![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
B DNA-Binding Activity by a Redox Mechanism in Human Leukemia and Colon Adenocarcinoma Cells1
Institute of General Pathology, Catholic University, Rome, Italy and * Section of Toxicology and Biomedicine, Ente per le Nuove Tecnologie, l’Energia e l’Ambiente, C.R. Casaccia, Rome, Italy
2To whom correspondence should be addressed. E-mail: p.palozza{at}rm.inicatt.it.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
B may be involved in the growth-inhibitory and proapoptotic effects of the carotenoid. To test this hypothesis, human leukemic cells (HL-60) and colon adenocarcinoma cells (LS-174 and WiDr) were treated with ß-carotene, alone or in combination with 
-tocopherol or N-acetylcysteine, and changes in 1) cell oxidative status, 2) cell growth and apoptosis, 3) DNA-binding activity of NF-B and 4) expression of c-myc, a NF-B target gene involved in apoptosis, were evaluated. In HL-60 cells, ß-carotene induced a significant increase in reactive oxygen species (ROS) production (P < 0.001) and in oxidized glutathione (GSSG) content (P < 0.005) at concentrations 
10 µmol/L. These effects were always accompanied by a sustained elevation of NF-B and by a significant inhibition (P < 0.002) of cell growth. NF-B DNA-binding activity increased at 3 h and persisted for at least 48 h. Colon adenocarcinoma cells displayed substantial differences in their sensitivity to ß-carotene, exhibiting increased ROS levels and activation of NF-B at concentrations much lower in LS-174 cells (2.5–5.0 µmol/L) than in WiDr cells (50–100 µmol/L). In all cell lines studied, -tocopherol and N-acetylcysteine inhibited the effects of ß-carotene on NF-B, cell growth and apoptosis, and normalized the increased expression of c-myc induced by the carotenoid. These data suggest that the redox regulation of NF-B induced by ß-carotene is involved in the growth-inhibitory and proapoptotic effects of the carotenoid in tumor cells.
KEY WORDS: • ß-carotene • NF-B • redox status • cell growth • apoptosis
Compelling evidence from epidemiologic studies (1
), clinical intervention trials (1
) and laboratory experiments (2
) has suggested that carotenoids may control cell growth and may also play a role in carcinogenesis. Whether they act as anticarcinogenic or as procarcinogenic agents is debatable. The majority of epidemiologic studies have consistently and clearly shown that increased consumption of fruit and vegetables rich in carotenoids is associated with a decreased incidence of various cancers (1
). In contrast, results from recent intervention studies, the Beta-Carotene and Retinol Efficacy Trial [CARET; (3
,4
)] and the Alpha-Tocopherol, ß-Carotene Cancer Prevention Study [ATBC; (5
)], indicated that exposure of subjects taking supplemental ß-carotene to cigarette smoke increased lung cancer incidence. A number of authors have attempted to explain why ß-carotene may exhibit such procarcinogenic activity under these conditions. In particular, Mayne and colleagues (6
) suggested that ß-carotene may act as a promoter of carcinogenesis through the generation of relatively high amounts of deleterious oxidation products (typically epoxides) of ß-carotene brought about by exposure to reactive oxygen species (ROS)3 found in tobacco smoke. Recently, Wang and Russell reported that ß-carotene may undergo oxidation, producing oxidative metabolites that modulate cytochrome P450 activity and abrogate retinoid signaling (7
). More recently, we hypothesized that carotenoids may modulate cell growth by acting as intracellular redox agents (8
). They could alternatively behave as antioxidant or as prooxidant molecules, depending on their redox potential and on the cellular environment. We linked ß-carotene exposure to production of ROS in both cultured human colon adenocarcinoma (9
) and leukemia (10
) cells and we reported that such a production was highly coincident with the ability of ß-carotene to induce apoptosis and cell cycle arrest (9
,10
).
ROS have been reported to control signal transduction cascades, acting as key regulatory switches in many cellular processes involved in cell growth. The delicate balance between oxidants and antioxidants ultimately determines the activity profile for many transcription factors. Although the precise molecular mechanism has yet to be elucidated, the nuclear factor (NF)-B pathway is generally thought to be a primary oxidative stress-response pathway (11
–16
). Several laboratories have demonstrated that treatment of cells with H2O2 can activate the NF-B pathway (17
). The observation that inducers of NF-B activity, such as tumor necrosis factor (TNF), interleukin (IL)-1, lipopolysaccharide (LPS), phorbol myristate acetate (PMA), UV and ionizing radiation, generated elevated levels of ROS prompted speculation that ROS may function as common mediators of NF-B activation. Further support for this model was provided by indirect evidence showing that nearly all pathways leading to NF-B activation could be blocked by a variety of antioxidants, including pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine (NAC), reduced glutathione (GSH) or by overexpression of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase or thioredoxin peroxidase (13
–15
).
