Download Drosophila - AOS-HCI-2011-Research

Investigation of the
Effects of St John’s Wort
on Cancer and Drosophila
AOS-HCI Combined Research Paper 2011
Arturo Neo Yong Yao (4S1-02)
Daniel Tan Han Jie (4S2-06)
Ashley Ferguson (AOS)
Caitlin Morris (AOS)
Hwa Chong Institution (High School)
Ms Sandra Tan
Abstract
Norepinephrine has been found to play a significant role in the progression of cancer
by promoting focal adhesion kinase which is a protein that helps facilitate cell life. Cancer
cells have also shown to have increased glycolysis for higher rate of production for ATP.
Hypericum perforatum has been found to inhibit the reuptake of norepinephrine in
cells, which should then slow the rate of cancer progression. It has also shown to be able to
inhibit or denature certain enzymes that are involved in the process of glycolysis, hence
slowing down the rate of which the cells are able to proliferate.
The purpose of our research is to develop a method to slow the growth and
metastasis of a mouse mammary cell line and that of a robust Drosophila cell line using the
herb extract Hypericum perforatum. An extract of its main active ingredient, hypericin, was
also tested on the mouse mammary cell line.
The mouse mammary cell line CRL 2539 was cultured and two concentrations of a
glycerol based liquid Hypericum perforatum extract (0.4%, 0.8%) as well as an extract of
hypericin. (0.001%) were applied to the cells. The concentration of hypericin applied was
based on typical amounts of hypericin found in a Hypericum perforatum plant. To gather
data, the cells’ growth was measured using microscope cell counts on intervals of 24 to 48
hours until they reached confluency. The robust Drosophila cell line was also cultured
similarly and the same glycerol based liquid Hypericum perforatum extract (2.5%, 5.0%,
12.5%, 50%) was applied to the cells. Cell counts were done using a haemocytometer daily
at 24 hour intervals. Each experimental trial was run alongside a control trial in order to
compare the growth of each.
In addition, Hypericum perforatum extract had also been tested on live Drosophila
melanogaster flies to observe if any immune-related proteins are affected by the extract. The
flies were fed varying concentrations of the extract and their protein profiles were observed
via SDS-PAGE.
So far, the hypericin trials have yielded inconclusive results but the Hypericum
perforatum extract has been found to slow the cancer’s growth in both the mouse mammary
cell line as well as the robust Drosophila cell line. After SDS-PAGE, 4 protein bands
observed in the control, which were missing in the experimental set ups, were then sent for
MALDI-TOF. 2 of these proteins had been identified to be enzymes essential for glycolysis.
In addition, samples of the mouse mammary cells’ media have been taken at the end
of each trial so that the norepinephrine levels can be measured in an ELISA kit. Likewise, an
ELISA can also be used to observe how the physiopathology of the robust Drosophila cells
are affected, especially in the areas of protein kinases and glycolytic enzymes.
Trials are continuing to evaluate the effect of hypericin on cell growth and measuring
the degree to which varying concentrations of Hypercium perforatum extract slow down the
growth of cancer cells.
Introduction
Hypericum perforatum is a yellow flowering plant found in various locations around
the world including West Asia, Europe, and North Africa. This herb is often used in
depression treatment. Other areas where Hypericum perforatum is applicable include
treating diseases, disorders, minor burns, and injuries (Linde, 2009). Hypericum perforatum
has been tested to treat depression in adults and attention deficit hyperactive disorder
(ADHD) in children (Weber et al., 2008). Cancer prevention research is one of the main
fields that the therapeutic effect of Hypericum perforatum can be investigated because it has
been found to inhibit the reuptake of norepinephrine, dopamine, and serotonin, which play a
role in the development and spread of cancer. Research has shown that the most significant
components of Hypericum perforatum in terms of treating disease are hypercerin and
hyperforin (Linde, 2009).
One of the older but still practiced methods of cancer treatment involving hypericin is
photodynamic therapy (PDT). PDT requires a photosensitizing agent (photosensitizer) and
the visible light of a wavelength which correlates with the absorption spectrum of the drug.
Alone the light and photosensitizer have no therapeutic effect, but when combined produce
cytotoxic products which trigger irreversible tumor destruction and cell damage. Hypericin is
probably nature’s stongest naturally photosensitizer so its use in PDT to induce cell
apoptosis and necrosis is immense (Anastasia Karloti et al. 2010). Out of 36 species of
Hypericum, 27 held hypericin, the most common being Hypericum perforatum (Agostinis,
Merlevede, Vantieghem, & Witte, 2002).
Cancer describes an immense group of related diseases. Each cancer case is
different in terms of how fast the cells grow and its set of genetics changes, which is why
cancer treatment is so difficult (Emory University, 2009). More specifically, cancer is “any
malignant growth or tumor caused by abnormal and uncontrolled cell division; it may spread
to other parts of the body through the lymphatic system or the blood stream” (Princeton
University, 2010). Breast cancer is “..cancer that affects the breast”. Most cases of breast
cancer occur in women although men have been found to develop it as well (Health
Information Publications, 2010).
The Drosophila melanogaster is commonly known as the fruit fly or vinegar fly.
