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2
*
Molecular Pharmacology Research Center at the New England Medical Center, Boston, MA 02111;
Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47906;
**
Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111; and the
Department of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111
2To whom correspondence should be addressed. E-mail: norman.krinsky{at}tufts.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSTransient transfection and...RESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • ß-carotene • excentric cleavage • breast tumor cells • activator protein-1 • retinoblastoma protein • E2F1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTransient transfection and...RESULTSDISCUSSIONLITERATURE CITED |
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) and therapeutic (2
–4
) effects of retinoids have been reported for various types of cancer. Retinoids and synthetic retinoid analogs have also been shown to inhibit breast tumor cell growth in vitro (5
) and in rat mammary cancer in vivo (6
). Unfortunately, the potential toxicity of these compounds (7
,8
) has limited their clinical use.
Animal and cell culture studies suggest that carotenoids may act as anticarcinogenic agents (9
–12
). Among the carotenoids, ß-carotene has been shown to induce differentiation and control proliferation of normal primary mammary epithelial cells in vitro, suggesting a protective role against breast carcinogenesis (13
).
ß-apo-carotenoic acids (ß-apo-CA)3
are random cleavage products of ß-carotene with chemical structures similar to that of all-trans-retinoic acid (atRA; Fig. 1
). ß-apo-14'-, and ß-apo-12'-CA, 2 and 4 carbon longer homologues of atRA, have anti-proliferative activity and induce the differentiation of leukemic HL-60 cells (14
). In this study, we extend those findings by examining the ability of synthetic excentric cleavage products of ß-carotene to inhibit breast tumor cell growth. Subsequently, we examined potential mechanisms by which these compounds could limit breast tumor cell growth in an in vitro model using human breast tumor cell lines that express both retinoic acid receptors (RAR) and estrogen receptors (ER) or only RAR, i.e., their effect on cell cycle regulators, their ability to suppress activator protein-1 (AP-1)-mediated gene activation, their ability to be converted into atRA and their ability to bind the RAR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTransient transfection and...RESULTSDISCUSSIONLITERATURE CITED |
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atRA, nonessential amino acids, HEPES, sodium pyruvate, gentamycin, insulin, diethyl pyrocarbonate and trypsin solution were obtained from Sigma Chemical (St. Louis, MO). Minimum essential medium-
(MEM), Dulbecco’s modified Eagle’s medium (DMEM), Ham’s medium and L-glutamine were obtained from Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT). ß-apo-12'-carotenal, ß-apo-10'-carotenal, methyl ß-apo-14'-carotenoate and methyl ß-apo-8'-carotenoate were kind gifts from Drs. U. Hengartner and K. Bernhard from Hoffmann-La Roche (Basel, Switzerland).
ß-apo-CA were obtained by oxidation of their corresponding aldehyde and/or hydrolysis of their methyl esters (15
). Apo-CA were then purified using amino propyl columns (Sep-Pak Vac Cartridges; Waters, Milford, MA). Ester residues were eluted using two washes with 4 mL chloroform/2-propanol (2:1, v/v). Pure apo-CA (99%) were recovered by co-elution with 4 mL diethyl ether-3% acetic acid, evaporated completely under N2, reconstituted in absolute ethanol and analyzed by HPLC against corresponding standards. All HPLC solvents were purchased from J. T. Baker Chemical (Phillipsburg, NJ) and were filtered through a 0.45-µm filter before use.
Cell culture.
The MCF-7 ER+ human breast tumor cell line was obtained from The Michigan Cancer Foundation (Detroit, MI). The Hs578T and MDA-MB-231, ER- human breast tumor cell lines, were obtained from the American Type Culture Collection (Rockville, MD). MCF-7 cells were grown in MEM containing 1X nonessential amino acids, 10 mmol/L HEPES, 1 nmol/L sodium pyruvate, 0.01 g/L insulin, 0.05 g/L gentamycin and 10% FBS. Hs578T cells were grown in DMEM with 4.5 g/L glucose, 0.01 g/L insulin and 10% FBS. MDA-MB-231 cells were grown in DMEM/Ham’s F-12 medium (1:1, v/v) with 1 mmol/L L-glutamine, 0.5 mmol/L sodium pyruvate, 0.03 g/L penicillin, 0.05 g/L streptomycin and 10% FBS. Routine passaging of 100-mm cell culture dishes was done weekly at a 1:3 ratio when cells were 80% confluent. All experiments were performed with cells at passages 23–27. All cells were incubated at 37° C in 5% CO2/air.
