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Sea Cucumber Holothuria
The Effects of Holothurin A on B16F10+GFP,
Mouse Melanoma, in a Living Chick Embryo
Table 1: Effects of Holothurin A on Swiss mice carrying an inoculation of 2X106 Krebs-2 cells
Mean wt.
No.
Mg.
No.
Mean
on 1st day
of Sex Holothurin Holothurin
Survival
of
Mice
Injection
Injection
Time (days)
Inoculation
M 13.7
M 21.7gm.
12
F
Controls
Controls
R 10-19
R 18-25
7 injections 8
M 8.5
M 21.6
6
F
0.01mg
days
R 5-10
R 19-23
7 injections 8
M 19
M 20.0
6
F
0.05mg
days
R 14-28
R 18.5-21
7 injections
M 23.5
M 22.7
6
F
0.10 mg
16 days
R 5-35
R 19-25
10 injections
M 34.6
M 21.9
6
F
0.10mg
20 days
R 8-51
R 19-25
Mean wt.
on 10th day
of
Inoculation
M 30.2gm.
R26-33
INTRODUCTION
•Next logical step in this research is to
move the methods into a living organism
•Finding out if B16F10 melanoma cancer
cell line, grown in vivo, will be killed by
M 23.2
Holothurin A
R 19.3-25.5
M 20.6
•Using a chick embryo because it is
R 16.7-25.2
immuno-deficient, easy to culture,
M 20.6
inexpensive and a commonly used
R 18-22.5
* M is the mean day of death or mean weight of mice; R is the range of variation
animal model
(Sullivan, Laude and Nigrelli 1955)
•B16F10 is transfected with Green
HYPOTHESIS
Fluorescent Protein (GFP) so it will be
•The concentration of medicine will not effect the
observable on the chick and so the
average weight of the chick embryos (Figure 3).
number of cells can be easily quantified •The Holothurin A will kill the B16F10 cancer cell line
(Figure 2).
more effectively than Dacarbazine with in vivo
Figure 1: Structure of Holothurin (? Et. al)
inoculations.
•The growing cancer will be directly observable and
quantifiable by looking at the skin under black light and
using a fluorescent plate reader after the chick has
been sacrificed (Figure 4).
Figure 2
Effects of Increasing Concentrations of Blasticidin
on B16F10 Mouse Melanoma Cells
Percent Viable Cells
125
100
75
50
25
0
0
5
10
15
20
25
Blasticidin mg/mL
Figure Legend 2: Blasticidin kills the most B16F10 cells at 15ug/ml. This is the conc.
used as the selecting agent when transfecting the GFP.
Figure 3
Chick Weights Vs. Concentration
of Medicine Added
20.0
Chick Embryo Weight (g)
Department of Biological Sciences, York College
REASEARCH DESIGN
REVIEW OF LITERATURE
•The dosage difference between “no
effect” and “complete killing” is very small.
Eggs
(Nigrelli and Zahl 1954)
•Toxic limit of Holothurin A, injected is
between 0.1 and 0.2 mg in mice.
B16F10+GFP is added
In vivo – Day 10
•The Glycoside Holothurin A has 3
effects:1) Decreased ATPase activity 2)
Experimental eggs
Control eggs
Increased permeability of membrane of
5 Groups of 4
8 total
Holothurin A
Saline
the SR and uncouple calcium, 3) increase
Day 12
Day
12
Experimental eggs
permeability of the lipid layer of the
5 Groups of 4
Dacarbazine
liposomes. (Rubtsov et. al 1981)
Day 12
•Sea Cucumber glycosides such as
Holothurin A are saponins whose
mechanism comes from disrupting
membrane structure and function.
Embryos sacrificed 5 days after
http://www.ext.vt.edu/resources/4h/virtualfarm/poultry/poultry_development.html
OBSERVED RESULTS
Jared J. Meehan and Dr. Jeffrey P. Thompson Ph.D.
PROJECT SUMMARY
There have been many medical
breakthroughs in the field of cancer
research within the past fifty years.
Some treatment methods were derived
from marine organisms. Previous
research has shown that Holothurin A
(Figure 1), a steroid glycoside released
from a Bahamian sea cucumber
(Actinopyga agassizi), can kill certain
cancer cell lines. Older research
focused mainly on experimenting with
Krebs-2 ascites and sarcoma 180
tumors. However, recent research done
by Jonathan Trager demonstrated that
Holothurin A will induce apoptosis in the
B16F10 mouse melanoma cancer cell
line.
Candled Chick egg day 9
http://departments.oxy.edu/tops/marinebio/organisms/PH24seacucumber.htm
Holothurin
Dacarbazine
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
0
1
2
3
4
5
6
Concentration in mM
Figure Legend 3: Control run of testing the viability of chick eggs to the treatment of
Holothurin A and dacarbazine. Non-injected control eggs average weight was 22.67g with
a standard deviation of 1.06g.
original inoculation - Day 17
EXPECTED RESULTS
Figure 4
Percent of cells showing GFP
Fluorescence after Treatment
Weigh embryos to find
mean weight
Observe all under black light as
well as under fluorescent plate
reader to see if any cancer growth
occurred
•Each day eggs are candled to make
sure the embryo is still viable
•Day 12 of growth is when the eggs are
originally inoculated with the medicines
•Day 17 eggs are put in freezer to be
sacrificed
•Eggs are cracked open and chicks
mass is measured
•Percentage scale measures how much
of the body is covered by cancer under
black light.
100
% of Fluorescent Cells
B16F10 cells bright field microscope
GFP producing B16F10 Cells Under GFP GFP producing
Fluorescence Microscope
Holothurin A
Dacarbazine
75
50
25
0
0
1
2
3
4
5
6
Concentration in mM
Figure legend 4: Holothurin A will kill the GFP expressing B16F10 cells. This
graph shows the percent fluorescence at different concentrations. The
Dacarbazine has little effect on the B16F10 cell line based on previous in vito
data.
LITERATURE CITED
Friess, S. L., Standaert, F. G., Whitcomb, E. R., Nigrelli, R. F., Chanley, J. D., and
Sobotka, H., 1960. Some pharmacological properties of Holothurin A, a glycosidic
mixture from the Sea Cucumber. Annals New York Academy of Sciences.
Kitagawa, I., Nishino, T., Kobayashi, M., and Kyogoku, Y., 1951. Bioactive
Triterpene-Oligoglycosides from the Sea Cucumber Holothuria leucospilota.
Chemical Pharmacy Bulletin. 29(7).
Nigrelli, R. F., Stempien, M. F. Jr., Ruggieri, G. D., Liguorori, V. R., and Cecil, J. T.
1967. Substances of potential biomedical importance from marine organisms.
Federation Proceedings. 26(4).
Nigrelli, R. F., and Zahl, P. A., 1954. Some biological characteristics of Holothurin.
New York Zoological Society.
Popov, A. M., 2002. A comparative study of the hemolytic and cytotoxic activities of
triterpenoids isolated from Ginseng and Sea Cucumbers. Biology Bulletin. 29(2).
Rubtsov, B. V., Ruzhitskil, A. O., Klebanov, G. I., Sedov, A. M., and Vladimirov, Y.
A., 1979. Effect of Triterpene Glycosides of marine invertebrates on permeability
of biological and artificial membranes. Central Institute for Advanced Training of
Physicians, Moscow. 3.
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: New York Zoological Society. 40(5).
ACKNOWLEDGMENTS
Dr. Thompson, Jonathan Trager, and the York College Biology Department.