According to Lawrence Reiter et al. (2001) 714 distinct human disease genes (77% of
disease genes searched) match 548 unique Drosophila sequences. Of the 548 Drosophila
genes related to human disease genes, 153 are associated with known mutant alleles and
56 more are tagged by P-element insertions in or near the gene. Cross-genomic analysis
with significant sequence matches can be carried out to prove the effects of drugs on
diseases.
In recent studies, researchers have been examining the effect of stress on the
progression of cancer. The body responds to stress by releasing catecholamines such as
norepinephrine and epinephrine. These catecholamines prevent the anoikis (programmed
cell death) of cancer tumor cells by increasing focal adhesion kinase. Focal adhesion kinase,
when activated, is a protein that promotes cell life. In cancer research when different factors
are looked at in isolation, stress has been found to play a main role in tumor cell proliferation.
This is because the cancer cells reuptake the catecholamines released by the body
furthering the cells’ ability to live (Sood et al., 2010).
Norepinephrine is one of the major catecholamines that plays a role in developing
and spreading cancer. There are many norepinephrine receptors on organs and in the
blood stream, and in the brain, which, when activated, may be used as message systems to
spread cancer because of the intracellular processes taking place in the organs or blood
stream (Fitzgerald, 2008). One field of research that has been focusing on a way to prevent
the cancer cell reuptake of the catecholamines is complementary and alternative medicine
(CAM). One of the main herbs that have been looked into is for its anticancer abilities is
Hypericum perforatum (Sood et al. 2010).
Glycolysis has also proven to be a new target for therapy in cancer. (Gatenby et. al.
2007) According to Rui-hua Xu et. al. (2005), the Warburg Effect has shown that cancer
cells frequently exhibit increased glycolysis and depend largely on this metabolic pathway
for generation of ATP to meet their energy needs. Under physiologic conditions, generation
of ATP through oxidative phosphorylation in the mitochondria is an efficient and preferred
metabolic process, which produces far more ATP molecules from a given amount of glucose
compared with glycolysis. However, when the ability of cells to generate ATP through
mitochondrial oxidative phosphorylation is compromised, cells are able to adapt alternative
metabolic pathways, such as increasing glycolytic activity, to maintain their energy supply.
Mitochondrial respiratory function can be negatively affected by multiple factors, including
mutations in mitochon- drial DNA (mtDNA), malfunction of the electron transport chain,
aberrant expression of enzymes involved in energy metabolism, and insufficient oxygen
available in the cellular micro-environment. It is known that mtDNA contains a displacement
loop, and the coding gene sequence for 13 important protein components of the
mitochondrial respiratory complexes without introns. Mutations in mtDNA are likely to cause
alterations of the encoded protein and compromise the respiratory chain function. Thus, the
frequent mtDNA mutations observed in a variety of human cancers are thought to contribute
to respiratory malfunction in cancer cells. The constant generation of reactive oxygen
species within the mitochondria and the increased free radical stress in cancer cells may
cause further damage to both mtDNA and the electron transport chain, thus amplifying
respiratory malfunctions and dependency on glycolysis.
Hypoxia is another important factor that contributes to the Warburg effect. The fast
growth of cancer cells and rapid expansion of the tumor mass usually outpace new vascular
generation, resulting in an insufficient blood supply to certain area of the tumor tissues. Such
a hypoxic environment within the tumor mass limits the availability of oxygen for use in
mitochondrial respiration and synthesis of ATP and forces the cancer cells to up-regulate the
glycolytic pathway as the main route of energy production. The ability of oxygen to regulate
glucose metabolism is know as the Pasteur effect and is mediated through several pathways
involving various kinases. In this case, the increased glycolytic activity in cancer cells is not
necessarily due to intrinsic mitochondrial defects but is induced by the tumor
microenvironment through a series of metabolic adaptation processes, including
preferentially increased expression of enzymes required for glycolysis.
Also, according to Gatenby et. al.(2007), the upregulation of glycolysis in cancer cells
results in increased glucose consumption, which leads to micro-environment acidosis. This
allows for the cells to develop phenotypes which make them resistant to acid-induced
toxicity, giving them a powerful growth advantage which ultimately leads to uncontrolled
proliferation.
However, therapies that involve glycolytic inhibitors have yet to yield any results in a
human trials and further research is still being carried out at the present moment. The
immune related proteins found in the Drosophila melanogaster are being further investigated
in our experiments.
Rationale and Objectives
This project explores the possibility of using Hypericum perforatum as a treatment for
cancer or a supplement for cancer therapy because while it has been most prominently used
in the treatment of depression. About 1,596,670 new cancer cases are expected to be
diagnosed in 2011 in the United States of America (Cancer Facts & Figures 2011) of which
about 571,950 are expected to die of cancer. This numbers are astonishing and while
survival rate has increased from 50% in 1975 – 1977 to 68% in 1999 – 2006, the number of
people contracting cancer has been increasing as well. Hence we have the following
objectives for our project:
For the team in Hwa Chong, we aim: 1. To determine which proteins (if any) are affected by Hypericum perforatum
2. To determine the effects of Hypericum perforatum extract on a Drosophila cell
line
For our partners in Academy of Science (Virginia): 1. To determine the effects of Hypericum perforatum extract and hypericin on a
mouse mammary cell line
2. To determine whether hypericin is the main active chemical constituent that
gives Hypericum perforatum its medicinal properties
This project would thus allow us to compare the effects of the Hypericum perforatum on the
Drosophila melanogaster and the mouse mammary cell line, both of which are able to make
cross-genetic comparisons to human diseases. Also, it allows us to compare the effects of
the extract on a vertebrate (mouse mammary) and an invertebrate (robust Drosophila) cell
line.
