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. 0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences. Manuscript received 10 July 2000. Initial review completed 14 August 2000. Revision accepted 17 January 2001. 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.