Download Effects of Oral Contraceptives on MCF

Effects of Oral Contraceptives on MCF-7 Human Breast Cancer Cells
Jane Clark, Mindy Heyano, Sam Johnson, Jami Marzano and Kaitlin Waters
Biology 220: Dr. Ian Quitadamo, Page Wooller: Gryffindor Group 5
The 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.
DISCUSSION
EXPERIMENTAL DESIGN
A 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 resuspended 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
37C 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
Initial Cell Count
Cell Count after 6 hours
160,000
140,000
120,000
100,000
80,000
60,000
122,500
95,000
100,000
12,666
12,666
12,666
12,666
Control
Combination Pill
Plan B
Mini Pill
77,500
40,000
20,000
0
Oral Contraceptives
RESEARCH QUESTION
How will Plan B, Combination Pill and Mini Pill affect growth rate of the MCF-7 human
breast cancer cells?
RESEARCH HYPOTHESIS
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.
CONTROL
BEFORE AFTER
PLAN B
BEFORE AFTER
Null 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.
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).
CONCLUSION
MCF-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.
REFERENCES
RESEARCH PREDICTIONS
MCF-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.
The 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.
Effects of Oral Contraceptives on Cell Growth Rate
Cell Count per mL
INTRODUCTION
COMBO
BEFORE AFTER
MINI PILL
BEFORE AFTER
Cancer 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.
ACKNOWLEDGEMENTS
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.
We 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.