Materials and Methodology
Rate of Proliferation of Mouse Mammary Cell Line (AOS)
Mouse models of human breast cancer are used widely to study molecular pathways of
tumorigenesis. The overexpression of oncogenes known to be involved in human breast
cancer, such as c-Myc, HER2/Neu, and Ccnd1, with tissue-specific promoters, induces
tumors in the mammary gland of transgenic mice. Likewise, the inactivation of tumor
suppressor genes via introduction of viral oncogenes or by conditional knockout using
CreLox technology canpromote tumorigenesis also contributes to cancer. (Cristina
Montagna et al. 2003).
The procedure for our preliminary test for cancer cell binding is summarised in Figure 1.
Culture 6 flasks (25cc) CRL 2539 Mouse
Mammary Cells with 10,000 cells each
Hypericum perforatum extract (0.4% & 0.8%)
and Hypericin were added to the culture medium
and grown. 3 other flasks were kept as controls
Cells were counted every 24 hours through the
use of a microscope
A haemocytometer was used to count the cells
after reaching confluency
1 ml of sample is frozen after each trial so that
an ELISA kit measuring norepinephrine levels
can be run in the future
Fig. 1: Procedure for Rate of Proliferation of Mouse Mammary Cell Line (AOS)
Materials:
-
Liquid Hypericum perforatum extract
-
Hypericin extract
-
CRL 2539 Mouse Mammary Cell Line
-
Cell Culture Medium (10% Fetal Bovine Serum)
Independent Variable(s)
-
Constant Variables
Number of mouse
-
Temperature
mammary cells
-
Growth Medium Used
or Hypericin)
-
Volume of extract used
Concentration of extract
-
Type of cell line
Extract added in medium
(Hypericum perforatum
-
Dependent Variable(s)
-
used
Rate of Proliferation of Robust Drosophila Cell Line (HCI)
The Drosophila genes have a 77% match with the human diseases genes and through the
investigation of the effects of the Hypericum perforatum extract on the robust Drosophila cell
line, we would be able to gauge the effects it would have on human cancer cells. A
summary of the experimental procedure is found in Figure 2.
Sub culture 2 flasks of Drosophila cells into 5 flasks (25cc)
Apply varying concentrations of Hypericum perforatum extract (2.5% - 50%) to the cell
culture medium
Cell counts at 24 hour intervals with the aid of a haemocytometer
Check the rate of growth of the cells by plotting a graph showing the cell count against
time & comparing it across concentrations
Fig. 2: Procedure for Proliferation of Robust Drosophila Cell Line (HCI)
Materials:
-
Robust Drosophila Cell Line (Cell Line Serial Number?)
-
Hypericum perforatum Extract (2.5%, 5.0%, 12.5%, 50.0%)
-
Cell Culture Medium
-
Sterile Water (for control)
Independent Variable(s)
-
Concentrations of
Dependent Variable(s)
-
Hypericum perforatum
Constant Variables
Number of robust
-
Temperature
Drosophila cells
-
Growth Medium Used
-
Volume of extract
Extract
added
-
Duration of incubation
-
Age and type of cell line
Effects on Immune-Related Proteins of the Drosophila melanogaster (HCI)
There are 3 phases to this experiment:
Preparing
Drosophila Samples
SDS-PAGE
MALDI-TOF
Fig. 3: Outline of experiments for tests on Drosophila Melanogaster (HCI)
Phase 1 involves preparing the Drosophila melanogaster by feeding them the Hypericum
perforatum extract that was commercially purchased.
Materials:
-
Wild Type Drosophila melanogaster Flies
-
Hypericum perforatum Extract
-
Sterile Saline
-
Sterile Water
Independent Variable(s)
-
Concentrations of
Dependent Variable(s)
-
Immune related protein
Hypericum perforatum
which strengthens the
Extract Applied
immune system of
Constant Variables
-
Volume of extract
added
-
Drosophila
Number of Drosophila
melanogaster
-
Duration of exposure
This was done so with the following procedure:
Varying concentrations of
Hypericum perforatum
were coated on the inner
surface of the falcon tube
(Control contains only
sterile water
Flies are transferred from
original containers using
chlorofoam
Centrifuge the tubes at
10oC, 7000rpm for 10
minutes before washing
the Hypericum perforatum
extract using sterile saline
Flies are placed in the
coated falcon tubes for
24h before extraction
Flies are crushed in 200µl
of sterile saine and put on
ice to ensure all bodily
fluids are realsed into the
solution
Fig. 4: Procedure for preparing the Drosophila fly samples
Phase 2 of the experiments would be Sodium Dodecyl Sulfate Poly-Acrylamide Gel
Electrophoresis (SDS-PAGE). It is a technique used to separate proteins according to their
electrophoretic ability in order for us to identify the unique protein profiles of the flies if they
are any apparent. As suggested by the name of the procedure, a gel electrophoresis is run
with the solutions prepared from the previous phase in order for us to determine whether any
immune-related proteins of the fly were affected by the introduction of Hypericum perforatum
extract.
