Transcript (PBS). Error bars represent SD. n=4

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
http://www.platinumsoursopgraviola.net/wp-content/uploads/2015/05/order-Platinum-Soursop1.jpg
U251 @ 10X
DISCUSSION
INTRODUCTION
METHODS
• 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).
• 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% CO2.
• For testing, 50,000 viable cells were plated per well, then treated
with increasing doses of soursop extract for 48 hours.
• CellTiter 96 AQueous 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.
• 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.
FUTURE STUDIES
•
•
Preparation of GFE
OBJECTIVE/HYPOTHESIS
•
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 temozolomideresistant 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
•
HA: Soursop will inhibit the growth of Glioblastoma cell lines
(U251 and U87-mg) without negatively affecting nontumorigenic Human Umbilical Vein Endothelial Cells
(HUVEC).
•
Cytotoxicity Assay
•
Statistical Analysis
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.
RESULTS
100
1.5
2.0
****
0.9
0.6
0.3
1.5
*
1.0
****
6%
4.5%
3%
1.5%
PBS
4.5%
3%
1.5%
PBS
Concentration of GFE (% v/v)
Concentration of GFE (% v/v)
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.
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.
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
HUVECS
U251
U87-mg
*
****
60
****
****
****
6%
**
***
40
0.5
0.0
0.0
****
80
% survival
1.2
A b s o rb a n c e (4 9 0 n m )
Absorbance (490nm)
•
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.
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.
Seeing the effect of soursop fruit extract on Glioblastoma cells.
U87-mg @ 10X
****
****
****
****
20
0
2
4
6
8
Concentration of GFE (% v/v)
Figure 4.
Comparing mean % survival of different cell types based on soursop
(GFE) concentration. Cells were treated for 48 hrs. Significant
difference (*) were determined comparing the mean of the treatment
to the control treatment (0% v/v GFE). Error bars represent SEM.
n=4 for HUVECs