This study examined the effects of ß-carotene and known antioxidants, such as -tocopherol and NAC, on DNA-binding activity of NF-B in tumor cells with differing origins and differing degrees of differentiation. Knowing that ß-carotene treatment influences the intracellular oxidation state in cultured cells and that NF-B is redox regulated, we sought to determine whether ß-carotene stimulates the binding activity of this transcription factor and whether antioxidants can influence this effect. Moreover, we also evaluated the effect of the carotenoid on the known NF-B target gene c-myc, which has been implicated in apoptosis (18
).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The human promyelocytic leukemia HL-60 cell line was maintained in log phase by seeding twice a week at density of 3 x 108 cells/L. Cells were routinely cultured in RPMI 1640 medium (Sigma Italia, Milan, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies Italia, Milan, Italy) at 37°C in 5% CO2/air atmosphere. HL-60 cells were induced to differentiate to neutrophil granulocytes by culturing the cells in a medium containing 13 g/L dimethyl sulfoxide (DMSO; Sigma) for 5 d (19
). The agent was subsequently removed and cell cultures utilized after 24 h. The differentiated status was verified by morphological inspection (smaller nucleus-to-cytoplasm ratio and a more irregular shape) and by a respiratory-burst assay performed by measuring superoxide production after stimulation with PMA (results not shown) (20
). In our preparations, 
95% of cells were considered fully differentiated. In contrast, no evidence of spontaneous terminal differentiation was observed in DMSO-untreated HL-60 cells. WiDr (kindly provided by Dr. S. D. Showalter, National Cancer Institute, Frederick, MD) and LS-174 (American Type Culture Collection, CCL 220.1, Rockville, MD) human adenocarcinoma cancer cell lines were cultured in RPMI 1640 medium (Gibco Biocult, Paisley, UK), without antibiotics supplemented with 10% fetal calf serum (Flow, Irvine, UK) and 2 mmol/L glutamine. They were maintained in log phase by seeding twice a week at density of 3 x 108 cells/L at 37°C under 5% CO2/air atmosphere. ß-Carotene (Fluka Chemika-bioChemika, Buchs, Switzerland) and dl--tocopherol (Fluka) were delivered to the cells (109 cells/L) using tetrahydrofuran (THF) as a solvent, containing 0.25 g/L BHT to avoid the formation of peroxides (21
). The purity of ß-carotene and that of -tocopherol was verified to be 97 and 98%, respectively. The stock solutions of ß-carotene (2 and 20 mmol/L) and those of -tocopherol (10, 25 and 50 mmol/L) were prepared immediately before each experiment. From the stock solutions, aliquots of ß-carotene and/or -tocopherol were rapidly added to the culture medium to give the final concentrations indicated. In both the ß-carotene and -tocopherol experiments, the amount of THF added to the cells was not >0.5% (v/v). Control cultures received an amount of THF equal to that present in ß-carotene– and/or -tocopherol–treated culture media. Because no differences were found between cells treated with different amounts of THF containing BHT and untreated cells in terms of cell number, viability, ROS production and NF-B DNA-binding activity, controls referred to untreated cells. After the addition of ß-carotene, -tocopherol or NAC, the medium was not further replaced throughout the experiments. Experiments were routinely carried out on triplicate cultures. At the times indicated, cells were harvested and quadruplicate hemocytometer counts were performed. The trypan blue dye exclusion method was used to evaluate the percentage of viable cells. All of the experiments were performed at ß-carotene and antioxidant concentrations that did not affect cell viability.
Measurement of ROS.
Cells (109 cells/L) treated with varying ß-carotene concentrations for 24 h were harvested to evaluate cellular peroxides and hydroxyl radical levels using the di(acetoxymethyl ester) analog (C-2938) of 6-carboxy-2',7'-dichlorofluouescein (DCF) (Molecular Probes, Eugene, OR) as described (22
). Before the addition of the fluorescent probes, media were removed to eliminate the amounts of ß-carotene and/or antioxidants that were not cell associated. Fluorescent units were measured in each well after a 30-min incubation with DCF (10 µmol/L) by use of a Cytofluor 2300/2350 Fluorescence Measurement System (Millipore, Bedford, MA). ß-Carotene and the antioxidants without cells, alone or in combination, did not alter the basal fluorescence of DCF.
Glutathione assay.
For the analysis of total and oxidized glutathione, 10–50 x 106 cells were homogenized in ice with HClO4 (1 mol/L)-EDTA (2 mmol/L) and centrifuged at 1000 x g for 10 min at 4°C. The supernatant, neutralized with NaOH, was extracted and analyzed by HPLC as indicated (23
). Chromatography was carried out with a LC-18 Supelcosil column, 25 cm x 0.46 cm, 5-µm particle size (Supelco, Bellefonte, PA). A C-18 Supelcosil precolumn, 2 cm x 0.46 cm, 5-µm packing, was used. The mobile phase was 7.5% methanol/92.5% acetate buffer (0.15 mol/L, pH 7). The flow rate was 1.5 mL/min. Peaks were detected by fluorescence measurement at 420 nm after excitation at 340 nm. GSH and oxidized glutathione (GSSG) concentrations in samples were derived by comparing the derivative peak area to a standard curve generated by derivatizing known amounts of GSH.
Extraction and analysis of ß-carotene and -tocopherol.
-Tocopherol and ß-carotene were extracted from 10 x 106 cells and analyzed by HPLC, as described earlier (24
).
Nuclear extracts and electrophoretic mobility-shift assay.
