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Nutrition and Cancer Biology Laboratory, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA
2 To whom correspondence should be addressed. Email: chun.liu{at}tufts.edu.
ABSTRACT
Cigarette smoking increases the risk for gastric cancer. Higher intakes or blood levels of lycopene are associated with a decreased risk of gastric cancer. However, the biological mechanisms by which lycopene may protect against gastric carcinogenesis are poorly understood. We evaluated the effects of lycopene supplementation on smoke-induced changes in protein levels of p53, p53 target genes (p21Waf1/Cip1 and Bax-1), cell proliferation, and apoptosis in the gastric mucosa of ferrets. Ferrets were assigned to cigarette smoke exposure or to no exposure and to no, low-dose, or high-dose lycopene supplementation (2 x 3 factorial design) for 9 wk. Lycopene concentrations were significantly elevated in a dose-dependent manner in the gastric mucosa of ferrets supplemented with lycopene alone, but were markedly reduced in ferrets supplemented with lycopene and exposed to smoke. Although ferrets were given lycopene containing 95% all-trans isomers, cis isomers were the predominant forms in the gastric mucosa. Total p53 and phosphorylated p53 levels were greater in ferrets exposed to smoke alone than in all other groups. Levels were 
300 and 500% of the controls, respectively. However, smoke-elevated total p53 and phosphorylated p53 were markedly attenuated by both doses of lycopene. p21Waf1/Cip1, Bax-1, and cleaved caspase 3 were substantially decreased, whereas cyclin D1 and proliferating cellular nuclear antigen (PCNA) were increased in ferrets exposed to smoke alone. Lycopene prevented smoke-induced changes in p21Waf1/Cip1, Bax-1, cleaved caspase 3, cyclin D1, and PCNA in a dose-dependent fashion. These data indicate that lycopene may prevent smoke exposure–induced changes in p53, p53 phosphorylation, p53 target genes, cell proliferation, and apoptosis in the gastric mucosa of ferrets.
KEY WORDS: • lycopene • smoke • cell proliferation • apoptosis • ferrets
Gastric cancer is the second leading cause of cancer deaths worldwide (1). Most studies have reported a positive association between cigarette smoking and the risk of gastric cancer (1). Metabolically activated carcinogens contained in tobacco smoke can directly affect the p53 tumor suppressor gene, which plays a pivotal role in the balance of cell proliferation and apoptosis, in the cellular responses to various stresses (2), and in suppressing gastric carcinogenesis (3). p53 is overexpressed in gastric cancer (4,5). Reactive oxygen intermediates generated from cigarette smoke interact with DNA and produce oxidative DNA damage with potentially mutagenic consequences (6). Upon DNA damage, p53 is activated, leading to the induction of p53 target genes, p21waf1/cip1 and Bax-1 (7,8). p21waf1/cip1, a cyclin-dependent kinase (CDK)3 inhibitor, is a key component in the cell cycle arrest in G1. Bax is a pro-apoptotic member of the Bcl1–2 family. p21waf1/cip1 and Bax-1 function as mediators to promote p53-dependent apoptosis (7–9). p53 phosphorylation, especially at serine 15, is an early cellular response to various genotoxic carcinogens and stresses that produce reactive oxygen species, and facilitates both accumulation and functional activation of p53 (10,11). Continued exposure to genotoxic carcinogens and reactive oxygen species can damage p53 and lead to its loss of function and mutations, resulting in the inactivation of the p53 stress-response pathway (7). Such changes would provide precancerous cells with a selective advantage for unregulated growth and the acquisition of genetic instability (7).
