ANTICANCER RESEARCH 34: 1377-1386 (2014) Lycopene and Beta-carotene Induce Cell-Cycle Arrest and Apoptosis in Human Breast Cancer Cell Lines NATHALIE FONSECA GLORIA1, NATHALIA SOARES2, CAMILA BRAND2, FELIPE LEITE OLIVEIRA2, RADOVAN BOROJEVIC3 and ANDERSON JUNGER TEODORO1 1Laboratory of Nutritional Biochemistry, Program of Food and Nutrition, UNIRIO, Rio de Janeiro, Brazil; 2Laboratory of Cell Proliferation and Differentiation, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 3Excellion Biomedical Services, Petrópolis, Rio de Janeiro, Brazil Abstract. Lycopene and beta-carotene are carotenoids Breast cancer is the most frequently diagnosed type of widely distributed in fruits and vegetables, with potential cancer globally (1), and is a complex disease caused by anticancer activity. Epidemiological trials rarely provide progressive genetic mutations, associated with other factors. evidence for the mechanisms of action of these compounds, The increased incidence of breast cancer follows the and their biological effects at different times of treatment are behavioral changes of urbanization, such as a sedentary still unclear. The aim of the present study was to determine lifestyle, excess of processed food in the diet, overweight the effect of carotenoids on the cell cycle and cell viability and obesity (2, 3). in human breast cancer cell lines. Human breast cell lines Diets enriched in fruits and vegetables are associated with were treated with carotenoids (0.5-10 μM) for 48 and 96 h. reduced rates of cancer and coronary heart disease. Bioactive Cell viability was monitored using the MTT method (3-[4,5- compounds present in the diet have been reported as dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; substances responsible for these effects. In this context, thiazolyl blue). The cell cycle was analyzed by flow carotenoids, such as lycopene and beta-carotene, are being cytometry, and apoptotic cells were identified by studied to correlate diet and decreased risk of breast and annexin/propidium iodide (PI) biomarkers. Our data showed other types of cancer (4, 5). a significant decrease in the number of viable breast cancer Lycopene, the major carotenoid component of tomato, cells on treatment with carotenoids. Carotenoids also exhibited potential anticancer activity in distinct types of promoted cell-cycle arrest followed by decreased cell cancer (6, 7). Epidemiological studies have reported a viability in the majority of cell lines after 96 h, compared to statistically significant inverse association between tomato controls. Furthermore, an increase in apoptosis was consumption and risk of several types of cancer, including observed in cell lines when cells were treated with lung, prostate and colon cancer (8-10). carotenoids. Our findings show the capacity of lycopene and On the other hand, beta-carotene, a carotenoid precursor beta-carotene to inhibit cell proliferation, arrest the cell of vitamin A, an essential nutrient with antioxidant activity, cycle in different phases, and increase apoptosis. These is suggested to be an important factor in reduction of cancer findings indicate that the effect was cell type-dependent and risk. It has been demonstrated that human cancer risks are that carotenoids are potential agents for biological inversely correlated with high blood levels of retinol or beta- interference with cancer. carotene and intake of food containing retinol (11-13). Even when administered at high doses during long periods of time, beta-carotene does not cause toxicity, whereas high doses of vitamin A and retinoids, as possible prophylactic agents for cancer prevention, can lead to acute hepatotoxicity and cause Correspondence to: Anderson Junger Teodoro, Universidade other adverse effects (14). Federal do Estado do Rio de Janeiro, Escola de Nutrição, Beta-carotene inhibited the growth of many human colon Departamento de Tecnologia de Alimentos CEP 22290-240, Rio de adenocarcinoma cell lines, inducing cell-cycle arrest and Janeiro, RJ, Brazil. Tel: +55 2125427276, Fax: +55 2125427752, e-mail: [email protected] apoptosis. It has been reported that beta-carotene was responsible for reducing cyclin A, an important regulator of Key Words: Lycopene, beta-carotene, breast cancer, bioactive cell-cycle progression (15). In vitro studies support the compounds, cell cycle. hypothesis that this carotenoid is chemopreventive or 0250-7005/2014 $2.00+.40 1377 ANTICANCER RESEARCH 34: 1377-1386 (2014) chemotherapeutic against some kinds of cancer, including of 2 g/l HEPES buffer, pH 7.4, under a 5% CO2 atmosphere. Cells the prostate (16) and melanoma (17). were passaged by trypsinization when reaching 70-80% of Lycopene has an anti-proliferative effect on prostate and confluence, about twice a week. For each experiment, all cells were seeded at a density of 104 cells/cm2 in 6- and 96-multiwell plates breast cancer cell-lines. In breast cancer models, reduced for cell cycle and cell