Frozen cell pellets were processed to obtain nuclear extracts. Briefly, 12 x 106 cells were collected, washed twice and pelleted by 200 x g centrifugation for 10 min. The pellet was resuspended in 440 µL cold buffer A [20 mmol/L HEPES, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT), 1 mmol/L Na4P2O7, 20 mmol/L NaF and 1 mmol/L polymethylsulfonyl fluoride, 1.5 mmol/L aprotinin] by gentle pipetting. The cells were allowed to swell on ice for 15 min; then 25 µL 10% NP40 was added and the tube was vortexed vigorously for 10 s. The homogenate was centrifuged for 30 s at 20,000 x g. The nuclear pellet was resuspended in 50 µL ice-cold buffer B (20 mmol/L HEPES, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L Na4P2O7, 20 mmol/L NaF and 1 mmol/L polymethylsulfonyl fluoride, 1.5 mmol/L aprotinin, 20% v/v glycerol) and the tube was vigorously rocked for 15 min at 4°C on a shaking platform. The nuclear extract was centrifuged for 5 min at 20,000 x g at 4°C.
Binding reactions containing 5 µg nuclear extracts, 10 mmol/L Tris-HCl (pH 7.6), 5% glycerol, 1 mmol/L EDTA, 1 mmol/L DTT, 50 mmol/L NaCl, and 3 mg poly(dI-dC) were incubated for 30 min with 5000 cpm of 32P-end-labeled double-stranded oligonucleotide in a total volume of 20 mL. The probe was 5'-AGTTGAGGGGACTTTCCCAGGC3'. Labeling of the probe was obtained by incubating 5 pmol of oligonucleotide with 10 pmol [
-32P]ATP and 3 UT4 polynucleotide kinase for 30 min at 37°C. The probe was then purified with MicroBIO-Spin P-30 columns. Complexes were separated on 60 g/L polyacrylamide gels with 45 mmol/L Tris-borate, 1 mmol/L EDTA, pH 8 buffer. After fixation and drying, gels were exposed on phosphor screens, which were then analyzed by phosphor/fluorescence imager STORM 840 (Molecular Dynamics, Sunnyvale, CA). The intensity of the revealed bands was directly quantified by Image QuaNT software (Molecular Dynamics).
Analysis of NF-B (p50 and p65)-associated proteins.
Nuclear extracts, 25–30 µg of protein, were separated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis with the use of 40–120 g/L Bis-Tris gels (NOVEX, San Diego, CA) and transferred to Immobilon-P membranes (Millipore) with the use of a semidry system. Immunoblots were blocked overnight at 4°C in 50 g/L dried milk in PBS, pH 7.4 plus 0.05% Tween 20. Blots were incubated with polyclonal primary antibodies to p-50 (NLS, sc 114, lot # E030, Santa Cruz Biotechnology, Santa Cruz, CA) and p-65 (C20, sc 372, lot # J250, Santa Cruz Biotechnology) in PBS plus 0.05% Tween 20 for 1–2 h at room temperature. The blots were washed and exposed to a horseradish peroxidase–labeled secondary antibody (Amersham Pharmacia Biotech, Arlington Heights, IL) for 45 min at room temperature. After incubation with secondary antibodies, the immunocomplexes were visualized by the enhanced chemiluminescence detection system and quantified by densitometric scanning.
Apoptosis detection.
Apoptosis was detected with an annexin V-fluorescein isothiocyanate (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 (300 x g). The cell pellet was resuspended in ice-cold binding buffer. After that, annexin V-FITC (1.25 µL/0.5 mL) and propidium iodide (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.
Analysis of c-myc 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 mmol/L MgCl2, 350 mmol/L NaCl, 20 mmol/L HEPES, 0.5 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DDT, 1 mmol/L Na4P2O7, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L aprotinin, 1.5 mmol/L leupeptin, 20% glycerol, 1% NP40). Cell lysates were centrifuged for 10 min at 4°C (10,000 x g) to obtain the supernatants, which were used for Western blot analysis with anti-c-myc monoclonal antibody (SC-40, lot # K141, Santa Cruz Biotechnology). The blots were washed and exposed to a horseradish peroxidase–labeled secondary antibody (Amersham Pharmacia Biotech) for 45 min at room temperature. After incubation with the secondary antibody, the immunocomplexes were visualized by the enhanced chemiluminescence detection system and quantified by densitometric scanning.
Statistical analysis.
Three separate cultures per treatment were utilized for analysis in each experiment. Values were presented as means ± SEM. Multifactorial two-way ANOVA was adopted to assess any differences among the treatments and among the concentrations (Fig. 1
, Tables 1
and 2
) and between the presence and absence of different treatments (Figs. 4
and 7)
. When the F-tests were significant (P < 0.05), post-hoc comparisons of means were made using Tukey’s Honestly Significant Differences test. One-way ANOVA was used to determine differences in ß-carotene or -tocopherol concentrations in Figure 2
. When the F-tests were significant (P < 0.05), post-hoc comparisons of means were made using Fisher’s test. Data were analyzed using Minitab Software (Minitab, State College, PA).
|
|
|
|
|
|
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
B activity in HL-60 leukemic cells.
