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Carcinogenesis Advance Access originally published online on April 9, 2007
Carcinogenesis 2007 28(7):1567-1574; doi:10.1093/carcin/bgm076
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Apo-10'-lycopenoic acid inhibits lung cancer cell growth in vitro, and suppresses lung tumorigenesis in the A/J mouse model in vivo

Fuzhi Lian, Donald E. Smith, Hansgeorg Ernst1, Robert M. Russell and Xiang-Dong Wang*

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
1 BASF, Inc, Ludwigshafen D-67056, Germany

* To whom correspondence should be addressed. 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
 
High intake of lycopene has been associated with a lower risk of a variety of cancers including lung cancer. We recently showed that lycopene can be converted to apo-10'-lycopenoids [Hu et al. (2006). J. Biol. Chem., 281, 19327–19338] in mammalian tissues both in vitro and in vivo, raising the question of whether apo-10'-lycopenoids have biological activities against lung carcinogenesis. In the present study, we report that apo-10'-lycopenoic acid inhibited the growth of NHBE normal human bronchial epithelial cells, BEAS-2B-immortalized normal bronchial epithelial cells and A549 non-small cell lung cancer cells. This inhibitory effect of apo-10'-lycopenoic acid was associated with decreased cyclin E, inhibition of cell cycle progression from G1 to S phase and increased cell cycle regulators p21 and p27 protein levels. In addition, apo-10'-lycopenoic acid transactivated the retinoic acid receptor ß (RARß) promoter and induced the expression of RARß. We further examined the effect of apo-10'-lycopenoic acid treatment on 4-(N-methyl-N-nitrosamino)-1-(3-pyridal)-1-butanone (NNK)-induced lung tumorigenesis in the A/J mouse model. We found that the lung tumor multiplicity was decreased dose dependently from an average of 16 tumors per mouse in the NNK injection alone group, to an average of 10, 7 and 5 tumors per mouse in groups injected with NNK and supplemented with 10, 40 and 120 mg/kg diet of apo-10'-lycopenoic acid, respectively. These observations demonstrate that apo-10'-lycopenoic acid is a biological active metabolite of lycopene and suggest that apo-10'-lycopenoic acid is a potential chemopreventive agent against lung tumorigenesis.

Abbreviations: BEGM, bronchial epithelial growth medium; FBS, fetal bovine serum; i.p., intra-peritoneal; LDH, lactate dehydrogenase; NNK, 4-(N-methyl-N-nitrosamino)-1-(3-pyridal)-1-butanone; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCR, polymerase chain reaction; RAR, retinoic acid receptor; RARE, retinoic acid response element; RLU, relative light unit; RXR, retinoid X receptor; SEM, standard error of mean; THF, tetrahydrofuran


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
High intake of lycopene, a major non-pro-vitamin A carotenoid in tomato and tomato-based products, has been associated with a lower risk of lung cancer (1). For example, analyses from the combination of the Nurses' Health Study and Health Professional Follow-Up Study (2) and from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (3) showed that high intake of lycopene was associated with an approximately 20–30% lower risk of lung cancer. In animal studies, Kim et al. (4) showed that lycopene given in drinking water decreased the incidence and multiplicity of lung tumors induced by the combination of diethylnitrosamine, N-methyl-N-nitrosourea and 1,2-dimethylhydrazine in B6C3F1 mice. Our previous study showed that lycopene supplementation prevents smoke-induced cell proliferation and the reduction of apoptosis in the lungs of smoke-exposed ferrets (5). However, other animal studies investigating the chemopreventive effect of lycopene against lung cancer have yielded conflicting results. Hecht et al. (6) showed that supplementation with lycopene-enriched tomato oleoresin did not affect benzo[a]pyrene and 4-(N-methyl-N-nitrosamino)-1-(3-pyridal)-1-butanone (NNK)-induced lung carcinogenesis in A/J mince. Guttenplan et al. (7) observed an enhancement of benzo[a]pyrene-induced mutagenesis by lycopene in the lung of LacZ mice. Although these discrepancies need further investigation, it has been demonstrated that lycopene may function as a natural antioxidant (8), enhance cellular gap junction communication (9), induce phase II enzymes involved in the activation of the antioxidant response element transcription system (10), suppress insulin-like growth factor 1-stimulated cell proliferation by inducing insulin-like growth factor-binding protein (5,11) and inhibit neoplastic transformation of normal cells (12). In addition, several reports including ours suggest that the biological activities of carotenoids could be related to the function of their metabolic products, which can possess either more or less activity than their parent compounds, or have entirely different functions (13,14).

Lycopene is a highly unsaturated lipophilic carotenoid with 11 conjugated double bonds and two unconjugated double bonds (Figure 1), which make it susceptible to oxidation. It has been reported that several auto-oxidative products of lycopene by in vitro chemical oxidation (15–17) can inhibit cancer cell proliferation (18), induce cell apoptosis (16,18,19), and enhance gap junction communication (15,20). However, the physiological roles of these lycopene products remain unknown since none of these metabolites have been detected in biological systems. We, as well as others, have recently showed that lycopene can be converted into apo-10'-lycopenal by carotene 9',10'-oxygenase both in vitro and in vivo (21,22). The cleavage product, apo-10'-lycopenal, can be further converted to apo-10'-lycopenoic acid and apo-10'-lycopenol in liver and lung tissues (22). Interestingly, apo-lycopenoids can be detected in the lungs of ferrets (22) and the liver of rats (23) after lycopene treatment. However, the biological properties of apo-10'-lycopenoids as well as their roles in the lung cancer prevention are unknown.


