Nutrition and Cancer

In Vitro Inhibition of Proliferation of Estrogen-Dependent and Estrogen- Independent Human Breast Cancer Cells Treated with Carotenoids or Retinoids1 Pankaj Prakash,*2 Robert M. Russell† and Norman I. Krinsky*† *Department of Biochemistry, School of Medicine and the †Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111-1837

ABSTRACT Both estrogen-receptor (ER) positive MCF-7 and ER-negative Hs578T and MDA-MB-231 human breast cancer cells were treated with carotenoids (␤-carotene, canthaxanthin and lycopene) and retinoids (all-trans-, 9-cis- and Downloaded from 13-cis-retinoic acid and all-trans-retinol). Among carotenoids, ␤-carotene significantly reduced the growth of MCF-7 and Hs578T cells, and lycopene inhibited the growth of MCF-7 and MDA-MB-231 cells. Canthaxanthin did not affect the proliferation of any of the three cell lines. All-trans- and 9-cis-retinoic acid significantly reduced the growth of both MCF-7 and Hs578T cells, whereas 13-cis-retinoic acid and all-trans-retinol had a significant effect only on MCF-7 cells. MCF-7 and Hs578T cells treated with all-trans-retinoic acid (all-t-RA) were further studied for the mechanism behind

growth inhibition. Retinoic acid receptors ␣ and ␥ (RAR␣, ␥) in MCF-7 cells and RAR␣, ␤ and ␥ in Hs578T cells were not jn.nutrition.org induced by all-t-RA treatment at either the protein or mRNA level. Hs578T cells treated with all-t-RA had signiﬁcantly more cells in the G0/G1 stage of the cell cycle, but the same was not observed for MCF-7 cells. All-t-RA induced a dose-dependent cell death in MCF-7 cells, which may be a necrotic phenomenon. These results demonstrate that ER status is an important, although not essential factor for breast cancer cell response to carotenoid and retinoid treatments, and the mode of action of all-t-RA in MCF-7 and Hs578T cells is not through the induction of RAR. Other at USDA, National Agricultural Library on April 28, 2009 mechanistic pathways that are either followed by or concomitant with growth inhibition are possible. J. Nutr. 131: 1574–1580, 2001.

KEY WORDS: ● ␤-carotene ● retinoic acid ● breast cancer cells ● cell cycle ● retinoic acid receptors

Cancer of the breast is the most common incident cancer ciated with an increased risk of breast cancer (Comstock et al. and cause of death from cancer in women (WHO 1997). The 1992, Potischman et al. 1990, Weisburger 1991). offspring of emigrants from countries with low breast cancer Carotenoids may act as chemoprotective agents through incidence to countries with high breast cancer incidence ac- biological activities such as metabolism to retinoids, antioxi- quire rates close to those of the new country, suggesting that dation, immunoenhancement, protection against cellular mu- environmental and lifestyle inﬂuences are important in the tagenesis and malignant transformation, and inhibition of etiology of breast cancer (McMichael and Giles 1988). This tumorigenesis (Bendich 1989, Krinsky 1989 and 1993, Shultz could be the result of noninherited factors including, among et al. 1992). Carotenoids also function as radical-trapping other possibilities, diet (Trichopoulos and Willett 1996). antioxidants in vitro and may have the same activity in vivo Epidemiologic and laboratory investigations exploring the (Burton and Ingold 1984, Krinsky 1989). Retinoids, on the other hand, exert a variety of effects on basic biological pro- relationship between diet and disease suggest an inverse cor- cesses such as growth, differentiation, development and ma- relation between consumption of fruits and vegetables rich in lignant transformation, in addition to receptor-induced signal carotenoids and certain cancer incidence rates (Block et al. transduction. Knowledge of the effect of these compounds has 1992, Ziegler 1989). In a review of possible environmental led to the hypothesis that retinoids may act as chemopreven- determinants of cancer, it was estimated that 35% of cancers tive agents as well as inhibitors of the growth of established in the United States might be attributable to dietary factors tumors (Hill and Grubbs 1992, Peto et al. 1981). (Doll and Peto 1981). Several studies have shown that low This study was designed to determine the effects of carote- levels of either dietary intake or plasma carotenoids are asso- noids and retinoids on the growth of human breast cancer cells. Brieﬂy, the growth of estrogen-receptor positive (ERϩ)3 MCF-7 and estrogen-receptor negative (ERϪ) Hs578T and 1 Supported in part by National Institutes of Health grant 5R01 CA 66914–03 MDA-MB-231 human breast cancer cells was determined after and by a fellowship from the Cancer Research Foundation of America. P.P. was supported by a fellowship from the Cancer Research Foundation of America. 2 To whom correspondence should be addressed at Silico Insights, Incorpo- 3 Abbreviations used: all-t-RA, all-trans-retinoic acid; ER(ϩ), estrogen recep- rated, 400 W. Cummings Park, Suite 6475, Woburn, MA 01801. E-mail: tor positive; ER(Ϫ), estrogen receptor negative; RAR, retinoic acid receptor; RXR, [email protected]. retinoid X receptor; THF, tetrahydrofuran.

