1. INVESTIGATING THE EFFECTS OF ST JOHN’S WORT ON COLORECTAL CANCER AND DROSOPHILA Ashley Ferguson Caitlin Morris Arturo Neo Daniel Tan

2. 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.

3. Background Norepinephrine as a product of stress. Norepinephrine contributes to cell growth. Hypericum perforatum thought to inhibit the reuptake of norepinephrine.

4. 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

5. 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)

6. Overview Research AOS (colorectal cancer) St. John’s Wort Effects on norepinephrine levels Rate of proliferation of cancer cells Hypericin HCI (drosophila) St. John’s WortHypericin Effects on immune-related proteins Rate of proliferation of robust cell line

7. 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

8. 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.

9. 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.

10. 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.

11. Link St Johns Wort Hypericin VertebrateInvertebrate Colorectal cancer cell line Robust cell line of drosophila AOSHCI

12. Link St Johns Wort Hypericin Cancer cell physiologyImmune system Effects on norepinephrine Effects of immune- related proteins AOSHCI

13. Extension Work  Conduct SDS-PAGE and send for MALDI-TOF the samples from the investigation of the rate of proliferation of cells.

14. 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

15. 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.