Figure 1
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  		Fig. 1. Chemical structures of lycopene, acycloretinoic acid, apo-10'-lycopenoic acid and retinoic acid.

 
Retinoid receptors (retinoic acid receptors: RAR{alpha}, ß and {gamma}; and retinoid X receptors: RXR{alpha}, ß and {gamma}) are transcription factors that act mainly as RAR–RXR heterodimers, and bind to the retinoic acid response element (RARE) in the promoter region of target genes. Upon retinoid binding, RAR–RXR heterodimers can recruit transcription co-activator complexes and activate transcription (24). RARß, which is regulated by retinoic acid via RARs (25), has been suggested to be a tumor suppressor gene, playing a critical role in mediating the growth inhibitory effects of retinoids in various cancer cells (24). We have shown previously that ß-carotene and its metabolite, apo-14'-carotenoic acid (26) and ß-cryptoxanthin (27) induced RARß expression, probably through their metabolites, such as retinoic acid (26,27). Interestingly, acycloretinoic acid (Figure 1), a centric cleavage product of lycopene identified by in vitro chemical oxidation at the lycopene 15,15' double bond, but not confirmed in biological tissues, was shown to transactivate the RARß promoter (18,19). Although similarity in chemical structure among retinoic acid, acycloretinoic acid and apo-10'-lycopenoic acid (Figure 1) exists, it is unclear whether apo-10'-lycopenoic acid can also activate retinoid signaling.

In the present study, we tested our hypothesis that apo-10'-lycopenoic acid, a relative stable derivative of apo-10'-lycopenoids, may be an active component that mediates the chemopreventive effect of lycopene against lung cancer. First, we investigated the growth inhibition and the underlying mechanisms of apo-10'-lycopenoic acid using in vitro cultured lung cells derived from humans. We then carried out an in vivo study using the A/J mouse, a widely used animal model for lung cancer, to determine the potential chemopreventive effect of apo-10'-lycopenoic acid.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
NHBE (a normal human bronchial epithelial cell line) was purchased from Cambrex Corporation (East Rutherford, NJ). BEAS-2B (an adenovirus-12 SV40 hybrid virus transformed, immortalized human bronchial epithelial cell line), A549 (a non-small cell lung cancer cell line) and HeLa (a cervix epithelial adenocarcinoma cell line) were purchased from American Type Culture Collection (Manassas, VA). A549 and HeLa cells were maintained in F12K medium (Invitrogen, Carlsbad, CA) or Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, from HyClone, Logan, UT), 4 mmol/l L-glutamine and antibiotics (40 mg/l streptomycin and 40 000 U/l penicillin). NHBE and BEAS-2B cells were grown in serum-free bronchial epithelial growth medium (BEGM, Cambrex Corporation) supplemented with growth factors, all-trans retinoic acid and hormones, as the manuals indicated. BEAS-2B cells were cultured in tissue culture plates coated with bovine serum albumen/collagen/fibronectin as previously reported (28). All cells were kept at 37°C in a humidified atmosphere containing 5% CO2. Unless specifically mentioned, A549 and HeLa cells were changed to the basic mediums containing 1% FBS, and NHBE and BEAS-2B cells were switched from fully supplemented BEGM medium to BEGM medium without all-trans retinoic acid before apo-10'-lycopenoic acid treatment. A stock solution (10 mmol/l) of apo-10'-lycopenoic acid (>99% purity, synthesized and provided by BASF, Germany) was prepared by dissolving apo-10'-lycopenoic acid in tetrahydrofuran (THF, Sigma, St. Louis, MO, containing 0.025% butylated hydroxytoluene as an antioxidant) and stored at –80°C. Upon treatment, aliquots from the stock solution of apo-10'-lycopenoic acid were added to the cell culture medium to the desired working concentration, and stirred vigorously. The final THF concentration in cell culture medium was 0.1%. The control culture received only THF. The cell culture medium containing apo-10'-lycopenoic acid was changed every other day. All procedures, including the incubation analysis, were performed under red light.

Measurement of cell growth
Cell counting and an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay were used to measure the effect of apo-10'-lycopenoic acid on cell growth, as described elsewhere (27). For cell counting, 1.2 x 105 cells per well in six-well plates were incubated in medium containing different concentrations of apo-10'-lycopenoic acid for 5 days. Then, cells were trypsinized, and the total cell numbers were counted using a hemacytometer. For the MTT assay, cells were seeded at 5 x 103 cells per well in 96-well plates. After 1 day, cells were treated with varying concentration of apo-10'-lycopenoic acid for indicated periods, and assayed using an MTT assay kit (Roche Applied Science, Indianapolis, IN). Cell viability is expressed as OD595. All measurements were done in triplicate.