1574 CAROTENOIDS, RETINOIDS AND BREAST CANCER CELLS 1575 treatment with a provitamin A carotenoid, ␤-carotene, and Cell proliferation. For the determination of cell proliferation, the nonprovitamin A carotenoids, canthaxanthin and lyco- MCF-7, Hs578T and MDA-MB-231 cells were incubated with ␤-car- pene, and the retinoids, all-trans-, 9-cis-, and 13-cis-retinoic otene, canthaxanthin, lycopene, all-trans-, 9-cis- and 13-cis-retinoic acid and all-trans-retinol. The mechanism underlying growth acid and all-trans-retinol solutions for up to 9 d. Proliferating viable inhibition caused by all-trans-retinoic acid (all-t-RA) in cells attached to the plastic wells were harvested and their numbers counted on d 2, 5 and 9 (data are shown for d 9 only, i.e., when the MCF-7 and Hs578T cells was further studied by determining cells reached confluency). For cell harvesting, the medium was aspi- the retinoic acid receptor (RAR) expression, cell cycle arrest rated from the wells; 0.5 mL of 0.25% trypsin solution was added to and apoptosis induction in these cells. each well and incubated at 37°C for 5, 3 and 2 min, for MCF-7, MDA-MB-231 and Hs578T cells, respectively. The reaction was stopped with 1.5 mL of the growth medium at room temperature. The MATERIALS AND METHODS cells were collected and counted in duplicate using an electronic Coulter Counter (model Z1; Coulter, Miami, FL). Cell morphology Chemicals. Growth media and other growth regulators were was monitored by periodic evaluation of the cells under a phase purchased from Gibco Life Technologies (Gaithersburg, MD). Crys- contrast microscope throughout the experiments. talline ␤-carotene, canthaxanthin, all-trans-, 9-cis-, and 13-cis-reti- Northern blot analysis of RAR(␣, ␤ and ␥) mRNA expression. noic acid and all-trans-retinol were purchased from Sigma Chemical MCF-7 and Hs578T cells were incubated with all-t-RA for 2, 6, 8, 24 (St. Louis, MO). Crystalline lycopene was kindly provided by and 72 h. RAR␣, ␤ and ␥ expression was examined at the transcrip- LycoRed, Beer-Shiva, Israel. Tetrahydrofuran (THF) stabilized with tion level in the control and treatment cells by Northern blot analysisDownloaded from BHT and acetone was purchased from Fisher Scientific, Pittsburgh, of total cellular RNA, which was extracted from the cells using the PA. The retinoid receptor cDNA probes were obtained as a kind gift cesium chloride gradient method (Tsou et al. 1994). RNA from each from the laboratory of Dr. Monica Peacocke, Columbia University, sample (30 ␮g) was subjected to electrophoresis on a 1.2% agarose gel New York, NY. containing 5% formaldehyde. The gel was transferred to a Nytran Cells and cell culture conditions. MCF-7 human breast cancer Plus nylon membrane using a Turboblotter system (Schleicher & cells were obtained from the Michigan Cancer Foundation (Detroit, Schuell, Keene, NH). The membrane(s) were subjected to UV MI). Hs578T and MDA-MB-231 human breast cancer cells were crosslinking and then hybridized to [␣-32P]-dCTP-labeled retinoidjn.nutrition.org obtained from American Type Culture Collection (Rockville, MD). receptor cDNA probes (Tsou et al. 1994). pHE7 was used as a control MCF-7 cells were maintained in ␣-MEM medium containing HEPES, gene to monitor equivalent loading of RNA in each lane because the MEM nonessential amino acids, sodium pyruvate, L-glutamine, insu- levels of pHE7 are not affected by retinoic acid (Tsou et al. 1994). lin, gentamycin and 10% fetal bovine serum (5% serum for the After high stringency washing (0.1X standard saline citrate at 60°C), experiments). Hs578T cells were maintained in Dulbecco’s modified the membranes were exposed to photographic film using an intensi- at USDA, National Agricultural Library on April 28, 2009 Eagle’s medium with 4.5 g/L glucose, 10 mg/L insulin and 10% fetal fying screen at Ϫ80°C for various lengths of time (between 1 and bovine serum. MDA-MB-231 cells were maintained in a 1:1 mixture 7 d). The films were developed using an RG II film processor (Fuji, of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium Elmsford, NY). containing 2 mmol/L L-glutamine, and 10% fetal bovine serum. Cells Semiquantitative evaluation of Northern blots. The films of were grown in 100-mm culture dishes and incubated at 37°C in a Northern blot analysis were quantified by densitometric scanning humidified atmosphere of 5% CO2 in air. Cells were seeded at a 2 using a PD model densitometer (Molecular Dynamics, Sunnyvale, concentration of ϳ5000 cells/cm for the experiments, unless other- wise indicated. The trypan