ß-Carotene exacerbates DNA oxidative damage and modifies p53-related pathways of cell proliferation and apoptosis in cultured cells exposed to tobacco smoke condensate

Paola Palozza1,5, Simona Serini1, Fiorella Di Nicuolo1, Alma Boninsegna4, Angela Torsello1, Nicola Maggiano2, Franco O. Ranelletti3, Federica I. Wolf1, Gabriella Calviello1 and Achille Cittadini3

1 Institute of General Pathology, 2 Institute of Pathology and 3 Institute of Histology, Catholic University, Rome, Italy and 4 Centro di Ricerche Oncologiche Giovanni XXIII, Catholic University, Rome, Italy

5 To whom correspondence should be addressed Email: p.palozza{at}rm.unicatt.it


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human intervention trials have suggested that supplemental ß-carotene resulted in more cancer in smokers, whereas it was protective in non-smokers. However, the mechanisms underlying these effects are still unknown. The aim of this study was to evaluate the effects of an association of cigarette smoke condensate (tar) and ß-carotene on DNA oxidative damage and molecular pathways involved in cell cycle progression and apoptosis in cultured cells. In RAT-1 fibroblasts, tar caused increased levels of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and this effect was enhanced by the concomitant presence of ß-carotene (0.5–4.0 µM) in a dose- and time-dependent manner. In contrast, ß-carotene alone did not significantly modify it. Fibroblasts treated with tar alone decreased their cell growth with respect to control cells through an arrest of cell cycle progression in the G0/G1 phase and an induction of apoptosis. These effects were accompanied by an increased expression of p53, p21 and Bax and by a decreased expression of cyclin D1. In contrast, fibroblasts treated with tar and ß-carotene, after an initial arrest of cell growth at 12 h, re-entered in cell cycle and were unable to undergo apoptosis at 36 h. Concomitantly, their p53 expression, after an increase at 12 h, progressively returned at basal levels at 36 h by a mechanism independent of Mdm2. Such a decrease was followed by a decrease in p21 and Bax expression and by an increase in cyclin D1 expression. Moreover, the presence of the carotenoid remarkably enhanced cyclooxygenase-2 expression induced by tar. During tar treatment, a depletion of ß-carotene was observed in fibroblasts. The effects of tar and ß-carotene on 8-OHdG levels, cell growth and apoptosis were also observed in Mv1Lu lung, MCF-7 mammary, Hep-2 larynx and LS-174 colon cancer cells. This study supports the evidence for potential detrimental effects of an association between ß-carotene and cigarette smoke condensate.

Abbreviations: ATBC, Alpha-Tocopherol, Beta-carotene Cancer Prevention Trial; CARET, ß-Carotene and Retinol Efficacy Trial; DMSO, dimethyl sulfoxide; 8-OHdG, 8-hydroxyl-2'-deoxyguanosine; THF, tetrahydrofuran


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A consistent body of epidemiological data shows that increased consumption of ß-carotene-rich foods and higher blood levels of ß-carotene are associated with a reduced risk of cancer (1). Although ß-carotene supplementation has been shown to protect from cancer risk in non smokers (2), results from two intervention trials, the Alpha-Tocopherol, Beta-carotene Cancer Prevention Trial (ATBC) in Finland (3) and the ß-Carotene and Retinol Efficacy Trial (CARET) in USA (4), suggested that supplemental ß-carotene may result in more cancer in smokers. These findings aroused widespread scientific debate and raised the suspicion that this compound may even have carcinogenic properties. Several hypotheses have been proposed to explain the negative results of these clinical trials. In particular, ß-carotene has been suggested: (i) to be a potential promoter of pre-existing latent lung cancer in smokers (5); (ii) to decrease the absorption of other carotenoids with better antioxidant profile (5); (iii) to enhance lung function in smokers and therefore to permit more of the carcinogens and the reactive oxygen species to reach the smokers' lungs (6); (iv) to induce at high doses the formation of metabolites, which may cause diminished retinoic signaling by down-regulating RARß expression and up-regulating AP-1 with consequent possible acceleration of lung tumorigenesis (7). A further attractive hypothesis that may be put forth to explain the results of the CARET and ATBC trials is that ß-carotene really acts as a redox agent, behaving as an antioxidant in some circumstances (8) and as a prooxidant in others (913). According to this, conditions in the lungs of heavy smokers may tip the ß-carotene antioxidant-prooxidant balance toward a prooxidant state. Such a hypothesis is strongly supported by the fact that ß-carotene diminished its antioxidant properties or even exhibited a prooxidant character at high oxygen pressure (9,10). The relatively high partial pressure in the lung combined with the reactive oxygen species from tobacco smoke may be conducive for both ß-carotene oxidation and the formation of oxidative metabolites, which can act as propagators of free-radical formation (14). In this view, unoxidized ß-carotene may exert its anticarcinogenic effects at least partly by acting as an antioxidant, while ß-carotene oxidation products either may have no protective effects or actually may facilitate carcinogenesis. Since smokers have a greater flux of radicals in their lung tissues, it is probable that they would have a higher ratio of oxidized ß-carotene isomers to unoxidized ß-carotene, as suggested by Salgo et al. (15). Several findings are consistent with this hypothesis. Cigarette smoke contains an abundance of free radical species (1619). Exposure of plasma to cigarette smoke led to the destruction of carotenoids and {alpha}-tocopherol (6,20). The oxidation of ß-carotene by smoke has been reported to generate various oxidation products, including 4-nitro-ß-carotene, ß-apocarotenals and ß-carotene epoxides (7,21,22), some of which are unstable under conditions of oxidative stress and, therefore, can contribute to a further oxidation. Moreover, a prooxidant character of ß-carotene may be enhanced by a high carotenoid concentration. Mayne and colleagues observed that the high dose given as a supplement in ATBC and CARET trials resulted in carotenoid blood levels much higher (3.0 and 2.1 mg/l in ATBC and CARET, respectively) than those reported for the US population (0.05–0.5 mg/l) (1). Overproduction of free radical species by ß-carotene itself has been demonstrated in vitro as a consequence of high carotenoid concentrations (12). This effect may occur through different mechanisms, such as changes in cytochrome P450 enzymes (23) and in iron levels (24). However, at the moment, no clear-cut data are available to establish that a prooxidant mechanism may be directly implicated in the promotion of cell proliferation by a combination of ß-carotene and tobacco smoke.

