Methyl-Donor Nutrients Inhibit Breast Cancer Cell Growth

Written By: Sam Savage

Published Date: October 8, 2008 Last Edited: October 8, 2008

By Park, Chung S Cho, Kyongshin; Bae, Dong R; Joo, Nam E; Kim, Hyung H; Mabasa, Lawrence; Fowler, Andrea W

Abstract Lipotropes (methyl group containing nutrients, including methionine, choline, folate, and vitamin B12) are dietary methyl donors and cofactors that are involved in one-carbon metabolism, which is important for genomic DNA methylation reactions and nucleic acid synthesis. One-carbon metabolism provides methyl groups for all biological methylation pathways and is highly dependent on dietary supplementation of methyl nutrients. Nutrition is an important determinant of breast cancer risk and tumor behavior, and dietary intervention may be an effective approach to prevent breast cancer. Apoptosis is important for the regulation of homeostasis and tumorigenesis. The anti-apoptotic protein Bcl-2 may be a regulatory target in cancer therapy; controlling or modulating its expression may be a therapeutic strategy against breast cancer. In this study, the effects of lipotrope supplementation on the growth and death of human breast cancer cell lines T47D and MCF-7 were examined and found to inhibit growth of both T47D and MCF-7 cells. Furthermore, the ratios of apoptotic cells to the total number of cells were approximately 44% and 34% higher in the lipotrope-supplemented treatments of T47D and MCF-7 cancer cells, respectively, compared with the control treatments. More importantly, Bcl-2 protein expression was decreased by approximately 25% from lipotrope supplementation in T47D cells, suggesting that lipotropes can induce breast cancer cell death by direct downregulation of Bcl-2 protein expression. Cancer treatment failure is often correlated with Bcl-2 protein upregulation. These data may be useful in the development of effective nutritional strategies to prevent and reduce breast cancer in humans. Keywords Lipotropes * Apoptosis * MCF-7 * T47D

Lipotropic nutrients (methionine, choline, folate, and vitamin B12) are important dietary methyl donors and cofactors that play key roles in one-carbon metabolism; one-carbon metabolism, which provides methyl groups for all biological methylation pathways, is highly dependent on dietary supplementation of methyl nutrients (Newbeme and Rogers 1986; Institute of Medicine 1998). Methyl nutrients are essential for epigenetic changes (DNA methylation and demethylation, methyl CpG recognition, histone modification, and chromatin remodeling), which are required for cell proliferation and maintenance of tissue integrity (Jones and Laird 1999; Shrubsole et al. 2001). Methyl groups needed for DNA methylation are acquired through the folate and methionine pathways, and DNA methylation patterns may be altered by changes in diet, genetic polymorphisms, and environmental chemicals (Waterland and Jirtle 2004). Dietary lipotropes influence the availability of the chief biological methyl donor, S-adenosylmethionine, and therefore may change genomic DNA methylation patterns and the expression of multiple cancer-related genes (Jones and Laird 1999; Ross 2003). Deficiency of methyl nutrients has been shown to increase chemical carcinogenesis in rodents (Newberne and Rogers 1986; Choi et al. 1993).

While there are some studies on individual dietary methyl nutrients and breast cancer, there are few that address the interplay among these methyl nutrients and their effects on the reduction of breast cancer. We have shown that lipotrope supplementation decreases MCF-7 human breast cancer cell growth by downregulation of Bci-2 gene expression (Kim and Park 2002). This study investigated whether or not lipotrope supplementation changes cancer cell growth and apoptosis. We found that lipotrope supplementation decreased Bcl-2 protein level and, consequently, increased apoptosis of T47D cancer cells.

Three cell lines (two breast cancer cell lines, T47D and MCF-7, and a normal mammary cell line, MCF-10A) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in basal media consisting of Dulbecco’s modified Eagle’s medium (Gibco Invitrogen, Carlsbad, CA) and F12 medium (Gibco Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Gibco Invitrogen), 1% antibiotic-antimycotic (Gibco Inviteogen), and 10 [mu]g/ml insulin (Gibco Invitrogen) as recommended by the supplier. For MCF-10A, 20 ng/ml epidermal growth factor (Gibco Invitrogen) and 100 ng/ml cholera toxin (Gibco Invitrogen) were also added. Cells were seeded in 25-cm^sup 2^ culture flasks at a density of 2 x 10^sup 4^ cells/ml and incubated in a 5% CO2-humidified atmosphere at 37[degrees] C in culture media. At 50% confluency, cells were then switched to basal control (normal levels of lipotropes in basal culture medium: 17 mg/1 L-methionine, 9 mg/1 choline, 3 mg/1 folic acid, and 2 mg/1 vitamin B12) and Hpotrope-supplemented media (ten times the level of lipotropes found in the basal control). The chosen dose of lipotropes was based on previous studies (Kim and Park 2002, 2003).

For the cell proliferation assay, cells were seeded into 96-well microplates at a density of 1.0 x 10^sup 5^ cells/ml and grown in culture media until 50% confluent. The cells were then switched to the two treatment media. On days O through 4, growth curves were obtained by a colorimetric [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] cell proliferation assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI) according to the manufacturer’s instructions. Briefly, 20 [mu]l of AQueous One Solution reagent was added to each well containing 100 [mu]l of cell culture medium. After a 4-h incubation period at 37[degrees] C, the amount of soluble formazan product was measured spectrophotometrically at an absorbance of 490 nm with a plate reader.

