Effects of Etoposide on the Apoptosis of HL-60 CellsStefanos 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.

Conclusion References

Acknowledgements

Introduction Methods

Objectives

Results

UFPB

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.

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.

FLOW CYTOMETRY

Channels (RED-HLin-Red Fluorescence (RED-HLin))0 40 80 120 160

Num

ber

06

01

20

18

02

40 G1

G2Fragmented DNA

MW 1 2 3 4

DNA ASSAY

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)

CITOTOXICITY: CELL VIABILITY – MTT

HL-60 cells x Etoposide

control 0,25 0,5 1,0 2,0 4,00

102030405060708090

100110

*

**

Etoposide [uM]

Cell

ula

r V

iab

ilit

y (

%)

Legend* = p < 0.001** = p < 0.0001

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)-2,5-diphenyltetrazolium bromide (MTT) to a colored blue

formazan (Kim et al., 2009).

FLOW CYTOMETRY

Figure 1. A graph of the statistical analysis of the effects of various concentrations of etoposide on HL-60 cells.

Control Etoposide 0.5 µM

Etoposide 5 µM

Etoposide 1 µM Etoposide 2.5 µM

(A) (B)

(C) (D)

(E)

12.25%16.09%

22.01%

37.35%

8.78%

1.14%

64.87%

7.79%

57.65%

0.69%

14.33%

88.26%

1.30%

4.17%

6.48%

3.50%

30.87%

2.00%

2.37%91.99%

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)

 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-americans-under-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.

DNA ASSAY

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.