In HL-60 cells, the treatment with ß-carotene alone significantly increased ROS levels (P < 0.001) and decreased cell growth (P < 0.002) in a dose-dependent manner (Fig. 1A and B
). From a mechanistic point of view, the growth-inhibitory effects of ß-carotene were highly coincident with the carotenoid-mediated ROS production. In contrast, growth and ROS levels of HL-60 cells were unaffected by treatment with -tocopherol alone (Fig. 1
C and D). When ß-carotene, at concentrations ranging from 10 to 30 µmol/L, was added in combination with -tocopherol, its growth-inhibitory and prooxidant effects were prevented. This prevention was complete at -tocopherol concentrations >5 µmol/L. Interestingly, the increase in ROS production by ß-carotene in HL-60 cells occurred very early because it was already evident at 3 h. It was also long lasting, persisting for at least 48 h and paralleling the effects of the carotenoid on cell growth (data not shown).
The treatment with ß-carotene was also able to decrease GSH content significantly (P < 0.001) and increase (P < 0.005) that of GSSG (Table 1
). These changes were higher at 20 than at 10 µmol/L ß-carotene. The combined addition of ß-carotene and -tocopherol completely prevented the changes in GSH status induced by the carotenoid at the concentrations tested.
ß-Carotene and -tocopherol were both incorporated into HL-60 cells after a 24 h-treatment. At this time, the amount of ß-carotene was 0.75 ± 0.05 nmol/106 cells, using 20 µmol/L ß-carotene. This concentration represented 4% of the total amount of the carotenoid present in the medium. The concomitant presence of -tocopherol significantly (P < 0.05) increased ß-carotene concentration in the cells (Fig. 2A
). On the other hand, we found that after a 24 h-treatment, the amount of -tocopherol in the cells was 0.67 ± 0.05 nmol/106 cells, using the antioxidant at 25 µmol/L. The concomitant presence of ß-carotene induced a significant (P < 0.05) dose-dependent decrease in the concentration of intracellular -tocopherol (Fig. 2
B).
DNA binding of nuclear extracts of HL-60 cells to the NF-B consensus sequence was elevated by a 24-h treatment with ß-carotene. This effect was dose dependent and occurred at high concentrations of the carotenoid (Fig. 3A
). Doses of ß-carotene up to 5 µmol/L had no stimulatory effects on DNA binding. NF-B DNA-binding activity was induced by 20 µmol/L ß-carotene at 3 h and remained elevated for at least 48 h (Fig. 3
B), paralleling the increase in ROS production induced by the carotenoid (data not shown). It should be also pointed out that DNA-binding activity by tumor necrosis factor- at the NF-B site in HL-60 cells was similar in magnitude to that caused by high concentrations of the carotenoid (data not shown). The addition of -tocopherol lowered ß-carotene–induced DNA binding at the NF-B site in HL-60 cells treated for 24 h (Fig. 3
C). On the other hand, ß-carotene, at concentrations of 5–20 µmol/L, did not increase the levels of NF-B (p50 and p65)-associated proteins in HL-60 cells treated for 24 h (Fig. 3
D), suggesting that the increased DNA-binding was due to a change in activity and not to an increased protein level.
|
), NF-B DNA-binding activity (Fig. 4
B) and cell growth (Fig. 4
C) were also inhibited by the preexposure of cells to NAC. Interestingly, the extent of inhibition of ROS production seems to be proportional to the extent of inhibition of cell growth and NF-B DNA-binding activity.
Redox-regulation of NF-B activity in other tumor cell lines.
Even in DMSO-differentiated HL-60 cells, ß-carotene treatment was able to enhance the intracellular ROS production, although this effect was seen only at the concentration of 40 µmol/L (Table 2
). The addition of -tocopherol completely reversed the increase in ROS production induced by the carotenoid. The prooxidant effect of ß-carotene was also accompanied by NF-B activation, which was prevented by -tocopherol addition (Fig. 5
).
|
B was observed at ß-carotene concentrations responsible for prooxidant effects (Fig. 6
).
|
B target gene c-myc and its relationship with apoptosis.
In HL-60 cells, a 24-h carotenoid treatment was also able to increase c-myc expression (Fig. 7A
) and to induce apoptosis (Fig. 7
B). The increased expression of c-myc occurred very early because it was already apparent at 3 h. It was also long lasting, persisting for at least 48 h and paralleling the effects of the carotenoid on NF-B activation (data not shown). The concomitant addition of -tocopherol blocked the effects of ß-carotene on both c-myc expression and apoptosis.
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
B. Moreover, we reported that such an effect was abrogated in cells loaded with known antioxidants, such as -tocopherol and NAC.
