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Carcinogenesis Advance Access originally published online on July 16, 2008
Carcinogenesis 2008 29(11):2153-2161; doi:10.1093/carcin/bgn018
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The sensitivity to β-carotene growth-inhibitory and proapoptotic effects is regulated by caveolin-1 expression in human colon and prostate cancer cells

Paola Palozza*, Rosanna Sestito, Nevio Picci1, Paola Lanza2, Giovanni Monego3 and Franco O. Ranelletti4

Institute of General Pathology, Catholic University School of Medicine, Largo F. Vito 1, Rome 00168, Italy
1 Department of Pharmaceutical Sciences, University of Calabria, Arcavacata of Rende, Cosenza 87036, Italy
2 Institute of Pathology
3 Institute of Anatomy
4 Institute of Histology, Catholic University School of Medicine, Largo F. Vito 1, Rome 00168, Italy

* To whom correspondence should be addressed. Tel: +39 06 3016619; Fax: +39 06 3386446; Email: p.palozza{at}rm.unicatt.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Although several mechanisms have been proposed to explain the putative role of β-carotene in cancer, no studies have investigated a possible influence of β-carotene on caveolin-1 (cav-1) pathway, an important intracellular signaling deregulated in cancer. Here, different human colon and prostate cancer cell lines, expressing (HCT-116, PC-3 cells) or not (Caco-2, LNCaP cells) cav-1, were treated with varying concentrations of β-carotene (0.5–30 µM) for different periods of time (3–72 h) and the effects on cell growth were investigated. The results of this study show that (i) β-carotene acted as a growth-inhibitory agent in cav-1-positive cells, but not in cav-1-negative cells; (ii) in cav-1-positive cells, the carotenoid downregulated in a dose- and time-dependent manner the expression of cav-1 protein and messenger RNA levels and inhibited AKT phosphorylation which, in turn, stimulated apoptosis by increasing the expression of β-catenin and c-myc and the activity of caspases-3, -7, -8 and -9; when the carotenoid was removed from culture medium, a progressive increase in cell growth was observed with respect to β-carotene-treated cells and (iii) the transfection of cav-1 in cav-1-negative cells increased cell sensitivity to β-carotene by inducing apoptosis. This effect was accompanied by a reduction of both cav-1 and AKT phosphorylation and by an increase of c-myc and β-catenin expression. Silencing of c-Myc attenuated β-carotene-induced apoptosis and β-catenin expression. All together, these data suggest that the modulation of cav-1 pathway by β-carotene could be a novel mechanism by which the carotenoid acts as a potent growth-inhibitory agent in cancer cells.

Abbreviations: Adcav-1, caveolin-1-expressing adenoviral vector; AdRSV, Ad-Rous sarcoma virus; Cav-1, caveolin-1; FAS, apoptosis stimulating fragment; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT, reverse transcription; SiRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The biological functions of caveolin-1 (cav-1) in cancer are complex, multifaceted and controversial (1,2) and seem to depend on cell type, tumor stage and its functional inactivation (i.e. tyrosine or serine phosphorylation and dominant-negative point mutation) in tissues. In fact, although cav-1 has been reported to act in some cases as a tumor suppressor gene (3,4) and cav-1–/– genotype has been demonstrated to accelerate mammary dysplasia in tumor-prone transgenic mice (5), there is substantial evidence that cav-1 is overexpressed in several tumor cells and promotes cell survival in prostate cancer as well as in other malignancies. In particular, cav-1 has been identified as a marker of aggressive disease in prostate (6–9), bladder (10), thyroid (11), lung (12), pancreatic (13) and esophageal (14) carcinoma. In cell model systems, cav-1 has also been demonstrated to promote progression to the metastatic phenotype (15) and the acquisition of drug resistance (16). Moreover, cav-1 has been shown to colocalize transiently with androgen (17) and estrogen (18) receptors at the plasma membrane and may be a direct mediator of hormone action, suggesting that growth and survival signals might be routed through caveolar rafts in hormone-responsive cancers. Some mediators of tumor growth, such as AKT, which is constitutively active in many human cancers due to amplification of AKT gene or as a result of amplification or mutations in components of the signaling pathway that regulate AKT activities, has been reported to be positively regulated by cav-1 (19).

Recently, much attention has been devoted to identify cancer chemopreventive agents of dietary origin. In particular, evidence from epidemiological studies has shown that a high dietary intake of fruits and vegetables, rich in β-carotene and other carotenoids, is associated with a low risk for neoplasia (20). Although some human intervention trials failed to demonstrate prevention of cancer by β-carotene supplements (21–25), other intervention studies on gastrointestinal cancer (26) or prostatic cancer (27) showed a significant protective effect of β-carotene, alone and/or in combination with other dietary compounds. Moreover, β-carotene supplementation reduced the rate of colon cell proliferation in patients with adenomatous polyps (28). Concomitantly, there is accumulating evidence that β-carotene may exert a marked protective effect against carcinomas of oral, skin, colon, urinary bladder, mammary and salivary gland carcinomas in animal models (29). Such data in vivo are reinforced by studies in vitro (30,31) showing the inhibitory effects of β-carotene on cell growth in several cancer cell lines.

