational modifications of p53 by
phos-phorylation have been implicated in its stabilization and
transcriptional activation (68). In response to stress, p53
undergoes rapid phosphorylation on several residues,
includ-ing serine-6, 9, 20, 37, 46, 81, 389 and 392. At later time points
following DNA damage, p53 is phosphorylated on residues 15
and 372 (69). Although we showed higher phospho-p53
expression in the SM
+ NNK group than in the control
group, there are no reports of higher p53 phosphorylation
in the tumor regions compared with the non-tumor regions.
Therefore, localization of p53 phosphorylation (serine-15)
was studied using immunohistochemistry in tumor regions
(Figure 3) in order to examine whether the increased
phos-phorylation of p53 in tumor regions may contribute to the
higher levels of phospho-p53 seen in the SM
+ NNK group
(Figure 2B). This change in phosphorylation may regulate
downstream events such as apoptosis by activation of Bax
in the tumor progress. Our findings point to a general shift
in the regulation of p53 phosphorylation, which takes place
during tumor development and/or progression.
As one of the downstream transcriptional targets of p53,
Bax is a Bcl-2 related protein promoting apoptosis. Bax has
been shown to contain p53-binding sites in its promoter and is
upregulated in response to DNA damage (70). The level of Bax
was higher in SM
+ NNK group compared with that of the
control group. Furthermore, AOX supplementation (SM
+
NNK
+
AOX)
prevented
the
upregulation
of
Bax
induced by SM
+ NNK (Figure 2C). This result indicates
that SM
+ NNK treatment may induce apoptosis as well as
proliferation. Since normal cells must maintain a homeostatic
balance between cell proliferation and apoptosis, the loss of
this balance may contribute to lung carcinogenesis. This would
particularly be the case if abnormal cell proliferation increased
much more than apoptosis, even if both were increased by
smoke exposure and NNK treatment.
In this study, we showed that BC (equivalent to 12 mg/day in
humans) in the presence of AT and AA prevented both
preneoplastic lesions and neoplasia induced by 6 months of
tobacco smoke exposure and NNK treatment (Table III).
Although this effect on lung lesions is not statistically
signi-ficant due to the small sample size, our biochemical and
molecular mechanistic analyses as well as our short-term
study data (Table I) fully support the beneficial effects of
AOX against lung carcinogenesis. Interestingly, in contrast
to our previous observation that lung squamous metaplastic
lesions were increased in ferrets exposed to high dose BC
(equivalent to 30 mg/day in humans) (6), the same dose of
BC supplementation in the presence of AT and AA in
smoke-exposed ferrets reduced the number of ferrets having lung
squamous metaplasia, as compared with the unsupplemented
smoke exposed group (1/9 versus 4/9, Table I). In addition, we
showed that a low dose of BC (equivalent to 6 mg/day in
humans) in the presence of AT and AA had a significant
pro-tective effect on lung squamous metaplasia induced by tobacco
smoke (Table I). However, we only observed a trend for the
protection of AOX supplementation against lung lesions in the
ferrets exposed to both tobacco smoke and NNK (Table III). A
possible explanation for the difference between these two
studies is that the ferrets were only exposed to smoke in the
short-term study and only squamous metaplasia was detected,
whereas the combined smoke-exposure and NNK injection
were used in the long-term study and preneoplastic lesions
including squamous metaplasia, squamous dysplasia and
atyp-ical adenomatous hyperplasia were detected. Therefore, the
NNK injection in addition to smoke-exposure in the ferrets
may have overwhelmed the ability of antioxidants to provide
strong protection. Another possible explanation is that the
AOX supplementation prevented against the cancer promoting
effects of smoke-exposure, such as smoke-induced
inflamma-tion and free radical damage, while not offering protecinflamma-tion
against NNK chemically induced lung carcinogenesis. The
investigation as to whether antioxidants can protect against
lung cancer induced by NNK alone is currently underway
in this laboratory.
In summary, this in vivo study showed that vitamin E and
vitamin C acting together play an important role in terms
of inhibiting oxidation of BC and facilitating conversion of
BC into RA in smoke-exposed lung tissue. AOX
supplementa-tion may exert its protective effect against lung carcinogenesis
by maintaining normal concentrations of RA and inhibiting
phosphorylation of JNK, ERK and p53. These studies and
the known biochemical interactions of BC, vitamin E and
vitamin C suggest that this combination of nutrients, rather
than individual agents, could be an effective chemopreventive
strategy against lung cancer in smokers.
Acknowledgements
Y.K. was supported by NIH training grant T32 DK62032-11. The authors thank both Cognis, Corp. and DSM Corp. for funding our pilot study and Heather Mernitz for help in the preparation of the manuscript. Supported by the
NIH grant R01CA49195 and U.S. Department of Agriculture, under agreement NO. 1950-51000-064. Any opinions, findings, conclusion or recommendations expressed in this publication are those of the author(s) and do not necessarily reflects the views of the U.S. Department of Agriculture.
Conflict of Interest Statement: None declared.
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Received August 24, 2005; revised December 18, 2005; accepted January 3, 2006