blue exclusion method using a hemocy- CA) and ImageQuant software. The pixel value obtained for each tometer was used to distinguish viable from dead cells. individual band was normalized to the value of its relative pHE7 Preparation of carotenoid and retinoid solutions. Solutions of expression. The values obtained after normalization were used to ␤-carotene, canthaxanthin and lycopene were prepared in THF con- compare the expression of retinoid receptors against their respective taining 0.25 g/L BHT as a preservative. Fresh solutions of the caro- controls in the cell lines at different time points. Data are expressed tenoids were prepared in a nitrogen environment in a plastic glove as fold induction, considering the value of the control to be 1. bag (Aldrich, Milwaukee, WI) on each day of the experiment as Flow cytometry. MCF-7 and Hs578T cells were incubated with needed. The required amount of THF was withdrawn from the sealed all-t-RA for 6 and 9 d. After exposure to experimental conditions, reagent bottle by a syringe purged with nitrogen to prevent oxidation cells were collected and washed twice with cold PBS and suspended of the solvent. Crystalline synthetic ␤-carotene, canthaxanthin and again in PBS. Absolute ethanol was added dropwise with vortexing at lycopene were stored in the dark at Ϫ70°C between experiments to low speed to bring the ethanol concentration to 70%; this suspension minimize oxidation and decay. Stock solutions of the carotenoids was fixed at 4°C for 30 min. Cells were again washed twice with PBS were prepared to result in concentrations of 1, 3, 7, 10 and 20 after removal of ethanol and incubated with 10 mg/L RNase solution mmol/L. The appropriate stock solution (1 ␮L) was added to each in PBS at 37°C for 30 min, with mixing several times during incu- bation. After being washed twice with PBS, cells were suspended in milliliter of the media to result in a final carotenoid concentration of 6 1, 3, 7, 10 and 20 ␮mol/L in the media. In this way, the concentration propidium iodide solution at 1 ϫ 10 cells/0.5 mL concentration. of THF in the media was 0.1%, which did not cause any toxicity to Cells were then analyzed for their cell cycle distribution using a Flow the cells as determined by cell growth (data not shown). All-trans-, cytometer (Beckton Dickinson, Franklin Lakes, NJ) and ModFit LT 9-cis- and 13-cis-retinoic acid and all-trans-retinol were dissolved in 2.0 software (Beckton Dickinson) for data analysis. dimethyl sulfoxide to produce stock solutions of 0.1 mol/L. Further Morphological analysis. MCF-7 cells were incubated with all- dilutions were made in acetone. Retinoids were then dissolved in the t-RA for 7 d. Cells were grown on chamber slides for the morphologic growth media to reach final concentrations of 10 nmol/L, 100 nmol/L analysis. After exposure to experimental conditions, cells were and 1 ␮mol/L at a 0.25% concentration of acetone that was not toxic washed with PBS and stained with acridine orange (25 mg/L) and to the cells (data not shown). The purity of the carotenoid and propidium iodide (25 mg/L) for 15 min in the dark. Cells were then retinoid stocks was determined by HPLC and the concentration was washed in PBS, fixed in 10% formalin for 30 min in the dark and determined by spectrophotometric analysis. Red light was used during washed again in PBS. Dual-stained cells were viewed using a fluores- preparation of carotenoid and retinoid solutions to prevent photo- cence microscope. Cells were coded as either early apoptotic (bright damage to these compounds. green, highly condensed chromatin), late apoptotic (bright orange, Treatment of cells. Treatments were applied 24 h after cultures highly condensed chromatin) or necrotic cells (bright orange nucleus had been seeded to ensure proper attachment of the cells to the without condensed chromatin) (Broaddus et al. 1996). plastic culture plates or dishes. MCF-7, Hs578T and MDA-MB-231 Statistics. Results are expressed as means Ϯ SD of three deter- cells were incubated with different carotenoids or retinoids for the minations. Comparisons of mean values of control and treatment periods mentioned in the respective experiments. Control cells re- cells were made using ANOVA on the whole population followed by ceived medium supplemented with THF or acetone only. Control and two-sample Student’s t test between control and each treatment treatment media were changed every other day. group. The statistical significance of difference (P Ͻ 0.05) for the 1576 PRAKASH ET AL. Downloaded from jn.nutrition.org