Although several mechanisms have been proposed to explain the putative role of β-carotene in the modulation of cell growth, including its provitamin A activity, its ability to act as a redox agent, to enhance immune response and gap junction communication (32), at the moment, no studies have directly investigated a possible influence of β-carotene on cav-1 pathway and the possible consequent effects on tumor cell growth. In this study, these questions were addressed by assessing the role of β-carotene on cell growth in different colon and prostate cancer cell lines, expressing (HCT-116 and PC-3 cells) or not (Caco-2 and LNCaP cells) cav-1. The results presented here show that (i) β-carotene acted as a potent growth-inhibitory agent in cav-1-positive cells, but not in cav-1-negative cells; (ii) in cav-1-positive cells, the carotenoid downregulated in a dose- and time-dependent manner the expression of cav-1 at protein and at messenger RNA (mRNA) levels and inhibited AKT phosphorylation which, in turn, stimulated apoptosis by increasing the activity of caspases, including caspases-3, -7, -8 and -9 and the expression of β-catenin and c-myc; these effects were reversible, as observed by removing β-carotene from culture medium and (iii) the transfection of cav-1 in cav-1-negative cells increased cell sensitivity to β-carotene by inducing apoptosis. This effect was accompanied by a reduction of both cav-1 and AKT phosphorylation, evidencing the specific action of β-carotene in this signaling way. All together, these data suggest that the modulation of cav-1 pathway by β-carotene could be a novel mechanism by which β-carotene acts as a growth-inhibitory agent in cancer cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell culture
The human colon carcinoma HCT-116 cell line (American Type Culture Collection, Rockville, MD) was grown in McCoy's 5a (Laboratoires Eurobio, Courtaboeuf, France) medium, supplemented with 10% heat-inactivated fetal bovine serum (Biowest, Nuaillé, France), glutamine 2 mM/l, penicillin (100 U/ml) and streptomycin (100 µg/ml); human prostate cancer PC-3 cell line (American Type Culture Collection) was grown in dulbecco's modified eagle medium (Laboratoires Eurobio) medium supplemented with fetal bovine serum 10%, glutamine 2 mM/l, penicillin (100 U/ml) and streptomycin (100 µg/ml); human colon carcinoma Caco-2 cell line (American Type Culture Collection) was grown in D-MEM supplemented with con fluorescence correlation spectroscopy (FCS) 20% (Flow, Irvine, UK), glutamine 2 mM, penicillin (100 U/ml), streptomycin (100 µg/ml) and non-essential aminoacids 1%; human prostate cancer LNCaP cell line (American Type Culture Collection) was grown in RPMI 1640 (Laboratoires Eurobio) medium supplemented with fetal bovine serum 10%, glutamine 2 mM, penicillin (100 U/ml) and streptomycin (100 µg/ml). 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 and 99% humidity. β-Carotene (Fluka Chemika-Bio-Chemika, Buchs, Switzerland) was delivered to the cells (109 cells/l) using tetrahydrofuran (Sigma–Aldrich, Milano, Italy) as a solvent. The purity of β-carotene was verified to be 97%. The stocks solutions of β-carotene (2 and 20 mmol/l) were prepared immediately before each experiment. From the stock solutions, aliquots of the carotenoid were rapidly added to the culture medium to give the final concentrations indicated. The amount of tetrahydrofuran added to the cells was not >0.5% (vol/vol). Control cultures received an amount of tetrahydrofuran equal to that present in β-carotene-treated culture media. 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.

Analysis of proteins expression
Cells (10 x 106) were harvested, washed once with ice-cold phosphate-buffered saline (PBS) and gently lysed for 30 min in ice-cold lysis buffer (1 mmol/l MgCl2, 350 mmol/l NaCl, 20 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.5 mmol/l ethylenediaminetetraacetic acid, 0.1 mmol/l ethylene glycol tetraacetic acid, 1 mmol/l p,p'-dichlorodiphenyltrichloroethane, 1 mmol/l Na4P2O7, 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l aprotinin, 1.5 mmol/l leupeptin, 20% glycerol and 1% NP40). Cell lysates were centrifuged for 10 min at 4°C (10 000g) to obtain the supernatants, which were used for western blot analysis with anti-cav-1 (clone N-20, catalog no. sc-894, Santa Cruz Biotechnology), anti-AKT-1 (clone B-1, catalog no. sc-5298, Santa Cruz Biotechnology, Heidelberg, Germany), anti-P-AKT1/2/3 (clone Thr 308-R, catalog no. sc-16646-R, Santa Cruz Biotechnology.), anti-Bax (clone P-19, catalog no. sc-526, Santa Cruz Biotechnology), anti-Bcl-2 (clone C-2, catalog no. sc-7382, Santa Cruz Biotechnology), anti-BcL-x S/L (clone L-19, catalog no. sc-1041, Santa Cruz Biotechnology), anti-c-Myc (clone 9E10, catalog no. sc-40, Santa Cruz Biotechnology) and anti-β-catenin (clone H-102, catalog no. sc-7199, Santa Cruz Biotechnology) monoclonal antibodies. The blots were washed and exposed to a horseradish peroxidase-labeled secondary antibody (Amersham Pharmacia Biotech, Munich,Germany) 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.