Cell cycle analysis
A549 cells were plated on six-well plates (3 x 105 cells per well) and grown in F12K medium containing 10% FBS overnight to allow for cell attachment. Cells were then changed to cell culture medium containing 1% FBS, and treated with apo-10'-lycopenoic acid for 48 h. Then, both detached and attached cells were harvested and fixed with 70% ethanol. After fixation, cells were washed with PBS, re-suspended in 1 ml of PBS containing 0.2 mg/ml RNase, 0.1 mg/ml propidium iodide and incubated at 37°C for 15 min. Samples of 10 000 cells were then analyzed for DNA content by a flow cytometer using a FACSCalibur instrument (Becton Dickinson, Franklin Lakes, NJ), and cell cycle phase distributions were analyzed by Modfit LT 3.0 software (Verity Software, Topsham, ME).

Measurement of apoptosis and cytotoxicity
Cell apoptosis was evaluated by measuring cytoplasmic histone-associated DNA fragments using a cell death detection ELISA kit (Roche Applied Science). In brief, A549 cells were treated with different concentrations of apo-10'-lycopenoic acid for 48 h; cells were collected, cell lysates were prepared by incubating cells in incubation buffer for 30 min at room temperature and then centrifuging at 20 000g for 10 min, and collecting the supernatant containing the cytoplasmic fraction for immunoassay. The immunoassay of histone-associated DNA fragments was performed as described in the manufacturer's manual. All measurements were done in triplicate. The results were further confirmed by examining the distribution of cells in sub-G1 phase in cell cycle analysis.

Cytotoxicity was measured by lactate dehydrogenase (LDH) release assays. In brief, A549 cells were treated with different concentrations of apo-10'-lycopenoic acid for 48 h, and then the cell culture medium was collected. LDH activities were determined with an LDH assay kit (Sigma) as described in the manufacturer's manual. All measurements were done in triplicate.

Western blot
Cells were lysed with whole-cell lysate buffer [20 mmol/l Tris–HCl (pH 7.5), 150 mmol/l NaCl, 1% Triton X-100, 1 mmol/l EDTA, 1 mmol/l EGTA, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l ß-glycerophosphate, 1 mmol/l Na3VO4, 25% glycerol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstain and 1 mmol/l phenylmethylsulphonylfluoride]. Cell lysates (50 µg per lane) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA). The membranes were blocked with 5% non-fat milk in TBST buffer [25 mmol/l Tris-HCl (pH 8.0), 150 mmol/l NaCl, 0.05% Tween 20] and incubated with a primary antibody. The protein was detected by a horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA). The specific bands were visualized by a SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL) according to the manufacturer's instructions. Anti-human ß-actin antibody (Sigma, St Louis, MO) was used to detect human ß-actin, which was used as a protein loading control. Anti-human cyclin E and p27 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-human p21, cyclin D1 and cleaved caspase-3 antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Real-time quantitative polymerase chain reaction
mRNA levels were measured by real-time quantitative polymerase chain reaction (PCR) after reverse transcription of RNA. Total RNA was extracted using TriPure reagent (Roche Applied Science). cDNA was generated with M-MLV reverse transcriptase (Invitrogen) as indicated in the manufacturer's manual. Primers were designed using Primer Express version 2.0 (Applied Biosystems, Foster City, CA) software. The sequences for the primers are shown in the Table I.


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  		Table I. Primers for quantitative PCR

 
Real-time PCRs were performed on an Applied Biosystems 7000 sequence detection system using Platinum SYBR Green qPCR Kit (Invitrogen) according to the manufacturer's instructions. The mRNA levels of the measured genes relative to ß-actin mRNA were determined using the 2–{Delta}{Delta}CT method (29). The mRNA levels were expressed as fold induction, relative to the control group.

Luciferase reporter assay
A series of reporter constructs (pGL2/RARß2-D2, pGL2/RARß2-D3, pGL2/RARß2-D5, pGL2/RARß2-D7) were provided by Dr Monica Peacocke (Columbia University) and have been described previously (30). In brief, the constructs were generated by inserting different fragments of the RARß2 promoter into a pGL2-basic vector (Promega Corp., Madison, WI), which contained a cDNA encoding the firefly luciferase gene. A pGL2/{Delta}RARß2-D7 vector was generated by mutating the RARE of RARß2 promoter (–53 to –37 bp) in the pGL2/RARß2-D7 vector from GGTTCACCGAAAGTTCA to GcTTacCCGAAAGTTCA, which abolished the binding of the RAR. A pRL-TK vector containing a Renilla luciferase gene driven by a SV40 promoter was obtained from Promega Corp.

HeLa cells (2 x 105 cells per well) in a six-well plate were co-transfected with 1 µg of firefly luciferase reporter vector and 0.1 µg of pRL-TK vector using Fugene 6 transfection reagent (Roche Applied Science), as indicated by the manufacturers. After transfection for 24 h, cells were trypsinized, re-plated by 1:8 in 24-well plate and incubated overnight. Then, cells were treated with apo-10'-lycopenoic acid or all-trans retinoic acid for 24 h. The cells were washed and lysed. The luciferase activities were measured using the dual-luciferase reporter system (Promega Corp.) and a Lumi-Scint luminometer (BioScan, Washington, DC), and normalized by the transfection efficiency. The promoter activities were calculated as relative light unit (RLU) of firefly luciferase activity/RLU of Renilla luciferase activity (RLUfirefly/RLUrenilla), and are expressed as fold induction relative to control. Means ± standard error of means (SEMs) from three experiments are given.