In our cell models, the ability of ß-carotene to act as a redox agent was clearly shown by an increase in both ROS production and in GSSG/GSH ratio. These effects were dose dependent and were accompanied by an inhibition of cell growth. The ability of ß-carotene to act as an oxidant stress agent was reported previously in several cell models (25
–28
). The carotenoid was able to increase H2O2-induced chromosomal aberrations in Chinese hamster ovary cells (29
). In addition, it altered susceptibility to H2O2 in Caco 2 cells (30
). Moreover, it was able to enhance the endogenous oxyradical production in human adenocarcinoma cells (9
). In all of these studies, prooxidant effects were observed only at relatively high concentrations of the carotenoid. Also in this study, the ß-carotene concentrations in the media exceeded the range of serum concentrations observed in humans supplemented with low doses of the carotenoid (3
–5
). However, it should be noted that the concentrations responsible for the prooxidant and the growth-inhibitory effects of ß-carotene in this in vitro study may be reached in vivo in the serum of humans supplemented with high doses of the carotenoid (31
,32
). In particular, Nierenberg and co-workers (31
) reported that supplementation with 50 mg/d of ß-carotene in humans resulted in plasma ß-carotene concentrations of up to 16.1 µmol/L. Moreover, in another study, Prince and Frisoli demonstrated that human subjects ingesting various doses of ß-carotene as supplements (from 51 mg/d to 102 mg three times each day) could have serum steady-state concentrations of the carotenoid that ranged from 2-13.2 µmol/L (32
). Moreover, our data show that the concentrations of ß-carotene responsible for increased oxyradical production depend mainly on cell type. In fact, ß-carotene acted as a prooxidant in LS-174 cells at concentrations ranging from 2.5 to 5 µmol/L. In WiDr cells, the same behavior was observed, but at concentrations of the carotenoid ranging from 50 to 100 µmol/L. Interestingly, in these cells, the carotenoid exhibited antioxidant properties at concentrations lower than those reported above (9
). The different cell sensitivity to ß-carotene may be explained by the different ability of the cells to incorporate the carotenoid. We recently reported that LS-174 cells incorporated greater amounts of ß-carotene into their membranes with respect to WiDr cells (33
). In addition, other factors may enhance the prooxidant character of the molecule. It should be noted that DMSO-differentiated HL-60 cells were less sensitive to the prooxidant effects of the carotenoid than the respective undifferentiated cells. It has been reported that during differentiation, HL-60 cells became more resistant to oxidative stress because they caused an increase in the content and/or the activity of antioxidants or modified the cell distribution of antioxidants (34
–36
). Interestingly, DMSO-differentiated cells have been reported to possess oxidative properties similar to those found in normal neutrophils (19
).
This observation suggests that the prooxidant character of ß-carotene could outweigh its antioxidant character in highly malignant tumor cells. In agreement with the hypothesis that an impairment of endogenous antioxidants may favor the prooxidant character of the carotenoid in the cells, the addition of -tocopherol and NAC at sufficient doses prevented the prooxidant effects of the carotenoid. Our finding suggests that -tocopherol may be consumed to retard the degradation of ß-carotene to oxidation products. Interactions between ß-carotene and tocopherols in oxidative processes have been previously reported. -Tocopherol may protect ß-carotene from autooxidation, as demonstrated by Handelman et al. (37
), and suggested by Kennedy and Liebler (38
). Moreover, as reported by Terao et al., 
-tocopherol enhanced the protective effects of ß-carotene on 1O2-initiated photooxidation of methyl linoleate (39
). Similarly, -tocopherol prevented the prooxidant effects of ß-carotene in a purified triacylglycerol fraction of rapeseed oil exposed to light (40
). Finally, -tocopherol may protect ß-carotene during free radical–induced lipoperoxidation in microsomal membranes (41
). Although little is known about the mechanisms of these interactions, it has been demonstrated that tocopherols may reduce carotenoid radical cations formed during oxidative processes (42
).
Our study clearly showed that the cell growth–inhibitory effects of ß-carotene may occur via a mechanism involving increased production of ROS and activation of NF-B. Increasing evidence has indicated that intracellular redox status and ROS can function as components of signal transduction cascades, acting as key regulatory switches in many cellular processes involved in cell growth (43
). The delicate balance between oxidants and antioxidants, such as glutathione and thioredoxin, ultimately determines the activity profile for many transcription factors involved in cell proliferation and apoptosis. The NF-B pathway is generally thought to be a primary oxidative stress-response pathway (11
–16
). In agreement with the hypothesis of a redox-regulation of NF-B, in this study we demonstrated that ß-carotene, administrated at doses found to induce growth-inhibitory and prooxidant effects, increased the DNA-binding activity of nuclear proteins at NF-B site in leukemic as well as in colon adenocarcinoma cells. Interestingly, consonant with ROS production, NF-B activation occurred at carotenoid concentrations higher in differentiated than in undifferentiated HL-60 cells and in WiDr than in LS-174 cells. Moreover, decreased NF-B DNA-binding activity was observed at carotenoid concentrations responsible for antioxidant effects in LS-174 cells.