Immunofluorescence
HCT-116 cells were washed three times with PBS and fixed for 30 min at room temperature with 2% paraformaldehyde in PBS. Fixed cells were rinsed with PBS and permeabilized with 0.1% Triton X-100/0.2% bovine serum albumin for 10 min. Cells were treated with 25 mM NH4Cl in PBS for 10 min at room temperature to quench free aldehyde groups. The cells were rinsed with PBS and incubated with anti-cav-1 IgG (clone N-20, catalog no. sc-894, Santa Cruz Biotechnology), (1:300 dilution). After three washes with PBS (10 min each), cells were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (1:200 dilution). Cells were washed three times with PBS (10 min for each wash). Slides were mounted with VECTASHIELD mounting medium for fluorescence (Vector laboratories, Burlingame, CA).

Immunohistochemistry
Fixed HCT-116 were rinsed with PBS and permeabilized with H2O2/methanol (1:100) for 7 min. Cells were incubated with anti-β-catenin antibody (clone H-102, catalog no. sc-7199, Santa Cruz Biotechnology) (1:100 dilution). After three washes with PBS (10 min each), cells were incubated with streptavidine–biotine–peroxidase complex for 30 min at room temperature. The sites of peroxidase binding were detected with diaminobenzidine.

The percentage of apoptotic cells was determined by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Briefly, cells were centrifuged, fixed with acetone and incubated for 5 min with the hybridization buffer (Boehringer Mannheim, Monza (MI), Italy). Then 2.5 U of terminal deoxynucleotidyltransferase and 100 pmol biotin–(2'-deoxyuridine 5'-triphosphate) in hybridization buffer were added and incubated for 1 h at 37°C. Thereafter, the cells were incubated with streptavidine-biotine-peroxidase complex for 30 min at room temperature. The sites of peroxidase binding were detected with diaminobenzidine. The percentage of TUNEL-positive apoptotic cells (labeling index, LI%) was counted at x400 magnification. In the absence of terminal deoxynucleotidyltransferase, no unspecific staining was observed. For each slide, three randomly selected microscopic fields were observed and at least 100 cells per field were evaluated.

Reverse transcription–polymerase chain reaction
Total RNA was extracted from tissue samples using Trizol according to the manufacturer's protocols (Invitrogen Life Technologies, Paisley, UK). The RNA was eluted in diethylpyrocarbonate-treated water (0.01% diethylpyrocarbonate) and stored at –80°C until reverse transcription (RT)–polymerase chain reaction (PCR) analysis. Nucleic acid concentrations were measured by spectrophotometry [Hewlett-Packard HP (Geneva, Switzerland) UV/VIS (Ultra-Violet/Visible) spectrophotometer 8450]. Five hundred nanograms of total RNA was treated with DNase I amplification grade (Invitrogen Life Technologies) following the manufacturer's instructions, in order to eliminate genomic DNA from each sample, and then the DNase I digested RNA was employed for cDNA synthesis.

RT–PCR assay was performed using the two-step method. For the first-step RT reaction, we used Superscipt III First-Strand Synthesis system for RT–PCR (Invitrogen Life Technologies), as described in the manufacturer's procedure for cDNA synthesis with oligo(dT).

For the second-step PCRs, we employed QuantiTect SYBR® Green Kits (Qiagen, Hilden, Germany) and QuantiTect® Primer Assays (Qiagen) for human β-actin, apoptosis stimulating fragment (FAS) and caveolin-1, according to the manufacturer’s protocol described for the real-time thermal cycler LightCycler (Roche, Monza (MI), Italy). PCR data were analyzed by Relative Quantification Software (Roche) and expressed as target to reference ratios.

Caspase activity
The activity of caspase-3, -7, -8 and -9 was measured by the fluorimetric assay as described. Briefly, cells were incubated for the indicated times and then harvested. Cells (2 x 106) were lysed in 50 mM Tris–HCl buffer, pH 7.5, containing 0.5 mM ethylenediaminetetraacetic acid, 0.5% octylphenoxy polyethyleneoxyethanol, branched and 150 mM NaCl. Cell lysates were incubated with 50 µM of fluorogenic substrates, such as Ac-DEVD-7-amido-4-methylcoumarin (AMC) (caspase-3; Alexis Biochemicals, San Diego, CA), Ac-IETD-AMC (caspase-8; Alexis Biochemicals) for caspase-9 (Alexis Biochemicals) and for caspase-7 (Alexis Biochemicals) in a reaction buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, containing 50 mM NaCl and 2.5 mM dithiothreitol) for 120 min at 37°C. The release of AMC was measured with excitation at 380 nm and emission at 460 nm using a fluorescence spectrophotometer.