Animals and study design
The A/J mouse model was used to examine the chemopreventive effect of apo-10'-lycopenoic acid. We used apo-10'-lycopenoic acid doses ranged from 10 to 120 mg/kg diet which was approximately 1/50 to 1/5 of the lycopene doses used in previous studies in order to further determine whether lycopene metabolite mediates the chemopreventive activity of lycopene (31). Male strain A/J mice (4–5 weeks old, The Jackson Labs, Bar Harbor, ME) were weight matched and assigned to one of five experimental groups (n = 13–14 per group) and treated as follows: (i) CNTL (Control): no agent plus intra-peritoneal (i.p.) injection of 100 mg normal saline/kg body weight; (ii) NNK: no agent plus i.p. injection of 100 mg NNK/kg body weight (>98% purity, from Toronto Research Chemical, Toronto, Canada); (iii) LYA10: 10 mg/kg diet of apo-10'-lycopenoic acid plus i.p. injection of 100 mg NNK/kg body weight; (iv) LYA40: 40 mg/kg diet of apo-10'-lycopenoic acid plus i.p. injection of 100 mg NNK/kg body wt and (v) LYA120: 120 mg/kg diet of apo-10'-lycopenoic acid plus i.p. injection of 100 mg NNK/kg body weight. Apo-10'-lycopenoic acid in a powder form was mixed directly into a powdered semi-purified diet AIN-93M (Dyets, Bethlehem, PA). Administration of apo-10'-lycopenoic acid supplemented diets began 2 weeks prior to NNK exposure. Animals were maintained on the experimental diets for 14 weeks after injection with daily feeding and observation and weekly weighing. Mice were fasted for 12 h prior to terminal exsanguination under Aerrane (Barter, Deerfield, IL) deep anesthesia; blood was collected in heparinized tubes and stored as plasma. All animal protocols were approved by the Institutional Animal Care and Use Committee at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University.

Pathology
Tumor nodules on the lung surface were carefully examined and counted as described previously (32). Lung tumors were quantified by determining the incidence and multiplicity of pulmonary pleural surface tumors of each mouse. Tumor incidence was defined as the number of mice in each group bearing one or more tumors divided by the total number of mice examined. Tumor multiplicity was defined as the total number of pulmonary tumors in each group divided by the total number of mice examined. Lung tumors were visually counted by two researchers blinded to treatment group and confirmed by microscopic examination of hematoxylin–eosin-stained lung sections.

High-performance liquid chromatography analysis
Plasma levels of apo-10'-lycopenoic acid were measured by high-performance liquid chromatography as described previously (22) with minor modifications. Briefly, 150 µl of plasma from two or three mice in the same group was pooled together. Then 2 ml of absolute ethanol and 100 µl of retinyl acetate (internal standard) were added and mixed for 1 min. The mixture was extracted with 4 ml of hexane:ether (1:1, vol/vol) twice. The hexane layer was combined and evaporated to dryness under a stream of nitrogen. The residue was reconstituted with 100 µl of ethanol; 50 µl was injected into the high-performance liquid chromatography system, and was separated as described previously (22). Apo-10'-lycopenoic acid was quantified relative to the internal standard by determining peak areas calibrated against known amounts of standards.

Statistical analysis
Results were expressed as means ± SEMs unless specifically indicated. Comparison of mean values of control and treatment cells was made using one-way analysis of variance with Fisher's least significant difference post-hoc procedure. A difference between groups was considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effect of apo-10'-lycopenoic acid on lung cell growth
Apo-10'-lycopenoic acid treatment at 3 µmol/l for 4 days significantly decreased the cell number of NHBE and BEAS-2B cells, which were kept in a serum-free medium, by 40 and 60%, respectively (Figure 2A). Further decrease in cell number of NHBE and BEAS-2B cells was observed after treatment with apo-10'-lycopenoic acid at higher concentrations. Treatment with 5 and 10 µmol/l of apo-10'-lycopenoic acid significantly decreased the total number of A549 cells cultured in 1% FBS by 30 and 50%, respectively (Figure 2A). This inhibition was not observed in the A549 cells cultured in 10% FBS (data not shown).


Figure 2
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  		Fig. 2. Effect of apo-10'-lycopenoic acid on lung cell growth. (A) Effect of apo-10'-lycopenoic acid on lung cell growth. In all, 5 x 104 NHBE, BEAS-2B and A549 cells were treated with the indicated concentration of apo-10'-lycopenoic acid for 4 days. Cells were collected and cell numbers were counted using a hematometer. (B) Effect of apo-10'-lycopenoic acid on viability of BEAS-2B cells. Cells (5 x 103) were treated with indicated concentration of apo-10'-lycopenoic acid for 48 or 72 h. Cell viability was measured by MTT assay. (C) Effect of apo-10'-lycopenoic acid on viability of A549 cells. Cells (5 x 103) were treated with the indicated concentration of apo-10'-lycopenoic acid for 48 or 72 h. Cell viability was measured by MTT assay. Values are means ± SEMs of three replicate assays. Means in the same group that do not share a letter differ, P < 0.05.