In this study, we examined the effects of cigarette smoke condensate, alone and in association with ß-carotene, on DNA oxidation and cell growth. In particular, various cell lines of different origin, including immortalized fibroblasts, lung, mammary, larynx and colon cancer cells, were exposed to a combination of ß-carotene and tar (cigarette total particulate matter). Tar contains high concentrations of stable radicals; it is continually being deposited in the lungs of smokers and associates with DNA damage (19). Therefore, cells exposed to this mixture represent a suitable model to evaluate the detrimental effects of cigarette tar and the concomitant interactions with ß-carotene. After the exposure, several parameters were evaluated: (i) DNA oxidative damage measured as 8-hydroxyl-2'-deoxyguanosine (8-OHdG) formation; (ii) cell growth; (iii) cell cycle progression and cell cycle-related proteins, such as p53, p21, CD1 and Rb; (iv) apoptosis induction and apoptosis related proteins, such as Bax, Bcl-2 and Bcl-xL; (v) expression of cyclooxygenase-2 (COX-2), which has been recently proposed as a suitable marker of smoke-related carcinogenesis (25,26); and (vi) ß-carotene oxidation.

Our study suggests a new mechanism through which the potential carcinogenic role of an association between ß-carotene and cigarette smoke condensate may be explained. In particular, it indicates that ß-carotene may act as a prooxidant in tar-exposed cells by exacerbating DNA oxidative damage caused by tar alone. Such damage induces changes in p53-related pathways involved in cell proliferation and apoptosis and leads on to an increased cell growth.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
RAT-1 murine immortalized fibroblasts (American Type Culture Collection, Rockville, MD) and MCF-7 human mammary cancer cells (American Type Culture Collection) were grown in MEM medium without antibiotics supplemented with 10% fetal calf serum and 2 mM glutamine. Hep-2 human larynx carcinoma cells (American Type Culture Collection), LS-174 human colon adenocarcinoma cancer cell line (American Type Culture Collection) was cultured in RPMI 1640 medium (Gibco Biocult, Paisley, UK) without antibiotics supplemented with 10% fetal calf serum (Flow, Irvine, UK) and 2 mM glutamine. Mv1Lu lung carcinoma cells (American Type Culture Collection) were cultured in D-MEM medium without antibiotics supplemented with 10% fetal calf serum (Flow) and 2 mM glutamine. Cells were maintained in log phase by seeding twice a week at density of 3 x 105 cells/ml at 37°C under 5% CO2/air atmosphere. Tar was delivered to the cells as dimethyl sulfoxide (DMSO) solutions. The amount of DMSO given to the cells was not >0.1% (v/v). The final concentration given to the cells was 25 µg/ml. Such a concentration was the maximum one that did not induce necrotic effects in all the cells analyzed. ß-Carotene (Fluka Chemika-bioChemika, Buchs, Switzerland) was delivered to the cells (106 cells/ml) using tetrahydrofuran (THF) as a solvent, containing 0.025% butylated hydroxytoluene to avoid the formation of peroxides (23). The purity of ß-carotene was verified to be 97%. The stock solutions of ß-carotene were prepared immediately before each experiment. From the stock solutions, aliquots of ß-carotene were rapidly added to the culture medium to give the final concentrations indicated. The amount of THF added to the cells was not >0.1% (v/v). Control cultures received an amount of solvent (DMSO and THF) equal to that present in tar and ß-carotene-treated ones. No differences were found between cells treated with DMSO plus THF and untreated cells in terms of cell number, viability and 8-OHdG levels. After the addition of ß-carotene, the medium was not further replaced throughout the experiments, with the only exception of the experiments shown in Figure 7. Experiments were routinely carried out on triplicate cultures. At the times indicated, cells were harvested and quadruplicate haemocytometer counts were performed. The trypan blue dye exclusion method was used to evaluate the percentage of viable cells.



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Fig. 7. Relationship between p53 and Mdm2 expression in RAT-1 fibroblasts treated with tar, alone (A) and in combination with ß-carotene (B) for different periods of time. Tar was added to the cells at the concentration of 25 µg/ml and the carotenoid at the concentration of 1 µM.