Transfection and viral infection
The plasmid vector expressing wild-type cav-1 (pcav-1) was constructed by inserting the human cav-1 cDNA into pcDNA3.1 (Invitrogen Life Technologies). Mutant cav-1 with the scaffolding domain deleted (cav-1{Delta}82-101) was generated by PCR mutagenesis using pcav-1 as a template (as indicated in ref. 33). In the first-step, intermediate PCR product A was produced using T7 promoter primer 5'-CTGAGTGATATCCC-3' and primer A carrying an adjacent DNA sequence from both sides of the region deleted (5'-CAGACAGCAAAAAACTGTGT-3'), and intermediate PCR product B was produced using primer B which also carries a DNA sequence adjacent to both sides of the region deleted (5'-TGTGTCAAAAAACGACAGAC-3') and pcDNA3.1/BGH reverse primer (5'-TAGAAGGCACAGTCGAGG-3'). These two intermediate PCR products which carry 20mer overlapped DNA sequence were annealed and amplified using T7 promoter primer and pcDNA3.1/BGH reverse primer. The resulting PCR product was digested with EcoRI and inserted into pcDNA3.1 (+) to generate pcav-1{Delta}82-101. Recombinant adenoviral vectors cav-1-expressing adenoviral vector (Adcav-1) and Ad-Rous sarcoma virus (AdRSV) were generated (as indicated in ref. 34,35). Subconfluent cells were trypsinized, collected by centrifugation and resuspended in regular medium. A single-cell suspension was then seeded at 5 x 105 cells per well (six-well plates) or 2 x 106 cells/10 cm diameter plate. Cells were infected or transfected the next day. Typically, cells were infected with Adcav-1 or AdRSV in serum-free medium at a multiplicity of infection of 10, which was indicated to produce an optimal level of cav-1 for cell survival (36). The infection medium was removed and replaced with complete medium 3 h after the infection. For transfection, 2 µg of DNA was used for each transfection in the six-well plates using Promega Tfx-50 reagent (Promega, Milano, Italy) at the Tfx-50 to DNA ratio of 2:1 in 1 ml of serum-free medium, and 12 µg of DNA was used to transfect cells in a 10 cm diameter plate with 5 ml of serum-free medium. One hour after the transfection, 2 ml of RPMI 1640 medium with 15% FCS was added to each well (six-well plate) or 5 ml of RPMI 1640 medium with 20% FCS was added to each 10 cm diameter plate. For experiments with both infection and transfection, cells were incubated for 16 h (overnight) after the viral infection.

c-Myc silencing by siRNA
Inhibition of c-Myc expression in cav-1-transfected LNCaP cells was performed using SureSilencing Human MYC small interfering RNA (siRNA) and Antibody Kit (SuperArray Biosciences, Frederick, MO). Cells were transfected with Myc-specific siRNA population using Lipofectamine2000 (Invitrogen Life Technologies) as recommended by the manufacturer. Non-specific siRNA was used as negative control. Cells were harvested 24 h after transfection.

Extraction and analysis of β-carotene
β-Carotene was extracted by washing cells (10 x 106 cells) twice with PBS, resuspending them in 1 ml ethanol, containing 0.1% butylated hydroxytoluene, and subsequently lysing them with an Ultra-turrax procedure. All the samples were extracted with hexane:diethyl ether (1:1) and evaporated to dryness under nitrogen. After the extraction, the samples were redissolved in methanol and a 20 µl aliquot was analyzed by reverse-phase high-performance liquid chromatography with spectrophotometric detection on a PerkinElmer LC-295 detector at 450 nm. The column was packed with Alltech C18 Adsorbosphere HS material, 3 µm particle size, in a 15 x 0.46 cm cartridge format (Alltech Associates, Deerfield, IL). A 1 cm precolumn containing 5 µm C18 Adsorbosphere packing was used. The mobile phase was 60% acetonitrile/10% methanol/30% isopropanol at a flow rate of 1 ml/min. Ammonium acetate, high-performance liquid chromatography grade, 0.01% was added to the mobile phase. The entire analytical procedure (extraction and chromatographic run) was carried out in dim light to avoid carotenoid photoautooxidation. For carotenoid quantitation, a calibration curve was periodically built from pure standard β-carotene (Fluka Chemika-BioChemika).

Statistical analysis
Three separate cultures per treatment were utilized for analysis in each experiment. Values were presented as means ± SEMs. Multifactorial two-way analysis of variance was adopted to assess any differences among the treatments and the times (Figures 1B and D, 2B and C, 3D and 6AGoGoGo). 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 analysis of variance was used to determine differences between different concentrations and treatments in Figures 2A, 4B, D, F and G and 5A–EGoGo. 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, State College, PA).