 
The growth inhibitory effect of apo-10'-lycopenoic acid on BEAS-2B and A549 cells was further examined by the MTT assay, a more sensitive measurement of viable cells, after cells were treated for 48 and 72 h. The viabilities of both BEAS-2B (Figure 2B) and A549 cells (Figure 2C) were decreased by treatment with apo-10'-lycopenoic acid at both time points. Similar to the results shown by cell counting, A549 cells were less sensitive to apo-10'-lycopenoic acid treatment, as compared with BEAS-2B cells. The viability of A549 cells decreased by 20% after treatment with 0.5 µmol/l of apo-10'-lycopenoic acid for 48 h, whereas the same treatment decreased the viability of BEAS-2B cells by ~80%.

We further examined apoptosis by measuring histone complex DNA fragments and cytotoxicity by LDH release assay in A549 cells treated with 5 and 10 µM of apo-10'-lycopenoic acid for 48 h. There were no changes in any of these markers among the treatment groups (data not shown), indicating the inhibition of cell growth by apo-10'-lycopenoic acid was not due to the induction of apoptosis or cytotoxicity.

Effect of apo-10'-lycopenoic acid on cell cycle and cell cycle regulators
We examined the effect of apo-10'-lycopenoic acid on cell cycle distribution by fluorescent-activated cell sorting analysis. Treatment with apo-10'-lycopenoic acid for 48 h significantly decreased A549 cells in S-phase from 31% in cells treated with THF alone to 24 and 21% in cells treated with 3 and 5 µmol/l of apo-10'-lycopenoic acid, respectively (Figure 3A). Accordingly, A549 cells in G1/G0 phase were significantly increased by the apo-10'-lycopenoic acid treatment from 61% in cells treated with THF alone to 69 and 71% in cells treated with 3 and 5 µmol/l of apo-10'-lycopenoic acid, respectively (Figure 3A). Treatment with apo-10'-lycopenoic acid did not affect the proportion of A549 cells in sub-G1 phase (data not shown), confirming that inhibition of cell growth by apo-10'-lycopenoic acid was not due to the induction of apoptosis.


Figure 3
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  		Fig. 3. Effect of apo-10'-lycopenoic acid on cell proliferation. (A) Effect of apo-10'-lycopenoic acid on cell cycle distribution. A549 cells (5 x 105) were treated with indicated concentrations of apo-10'-lycopenoic acid for 48 h. Then, both detached and attached cells were harvested, fixed and analyzed for DNA content. Cell cycle distribution was analyzed using Modfit software. Values are means ± SEMs of three replicate assays. Means that do not share a letter differ, P < 0.05. (B) Effect of apo-10'-lycopenoic acid on mRNA levels of cyclin D, cyclin E, p21 and p27. A549 cells (5 x 104) were treated with the indicated concentration of apo-10'-lycopenoic acid for 2 days. Total RNA was extracted, and the mRNA levels of genes were measured by quantitative reverse transcription PCR. Values are means ± SEMs of three replicate assays. Means that do not share a letter differ, P < 0.05. (C) Effect of apo-10'-lycopenoic acid on protein levels of cell cycle regulators. A549 cells (5 x 104) were treated with the indicated concentration of apo-10'-lycopenoic acid for 4 days. Total cellular protein was separated on 12% sodium dodecyl sulphate–polyacrylamide gels and blotted with anti-cyclin D1, cyclin E, p21, p27 and ß-actin antibodies. The figure shows a representative western blot photograph of three experiments.

 
We further examined the effect of apo-10'-lycopenoic acid on the expression of several genes regulating cell proliferation. The mRNA levels of cyclin E, but not cyclin D1, were dose dependently decreased in A549 cells treated with apo-10'-lycopenoic acid for 48 h (Figure 3B). Consistent with mRNA levels, western blot showed that protein levels of cyclin E were decreased and cyclin D1 levels were not changed in A549 cells treated with apo-10'-lycopenoic acid for 72 h (Figure 3C).

mRNA levels of p21 and p27 were not affected by treatment with apo-10'-lycopenoic acid (Figure 3B). However, the treatment with 1 µmol/l of apo-10'-lycopenoic acid increased the p21 protein levels, as compared with control cells (Figure 3C). There was no further increase of p21 in A549 cells treated with higher concentrations of apo-10'-lycopenoic acid. In contrast to p21 protein, p27 protein was barely detected in A549 cells without apo-10'-lycopenoic acid treatment, but its expression became apparent after treatment with as low as 1 µmol/l of apo-10'-lycopenoic acid for 72 h and increased in cells treated with higher concentrations.

Although the expression of p27 was undetectable in BEAS-2B cells (27), we have examined the effect of apo-10'-lycopenoic acid on cell cycle and cell cycle-related proteins in BEAS-2B cells. Similar to the results observed in A549 cells, the treatment of apo-10'-lycopenoic acid down-regulated cyclin E, up-regulated p21 and did not change cyclin D1 levels in BEAS-2B cells (data not shown).