Higher consumption of tomato and tomato-based products (major sources of lycopene), and higher intakes or plasma levels of lycopene are associated with a reduced risk of gastric cancer in epidemiologic studies in various populations (12–14), suggesting a protective effect of lycopene against the development of gastric cancer. However, the potential biological mechanisms by which lycopene might inhibit gastric carcinogenesis are poorly understood. Previous experimental studies from the same research group reported that administration of lycopene increases antioxidant activities and apoptosis, and inhibits the development of carcinogen-induced gastric cancer and lipid peroxidation in rats (15–17). Due to the lack of assessment of lycopene in blood and gastric tissue in these studies, it is difficult to judge whether there were significant changes in lycopene status across the treatment groups that would result in those biologic changes described above. To date, no animal or human studies have evaluated the effects of lycopene supplementation on cigarette smoke exposure–related gastric carcinogenesis. Furthermore, there are no available data concerning the effect of lycopene on changes in p53, p21waf1/cip1, Bax-1, apoptosis, and cell proliferation in gastric tissues associated with cigarette smoke exposure.
The ferret is an excellent model with which to study lycopene supplementation and cigarette smoke exposure (18). In addition, ferrets are naturally infected with Helicobacter mustelae, which has many biochemical and molecular characteristics and gastric pathologic changes similar to those of Helicobacter pylori in humans (19–21). Helicobacter pylori is an established risk factor for the development of gastric cancer (1).
In the present study, we evaluated the effects of lycopene supplementation on protein levels of p53, p53 target genes (p21waf1/cip1, a CDK inhibitor, and Bax-1), cell proliferation [cyclin D1 and proliferating cellular nuclear antigen (PCNA)], and apoptosis (cleaved caspase 3) in the gastric mucosa of ferrets with or without cigarette smoke exposure.
MATERIALS AND METHODS
Animals, diet and study groups. Male adult ferrets (1.0–1.2 kg) from Marshall Farms consumed a semipurified ferret diet (Research Diets) that contained no lycopene, as determined by HPLC analysis in our laboratory, and water ad libitum. Before the experiment started, all ferrets were quarantined for at least 2 wk to evaluate their health. Male ferrets (n = 36) were randomly assigned to 6 groups (n = 6/group) for 9 wk as follows: 1) control; 2) smoke exposed (SM); 3) low-dose lycopene [LLyco; 1.1 mg/(kg·d)]; 4) high-dose lycopene [HLyco; 4.3 mg/(kg·d)]; 5) SM + LLyco; 6) SM + HLyco. During the 9-wk experimental period, ferret body weights were recorded weekly. All ferrets were terminally exsanguinated under deep isoflurane anesthesia after the 9-wk experimental period. Tissues and plasma were collected and stored at –80°C until analyzed. All experimental procedures were approved by the Animal Care and Use Committee at the Human Nutrition Research Center on Aging and conducted under the supervision of the Animal Care and Use Committee.
Cigarette smoke exposure.
The cigarette smoke-exposure procedure used in the present study was described elsewhere (18). Briefly, the ferrets were put in a chamber connected with a smoking device. Cigarette smoke was drawn out of the cigarettes (Standard Research Cigarettes, Type 1R4F, Tobacco and Health Research Institute, University of Kentucky) with a vacuum and then exhausted into the chamber. During the first 2 wk of study, the number of cigarettes was increased progressively to a rate of 10 cigarettes/30 min twice in the morning and twice in the afternoon and then maintained for the rest of the 9-wk experimental period. In our previous study, we demonstrated that the concentration of urinary cotinine equivalents (0.1 mmol/L urine) of this amount of smoke-exposure to ferrets is similar to that in humans smoking 2 packages of cigarettes/d (18).
Lycopene supplementation.
Because the total absorption of ß-carotene by the ferret is 80% less than that by humans (22,23) and lycopene is similar in chemical structure to ß-carotene, the calculation for lycopene dosage was based upon the assumption that ferrets absorb lycopene 80% less efficiently than humans. Lycopene 10% dry power (Lycovit® 10%, BASF) was dissolved in 0.5 mL corn oil and given orally to ferrets (not gavage) at either a low or high dose every morning for 9 wk. This lycopene product contained 95% all-trans isomers in the HPLC analysis. The ferrets that were not supplemented with lycopene were given corn oil only [(0.5 mL/(kg·d)]. Ferrets in the LLyco group were supplemented with 1.1 mg/(kg·d), which corresponds to 15 mg of lycopene/d intake in a 70-kg person. This dose of lycopene is higher than the average intake of lycopene (9.4 ± 0.3 mg/d) in men and women in the United States (24). Ferrets in the HLyco group were supplemented with 4.3 mg/(kg·d), which corresponds to 60 mg of lycopene/d intake in a 70-kg person and is attainable in a diet enriched with tomato products or supplements. The dose of lycopene administrated in a human Phase II randomized clinical trial of lycopene for the prevention of prostate cancer progression was 15 mg 2 times/d (25,26). This dose of lycopene was within the range of lycopene doses used in the present study.
