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Nutrition and Cancer Biology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging and * Department of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111
2To whom correspondence should be addressed. E-mail: xiang-dong.wang{at}tufts.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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5–9 servings of fruits and vegetables, has no apparent detrimental effects, but rather appears to have a protective effect against cigarette smoke–induced lung lesions in ferrets. In the present study, we investigated the effects of BC, ß-apo-14'-carotenoic acid (14'CA), or benzo[a]pyrene (BP; a primary lung carcinogen from cigarette smoke) treatments, either alone or in combination, on cell growth and expression of the retinoic acid receptor (RAR) of normal human bronchial epithelial (NHBE) cells. We found that both BC and 14'CA inhibited the growth of NHBE cells (P < 0.05) with or without BP. The level of RARß, a tumor suppressor, but not RAR
or RAR
, was reduced by 50% in the NHBE cells treated with BP. However, treatment with either BC or 14'CA significantly induced the expression of RARß in the NHBE cells, and prevented the reduction of RARß by BP. Furthermore, 14'CA transactivated the RARß promoter primarily via its conversion to retinoic acid (RA). In the presence of 3-mercaptopropionic acid, an inhibitor of fatty acid oxidation, both RA formation and transactivation activity from 14'CA were decreased. These observations indicate that the growth inhibitory effects of BC and ß-apo-carotenoic acid are through their conversion to RA and upregulation of RARß.
KEY WORDS: • ß-carotene • ß-apo-carotenoids • retinoic acid receptor • lung cancer
Beneficial effects of fruits and vegetables rich in ß-carotene (BC)3 on risk reduction of lung cancer have been found in a number of observational studies (1). In contrast, clinical intervention trials conducted to determine the effect of BC supplementation on the incidence of lung cancer found either no protective effect (2) or a negative effect (3,4). However, supporting evidence for a protective role of fruits and vegetables rich in BC in cancer prevention continues to be reported in observational studies (5,6), intervention studies (7,8), animal studies (9–11), and cell studies (12,13). Although the reasons for these discrepant findings are unclear, we proposed that the harmful effect of BC supplementation in smokers is associated with the pharmacologic doses of BC used in the human intervention studies and the free radical–rich atmosphere in the lungs of cigarette smokers (14–16). With pharmacologic (high) dose BC supplementation, the environment of the lungs of cigarette smokers enhances BC breakdown to produce oxidative by-products, such as, ß-apo-carotenals and BC-epoxides. These oxidative metabolites of BC may promote lung carcinogenesis by several mechanisms, e.g., enhancement of retinoic acid (RA) catabolism (17), downregulation of retinoic acid receptor (RAR)ß, which functions as a tumor suppressor, and upregulation of protooncogene gene (c-Jun and c-Fos) expression (14,16). Perocco et al. (18) showed that induction of BALB/c 3T3 cell transformation by benzo[a]pyrene (BP), an important carcinogen found in cigarette smoke, was markedly enhanced by the presence of BC. Further, Salgo et al. (19) reported that BC metabolites, but not BC, increase the binding of metabolites of BP to DNA. In contrast, we showed that in ferrets, low-dose BC supplementation, such as would be provided by consuming 5–9 servings of fruits and vegetables/d, had no detrimental effects, but rather a protective effect against cigarette smoke–induced lung damage (16). These findings indicated that BC at low dose or its metabolites at a low concentration can act as anticarcinogenic agents. However, this hypothesis requires additional supporting evidence.
Increased dietary BC or other provitamin A carotenoids may affect the steady-state concentration of carotenoids in body fluids or tissues and serve as localized substrates for retinoid formation. RA, which exerts striking effects on diverse processes such as growth, development, and differentiation (20), can be produced from BC during intestinal metabolism in both animals (21–23) and humans (24,25). Two families of nuclear receptors [RAR and retinoid X receptor (RXR)] were shown to be active in receptor-mediated regulation of gene transcription (26). These receptors have several discrete functional domains, which in the presence of retinoid ligands, can bind to cognate DNA sequences through the DNA-binding domain, thereby modulating gene expression. The ligand for the RARs is either all-trans-RA or 9-cis-RA, whereas the ligand for the RXRs is 9-cis-RA. Both in vitro and in vivo studies demonstrated that all-trans-RA is a metabolite of all-trans-BC, but both 9-cis-RA and all-trans-RA are metabolites of 9-cis-BC (23,24). Thus, gene expression may be regulated by the conversion of BC to RA isomers. The conversion of BC into RA may involve either the well-known central cleavage pathway (27,28) or an excentric cleavage pathway (29,30). Although excentric cleavage metabolites (e.g., ß-apo-carotenoic acids) other than RA were shown to have biological activity (31,32), it is not known whether this activity is a function of binding to nuclear receptors and transcriptionally activating genes, or of metabolism to RA.
