investigating the effects of st john’s wort on colorectal cancer and drosophila ashley ferguson...
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INVESTIGATING THE EFFECTS OF ST JOHN’S WORT ON
COLORECTAL CANCER AND DROSOPHILA
Ashley FergusonCaitlin MorrisArturo NeoDaniel Tan
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Background
• Hypericum perforatum (St. John’s Wort)
• Claimed by alternative medical practitioners to be able to treat burns, wounds and inflammation.
• Hypericin thought to be the main active constituent.
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Background
• Norepinephrine as a product of stress.
• Norepinephrine contributes to cell growth.
• Hypericum perforatum thought to inhibit the reuptake of norepinephrine.
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Objectives (HCI)
• To determine whether hypericin is the main chemical constituent responsible for the chemical properties that the St. John’s Wort possesses.
• To determine which proteins (if any) are affected by St. John’s Wort or hypericin.
• To determine the effects of St. John’s Wort on a drosophila cell line (invertebrate)• Whole extract (liquid)• Hypericin
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Objectives (AOS)
• To determine whether hypericin is the main chemical constituent responsible for the chemical properties that the St. John’s Wort possesses.
• To determine whether there is a correlation between the amount of norepinephrine secreted by the cells and the concentration of St. John’s Wort and hypericin applied.
• To determine the effects of St. John’s Wort on a colorectal cancer cell line (vertebrate)• Whole extract (liquid)• Hypericin• Whole extract (prepared from fresh St. John’s Wort)
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Overview
Research
AOS(colorectal
cancer)
St. John’s Wort
Effects on norepinephri
ne levels
Rate of proliferation
of cancer cells
Hypericin
HCI(drosophila)
St. John’s Wort Hypericin
Effects on immune-related proteins
Rate of proliferation
of robust cell line
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Methods (effects on immune-related proteins)
• Feed drosophila flies• One group with St. John’s Wort added to
food• One group with hypericin added to food• One group with regular food
• Grind up the 3 sets of flies.• Conduct SDS-PAGE.• Send to NUS for MALDI-TOF.
Overview of SDS-PAGE.Source: http://www.imb-jena.de/~rake/Bioinformatics_WEB/gifs/sds_page1.gif
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Methods (rate of proliferation of robust cell line)
• Culture drosophila cell line.• Apply varying concentrations of St.
John’s Wort (4 concentrations) to media of cells.
• Apply varying concentrations of hypericin (4 concentrations) to media of cells.
• Conduct a cell count to check the rate of proliferation of drosophila cells.
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Methods (rate of proliferation of cancer cells)
• Culture colorectal cancer cells.• Apply varying concentrations of St.
John’s Wort (4 concentrations) to media of cells.
• Apply varying concentrations of hypericin (4 concentrations) to media of cells.
• Conduct a cell count to check the rate of proliferation of colorectal cancer cells.
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Methods (effects on norepinephrine levels)
• Add samples of media into ELISA plate which has wells attached with antibodies. Rinse.
• Add fluorescent antibodies into the wells.• Put the wells through an absorbance
plate reader.• Determine the amount of norepinephrine
in the wells by observing the amount of fluorescence.
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Link
St Johns WortHypericin
Vertebrate Invertebrate
Colorectal cancer cell
line
Robust cell line of drosophila
AOS HCI
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Link
St Johns WortHypericin
Cancer cell physiology Immune system
Effects on norepinephr
ine
Effects of immune-related
proteins
AOS HCI
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Extension Work
Conduct SDS-PAGE and send for MALDI-TOF the samples from the investigation of the rate of proliferation of cells.
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References
Mechanisms of mitotic cell death induced by chemotherapy-mediated g2 checkpoint abrogation. (2007). Cancer Res,67(339), Retrieved from http://cancerres.aacrjournals.org/content/67/1/339.full.pdf+html doi: 10.1158/0008-5472.CAN-06-2548
St John's wort. (2010). Wikipedia. Retrieved November 3, 2010, from http://en.wikipedia.org/wiki/St_John's_wort
Pressman, A H. (1998). St. john's wort - the miracle medicine. US: The Philip Lief Group, Inc.
St. John's Wort Side Effects, Interactions and Warnings . (2010). Personal health zone. Retrieved November 4, 2010, from http://www.personalhealthzone.com/stjohnswort.html
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Acknowledgements
Project mentors from both AOS and HCI, Ms Jackie Curley and Ms Sandra Tan.
Other teachers and advisors who have provided valuable advice, especially Mr George Wolfe.