Tissue extraction and HPLC analyses. The stomach was washed with ice-cold isotonic saline (8.5 g/L NaCl). While on ice, the mucosa was gently scraped off with a razor blade and the wet weight was recorded. The gastric mucosa (0.1 g wet weight) was homogenized with 5 mL of CHCl3:CH3OH (2:1, v:v) and 50 µL of internal standard (echinenone). After the addition of 500 µL of normal saline, the mixture was mixed on a vortex and centrifuged for 10 min at 800 x g at 4°C. After the lower layer had been collected, hexane (5 mL) was added; the mixture was again mixed on a vortex and centrifuged for another 10 min at 800 x g at 4°C. The chloroform and hexane layers were combined and evaporated under N2, and a 50-µL aliquot of the extract reconstituted with ethanol was injected onto the HPLC. The HPLC system, mobile phase, and gradient procedure were described elsewhere (18). In this HPLC system, 13-cis-lycopene, 9-cis-lycopene, all-trans lycopene, and 5-cis-lycopene eluted at 29.7, 33.4, 36.1, and 36.5 min, respectively. Lycopene isomers were quantified relative to the internal standard by determining peak areas calibrated against known amounts of standards.
Western blot analysis. Western blot analysis for total p53, phosphorylated-p53 at serine 15, cyclin D1, and PCNA was performed using nuclear protein extracts from the gastric mucosa of ferrets, which were prepared as previously described (27). Western blot for Bax-1, cleaved caspase 3, and p21Cip1/Waf1 was conducted using the whole-tissue lysates of the gastric mucosa from 6 ferrets in each group, which were prepared as described previously (18). Antibodies against p53 were purchased from Calbiochem. Antibodies against phosphorylated-p53 at serine 15, Bax-1, cleaved caspase 3, and p21Cip1/Waf1 came from Cell Signaling. Antibodies against cyclin D1and PCNA were from Santa Cruz Biotechnology. The intensity of the protein signal was quantified by Densitometry (GS-710 calibrated imaging densitometer, Bio-Rad) and expressed as a relative value. Relative values were defined as the intensity of the signal of each sample of the 5 treatment groups divided by the intensity of the signal of the control sample in each run.
Statistical analysis. Results were expressed as means ± SD; data were analyzed by 2-way ANOVA followed by Tukey's honestly significant difference test. Differences were considered significant at P < 0.05.
RESULTS
Lycopene concentrations in the gastric mucosa of ferrets after 9-wk treatment. Lycopene supplementation at either the low or high dose for 9 wk significantly increased the concentrations of lycopene isomers in the gastric mucosa of the ferrets; the increases were dose dependent (Table 1). Because the semipurified ferret diet used in this study contained no lycopene, we did not detect lycopene in the gastric mucosa in the control and SM only groups of ferrets. Lycopene concentrations in the gastric mucosa in the HLyco groups that were or were not exposed to smoke were significantly higher than those in both of the LLyco groups. Smoke exposure decreased gastric mucosa lycopene concentrations in both the LLyco (–70%) and HLyco (–34%) groups (Table 1). Although ferrets were given lycopene containing 95% all-trans isomers, cis isomers of lycopene were the predominant forms in the gastric mucosa of ferrets: 13-cis-, 9-cis-, all-trans- and 5-cis-lycopene accounted for 6–13, 2–10, 8–9, and 69–82% of total lycopene, respectively.
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300% of controls; Fig. 1A) and phosphorylated p53 (500% of controls; Fig. 1B) protein levels. Total p53 and phosphorylated p53 protein levels did not differ from the control group in ferrets supplemented with lycopene alone or when combined with smoke exposure.