The present study was designed to determine whether an interaction exists between BP and BC, or one of its metabolites, [ß-apo-14'-carotenoic acid (14'CA)], to affect the growth regulation and the expression of RAR of normal human bronchial epithelial (NHBE) cells. The derivation of this cell line from normal human lungs and the basal expression of RAR, ß, and genes make NHBE cells a good model for this study. Furthermore, we evaluated the ability of BC metabolites (14'CA, which is two carbons longer than RA, and ß-apo-13-carotenone, which is two carbons shorter than RA) to activate transcription of the RARß2 promoter (Fig. 1).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell growth inhibition.
NHBE cells, derived from normal human lung, were obtained from Clonetics. Cell cultures were maintained in the media kit obtained from Clonetics. Cells were grown in 100-mm culture dishes and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were seeded at a concentration of 5000 cells/cm2 for these experiments. Treatments were applied 24 h after cultures had been seeded to ensure proper attachment of the cells to the plastic wells. NHBE cells were incubated with BP, BC, 14'CA or the combination of carotenoids and BP, dissolved in THF (0.1%). The concentrations of BC (30 µmol/L) and 14'CA (1 and 10 µmol/L) that could induce RARß expression in NHBE cells in our preliminary study were used in the present study. In our preliminary study, we also found that the intracellular concentration of BC of the cells was only 3% (0.9 µmol/L) of the BC concentration added to the cell medium (30 µmol/L). The concentration of apo-carotenoid used in the study was much less than that of BC because 14'CA is an intermediate compound during the conversion of BC into RA (29, 30). Further, we demonstrated previously (22) that the incubation of ferret tissue with BC or apo-carotenoid at similar concentrations can produce RA. The concentration of BP (10 µmol/L) that can reduce RARß level in NHBE cells was used in the present study. Control cells received medium supplemented with THF only. The medium was changed every other day. Cell morphology was monitored by periodic evaluation of the cells under a phase contrast microscope during the entire course of the experiments. The cells were harvested at d 8–9 using a media kit (Clonetics), and counted in duplicate using an electronic Coulter Counter (Model Z1, Coulter).
Expression and reporter constructs. The expression construct used was a 1.4-kb cDNA piece of the coding sequence of the RARß isoform (kindly supplied by Prof. Pierre Chambon, Strasbourg, France) cloned into the vector, PREP9 (Invitrogen). The PREP9 construct was driven by an RSV promoter and used an SV40-derived poly A tail. For the RARß2 reporter construct, pXP-D2 containing 1.6 kb of the 5' regulatory sequence of the RARß2 promoter [-1.6 kb to +156 (PstI-BamHI) including the RARE and the TATA box] was inserted into the XhoI-Bg/II site of the promoterless luciferase reporter plasmid, pXP2 (33,34).
Transient transfection assay. Rat embryo fibroblasts cells from the American Type Culture Collection were grown in DMEM with 10% fetal calf serum (FCS); the medium was replaced every other day. Cells at low passage number were grown in DMEM with 10% FCS at 37°C, split into 60-mm dishes, and grown to 75% confluence (3 x 105 cells/60-mm dish). The medium of the cells was changed 2–4 h before transformation. The assays were conducted using the calcium phosphate coprecipitation technique. Plasmid DNA (5 µg/plate) for both the RARß expression and RARß2 reporter constructs was mixed with 10 µg pBSK carrier DNA in a small volume, mixed on a vortex, then allowed to sit at room temperature for 20 min. The relevant plasmid DNA-carrier DNA mixture was added dropwise to the plated cells in the medium and swirled. This DNA mixture was allowed to incubate with the cells for 18 h at 37°C. The cells were rinsed and refed with DMEM and 0.5% FCS for 24 h. After 24 h of serum starvation, the cells were stimulated with all-trans-RA, 14'CA, or ß-apo-13-carotenone [dissolved in 10 µL dimethyl sulfoxide (DMSO)]. Control cells were treated with 10 µL DMSO alone. After 18 h of incubation, the plates were washed twice with cold PBS, then harvested and lysed with reporter lysis buffer (Luciferase Assay System, Promega). Protein quantitation was performed using the bicinchoninic acid protein assay kit (Pierce). The transfection efficiency was not normalized using other expressed DNA constructs because of the small variation in transactivation activity among the experiments.
Luciferase activity. Luciferase activity was determined using equal amounts of protein from each sample and assayed with the Luciferase Assay System (Promega). The sample extracts were placed in a luminometer (Monolight 2010, Analytical Luminescence Laboratory). The reaction was initiated with 20 µL of 1 mmol/L luciferin. The peak light emission was recorded for each sample within 10 s. The luciferase activity was expressed as the fold increase compared with the control cells (DMSO only). RA, 14'CA, and ß -apo-13-carotenone concentrations giving half-maximal activation were determined from the plots of dose-response experiments.
