Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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