NF-B exists in an inactive form bound to inhibitors of B (IB) proteins in the cytoplasm and thus does not require de novo protein synthesis for activation (17
). Upon activation, IB is degraded and the active NF-B complex is translocated to the nucleus (12
). Redox regulation of NF-B is thought to be modulated in part through a conserved cysteine residue in the p50 subunit and through IB release. Western blot analyses indicated no substantial change in p50 or p65 protein levels in the nuclear extracts of ß-carotene–treated cells, indicating that the increased DNA-binding by the carotenoid was due to a change in activity and not to an increased protein level. Therefore, the possible explanation for ß-carotene–induced increases in NF-B DNA-binding activity is that the carotenoid induced oxidative alteration of the proteins themselves (e.g., by oxidative modulation of sulfydryl groups or phosphorylation status). The ability of treatment with -tocopherol and NAC to diminish both ß-carotene–induced NF-B DNA-binding activity and DCF fluorescence in tumor cells further suggests that oxidative stress contributes to the regulation of this activity. Our observations are in agreement with other studies, showing that nearly all pathways leading to NF-B activation were blocked by a variety of antioxidants, including PDTC, NAC, glutathione, thioredoxin, or by overexpression of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase or thioredoxin peroxidase (13
–15
). NF-B is not sensitive to superoxide radical, but it is most potently induced by hydrogen peroxide (17
). In addition, DCF fluorescence has been reported to detect mainly H2O2 (44
). These observations suggest that H2O2 may be deeply involved in the NF-B activation by ß-carotene. However, other oxyradical species may be involved in the activation of NF-B by the carotenoid. Indeed, it has been suggested that DCF is able to detect a broad range of oxidizing free radical species (45
). On the other hand, Shi et al. (46
) recently demonstrated that hydroxyl radicals were primarily responsible for activation of NF-B in Jurkat cells. Therefore, further studies are required to elucidate the species of ROS responsible for ß-carotene–induced NF-B activation and to establish the subcellular localization of ROS generation by the carotenoid. Interestingly, it was suggested recently that ß-carotene was also able to modulate the activation of AP-1 (7
), another redox-sensitive transcription factor involved in the regulation of cell growth (11
). It was reported recently that NF-B is activated by certain apoptotic stimuli and that some of NF-B target genes, such as c-myc, are involved in apoptosis induction (18
). Our data showing the activation of NF-B and the increased expression of c-myc by ß-carotene seem to be particularly interesting in view of our previous findings of proapoptotic effects of the carotenoid in tumor cells (9
,10
,33
).
In summary, our study demonstrates that one of the possible mechanisms responsible for the control of cell growth by ß-carotene may be the redox regulation of NF-B and the consequent expression of proteins, such as c-myc, involved in apoptosis induction.
|
FOOTNOTES |
|---|
3 Abbreviations used: DCF, dichlorofluorescein; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GSH, reduced glutathione; GSSG, oxidized glutathione; IB, inhibitory B; IL, interleukin; LPS, lipopolysaccharide; NAC, N-acetylcysteine; NF-B, nuclear factor B; PDTC, pyrrolidine dithiocarbamate; PI, propidium iodide; PMA, phorbol myristate acetate; ROS, reactive oxygen species; THF, tetrahydrofuran; TNF, tumor necrosis factor.
Manuscript received 5 September 2002. Initial review completed 3 October 2002. Revision accepted 4 November 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Mayne, S. T. (1996) ß-Carotene, carotenoids, and disease prevention in humans. FASEB J. 10:690-701.[Abstract]
2. Gerster, H. (1995) ß-Carotene, vitamin E and vitamin C in different stages of experimental carcinogenesis. Eur. J. Clin. Nutr. 49:155-168.[Medline]
3. Omenn, G. S., Goodman, G. E., Thornquist, M. D., Balmes, J., Cullen, M. R., Glass, A., Keogh, J. P., Meyskens, F. L., Jr, Valanis, B., Williams, J. H., Jr, Barnhart, S. & Hammar, S. (1996) Effects of a combination of ß-carotene and vitamin A on lung cancer and cardiovascular disease. N. Engl. J. Med. 334:1150-1155.
4. Omenn, G. S., Goodman, G. E., Thornquist, M. D., Balmes, J., Cullen, M. R., Glass, A., Keogh, J. P., Meyskens, F. L., Jr, Valanis, B., Williams, J. H., Jr, Barnhart, S. & Hammar, S. (1996) Risk factors for lung cancer and for intervention effects in CARET, the Beta-Carotene and Retinol Efficacy Trial. J. Natl. Cancer Inst. 88:1550-1559.
5. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group (1994) The effect of vitamin E and ß-carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med. 330:1029-1035.
6. Mayne, S. T., Handelman, G. J. & Beecher, G. (1996) ß-Carotene and lung cancer promotion in heavy smokers—a plausible relationship?. J. Natl. Cancer Inst. 88:1513-1515.
7. Wang, X. D., Liu, C., Bronson, R. T., Smith, D. E., Krinsky, N. I. & Russell, R. M. (1999) Retinoid signaling and Activator Protein-1 expression in ferrets given ß-carotene supplements and exposed to tobacco smoke. J. Natl. Cancer Inst. 91:60-66.
8. Palozza, P., Serini, S., Di Nicuolo, F. & Calviello, G. (2001) Mitogenic and apoptotic signaling by carotenoids: involvement of a redox mechanism. IUBMB Life 52:1-5.