Figure 1
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  		Fig. 1. Regulated expression of caveolin-1 (cav-1) by β-carotene in HCT-116 cells. (Panel A) Representative western blot analyses. (Panel B) Densitometric analyses of the immunoblots. (Panel C) Immunolocalization. A cav-1-specific antibody probe was used to localize cav-1 after 24 h of incubation in the absence (A) or in the presence (B) of 5 µM β-carotene; (panel D) mRNA levels. The treatment–time interactions (panels C and D) were significant (P < 0.05). Values not sharing the same letter were significantly different [(C) P < 0.001; (D) P < 0.005, Tukey's test].

 


Figure 2
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  		Fig. 2. Effects of β-carotene on the growth of HCT-116 cells. (Panel A) Dose-dependent effects of β-carotene after 24 h. (Panel B) Time-dependent effects. (Panel C) Effects of β-carotene removal from culture medium on cell growth. (Panel D) Cav-1 expression. In (panels C and D), β-carotene was added for 72 h or added only for 24 h and then the medium was replaced with fresh medium not containing the carotenoid until 48 and 72 h. The values were the means ± SEMs, n = 5. In (panel A), values not sharing the same letter were significantly different (P < 0.05, Fisher’s test). The treatment–time interactions (panels B and C) were significant (P < 0.05). Values not sharing the same letter were significantly different [(B) P < 0.005; (C) P < 0.005, Tukey's test].

 


Figure 3
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  		Fig. 3. Cav-1 expression and growth-inhibitory effects of β-carotene in tumor cell lines. (Panel A) Basal levels of cav-1 in HCT-116, CaCo-2, PC-3 and LNCaP cells. (Panel B) Growth-inhibitory effects of β-carotene. (Panel C) Downregulation of cav-1 expression by β-carotene in PC-3 cells. (Panel D) Growth-inhibitory effects of 20 µM β-carotene in AdRSV- and AdCav-1-transfected cells. In (panel B), the values were the means ± SEMs, n = 5. In panel D, the values were the means ± SEMs, n = 3; the treatment interactions were significant (P < 0.05). Values not sharing the same letter were significantly different (P < 0.005, Tukey's test).

 


Figure 4
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  		Fig. 4. Effects of β-carotene on AKT, c-myc, β-catenin and FAS levels in HCT-116 cells. (Panels A, C and D) Representative western blot analysis. (Panels B, D–F) Densitometric analyses. Cells were treated with varying β-carotene concentrations (1–5 µM) for 24 h. (Panel G) Immunostaining of β-catenin after 24 h of incubation in control (a) and in β-carotene (5 µM)-treated (b) cells. (Panel H) Densitometric analysis of FAS obtained by northern blots. Values were the means ± SEMs, n = 5. Values not sharing the same letter were significantly different (P < 0.05, Fisher's test).

 


Figure 5
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  		Fig. 5. Effects of β-carotene on apoptosis and apoptosis-related proteins in HCT-116 cells. Apoptosis was measured as TUNEL-positive cells (panel A), caspase-9 (panel B), caspase-8 (panel C), caspase-7 (panel D) and caspase-3 (panel E) activation. (Panel F) Representative western blot analyses of Bcl-2, Bax and Bcl-xL. Cells were treated with varying β-carotene concentrations (1–5 µM) for 24 h. Values were the means ± SEMs, n = 5. Values not sharing the same letter were significantly different (P < 0.05, Fisher's test).

 


Figure 6
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  		Fig. 6. Effects of β-carotene on apoptosis (panel A), cav-1, AKT, c-myc and β-catenin expression (panel B) in AdRSV- and AdCav-1-transfected cells. Silencing of c-Myc attenuates β-carotene-induced apoptosis (panel C) and β-catenin expression (panel D) in AdCav-1-transfected cells. Cells were treated with β-C (20 µM) for 24 h. Values were the means ± SEMs, n = 3. In panels A and C, the treatment interactions were significant (P < 0.05). Values not sharing the same letter were significantly different [(A) P < 0.005, Tukey's test; (C) P < 0.05, Fisher's test].

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Effects of β-carotene on cav-1 expression and its relationship with cell growth
In order to investigate a possible role of β-carotene on cav-1 expression, HCT-116 cells, reported previously (37) to highly express the protein, were treated with varying concentrations of the carotenoid (1–5 µM) for different periods of time (24–72 h) (Figure 1). The expression of cav-1 was assessed at protein level by western blotting (A and B) and immunofluorescence (C) analysis and at mRNA level (D) in cells treated with β-carotene for 24 h (A–D) and for 3 h (D). The carotenoid was able to downregulate cav-1 protein and mRNA transcript levels in a dose- and time-dependent manner.

Next, the number of viable cells was analyzed at the concentrations and at the time points indicated. Figure 2A shows the effects of the carotenoid at 1–5 µM for 24 h and Figure 2B shows the effects of the carotenoid at 5 µM for different periods of time (24–72 h) in HCT-116 cells. In this cell line, β-carotene acted as a potent growth-inhibitory agent, significantly decreasing cell number in a dose- and time-dependent manner with respect to vehicle-control cells.

