You are on page 1of 5

Research Description

The summer after my sophomore year of high school I wanted to gain experience in lab
techniques in gastroenterology and joined the lab of Dr. Jean-Pierre Raufman at University of
Maryland Medical Center in Baltimore, MD. I primarily worked with Dr. Anan Said for my
project. The lab's focus was on MMP1, a matrix metalloproteinaise produced from a M3R
muscarinic receptor in colon cancer cells. It had been discovered that muscarinic receptors in
colon cancer take an active role in aiding with metastasis and could hold the key to therapy of the
disease. Various agonists like acetylcholine activate signaling complexes that produce various
metalloproteinases or MMPs. In our study, the attributes of MMP1, a protein that breaks down
collagen and thus allows cancer to metastasize, were in special focus. Further study of the
products of muscarinic receptors will help scientists understand the signaling pathways that
allow cancer cells to proliferate.
In this experiment, cell migration correlates to MMP1 production as it is the facilitator of
cell migration. This is the rationale behind pursuing a migration assay as the method of learning
more about the MMP1 pathway.

The pathway proposed by the above image was crucial for the basis of the experiments.
The inhibitors and agonists to various parts of the pathway helped us elucidate which part of the
pathway was most important to MMP1 production and thus cell migration.
The main method behind this project was the use of cell migration assays. Six well plates
of HT29 colon cancer cells were grown as confluent monolayers in 6-well plates supplied with
10% fetal bovine serum. Cell monolayers were then wounded by scraping with a disposable 200µl pipette tip and then washed twice with fresh serum-free medium, and incubated in serum-free
medium in the absence or presence of test agents. Cells were also provided with mitomycin C, an

anti-proliferative agent. The use of mitomycin was important because this project measures cell
migration, which could be tough to measure with rapid cell growth. I personally used the pipette
tip to scrape the cells and also added serum and test agents to the cells.
Using an Axiovert 200 microscope (total magnification, 100X), rates of migration of cells
at the edge of wounded monolayers were determined from photographs taken 24 to 48 hours
after wounding. I assisted my mentor in taking photographs by editing and working with the
photos on the computer as soon as the microscope took the photos. I also analyzed the photos
and told the mentor if photos needed to be re-taken or adjusted. Twelve measurements were
performed per field by placing a transparent grid over the photograph and measuring the distance
that cells in the leading edge moved from the original wound line. When chemical inhibitors are
antibodies were used, these were incubated with cells for 30 minutes before wounding.
Incubation with EGF (epidermal growth factor) and serum-free media were used as positive and
negative controls, respectively. After these steps, my mentor then used a computer program to
determine mean distance migrated in arbitrary units. I was unfortunately not allowed access to
this program as a student and thus had to rely on my mentor for the final calculations.
The experiment was kept fair by using same volumes, temperatures, and methods for all
trials. Controls were established with the use of no serum plates and with plates which contained
only acetylcholine, a known agonist for colon cancer.
The main purpose of this experiment was to test how migration differed with various inhibitors
and agonists of the MMP1 production pathway. The inhibitors and agonists used are listed
respectively below. (Note: inhibitor is followed by its agonist)

Atropine: Acetylcholine

G06979: PKC isozyme

AG1478: EGFR

PD98059: MEK

PP2: Src

SB203580: p38MAPK

Although atropine is known to inhibit acetylcholine, an agonist for MMP1 production, atropine
produces harmful effects when used clinically, thus giving a reason to explore other parts of the
pathway.
After my mentor calculated the raw data from the computer program, I modified and analyzed it
onto Excel graphs and figures, which are discussed below.

Figure 1: Relative migration with inhibitors of the MMP1 production pathway and acetylcholine

Figure 2: Relative migration with agonists of the MMP1 production pathway (control)
Figure 1 shows the comparative levels of migration for the different inhibitors, with no
serum and acetylcholine serving as controls. The p38 inhibitor caused the greatest decrease in
migration, indicating the importance of it in the pathway. The results also indicate that other parts
of the pathway such as MEK (inhibitor is PD98069) and PKC (inhibitor is G06976) are crucial in
cell migration.
Figure 2 indicates the importance of epidermal growth factors and PMA in the signaling
pathway to produce MMP1. The combination of multiple agonists can yield greater migration.

Figure 3: A comparison of p38 inhibitor with acetylcholine
After promising results with p38 inhibitor in Figure 1, the previous steps were repeated in
another trial with just p38 inhibitor and acetylcholine to ensure proper results. In the presence of
both acetylcholine (an agonist) and p38 inhibitor, migration significantly decreased.
The projections above the bars in Figures 1-3 represent standard error of the mean and
are used to account for variances and errors in data.
The results indicate that the p38 inhibitor is most promising as far as a potential target for
decreasing MMP1 production and thus migration. Acetylcholine certainly caused an increase in
migration but the addition of various inhibitors confirms that the proposed pathway discussed
earlier is correct. Knowing what combination of inhibitors decreases migration the most can
allow for targeted therapy involving different receptors and parts of the signaling pathway. This
bodes huge implications for the pharmaceutical industry. In addition, the data in this experiment
can be supported in the future by protein blots and knockout mice. The data from this experiment
shows that MMPs and muscarinic receptors serve as crucial keys in curing colon cancer, a feat
which would benefit humanity for ages. I was immensely proud to have worked in the scraping,
preparation, and photography of the cells, as well as in the analyzing and writing of the data and
project. After my work on the project that summer, I was offered the chance to take a course in
basic research at the University of Maryland Medical School and completed the graduate level
course in two weeks.
I entered my project into the Howard County Science Fair, the Baltimore Science Fair
(ISEF affiliated), and the Google Science Fair. I was honored to receive the MIT Alumni Award
for Excellence in High School Research for my project.
This past summer I hoped to continue my work in Dr. Raufman’s lab, but was unable to
do so because of new lab regulations for minors at the University of Maryland Medical Center.

Instead I capitalized on the opportunity to investigate a disease that was a part of my life:
Crohn’s disease. I joined Dr. Xuhang Li’s lab at the Johns Hopkins School of Medicine, which
focused on biomarkers and the role of the CLC5 gene in metabolism and Crohn’s disease. I
focused on learning a variety of lab techniques such as PCR, genotyping, gel electrophoresis,
blood processing, and animal dissections. I extracted tissues and samples from knockout mice
and genotyped samples of mice, trying to determine wild type or CLC5 genotypes. Finally, my
internship consisted of writing a to-be published literature review on the effectiveness of
azathioprine as a Crohn’s treatment. I specifically focused on researching the effect of the drug
on pregnant women and am working with my mentor to synthesize it into a paper.
My research experience over the past two summers have allowed me to deeply explore
gastroenterology and science research, cultivating an appreciation for the sciences that I will
carry for the rest of my life and career.