Cell treatment with ß-apo-CA.
Cells were plated on 6-well plates (5000 cell/cm2) in 10% FBS medium for 24 h. atRA and ß-apo-CA treatments were prepared by dilution of ethanol stock solutions in appropriate growth medium (final ethanol content = 0.3%). For the growth inhibition assays, the medium was removed, and experimental medium containing 5% FBS and one of three concentrations (10 nmol/L, 100 nmol/L or 1 µmol/L) of ß-apo-CA, atRA or 0.3% ethanol alone was added to the cells for 1–9 d. This level of ethanol did not affect cell growth significantly (data not shown). The experimental medium was replaced daily. Cells were trypsinized with 1X trypsin/EDTA solution (0.5 g/L trypsin, 0.53 mmol/L EDTA.4Na; Life Technologies), neutralized with growth medium containing 5% FBS and counted at each time-point using a Coulter Counter (Coulter Corp., Miami, FL). The data of the growth assays represent the average of triplicate samples ± SD. For Northern and Western blotting, cells were grown on 100-mm culture dishes (5000 cell/cm2) in 10% FBS medium for 24 h, and then the medium was removed and experimental medium containing 5% FBS and 1 µmol/L ß-apo-CA, atRA or ethanol was added to the cells for 24 or 72 h or for 1, 6 or 9 d.
HPLC analysis of retinoid metabolites.
Samples of fresh growth medium, conditioned growth medium and cell pellets from growth inhibition assays were examined for metabolic product formation at 5 h or at d 9 of incubation. Retinoids and carotenoids were extracted from these samples using 6 mL of CHCl3/CH3OH (2:1, v/v), followed by a second extraction with 2 mL hexane. Retinyl acetate was added as an internal standard. After centrifugation of the mixtures (3200 x g at 4°C for 10 min), the chloroform layer was saved and the aqueous phase was extracted again with hexane. The chloroform and the hexane layers were then pooled and evaporated completely under N2. The residue was redissolved in 100 µL ethanol. The final extract (50 µL) was injected onto a HPLC system that consisted of a Waters 616 pump, a Waters 600s controller, a Waters 996 photodiode array detector, a Vydac 201TP54, C18 reverse-phase column (0.46 i.d. x 25 cm; Vydac, Hesperia, CA), and a Waters 840-Digital 350 data station (Waters Chromatography Division/Millipore Corporation, Bedford, MA). The HPLC mobile phase was methanol/1% ammonium acetate in water (solvent A: 70:30, v/v), and 100% methanol (solvent B), flow rate: 1.7 mL/min. A gradient procedure was used as follows: 100% solvent A was used for 8 min followed by a 10-min linear gradient to 20% solvent A and 80% solvent B, a 10-min linear gradient to 100% solvent B, a 20-min hold at 100% solvent B, and a 4-min gradient back to 100% solvent A. Retinoids and ß-apo-14'-CA were analyzed at 340 nm; ß-apo-10'-, ß-apo-12'-CA at 400 nm; and ß-apo-8'-CA at 450 nm. ß-apo-carotenoids and retinoids were identified by co-elution with corresponding standards and absorption spectra analyses.
Western blots.