To evaluate a possible relationship between cav-1 expression and cell growth, the carotenoid was removed from culture medium after 24 h of incubation and cell number (Figure 2C) and cav-1 levels (Figure 2D) were evaluated in HCT-116 cells. In the presence of β-carotene (5 µM), a time-dependent inhibition of cell growth was observed. Such an effect was evidenced at 24 h and it remained pronounced by prolonging carotenoid treatment for 72 h. When the carotenoid was removed from culture medium after 24 h of incubation, a progressive increase in growth of HCT-116 cells was observed with respect to β-carotene-treated cells (panel C). Concomitantly, while carotenoid maintained the low level of cav-1 during the 72 h of incubation, its removal from culture medium after 24 h strongly induced a progressive cellular increase of cav-1 expression (panel D). These findings suggest that β-carotene inhibits cell growth and downregulates cav-1 in a reversible way.

To evaluate a possible relationship between inhibition of cell growth by β-carotene and cell expression of cav-1, we examined the effects of β-carotene on the growth of human colon and prostate cancer cells either expressing (HCT-116 and PC-3) or not expressing (Caco-2 and LNCaP) the cav-1 protein (Figure 3A). The ability of the carotenoid to inhibit cell growth was remarkably correlated with the capacity of the cells to express cav-1. In HCT-116 and in PC-3 cells, both expressing cav-1, the carotenoid strongly acted as a growth-inhibitory agent (Figure 3B). On the other hand, it was almost ineffective, even at very high concentrations (30 µM), in inhibiting the growth of Caco-2 and LNCaP cells, in which cav-1 was undetectable (Figure 3B).

An inhibition of cav-1 by β-carotene was also observed in PC-3 cells (Figure 3C). Interestingly, the downregulation of the protein was also associated with an inhibition of cell growth, as it was observed in HCT-116 cells.

On the other hand, we were not able to find remarkable differences in the incorporation and/or cell association of the carotenoid among the cells, when equal amounts of β-carotene were added. Experiments showed that, after 24 h of incubation with 5 µM β-carotene, carotenoid accumulation was 0.20 ± 0.02 nmol/106 cells and 0.21 ± 0.02 nmol/106 cells in HCT-116 cells and in Caco-2 cells, respectively.

To a better understanding of the relationship existing between cav-1 expression and β-carotene, we infected LNCaP cells (cav-1 negative) with Adcav-1. Forty-eight hours after the infection, cells were treated with 20 µM β-carotene. After 24 h of treatment with the carotenoid, cell growth was measured (Figure 3D). AdCav-1-transfected LNCaP cells became sensitive to the growth-inhibitory effects of 20 µM β-carotene, whereas AdRSV-transfected LNCaP cells, at this concentration, remained completely insensitive to the carotenoid. All these data suggest a key role of cav-1 in the cell growth-inhibitory effects of β-carotene.

Modulation of AKT phosphorylation by β-carotene in cav-1-expressing cells: effects on β-catenin and c-myc
To determine possible mechanisms by which β-carotene controls cancer cell growth in the cav-1-expressing cells, we examined AKT expression and its phosphorylation in HCT-116 cells (Figure 4A and B). It has been reported that AKT/PI3K pathway may be modulated by cav-1 in cancer cells. Increasing evidences suggest that cav-1 maintains the phosphorylation state of AKT through scaffolding binding site interactions with and inhibition of two major serine–threonine protein phosphatases, PP1 and PP2A (33). Treatment of HCT-116 cells with β-carotene for 24 h decreased phosphorylation of AKT in a dose-dependent manner, with substantial inhibition already evident at 1 µM β-carotene. A reduced phosphorylation of AKT was also observed in PC-3 cells, the other cell line expressing cav-1, following a 24 h treatment with the carotenoid (data not shown).

It has been reported that cav-1 may regulate multiple AKT downstream targets, including serine/threonine kinase 3 {alpha}/β (33). Inactivation of serine/threonine kinase 3 {alpha}/β by cav-1 would induce the expression of its specific substrates, including β-catenin and c-myc, which affect cell survival through different mechanisms and induce apoptosis. Therefore, we measured the expression of these two proteins after the addition of varying β-carotene concentrations in HCT-116 cells (Figure 4C–F). After 24 h of incubation with the carotenoid, a strong increase in c-myc (Figure 4C and D) and β-catenin (Figure 4E and F) protein levels was observed by western blotting. In particular, the cellular increase of β-catenin was found in nucleus, in perinuclear area as well as in the membrane, as assessed by immunohistochemical analysis in HCT-116 cells treated for 24 h with 5 µM β-carotene (Figure 4G). Concomitantly, the increase in c-myc expression by β-carotene was also followed by the enhanced expression of FAS at mRNA levels (Figure 4H).

Induction of apoptosis in cav-1-expressing cells
Consistent with the observation that AKT-pathway signaling is required for cell survival, the dose-dependent reduction in AKT phosphorylation induced by β-carotene was also accompanied by a dose-dependent increase in apoptosis, measured by TUNEL method in HCT-116 cells (Figure 5A).