Effect of apo-10'-lycopenoic acid on the expression of RARß and transactivation of RARß promoter
To determine whether the inhibitory effect of apo-10'-lycopenoic acid on cell growth was associated with the induction of RARß, we examined mRNA levels of RARß in all three cell lines after the treatment with apo-10'-lycopenoic acid for 48 h (Figure 4). In NHBE cells, 3 µmol/l of apo-10'-lycopenoic acid increased RARß mRNA levels by 6-fold, comparable to RARß induction by 1 µmol/l of all-trans retinoic acid (Figure 4, insert). Similarly, treatment with 3 and 5 µmol/l of apo-10'-lycopenoic acid significantly increased RARß mRNA levels in BEAS-2B cells by 3- and 5-fold, respectively. However, in A549 cells, treatment with apo-10'-lycopenoic acid only slightly increased RARß levels (1.4-fold in cells treated with 5 µmol/l of apo-10'-lycopenoic acid, Figure 4). As compared with NHBE and BEAS-2B cells, A549 cells were much less sensitive to all-trans retinoic acid treatment in terms of RARß expression (~2-fold increase in cells treated 1 µmol/l of all-trans retinoic acid compared with control, Figure 4, insert).


Figure 4
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  		Fig. 4. Effect of apo-10'-lycopenoic acid on the expression of RARß. In all, 3 x 105 NHBE, BEAS-2B and A549 cells were treated with the indicated concentration of apo-10'-lycopenoic acid for 2 days. Total RNA was extracted, and RARß mRNA levels were measured by quantitative real-time PCR. Values are means ± SEMs of three replicate assays. Means that do not share a letter differ, P < 0.05. Insert: RARß induced by 1 µmol/l of all-trans retinoic acid as a positive control.

 
We next investigated whether apo-10'-lycopenoic acid transactivates retinoid receptor promoter by using a series of reporter vectors containing different fragments of the RARß promoter (Figure 5A). HeLa cells transfected with a reporter vector and an internal control vector (pRL-TK) were treated with apo-10'-lycopenoic acid or all-trans retinoic acid for 24 h. Treatment with 5 µmol/l of apo-10'-lycopenoic acid increased luciferase activity of all RARß promoter constructs, although the magnitude of the increase was less than that induced by 1 µmol/l of all-trans retinoic acid (Figure 5A). The depletion of the region between –1500 and –124 bp of the RARß promoter did not significantly affect the transactivation activity of apo-10'-lycopenoic acid (Figure 5A).


Figure 5
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  		Fig. 5. Effect of apo-10'-lycopenoic acid on retinoid receptor signaling. (A) Effect of apo-10'-lycopenoic acid and all-trans retinoic acid on RARß promoter activity. Upper panel shows a diagram of RARß promoter constructs. (B) The involvement of RARE on apo-10'-lycopenoic acid-transactivated RARß promoter activity. Upper panel shows a diagram of the generation of the mutated RARß promoter. HeLa cells transfected with an RARß reporter vector and an internal control vector were treated with 5 µmol/l of apo-10'-lycopenoic acid or 1 µmol/l of all-trans retinoic acid for 24 h. Luciferase activities were measured by dual-luciferase reporter system. Values are means ± SEMs of three replicate assays.*Statistically significantly different, as compared with control in the same group, P < 0.05.

 
To further investigate the involvement of the RARE that locates between –53 and –37 bp of the RARß promoter in the induction of RARß by apo-10'-lycopenoic acid, we mutated the RARE sequence of pGL2/RARß-D7 from GGTTCA to GcTTac (pGL2/RARß/{Delta}RARE-D7), which abolished the RAR binding site (Figure 5B). The luciferase assay showed that both all-trans retinoic acid and apo-10'-lycopenoic acid treatment significantly induced the promoter activity of RARß/D7 in HeLa cells. However, the induction was abolished in HeLa cells transfected with the mutated pGL2/{Delta}RARß2-D7 promoter (Figure 5B).

Effect of apo-10'-lycopenoic acid on NNK-induced lung tumorigenesis in A/J mice
We performed an in vivo study to determine whether apo-10'-lycopenoic acid could inhibit lung carcinogenesis in the A/J mouse model of lung cancer. Apo-10'-lycopenoic acid supplementation did not significantly affect mouse body weight during the entire study (Figure 6A). After supplementation with apo-10'-lycopenoic acid for 16 weeks, there was a dose-dependent increase in plasma levels of apo-10'-lycopenoic acid in mice receiving supplementation (Figure 6B), whereas no apo-10'-lycopenoic acid was detected in the plasma of mice without supplementation. The plasma concentration of apo-10'-lycopenoic acid was positively correlated with the levels of apo-10'-lycopenoic acid in the diet (R2 = 0.9986, P < 0.001).


Figure 6
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  		Fig. 6. Effect of apo-10'-lycopenoic acid supplementation on NNK-induced lung tumor development in A/J mice. (A) Effect of apo-10'-lycopenoic acid supplementation on mouse body weight. (B) The plasma concentration of apo-10'-lycopenoic acid in mice supplemented with apo-10'-lycopenoic acid for 16 weeks. (C) Effect of apo-10'-lycopenoic acid on lung tumor multiplicity. Tumor nodules on the surface of mouse lung tissues were counted and recorded as tumor multiplicity (tumors per mouse). Values are presented as means ± SEMs, n = 12–14. Groups that do not share a letter are significantly different, P < 0.05.