Nuclear proteins were obtained following protocols previously described with some modifications (16
,17
). Briefly, after treatment cells were washed with cold PBS and scraped into a buffer containing 10 mmol/L HEPES (pH 7.8), 10 mmol/L KCl, 2 mmol/L MgCl2, 1 mmol/L dithiothreitol (DTT), 0.1 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 0.05 g/L antipain and 0.05 g/L leupeptin. Cells were lysed by adding 1% Igepal (Sigma) followed by a 15-s vortex mixing. Nuclei were collected by centrifugation at 16,000 x g for 30 s. Nuclear proteins were extracted from the pellets with a high salt buffer containing 50 mmol/L HEPES (pH 7.8), 50 mmol/L KCl, 300 mmol/L NaCl, 1 mmol/L DTT, 0.1 mmol/L EDTA, 1 mmol/L PMSF, 10% glycerol (v/v) by shaking at 4°C for 20 min, followed by centrifugation at 16,000 x g for 5 min. Nuclear extracts were stored at -80°C until used. Proteins were separated by 10% polyacrylamide gel electrophoresis. Nuclear protein (40 µg) was loaded per lane in all cases. Proteins were transferred to an Immobilon-P membrane (Millipore) using a semidry transfer cell (Bio-Rad, Hercules, CA) set at 10 V at room temperature for 45 min. The transferred membranes were incubated at 4°C overnight in blocking solution containing 10% nonfat dry milk in TBS-T buffer (24.8 mmol/L Tris, 162.4 mmol/L NaCl and 0.1% Tween 20). The membranes were then incubated with rabbit polyclonal antibodies against retinoid receptor isoforms (Biomol, Plymouth Meeting, PA) at a final concentration of 0.002 g/L at room temperature for 1 h. The membranes were washed for 30 min in TBS-T buffer and incubated with HRP-conjugated secondary antibodies (Bio-Rad) at room temperature for 30 min at a dilution of 1:5000 in TBS-T. Bands were visualized by chemiluminescent detection (ECL; Amersham, Piscataway, NJ) and the membrane was exposed to X-ray film to yield an image. Densitometric analysis of blots was performed by scanning autoradiographs using the Molecular Dynamics Image Quant software, Version 3.3 (Molecular Dynamics, Sunnyvale, CA). Band intensities were normalized to the level of ß-actin in each sample. ß-actin expression was determined using rabbit anti-actin primary antibody and horseradish peroxidase labeled rabbit anti-goat IgG secondary antibody (Sigma-Aldrich, St. Louis, MO).
RNA isolation and Northern blotting.
Cells were incubated with 1 µmol/L ß-apo-CA or atRA as described above. The cells were washed twice with PBS, lysed with guanidine isothiocyanate solution followed by centrifugation through a cesium chloride layer as described previously (18
). RNA was quantitated by measuring absorbance at 260 nm, and the sample integrity was determined by agarose gel electrophoresis. Total cellular RNA (30 µg) was size-fractionated on a 1.2% agarose gel containing 2.2 mol/L formaldehyde and transferred to nylon membranes using a Turboblotter (Schleicher & Schuell, Keene, NH) rapid downward transfer system. The membranes were probed with 32P-labeled RAR, RARß and RAR
cDNA (19
). Autoradiography was performed on Kodak XAR film, with one intensifying screen at -80°C. Densitometric analysis of blots was performed by scanning of autoradiographs using the Molecular Dynamics Image Quant software, Version 3.3 (Molecular Dynamics). RAR mRNA signals were normalized to the expression of pHE7 mRNA. The pHE7 gene is constitutively expressed and is not induced by atRA (19
). The RAR, RARß, RAR and pHE7 cDNA probes were provided by Dr. Monica Peacocke (Columbia University, NY). RAR cDNA is a 1.6-kb fragment in the KpnI-EcoRI site of the plasmid p63 (20
). RARß cDNA is a 0.6-kb fragment in the EcoRI site of plasmid B1-RARe (21
). RAR cDNA is a 1.6-kb insert in the EcoRI site of plasmid pS65 (22
). pHE7 is a 300-bp insert in the Pst1 site of pGEM-1 (23
).
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Transient transfection and chloramphenicol acetyl transferase (CAT) assay |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTransient transfection and...RESULTSDISCUSSIONLITERATURE CITED |
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The pBLCAT3 plasmid containing the -1200/+63 collagenase promoter in front of the CAT reporter was a gift from Dr. Ronald M. Evans (Howard Hughes Medical Institute, The Salk Institute, CA). The collagenase promoter contains an AP-1-binding site responsive to the tumor promoter 12-O-tetradecanoyl-phorbol-13 acetate (TPA) (24
). The RSV-ß-galactosidase (RSV-ß-gal) expression plasmid was a gift from Dr. Erick Paulson (HNRC at Tufts University, Boston, MA) and was used as an internal control for transfection efficiency.