Similar results were found when we determined the effects of β-carotene on caspase activation, measuring the activity of two upstream executors of apoptosis, caspase-9 (Figure 5B) and caspase-8 (Figure 5C) and that of two downstream executors of apoptosis, caspase-7 (Figure 5D) and caspase-3 (Figure 5E) in HCT-116 cells. The activity of the four caspases increased markedly in response to a 24 h treatment with varying β-carotene concentration. Such an effect was clearly dose-dependent.

According with these data, the expression of the antiapoptotic proteins Bcl-2 and Bcl-xL was remarkably decreased by a 24 h β-carotene treatment (Figure 5F). On the other hand, the carotenoid did not significantly modify the expression of the proapoptotic protein Bax (Figure 5F).

These observations strongly suggest that β-carotene may act as a potent proapoptotic agent in cancer cells expressing cav-1.

Effects of β-carotene on cav-1, AKT, c-myc, β-catenin and apoptosis in cav-1-transfected cells
To a better understanding of the relationship existing between cav-1 expression and β-carotene, we measured apoptosis by TUNEL method (Figure 6A) and the expression of cav-1, AKT, c-myc and β-catenin (Figure 6B) in LNCaP cells (cav-1 negative) transfected with the Adcav-1. Forty-eight hours after the infection, cells were treated with 20 µM β-carotene. After 24 h of treatment with the carotenoid, AdCav-1-transfected LNCaP cells became sensitive to the proapoptotic effects of 20 µM β-carotene, whereas AdRSV-transfected LNCaP cells, at this concentration, remained completely insensitive to the carotenoid. Moreover, the carotenoid decreased cav-1 expression and AKT phosphorylation and increased c-myc and β-catenin expression in AdCav-1-transfected cells, confirming that caveolin-mediated AKT pathway is strongly modified by β-carotene and that both c-myc and β-catenin are possible downstream effectors of this pathway. According with this, silencing of c-Myc attenuated β-carotene-induced apoptosis (panel C) and β-catenin expression (panel D) in AdCav-1-transfected cells.


   Discussion
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Currently, the role of cav-1 in oncogenic transformation, cancer and metastasis has been extensively studied as well as its role in the modulation of cellular signaling cascades involved in cell proliferation and apoptosis (38). Its role as an oncogene or as a tumor suppressor gene seems to strictly depend on cell type. According with this, we found a dramatic difference in the endogenous levels of cav-1 protein in colon as well as in prostate human cancer cells. The colon adenocarcinoma HCT-116 cells and the prostate carcinoma PC-3 cells expressed the protein, whereas the colon adenocarcinoma Caco-2 cells and the prostate cancer LNCaP cells did not express it. In agreement with our study, HCT-116 cells have been reported to possess remarkable levels of cav-1 protein with respect to other colon cancer cell lines, including HT-29 and Caco-2 and such an effect was directly associated with the elevated growth rate of these cells (37). Treatment with β-carotene also resulted in remarkable differences in the growth of the different cancer cell lines. Interestingly, we found that the carotenoid acted as a potent growth-inhibitory agent only in cav-1-expressing cells (HCT-116 and PC-3), but not in cells lacking the protein (Caco-2 and LNCaP) and a clear relationship appears to exist between cav-1 expression and β-carotene efficiency in inhibiting tumor cell growth. Growth-inhibitory effects of β-carotene in colon and in prostatic cancer cells have been reported previously by us (39) and by other authors (30,40), but never directly related to cav-1 expression. In a previous study (39), we suggested that the different response of colon cancer cells to β-carotene could be related to its different cell incorporation. However, we were not able to find remarkable differences in the incorporation and/or cell association of the carotenoid between Caco-2 cells and HCT-116 cells, when equal amounts of β-carotene were added. Therefore, the results of this study seem to suggest that other mechanisms could be implicated in the antitumoral effects of β-carotene. In fact, although we challenged Caco-2 and LNCaP cells with very high concentrations of the carotenoid (up to 30 µM), we were not able to obtain remarkable growth-inhibitory effects by β-carotene. Such a difference seems, at least in part, to be dependent on the lack of cav-1 expression. It is interesting to note that HCT-116 cells expressing high levels of cav-1 were more sensitive to the cav-1-reducing effect of β-carotene than PC-3 cells possessing lower levels of cav-1. Furthermore, LNCaP-transfected cells with cav-1 became sensitive to lower concentrations of β-carotene (20 µM) and their growth was significantly inhibited by the carotenoid. The main action of β-carotene on cav-1 expression was related to its downregulation, as shown by western blotting, northern blotting and immunohistochemical analyses. Such an effect was observed at both protein and mRNA levels and it was clearly dose and time dependent. Moreover, it was observed in both the cell lines expressing cav-1. The removal of the carotenoid from cell culture medium after 24 h of incubation, progressively increased the levels of cav-1 and cell growth with respect to β-carotene-treated cells, suggesting that the downregulation of cav-1 expression and the inhibition of cell growth are events strictly related to the presence of the carotenoid. Furthermore, the observation that β-carotene was able to decrease constitutively both the expression of cav-1 and that from transfected cells seems to suggest that carotenoid may exert this effect at posttranscriptional levels.