 
Pulmonary pleural surface tumors induced in A/J mice were quantified and confirmed by examining hematoxylin–eosin-stained lung tissue sections under the microscope, based upon established criteria (33). At the time of sacrifice, only one lung tumor in each mouse was found in 2 of 13 mice in the CNTL group (tumor incidence = 15.4%, tumor multiplicity = 0.15 ± 0.38 tumors per mouse). Compared with the CNTL group, the group receiving the NNK injection alone had significantly higher tumor incidence (100%) and tumor multiplicity (15.8 ± 5.8 tumors per mouse) (P < 0.001). Treatment with apo-10'-lycopenoic acid did not affect NNK-induced lung tumor incidence; however, tumor multiplicity was dose dependently decreased from an average 16 tumors per mouse in the NNK injection alone group to an average 10, 7 and 5 tumors per mouse in groups injected with NNK and supplemented with apo-10'-lycopenoic acid at doses of 10, 40 and 120 mg/kg, respectively (Figure 6C), which represent 32.7, 53.6 and 65.4% declines in tumor number, as compared with the non-supplemented group (P < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, we explored the role of apo-10'-lycopenoic acid, a relatively stable derivative of apo-10'-lycopenal on the growth of cells derived from lung tissues. In order to study the effects of apo-10'-lycopenoic acid on different stages of lung carcinogenesis, we used three cell lines: NHBE, a primary human bronchial epithelial cell, BEAS-2B, a virus transformed, immortalized human bronchial epithelial cell line and A549, a human adenocarcinoma cell line. We found that apo-10'-lycopenoic acid inhibited the growth of all three cell lines, suggesting that growth inhibitory activity is an intrinsic property of apo-10'-lycopenoic acid. This growth inhibitory activity is largely due to the inhibition of cell proliferation, since treatment with apo-10'-lycopenoic acid blocked cell cycle progress in G1 phase, decreased expression of cyclin E and increased protein levels of p21 and p27. Cyclin E is a cell cycle regulator that binds and activates cyclin-dependent protein kinase 2, and therefore induces DNA synthesis and drives cell transition from G1 to S phase. In contrast, p21 and p27 negatively regulate cell cycle progress from G1 to S phase by binding to cyclin-dependent protein kinase 2 and inhibiting cyclin E–cyclin-dependent protein kinase 2 activity (34,35). We did not observe the induction of apoptosis or cytotoxicity by apo-10'-lycopenoic acid, which further supports the role of apo-10'-lycopenoic acid in inhibition of cell proliferation.

Although the more effective concentrations of apo-10'-lycopenoic acid with regard to inhibition of cell growth in vitro study were > 3 µmol/l, which are higher than the reported plasma lycopene concentration in humans [~1.5 µmol/l after the supplementation with lycopene or tomato product (36,37)], we have observed the growth inhibitory effects of apo-10'-lycopenoic acid at lower concentrations (0.5–1 µmol/l) when using the MTT method, a more sensitive method than the cell number counting. This suggests that the concentrations of apo-10'-lycopenoic acid used to inhibit cell growth in our in vitro study are ‘physiologically’ relevant. It has been shown that lycopene treatment down-regulates cyclin D but not cyclin E in different cell types (38–40). The discrepancies between lycopene and apo-10'-lycopenoic acid on cyclin D and E could be due to either the function of either lycopene itself or lycopene metabolites other than apo-10'-lycopenoic acid. For example, lycopene can be metabolized to apo-8'- and 12'-lycopenoids in rat liver (23) and other unknown compounds in ferret lungs after the lycopene supplementation (5,41). In addition, the different experimental designs and cell culture conditions may contribute to the discrepancies. For example, previous models that examined the effect of lycopene treatment on cyclin D and cyclin E expression were either induced by growth factor or stimulated by tobacco smoke condensate (38–40), whereas our study examined the effect of apo-10'-lycopenoic acid on expression of cell cycle regulators without those stimulators.

RARß tumor suppressor gene, containing an RARE in its promoter region, is a target gene of retinoid receptor signaling (25). RARß has been shown to partially mediate the functions of pro-vitamin A carotenoids (26,27) by interacting with carotenoid metabolites, such as retinoic acid (42) and apo-carotenoids (26). For example, apo-carotenoic acids from ß-carotene conversion inhibit lung cell proliferation by increasing RARß transcription via their further oxidation to retinoic acid (26). Lycopene is not a retinoid precursor; however, acycloretinoic acid, an open-chain analog of retinoic acid and a centric cleavage product of lycopene, was shown to transactivate RARs (19,20). In the present study, we showed that treatment with apo-10'-lycopenoic acid increased the mRNA levels of RARß in NHBE, BEAS-2B and A549 cells, which was associated with the increased expression of p21 and p27. Since the activation of RARß has been shown to inhibit cell growth and increase the expression of p21 (43–45) and p27 (46–48), we proposed that apo-10'-lycopenoic acid may function as retinoic acid analogue. Using reporter vectors containing different sizes of the RARß promoter, we consistently demonstrated that apo-10'-lycopenoic acid increased the transcriptional activity of the RARß promoter, which was not affected by deleting the region between –1500 and –124 bp. Our further observation that the mutation of the RARE motif located between –53 and –37 bp (25) totally abolished the transactivation activity of apo-10'-lycopenoic acid provides more evidence that lycopene metabolites may function as agonists for the retinoid receptors.