 
Tar preparation
The particulate phase of cigarette smoke condensate (tar) was provided by the Ente Tabacchi Italiano (ETI, Rome, Italy). It was obtained by mechanically smoking cigarettes, using a smoking machine (Cerulean, ASM 516 model). Cigarettes were smoked using the puff profile (one 35-ml puff/min) to a butt length of 2.3 cm, as indicated by ETI protocol. The experimental conditions during smoking were: 22°C (temperature), 60% humidity. The tar from 20 cigarettes was trapped on filters and then extracted with deionized water. The aqueous solutions were filtered through a Whatman (0.2 µm) filter and dried in vacuo at room temperature. The dried cigarette total particulate matter was re-dissolved in DMSO and stored at –20°C.

Clonogenic assay
Clonogenic assay was performed as described by Conneally et al. (27). Briefly, the cells (8 x 103/ml) were suspended in a medium containing 0.8% methylcellulose (MethcultTM H4100, Stem Cell Technologies, Paris, France), 10% fetal calf serum, 2 mM L-glutamine in the absence or in the presence of the compounds indicated (1 µM ß-carotene, 25 µg/ml tar or a combination of both of them) and then plated in 35 mm Petri dishes. Colonies (aggregation of 30 or more cells) were scored in situ after 14 days of incubation at 37°C in a humidified atmosphere of 5% CO2 in air.

Assays for 8-OhdG
Cytospin samples were prepared as follows: cells were diluted in sucrose buffer (0.25 M sucrose, 1.8 mM CaCl2, 25 mM KCl, 50 mM Tris, pH 7.5) at a density of ~3.5 x 106 cells/ml. A total of 50 µl was added to carbowax–ethanol buffer (carbowax stock: 77 ml of PEG-1000 in 50 ml of water, 1 ml of stock in 74 ml of 70% ethanol) (Sigma, Italia, Milan, Italy) and mixed. Aliquots of 150 µl were placed into cytospin funnels and centrifuged at 300 r.p.m. for 5 min on slides coated with aminopropyl-triethoxysilane (Kindler, Freiburg, Germany). Samples were air-dried for 10–30 min, fixed in 95% cold ethanol (–20°C) for 10 min and stored at –20°C.

Detection of 8-OHdG by immunohistochemistry coupled with DAB (Vector, Burlingham, NY) was carried out essentially as described by Yarborough et al. (28). 1F7 monoclonal antibody for 8-OHdG was kindly provided by Dr R.M.Santella, Columbia School of Public Health, NY. Semi-quantitative evaluation of the staining was carried out by an optical microscope (ECLIPSE E600, Nikon, at 400x) connected to an Image-Pro plus Version 4.1 (Media Cybernetics, Silver Spring, MD). Nuclear staining was evaluated in ~100 cells of randomly chosen images by operators who were blind to the status of cell treatment, as recommended in ref. (28). Negative and positive controls (untreated and 0.5 mM H2O2-treated cells, respectively) were included within each batch of slides. Data are reported as units of optical density. Detection of 8-OHdG by a HPLC–ECD method (29) in preliminary experiments validated the results obtained by immunohistochemical analysis.

Cell cycle analysis
Cell cycle stage was analyzed by flow cytometry. Aliquots of 106 cells were harvested by centrifugation, washed in phosphate-buffered saline (PBS), fixed with ice-cold 70% ethanol and treated with 1 mg/ml RNAse for 30 min. Propidium iodide (PI) was added to a final concentration of 50 mg/ml. Data were collected, stored and analyzed using Multicycle software.

Apoptosis detection
Apoptosis was detected by an annexin V-FITC kit (Oncogene Research, Cambridge, MA), according to the manufacturer's instructions. Briefly, at the time indicated, cells were collected, washed with ice-cold PBS and centrifuged. Cells included both those harvested by tripsinization and those floating in the medium. The cell pellet was re-suspended in ice-cold binding buffer. After that, annexin V-FITC (1.25 µl/0.5 ml) and PI (10 µl/0.5 ml) solutions were added. The tube was incubated for 15 min in the dark, before being analyzed by flow-cytometry (Coulter Epics XL-MCL, Miami, FL) with a 620 nm-filter.

Western blot analysis of p53, Rb, p21WAF-1/CIP-1, Cyclin D1, Bax, Bcl-2, Bcl-xL, Mdm2, COX-2 expression
Cells (10 x 106) were harvested, washed once with ice-cold PBS and gently lysed for 30 min in ice-cold lysis buffer (1 mM MgCl2, 350 mM NaCl, 20 mM HEPES, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM Na3VO4, 1 mM PMSF, 1 mM aprotinin, 1.5 mM leupeptin, 20% glycerol, 1% NP-40). Cell lysates were centrifuged for 10 min at 4°C (10 000 g) to obtain the supernatants, which were used for western blot analysis with anti-p53 (clone DO-1, catalog no. SC-126, YLEM, Rome, Italy), anti-Rb (clone C15, catalog no. SC-50, Santa Cruz Biotechnology, Santa Cruz, CA), anti-p21 WAF-1/CIP-1 (clone F-5, catalog no. SC-6246, Santa Cruz Biotechnology), anti-CD1 (clone 72-13G, catalog no. SC-450, Santa Cruz Biotechnology), anti-Bcl-2 (clone Bcl-2/100/D5 YLEM), anti-Bcl-xL S/L (clone L-19, catalog no. SC-1041, Santa Cruz Biotechnology), anti-Bax (clone P-19, catalog no. SC-526, Santa Cruz Biotechnology), anti Mdm-2 (clone SMP14, catalog no. SC-965, Santa Cruz Biotechnology) and anti-Cox-2 (clone C-20, catalog no. 1745, Santa Cruz Biotechnology) monoclonal antibodies. The blots were washed and exposed to horseradish peroxidase-labeled secondary antibodies (Amersham Pharmacia Biotech, Arlington Heights, IL) for 45 min at room temperature. The immunocomplexes were visualized by the enhanced chemiluminescence detection system and quantified by densitometric scanning.

