1. Asiatic acid, a derivative of Centella asiatica, induced apoptosis and decreased levels of free Ca 2+ in A-375 human melanoma Jason Aloisio, Department of Biological Sciences, York College of Pennsylvania INTRODUCTION  Currently non-cytotoxic treatments for skin cancer are unavailable. This AFFECTS more than 1 MILLON new people in the United States every year and of those approximately 60,000 are melanoma, the most serious type 1,2.  Asiatic acid (AA), derived from Centella asiatica, is non-cytotoxic to the immune system and reduces tumor size making it an ideal therapeutic agent to study 2.  SK-MEL-2 human melanoma was shown to undergo APOPTOSIS when treated with AA, however NO DATA have been presented on the reaction of A-375 human melanoma with AA, thus making it an ideal candidate for further study 3.  FREE Ca 2+ released from the ER regulates apoptosis in multiple ways (1)accumulation in mitochondria leading to CELL DEATH and (2) necessary chelator for PKC activation eventually leading to CASPASE-3 activity, a well known regulator of apoptosis.  APOPTOSIS can be characterized by the following hallmarks: cell shrinkage, nuclear condensation membrane bleeding, and fragmentation into apoptotic bodies 4,5.  PROPOSED MECHANISM: AA activates Phospholipase-C (PLC) which generates increasing IP3 concentrations thus releasing intracellular Ca 2+ stores into the cytoplasm 6,7 Consequently, free Ca 2+ induces mitochondria to release Cytochrome c, a pro-apoptotic factor 8 which is an upstream regulation of caspase- 3, thus triggering apoptosis. LITERATURE CITED 1) American Cancer Society. 2007. Why you should know about melanoma. Accessed August 27, 2007 2) Babu, T.D., Kuttan, G., and Padikkala, J. 1995. Cytotoxic and anti-tumour properties of certain taxa of Umbelliferae with special preference to Centella asiatica (L.) Urban. Journal of Thno-Pharmacology. 48:53-57 3) Park, B., Bosire, K., Lee, E., Lee, Y., and Kim, J. 2004 Asiatic acid induces apoptosis in SK-MEL-2 human melanoma cells. Cancer Letters 218: 81-90 4) Savil, J., Fadock, V., Henson, P., Haslett, C. 1993. Phagocytic recognition of cells undergoing apoptosis. Immunology Today 14:131-136 5) Wyllie, A.H., Kerr, J.F.R., Currie, A.R. 1980. Cell death: The significance of apoptosis. International Review of Cytology 68:251-305 6) Mikoshiba, K. 2006. Inositol 1,4,5-trisphosphate IP(3) receptors and their role in neuronal cell function. Journal of Neurochemistry 97:1627–1633 7) Patel, S., Joseph S.K. & Thomas, A.P. 1999. Molecular properties of inositol 1,4,5-trisphosphate receptors. Cell Calcium 25:247–264 8) Joseph, S.K. & Hajn´oczky, G. 2007. IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond. Apoptosis. 12:951–968 QUESTIONS ASKED (1)Does AA induce apoptosis and cell death in A-375 human melanoma cells? (2) Is there a relationship among free Ca 2+, caspase-3 and apoptosis in AA-treated A-375? Cultured A-375 Cells Passaged/Split Cells Plated in 96-well plate Caspase-3 Cell Viability Ca 2+ and Gelstar 30,000 cells/well (Ca 2+ and Viability) 24 h 24, 48 h24 h 10,000 cells/well (Caspase-3) METHODOLOGY (+) Control: Taxol (-) Control: No treatment Exp: AA: 1,3.3, 10, 33.3, 100, 333, 666, 1000  M. (N=3) ACKNOWLEDGMENTS I’d like to thank Dr. Kaltreider for sharing his knowledge and for his guidance in the laboratory and Dr. Rehnberg for his insight into poster construction. CONCLUSIONS FUTURE WORK RESULTS  Cell Viability decreases significantly (p<0.05) at 100  M (AA) for 24h and 48h treatments (Fig 1)  Apoptosis increased in a dose dependent manner (Fig 2)  Caspase-3 is involved in apoptotic induction (Fig 2)  Free Ca 2+ reduces significantly at apoptotic threshold (Fig 3) Analysis (Two-Tailed T-Test) (1)A-375 human melanoma undergo apoptosis in 48h when treated with AA, LD50=186.9 (Fig 2). (2)After 48h, free Ca 2+ binds to PKC which activates Bax and Bcl-2 which mediate apoptosis in A-375 cells treated with 100  AA (Fig 3). Isoform-specific analysis of PKC activity to further illuminate apoptotic pathway Determine whether AA is cell-permanent or if it binds to a cell surface receptor http://www.ics.trieste.it/MedicinalPlant/MedicinalPlantImage.aspx?id=165 http://www.sigmaaldrich.com/catalog/search/ProductDetail/ALDRICH/546712 PLC PKC DAG Mitochondria IP 3 Bax Ca 2+ Bcl-2 Caspase-3 Cyt c APOPTOSIS Anti-Apoptosis ER Asiatic Acid CELL Figure 2: A375 human melanoma cells were treated with Asiatic Acid at 1, 3.3, 10, 33.3, 100, 333.3, 666, 1000uM for 48H at 37 o C with 5% CO 2. Taxol, the positive control, is indicated by the dashed line and has a fluorescence value = 20860.7, EC50 = 186.9. * indicates a statistically significant difference when compared to taxol the positive control (P<0.05). Figure 3: A-375 was treated with various concentrations of AA for 48h. Free Ca 2+ was measured and compared as a ratio of cell count using gelstar green. * indicates a statistical difference compared to no treatment (P<0.05) followed by subsequent Ca 2+ increase. * * * * * * Figure 1: Cell Viability of A375 human melanoma was measured when treated with AA for 24 h at 37 C in 5% CO2 with concentrations of 0, 1, 3.3, 10, 33.3, 100, 333.3, 666, 1000uM. Taxol the positive control was measured at 0.49825 for 24h and EC50=295.7. * indicates a statistically significant difference when compared to the negative control (P<0.05). * *