FIGURE 2 Effect of all-trans-, 9-cis- and 13-cis-retinoic acid (RA), dissolved in acetone (0.25%), on the growth of Hs578T human breast cancer cells grown at a density of 5000 cells/cm2. Cells were treated with the retinoids at concentrations of 10 nmol/L, 100 nmol/L and 1 ␮mol/L in the media for 9 d. Values are means Ϯ SD of three replicate at USDA, National Agricultural Library on April 28, 2009 assays; *signiﬁcantly different from control, P Ͻ 0.05.

icantly inhibited the growth of MCF-7 cells, stimulated the growth of MDA-MB-231 cells and had no effect on Hs578T FIGURE 1 Effect of ␤-carotene (BC; panel A) and lycopene (Lyco; cells ond9oftreatment (P Ͻ 0.05; Fig. 3). Signiﬁcant effects panel B) in tetrahydrofuran (THF; 0.1%) on the growth of MCF-7, Hs578T and MDA-MB-231 human breast cancer cells grown at a density of 5000 cells/cm2. Cells were treated with ␤-carotene at concentrations of 1, 3 and 10 ␮mol/L and lycopene at concentrations of 7, 10 and 20 ␮mol/L in the media for 9 d. Values are means Ϯ SD of three replicate assays; *signiﬁcantly different from control, P Ͻ 0.05. treatment groups was determined relative to their respective control group.

RESULTS Effect of carotenoids on cell growth. The effects of ␤-carotene at 1, 3 and 10 ␮mol/L and lycopene at 7, 10 and 20 ␮mol/L, dissolved in THF (0.1%), on the growth of MCF-7, Hs578T and MDA-MB-231 cells are shown in Figure 1. ␤-Carotene significantly inhibited the growth of MCF-7 and Hs578T cells ond9(P Ͻ 0.05) at concentrations Ն3 ␮mol/L, whereas it significantly increased the growth of MDA-MB-231 cells at Ն1 ␮mol/L (Fig. 1A). Lycopene significantly inhibited the growth of MCF-7 at 10 and 20 ␮mol/L and MDA-MB-231 cells at 20 ␮mol/L ond9(P Ͻ 0.05; Fig. 1B). Canthaxanthin, at concentrations up to 20 ␮mol/L, did not affect cell growth (data not shown). Effect of retinoids on cell growth. The effects of all-trans-, 9-cis- and 13-cis-retinoic acid, dissolved in acetone (0.25%), FIGURE 3 Effect of all-trans-retinol (all-t-ROL), dissolved in ace- on the growth of Hs578T cells are shown in Figure 2. All- tone (0.25%), on the growth of MCF-7, Hs578T and MDA-MB-231 trans- and 9-cis-retinoic acid significantly inhibited the growth human breast cancer cells grown at a density of 5000 cells/cm2. Cells of Hs578T cells at 100 nmol/L and 1 ␮mol/L ond9(P were treated with retinol at concentrations of 10 nmol/L, 100 nmol/L Ͻ 0.05), whereas 13-cis-retinoic acid had no effect on cell and 1 ␮mol/L in the media for 9 d. Values are means Ϯ SD of three growth. All-trans-retinol at 100 nmol/L and 1 ␮mol/L signif- replicate assays; *significantly different from control, P Ͻ 0.05. CAROTENOIDS, RETINOIDS AND BREAST CANCER CELLS 1577

