Re: Methyl-Donor Nutrients Inhibit Breast Cancer Cell Growth
trigonometry1972@gmail.com |
Plus you should have included the PMID numbers and
a little application might be nice as well.
Eat those eggs, consume some lecithin, take some
betaine aka TMG. Don't just follow one metabolic
pathway for methyl donors.
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Methyl-Donor Nutrients Inhibit Breast Cancer Cell Growth
http://www.redorbit.com/news/health/1581618/methyldonor_nutrients_inh...
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 <0.05.
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<0.05) and MCF-7 (P<0.05) cancer cells, although the pattern
of
these two cell lines were different (Fig. 1). The proliferation of
T47D and MCF-7 breast cancer cell lines was significantly reduced by
lipotropes, while the proliferation data showed no difference on a
normal breast cell line, MCF-10A. These data indicate that lipotropes
have an inhibitory effect on cancer cell growth, but not on normal
mammary cell growth. The cell proliferation data as well as the
apoptosis observation suggest cancer suppressive effects of
lipotropes. The difference in proliferation data from the two
tumorigenic cell lines, T47D and MCF- 7, and a non-tumorigenic cell
line, MCF-1OA1 may be due to one or several reasons. One possible
explanation is the presence or absence of estrogen receptors. T47D
and
MCF-7 breast cancer ceil lines are estrogen receptor positive, but a
normal breast cell line, MCF-IOA, is estrogen receptor negative (Soto
et al. 1986; Soule et al. 1990). For example, silencing estrogen
receptor expression correlates with transcriptional inactivation of
estrogen receptor genes in some human breast cancers (Ottaviano et
al.
1994). This implies that lipotropes may influence not only hormone
resistance but also a transcriptional cascade in estrogen receptor
positive breast cancer cells. The relationship between lipotropes and
estrogen receptors may require further investigation, including the
analysis of cell cycle kinetics to define the effects of lipotropes
on
cell cycle. The Bcl-2 protein assay revealed that Iipotrope
supplementation significantly (P<0.05) downregulated BcI-2 protein
expression in the T47D cell line (Fig. 2); the BcI2 protein level in
the Iipotrope group was approximately 25% lower than that in the
control group. However, there is no significant difference in the
MCF-7 cell line (data not shown), which may be due to different
apoptosis pathways. Several classes of cell autonomous proteins have
been identified as important mediators of apoptosis. BcI-2 regulates
apoptosis and has been shown to directly affect the sensitivity of
cancer cells to chemotherapy agents (Dole et al. 1994). For example,
tamoxifen induces apoptosis of breast cancer cells by decreasing both
expression of Bcl-2 and the interaction of estrogen/estrogen
receptors
(Zhang et al. 1999). Moreover Bcl-2 protein is overexpressed in up to
70% of all breast cancer cases (Silvestrini et al. 1994). In vitro
studies show that overexpression of BcI-2 results in resistance to
cancer cell death, suggesting that Bcl-2 can inhibit apoptosis
mediated by chemotherapy (Dole et al. 1994). Bcl-2 blocks both p53-
dependent and p53-independent cell death pathways (Dole et al. 1994).
Sumantran et al. (1995) suggested that strategies designed to
downregulate Bcl-2 protein may be useful in reducing breast cancer.
The Bcl-2 family of proteins plays a critical role in controlling
cancer cell apoptosis. Some members exhibit a pro-apoptotic activity,
whereas others prevent cell death. The comparison of the two major
groups in the Bcl-2 family may be useful to explain the role of
lipotropes in the upregulation of cancer cell apoptosis.
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.