Effects of Folic Acid Supplementation on MCF 7

Effects of Folic Acid Supplementation on MCF 7 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 B 9 (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 MCF 7 breast cancer cells concentrated folic acid affect their rate of growth and morphology? Research Hypothesis: 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 MCF 7 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 Growth Rate (cells/hour) Introduction: 0 0, 25 0, 75 1 1, 25 1, 75 2 -5000 -8838 -10000 -15000 -20000 -21591 -24545 -24606 -25000 -30000 Concentration of Folic Acid (pg/m. L) 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. 25 pg/m. L 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) Null Hypothesis: Folic acid concentration does not affect MCF 7 breast cancer growth rate and proliferation. As folic acid concentration decreases, MCF 7 breast cancer growth and proliferation accelerates. Experimental Design: An adherent MCF 7 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 1 X phosphate-buffered saline or PBS (Sigma), removing cells with 1 X 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 m. L complete Dulbecco’s Modified Eagle Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (Invitrogen), and sterile-filtered and cell-culture-tested penicillin/streptomycin 100 X (Sigma). ). Next 0. 5 m. L of the resuspended cell mixture was added to each of the two tissue culture flasks, along with 4. 5 m. L of the supplemented DMEM. The two identical flasks were grown in an incubated environment at 5% CO 2 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 MCF 7 cultures were passaged with a mixture of 5 m. L of folic acid solutions in DMEM at 0. 25 pg/m. L, 1. 0 pg/m. L, and 1. 75 pg/m. L 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 MCF 7 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 5 m. L 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/m. L), 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, MCF 7 breast cancer growth rate and proliferation slows Feeding MCF 7 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/m. L, 1. 0 pg/m. L, and 1. 75 pg/m. L concentrations had -8838, -24545, and -21691 cell growth rates per hour respectively. The results indicate that the 1. 0 pg/m. L and 1. 75 pg/m. L concentrations of folic acid had a more substantial negative effect on cell proliferation when compared with the 0. 25 pg/m. L 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 MCF 7 breast cancer growth rate and proliferation. Research Prediction: Discussion: References: Figure 2. Effect of folic acid supplementation on MCF-7 cell viability. Figure 2 a shows percentage of cell viability in the control group. Figures 2 b-d show percentage of cell viability among treatment groups (0. 25 pg/m. L, 1. 0 pg/m. L, and 1. 75 pg/m. L of folic acid in DMEM respectively). There is little variability 12. 64 among the control group (92. 7% alive) and the groups treated with 0. 25 pg/m. L (94. 2% alive) and 1. 75 pg/m. L (92. 2% alive). However the cells treated with 1. 0 pg/m. L had a significantly lower percentage of cell viability at 85. 2% alive. a) Control BEFORE AFTER b) 0. 25 pg/m. L BEFORE AFTER c) 1. 0 pg/m. L BEFORE AFTER d) 1. 75 pg/m. L 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. PLo. S ONE 9: e 84635. 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 MCF 7 cells are densely grouped and appear angular in shape and no longer appear smooth or circular. The 0. 25 pg/m. L and 1. 0 pg/m. L treatments of MCF 7 cells grouped into small pods and were angular in shape. The 1. 75 pg/m. L treatment of MCF 7 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.