(P Ͻ 0.05) of all-trans-, 9-cis- and 13-cis-retinoic acid were observed on the growth inhibition of MCF-7 cells and growth stimulation of MDA-MB-231 cells. These data are not repre- sented here because similar results have previously been published elsewhere (Marth et al. 1993, Van heusden et al. 1998). Effect of cell concentration and growth on carotenoid- and retinoid-induced growth inhibition. Several of the growth experiments were also conducted at high and low initial den- sities of MCF-7 cells. Figure 4A shows the growth of the cells treated with ␤-carotene and all-t-RA, when grown at a high initial density (15,000 cells/cm2). ␤-Carotene and all-t-RA at 10 and 1 ␮mol/L, respectively, did not inhibit growth under these conditions, whereas these concentrations signiﬁcantly reduced the cell growth when the initial cell density was 5000 cells/cm2 (Figs. 1, 4). In the control cultures of MCF-7 cells, a rapid increase in

cell proliferation was observed between d 5 and 9 of growth. Downloaded from The cells grew from ϳ8 ϫ 105 cells/well ond5toϳ5 ϫ 106 cells/well on d 9 (not shown). To determine whether the growth inhibitory effect of all-t-RA was due to the rapid cell doubling between these two time points, one set of cells was treated with 1 ␮mol/L all-t-RA from d 5 onward and compared with another set of cells supplemented with 1 ␮mol/L all-t-RA from d 1 onward. The cell numbers in the group supplemented jn.nutrition.org from d 5 onward were not different from the control group (Fig. 4B), indicating that an accumulation of retinoic acid in at USDA, National Agricultural Library on April 28, 2009

FIGURE 5 Northern blot analysis of the expression of retinoic acid receptors ␣ and ␥ (RAR␣ and ␥) in MCF-7 and RAR␣, ␤ and ␥ in Hs578T human breast cancer cells treated with 1 ␮mol/L all-trans-retinoic acid (RA) dissolved in acetone (0.25%) for 2, 6, 8, 24 and 72 h. The fold induction of the receptors is shown in densitometric scanning of the bands after normalizing to pHE7 (Table 1). The observed induction of receptors due to retinoid treatment was not found to be consistent in other replicate analyses.

the cells before this time point is necessary for the retinoid to be effective. The other possibility could be that the cell density on d 5 was too large for the retinoid to have an effect. Effect of all-t-RA on the expression of RAR. The expression of RAR␣ and ␥ in MCF-7 cells as a result of all-t-RA treatment is shown in Figure 5. Cells were treated with 1 ␮mol/L all-t-RA for 2, 6, 8, 24 or 72 h and investigated for the induction of retinoid receptors at the mRNA level using Northern blot analysis. The bands were compared using densitometric scanning after normalizing with the pHE7 control to determine the fold induction under retinoid treatment (Table 1). Small inductions of RAR␣ and ␥ were observed at some time points, but these inductions were not consistent. Similar results of Northern blot analysis were observed in Hs578T cells (Fig. 5 and Table 1) using similar treatments and duration. RAR(␣, ␤ and ␥) were not found to be induced consistently. Similarly, no induction of retinoic acid receptors was found at the protein level in MCF-7 and Hs578T cells FIGURE 4 Effect of an alteration in culture and treatment condi- treated with 1 ␮mol/L all-t-RA using Western blot analysis tions on carotenoid- and retinoid-induced growth inhibition of MCF-7 (data not shown). human breast cancer cells. (A) Cells were grown at a density of 15,000 2 Effect of all-t-RA on the cell cycle stage distribution. cells/cm and treated with either 10 ␮mol/L ␤-carotene (BC) or 1 Figure 6 shows the cell cycle distribution of Hs578T cells ␮mol/L all-trans-retinoic acid (All-t-RA) in the media for 9 d. (B) Cells were grown at a density of 5000 cells/cm2 and treated with 1 ␮mol/L treated with 1 ␮mol/L all-t-RA for 6 and 9 d. A signiﬁcant retinoic acid supplied from either d 1 onward (hatched bar) or d 5 increase in cell number in the G0/G1 stage of the cell cycle onward (open bar) in the media for 9 d. Values are means Ϯ SD of three after treatment with all-t-RA was observed for d 6 and 9. No replicate assays; *signiﬁcantly different from control, P Ͻ 0.05. Abbre- increase was observed for MCF-7 cells under similar conditions viation: THF, tetrahydrofuran. (data not shown). 1578 PRAKASH ET AL.

