Effects of Folic Acid Supplementation on MCF7 Breast Cancer Cell Viability and Proliferation
Cassie Weldon, Ryan Bernstein, Shannon Mallory, Sarah Tracy
Biology 220: Dr. Ian Quitadamo Group 10: Saved by the Cell
Fortification of the American food supply with folic acid may be harmful to a certain subset
of the population. In 1996, the FDA approved the addition of folic acid to all enriched grain
products. In 1998 the organization made compliance to this fortification strategy
mandatory. This fortification strategy was introduced as a solution to neural tube defects
occurring in infants born from mothers with dietary folate deficiency; however, this new
protective measure did not take into account the remaining population. New research
suggests that folic acid supplementation can have negative effects on the health of breast
cancer patients, cancer survivors, and postmenopausal women. Though proven to decrease
the risk of neural tube defects, folic acid supplementation in the presence of preneoplastic
lesions seems to accelerate the growth of existing cancerous tissue (Deghan 2014).
Mammary tumors appear to be most susceptible to this phenomenon. In light of the
prevalence of breast cancer in today’s society (approximately 232,670 new cases in 2014)
this new research is particularly troubling and deserves further examination.
Folic acid is the synthetic form of Vitamin B9 (Folate) and is used in processed foods for
enrichment and fortification. All B vitamins work to promote the conversion of
macronutrients to functional components in the cell. They are also responsible for proper
nervous system function. Folic acid specifically aids in the production of DNA and RNA, and
is especially important when cells and tissues are growing rapidly (Moens, 2008). Because
cancer is associated with the proliferation of genetic mutation and folic acid is involved in
the preservation of genetic integrity, it is considered preventative against cancer
development. However, once genetic mutations have occurred it is likely that folic acid
continues to preserve mutated genetic integrity during proliferation. Due to its role in
maintaining DNA production and preservation in rapidly developing tissue, folic acid
supplementation could be detrimental in the presence of rapidly developing cancerous
tissue.
Research Question:
Does feeding MCF7 breast cancer cells concentrated folic acid affect their rate of growth
and morphology?
Cell viability and density of suspension was tested using 0.4% Trypan Blue dye exclusion and
a disposable hemacytometer (VWR). Cells were passaged after 2 days and viability and
density was tested again using Trypan Blue as discussed previously. Cell viability and density
was measured and compared between initial and final cultures across folic acid
concentrations to determine the effect of folic acid concentration on MCF7 human breast
cancer cells. Cell viability and death were also assayed using a flow cytometer (Bio-Rad) by
resuspending and centrifuging cell pellet two times in PBS and adding 10 microliters of
propidium iodide to final pellet prior to flow analysis
Results:
Effects of Folic Acid Supplementation on MCF-7 Cell Proliferation
0
0
Growth Rate (cells/hour)
Introduction:
0.25
0.5
0.75
1
1.25
1.5
1.75
2
-5000
-8838
-10000
-15000
-20000
-21591
-24545
-24606
-25000
-30000
Concentration of Folic Acid (pg/mL)
Figure 1. Effect of folic acid supplementation on MCF-7 cell proliferation. The cells were
allowed to grow over a period of 22 hours. Both the control group and the treatment
groups had higher rates of cell death then cell growth. The control had the highest rate of
cell death at -24,606 cells per hour. The cells treated with 0.25pg/mL of folic acid in DMEM
had the lowest rate of cell death at -8838 cells per hour.
Effects of Folic Acid Supplementation on MCF-7 Cell Viability
a)
b)
Research Hypothesis:
Null Hypothesis: Folic acid concentration does not affect MCF7 breast cancer growth rate
and proliferation.
As folic acid concentration decreases, MCF7 breast cancer growth and proliferation
accelerates.
Experimental Design:
An adherent MCF7 human breast cancer culture growing in a Genessee T-25 tissue culture
flask was passaged into two new tissue culture flasks in a biological safety cabinet (Forma
Scientific). This was accomplished by washing cells with 1X phosphate-buffered saline or
PBS (Sigma), removing cells with 1X Trypsin/EDTA (Invitrogen), and finally centrifuging
resulting cell suspension at 7500 x g for 7 minutes at room temperature. The cell pellet
formed by centrifuging was resuspended in 2.5 mL complete Dulbecco’s Modified Eagle
Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (Invitrogen), and
sterile-filtered and cell-culture-tested penicillin/streptomycin 100X (Sigma). ). Next 0.5 mL
of the resuspended cell mixture was added to each of the two tissue culture flasks, along
with 4.5 mL of the supplemented DMEM. The two identical flasks were grown in an
incubated environment at 5% CO2 and 37°C. The cells were monitored and given fresh
DMEM when needed and passaged if necessary for 2-3 weeks. After this growth period the
MCF7 cultures were passaged with a mixture of 5mL of folic acid solutions in DMEM at 0.25
pg/mL, 1.0 pg/mL, and 1.75 pg/mL of folic acid for a specified length of time. The negative
control was DMEM without folic acid and was passaged with the variable flasks. The effects
of MCF7 breast cancer cell proliferation under different folic acid concentrations were
observed, and samples of the different concentrations of treatment were observed in their
flasks under inverted light microscope.