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350% (Fig. 3B) and PCNA 400% (Fig. 3C) in the SM alone group. None of the protein levels differed among the other 5 groups.
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DISCUSSION
Cigarette smoke exposure reduces circulating concentrations of lycopene in humans (28) and ferrets (18). In the present study, we also demonstrated that cigarette smoke exposure substantially lowered lycopene concentrations in the gastric mucosa of ferrets (Table 1). In addition, cis isomers of lycopene predominated in the gastric mucosa, although ferrets were given lycopene containing 95% all-trans isomers. This finding is consistent with previous studies, suggesting that isomerization of lycopene occurs in the gastrointestinal tract, particularly in the stomach (29), and cis isomers of lycopene are more bioavailable than all-trans lycopene (30).
Smoke carcinogens have an effect on the p53 tumor suppressor gene, a critical component in regulating the balance between cell proliferation and apoptosis and the responses to cellular stresses (2) as well as in suppressing gastric carcinogenesis (3). p53 overexpression was demonstrated in human gastric cancer (4,5). The elevated level of p53 was also shown in the gastric mucosa of rats exposed to cigarette smoke (31). Because malfunction of p53 may result in increased cell proliferation and decreased apoptosis and eventually tumor development and progression, elevated levels of p53 accumulation and phosphorylation may serve as markers for monitoring genotoxic effects and tumorigenesis.
However, no animal or human studies have examined the effects of lycopene on p53 and p53 phosphorylation in the gastric mucosa. In the present study, smoke exposure markedly increased total p53 and phosphorylated p53 protein levels in the gastric mucosa of ferrets. More importantly, supplementation with either a low or a high dose of lycopene substantially reduced the excess increase in total p53 and phosphorylated p53 protein levels due to smoke exposure. These data provide evidence that lycopene supplementation prevents the changes in p53 and p53 phosphorylation induced by cigarette smoke exposure in the gastric mucosa of ferrets.
p53 controls cell cycle checkpoints in response to a variety of cellular stresses (9). The p21waf1/cip1 CDK inhibitor is directly regulated by p53. Once activated, p21waf1/cip1 protein binds to both the cyclin-CDK complex and PCNA, subsequently suppressing their activities and cell proliferation (9). Previous studies showed that in contrast to p53 expression, the expression of p21waf1/cip1 is lower in gastric tumors than in the normal gastric mucosa (5). In addition, negative expression of p21waf1/cip1 and overexpression of p53 are associated with aggressive behavior of gastric tumors and poor survival among patients with gastric cancer (5,32,33). There are few studies evaluating the relation of smoke exposure to p21waf1/cip1 expression. In particular, there are no reports that evaluated smoke exposure in relation to p21waf1/cip1 expression using gastric cell lines or gastric tissues. Components of smoke exposure were shown to stimulate gastric cell proliferation and promote gastric tumor growth in mice (34) and in human gastric cancer cells (35). Although lycopene supplementation inhibits proliferation of several types of cancer cells such as prostate, breast, endometrium, and lung (36,37), there are no available data for gastric cancer. In the present study, smoke exposure substantially decreased levels of p21Waf1/Cip1 accompanied by increased levels of cyclin D1 and PCNA, cell proliferation indices, in the gastric mucosa of ferrets. Supplementation with either low or high doses of lycopene prevented the changes in p21Waf1/Cip1, cyclin D1, and PCNA caused by smoke exposure in a dose-dependent fashion. Findings from the present study provide the first evidence that the inhibitory effect of lycopene supplementation on smoke-induced cell proliferation is mediated in part by p21Waf1/Cip1, a p53 target gene.