Materials:
-
Acrylamide Gel (5% Stacking Gel, 10% Running Gel)
-
Loading Dye
-
Protein Ladder for Electrophoresis
-
Solution of crushed Drosophila melanogaster flies
Independent Variable(s)
-
Dependent Variable(s)
-
Solution of crushed
Protein Profiles of each
Constant Variables
-
sample
Drosophila (placed in
Concentration of
Acrylamide Gel
-
varying concentrations
Volume of solution
added into each well
Hypericum Perforatum
Extract
The samples were first prepared by mixing 2 units (10 µl) of the crushed Drosophila solution
with 1 unit (5 µl) of loading dye in an eppendorf tube before placing them in a hot water bath
(50oC) for 10 minutes, following which the standard SDS-PAGE procedure was carried out
as follows:
Load first well with
10µl of protein ladder,
subsequent wells to
be filled with
prepared Drosophila
solutions
Set the
electrophoresis kit to
25mA and allow the
gel to run until the
protein column runs
to the end of the gel
Stain the gel using
Coomassie Blue & if
any significant
proteins are
identified, they will be
sent for MALDI-TOF
Fig. 5: Procedure for SDS-PAGE
M
ctrl
50%
37.5%
25%
12.5%
2.5%
2%
1.5%
1%
1
2
1
3
1
4
1
Fig. 6: SDS-PAGE Gel showing the 4 samples of unknown proteins to be tested
Phase 3 is the experimental procedure known as Matrix Assisted Laser
Desorption/Ionization – Time-Of-Flight Mass Spectrometry (MALDI-TOF). This procedure
allows for the analysis of biomolecules or large organic molecules. A co-precipitate of a UVlight absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. Most of
the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the
peptides. The ionized peptides are accelerated in an electric field and enter the flight tube.
During the flight in this tube, different peptides are separated according to their mass to
charge ratio and reach the detector at different times. In this way each molecule yields a
distinct signal. The intensity of the signal is directly related to the abundance of that specific
peptide.
The experiments were carried out in a laboratory located in Australia.
Rate of Proliferation of Robust Drosophila Cell Line in a High Glucose Content
Environment (HCI)
In order to test whether Hypericum perforatum slows down the proliferation of the
cells by retarding the rate of glycolysis by the cells, flasks with glucose added to the cell
culture medium were tested.
Materials
-
Robust Drosophila Cell Line
-
Hypericum perforatum Extract (50%)
-
10% Glucose Solution
-
Cell Culture Medium
-
Sterile Water
Independent Variable(s)
-
Presence of Hypericum
perforatum extract
-
Presence of glucose
Dependent Variable(s)
-
Constant Variables
Number of robust
-
Temperature
Drosophila cells
-
Growth Medium Used
-
Volume of
extract/glucose added
solution
-
Age and type of cell line
A procedure similar to that shown in Figure 2 was used. We have tested the rate of
proliferation of cells in high glucose content media in both the presence and the absence of
Hypericum perforatum, as well as having a control set with only sterile water and another
with only Hypericum perforatum added. Triplicates were conducted and cell counts are
collected every 24 hours.
Results & Discussion
Introduction of Hypericum perforatum Extract slows down the rate of proliferation of
Mouse Mammary Cell Line; Hypericin does not encompass the full effect of the
Hypericum perforatum (AOS)
Control
0h
24h
96h
144h
Flask 1
10000
22919
347311
4900000
Flask 2
10000
29971
386097
4300000
Flask 3
10000
24682
281492
4550000
0h
24h
96h
144h
168h
264h
Flask 1
10000
26445
42900
91088
145154
4800000
Flask 2
10000
24682
72871
124585
229190
4050000
Flask 3
10000
18218
74046
135751
257398
4350000
0h
48h
72h
120h
Flask 1
10000
23507
23507
270000
Flask 2
10000
29971
29971
280000
Flask 3
10000
28208
28208
250000
0.4% Hypericum perforatum
0.001% Hypericin
Tables 1.1 – 1.3: Showing the number of cells counted in the flasks at fixed time
intervals
After data of cell populations in the flasks were collected, graphs comparing the
control and experimental trials at the time were made to compare how the cells were
growing.
Experimental Hypericum perforatum (0.4%) Growth vs.
Conrol Growth
Number of Cells
5000000
Experimental
0.4%
Hypericum
Perforatum
Data
Control Data
4000000
3000000
2000000
1000000
0
0
100
200
Time (hrs)
Graph 1: Graph comparing 0.4% Hypericum perforatum Data and Control Data
Number of Cells
Experimental Hypericin (0.001%) Growth vs. Control
Growth
900000
800000
700000
600000
500000
400000
300000
200000
100000
0
Control Data
Experimental
Hypericin Data
0
50
100
Time (hrs)
Graph 2: Graph comparing 0.001% Hypericin Data and Control Data
Graphs 1 and 2 show the number of cells that were in each of the three control or
experimental flasks at certain times. After certain times (around 96 hours for the 0.4%
Hypericum perforatum and around 72 hours for 0.001% hypericin) it is clear that the
experimental cells are growing slower than the control cells. The difference between the cell
populations between the experimental and control flasks increase as time goes on.
