The Effects of Holothurin A on B16F10+GFP, Mouse Melanoma, in a Living Chick Embryo Jared J. Meehan and Dr. Jeffrey P. Thompson Ph.D. Department of Biological Sciences, York College 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. 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 Holothurin A Using a chick embryo because it is immuno-deficient, easy to culture, inexpensive and a commonly used animal model B16F10 is transfected with Green Fluorescent Protein (GFP) so it will be observable on the chick and so the number of cells can be easily quantified (Figure 2). REVIEW OF LITERATURE The dosage difference between “no effect” and “complete killing” is very small. (Nigrelli and Zahl 1954) Toxic limit of Holothurin A, injected is between 0.1 and 0.2 mg in mice. The Glycoside Holothurin A has 3 effects:1) Decreased ATPase activity 2) Increased permeability of membrane of the SR and uncouple calcium, 3) increase permeability of the lipid layer of the liposomes. (Rubtsov et. al 1981) Sea Cucumber glycosides such as Holothurin A are saponins whose mechanism comes from disrupting membrane structure and function. No. of Mice Sex Mg. Holothurin Injection No. Holothurin Injection Mean Survival Time (days) Mean wt. on 1 st day of Inoculation Mean wt. on 10 th day of Inoculation 12FControls M 13.7 R M 21.7gm. R M 30.2gm. R F0.01mg 7 injections 8 days M 8.5 R 5-10 M 21.6 R F0.05mg 7 injections 8 days M 19 R M 20.0 R M 23.2 R F0.10 mg 7 injections 16 days M 23.5 R 5-35 M 22.7 R M 20.6 R F0.10mg 10 injections 20 days M 34.6 R 8-51 M 21.9 R M 20.6 R HYPOTHESIS The concentration of medicine will not effect the average weight of the chick embryos (Figure 3). The Holothurin A will kill the B16F10 cancer cell line more effectively than Dacarbazine with in vivo 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). * M is the mean day of death or mean weight of mice; R is the range of variation (Sullivan, Laude and Nigrelli 1955) REASEARCH DESIGN Eggs Experimental eggs 5 Groups of 4 Holothurin A Day 12 Embryos sacrificed 5 days after original inoculation - Day 17 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 Table 1: Effects of Holothurin A on Swiss mice carrying an inoculation of 2X10 6 Krebs-2 cells OBSERVED RESULTS 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. EXPECTED RESULTS LITERATURE CITED Friess, S. L., Standaert, F. G., Whitcomb, E. R., Nigrelli, R. F., Chanley, J. D., and Sobotka, H., 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., 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 Substances of potential biomedical importance from marine organisms. Federation Proceedings. 26(4). Nigrelli, R. F., and Zahl, P. A., Some biological characteristics of Holothurin. New York Zoological Society. Popov, A. M., 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., 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., 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. Figure 4 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 Sea Cucumber HolothuriaCandled Chick egg day 9 GFP producing B16F10 Cells Under GFP GFP producing B16F10 cells bright field microscope Fluorescence Microscope Figure 2 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. B16F10+GFP is added In vivo – Day 10 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. Experimental eggs 5 Groups of 4 Dacarbazine Day 12 Control eggs 8 total Saline Day 12 Figure 1: Structure of Holothurin (? Et. al)