MCF-7 and Hs578T cells were seeded at 1 million cells/100 mm cell culture dish in medium containing 10% charcoal-treated FBS. The next day, the cells were fed fresh growth medium 3 h before transfection. The cells were then transfected by the calcium phosphate precipitation method using 2 µg pBLCAT3 reporter plasmid and 2 µg of RSV-ß-gal. The total amount of transfected DNA was adjusted to 20 µg with pUC18 (Life Technologies). The cells were exposed to the precipitate for 18 h. After this incubation, the cells were fed 0.5% charcoal-treated FBS medium for 24 h. Then, fresh growth medium containing either ethanol, TPA (0.0001 g/L) alone or in combination with 1 µmol/L apo-CA or atRA in 0.3% ethanol and 0.5% charcoal-treated FBS was added to the cells. After 12 h, the cells were collected and CAT levels were determined using a CAT-ELISA (Boehringer Mannheim, GmbH, Germany).
Competitive binding assays.
Protein extracts containing recombinant RARs were kindly provided by Dr. M. Clagett-Dame (Department of Biochemistry, University of Wisconsin, Madison, WI). Recombinant mouse RARß (mRARß) and human RAR (hRAR) proteins were obtained using the lytic virus Autographa californica nuclear polyhedrosis virus baculovirus insect cell system (25
). Human RAR (hRAR) protein was generated as a pATH (plasmids that are amenable for making Trp hybrids) fusion protein using the pATH expression vector (26
) in Escherichia coli (27
). Equilibrium saturation binding analyses were done as described previously (27
). For competitive binding assays purified RAR/pATH fusion (denatured and refolded), mRARß, and hRAR proteins were incubated with 5 nmol/L all-trans-[3H]RA in the absence or presence of different concentrations of unlabeled atRA or apo-CA (0.5 nmol/L to 50 µmol/L) or ethanol (4% final concentration). Ligand-bound receptors were separated from free-ligand by use of hydroxyapatite. Quantitation of the all-trans-[3H]RA ligand bound to the retinoid receptors was determined by liquid scintillation counting. Radioligand competition and agonist/antagonist concentration-response curves from at least two independent experiments performed in triplicates were calculated based on sigmoidal data fits using the Graph Pad Prism software (Version 3.0). All incubation reactions were performed under red light on ice.
Statistics.
In the growth inhibition studies, differences between treatments at d 9 were analyzed by ANOVA followed by Fisher’s Protected Least Significant Difference (StatView, Version 4.5; Abacus Concepts, Berkeley, CA). For Northern blots, Western blots and collagenase-CAT transactivation data, differences between means of triplicate samples from two or three independent experiments from treated cells and controls were determined by two-sided t tests. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTransient transfection and...RESULTSDISCUSSIONLITERATURE CITED |
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The identity of the 99% pure apo-CA synthesized was confirmed by comparison against corresponding standards of ß-apo-14'-CA, ß-apo-12'-CA, ß-apo-10'-CA and ß-apo-8'-CA (data not shown). The synthesized products matched perfectly the retention times and spectra of the standards with absorption maxima of 378, 408, 424 and 441 nm, respectively.
ß-apo-CA and all-trans-retinoic acid inhibit the growth of breast tumor cells.
We examined the effect of three different concentrations of four synthetic excentric cleavage products of ß-carotene and atRA on the growth of the ER+ breast tumor cell line MCF-7 and the ER- breast tumor cell lines Hs578T and MDA-MB-231. The ß-apo-CA tested were similar in structure to atRA but had 5–8 double bonds in their polyene chain (Fig. 1)
. None of the compounds tested induced growth inhibition after 2 or 6 d of treatment (data not shown), but longer-term exposure induced growth inhibition. Control cultures in the absence of retinoids or carotenoids continued to grow exponentially up to d 13 (data not shown). The effect of continuous 9-d treatment of MCF-7 ER+ and Hs578T ER- breast tumor cells to ß-apo-CA and atRA is depicted in Figure 2
. atRA induced a concentration-dependent inhibition of cell proliferation in both MCF-7 and Hs578T cell lines (9%, 32% and 64% inhibition in MCF-7 and 26%, 36%, and 46% inhibition in Hs578T at 10 nmol/L, 100 nmol/L and 1 µmol/L, respectively). In MCF-7 cells, ß-apo-14'-CA induced concentration-dependent inhibition of cell proliferation (100 nmol/L, 14% inhibition and 1 µmol/L, 34% inhibition; P < 0.05). ß-apo-12'-CA at 1 µmol/L inhibited cell proliferation (31%; P < 0.05). Neither ß-apo-10'-CA nor ß-apo-8'-CA inhibited cell proliferation except for a small but statistically significant inhibition at 1 µmol/L (14%; P < 0.05) by ß-apo-8'-CA (Fig. 2)
. Using Hs578T cells, only ß-apo-14'-CA induced an anti-proliferative response (1 µmol/L, 23% inhibition, P < 0.05). Only ß-apo-14'-, ß-apo-12'-CA, and atRA were tested in MDA-MB-231 cells. They had no antiproliferative effects in this cell line (data not shown).