Control
Experimental
Mean
25857.33
23115
Variance
13468732
18762499
Observations
3
3
Hypothesized Mean Difference
0
P(T<=t) two-tail
0.4499
Table 2.1: t-Test: Two-Sample at 24 Hours 0.4% Hypericum perforatum
Control
Mean
Experimental
338300
63272.3333
2.8E+09
311619130.3
Observations
3
3
Hypothesized Mean Difference
0
Variance
P(T<=t) two-tail
0.0134
Table 2.2: t-Test: Two-Sample at 96 Hours 0.4% Hypericum perforatum
Control
Mean
Experimental
4583333
117141.3333
9.08E+10
540252022.3
Observations
3
3
Hypothesized Mean Difference
0
Variance
P(T<=t) two-tail
0.0015
Table 2.3: t-Test: Two-Sample at 144 Hours 0.4% Hypericum perforatum
Control
Mean
Variance
Experimental
24682
27228.67
3108169
11165144
Observations
3
3
Hypothesized Mean Difference
0
P(T<=t) two-tail
0.32735
Table 2.4: t-Test: Two-Sample at 48 hours 0.001% Hypericin
Control
Experimental
Mean
86974.67
22918.67
Variance
4.86E+08
4144225
Observations
3
3
Hypothesized Mean Difference
0
P(T<=t) two-tail
0.037581
Table 2.5: t-Test: Two-Sample at 72 hours 0.001% Hypericin
Control
Mean
Variance
Experimental
810000
266666.7
6.3E+09
2.33E+08
Observations
3
3
Hypothesized Mean Difference
0
P(T<=t) two-tail
0.007296
Table 2.6: t-Test: Two-Sample at 120 hours 0.001% Hypericin
To analyze the data, t-tests were run to compare the mean number of cells at various
times between the control and experimental flasks. The purpose of this test is to determine if
there is a statistically significant difference between the cell populations at the set times,
shown in the resulting p-value. P-values of less than 0.05 demonstrate statistically
significant data at 95% confidence when used in comparing biology related data. As shown
in tables 2.2 and 2.3, p-values of less than 0.05 were observed for the control compared to
the Hypericum perforatum (0.4%) at times 96hrs (p-value 0.013) and 144hrs (p-value
0.0015). As shown in table 2.1, at 24hrs the p-value wasn’t less than 0.05 (p-value 0.45). As
shown in tables 2.5 and 2.6, p-values of less than 0.05 were also observed for the control
compared to the hypericin (0.001%) at times 72 hrs (p-value 0.037) and 120 hrs (p-value
0.007). As shown in table 2.4, 48 hours the p-value wasn’t less than 0.05 (p-value .016).
Graph 3.1: Normal Distribution Comparing the Growth of 0.4% Hypericum perforatum and
Control Cells at time 24 Hours
Graph 3.2: Normal Distribution Comparing the Growth of 0.4% Hypericum perforatum and
Control Cells at time 96 Hours
Graph 3.3: Normal Distribution Comparing the Growth of 0.4% Hypericum perforatum and
Control Cells at time 144 Hours
Graph 3.4: Normal Distribution Comparing the Growth of 0.001% Hypericin and Control Cells
at time 48 Hours
Graph 3.5: Normal Distribution Comparing the Growth of 0.001% Hypericin and Control Cells
at time 72 Hours
Graph 3.6: Normal Distribution Comparing the Growth of 0.001% Hypericin and Control Cells
at time 72 Hours
The normal distribution curves for the Hypericum perforatum vs. control at 96hrs
(graph 3.2) and 144hrs (graph 3.3) as well as hypericin vs. control at 72hrs (graph 3.5) and
120hrs (graph 3.6) provide a visual demonstration of the statistically significant difference
between the experimental and control means. Because neither of the graphs had
overlapping of either data set, the mean values of the control and experimental flasks at
times 96hrs and 144hrs for the Hypericum perforatum and at times 72 hrs and 120 hrs for
the hypericin were found to be statistically different. The normal distribution curves for the
Hypericum perforatum vs control 24 hrs (graph 3.1) and the hypericin vs control at 48 hrs
(graph 3.4) showed two data set overlapping demonstrating that there wasn’t a statistically
significant difference between the experimental and control means at these times.