Western blotting analysis.
The cell plates were washed twice with cold PBS. The cells were collected by scraping and lysed in a solution containing 50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 5 mmol/L EGTA, 1% Triton X-100, 10% glycerol and protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 10 mg aprotinin/L and 10 mg leupeptin/L). Immunoblot analysis for the detection of RAR proteins was performed using antibodies against RAR, ß, and (Santa Cruz), as described (14,16).
Sample extraction and HPLC analysis.
The sample extraction was done as previously described (35). RA was identified by co-elution with standards and spectral properties (matching both the retention time, 5.6 min, and the 
max, 342 nm, of a RA standard), and quantified relative to an internal standard, retinyl acetate, by determining peak areas calibrated against known amounts of standards (35).
Statistics. Results are expressed as means ± SD of at least three determinations. Data were analyzed by 1-way ANOVA followed by Tukey’s Honestly Significant Difference test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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ß-Carotene (30 µmol/L) with or without BP inhibited cell growth 28% (P < 0.05) (Fig. 2, upper panel). HPLC analysis of BC or its metabolites (e.g., apo-carotenoids and RA) in NHBE cells was not undertaken in this investigation because of the large number of cells required and the problem of "leakage" of BC metabolites from cells into the cell media. Because it is possible that BC is converted to ß-apo-carotenoids after its uptake by NHBE cells (Fig. 1), we tested the possibility that ß-apo-carotenoid acid may have independent effects on NHBE cell growth. 14'CA at both 1 and 10 µmol/L inhibited cell growth (51 and 65% inhibition, respectively) and was significantly more growth inhibitory than BC (Fig. 2). A dose-dependent cell growth inhibition was observed using 14'CA with significant inhibition at 1 and 10 µmol/L (P < 0.05); however, the inhibitory effects of 14'CA were reduced significantly in the present of BP (Fig. 2, lower panel).
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The densitometry scanning of the bands revealed that RARß protein expression was downregulated by BP (50%) and upregulated by BC (259%) (Fig. 3) and 14'CA (by 278%) (Fig. 4) (P < 0.05). Combining BP with BC or 14'CA reversed the reduction of RARß by BP alone (Figs. 3, 4). The densitometric scanning of RAR and RAR, c-Fos and c-Jun proteins (data not shown) did not differ among any of the treatment groups.
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Both 14'CA and all-trans-RA (as a positive control) at 1 µmol/L transactivated the reporter construct in the presence of the RARß2 receptor (Table 1). However, the activity of 14'CA was 10% of all-trans-RA (Fig. 5). ß-Apo-13-carotenone, another excentric cleavage metabolite of BC (36), induced little transcriptional activity of the RARß2 promoter (Table 1). Similarly, retinoyl-ß-glucuronide had little transcriptional activity (Table 1).
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0.1 µmol/L, whereas the half-maximum activation for 14'CA required at least 1 µmol/L (Fig. 5, lower panel). The time course experiment was carried out at 0, 1, 3, 9, and 18 h after the addition of ligand. RA (0.1 µmol/L) or 14'CA (0.5 µmol/L) transactivated the reporter construct in a time-dependent manner (Fig. 5, upper panel). There appeared to be a lag period in the transactivation by 14'CA. To determine whether there was any difference in the level of RARß2 protein in the rat embryo fibroblast cells after the incubation of either 14'CA or RA, we examined RARß expression using Western blot analysis in the cell extracts after an 18-h transactivation assay. There was no difference in the level of RARß protein expressed after treatment with either RA or 14'CA (data not shown). We did not detect any endogenous RARß in either untransfected or vector-transfected cells. Effect of an inhibitor of 14'CA metabolism on RARß2 promoter transactivation.
