INVESTIGATING THE EFFECTS OF ST JOHN’S WORT ON

COLORECTAL CANCER AND DROSOPHILA

Ashley FergusonCaitlin MorrisArturo NeoDaniel Tan

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.

Background

• Norepinephrine as a product of stress.

• Norepinephrine contributes to cell growth.

• Hypericum perforatum thought to inhibit the reuptake of norepinephrine.

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

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)

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

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

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.

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.

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.

Link

St Johns WortHypericin

Vertebrate Invertebrate

Colorectal cancer cell

line

Robust cell line of drosophila

AOS HCI

Link

St Johns WortHypericin

Cancer cell physiology Immune system

Effects on norepinephr

ine

Effects of immune-related

proteins

AOS HCI

Extension Work

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

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

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.