For the determination of Bcl-2 protein levels, cells were cultured and treated as described above. On day 4, cells were collected, pelleted (2 x 10^sup 6^ cells/pellet), and stored at 0 – 80[degrees] C until analysis of Bcl-2 protein by enzyme immunometric assay (EIA, TiterZyme EIA kit, Assay Designs, Ann Arbor, MI) according to the manufacturer’s suggestions. Briefly, cell pellets were lysed in a lysis buffer with phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The protein concentrations in the supernatant were quantified by bicinchoninic acid protein assay (BCA Protein kit, Sigma-Aldrich). The EIA consists of binding the Bcl-2 in the supernatant to a monoclonal antibody specific to human Bcl-2 protein that was immobilized on a microtiter plate. Then, a biotinylated monoclonal antibody to Bcl-2 was added, followed by streptavidin-conjugated horseradish peroxidase to bind to the biotinylated antibody, and the substrate was added to generate color. The absorbance was measured spectrophotometrically at 450 nm with a plate reader.

4,6-Diamidino-2-phenylindole (DAPI) fluorescent staining was used for observation of apoptotic nuclear morphological changes. Cells (1 x 10^sup 4^ cells/ml) were seeded in polystyrene Labtek-4-chamber slides (Nalgene Nunc, Rochester, NY) and cultured in pre-incubation medium until 50% confluent as described above. The cells were then switched to the two treatment media. On day 4, cells were washed twice and fixed by incubation with DAPI-methanol (1 [mu]g/ml) for 15 min at 37[degrees] C. After the second washing, cells were incubated at room temperature in 1 [mu]g/ml of DAPI solution for 30 min in the dark. The cells were washed with methanol, and a cover-slip was secured to each slide using a fluorescent mounting medium (Gel/ Mount, Biomeda, Foster City, CA). Microscopic analyses were carried out on a Nikon Microphot-FX upright microscope (Nikon, Melville, NY) with Image Pro-Plus Software (Media Cybernetics Inc., Silver Springs, MD). Photomicrographs of five different fields per treatment for each cell line were taken, and the percentage of apoptotic cells to normal cells was evaluated. Apoptotic cells were defined by features such as condensed, fragmented, or hyper- segmented nuclei, and nuclear and cytoplasmic shrinkage (Collins et al. 1997).

One-way analysis of variance followed by Tukey test was conducted to detect differences among groups. All statistical analyses were performed using the SAS program (SAS Institute, Gary, NC). Statistical differences were considered significant at P

This study determined the effect of lipotropes on growth and apoptosis of established human breast cancer cell lines, T47D and MCF-7. Lipotrope supplementation significantly inhibited the growth of both T47D (P

Apoptosis is morphologically defined by cell shrinkage, membrane blebbing, chromatic condensation, and formation of apoptotic bodies (Mooney et al. 2002). The effect of lipotropes on apoptosis in T47D and MCF-7 breast cancer cell lines was assessed by DAPI staining and the observation of nuclear morphology (Fig. 3). In T47D cells, the ratio of apoptotic dead cells was calculated to be approximately 44% higher, compared with that of the control treatment. In MCF-7 cells, the ratio of apoptotic dead cells was approximately 34% higher, compared with that of the control treatment. The increase in apoptotic cells suggests that lipotropes may induce cancer cell death.

DNA methylation is accepted as one of the most important underlying biological mechanisms regulating epigenetic modification of the expression of heritable genes, and transcriptional control by DNA methylation is an important epigenetic mechanism and essential in maintaining cellular function (Jones and Laird 1999). Changes in methylation patterns may contribute to the development of cancer (Davis and Uthus 2004). Evidence indicates that nutrition is an important determinant of cancer risk and tumor behavior, and dietary intervention may be an effective approach to reduce cancer.

Even though further study of DNA methylation of T47D and MCF-7 breast cancer cell lines for a detailed mechanism of anti-cancer effects is needed, lipotropes may alter the gene expression of cancer cells via DNA methylation. Altered DNA methylation is a useful mechanism for the suppression of cancer gene expression (Jones and Laird 1999). In an in vivo study, we found that dietary lipotropes decrease tumor incidence in the nitrosomethylurea- induced cancer model and significantly reduce the expression of ornithine decarboxylase, a marker of cancer cell proliferation (Moon et al. 1998). Moreover, the expression of Bcl-2 gene is decreased in MCF-7 human breast cancer cells growing in lipotrope-suppiemented medium (Kim and Park 2002). It appears that a methyl supplementation- mediated alteration of one-carbon metabolism may act on the expression of genes involved in proliferation and apoptosis thereby suppressing the progression of mammary tumorigenesis.

In summary, these findings have shown that lipotrope supplementation suppressed the growth of T47D and MCF7 breast cancer cell lines. Furthermore, lipotropes stimulated the induction of apoptosis and pro-apoptotic Bcl-2 protein expression in the T47D cell line. Cancer treatment failure is often correlated with Bcl-2 protein upregulation. Nutritional strategies for cancer prevention and therapy are low risk and cost effective. This study shows that lipotropes (methyl nutrients) may inhibit the growth of breast cancer cells and downregulate the Bcl-2 protein expression, suggesting that lipotropes may be useful in the development of nutritional strategies to prevent and reduce breast cancer in humans.

Acknowledgment This study was supported by a grant (1 R15 CA098016-01A1) from the National Institutes of Health-National Cancer Institute.

Received: 9 November 2007 /Accepted: 24 March 2008 /Published online: 23 May 2008 / Editor: J. Denry Sato

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C. S. Park (*) * K. Cho – L. Mabasa * A. W. Fowler

Department of Animal Science, North Dakota State University,

Fargo, ND 58105, USA

e-mail: [email protected]

D. R. Bae

Department of Animal Sciences, The Ohio State University,

Columbus, OH 43210, USA

N. E. Joo

Department of Periodontics/Prevention/Geriatrics,

School of Dentistry, University of Michigan,

Ann Arbor, MI 44109, USA

H. H. Kirn

Abramson Cancer Center, University of Pennsylvania,

Philadelphia, PA 19104, USA

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