Extraction and analysis of ß-carotene
ß-Carotene was extracted with 1 vol methanol and 3 vol hexane:diethyl ether (1:1) from 10 x 106 cells after 24 h treatment with 1 µM ß-carotene in the absence or in the presence of tar and analyzed by HPLC, as described earlier (12).

Statistical analysis
Three separate cultures per treatment were utilized for analysis in each experiment. Values were presented as means ± SEM. Multifactorial two-way analysis of variance (ANOVA) was adopted to assess any differences among the treatments, the times and/or the concentrations. When significant values were found (P < 0.05), post hoc comparisons of means were made using the Tukey's Honestly Significant Differences test. One-way ANOVA was used to determine differences between treatment with tar alone and in combination with ß-carotene in Table IV and Figure 2. When significant values were found (P < 0.05), post hoc comparisons of means were made using Fisher's test. Differences were analyzed using Minitab Software (Minitab, Inc., State College, PA).


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Table IV. Changes in cell number, 8-OHdG levels and apoptosis in different cell lines treated for 36 h with a combination of TAR (25 µg/ml) and ß-carotene (1 µM)

 


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Fig. 2. Effects of tar and ß-carotene, alone and in combination, on the growth of RAT-1 fibroblasts. (A) The growth of cells treated with tar at the concentration of 25 µg/ml and various ß-carotene concentrations for 36 h. (B) The percentage of the increase in cell number, as calculated by the values shown in (A). (C) The growth of cells treated with tar (25 µg/ml) and with 1 µM ß-carotene for different periods of time. The values were the means ± SEM, n = 10. In (A), values not sharing a letter were significantly different (P < 0.05, Fisher's test). In (C), the treatment/time interaction was significant (P < 0.05). Values not sharing a letter were significantly different (P < 0.002) (Tukey's test).

 

    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
8-OH-dG formation and cell growth
It is well known that cigarette smoking generates a large number of free radicals and 8-OHdG has been used frequently as a biomarker for oxidative DNA damage caused by smoke (30). In fact, the levels of 8-OHdG have been shown to correlate with mutagenesis and carcinogenesis in which oxidative damage is involved as causative mechanisms (31,32). To assess oxidative changes on DNA induced by tar and ß-carotene, we measured the levels of 8-OHdG in RAT-1 fibroblasts treated with tar (25 µg/ml) and various ß-carotene concentrations for 36 h (Figure 1A and B) and tar (25 µg/ml) and ß-carotene at the concentration of 1 µM for different periods of time (Figure 1C). A significant increase in 8-OHdG-specific immunoreactivity was detected in fibroblasts treated with tar. In contrast, ß-carotene alone did not significantly modify 8-OHdG levels. The combination of tar and ß-carotene significantly enhanced the formation of 8-OHdG with respect to tar alone (Figure 1A).



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Fig. 1. Levels of 8-OHdG in RAT-1 fibroblasts. (A) The levels of 8-OHdG in cells treated with tar (25 µg/ml) and various ß-carotene concentrations for 36 h. (B) The percentage of the increase in 8-OHdG by the combination of tar and different doses of the carotenoid with respect to tar, as calculated by the values shown in (A). (C) The levels of 8-OHdG in cells treated with tar (25 µg/ml) and ß-carotene at the concentration of 1 µM for different periods of time. The values were the means ± SEM, n = 10. The treatment/concentration (A) and treatment/time (C) interactions were significant (P < 0.05). Values not sharing a letter were significantly different (A, P < 0.001; C, P < 0.002) (Tukey's test).

 
The percentage of the increase in 8-OHdG by the combination of tar and different doses of the carotenoid with respect to tar alone is shown in Figure 1B. This effect was significantly dose-dependent in a range of carotenoid concentration from 0.75 to 4 µM (Figure 1B). The oxidative DNA damage was time-dependent and clearly observed at 6 h of treatment. It increased progressively up to 24 h and it remained evident up to 72 h of treatment (Figure 1C). To determine whether the association of smoke and ß-carotene also resulted in changes of cell growth, we investigated the effects of tar and ß-carotene, alone and in combination, on RAT-1 fibroblasts cell growth. Fibroblasts received tar at the concentration of 25 µg/ml. When indicated, they were also treated with various ß-carotene concentrations for 36 h (Figure 2A and B) and with 1 µM ß-carotene for different periods of time (Figure 2C). As shown in Figure 2A, fibroblasts treated with combinations of tar and ß-carotene increased their cell number with respect to cells treated with tar alone in a dose-dependent manner, at least in a range of carotenoid concentrations from 0.75 to 4 µM. Higher concentrations of the carotenoid (from 5.0 to 10 µM) in association with tar were toxic for the cells and a progressive dose-dependent loss of cell viability was observed (data not shown). On the other hand, the carotenoid alone up to 4 µM did not significantly modify cell growth with respect to vehicle control cells, while it inhibited it starting from 5 µM (data not shown). The percentage of the increase in cell number by the combination of tar and different doses of the carotenoid with respect to tar alone is shown in Figure 2B. As shown in Figure 2C, RAT-1 fibroblasts treated with tar alone decreased their cell growth with respect to control cells. Such an effect started at 12 h and was maintained for all the time of incubation (72 h). On the other hand, the combination of tar and ß-carotene induced a marked increase in cell growth, which became significant at 36 h and was maintained until 72 h (Figure 2C).