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The effects of ß-apo-14'-CA, ß-apo-12'-CA and atRA on the expression of two cell cycle regulators, Rb and E2F1 proteins, and on Rb phosphorylation in MCF-7 and Hs578T breast tumor cells are depicted in Figures 3
and 4
. Western blots of whole cell lysates showed two different forms of Rb protein, a hyperphosphorylated form of Rb (ppRb), and a hypophosphorylated form (pRb). Treatment with ß-apo-CA and atRA (1 µmol/L) inhibited (P < 0.05) the total Rb protein level and the level of ppRb at different time points in MCF-7 cells (Fig. 3)
. ß-apo-14'-CA inhibited the level of ppRb by 54% at d 6 (P < 0.05), whereas ß-apo-12'-CA inhibited it by 59% at d 1 (P < 0.05), and atRA by 53% (P < 0.05), and 70% (P < 0.05) at d 1 and 6, respectively. A reduction of pRb was observed at some time-points after treatment with ß-apo-12'-CA and atRA but not with ß-apo-14'-CA in this cell line. No significant effects were observed at d 9 (data not shown). In Hs578T cells, the level of Rb protein was not affected by any of the treatments (data not shown).
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A). In Hs578T cells, ß-apo-14'-CA reduced the expression of E2F1 by 50% at 24 h (P < 0.05) and by 60% at 72 h (P < 0.05; Fig. 4
B). In this cell line, atRA inhibited E2F1 expression by 60% at 24 h (P < 0.05) and by 40% at 72 h of treatment (P < 0.05). ß-apo-12'-CA did not affect the expression of E2F1 in either of these cell lines. Expression of the control protein ß-actin did not differ among treatments. ß-apo-CA suppress AP-1-mediated gene transactivation.
To determine whether the ß-apo-CA-induced growth inhibitory effect was due to an anti-AP-1 activity similar to that elicited by atRA, we examined the effect of ß-apo-14'-CA and ß-apo-12'-CA (1 µmol/L) on the TPA-induced transcriptional response of the collagenase CAT reporter gene. atRA was used for comparison. TPA induced a 20-fold increase of CAT activity in MCF-7 cells (Fig. 5
) and a 595-fold increase of CAT activity in Hs578T cells (Fig. 5)
. In both MCF-7 and Hs578T cells, all of the compounds tested significantly inhibited the TPA-induced reporter activity. atRA significantly inhibited TPA-induced CAT activity. ß-apo-14'-CA and atRA induced a stronger inhibitory effect in Hs578T cells (59% and 85%; P < 0.05, respectively; Fig. 5
). ß-Apo-12'-CA was not tested in Hs578T cells because this compound had no antiproliferative effect in our growth assays. These results showed that ß-apo-CA can effectively inhibit the collagenase promoter activity induced by TPA.
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ß-apo-CA are not metabolized to atRA or to more polar metabolites in MCF-7 cells.
The metabolism of 1 µmol/L atRA and 1 µmol/L apo-14'-CA was examined by HPLC after a 5-h incubation in MCF-7 cells. We detected some metabolism of atRA to more polar metabolites (Fig. 6A
) but did not find any metabolism of ß-apo-14'-CA to either atRA or to more polar metabolites (Fig. 6
B). No atRA or atRA metabolic product formation was detected in the growth medium or the cell pellets after 9 d incubation with any of the apo-CA in the cell lines tested (data not shown). The HPLC system had a detection limit of 0.2 nmol/L (80 pg) for atRA.