Introduction of Hypericum perforatum Extract slows down the rate of proliferation of
Robust Drosophila Cell Line (HCI)
24 hours
48 hours
72 hours
Control
13267
10967
113700
2.50%
10133
12600
103275
5%
16267
15900
124800
12.50%
12167
19267
73050
50%
18467
19567
41775
Table 2.1: Comparison of cell numbers of control setup and setups whereby varying concentrations of
Hypericum perforatum were added (newer cells set 1)
24 hours
48 hours
72 hours
96 hours
Control
13050
14550
21825
12750
2.50%
13050
10800
20025
16425
5%
13050
18150
28725
17550
12.50%
13050
17100
24450
10500
50%
13050
11325
8850
13125
Table 2.2: Comparison of cell numbers of control setup and setups whereby varying concentrations of
Hypericum perforatum were added (newer cells set 2)
96 hours
120 hours
144 hours
Control
83100
73050
33375
2.50%
83100
92700
72000
5%
83100
94950
44025
12.50%
83100
87225
58125
50%
83100
90975
38850
Table 2.3: Comparison of cell numbers of control setup and setups whereby varying concentrations of
Hypericum perforatum were added (older cells)
Graph 4.1: Comparison of cell numbers of control setup and setups whereby varying concentrations
of Hypericum perforatum were added (newer cells)
Graph 4.2: Comparison of cell numbers of control setup and setups whereby varying concentrations
of Hypericum perforatum were added (older cells)
Tables 2.1 to 2.3 show the daily cell counts of our robust Drosophila cell line. These
cells were subcultured and counted from a period of 0h – 72h. As can be observed, the peak
of growth occurs between 48h to 72h and the introduction of Hypericm perforatum has
resulted in a general slow down in the proliferation of the robust Drosophila cell line. This
effect was most significant when 50% Hypericum perforatum extract was added to the cell
culture medium as seen from graph 4.1.
However, when the extract was tested on the older set of cells (cells grown from 96h
– 144h), the Hypericum perforatum had a different effect on the proliferation of the cells. The
cell numbers had started decreasing rapidly in the control flask but those flasks containing
Hypericum perforatum extract showed a decrease in cell death rate as can be observed
from graph 4.2.
Observed
Expected
Chi-square value
2.50%
61650
64706.25
2588250
5%
76762.5
72262.5
1445250
12.50%
48750
58256.25
466050
50%
25312.5
46537.5
93075
Table 3.0: Chi-square values of the results of the newer cells
To analyze the data, chi-square tests were run to compare the mean number of cells
at 72h between the control and experimental flasks for the cells grown from 0h to 96h. The
purpose of this test is to determine if there is a statistically significant difference between the
cell populations after the introduction of Hypericum perforatum and reaching peak growth
rate. A table (Table 3.0) showing the calculations, proving that the results at 48h are
statistically significant.
However, the behavior of the cells during the growth cycle from 0h – 144h suggests
that Hypericum perforatum is only suitable to be used for treatment of cancer during the
early stages rather than the late stages as it slows down rate of proliferation early on and
slows down rate of cell death later on.
Consumption of Hypericum perforatum extract by the Drosophila melanogaster
results in the inhibition of glycolytic enzymes in the Drosophila (HCI)
As shown from Figure 6, there were 4 protein bands that were present in the flies of
the control setup when compared to those of the flies that have been fed the Hypericum
perforatum for a period of 24h. The same bands were also observed to be missing when
samples were tested with a low concentration of 0.5% Hypericum perforatum extract even
though the effects were less obvious (Figure 7). This shows that Hypericum perforatum is
dosage dependent at both high and low concentrations.
0.5% ctrl
M
Fig. 7: SDS-PAGE Gel showing how at 0.5% Hypericum Perforatum still shows an
inhibition of the same 4 protein bands
The 4 bands of protein were cut out from the SDS-PAGE gel and sent for MALDITOF for identification. The results that were obtained from the analysis were as follows:
Sample
Most-Highly
Matched Protein
Sequence
Sequence
Coverage
B4IS91
1
(unknown
27%
function)
2
Enolase
10%
B3LX89
3
(unknown
26%
function)
Fructose4
bisphosphate
aldolase
31%
Table 4.0: Table showing the results obtained from the 4 unknown protein bands after
MALDI-TOF
Proteins 1 and 3 have no functions whatsoever and are possibly contaminants that
were consumed by the fly. Proteins 2 and 4 have been identified to be Enolase and Fructose
Bisphosphate Aldolase respectively. Both these enzymes have been found to be enzymes
required to facilitate the process of glycolysis. (Figure 8)
Fig. 8: Flow chart showing glycolysis and the enzymes facilitating the process (Source:
http://en.wikipedia.org/wiki/File:Glycolysis.jpg)
Glycolysis is the metabolic pathway that converts glucose into pyruvate. The free
energy released in this process is used to form the high-energy compounds ATP (adenosine
triphosphate) and NADH (reduced nicotinamide adenine dinucleotide). The absence of the 2
enzymes, Enolase and Fructose Bisphosphate Aldolase, could lead to a slow down or even
stop the process of glycolysis entirely. With a slowed rate of glycolysis, the cancer cells will
be unable to maintain their growth advantage and the rate of proliferation will also start to
decrease. In order to prove this hypothesis, we tested the effects of adding glucose to the
cell culture medium of the robust Drosophila cell line.