9. Palozza, P., Calviello, G., Serini, S., Maggiano, N., Lanza, P., Ranelletti, F. O. & Bartoli, G. M. (2001) ß-Carotene at high concentrations induces apoptosis by enhancing oxy-radical production in human adenocarcinoma cells. Free Radic. Biol. Med. 30:1000-1007.[Medline]
10. Palozza, P., Serini, S., Torsello, A., Boninsegna, A., Covacci, V., Maggiano, M., Ranelletti, F. O., Wolf, F. I. & Calviello, G. (2002) Regulation of cell cycle progression and apoptosis by ß-carotene in undifferentiated and differentiated HL-60 leukemia cells: possible involvement of a redox mechanism. Int. J. Cancer 97:593-600.[Medline]
11. Schulze-Osthoff, K., Los, M. & Baeuerle, P. A. (1995) Redox signalling by transcription factors NF-B and AP-1 in the immune system. Biochem. Pharmacol. 50:735-741.[Medline]
12. Sen, C. H. & Packer, L. (1996) Antioxidant and redox regulation of gene transcription. FASEB J. 10:709-720.[Abstract]
13. Flohé, L., Brigelius-Flohé, R., Saliou, C., Traber, M. G. & Packer, L. (1997) Redox regulation of NF-kappa B activation. Free Radic. Biol. Med. 22:1115-1126.[Medline]
14. Ginn-Pease, M. E. & Whisler, R. L. (1998) Redox signals and NF-B activation in T cells. Free Radic. Biol. Med. 25:346-361.[Medline]
15. Mercurio, F. & Manning, A. M. (1999) NF-B as a primary regulator of the stress response. Oncogene 18:6163-6171.[Medline]
16. Bowie, A. & O’Neill, L. A. J. (2000) Oxidative stress and nuclear factor-B activation. Biochem. Pharmacol. 59:13-23.[Medline]
17. Baeuerle, P. A. & Henkel, T. (1994) Function and activation of NF-B in the immune system. Annu. Rev. Immunol. 12:141-179.[Medline]
18. La Rosa, F. A., Pierce, J. W. & Sonenshein, G. E. (1994) Differential regulation of the c-myc oncogene promoter by the NF-B Rel family of transcription factors. Mol. Cell. Biol. 14:1039-1044.
19. Klinder, J. F., Wenzel-Seifert, K. & Seifert, R. (1996) G-Protein-coupled receptors in HL-60 human leukemia cells. Gen. Pharmacol. 27:33-54.[Medline]
20. Scatena, R., Nocca, G., De Sole, P. & Giardina, B. (1996) Monocytes oxygenation activities are significantly influenced by non-enzymatic glycosylation of collagen. J. Biolumin. Chemilumin. 11:207-212.[Medline]
21. Bertram, J. S., Pung, A., Churley, M., Kappock, , IV, T. J., Wilkins, L. R. & Cooney, R. V. (1991) Diverse carotenoids protect against chemically induced neoplastic transformation. Carcinogenesis 12:671-678.
22. Kane, D. J., Sarafian, T. A., Anton, R., Hahn, H., Gralla, E. B., Valentine, J. S., Örd, T. & Bredesen, D. E. (1993) Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science (Washington, DC) 262:1274-1277.
23. Neuschwander-Tetri, B. A. & Roll, F. J. (1989) Glutathione measurement by high-performance liquid chromatography separation and fluorimetric detection of the glutathione-orthophtaldehyde adduct. Anal. Biochem. 179:236-241.[Medline]
24. Palozza, P., Calviello, G., Maggiano, N., Lanza, P., Ranelletti, F. O. & Bartoli, G. M. (2000) ß-Carotene antagonizes the effects of eicosapentaenoic acid on cell growth and lipid peroxidation in WiDr adenocarcinoma cells. Free Radic. Biol. Med. 28:228-234.[Medline]
25. Burton, G. W. & Ingold, K. U. (1984) ß-Carotene: an unusual type of lipid antioxidant. Science (Washington, DC) 224:569-573.
26. Palozza, P. (1998) Prooxidant actions of carotenoids in biologic systems. Nutr. Rev. 56:257-265.[Medline]
27. Schwartz, J. L. (1996) The dual roles of nutrients as antioxidants and prooxidants: their effect on tumor cell growth. J. Nutr. 126:1221S-1227S.
28. Young, A. J. & Lowe, G. M. (2001) Antioxidant and prooxidant properties of carotenoids. Arch. Biochem. Biophys. 385:20-27.[Medline]
29. Cozzi, R., Ricordy, R., Aglitti, T., Gatta, V., Perticone, P. & De Salvia, R. (1997) Ascorbic acid and ß-carotene as modulators of oxidative damage. Carcinogenesis 18:223-228.
30. Bestwick, C. S. & Milne, L. (2000) Effects of ß-carotene on enzyme activity, intracellular reactive oxygen and membrane integrity with post confluent Caco-2 intestinal cells. Biochim. Biophys. Acta 1474:47-55.[Medline]
31. Nierenberg, D. W., Stukel, T. A., Baron, J. A., Dain, B. S. & Greenberg, E. R. (1991) Determinants of increase in plasma concentrations of ß-carotene after chronic oral supplementation. Am. J. Clin. Nutr. 53:1443-1449.
32. Prince, M. R. & Frisoli, J. K. (1993) Beta-carotene accumulation in serum and skin. Am. J. Clin. Nutr. 57:175-181.
33. Palozza, P., Serini, S., Maggiano, M., Angelini, M., Boninsegna, A., Di Nicuolo, F., Ranelletti, F. O. & Calviello, G. (2002) Induction of cell cycle arrest and apoptosis in human colon adenocarcinoma cell lines by ß-carotene through down-regulation of cyclin A and Bcl-2 family proteins. Carcinogenesis 23:11-18.
34. Reiners, J. J., Jr, Thai, G., Pavone, A., Rupp, T. & Kodari, E. (1990) Modulation of catalase activities in murine epidermal cells as a function of differentiation and exposure to 12-O-tetradecanoylphorbol-13-acetate. Carcinogenesis 11:957-953.