Bax-1, a proapoptotic member of the Bcl-2 family of proteins that plays a key role in programmed cell death, is a downstream mediator of p53-dependent apoptosis (9). p53 upregulates the expression of Bax-1 gene through a p53 DNA-binding element within the promoter region of the Bax gene (9). The expression of Bax-1 was identified in normal tissues (38), adenomas (39), and tumor tissues (39,40) of the stomach. Studies showed that the expression level of Bax-1 (41,42) and caspase-3 (43,44), an apoptosis index, is higher in normal gastric tissues than in gastric carcinomas. In addition, most caspase-3 in gastric cancer cells is inactivated, suggesting that cancer cells may evade programmed cell death by inhibiting caspase 3 activation (43,45). An early study showed that short-term passive cigarette smoke exposure for up to 9 d upregulates apoptosis in the gastric mucosa of rats (31) and in human gastric epithelial cell lines (46). However, there is no report on how long-term cigarette smoke exposure influences apoptosis in the gastric mucosa. In the present investigation, exposing ferrets to smoke for 9 wk significantly decreased both cleaved caspase 3 and Bax-1 in the gastric mucosa, indicating that long-term smoke exposure downregulates p53-dependent apoptosis in the gastric mucosa.
Lycopene or tomato increases apoptosis in several cancer cell lines including the prostate (47,48), leukemia (49), and colon (50). Limited data exist concerning the effect of lycopene on apoptosis in gastric cancer. Additionally, no data exist concerning the effect of lycopene on smoke-induced changes in apoptosis in the stomach. In the only previous study of gastric tumors using a chemical carcinogen [N-methyl-N'-nitro-N-nitroso-guanidine (MNNG)] in rats, lycopene supplementation inhibited MNNG-reduced expression of Bax and caspase 3 in the gastric tissues (17); it is unclear, however, whether these changes in apoptosis are related to p53 because the expression of p53 and other p53-associated molecules was not examined. In the present study, lycopene dose dependently prevented smoke-reduced protein levels of Bax-1 and cleaved caspase 3 in the gastric mucosa of ferrets, suggesting that lycopene can overcome the adverse changes in apoptosis caused by cigarette smoke exposure. We demonstrated that smoke-reduced apoptosis, reflected by decreased levels of Bax-1 and cleaved caspase 3, was associated with smoke-elevated cell proliferation, indicated by increased levels of cyclin D1 and PCNA and decreased p21Waf1/Cip1, as well as with levels of total p53 and phosphorylated p53 increased by smoke exposure. Furthermore, we showed that lycopene dose dependently reversed smoke-induced changes in p21Waf1/Cip1, Bax-1, cleaved caspase 3, cyclin D1, PCNA, and p53. These data suggest that lycopene protects against the development of gastric cancer associated with cigarette smoke exposure at least in part through its effect on p53-dependent apoptosis and cell proliferation.
Helicobacter pylori infection was shown to upregulate the expression of p53 in human gastric mucosa and cell lines (51,52). Previously, we observed that p53 protein was detected in the gastric mucosa of ferrets infected with Helicobacter mustelae (6 of 6 ferrets) but not in that of the specific pathogen–free ferrets (0 of 5 ferrets) (Wang XD and Fox JG, unpublished data). In the present study, we also detected total p53 and phosphorylated p53 in control ferrets and observed that total p53 and phosphorylated p53 levels were slightly lower in the groups given lycopene supplementation alone compared with the controls, suggesting that lycopene might also alleviate Helicobacter mustelae–related p53 alterations.
In conclusion, this study demonstrates that lycopene supplementation at either low or high dose may prevent smoke exposure–related changes in p53, p53 phosphorylation, p53 target genes (p21Waf1/Cip1 and Bax-1), cell proliferation, and apoptosis in the gastric mucosa of ferrets. Our study provides evidence that lycopene may protect against the development of gastric cancer by modulating p53-dependent cell cycle control and apoptosis.
FOOTNOTES
1 Supported by the National Institutes of Health Grant ES11537 and the 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 reflect the views of the U.S. Department of Agriculture. 
3 Abbreviations used: CDK, cyclin-dependent kinase; HLyco, high-dose lycopene supplementation; LLyco, low-dose lycopene supplementation; MNNG, N-methyl-N'-nitro-N-nitroso-guanidine; PCNA, proliferating cellular nuclear antigen; SM, smoke-exposed.
Manuscript received 15 September 2005. Initial review completed 3 October 2005. Revision accepted 12 October 2005.
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