Transcript The Effects of H202,Tamoxifen, PAHs, and BAP on MDA-MB 231 Cells Britney Nugent, Sharnese Goodwin, and Dr.

The Effects of H202,Tamoxifen, PAHs, and BAP on MDA-MB 231 Cells
Britney Nugent, Sharnese Goodwin, and Dr. Yavuz Cakir
Benedict College, Columbia, SC 29204
Abstract
The kinetic analysis of the cell cycle of cancer cells provides a valuable link to
the study of the cancer cell's lifespan and progression. In this study, we
investigated the effects of H2O2 (Hydrogen Peroxide), Tamoxifen, Polycyclic
Aromatic Hydrocarbon (PAH), and Benzo-a-Pyrene (BAP) on the cell cycle
kinetics of the MDA-MB 231 human breast cancer cells. The cell cycle phases
were analyzed using flow cytometer. The cell morphology of MDA-MB-231
cells was observed under phase contrast microscopy. In our study, H2O2 at 25
µmole/ml concentrations caused morphological changes indicative of increased
sub-G1 peaks and also caused accumulation of cells in the G0/G-1, indicating a
G-1, cell cycle arrest concomitant with a depletion of cells from the S and G2/M
compartments. The treatment of MDA-MB-231 cells with 1ug/ml tamoxifen
resulted in a marked accumulation of cells in G-1 phase indicating a reduction
in the fraction of cells in S phase. Also, PAH at 500 ng/ml induced
morphological changes indicative of apoptosis. The cell cycle analysis
demonstrated that the PAH suppressed the cells at G-1 phase and also caused
marked increases in S and G2/M phases indicating a G2/ M cell cycle arrest.
Where as BAP at the same concentration did the opposite.
Introduction
Breast cancer is affecting many women in the developed nations. There are
several treatment options for the breast cancer, but none of them are effective
enough to eradicate the cancer cells. The MDA-MB-231 cells are triple
negative; they don’t express estrogen and progesterone receptors, and no
overexpression/amplification of the HER2-neu gene occurs. Therefore, this
subtype of breast cancer lacks the benefits of specific therapies that target these
receptors. Today chemotherapy is the only systematic therapy for patients with
triple-negative breast cancer. The H2O2 (hydrogen peroxide), one of the
reactive oxygen species, is produced by the normal cells in response to cellular
oxidative stress inducing cell death (apoptosis). PAHs are a group of organic
contaminants that form from the incomplete combustion of hydrocarbons, such
as coal and gasoline. Their toxicity causes them to be an environmental interest
and labeled as carcinogens. BAP, as one of the PAHs, can be labeled as a
cancer-causing agent in humans. It results from burning plants, wood, and
operation of vehicles. The major indoor sources of BAP in the air are woodburning fireplaces, stoves and tobacco smoke. On the other hand, tamoxifen is
antiestrogenic and inhibits proliferation of some breast cancer cells including
estrogen receptor positive breast cancer cells. For this reason tamoxifen is now
widely used for the treatment of patients with breast cancer.
References
Bekim Sadikovic and David I. Rodenhiser. Benzo-a-Pyrene exposure disrupts DNA methylation
and growth dynamics in breast cancer cells. Toxicol Appl Pharmacol. 2006 Nov 1;216(3):458-68.
Yubin Mao, Gang Song, Qiufeng Cai, Min Liu, Haohong Luo, Mingxin Shi, Gaoliang Ouyang ,
Shideng Bao. Hydrogen peroxide-induced apoptosis in human gastric carcinoma MGC803 cells
Cell Biology International 30 (2006) 332-337.
Materials and Methods
•Grow cells under sterile conditions until confluent in a
incubator at 37ºC.
Results
Normal Cells
Hydrogen Peroxide 25 µmole/ml
•Treat the cells with chemicals: H202,Tamoxifen, PAHs, and
BAP.
•Take the pictures of cells under phase contrast microscopy.
•Fix the cells with the 70% ethyl alcohol.
Benzo A Pyrene 500ng/ml
Tamoxifen 500ng/ml
• Add Propidium Iodide.
• Collect data using flow cytometer.
Conclusion
PAH 500ng/ml
The results collectively indicate that the chemicals caused growth
inhibition and apoptosis on MDA-MB-231 cells by inducing cell
cycle arrest at different phases of the cell cycle. The table shows the
changes on the cell cycle.
Discussion
Cancer is among the most serious diseases that affect developed
nations. In this study, the efficacy of various chemicals on the triple
negative MDA-MB-231 breast cancer cells was investigated using flow
cytometer. Triple negative cells are associated with early relapse and
poor survival. There is no targeted therapy for triple-negative breast
cancer. The cell cycle analysis by the flow cytometer provides valuable
information for the therapy and diagnosis of cancer. The cell viability
and proliferation were measured and the data show the effects of each
chemicals on the cell cycle of MDA-MB-231 breast cancer cells.
H202, which respiration and tamoxifen, a synthetic drug also caused
complete apoptosis due to its high level of concentration. Similarly,
PAHs and BAP have the same effect except they have some living
cells as well. The chemicals used in this study caused significant
changes on the cell cycle phases of MDA-MB-231 cells and could be
used as agents to control the cancer cell proliferation and progression.
Our in vitro studies demonstrate that targeting the triple negative cells
by various chemicals may be a therapeutic option which should be
evaluated in studies in vivo.
Treatments
%TOTAL
CELLS
% Sub G1
% G1
%S
% G2/M
Control
97.7  0.6
0.9  0.01
61.4  1.3
12.4  1.3
10.9  1.2
25 µmole/ml
H2O2
91.2  2.2
8.2  1.2
79.8  1.7
8.1  0.9
7.2  1.3
1 µg/ml
Tamoxifen
97.3  1.9
0.8  0.4
82.8  0.8
6.8  1.4
5.1  0.8
500 ng/ml BAP 97.3  1.9
0.5  0.34
79.8  1.7
7.8  1.7
6.1  0.7
500 ng/ml PAH 98.6  1.1
1.0  0.5
60.3  0.9
18.8  0.9
16.1  0.9
Acknowledgements
•Benedict College Summer Undergraduate Research 2011.
•Funding provided by National Nuclear Security Administration.
•Funding provided by Department of Energy.
•Dr. Samir Raychoudhury and Mr. Bill McAmis.