We then investigated possible mechanisms through which the downregulation of cav-1 by β-carotene may influence the cell growth. Our study indicates that in cav-1-expressing cancer cells, β-carotene was able to modulate AKT pathway, by decreasing AKT phosphorylation in a dose-dependent manner. Concomitantly, it was able to influence the expression of multiple AKT downstream targets, including β-catenin and c-myc. These results are in agreement with previous observations showing that cav-1 plays a key role as a positive regulator in the AKT-signaling pathway by maintaining the phosphorylated state of AKT (33). This is presumably related to its ability to interact and to inhibit two major serine/threonine proteins, the phosphatases PP1 and PP2A. As a consequence of such an inhibition, the reduced activities of PP1 and PP2A lead to higher phosphorylation levels of their specific substrates, including pyruvate dehydrogenase kinase 1, extracellular signal-regulated kinase1/2 and AKT.

Several studies have linked cav-1 to PI3-k/AKT-signaling pathway and to changes in cell ability to undergo apoptosis. Although it has been reported that cav-1 can play a proapoptotic role in specific model systems (41), several other studies demonstrated that cav-1 plays an important role in PI3-K/AKT-mediated cell survival activities (19,42). Disruption of caveolae, using cholesterol-sequestering agents, has been shown to block interleukin-6 and insulin-like growth factor-1-induced activation of the PI3-kinase/AKT-signaling pathway, suggesting that caveolae and cav-1 are required to mediate proper survival signals through PI3-kinase/AKT pathway (42). Moreover, cav-1 overexpression in Rat1A cells and human LNCaP clones renders these cells more resistant to apoptosis (8,35). In addition, antisense-mediated downregulation of cav-1 results in prostate cancer cells that are more sensitive to apoptosis (34,36,40). Our study also suggests a positive role for cav-1 in cell survival. β-carotene treatment was accompanied by a downregulation of cav-1 and by an increased ability of HCT-116 cells to undergo apoptosis. Concomitantly, LNCaP cells transfected with cav-1 enhanced p-AKT expression and became sensitive to the proapoptotic effects of β-carotene. It is interesting that in LNCaP cells AKT is constitutively active, as shown by the high expression of pAKT even in the absence of cav-1. This is presumably due to phosphatase and tensin homolog mutation. The observation that, in LNCaP cells, β-carotene was able to decrease pAKT and clearly increase both c-myc and β-catenin expression only in the presence of cav-1 strongly confirms the main role of cav-1 pathway in the proapoptotic effects of this compound with c-myc and β-catenin as down stream effectors. According with this, in the presence of β-carotene, β-catenin was remarkably increased in the nuclear area. Moreover, silencing of c-myc attenuated β-carotene-induced apoptosis and β-catenin expression in AdCav-1-transfected cells.

Proapoptotic effects of β-carotene and other carotenoids have been reported previously in cancer cells and different mechanisms have been implicated, including carotenoid's ability to induce caspase cascade (43,44), affect mitochondrial functions (43), modulate the expression of apoptosis-related proteins, including Bcl-2 and Bcl-xL (39,44), Bad (45), Bid (45) and Bax (46,47), and the levels of transcription factors involved in apoptosis induction (48). Accordingly, recent data show that β-carotene was able to induce apoptosis in HL-60 cells and that this effect was accompanied by an increased expression of c-myc (49). The upregulation of c-myc occurred very early and it was long lasting, paralleling the effect of the carotenoid on Nuclear Factor Kappa-B activation (49). The observation of the present study that the increased c-myc expression following β-carotene treatment was accompanied by increased mRNA levels of FAS is particularly interesting in view of findings showing that the expression of Fas ligand is regulated by c-myc (50,51). According to these studies, c-myc seems to be able to augment the apoptotic activity of cytosolic death receptor signaling.

In conclusion, the results presented here show that (i) β-carotene acted as a potent growth-inhibitory agent in cav-1-positive tumor cells, but not in cav-1-negative cells; (ii) in cav-1-positive cells, the carotenoid downregulated in a dose- and time-dependent manner the expression of cav-1 at protein and at mRNA levels and inhibited AKT phosphorylation, which, in turn, stimulated apoptosis by increasing the expression of β-catenin and c-myc and the activity of caspases and (iii) the transfection of cav-1 in cav-1-negative cells increased cell sensitivity to β-carotene by promoting apoptosis. Although, further studies are needed in vivo to verify the relevance of this finding, the downregulation of cav-1 by β-carotene may be a novel mechanism by which the carotenoid exerts growth-inhibitory effects in human cancer tissues.


   Funding
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
The Ministero Università e Ricerca (MIUR D1).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received June 4, 2007; revised December 21, 2007; accepted January 11, 2008.


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