The Effect of Oxaliplatin on c-Jun Levels in Colorectal Cancer Cells

Debbie Kim and Danielle Domini

Biology 241

Dr. Busch

12/06/2013
Introduction

Previous Research. Presently in the United States, cancer is the second leading cause of

death; therefore, researchers are pursuing knowledge in the field of chemotherapy and

chemoresistance (Leading Causes of Death 2013). Cisplatin, also known as CDDP, is a common

drug used to treat cancer. Researchers have discovered that the protein c-Jun is the regulator of

CDDP effectiveness. If there is a lot of active c-Jun, CDDP is less effective; however, if c-Jun is

inactive, CDDP is more effective. Researchers have conducted numerous experiments that

demonstrate the differing effects of cisplatin on cancer cells found in the brain, bone marrow, and

nervous system (Xia 2013). Cisplatin is not the only chemotherapy drug. Oxaliplatin is a

platinum-based drug that affects c-Jun levels colorectal cancer cells. Whether oxaliplatin has a

positive or negative effect on c-Jun levels in colorectal cancer cells, however, is unknown.

Research Hypothesis. Oxaliplatin decreases c-Jun levels in colorectal cancer cells.

Proposed Objective. In order for the hypothesis to be tested, both gel electrophoresis and

western blotting will be utilized. Gel electrophoresis uses both an electric current and agarose gel

to separate segments of protein for this particular experiment (Gel Electrophoresis 2008).

Western blotting uses gel electrophoresis to separate proteins which are then transferred onto a

membrane and stained with specific antibodies (US National Library of Medicine National

Institutes of Health 2012). Both gel electrophoresis and western blotting aid in the determination

of oxaliplatins effect on c-Jun levels in colorectal cancer cells.

Proposed Research

Methodology. HCT116 adenocarcinoma cancer cells line the tissue of the human colon,

and these colorectal cancer cells will be used in the experiment (US National Library of

Medicine National Institutes of Health 1993). The experimental system will consist of HCT116
cells at a density of 4 10 per 60-mm plate. The cells will be grown on an agarose plate for 16
5

hours and treated with 50 g/ml oxaliplatin (Xia 2013). Organizing the HCT116 cells at a density

of 4 10 per 60-mm plate and allowing the cells to be treated for 16 hours ensures sufficient cell
5

growth. Three plates will be treated with the solution of oxaliplatin. Three additional plates will

be used as a control group, treated without the solution of oxaliplatin. After 16 hours has passed,

a sample from each plate will be transferred into its own centrifuge tube and centrifuged for 10

minutes (US National Library of Medicine National Institutes of Health 2012). Centrifugation is

the spinning process used to separate materials suspended in a liquid medium. The pellet at the

bottom of the centrifuged tube contains c-Jun protein and will then undergo gel electrophoresis.

Gel electrophoresis will separate segments of protein based on size and charge. The protein

solutions will be placed in agarose wells, put into a box filled with buffer solution, and then

hooked up to an electric current. The buffer contains ions which allow the electricity to actually

pass through the apparatus, and the current causes the proteins to be pulled down the gel towards

the positive electric terminal (Gel Electrophoresis 2008). Upon completion of gel

electrophoresis, a western blot will be used. Western blotting enables the identification of c-Jun

from a complex mixture of proteins extracted from the pellet. The technique used to accomplish

western blotting began with gel electrophoresis which separated the proteins by size. Then the

obtained results will be transferred onto a membrane and incubated for 30 minutes on ice to

prevent the proteins from denaturing. Then specific antibodies are labeled to target the protein c-

Jun. The antibodies used for this experiment are Ser63, which is a primary antibody, and Ser73,

which is the secondary antibody. The primary antibody is added to the serum on the membrane

and incubated overnight in a cold environment. Then the membrane is washed and the secondary

antibody is injected into the serum and incubated for an hour. This incubation process ensures
that the targeted protein, c-Jun, is properly tagged. An enhanced chemiluminescence (ECL) mix

is prepared, which causes certain proteins to luminesce in dark conditions. The ECL is also

added to the serum and incubated for 1-2 minutes. Finally, the membrane is observed in a dark

room (US National Library of Medicine National Institutes of Health 2012).

The methodology is repeated twice more to ensure accurate results.

Expected Results. Following observation of the western blot in a dark environment, some

bands on the membrane will glow and other bands will not glow. The bands containing

oxaliplatin glow less or not at all because oxaliplatin decreases c-Jun levels in colorectal cancer

cells (Xia 2013). Therefore, the hypothesis that oxaliplatin decreases c-Jun levels in colorectal

cancer cells is supported.

References Cited

Gel Electrophoresis [Internet]. HudsonAlpha Institute for Biotechnology; [2008 Dec 12, cited

2013 Dec 5] . Available from:

http://www.hudsonalpha.org/education/kits/hnpcc/electrophoresis

Leading Causes of Death [Internet]. 2013 Jan 11. Hyattsville(MD): Centers for Disease Control

and Prevention; [2013 Jan 11, cited 2013 Dec 5]. Available from:

http://www.cdc.gov/nchs/fastats/lcod.htm

US National Library of Medicine National Institutes of Health [Internet]. 2. 1993. PubMed [cited

2013 Dec 5]. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8261574

US National Library of Medicine National Institutes of Health [Internet]. 9. 2012. PubMed [cited

2013 Dec 5]. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/

Xia Y, Yang W, Bu W, Ji H, Zhao X, Zheng Y, Lin X, Lu Z. 2013. Differential Regulation

of c-Jun Protein Plays an Instrumental Role in Chemoresistance of Cancer Cells. The

Journal of Biological Chemistry (288) [Internet]. [2013 Jul 5, cited 2013 Dec 5]. Available

from: www.jbc.org/content/288/27/19321.full