Addition of Hypericum perforatum and glucose to the cell culture medium causes
slow down in rate of proliferation of robust Drosophlia cells (HCI)
A
B
C
control
10462.5
9112.5
15412.5
glucose
13275
15075
15300
1237.5
10462.5
3262.5
glucose+ Hypericum
perforatum
Table 5.0: Comparison of cell numbers 24 hours after the start of experimentation
Graph 5.0: Comparison of cell numbers between the setup with glucose, the setup with both
glucose and Hypericum perforatum and the control setup
From table 5.0 and graph 5.0, we are able to see that even though it has only been a
period of 24 hours, the addition of glucose has significantly increased the proliferation of the
robust Drosophila cells. However, with the addition of 50% Hypericum perforatum extract,
the growth rate has shown to slow down and this has been proven statistically significant as
shown by a p-value of 0.0398 on the t-test.
While the results might not be conclusive at 24h, the experiment shows much
promise as Hypericum perforatum at high and regular dosages might be able to counter the
growth of cancer cells in humans if the Warburg effect is significant enough in cancer cell
growth. Further experiments will be carried out to test the effectiveness of the Hypericum
perforatum extract on robust Drosophila cells grown in a highly glucose-concentrated
environment.
Conclusion
When Hypericum perforatum and hypericin were added to the mouse mammary cell
line, the results had shown that cancer cells when exposed to the extract will grow at a
slower rate compared to the control cells. It was concluded with 95% confidence that
Hypericm perforatum and hypericin extracts did significantly slow the growth of the mouse
mammary cancer cells after times of 96hrs for the Hypericum perforatum extract and 72hrs
for the hypericin extract. With time, both extracts decreased cell growth at greater rates as
shown in the decreasing p-values over time (0.013 to 0.0015 from times 96hrs to 144hrs for
the Hypericum perforatum extract and 0.037 to 0.007 from times 72hrs to 129hrs for the
hypericin extract).
Hypericum perforatum has also shown to be able to slow down the growth rate of the
robust Drosophila cells when added in high concentrations. This effect is most obvious
during the 48h – 72h time period and it has also been proven to be statistically significant by
the chi-squared test. Following which, the effect of the Hypericum perforatum was not so
ideal when it slowed down the rate of cell death as compared to the control setup. Showing
that Hypericum perforatum is only suitable to be used for early cancer treatment.
One possible way that the Hypericum perforatum is affecting the proliferation of the
cells is by disturbing the glycolytic pathway of the cells as it denatures or inhibits the
production of 2 enzymes essential for glycolysis. Cancer cells have shown increased
glycolysis in order to produce more ATP and with Hypericum perforatum affecting this
pathway, the lack of energy could result in a higher rate of cell death or slow down in the
rate of proliferation of these cells.
Future Work
ELISA stands for enzyme-linked immunosorbent assay. The purpose of which is to
determine if a particular protein is present in a sample and if so, how much of it is present.
There are two main variations on this method: one can determine how much antibody is in a
sample, or determine how much protein is bound by an antibody. The distinction is whether
one is trying to quantify an antibody or another protein.
For the AOS side, an ELISA test will be run to determine the amount of
norepinephrine in each sample, representative of the amount of norepinephrine secreted by
the cells in each flask. The experimental samples should contain greater amounts of
norepinephrine than the control samples because Hypericum perforatum is thought to inhibit
the reuptake of norepinephrine in cells. Each sample of increasing concentrations of
Hypericum perforatum and hypericin should contain greater amounts of norepinephrine.
A SDS-PAGE gel can also be run to determine the proteins in the mouse mammary
cell line that have been inhibited or denatured by the Hypericum perforatum and further tests
by ELISA can determine how the concentration of the extract affects the amount of proteins
denatured or inhibited by the Hypericum perforatum.
For the Hwa Chong side, we aim to find out more about the physiopathology of the
Drosophila cell growth in the areas of protein kinases and the cell glycolysis pathway
through the use of an ELISA. Through this, we will be able to understand how the Hypericum
perforatum is causing a change in the growth rate of the cells and hence deduce how such
an effect can also be observed in human cells.
Also, both sides would continue to experiment on varying concentrations to
determine how the dosage and concentration of Hypericum perforatum extract would affect
the growth rates of both cell lines and ultimately determine what is the optimum for dosage
and concentration for usage as a possible clinical drug for trial.
References
Heck, D., Torshina, N., Zhang, J. (2007). Catalytic therapy of cancer with ascorbate and
extracts of medicinal herbs. eCam, 7(2), 203-212. Doi: 10.1093/ecam/nem159
Hulka, B., Moorman, P. (2008). Reprint of breast cancer: hormones and other risk factors.
Maturitas, 61(1), 203-213. Doi: 10.1016/j.maturitas.2008.11.016
Leuner, K., Kazanski, V., Muller, M., Essin, K., Henke, B., Gollasch, M., Harteneck, C.,
Muller, W., (2007). Hyperforin—a key constituent of St. John’s Wort specifically
activates TRPC6 channels. The FASEB Journal, 21, 4101-4111. Doi: 10.1096/fj.078110com
Linde, K. (2009). St. John’s Wort- an overview. Karger, 16(3), 146-155.