Since there is no specific trend in our data, it is more likely that the results are indicative of
an error during the procedure than a result of treatment. Certain variables were
uncontrollable. These include the fluctuation of cell temperature due to the opening and
closing of the cell incubator, the amount of time the cells were exposed to room
temperature, possible contamination due to opening and closing of flasks and supply
availability. Labs were frequently out of 5mL pipettes affecting the team’s ability to resuspend cells, leading to crowding in the control group. Variables that could be controlled
include proper technique for cell feeding and splitting, length of time trypsin was applied in
splitting, the time the cells spent outside of the incubator and the sterilization of the hood
and materials. Improper technique during the splitting and/or treatment of the cells likely
caused a decrease in cell viability unrelated to treatment.
Flaws in technique may have caused the DMEM and/or folic acid treatments to be
contaminated. To reduce these human errors in the future there should be improved
communication between researchers and increased attention to proper technique when
treating and splitting within the hood. It is also important to note that the concentrations of
folic acid used in treatment could have been unrealistic relative to what a small amount of
cells would be exposed to in a normal cell environment. Further research on the
development of more accurate concentrations for treatment may be necessary.
At all concentrations of folic acid supplementation, including the control group (0 pg/mL),
there were significant rates of cell death. Percentage of cell viability was fairly similar
among control and treatment groups. Morphology was altered in all groups including the
control. Because the control experienced similar changes to changes in the treatment
groups we can neither reject nor accept the null or alternate hypotheses on the basis of
treatment alone. Results are more likely indicative of procedural errors.
d)
c)
As folic acid concentration increases, MCF7 breast cancer growth rate and proliferation
slows
Feeding MCF7 breast cancer cells folic acid will affect their rate of growth and overall
proliferation.
This experiment was designed to investigate the effects of folic acid on MCF-7 human breast
cancer cell proliferation and viability. The results revealed a -24606 cell growth rate per
hour in the control group. The 0.25 pg/mL, 1.0 pg/mL, and 1.75 pg/mL concentrations had
-8838, -24545, and -21691 cell growth rates per hour respectively. The results indicate that
the 1.0 pg/mL and 1.75 pg/mL concentrations of folic acid had a more substantial negative
effect on cell proliferation when compared with the 0.25 pg/mL concentration; however, all
groups showed a negative effect on cell proliferation, with the highest rate of cell death
occurring in the control group. Considering the control group theoretically should not have
been effected so drastically, this suggests that outside factors may have impacted cell
proliferation. While these results may be occurring due to treatment, the data failed to
show a consistent pattern across concentrations making it difficult to discern the exact
cause of cell death. Despite high rates of cell death, there were still relatively high amounts
of viable cells as the majority of dead cells were washed away in PBS solution during the
culturing process. As seen from Figure 2, Over 90% of cells were alive in every group,
excluding the lowest concentration which contained 85% live cells. In terms of morphology,
MCF-7 cells were spherical in shape prior to treatment but became angular and more
closely packed post-treatment.
Conclusion:
Alternate Hypothesis: Folic acid concentration does affect MCF7 breast cancer growth rate
and proliferation.
Research Prediction:
Discussion:
References:
Figure 2. Effect of folic acid supplementation on MCF-7 cell viability. Figure 2a shows
percentage of cell viability in the control group. Figures 2b-d show percentage of cell
viability among treatment groups (0.25pg/mL, 1.0pg/mL, and 1.75pg/mL of folic acid in
DMEM respectively). There is little variability 12.64among the control group (92.7% alive)
and the groups treated with 0.25pg/mL (94.2% alive) and 1.75pg/mL (92.2% alive). However
the cells treated with 1.0pg/mL had a significantly lower percentage of cell viability at 85.2%
alive.
a) Control
BEFORE
AFTER
b) 0.25 pg/mL
BEFORE
AFTER
c) 1.0 pg/mL
BEFORE
AFTER
d) 1.75 pg/mL
BEFORE
AFTER
Deghan Manshadi S, Ishiguro L, Sohn KJ, Medline A, Renlund R, et al. (2014) Folic acid
supplementation promotes mammary tumor progression in a rat model. PLoS ONE 9:
e84635.
Honein MA, Paulozzi LA, Matthews TJ, Erickson JD, Wong LC. Impact of Folic Acid
Fortification of the US Food Supply on the Occurrence of Neural Tube Defects. JAMA,
November 14, 2001—Vol 286, No. 18 (Reprinted)
Moens, An L., Christiaan J. Vrints, Jean-Pierre Timmermans, Hunter C. Champion,
and David A. Kass. "Mechanisms and Potential Therapeutic Targets for Folic Acid in
Cardiovascular Disease." American Journal of Physiology - Heart and Circulatory
Physiology 294.5 (2008): 1971-977. Web.
"Folic Acid." Folic Acid. American Cancer Society, 07 Mar. 2011. Web. 12 Nov. 2014.
Acknowledgments
Figure 3. Prior to folic acid exposure the cells appeared smooth and were evenly dispersed
along the bottom of the flask. After 22 hours the control group MCF7 cells are densely
grouped and appear angular in shape and no longer appear smooth or circular. The 0.25
pg/mL and 1.0 pg/mL treatments of MCF7 cells grouped into small pods and were angular
in shape. The 1.75 pg/mL treatment of MCF7 cells were more densely packed, some cells
still appeared smooth.
Special thanks to Dr. Ian Quitadamo, Kristy Kappenman, Eric Foss, and Mark Young for their
constant support.