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Carcinogenesis Advance Access originally published online on January 9, 2006
Carcinogenesis 2006 27(7):1410-1419; doi:10.1093/carcin/bgi340
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Combined antioxidant (ß-carotene, {alpha}-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 1 and Xiang-Dong Wang 1, *

1 Nutrition and Cancer Biology Laboratory, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, USA

* 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{at}tufts.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Interactions among ß-carotene (BC), {alpha}-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, particularly against smoke-related lung cancer. In this investigation, 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 concentrations 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.

Abbreviations: AA, ascorbic acid; AT, {alpha}-tocopherol; BC, ß-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


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
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 addictive 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 ß-carotene (BC) levels have a lower risk of cancer, particularly lung cancer (1). In contrast, two intervention trials, the Alpha-Tocopherol, Beta-carotene Cancer Prevention Trial (ATBC) in Finland (2) and the ß-Carotene and Retinol Efficacy Trial in the United States (3) demonstrated 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 environment 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, {alpha}-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 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 phosphorylation 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 G1 phase 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 supplementation promotes the smoke-induced phosphorylation 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
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
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. According 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 at –80°C 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), n = 9; (ii) smoke exposed (SM) without any supplementation, n = 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, n = 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, n = 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 = 9; (ii) smoke-exposed plus NNK-treated (SM + NNK), n = 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)], n = 9; and (iv) SM + NNK treated plus combined AOX supplemented, n = 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, N'-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-{alpha}-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 is ~5 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 100 µl 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 4°C. Four milliliter of hexane was added after the lower layer had been collected. The chloroform and hexane layers were evaporated under N2 and a 50 µl 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 x 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 embedded in paraffin. The lung sections, 5 µm 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 investigators 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 immunohistochemistry, 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 mM ß-glycerophosphate and a mixture of protease inhibitors) with agitation at 4°C 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 centrifugation 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
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
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 combined 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 spontaneous occurrence. Although there were no significant differences 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 significantly 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).


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  		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

 
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.


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  		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

 
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 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. Furthermore, 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 phosphorylation. 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).


Figure 1
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  		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 (n = 9); SM + NNK, smoke-exposed plus NNK-treated (n = 10); AOX, antioxidant supplemented (BC, AT, AA) (n = 9); SM + NNK + AOX, smoke-exposed plus NNK-treated plus antioxidants (BC, AT, AA) supplemented (n = 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.

 
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 difference) (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 compared 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 significantly 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.


Figure 2
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  		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 (n = 9): SM + NNK, smoke-exposed plus NNK-treated (n = 10); AOX, antioxidant supplemented (BC, AT, AA) (n = 9); SM + NNK + AOX, smoke-exposed plus NNK-treated plus antioxidants (BC, AT, AA) supplemented (n = 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.

 

Figure 3
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  		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 (x 100). (B) Non-tumor region (higher magnification, 400x) from (A); (C), Tumor-region (higher magnification, 400x) from (A).

 
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 = 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 = 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).


Figure 4
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  		Fig. 4. Representative preneoplastic and neoplastic lesions seen in the lungs of ferrets after 6 months of SM + NNK treatment. (A) Squamous dysplasia, HE (400x). (B) Atypical adenomatous hyperplasia, HE (100x). (C) Squamous cell carcinoma, HE (200x). (D) Adenocarcinoma, HE (200x).

 

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  		Table III. The incidence of preneoplastic lesion and neoplastic lesions in the four groups of ferrets after 6 months of treatment

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
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 studying 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 formation 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. Interestingly, 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 apoptosis. 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 of JNK and ERK was higher in the SM + NNK-treated ferrets, as compared with the control group and this increased phosphorylation 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 supplementation 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-ß (58). Although the efficacy 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 suggests 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 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 functional 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 phosphorylation have been implicated in its stabilization and transcriptional activation (68). In response to stress, p53 undergoes rapid phosphorylation on several residues, including 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 phosphorylation 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 significant 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 protective 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 atypical 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 inflammation and free radical damage, while not offering protection 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 supplementation 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.


   Notes
 
2 Present address: Division of Nutrition, Department of Pediatrics, Faculty of Medicine at Ramathibodi Hospital, Mahidol University, Rama 6 Road, Bangkok, Thailand 10400 Back


   Acknowledgments
 
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.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 24, 2005; revised December 18, 2005; accepted January 3, 2006.