Whereas loss of RARß expression in various cancer cells has been associated with resistance to retinoic acid treatment (49,50), over-expression of RARß2 in retinoid-resistant human lung cancer cell lines sensitized these cells to retinoid-induced growth inhibition (51). It has been shown that cells decrease or lose their responsiveness to retinoid treatment as they progress from normal cells to premalignant and malignant cells (52,53). NHBE and BEAS-2B cells have been shown to be sensitive to the growth inhibitory effect of all-trans retinoic acid (54), whereas A549 non-small cell lung cancer cells were relatively resistant to the growth inhibitory effect of all-trans retinoic acid (50) and retinoic acid-induced RARß expression (49,50). Similarly, we showed a differential response of NHBE, BEAS-2B and A549 cells to the growth inhibitory effect of apo-10'-lycopenoic acid: A549 cells were more resistant to apo-10'-lycopenoic acid treatment as compared with NHBE and BEAS-2B cells. The lower sensitivity to the growth inhibitory effects was accompanied by the lower induction of RARß expression by apo-10'-lycopenoic acid treatment. These results further support our hypothesis that the inhibitory effect of apo-10'-lycopenoic acid on cellular proliferation may be mediated through retinoid signaling.

To further provide evidence that apo-10'-lycopenoic acid may be a potential chemopreventive agent against lung cancer, we carried out an in vivo study in the A/J mouse model to examine the effect of apo-10'-lycopenoic acid on tobacco-specific carcinogen NNK-induced lung tumorigenesis. We clearly demonstrated that supplementation with apo-10'-lycopenoic acid at 10, 40 and 120 mg/kg diet for 16 weeks dose dependently decreased the number of NNK-induced lung tumors in A/J mice. We did not examine the levels of apo-10'-lycopenoic acid in mouse lung tissues due to the lung tissue sample limitation (e.g. lung was used for the pathological examination). However, we did observe that the mouse plasma concentrations of apo-10'-lycopenoic acid at the time of sacrifice ranged from 1 to 17 nmol/l, and were correlated with the doses given in the diet. We have shown previously that lung levels of carotenoids accumulated after carotenoid treatment were much higher than those in plasma (5,55,56). This could help to explain why the concentration of apo-10'-lycopenoic acid to inhibit cell growth in our in vitro study was higher than that in plasma. On the other hand, the fact that apo-10'-lycopenoic acid is an enzymatic metabolite of lycopene (22) and that plasma levels of apo-10'-lycopenoic acid necessary to show a protective effect against lung tumors in mice were much lower than reported plasma lycopene concentrations in humans (0.1–1 µmol/l (57,58)] suggest that apo-10'-lycopenoic acid may, at least partially, mediate the chemopreventive activity of lycopene.

In the in vitro studies, we showed apo-10'-lycopenoic acid modulates the expression of p21, p27, cyclin D, cyclin E and RARß. We have also examined the mRNA expression of these genes in mouse lung tissues (data not shown). Due to relatively large variation of the data, we could not observe any statistically significant changes in gene expression among the five treatment groups. Two explanations for these negative results could be that (i) the anti-carcinogenic effect of apo-10'-lycopenoic acid may have occurred in the earlier stages of tumorigenesis, so that the changes on in these biomarkers could not be detected at the end of our experiment (14 weeks after NNK injection), and (ii) apo-10'-lycopenoic acid-induced changes in tumor regions could be masked by lack of change in the normal surrounding tissues when the whole-tissue homogenates were prepared for mRNA levels of these biomarkers. Meanwhile, we cannot exclude that anti-carcinogenic activity of apo-10'-lycopenoic acid in vivo may involve other mechanisms.

The potential use of lycopene or apo-10'-lycopenoic acid as chemopreventive agent against lung cancer demands careful investigation. The metabolism of lycopene in lung tissue is complicated, and may be affected by a number of environmental factors such as oxidative stress induced by cigarette smoking. For example, we have shown that high doses of ß-carotene in an oxidative environment (such as in the lungs of smokers) may result in excess levels of polar metabolites, which can promote carcinogenesis, whereas lower doses of ß-carotene have been shown to be protective (13,56). In the current study, we showed apo-10'-lycopenoic acid inhibits the growth of normal, premalignant and malignant lung cells in vitro and the formation lung tumors in vivo. Although no apparent adverse effects such as decrease in body weight or tissue damage were observed in apo-10'-lycopenoic acid-supplemented, NNK-treated A/J mice in this study, one previous study did show supplementation of lycopene enhanced benzo[a]pyrene-induced mutagenesis in mouse lung and colon tissues (7), suggesting that lycopene or lycopene metabolites may, as ß-carotene and its metabolites do, enhance lung carcinogenesis. Further investigation to study the dose effects of apo-10'-lycopenoic acid as well as the interaction between lycopene metabolism and cigarette smoke-exposure during lung carcinogenesis is needed.


   Acknowledgments
 
The authors thank Dr Heather Mernitz for help in the preparation of the manuscript. This work was supported by grant R01CA104932 from the National Institutes of Health, Bethesda, MD, and by the US Department of Agriculture, under agreement No. 58-1950-7-707. Any opinions, findings, conclusion or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the US Department of Agriculture.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 1, 2006; revised February 23, 2007; accepted March 28, 2007.


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C.-S. Huang, J.-W. Liao, and M.-L. Hu
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