proliferation analyses, respectively. Lycopene expression of cell cycle regulatory proteins, such as cyclins (WS) was dissolved in water at 50˚C within a range from 0.5 to (D1 and E) and cyclin-dependent kinases (-2 and -4), as well 10 μM and beta-carotene was dissolved in a solution with water as suppression of insulin-like growth factor (IGF-I) action, and ethyl alcohol. Carotenoids were then added to the plates. Cells have been correlated with the effects of lycopene on cell untreated (controls) were included on each plate. After 24 h, the proliferation (18, 19). Other studies reported that lycopene culture medium was changed. Cells were then incubated for 48 and had limited effect on cell proliferation of cancerous and non- 96 hours with daily medium replacement. cancerous cell lines in an in vitro system with doses within Cell viability assay. The status of cancer cell line viability was the physiological range, but only rarely have studies reported determined by of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- no effect of lycopene on cell proliferation (20, 21). diphenyltetrazolium bromide; thiazolyl blue) assay (Sigma, New Lycopene and beta-carotene have been proposed to York, USA). Exponentially growing cell lines were adjusted to negatively affect cancer cells or the development of cancer 1.0×104 cells/cm2 with DMEM, plated in 96-well plates at 200 by modulating cell-cycle progression and cell proliferation. μl/well and then incubated for 24 h according to routine procedure. The de-regulated cell cycle is one of the hallmarks of cancer After treatment with lycopene and beta-carotene (0.5-10 μM) and cells. These cells have disturbed cell cycle and inability to incubation for 48 h and 96 h (six wells for each sample), 20 μl of MTT (5 g/l) were added to each well. After 4 h of incubation, the control their rate of proliferation. A rate-limiting step in the medium was then removed and 100 μl/well sodium dodecyl sulfate cell cycle that is often disturbed in cancer is the progression (SDS) was added to dissolve the reduced formazan product. Finally, of cells through the first gap (G1) phase (22). Recently, the plate was read in an enzyme-linked immunosorbent microplate several reports have shown that lycopene can induce cell- reader (Bio-Rad 2550 at 490 nm. The cellular proliferation inhibition rate (CPIR) was calculated using the following formula: cycle arrest at the G1 phase in human hepatocarcinoma cells, CPIR=(1–average absorbance value of experimental group/average and G0/G1 arrest and S phase block in human prostate cancer cell lines LNCAP and PC3. absorbance value of control group)×100%. Although the role of carotenoids in the prevention of Cell cycle analysis. Cells were rinsed briefly with calcium- and breast cancer has been studied more extensively, human magnesium-free phosphate-buffered saline (PBS) and detached studies with lycopene and beta-carotene on cancer are now from cell culture flasks with trypsin at 37˚C. The cells were washed being undertaken. The majority of studies on the effects on twice with PBS 1×106 cells were resuspended in 1.0 ml of ice-cold the cell cycle were carried out after 48 h of carotenoid VindeLov solution containing 0.1% Triton X-100, 0.1% citrate treatment, which can lead to underestimation of the effect of buffer 0.1 mg/ml RNase and 50 μg/ml propidium iodide (Sigma these substances. The aim of the present study was to Chemical Co.). After 15 min incubation, DNA content was determine the effect of lycopene and beta-carotene on cell measured by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). The relative cycle and cell viability in three different human breast cancer proportions of cells with diploid DNA content G -G (2n), S phase cell lines at different time points. 0 1 (>2n but <4n), and G2/M phase (4n) were acquired and analyzed using CellQuest and WinMDI 2.9, software, respectively. The Materials and Methods percentage of the cell population at a particular phase was estimated with EXPO32 V1.2 Analysis software (Beckman Coulter, Cell culture reagents. All-trans lycopene and all-trans beta-carotene Inc., Brea, CA, USA). The cell dissociation procedure did not was purchased from Sigma Chemical Company (St. Louis, MO, affect fluorescence under the experimental conditions that were USA). Water-soluble (WS) lycopene (10%) and CWS beta-carotene used in this study. (1%) was provided by Roche (Rio de Janeiro, RJ, Brazil). Dulbecco’s cell culture medium (DMEM) and bovine serum Apoptosis assay. Human breast cancer cell lines (1.0×104 albumin were obtained from Sigma, and fetal bovine serum (FBS) cells/cm2) were treated with lycopene and beta-carotene at a from Laborclin (Campinas, SP, Brazil). Cell culture flasks and cell concentration of 5 and 10 μM in 6-well plates. After 48 and 96 h of scrapers were obtained from Nunc (Roskilde, Denmark). All incubation, the non-adherent cells were collected, and adherent chemicals were of analytical grade.