Similar effects of tar and ß-carotene, alone and in combination, on the growth of RAT-1 fibroblasts were obtained using clonogenic assay as indicated in the Materials and methods section. In the different treatments, the number of colonies was: 161 ± 10 (control cells); 150 ± 9 (ß-carotene-treated cells); 108 ± 10 (tar-treated cells) and 168 ± 10 (tar- and ß-carotene-treated cells). This number of colonies in tar plus ß-carotene-treated cultures was significantly (P < 0.01) higher than in cultures treated with tar alone.

Cell cycle progression
To elucidate the mechanism(s) responsible for the increase in cell number by the treatment with the combination of tar and ß-carotene, we first examined the effects of these compounds on cell cycle progression. Table I shows the cell cycle distribution of RAT-1 fibroblasts incubated in the absence or in the presence of tar (25 µg/ml), ß-carotene (1 µM) and a combination of both of them for 12 and 36 h. In the absence of tar and ß-carotene, most RAT-1 fibroblasts were in G0/G1 phase. No significant differences were observed in cells treated with ß-carotene alone. However, in the presence of tar, we observed a net increase in the percentage of cells in G0/G1 phase, which was maintained throughout the treatment (36 h). The G0/G1 phase accumulation was accompanied by a corresponding reduction in the percentages of cells in S phase. The combined addition of tar and ß-carotene to the cells induced an increase in the percentage of cells in G0/G1 only at 12 h of treatment, which was followed by a late decrease (36 h). The latter effect was accompanied by an increase in the percentage of cells in S phase, which was also observed prolonging the incubation time until 72 h (data not shown).


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Table I. Effects of ß-carotene and TAR on cell cycle distribution of RAT-1 fibroblasts

 
Apoptosis induction
Table II shows the percentages of apoptotic RAT-1 fibroblasts following the different treatments for 12 and 36 h, as measured by annexinV-FITC method. Tar (25 µg/ml) treatments significantly induced apoptosis, as evidenced by the increase in the percentage of apoptotic cells at 36 h of treatment. ß-Carotene (1 µM) alone did not significantly modify the percentage of apoptosis with respect to control cells. Surprisingly, the combination of tar and ß-carotene did not affect it. No differences in the percentage of apoptotic cells were found between control and ß-carotene-treated cells at the concentration of the carotenoid employed in this study.


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Table II. Effects of ß-carotene and TAR, alone or in combination, on apoptosis in RAT-1 fibroblasts

 
Expression of the cell cycle-regulating proteins p53 and pRb
Since the tumor-suppressor protein p53 has been shown to down-regulate cell proliferation following treatments with DNA-damaging agents (33,34), we measured the expression of p53 during the exposure to tar and ß-carotene in RAT-1 fibroblasts (Figure 3). Tar-treated cells exhibited a higher expression of p53 than control cells at both 12 and 36 h (Figure 3A). This effect started at 9 h, became remarkable at 12 h and it was maintained for all the period of incubation (Figure 3B). On the other hand, cells treated with the combination of tar and ß-carotene showed an increase in p53 expression, which started at 9 h, reached its maximum at 12 h, but was followed by a late decrease at 36 h (Figure 3B). Prolonging the incubation up to 48 h, no further changes were observed.



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Fig. 3. Effect of tar, alone and in combination with ß-carotene, on the expression of p53 in RAT-1 fibroblasts. Tar was added to the cells at the concentration of 25 µg/ml and the carotenoid at the concentration of 1 µM. (A) Representative western blot analysis. (B) Densitometric analysis of the autoradiographs. The values were the means ± SEM, n = 5. The interaction time/treatment was significant (P < 0.05). Values not sharing a letter differed, P < 0.005 (Tukey's test).

 
It has been reported that the phosphorylation of Rb is another key regulatory event during G1 phase (35). In its hypophosphorylated state, Rb binds and inhibits the E2F family of transcription factor, and this inhibition is released upon Rb phosphorylation, which in turn elicits S-phase initiation (35). Therefore, we evaluated Rb expression in RAT-1 fibroblasts following treatment with tar and ß-carotene. In these cells, both the active hypophosphorylated form and the inactive hyperphosphorylated (slower migrating) forms of Rb were present. No significant differences in the expression of both the forms of Rb were found among the different groups during the 48 h of treatment (data not shown), suggesting that this protein is not involved in the effects of tar, alone and/or in combination with the carotenoid, on cell growth.

Expression of p53-related proteins involved in cell cycle progression and apoptosis
In an attempt to explore the effects of tar and ß-carotene on cell cycle-regulating proteins, we examined the expression of p21WAF/CIP1, a cyclin-dependent kinase (CDK) inhibitor (36) and CD1, which plays a regulatory role during G1 phase of the cell cycle (37) (Figure 4). It has been suggested that the modulation of the levels of such proteins may contribute to p53-mediated G1 growth arrest (38). In RAT-1 fibroblasts, the modulation of p21WAF/CIP1 expression by both tar and tar plus ß-carotene was similar to that of p53 expression. In particular, the combined addition of tar induced an increase in the expression of these proteins at 12 h followed by a decrease at 36 h.