During the course of the transactivation assay, a ligand added to the cell may be metabolized to a more or less active form. We tested this hypothesis by analyzing RA in the cell extracts after the transactivation assay. RA was detected in the cell extracts after incubation with, 0.1 µmol/L RA, 1 µmol/L 14'CA (Fig. 6), or 1 µmol/L of each retinoid (Table 1). This result is consistent with our previous study, showing that 14'CA can be converted into RA via a ß-oxidative process in both rabbit liver mitochondria in vitro and perfused ferret liver (37). Because 3-mercaptopropionic acid (MPA) inhibits the oxidation of ß-apo-carotenoic acids (37), we compared the effect of adding 3-MPA on both RARß2 promoter transactivation activity (Fig. 6, upper panel) and RA level (Fig. 6, lower panel) in rat embryo fibroblasts that were treated with RA or 14'CA. We used a low dose (0.01 µmol/L) of 3-MPA because concentrations > 0.1 µmol/L caused cell morphologic changes in a preliminary study (data not shown). In cells exposed to 0.1 µmol/L RA (Fig. 6), 3-MPA treatment did not affect either RA concentrations or luciferase transactivation activities. This concentration of RA induced an eightfold increase in luciferase activity, and was thus sufficient to allow us to detect RA in the cell extract. Treatment of cells with 1 µmol/L 14'CA induced a 12-fold increase in luciferase activity and produced measurable quantities of RA (Fig. 6). When the cells were treated with a combination of 0.1 µmol/L RA and 1 µmol/L 14'CA, both the luciferase activities and RA levels were close to additive for the individual compounds (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We further observed that 14'CA can induce transcriptional activity of the RARß2 promoter, although it is 10- to 100-fold less potent than all-trans-RA in this activity (Fig. 5). RARß expression in NHBE cells after treatment with either all-trans-RA or 14'CA was similar (data not shown). This suggests that the difference in the transcriptional activity was not due to an effect on protein expression by all-trans RA or 14'CA. Rather, the activation by 14'CA appears to occur, in large part, via metabolism to the potent RAR ligand, all-trans-RA (Table 1 and Fig. 6). This is further supported by the current finding that the transactivation activity of 14'CA is reduced by 3-MPA, an inhibitor of ß-apo-carotenoic acid oxidation to RA (Fig. 6) and by the observation that 14'CA has very weak affinity for RARs (46). On the other hand, ß-apo-13-carotenone, as well as the RA metabolite, ß-retinoyl glucuronide, had very limited transactivation activities (Table 1). Neither of these compounds are intermediates in the conversion of BC to RA. The limited activity of ß-apo-13-carotenone agrees with findings from previous studies that reported that another metabolite, 5,8-endoperoxy-2,3-dihydro-ß-apo-carotene-13-one, had no transactivation activity of RAR (47).
It is intriguing that 3-MPA-treated cells that were cultured in combination with RA and 14'CA had greater transactivation than cells that were treated with a similar concentration of RA alone (Fig. 6). Recent studies showed that although ß-apo-12'-carotenoic acid can inhibit the growth of HL-60 cells (31); 14'CA can stimulate the differentiation of U937 leukemic cells (32) and inhibit the growth of breast cancer cells (46). Thus, it is possible that various breakdown products of BC play a role in regulating cell functions, apart from their ability to be metabolized to RA. This is supported by our present finding that there was relatively more luciferase activity from 14'CA than could be accounted for by the appearance of RA compared with the effect of RA alone (Fig. 6). It would be informative to investigate whether the biological activity of 14'CA is mediated through its interaction with RXR, similar to RXR-selective retinoids.
One of the important questions is whether the beneficial vs. detrimental effects of carotenoids are related to the carotenoid dose administered in vivo and the accumulation of carotenoids in a specific organ. Because the beneficial and harmful effects of carotenoids could be due to their metabolites or decomposition products (48), it is possible that the excentric cleavage products of BC are formed in small quantities in cells and increase the RA level by their conversion into RA at physiologic concentrations. They could also be formed in large quantities in the cell (e.g., due to supplementation with high-dose BC in the highly oxidative conditions of the lung) and enhance catabolism of RA by their induction of cytochrome P450 enzymes at a high concentration (17). In the present study, the intracellular concentration of BC of the cells was 3% (0.9 µmol/L) of the BC concentration (30 µmol/L) added in the cell medium (unpublished data). This intracellular concentration of BC is slightly higher than the normal plasma levels in U.S. adults aged 
19 y (0.4 µmol/L) but much less than the serum levels in the intervention groups (6 µmol/L) in the Alpha-Tocopherol, Beta Carotene Cancer Prevention Study (3). Therefore, the experimental conditions in our study were more relevant to physiologic effects achieved with dietary sources. As we demonstrated, the presentation of high doses of BC via supplements to the highly oxidative environment in the lung in smokers results in increased levels of oxidative metabolites of BC, which may have detrimental effects. On the other hand, we observed that low-dose BC (6 mg/d) supplementation partially prevented the decrease in lung RA levels in the group exposed to smoke. The present study indicates that 14'CA, an excentric cleavage metabolite of BC, can prevent the reduction of RARß caused by BP and induce transcriptional activity of the RARß2 promoter by its conversion into RA, thus providing more evidence to support the possibility that BC or its metabolites, when given at low doses, could act to supply adequate RA to induce RARß (48). More mechanistic insights for these observations are required to assist in the design of future clinical trials using carotenoids as cancer preventive agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BC, ß-carotene; BP, benzo[a]pyrene; 14'CA, ß-apo-14'-carotenoic acid; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; MPA, 3-mercaptopropionic acid; NHBE, normal human bronchial epithelial; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; THF, tetrahydrofuran.
Manuscript received 3 October 2003. Initial review completed 20 October 2003. Revision accepted 1 December 2003.
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