Transcript Pic. 1. - York College of Pennsylvania

The Mechanism of Suppression of Tumorigenicity in B16F10
Mouse Melanoma Cells by the Steroid Saponin Holothurin A
Jonathan D. Trager, BA & Jeffrey P. Thompson, Ph.D.
York College of Pennsylvania
Department of Biological Sciences
A number of substances have been isolated from
marine animals that demonstrate usefulness in
biomedical research. Holothurin A, a steroid glycoside
extracted from the Bahamian sea cucumber
Actinopyga agassizi, has shown promise in the field of
cancer research. Partially purified preparations of
Holothurin A have been shown to possess a variety of
pharmacologic properties including antitumor activity
(Friess et al. 1960, Nigrelli et al. 1967).
METHODS
RESULTS
B16F10
MOUSE CELL LINE
Grown to Confluency
Holothurin A was found to have an EC50 of ~97 µM
in B16F10 cells. The Dacarbazine had little to no
effect at any of the doses. Holothurin A appeared to
induce apoptosis in the B16F10 mouse melanoma
cell line.
Confluent B16F10 mouse melanoma cells
A confluent flask with melanic spots.
80
Figure 1.
CELL DEATH (%)
This study will examine a new role for the use of
Holothurin A as a cancer therapy by applying it to a
melanoma model. Previous research limited its scope
of experimentation to Krebs-2 ascites and Sarcoma
180 tumors.
Apoptosis
20000
Figure 3.
90
60
CELL DEATH (%)
Dose Response
EC50
OBJECTIVE 2
Figure 2.
100
HOLOTHURIN A
DACARBAZINE
70
OBJECTIVE 1
110
50
40
30
20
15000
80
Fluorescence
INTRODUCTION
70
60
50
40
30
10000
5000
20
10
Plate & Treat Cells
0-500μg
Holothurin A
Dacarbazine
The antitumor mechanism of action of Holothurin A
has not yet been determined. This study will aim to
advance the field of cancer research by ascertaining
the anti-neoplastic role of Holothurin A and perhaps
provide the information needed to develop a new
treatment for melanoma.
Plate & Treat Cells
0-500μg
Holothurin A
Dacarbazine
10
0
0
50
100
150
[DRUG] (M)
200
250
0
1.6
0
1.7
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
CTL
HOLO A
log [DRUG] (M)
Figure 1. The percent cell death (%) of B16F10 mouse melanoma cells after exposure to varying
concentrations (0-238μM) of Holothurin A and Dacarbazine in equivalent molar amounts. Each point
represents n = 32. Error bars represent SEM.
Figure 2. Dose-Response of B16F10 mouse melanoma cells after treatment with varying concentrations
(0-238μM) of Holothurin A. The EC50 for Holothurin A is ~97 μM. Each point represents n=32. Error bars
represent SEM.
Determine Cell
Viability
Cell Titer 96 assay
Colorimetric analysis
Determine Apoptosis
Induction
Caspase-3 activity
assay
DISCUSSION
Dacarbazine
OBJECTIVES
Figure 3. Detection of Caspase-3 activity in B16F10 cells using the EnzCheck Caspase-3 Kit #2 with ZDEVD-R110 substrate. The cells were treated with 238 μM of Holothurin A (HOLO A) for 24 hours at
37oC. Both treated and control cells (CTL) were harvested and assayed. The reactions were carried out at
room temperature. Fluorescence was measured using excitation at 485nm and emission detection at
530nm. Background fluorescence for a no-enzyme control, was subtracted from each value. Bars
represent the SEM. Amount of fluorescence is significantly different (P<0.05, Mann-Whitney).
Holothurin A was effective at killing the B16F10
mouse melanoma cell line in vitro.
Determine the dose-response relationship of
Holothurin A to B16F10 mouse melanoma cell lines
and the EC50.
Holothurin A was effective at inducing apoptosis
in B16F10 mouse melanoma cells.
Holothurin A
Determine whether Holothurin A induces apoptosis
in B16F10 mouse melanoma cell lines.
Pathway Leading to Apoptosis
Chemotherapeutic drugs cause DNA damage and other
signals that stimulate p53. As a result, Apaf-1 is activated,
followed by activation of Caspase-3, triggering apoptosis.
Disruption of this pathway allows pre-cancerous cells to
survive and proliferate, and eventually form a tumor.
http://www.cshl.org/public/releases/lowe011001.html
LITERATURE CITED
Friess S.L., Standaert F.G., Whitcomb E.R., Nigrelli R.F., Chanley J.D. and Sobotka H. 1960. Some
pharmacologic properties of holothurin A, a glycosidic mixture from the sea cucumber. Annals of the New York
Academy of Science. 90: 893-901.
Kitagawa I., Nishino T., Kobayashi M. and Kyogoku Y. 1981. Marine natural products: VIII. Bioactive
triterpene-oligoglycosides from the sea cucumber Holothuria leucospilota Brandt. Structure of holothurin A. Chemical
& Pharmaceutical Bulletin (Tokyo). 29:1951–1956.
Nigrelli R.F, Stempien M.F., Ruggieri G.D., Liguori V.R. and Cecil JT. 1967. Substances of potential biomedical
importance from marine organisms. Federation Proceedings. 26(4):1197–205.
Sullivan, T.D., Ladue, K.T. and Nigrelli, R.F.. 1955. The effects of holothurin, a steroid saponin of animal origin,
on Krebs-2 ascites tumors in Swiss mice. Zoologica. 40: 49-52
Kitigawa et al. 1981
Pic. 1
Pic. 2
Sullivan, T.D. and Nigrelli, R.F.. 1956. The antitumorous action of biologics of marine origin I. Survival of Swiss
mice inoculated with Krebs-2 ascites tumor and treated with holothurin, a steroid saponin from the sea cucumber,
Actinopyga agassizi. Proceedings of the American Association for Cancer Research. 2: 151
ACKNOWLEDGEMENTS
Pic. 3
Pic. 1. The Bahamian sea cucumber, Actinopyga agassizi. http://malhavoc.smugmug.com/gallery/86/1/2928
Pic. 2. B16F10 mouse melanoma cells. The arrow is pointing to the cell nucleus.
Pic. 3. B16F10 cells after treatment with Holothurin A. Notice how cells have condensed in size.
Holothurin is produced in the Cuvierian
tubules of the respiratory tree.
http://www-micro.msb.le.ac.uk/3035/kalmakoff/baculo/baculohostinteract.html
Pennsylvania Academy of Science
Dr. Jeffrey P. Thompson, Mentor
Dr. Ronald Kaltreider
Dr. Karl Kleiner
Dr. Bradley Rehnberg