Doi:10.1159/000209290
Martinez-Poveda, B., Medina, M., Quesada, A. (2005). Hyperforin, a bio-active compound of
St. John’s Wort, is a new inhibitor of angiogenesis targeting several key steps of the
process. International Journal of Cancer, 117 (5), 775-780. Doi: 10.1002/ijc.21246
Pomerantz, M., Ahmadiyeh, N., Jia, Li., Herman, P., Verzi, M., Doddapaneni, H., Beckwith,
C., Chan, J., Hills, A., Davis, M., Yao, K., Kehoe, S., Lenz, H., Haiman, C., Yan, C.,
Henderson, B., Frenkel, B., Barrentina, J., Bass, A., Tabernero, J., Baselga, J.,
Regan, M., Manak, J., Shivdasani, R., Coetzee, G., Freedman, M. (2009). The 8q24
cancer risk variant rs6983267 shows long-range interaction with MYC in colorectal
cancer. Nature, 41, 882-884. Doi: 10.1038/ng.403.
Rigel, D. (2008). Cutaneous ultraviolet exposure and its relationship to the development of
skin cancer. Journal of the American Academy of Dermatology, 58(5), S129-S132.
Doi: 10.1016/j.jaad.2007.04.034
Rothenburg, M. (n.d.). Corning Life Sciences: an introduction to cell culture: an introductory
lesson on how to culture and handle mammalian cells. Retrieved December 10,2010
from http://mmsrvolbintellor.com/corning/111209/corning.html?go=Launchvideo
Sood, A., , Halder, J., Nick, A., Stone, R., Hu, W., Carroll, A., Spannuth, W., Deavers, M.,
Allen, J., Han, L., Kamat, A., Shahzad, M., McIntyre, B., Diaz-Montero, C., Jennings,
N., Lin, Y., Merritt, W., DeGeest, K., Vivas-Mejia, P., Lopez-Berestein, G., Schaller,
M., Cole, S., & Lutgendorf, S. (2010). Adrenergic modulation of focal adhesion
kinase protects human ovarian cancer cells from anoikis. The Journal of Clinical
Investigation, 120(5), 1515-1523. doi:10.1172/JCI40802
The Trustees of Princeton University. (2010). Retrieved October 12, 2010 from:
http://wordnetweb.princeton.edu/perl/webwn?s=cancer
Wang, Y., Broderick, P., Webb, E., Wu, X., Vijayakrishnan, J., Matakidou, A., Qureshi, M.,
Dong, Q., Gu, X., Chen, W., Spitz, M., Eisen, T., Amos, C., Houlstoun, R. (2008).
Common 5p15.33 and 6p21.33 varients influence lung cancer risk. Nature, 40, 14071409. Doi: 10.1038/ng.273
Weber, W., Stoep, A., McCarty, R., Weiss, N., Biederman, J., McClellan, J. (2008). A
randomized placebo controlled test for attention deficit hyperactivity disorder in
children and adolescents. Journal of the American Medical Association, 299(22),
2633-2641. Doi:10.1001/jama.299.22.2633
Karloti, A., & Billia, A. R. (2010). Hypericins as potential leads for new therapeutics. Int. J.
Mol. Sci. 2010, 11, 562-594.
Reither, L. T., Potocki, L., Chien, S., Gribskov, M., & Bier, E. (2001). A systematic analysis
of human disease-associated gene sequences in drosophila melanogaster. Genome
Res. 2001, 11, 1114-1125.
Gatenby, R. A., & Gillies, R. J. (2007). Glycolysis in cancer: a potential target for therapy. Int
J Biochem Cell Biol. 2007, 39(7-8), 1358-1366.
Xu, R., Pelicano, H., Zhou, Y., Carew, J. S., Feng, L., Bhalla, K. N., Keating, M. J. & Huang,
P. (2005). Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug
resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res
2005, 65, 612-621.
Gatenby, R. A., & Gillies, R. J. (2004). Why do cancers have high aerobic
glycolysis?. Nature Reviews Cancer, 4, 891-899.
Pressman, AH. (1998). St. john's wort - the miracle medicine. New York: The Philip Lief
Group, Inc.
American Cancer Society. Cancer Facts & Figures 2011. Atlanta: American Cancer Society;
2011.
Vogel, C., Hager, C. & Bastians, H. (2007) Mechanisms of mitotic cell death induced by
chemotherapy-mediated g2 checkpoint abrogation. Cancer Res,67, 339-345
St. John's Wort Side Effects, Interactions and Warnings . (2010). Personal health zone.
Retrieved November 4, 2010, from
http://www.personalhealthzone.com/stjohnswort.htm
Acknowledgements
We would like to thank the following people, without whom our project would not have been
successful:
-
Ms Sandra Tan, our HCI mentor, for her unwavering support and excellent
mentorship of our project
-
Ms Jackie Curley, our AOS mentor, for her guidance and support throughout the
project
-
Mdm Lim CF, lab manager of the Science Laboratories at the Science Research
Centre, for her most helpful assistance and guidance when carrying out our
experiments
-
Hwa Chong Institution, for kindly allowing us the opportunity to collaborate with
Academy of Science, Virginia on this project as well as allowing us to conduct our
experiments in the science laboratories
-
Mr George Wolfe and Mr Duke Writer, for their generous help and advice during the
preparatory phase and finalization of the project
-
Any other person who has helped us along the way in one way or another