TABLE 1 TABLE 2 Fold induction of retinoid receptor subtypes in MCF-7 and Cell death induced by all-trans-retinoic acid (all-t-RA) Hs578T cells treated with all-trans-retinoic acid1 in MCF-7 cells1

Band2 2 Hour 6 Hour 8 Hour 24 Hour 72 Hour Green cells2 Orange cells2 Orange cells

MCF-7 n ϭ 200 cells % RAR ␣ Upper band 1.24 0.87 1.13 1.3 1.43 Control3 195.00 Ϯ 2.65 5.00 Ϯ 2.65 2.5 Ϯ 1.32 Lower band 1.13 0.86 1.15 1.44 1.7 All-t-RA,3 1 ␮mol/L 161.67 Ϯ 10.02 38.33 Ϯ 10.02 19.17 Ϯ 5.01* RAR ␥ 1.23 0.88 1.1 1.47 2.013 All-t-RA,3 10 ␮mol/L 149.33 Ϯ 15.31 50.67 Ϯ 15.31 25.33 Ϯ 7.65* Hs578T RAR ␣ 1 MCF-7 cells were treated with all-trans-retinoic acid at 1 and 10 Upper band 1.48 0.97 1.08 1.56 0.58 ␮mol/L concentration for 7 d. Lower band 1.77 0.72 0.97 1.52 0.57 2 Cells were stained with acridine orange and propidium iodide (25 RAR ␤ mg/L) and studied under ﬂuorescence microscope. Orange cells with Upper band 1.44 1.24 1.34 1.53 0.67 no nuclear fragmentation were considered dead and counted against Lower band 1.25 1.34 1.53 1.92 1.1 live green cells as a marker for necrosis. Downloaded from RAR ␥ 3 Values are means Ϯ SD of three replications with 200 cells counted Upper band 1.44 1.01 1.12 1.86 0.41 in each replication; * retinoid-induced cell death was signiﬁcant (P Lower band 1.42 1.42 1.12 1.48 0.34 Ͻ 0.05) and dose dependent.

1 MCF-7 and Hs578T cells were treated with 1 ␮mol/L all-trans- retinoic acid for 2, 6, 8, 24 or 72 h and investigated for the induction of retinoid receptors (RAR␣, ␤ and ␥) at the mRNA level using Northern

marker of apoptosis, when treated with 1 and 10 ␮mol/Ljn.nutrition.org blot analysis (Fig. 5). all-t-RA for 7 d and stained with acridine orange and pro- 2 The density of the bands was determined using densitometric pidium iodide for ﬂuorescent microscopy (data not shown). scanning. The values obtained were normalized with the values obtained for pHE7 control to determine the fold induction under retinoid However, all-t-RA treatment induced necrosis in the cells on treatment. the basis of orange-red staining of the cells. Orange cells with

