Transcript Effects of Etoposide on the Apoptosis of HL-60 Cells

Effects of Etoposide on the Apoptosis of HL-60 Cells
Stefanos F. Haddada, Glaucia V. Faheina-Martinsb,c, Demetrius A. M. Araújob,c
a
Department of Biology, State University of New York at Oswego, Oswego, NY, United States of America.
b Departamento de Biologia Molecular, Universidade Federal da Paraíba, João Pessoa,PB, Brazil.
c Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, João Pessoa, PB, Brazil.
Introduction
UFPB
Methods
FLOW CYTOMETRY
120
Number
180
240
G1
60
Objectives
G2
Fragmented DNA
0
Cancer is a group of diseases characterized by abnormal, uncontrolled cell
growth. Healthy cells grow, die, and are replaced in a very controlled way.
When the genetic material of a cell is damaged or altered by environmental or
internal factors, it may result in cells that do not die and continue to multiply
until a mass of cancer cells or a tumor develops. Great advancements have
been made to treat cancer but it is still the leading cause of death for people
under the age of 85 (DeNoon, 2005).
Chemotherapy is a treatment that uses drugs to treat cancer. More
specifically, it is the use of cytotoxic treatments to kill off cancer cells.
Cytotoxic treatments are treatments that kill both cancer cells and healthy
cells. Etoposide is a cytotoxic, anti-cancer agent. It inhibits topoisomerase II,
an enzyme that plays a major role in unwinding DNA. By inhibiting this
enzyme, etoposide causes DNA strands to break and triggers a mechanism
which eventually leads to apoptosis (Montecucco and Biamonti, 2006). Thus,
etoposide induces apoptosis. Apoptosis is a form of cell death in which a
programmed sequence of events lead to the elimination of cells without
releasing harmful substances into the surrounding area. Since cancer cells
replicate their genome and divide much more rapidly than normal, healthy
cells, they are more dependent on the proper functioning of the enzyme
topoisomerase II. As a result, etoposide can be used as a type of
chemotherapy treatment, killing cells that divide rapidly with one of its main
focuses on cancer cells (Montecucco and Biamonti, 2006).
The HL-60 cell line is Human promyelocytic leukemia cells. HL-60 cells
multiply continuously in suspension culture in nutrient medium supplemented
with fetal bovine serum; the doubling time is about two days. This study is
designed to test the effects of etoposide on the apoptosis of HL-60 cells.
0
40
80
120
160
C hannels (R ED -H Lin-R ed Fluorescence (R ED -H Lin))
This study assessed the cytotoxicity of
etoposide, a substance used in the standard
therapy against cancer. The techniques used in
this study are important for the identification of
new anticancer drugs that have potential to be
used for in vivo trials.
DNA ASSAY
Results
CITOTOXICITY: CELL VIABILITY – MTT
FLOW CYTOMETRY
(A)
Control
Cellular Viability (%)
HL-60 cells x Etoposide
110
100
90
80
70
60
50
40
30
20
10
0
(B)
Etoposide 0.5 µM
37.35%
Legend
* = p < 0.001
** = p < 0.0001
*
DNA ASSAY
1
2
3
4
1.14%
22.01%
16.09%
MW
7.79%
12.25%
8.78%
64.87%
**
(C)
control 0,25
0,5
1,0
2,0
4,0
(D)
Etoposide 1 µM
Etoposide 2.5 µM
Etoposide [uM]
1.30%
0.69%
Figure 1. A graph of the statistical analysis of the effects of
various concentrations of etoposide on HL-60 cells.
4.17%
14.33%
57.65%
30.87%
88.26%
6.48%
(E)
Figure 4. DNA Assay of extracted DNA from
HL-60 cells treated with various etoposide
concentrations. (MW, 100 bp ssDNA Ladder;
Lane 1, control; Lane 2, 1 µM etoposide;
Lane 3, 2.5 µM etoposide; Lane 4, 5 µM
etoposide)
Etoposide 5 µM
3.50%
2.00%
Figure 2. A photo of the MTT plate before it was
analyzed in the reader plate. Cell viability was evaluated
by measuring the mitochondrial-dependent reduction of
colorless 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) to a colored blue
formazan (Kim et al., 2009).
Conclusion
The MTT test revealed that increased concentrations of etoposide decreases cell viability.
The combined results of the Flow Cytometry and DNA Assay lead us to suggest that the
mechanism this happens through is apoptosis.
Flow Cytometry shows a dramatic shift of fluorescent cells being stopped in the sub G1
phase when treated with higher concentrations of etoposide. This is characteristic of
fragmented DNA and apoptosis. In addition, the DNA Assay shows increased DNA
fragmentation with higher concentrations of etoposide which is once again characteristic of
apoptosis. Thus, etoposide triggers a mechanism that leads to apoptosis (Montecucco and
Biamonti, 2006).
Etoposide is a good molecule for the positive control in studies that measure cytotoxicity
and apoptosis. Such studies are important for the future development of clinical trials
aiming to target cancer.
91.99%
2.37%
Figure 3. Flow Cytometry measures
the cell cycle analysis of HL-60 cells
after treatment with various
concentrations of etoposide. (A,
control; B, 0.5 µM etoposide; C, 1
µM etoposide; D, 2.5 µM etoposide;
E, 5 µM etoposide)
References
DeNoon, Daniel J. "Cancer Now Top Killer of Americans Under 85." WebMD - Better
Information. Better Health. WebMD Health News, 19 Jan. 2005. Web. 01 Aug. 2011.
<http://www.webmd.com/cancer/news/20050119/cancer-now-top-killer-of-americansunder-85>.
Kim, H., Yoon, S.C., Lee, T.Y. and Jeong, D. (2009). Discriminative Cytotoxicity
Assessment Based on Various Cellular Damages. Toxicology Letters. 184, 13-17.
Montecucco, A. and Biamonti, G. (2006). Cellular Response to Etoposide Treatment.
Cancer Letters. 252, 9-18.
Acknowledgements