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To examine whether ß-apo-CA can act as ligands for the RAR proteins, we performed in vitro incubations of purified recombinant RAR proteins with different concentrations of unlabeled ß-apo-CA. The Kd values of atRA for the RAR proteins were obtained by transformation of the equilibrium saturation data using Scatchard analysis. The equilibrium saturation data for RARß are depicted in Figure 7A
. The Kd values obtained were: RARß, 0.26 nmol/L (Fig. 7
A) and RAR , 1.48 nmol/L (data not shown). The RAR/pATH Kd of 0.4 nmol/L had been previously reported by Repa et al. (27
).
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B). The Ki values obtained were apo-14'-CA, 617 nmol/L; apo-12'-CA, 4494 nmol/L; apo-10'-CA, 388 nmol/L; and apo-8'-CA, 9052 nmol/L in contrast with unlabeled atRA, 0.32 nmol/L. Similar patterns of competitive binding were observed with RAR and RAR (data not shown) suggesting that these apo-CA are 3–4 orders of magnitude less effective than all-trans-[3H]RA for binding to the RARs. RAR gene expression.
We examined RAR gene expression at the mRNA and protein level and found no changes in the expression of mRNA after 2–72 h. We also did not find changes in the RAR, RARß or RAR protein levels after treatment with 1 µmol/L atRA or the apo-CA for 1–9 d (data not shown). RARß was neither expressed nor induced by the treatments in MCF-7 cells (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTransient transfection and...RESULTSDISCUSSIONLITERATURE CITED |
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,29
). We showed that both ß-apo-14'-, and ß-apo-12'-CA are effective inhibitors of ER+ MCF-7 breast tumor cell growth with 
50% the efficacy of atRA at 1 µmol/L. ß-apo-14'-CA has 44% the efficacy of atRA at 100 nmol/L. In Hs578T ER- cells, ß-apo-14'-CA is also one-half as effective as atRA at 1 µmol/L but has no effect at any other concentration tested. The longer-chain apo-CA are weak inhibitors or have no effect on breast tumor cell growth. Neither apo-carotenoids nor atRA inhibits the growth of ER- MDA-MB-231 cells. The ER+ MCF-7 cell line was the most sensitive to treatment with apo-CA and atRA, yet Hs578T, an ER- cell line, was significantly growth-inhibited by atRA treatments at 10 nmol/L as well as by 1 µmol/L ß-apo-14'-CA, suggesting that the growth inhibitory response may not be directly associated with the ER status of these cell lines.
Two of the apo-CA used in this study have been previously tested in the human acute promyelocytic leukemia cell model HL-60 (14
). In this cell line apo-12'-CA was also biologically active, whereas ß-apo-14'-CA had weak biological activity in terms of growth inhibition and cell differentiation. This suggests that the degree of responsiveness is cell-specific. Additionally, in a specific cell type, the length of the polyene chain of apo-CA may be correlated with their biological activity.
In an attempt to understand the mechanism of action of ß-apo-14'- and ß-apo-12'-CA, we studied their potential effect on cell cycle regulators. One of the mechanisms by which atRA inhibits proliferation of breast tumor cells is by down-regulation of total retinoblastoma protein (RB) protein and RB phosphorylation (30
). Changes in RB phosphorylation control cell cycle progression. Using Hs578T cells, we found no significant inhibition of total RB protein or any of its forms in response to the treatments tested. In MCF-7 cells, ß-apo-CA and atRA inhibited the expression of the hyperphosphorylated form of RB, in agreement with published studies using atRA (30
,31
). These data suggest that ß-apo-CA may induce breast tumor growth inhibition by down-regulating RB protein expression and changing its phosphorylation state.
Another important protein involved in cell cycle control, and a target of RB protein, is E2F1 (32
). We found a significant down-regulation of E2F1 protein expression in MCF-7 and Hs578T cells after treatment with ß-apo-14'-CA and atRA, occurring as early as 24 h (Fig. 4
, A and B). atRA-induced inhibition of E2F1 and RB protein expression in MCF-7 cells has also been reported by Zhu et al. (30
), although in this case the inhibition of E2F1 protein expression was not observed until 72 h.