3 A small induction of the retinoid receptor subtypes was observed no nuclear fragmentation were considered dead and counted at USDA, National Agricultural Library on April 28, 2009 at some time points, but this induction was not consistent. against live green cells as a marker for necrosis. All-t-RA– induced necrosis was significant (P Ͻ 0.05) and dose dependent in MCF-7 cells (Table 2). Effect of all-t-RA on the induction of apoptosis. Chroma- tin condensation was used as the criterion to determine the DISCUSSION induction of apoptosis in the cells as a result of all-t-RA treatment. MCF-7 cells did not contain fragmented nuclei, a Numerous studies have been conducted in recent years to evaluate the potential role of vitamin A in the prevention of breast cancer (Hunter and Willett 1994). The results of both case-control and prospective studies have shown a risk reduc- tion associated with diets high in vitamin A. Retinoids including all-trans-, 9-cis- and 13-cis retinoic acid have been studied by various investigators using cancer cells in culture as model systems of breast cancer (Prakash et al. 2000). Few investigators have tried to study the role of other retinoids, and to an even lesser extent, carotenoids, for their effects on breast cancer cell growth in culture. The growth assays conducted in this study were based on the time required for control cultures to reach confluency (d 9) at an initial seeding density of 5000 cells/cm2. Until d 9, cells demonstrated normal cell morphology as determined by phase contrast microscopy. Beyond this point, a change in the cell morphology and a rapid increase in cell detachment were observed. Therefore, d 9 was determined to be the point of termination of these growth studies. The cells were counted in both control and treatment cultures on d 2, 5 and 9. The cells that die during culture detach from the plastic of the culture plates and are automatically removed when the growth medium is changed. Hence, the cells counted at these time points were actually the proliferating viable cells attached to the plates. The results demonstrate that the number of these proliferating viable cells was altered in treatment groups compared with control groups ond9oftreatment. The inhibitory treatments led to more cell detachment from the plastic and FIGURE 6 Effect of all-trans-retinoic acid (All-t-RA) on the cell hence a lower cell count. Growth inhibition was observed to cycle distribution of Hs578T cells. Cells were treated with the retinoid at be maximal at d 9 of treatment; therefore the data are shown a1␮mol/L concentration for up to 9 d. Values are means Ϯ SD of three for this time point only. replicate assays; *significantly different from control, P Ͻ 0.05. Tetrahydrofuran (0.1%) and acetone (0.25%) were used in CAROTENOIDS, RETINOIDS AND BREAST CANCER CELLS 1579 this study to deliver carotenoids and retinoids to the cells and investigation. Thus, breast cancer cells may be sensitive to were not found to be toxic to the cells (data not shown). carotenoid and retinoid treatments irrespective of their ER Carotenoids and retinoids were supplied to the cells every status. other day in this study. Other investigations, however, have Retinoids exert their modulatory effects on cell growth by used from 1 (Zhu et al. 1997) to 4 d (Shultz et al. 1992) as the binding to the nuclear retinoid receptor proteins, of which time period between feeding the cells. Similarly, the entire there are two classes, the RAR and the retinoid X receptor duration of treatment before determining the effects on cell (RXR), each of which has three subtypes (␣, ␤ and ␥). growth also differs among studies. The reported duration of Retinoic acid is thought to first bind to its nuclear receptors; treatment has varied from 3 (Kaleagasioglu et al. 1993) to 13 d this ligand-receptor complex in the form of homodimers or (Shultz et al. 1992). heterodimers then binds to its respective response elements ␤-Carotene at 10 ␮mol/L is similar to the concentration (retinoic acid response elements) in the promoters of a variety found in the sera of humans supplemented with ␤-carotene of retinoic acid–responsive genes. Binding of the natural reti- (Nierenberg et al. 1991). In general, concentrations of 1 and noids all-t-RA and 9-cis-retinoic acid to the retinoid receptors 10 ␮mol/L for retinoids and carotenoids, respectively, resulted enhances receptor responses (Allenby et al. 1993, Heyman et in maximum inhibition of cell growth in this study. This is al. 1992, Levin et al. 1992). The RAR bind to both all-t-RA consistent with other studies in which retinoids have been and 9-cis-retinoic acid with high affinity, whereas RXR bind to

used at varying levels, with the 1 ␮mol/L dose resulting in 9-cis-retinoic acid only (Heyman et al. 1992, Levin et al.Downloaded from maximum growth cessation (Chen et al. 1997, Fanjul et al. 1992). RXR play an extremely important role in mediating 1996, Marth et al. 1993, Van heusden et al. 1998, Zhao et al. retinoic acid and vitamin D effects at the level of gene ex- 1995, Zhu et al. 1997). The growth inhibitory effect of lyco- pression by forming heterodimers with RAR and vitamin D pene in MCF-7 cells (at 10 ␮mol/L) observed in this study, receptor, respectively (Zhao et al. 1995). however, was markedly less than that observed by Levy et al. The Northern blot analyses revealed that all three cell lines (1995) (0.3 ␮mol/L), possibly due to the different culture used in this study express basal levels of RAR␣ and ␥, whereas conditions and the assay used. only Hs578T cells express RAR␤. The level of the basaljn.nutrition.org A signiﬁcant effect (P Ͻ 0.05) of all-trans- and 9-cis- expression of these receptor subtypes was also variable in the retinoic acid was observed on the growth inhibition of Hs578T three cell lines. We did not observe a consistent induction of cells (Fig. 2). Similarly, there was a signiﬁcant effect (P any of the RAR subtypes in MCF-7 and Hs578T cells either at