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Fig. 4. Representative western blot analysis of the expression of p21 and cyclin D1 in RAT-1 fibroblasts treated with tar, alone and in combination with ß-carotene, for 12 and 36 h. Tar was added to the cells at the concentration of 25 µg/ml and the carotenoid at the concentration of 1 µM.

 
Moreover, RAT-1 fibroblasts treated with tar alone decreased their CD1 expression, while fibroblasts treated with the combination of tar and ß-carotene showed an initial decrease of the cyclin expression at 12 h followed by a late increase at 36 h. In an effort to investigate the molecular pathway involved in apoptosis induction by tar and ß-carotene, we also examined the effects of these two compounds, alone and in combination, on the expression of the apoptosis-blocking proteins Bcl-2 and Bcl-xL, and the apoptosis promoter protein Bax, which is known to be modulated by p53 (Figure 5). Tar increased the expression of Bax at 36 h in RAT-1 fibroblasts, whereas ß-carotene alone was ineffective. However, the presence of the carotenoid completely abolished the ability of tar to induce Bax expression at 36 h in RAT-1 cells. In contrast, neither Bcl-2 nor Bcl-xL expression was significantly modified by tar and/or ß-carotene, alone and in association.



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Fig. 5. Representative western blot analysis of the expression of Bax, Bcl-2 and Bcl-xL in RAT-1 fibroblasts treated with tar, alone and in combination with ß-carotene, for 12 and 36 h. Tar was added to the cells at the concentration of 25 µg/ml and the carotenoid at the concentration of 1 µM.

 
Expression of the p53-regulating protein Mdm2
Since p53 expression is known to be regulated by Mdm2 protein, which blocks its transcriptional activity by exporting of the protein into the cytoplasm, and/or promotes its degradation (39,40), we measured Mdm2 expression of this protein in RAT-1 fibroblasts treated with tar (25 µg/ml) and ß-carotene, alone and in combination (Figure 6). In our investigation, no significant differences were found among the different groups at 12 h.



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Fig. 6. Representative western blot analysis of the expression of Mdm2 in RAT-1 fibroblasts treated with tar, alone and in combination with ß-carotene, for 12 and 36 h. Tar was added to the cells at the concentration of 25 µg/ml and the carotenoid at the concentration of 1 µM.

 
However, prolonging the time of incubation up to 36 h, an increase in the expression of Mdm2 was found only in ar-treated cells, but not in cells treated with a combination of the two compounds. The relationship existing between p53 and Mdm2 expression is shown in Figure 7. While tar-treated fibroblasts showed an over-expression of p53 followed by enhanced levels of Mdm2, tar plus ß-carotene-treated cells did not do it, suggesting that the decrease in p53 expression occurs by a mechanism independent of Mdm-2.

Reversibility of the effects of tar plus ß-carotene on cell growth and p53 status
In order to establish whether the effect of the combined addition of tar and ß-carotene on cell growth still persisted following a further exposure to tar alone, we re-treated RAT-1 cells, exposed previously to a 36-h treatment with tar, alone and in combination with ß-carotene, with a new addition of tar (25 µg/ml) for 12 h and we measured cell growth (panel A) and p53 expression (panel B) (Figure 8). Tar-treated fibroblasts showed a further arrest of cell growth, while tar plus ß-carotene-treated ones did not do it. These data support the hypothesis that cells treated with tar and ß-carotene in combination may undergo a persistent loss of some cellular regulatory functions of p53.



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Fig. 8. Effect of a new addition of tar (25 µg/ml) for 12 h on cell number (A) and p53 expression (B) in RAT-1 fibroblasts, exposed previously to a 36-h treatment with tar (25 µg/ml), alone and in combination with ß-carotene (1 µM). In (A), the values were the means ± SEM, n = 5. In (B), representative western blot analysis.

 
Expression of COX-2 as a marker of smoke-induced damage
Since it has been reported recently that cigarette smoke condensate induced the expression of COX-2 (25) and that the levels of COX-2 can be considered a suitable marker in vivo for cancer progression (26), we examined the expression of this protein in RAT-1 fibroblasts following treatment with ß-carotene and tar for 12 h (Figure 9). Tar alone increased the expression of COX-2, while ß-carotene alone decreased it. On the other hand, the combination of ß-carotene and tar markedly increased COX-2 expression. This effect was maintained prolonging the incubation time up to 72 h (data not shown).



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Fig. 9. Representative western blot analysis of the expression of Cox-2 in RAT-1 fibroblasts treated with tar, alone and in combination with ß-carotene, for 12 h. Tar was added to the cells at the concentration of 25 µg/ml and the carotenoid at the concentration of 1 µM.

 
ß-Carotene incorporation and oxidation
ß-Carotene was incorporated and/or associated to RAT-1 fibroblasts. This effect was clearly observed at 6 h of treatment (data not shown) and became maximum at 24 h. At this time, the carotenoid reached the concentration of 200 ± 18 pmol/106 cells. A loss of ß-carotene was observed in RAT-1 fibroblasts during the incubation. However, the concomitant presence of tar caused an increased significant depletion of the carotenoid levels (Table III).


