Effects of Oral Contraceptives on MCF-7 Human Breast Cancer CellsJane Clark, Mindy Heyano, Sam Johnson, Jami Marzano and Kaitlin Waters

Biology 220: Dr. Ian Quitadamo, Page Wooller: Gryffindor Group 5

COMBOBEFORE AFTER

CONTROLBEFORE AFTER

MINI PILLBEFORE AFTER

PLAN BBEFORE AFTER

INTRODUCTIONThe availability of contraceptives has completely transformed the lives of women around the world. Women are able to use contraceptives to control their reproduction rate. Not only do women use contraceptives to reduce the chance of unintended pregnancy, but use them to treat other health conditions. The increased usage of contraceptives has brought attention to different forms of birth control, the chemical make-up of these different forms and their relation to breast cancer. Oral contraceptives are the most common type of birth control and are taken daily. These contraceptives vary in Estrogen and Progesterone dosage, which are the hormones produced in the ovaries. Progesterone is required for breast development (Lange, 2013). Estrogen is a steroid hormone essential for female development (Siteri, 2006). Levonorgestrel, Desogestrel, Ethinyl Estradiol and Norethindrone are examples of synthetic forms of these important hormones used in the chemical make-up of contraceptives (Marchbanks, 2013). One type of oral contraceptive is the Combination Pill, which contains synthetic forms of Estrogen and Progesterone (Marchbanks, 2013), whereas the Mini Pill contains only the synthetic form of Estrogen. Another form of oral contraceptive is Plan B which contains a synthetic form of Estrogen in high dosage. Contraceptive use has become a more recognized cause of increased breast cancer growth (Ehsanpour, Fahime, Fariborz, 2013). One study proposed that women who take a form of oral contraceptive and have a first degree relative diagnosed with breast cancer are three times more likely to develop breast cancer (Marchbanks, 2013). For lab purposes, MCF-7 human breast cancer cells are used for testing the relationship between breast cancer and contraceptive use. MCF-7 cells are a type of breast cancer cell formed in the excess fluid surrounding the lungs of patients diagnosed with breast adenocarcinoma (MCF-7, 2013). The MCF-7 cells express a wide range of estrogen and progesterone receptors. The ability of the MCF-7 cells to synthesize estrogen differs from the synthesis abilities of mammary epithelium (breast tissue) (MCF-7, 2013). Given the fact that 62% of sexually active women in the United States alone use some form of contraceptive, (Jones, Mosher, Daniels, 2012) it is important to test the relationship between breast cancer and contraceptive use.

RESEARCH QUESTIONHow will Plan B, Combination Pill and Mini Pill affect growth rate of the MCF-7 human breast cancer cells?

RESEARCH HYPOTHESISNull Hypothesis: The Combination Pill, Mini Pill and Plan B Pill will have no effect on growth rate of MCF-7 breast cancer cells.Alternative Hypothesis: The Combination Pill, Mini Pill and Plan B Pill will all affect growth rate of MCF-7 breast cancer cells.

RESEARCH PREDICTIONSMCF-7 growth rate will increase the fastest when combined with the Combination Pill, the second fastest when combined with the Plan B Pill and the slowest when combined with the Mini Pill.

EXPERIMENTAL DESIGNA subconfluent MCF-7 cell culture was grown to prepare for contraceptive testing. Briefly, MCF-7 cells were grown in a Falcon T-25 tissue culture flask, washed using 1X phosphate buffered saline (PBS), detached from the T-25 using 1X Trypsin/EDTA (Sigma), quenched using 2mL Dulbecco Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum, and centrifuged at 750-1000x g for 5 minutes at room temperature. Resulting supernatant was aspirated and cell pellet re-suspended in fresh DMEM then plated at equal 0.25mL volumes in 12-well tissue culture plate (Nunc) and maintained in a humidified incubator (Forma Scientific) at 37C and 5% CO2. Three cultures contained MCF-7 cells (control) and were not treated with any additional substances. Three cultures were treated with each 0.15mg Desogestrel and 0.03mg Ethinyl Estradiol solution, 0.35 mg Norethindrone solution or 1.5mg Levonorgestrel solution. The duration of treatment was 6 hours. The cell growth rate of the control was compared to the 3 hormone solutions.

RESULTS

Figure 1. Effects of oral contraceptives on MCF-7 cell growth rate. The control group had a cell count of 77,500 after treatment with an initial cell count of approximently 12,666. Cells treated with a synthetic Estrogen hormone showed a significant increase in cell growth rate, with the Mini Pill having the highest growth rate.