35. Shen, Q., Chada, S., Whitney, C. & Newburger, P. (1994) Regulation of the human cellular glutathione peroxidase gene during in vitro myeloid and monocytic differentiation. Blood 84:3902-3908.
36. Steinkuhler, C., Sapora, O., Carri, M. T., Nagel, W., Marcocci, L., Circolo, M. R., Weser, U. & Rotilio, G. (1991) Increase of Cu, Zn-superoxide dismutase activity during differentiation of human K562 cells involves activation by copper of a constantly expressed copper-deficient protein. J. Biol. Chem. 266:24580-24587.
37. Handelman, G. J., van Kuijk, F.J.G.M., Chatterjee, A. & Krinsky, N. I. (1991) Characterization of products formed during the autooxidation of ß-carotene. Free Radic. Biol. Med. 10:427-433.[Medline]
38. Kennedy, T. A. & Liebler, D. C. (1991) Peroxyl radical oxidation of ß-carotene: formation of ß-carotene epoxides. Chem. Res. Toxicol. 4:290-295.[Medline]
39. Terao, J., Yamauchi, R., Murakami, H. & Matsushita, S. (1980) Inhibitory effects of tocopherols and ß-carotene on singlet oxygen-initiated photooxidation of methyl linoleate and soybean oil. J. Food Process Preserv. 4:79-93.
40. Haila, K. & Heinonen, M. (1994) Action of ß-carotene on purified rapeseed oil during light storage. Lebensm.-Wiss. Technol. 27:573-577.
41. Palozza, P. & Krinsky, N. I. (1992) ß-Carotene and -tocopherol are synergistic antioxidants. Arch. Biochem. Biophys. 297:184-187.[Medline]
42. Mortensen, A., Skibsted, L. H. & Truscott, T. G. (2001) The interaction of dietary carotenoids with radical species. Arch. Biochem. Biophys. 385:13-19.[Medline]
43. Nakamura, H., Nakamura, K. & Yodoi, J. (1997) Redox regulation of cellular activation. Annu. Rev. Immunol. 15:351-369.[Medline]
44. Vowells, S. J., Sekhsaria, S., Malech, H. L., Shalit, M. & Fleisher, T. A. (1995) Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of the fluorescent probes. J. Immunol. Methods 178:89-97.[Medline]
45. Hempel, S. L., Buettner, G. R., O’Malley, Y. Q., Wessels, D. A. & Flaherty, D. M. (1999) Dihydroflurescein diacetate is superior for detecting intracellular oxidants: comparison with 2', 7'-dichlorodihydrofluorescein diacetate, 5 (and 6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, and dihydrhodamine 123. Free Radic. Biol. Med. 27:146-159.[Medline]
46. Shi, X., Dong, Z., Huang, C., Ma, W., Liu, K., Ye, J., Chen, F., Leonard, S. S., Ding, M., Castranova, V. & Vallyathan, V. (1999) The role of hydroxyl radical as a messenger in the activation of nuclear transcription factor NF-kappa B. Mol. Cell. Biochem. 194:63-70.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. Palozza, R. Sestito, N. Picci, P. Lanza, G. Monego, and F. O. Ranelletti The sensitivity to {beta}-carotene growth-inhibitory and proapoptotic effects is regulated by caveolin-1 expression in human colon and prostate cancer cells Carcinogenesis, November 1, 2008; 29(11): 2153 - 2161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. K. Chun, S.-J. Chung, K. J. Claycombe, and W. O. Song Serum C-Reactive Protein Concentrations Are Inversely Associated with Dietary Flavonoid Intake in U.S. Adults J. Nutr., April 1, 2008; 138(4): 753 - 760. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. B. Janakiram, I. Cooma, A. Mohammed, V. E. Steele, and C. V. Rao -Ionone inhibits colonic aberrant crypt foci formation in rats, suppresses cell growth, and induces retinoid X receptor-{alpha} in human colon cancer cells Mol. Cancer Ther., January 1, 2008; 7(1): 181 - 190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Senesse, M. Touvier, E. Kesse, J. Faivre, and M.-C. Boutron-Ruault Tobacco Use and Associations of {beta}-Carotene and Vitamin Intakes with Colorectal Adenoma Risk J. Nutr., October 1, 2005; 135(10): 2468 - 2472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Nkondjock, P. Ghadirian, K. C. Johnson, D. Krewski, and the Canadian Cancer Registries Epidemiology Resear Dietary Intake of Lycopene Is Associated with Reduced Pancreatic Cancer Risk J. Nutr., March 1, 2005; 135(3): 592 - 597. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Satia, T. Keku, J. A. Galanko, C. Martin, R. T. Doctolero, A. Tajima, R. S. Sandler, and J. M. Carethers Diet, Lifestyle, and Genomic Instability in the North Carolina Colon Cancer Study Cancer Epidemiol. Biomarkers Prev., February 1, 2005; 14(2): 429 - 436. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Gao, O. I. Bermudez, and K. L. Tucker Plasma C-Reactive Protein and Homocysteine Concentrations Are Related to Frequent Fruit and Vegetable Intake in Hispanic and Non-Hispanic White Elders J. Nutr., April 1, 2004; 134(4): 913 - 918. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