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Table III. Consumption of ß-carotene by TAR in RAT-1 fibroblasts

 
Effects of tar plus ß-carotene on cell growth, DNA damage and apoptosis in other cell lines
The effects of tar and ß-carotene, alone and in combination, on cell growth, DNA oxidative damage and apoptosis were also studied in human tumor cells of epithelial origin, including lung, mammary, larynx and colon cancer cells (Table IV). In all of them, a 36-h treatment with the combination of tar (25 µg/ml) and ß-carotene (1 µM) induced an increase in cell number and in the levels of 8-OHdG with respect to the treatment with tar alone and a concomitant decrease in the percentage of apoptotic cells. On the other hand, the carotenoid alone did not modify cell growth and did not significantly protect cells from oxidative DNA damage when compared with control cells (data not shown). These results were in perfect agreement with those observed in RAT-1 fibroblasts.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mechanism(s) by which ß-carotene may result in an increased lung cancer incidence and mortality among smokers (3,4), but not in non-smokers (2) has yet to be defined. In this study, we suggest a new mechanism through which the potential carcinogenic role of an association between ß-carotene and cigarette smoke may be explained. In particular, we hypothesized that the deleterious effects of ß-carotene and cigarette smoke observed in clinical trials may be due to a possible prooxidant action of the carotenoid, which exacerbates DNA oxidative damage caused by cigarette smoke condensate and which induces changes in p53-related pathways involved in cell proliferation and apoptosis. According to this hypothesis, our work with different cell lines demonstrates that tar caused increased levels of 8-OHdG, one of the most critical lesions generated from deoxyguanosine in DNA by free radical species (30,31), and that this effect was significantly more pronounced in the presence of ß-carotene. In the presence of tar, the carotenoid was able to significantly increase DNA damage at the concentrations of 0.75–4.0 µM. Such concentrations are exactly in the range of ß-carotene levels found in the serum of subjects who received ß-carotene as a supplement. In particular, Mayne and colleagues observed that subjects from ATBC and CARET trials reached blood levels of ß-carotene of 5.6–3.9 µM, respectively (1). On the other hand, in our investigation, ß-carotene per se up to 4.0 µM did not induce any oxidative DNA damage, although an increased formation of 8-OHdG was observed at higher carotenoid concentrations (from 5 to 10 µM). These data are in agreement with a previous study (41). ß-Carotene and lycopene at relatively low concentrations (1–3 µM) increased DNA resistance to oxidative damage in HepG2 cells, while they completely lost such an ability and even became prooxidant agents at higher concentrations (4–10 µM) (41). The observation that, in our study, ß-carotene in combination with tar became a prooxidant at relatively low concentrations with respect to the carotenoid alone may be explained by an impairment of the endogenous antioxidant defences caused by tar. It is well known that cigarette smoke lowers levels of antioxidants in plasma and cells (20,42,43) and that the carotenoid may be protected from its oxidation by the concomitant presence of endogenous antioxidants (10). In our study, it is clearly evidenced a decreased cell content of ß-carotene in the presence of tar. Although it is possible that tar affects the concentration of the carotenoid simply reducing its entry into the cells, several studies show that tobacco smoke is able to oxidize carotenoid molecules, forming different oxidation products (7,21,22). Some of these could be responsible for the tumor-promoting activity observed in our experimental conditions. Recently, it has been reported that ß-carotene, in its oxidized form, may be an inducer of oxidative DNA damage and may act as a procarcinogenic agent (15,44). This hypothesis has been also proposed by Wang and Russell with respect to the apparent procarcinogenic effects of high-dose ß-carotene supplements in ferrets (45). Carotenoid oxidation products could arise in the free radical-rich environment in the lungs of cigarette smoke-exposed ferrets.

In a subsequent paper, the same group observed that low-dose ß-carotene supplementation in ferrets appeared to be protective with respect to squamous cell proliferation in the lung (46), whereas high-doses of ß-carotene increased lung metaplasia (7). The tumor-promoting activity of ß-carotene in combination with tar did not seem to be related only to the presence of dimethylbenz[a]anthracene, an important initiator of carcinogenesis in tar mixture. In fact, when this compound was used alone, in the absence of 12-tetradecanoylphorbol 13-acetate, ß-carotene failed to act as a tumor promoter agent in a two stage-model of skin carcinogenesis (47). A consensus has emerged that p53 responds to the types of stress signals that can cause oncogenic alterations, such as DNA damage or conditions that arise in developing tumor cells, such as abnormal proliferation or hypoxia (32,33). Activation of p53 by these stress signals rapidly inhibits cell growth, by arresting proliferation and/or inducing apoptosis, thereby preventing the propagation of cells that may be undergoing malignant transformation. In our work, a clear relationship appeared to exist between the extent of oxidative DNA damage induced by tobacco smoke and expression of p53. Cells exposed to tar significantly increased their p53 content and such an effect was maintained for all the time of incubation. An association between p53 over-expression and smoking has been described in the early stage of some cancers (48,49). In addition, it has been reported that hamster tracheal epithelial cells exposed to benzo[a]pyrene, a well-known carcinogen present in tobacco, deeply increased their p53 expression (50). However, our investigation shows that cells exposed to tar in combination with ß-carotene increased their DNA damage and progressively lost their ability to maintain high levels of p53. Such effects still persisted following a second exposure to tar alone.