DISCUSSIONThe purpose of our study was to determine the effect oral contraceptives have on the growth rate of MCF-7 human breast cancer cells after a 6 hour treatment. The results indicated all three types of oral contraceptives tested increased the cell count per mL. The Mini Pill had the most growth with a 9.67% increase in cell count per mL. Plan B had a 7.90% increase in cell count per mL. The Combination Pill had a 7.50% increase in cell count. From our results, we are able to determine synthetic estrogen in the absence of synthetic progesterone has the greatest growth rate. Initially, we believed Plan B would have the greatest cell count per mL after treatment due to the highest dosage of hormones. However, the Mini Pill showed the greatest growth rate, contradicting our predictions. We believe this occurrence is due to the dosage of synthetic estrogen alone, compared to a synthetic estrogen paired with synthetic progesterone. Based on these findings, and the understanding of oral contraceptives effects on MCF-7 human breast cancer cells on the cellular level, women diagnosed with breast cancer need to make educated decisions on usage of oral contraceptives (Jones, Mosher, Daniels, 2012). The controlled variables in our experiment were our oral contraceptive concentrations, incubator temperature, sterilization of Fume Hood, steps for cell counting, amount of media used and treatment time. The uncontrolled variables were viability of MCF-7 human breast cancer cells when treated with ethanol, temperature inconsistency outside of the incubator, and effects of the external environment when treated in Fume Hood. Our results showed a positive correlation between oral contraceptives and breast cancer cell growth. If the experiment were to be repeated, we would extend the duration of treatment from 6 hours to 24 hours while tracking the cell growth. An alteration to future experiments would be to vary concentration levels of contraceptive treatment. Understanding the dosage associated with the highest growth rate would further our understanding of oral contraception on breast cancer cells. All women should acknowledge the risk of breast cancer cell growth rate through oral contraceptive use (Ehsanpour, Fahime, Fariborz, 2013).

CONCLUSIONMCF-7 human breast cancer cells were directly affected by Plan B, Combination Pill and Mini Pill. All three oral contraceptive concentrations increased the growth rate of the MCF-7 breast cancer cells over a 6 hour time period. We reject our null hypothesis because a positive growth rate occurs when the MCF-7 breast cancer cells are treated with oral contraceptives.

REFERENCESCancer demographics: "National Cancer Institute." Oral Contraceptives and Cancer Risk -.N.p., n.d.

Web. 08 Oct. 2013. Marchbanks, Polly A. "The New England Journal of Medicine." Oral Contraceptives and the Risk of

Breast Cancer — NEJM. N.p., n.d. Web. 08 Oct. 2013 Siiteri, Pentti K. Online Library. New York Academy of Science, 2 Dec. 2006. Web. 5 Oct. 2013. Ehsanpour, Soheila, Fahime S A Nejad, Fariborz M. Rajabi, and Fariba Taleghani. "Investigation on the

Association between Breast Cancer and Consumption Patterns of Combined Oral Contraceptive Pills in the Women of Isfahan in 2011." PUBMED. N.p., May-June 2013. Web. 08 Oct. 2013.

Lange, Carol A. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 08 Oct. 2013.

Jones J, Mosher WD and Daniels K, Current contraceptive use in the United States, 2006–2010, and changes in patterns of use since 1995, National Health Statistics Reports, 2012, No. 60, <http://www.cdc.gov/nchs/data/nhsr/nhsr060.pdf>, accessed Mar. 20, 2013.

"MCF-7." Human Tumor Cells. Creative-Bioarray, n.d. Web. 08 Oct. 2013.

ACKNOWLEDGEMENTSWe would like to thank Dr. Ian Quitadamo and Page Wooller for the opportunity to cultivate and treat human breast cancer cells which is a unique skill set we will bring to each of our scientifically geared fields. Thank you to the Media Prep Faculty for equipping our Cell Biology Lab. We extend our sincerest gratitude to Central Washington University for funding a course which makes Central Washington University students unique assets in the work force.

Figure 2. Visual growth rate of MCF-7 human breast cancer cells observed under a microscope before and after treatment. Mini Pill showed the most growth over a 6 hour time period.

Control Combination Pill Plan B Mini Pill0

20,000

40,000

60,000

80,000

100,000

120,000

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160,000

12,666 12,666 12,666 12,666

77,50095,000 100,000

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Effects of Oral Contraceptives on Cell Growth RateInitial Cell Count Cell Count after 6 hours

Oral Contraceptives

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