Ͻ 0.05) of all-trans-, 9-cis- and 13-cis-retinoic acid on the the mRNA level using Northern blot analysis (Fig. 5) or at the at USDA, National Agricultural Library on April 28, 2009 growth inhibition of MCF-7 cells and growth stimulation of protein level using Western blot analysis (data not shown). MDA-MB-231 cells. These data are not presented here, how- Several studies have tried to correlate the retinoid receptors ever, because similar results have previously been published to the retinoid sensitivity, and a majority of these studies have elsewhere (Marth et al. 1993, Van heusden et al. 1998). On used different receptor-selective retinoids for this purpose. the basis of a report by Repa et al. (1993) that indicated that RAR␣-selective retinoid agonists inhibited the anchorage- all-trans-retinol is a ligand for the RAR and only four- to dependent growth of MCF-7 cells after a 7-d treatment, sug- sevenfold less potent than all-t-RA in binding to RAR(␣, ␤ gesting that RAR␣ is the retinoid receptor involved in the and ␥) proteins in competitive binding assays, we set out to inhibition of adherent cell growth by retinoids (Dawson et al. test the efficacy of this compound on the growth of MCF-7, 1995). In another study, retinoic acid strongly inhibited the Hs578T and MDA-MB-231 cell lines. We found that a 1 proliferation of ER(ϩ) MCF-7 cells but not of ER(Ϫ) Hs578T ␮mol/L dose of all-trans-retinol induced growth inhibition and MDA-MB-231 cells, and RAR␣ mRNA was highly ex- almost similar to that observed with the same dose of all-t-RA pressed in the RA-responsive lines, but not in the unrespon- in both MCF-7 (not shown) and Hs578T cells. sive lines (van den Burg et al. 1993). The authors suggested The initial cell density appears to be of paramount impor- that the loss of functional RAR may be a frequent event, tance in determining the effects of carotenoids and retinoids leading to RA unresponsiveness of ER(Ϫ) breast cancer cells. on cell proliferation. In the series of growth experiments RAR␤-negative cell lines MCF-7 and MDA-MB-231 under- conducted in our laboratory, we found that the cells must be at went growth inhibition associated with G1 arrest when treated an appropriate density to be sensitive to retinoid and carot- with 1 ␮mol/L all-t-RA after a human RAR␤ gene was intro- enoid treatments. When the starting cell numbers of MCF-7 duced into these cell lines using a retroviral vector-mediated cells were increased to ϳ15,000 cells/cm2 (Fig. 4), neither gene transduction (Seewaldt et al. 1995). Similarly, RAR␤ ␤-carotene nor all-t-RA inhibited growth at concentrations of induction in MCF-7 cells by all-t-RA was demonstrated using 10 and 1 ␮mol/L, respectively. However, significant growth different receptor-selective retinoids. By using an RAR␣-se- inhibition was achieved using the same levels of these com- lective antagonist (Ro 41–5253), RAR␤ expression was in- pounds when the starting cell numbers were reduced to almost duced by all-t-RA through an RAR␣-dependent signaling one third (Figs. 1, 4). We also observed that an uptake of pathway in MCF-7 cells (Shang et al. 1999). Receptor selec- all-t-RA by the cells in the early phase of cell culture is tive retinoids induced apoptosis in MCF-7 cells, indicating essential for this retinoid to inhibit growth. Cells treated with that RAR␣, RAR␥ and RXR␣ can mediate programmed cell retinoic acid from the middle phase of culture, i.e., when the death (Toma et al. 1998). These observations, which have cells achieved a count of ϳ98,000 cells/cm2, showed no followed an indirect approach of relating retinoid receptors to growth inhibition (Fig. 4). the cell growth inhibitory mechanism, suggest the involve- Among patients with breast cancer, tumors that express ment of all three subtypes of RAR in the growth inhibition of estrogen receptors (ER) are associated with improved survival breast cancer cells. The results of our study, which involved a and better response to hormone therapy than those not ex- direct method of determining the expression of RAR as a pressing these receptors (Rutqvist 1990). Despite the general result of treatment with their natural ligand all-t-RA, indicate observation that ER(Ϫ) breast cancer cells are not sensitive to that altering RAR expression is not involved in the growth retinoids (Lacroix and Lippman 1980, van den Burg et al. inhibition of breast cancer cells caused by this retinoid. Sim- 1993), the growth of ER(Ϫ) Hs578T cells was inhibited by ilarly, RAR␥ selectively binding retinoids (CD2325, CD666 all-trans- and 9-cis-retinoic acid as well as by ␤-carotene in this and CD437) inhibited the proliferation of MCF-7 cells similar 1580 PRAKASH ET AL. to all-t-RA and no correlation was found between expression Kaleagasioglu, F., Doepner, G., Biesalski, H. K. & Berger, M. R. (1993) Anti- proliferative activity of retinoic acid and some novel retinoid derivatives in of RAR and antiproliferative effects of the retinoids (Widsch- breast and colorectal cancer cell lines of human origin. Arzneim.-Forsch. 43: wendter et al. 1997). 487–490. The flow cytometry results of this investigation indicate an Krinsky, N. I. (1989) Antioxidant functions of carotenoids. Free Radic. Biol. arrest of Hs578T cells in the G0/G1 stage of the cell cycle as Med. 7: 617–635. Krinsky, N. I. (1993) Actions of carotenoids in biological systems. Annu. Rev. a result of all-t-RA treatment (Fig. 6). 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