Recent studies have indicated that the modulation of CD1 and p21WAF/CIP1 expression may contribute to p53-mediated G1 growth arrest (38). CD1 plays a key regulatory role during G1 phase of the cell cycle and its gene is amplified and over-expressed in many cancers (37). p21WAF/CIP1, a CDK inhibitor and downstream effector of p53-mediated growth suppression, is capable of linking the DNA damage response pathway with the cell-cycle machinery, contributing to DNA repair and maintaining genomic integrity (36). In this study, the over-expression of p53 by tar was accompanied by an increase in p21 expression, suggesting that in our model p21 is regulated through a p53-dependent pathway. On the other hand, a negative relationship between p53 and CD1 expression was observed in tar-treated cells. The discovery of substantial and sustained decreased cyclin D1 expression as well as the observation of increased levels of p21 as a consequence of tobacco smoke exposition have been reported recently (51,52). In contrast, in agreement with p53 expression, the p53-related genes p21 and CD1 progressively became unresponsive in cells treated with the combination of tar and the carotenoid. However, the treatment with ß-carotene alone, at least at the concentrations used in this study, did not significantly modify the expression of both these proteins, although some authors reported a modulation of them by carotenoids (51). In particular, it has been reported in neoplastic cultured cells that lycopene at low concentrations (2–3 µM) down-regulates cyclin D and that a 1-week-treatment with ß-carotene over-expressed cyclin D1/2 in lung of mice (51). Moreover, in normal human fibroblasts, ß-carotene induced an increase in the amount of p21 accompanied by an inhibition of cyclin D1-associated cdk4 kinase activity (53). Similarly, the carotenoid increased p21 expression in HL-60 cells (13).

We reported that, in our model, tar was also able to modulate the expression of the Bcl-2 family proteins, which are known to control apoptosis. In particular, it increased the expression of the pro-apoptotic protein Bax, without affecting the expression of both the anti-apoptotic proteins Bcl-2 and Bcl-xL. It has been reported recently that acute cigarette smoke exposure induces apoptosis of alveolar macrophages, through over-expression of Bax, although this effect seems to be independent of p53 (54). On the other hand, the concomitant presence of tar and ß-carotene into the cells did not influence Bax expression, presumably because of the low levels of p53 protein, which is known to regulate it.

As a consequence of the changes in the above-mentioned cell growth-related genes, cells exposed to tar exhibited a different growth with respect to cells treated with the combination of tar and ß-carotene. The former were altered in their DNA and first blocked their cell cycle progression in G0/G1 phase and then increased their apoptosis. The latter, although damaged in their DNA, after an initial block in G0/G1, re-entered in cell cycle and were unable to undergo apoptosis. Moreover, they increased their risk for cancer development, as demonstrated by their enhanced expression of COX-2. Preliminary data from our laboratory seems to show that the promotion of cell growth by an association of tar and ß-carotene was specific for this carotenoid, since lycopene strongly enhanced the inhibitory effects of tar on the growth of RAT-1 fibroblasts (data not shown). This observation is in agreement with recent findings, showing that ß-carotene (7), but not lycopene (55) enhanced the potential harmful effects of tobacco smoke in ferrets.

Although it seems clear from our study that p53 plays a key role in modulating molecular pathways involved in cell cycle arrest and in apoptosis induction as a consequence of the oxidative DNA damage induced by tar exposure, it still remains unclear why such a protein undergoes a decrease following the treatment with the combination of tar and ß-carotene, even though cells still showed sustained DNA oxidative damage. A complex network of proteins has been reported to regulate p53 levels (39). One of these proteins is Mdm-2, which is known to be responsible for p53 degradation through a feedback mechanism dependent on high p53 levels (40). While in our study tar-treated fibroblasts showed an over-expression of p53 followed by enhanced levels of Mdm2, tar plus ß-carotene-treated cells did not exert the same effect, suggesting that the decrease in p53 expression occurs by a mechanism independent of Mdm-2. Since it has been reported recently that NAD(P)H-quinone oxidoreductase-1 is involved in enhancing the stability of p53, especially in response to oxidative stress (56), it could be extremely interesting to verify the role of such a protein in cells exposed to a combination of cigarette smoke condensate and ß-carotene. Our study provides a rationale for the surprising results of human trials of ß-carotene in smokers. In those trials, smokers who took ß-carotene had a higher relative risk for lung cancer than those on placebo. We reported that ß-carotene might act as a prooxidant in tar-exposed cells by exacerbating DNA oxidative damage caused by tar alone. Such damage induces changes in p53-related pathways involved in cell proliferation and apoptosis and leads on to an increased cell growth. However, we wish to point out some important caveats in using our data in this way. First, our study was done in cell cultures using a specific preparation of cigarette tar (cigarette total particulate matter) and, therefore, our results should not be extrapolated to humans, without more work with in vitro and in vivo models. Secondly, different results from ours could be obtained by using carotenoids other than ß-carotene, such as lycopene and lutein.

Thus, this study may represent the starting point for further work on the relationship between ß-carotene and cigarette smoke in cancer.


    Acknowledgments
 
This work was supported by MURST-ex 60%. We wish to thank Dr Alfredo Nunziata of Ente Tabacchi Italiano (ETI) for kindly providing cigarette tar and Prof. N.I.Krinsky for his helpful suggestions in revising the manuscript.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 15, 2003; revised March 3, 2004; accepted March 7, 2004.