Another potential mechanism of growth inhibition by ß-apo-CA and atRA is the anti-AP-1 activity of these compounds. RAR-dependent inhibition of AP-1 function is one of the mechanisms by which retinoids may inhibit cell proliferation (33
). Different lines of evidence suggest that RAR anti-AP-1 function is independent of RAR transcriptional activation (34
,35
) and that the regions of the RAR involved in transcriptional activation may be different from those involved in RAR-AP-1 antagonism (34
). We found that ß-apo-CA inhibited AP-1 transcriptional up-regulation of an AP-1 reporter gene in response to the tumor promoter TPA in MCF-7 and Hs578T cells (Fig. 5)
. atRA inhibited the TPA-induced transcriptional activation of the collagenase-CAT reporter in agreement with published studies (36
,37
), yet it did not consistently alter the basal expression of AP-1 proteins. Although the atRA anti-TPA-induced AP-1 activity was stronger than that of ß-apo-14'- and ß-apo-12'-CA, the magnitude of the inhibitory response induced by the latter compounds demonstrates their appreciable anti-AP1 activity.
Because retinoid-induced growth inhibition is mediated by the RAR and RXR families of nuclear receptors (38
), we examined the potential role of ß-apo-CA as ligands for the RAR receptors. Our competitive binding data indicated that ß-apo-CA are very weak competitors of [3H] atRA for binding to recombinant RAR receptors (Fig. 7
B). The lower binding affinity compared to unlabeled atRA is probably due to the length of the side chain of ß-apo-CA. These results are similar to studies with different synthetic atRA analogs with modified chemical structures (39
,40
). The low binding affinities of the ß-apo-CA at the recombinant RAR led us to hypothesize that the ß-apo-CA-induced growth inhibition (Fig. 2)
may be due to indirect upregulation of RAR. Northern and Western blots demonstrated that neither atRA nor apo-CA up-regulated RAR receptors in MCF-7 and Hs578T (data not shown), ruling out this possibility.
Our studies on the potential metabolic conversion of apo-CA to atRA clearly showed that neither atRA nor more polar metabolites are formed from apo-CA under the conditions studied, as shown by HPLC (atRA detection limit = 0.2 nmol/L or 80 pg/L). Our control assays showed the formation of atRA polar metabolites after a 5-h incubation of MCF-7 cells with atRA, which is in agreement with published studies (41
). Because we could not detect atRA or its metabolites even after 5 h incubation with ß-apo-14'-CA (Fig. 6)
, it is very unlikely that the antiproliferative response induced by apo-14'-CA was due to its conversion to atRA.
Collectively, our data indicate that some synthetic excentric cleavage products of ß-carotene are biologically active in breast tumor cells and that their growth inhibitory effect is not due to their conversion to atRA. Our data also suggest that the breast tumor antiproliferative activity of ß-apo-CA may be partially related to their ability to down-regulate cell cycle control proteins and to inhibit transactivation of AP-1 responsive genes. Similarly, a recent study demonstrated that acyclo-retinoic acid, an open-chain atRA analog, seems to also affect breast tumor cell growth through down-regulation of a cell cycle regulatory protein, in this case, cyclin D, independently of RAR transactivation (42
).
Because of the lack of apo-CA’s up-regulation of, and their poor interaction with RAR, we speculate that in contrast to retinoids, ß-apo-CA down-regulate AP-1 activity through different mechanisms (e.g., through proteins other than RAR). There is ample evidence that retinoids, which act specifically through inhibition of AP-1-dependent gene expression, have limited pleiotropic effects, and as such, have a greater clinical potential (34
,43
,44
). Therefore, the anti-proliferative effect along with the RAR-independent anti-AP-1 activity of ß-apo-CA, support further study of these compounds as potential chemotherapeutic agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AP-1, activator protein-1; atRA, all-trans-retinoic acid; ß-apo-CA, ß-apo-carotenoic acid; CAT, chloramphenicol acetyl transferase; DTT, dithiothreitol; ER, estrogen receptor; FBS, fetal bovine serum; MEM, minimum essential medium-; PMSF, phenylmethylsulfonyl fluoride; RAR, retinoic acid receptor; RB, retinoblastoma protein; TPA, 12-O-tetradecanoyl-phorbol-13 acetate.
Manuscript received 14 November 2001. Initial review completed 16 January 2002. Revision accepted 4 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTransient transfection and...RESULTSDISCUSSIONLITERATURE CITED |
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