1. Fruit Against Cancer: The Effects of Soursop (Annona muricata) Fruit Extract on Glioblastoma Cell Lines Fabiola Ngalle Ehabil Department of Biological Sciences, York College of Pennsylvania INTRODUCTION Glioblastoma (GB) is one of the most aggressive intracranial cancers which in 2015, killed 15,320 people (National Cancer Institution). The usage of Alternative Medicine in cancer treatment from 1977 to 1997 increased from 9% to 40 % in countries like Finland, Australia and US (Ernst and Cassileth, 1998). Complementary and Alternative medicine (CAM) is a health care practice that is sometimes used with conventional medicine to increase the effects of the treatment and help with side effects (Armstrong 2006, Burstein 1999). Examples are Reiki, meditation, chiropractic. Soursop (Annona muricata), also known as Graviola or Corosol, is a tropical fruit which is used as a herbal medicine in some countries for health promoting effect as well as supporting healthy cell growth and immune function (Dai, 2011). Soursop is effective in inhibiting the growth of breast, colon, and pancreatic cancer cells (Dai 2011, Moghadamtousi 2014, Torres 2012). OBJECTIVE/HYPOTHESIS Seeing the effect of soursop fruit extract on Glioblastoma cells. HA: Soursop will inhibit the growth of Glioblastoma cell lines (U251 and U87-mg) without negatively affecting non- tumorigenic Human Umbilical Vein Endothelial Cells (HUVEC). METHODS Soursop extract (GFE) was prepared with PBS and vacuum filtered, centrifuged, and then sterilized with a 0.2um syringe filter. The absorbance of GFE was checked using a Nano drop spectrometer (figure 1). The media for each line of cells was changed when there was a lot of debris in the flasks as well as a sign of less or no more nutrient in the media from the pH indicator (the flask looked orange instead of red). The media of U251 and U87-mg consisted of DMEM, 10% Fetal Bovine Serum (FBS), 1x Glutamax, Pen/strep, 1x non-essential amino acids (NEAA), and 1x Na-Pyruvate (NaP) The media for HUVECs was F-12k nutrient mixture, 0.03mg/ml endothelial cell growth supplement (ECGS), 0.1mg/ml heparin, 1x Glutamax and pen strep, and 10% FBS. The cells grew in a 37°C incubator containing 5% CO 2. For testing, 50,000 viable cells were plated per well, then treated with increasing doses of soursop extract for 48 hours. CellTiter 96 AQ ueous Solution Cell Proliferation Assay, a cytotoxicity assay, with a plate reader was used to determine the amount of viable cells remaining in each well A two way ANOVA was used for statistical analysis. Preparation of GFE Cell Culturing Treatment of Cells with 1.5% v/v, 3% v/v, 4.5% v/v, 6% v/v GFE and PBS for 48hrs. Cytotoxicity Assay Statistical Analysis RESULTS DISCUSSION The wavelength of maximum absorbance of GFE was lower than the wavelength of maximum absorbance for the assay (figure 1). Soursop does inhibit the growth of GB cell lines; U251, and U87-mg (figures 2&3), similar to its inhibition of breast, colon and pancreatic cancer (Dai 2011, Moghadamtousi 2014, Torres 2012) Soursop effectiveness in inhibiting GB cells is greater in U251 than it is in U87-mg (figure 4; Zhang et al. 2013, and Cho et al. 2012). The results also show soursop to kill a more significant amount of GB cells than HUVECs, through out the different doses (figure 4). Soursop can be a potential CAM for the treatment of cancer. More research on how soursop works in vivo instead of in vitro will be needed. ACKNOWLEDGEMENT I would like to thank Dr. Jeffrey Thompson for guiding me throughout the project, Joan Carpenter for providing supplies, Dr. Bridgette Hagerty for helping with the statistical analysis, and for my parents for the support and giving me the idea to work with Soursop. LITERATURE CITED Dai, Y., Hogan, S., Eva, S., Ju, Y., Canning, C., and Zhou, K. 2011. Selective Growth Inhibition of Human Breast Cancer Cells by Graviola Fruit Extract In Vitro and In Vivo Involving Downregulation of EGFR Expression. Nutrition & Cancer [serial online] 63(5):795-801 Ernst, E., and Cassileth, B. 1998. A systematic review: The Prevalence of Complementary/Alternative Medicine in Cancer. American Cancer Society [serial online] 83(4):777-782 National Cancer Institution. http://www.cancer.gov/about-cancer/what-is-cancer/statistics. Accessed 2016 January Torres, M., Rachagani, S., Purohit, V., Pandey, P., Joshi, S., Moore, E., Johansson, S., Singh, P., Ganti, A., and Batra, S. 2012. Graviola: A novel promising natural-derived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism. Cancer Letters [serial online] 323(1):29-40 Zhang, X., Li, W., Wang, C., Leng, X., Lian, S., Feng, J., and Wang, H. 2014. Inhibition of autophagy enhances apoptosis induced by proteasome inhibitor bortezomib in human glioblastoma U87 and U251 cells. Molecular and cellular biochemistry [serial online] 385(1-2):265-275. Cho, H. Y., Wang, W., Jhaveri, N., Torres, S., Tseng, J., Leong, M. N., and Louie, S. G. 2012. Perillyl alcohol for the treatment of temozolomide- resistant gliomas. Molecular cancer therapeutics [serial online] 11(11):2462-2472. Moghadamtousi, Z.S, Karimian, H., Rouhollahi, E., Paydar, M., Fadaeinasab, M., and Kadirn, A.H. 2014. Annona muricata leaves induce G1 cell cycle arrest and apoptosis through mitochondria-mediated pathway in human HCT-116 and HT-29 colon cancer cells. Journal of Ethnopharmacology [serial online] 156: 277-289 http://www.platinumsoursopgraviola.net/wp-content/uploads/2015/05/order-Platinum-Soursop1.jpg U251 @ 10XU87-mg @ 10X 6%4.5%3%1.5%PBS 0.0 0.3 0.6 0.9 1.2 1.5 Concentration of GFE (% v/v) A b s o r b a n c e ( 4 9 0 n m ) Figure 2. Effects of soursop on U251 is shown above. Cells were incubated for 48hrs and checked with an absorbance reading at 490nm. Decrease in the absorbance reading @ 490nm indicate decreasing cell activity. Having 4 asterisks represent a greater significant difference from the control (PBS). Error bars represent SD. n=5. **** 6%4.5%3%1.5%PBS 0.0 0.5 1.0 1.5 2.0 Concentration of GFE (% v/v) A b s o r b a n c e ( 4 9 0 n m ) Figure 3. Effect of soursop on U87-mg is shown above. U87-mg were treated for 48 hrs. The results of viable cells were obtained from an absorbance reading of 490nm. Decrease absorbance reading @ 490nm indicates decreasing cell activity. Increasing amount of asterisks represents an increasing significant difference from the control group (PBS). Error bars represent SD. n=4 **** * FUTURE STUDIES Figuring why cells treated at a higher concentration of GFE will survive more than cells at a lower dose Looking at how GFE acts in an environment filled with both HUVECs and glioblastoma cells. Figure 1. Absorbance spectrum of GFE. This is showing that the absorbance reading for the cytotoxicity assay is not affected by the color of the soursop extract.