Capstone Research Journal Article
(This manuscript fulfills the requirements for the North Carolina Agricultural and
Technical State University Department of Biology Senior Project BIOL 501)
Title of Article
A Literary Review of Stilbenoids’ Chemotherapeutic Properties
Author’s Full Name
Annie Jovita McPherson
Semester and Year
Spring 2012
Signature of Student Author: _________________________ Date: _____________
Signature of Faculty Advisor: _________________________ Date:______________
Student Demographics
My name is Annie J. McPherson, and I was raised in High Point, NC. For as long as I
can remember, I have had a passion for experiments. I would ask what would happen if I did
this, or that, and what changed. I even went as far as taking my little sister as a test subject, with
her permission. When I entered high school at The Early College at Guilford College, I loved
and excelled in my science classes. This inspired me to become a biology major at North
Carolina Agricultural and Technical State University. I knew I wanted to teach biology, but not
how to achieve this goal. As luck would have it, the summer before I entered university, I
participated in the Research Initiative for Scientific Enhancement (RISE) Pre—matriculation
program. It was here that I learned what qualification I needed to have to teach at the college
level, a Ph.D. Since then, with the guidance of the RISE program and my many mentors, I have
been preparing myself, with numerous research experiences, for this next step.
After graduation, I will be enrolling in Emory University’s Genetics and Molecular Biology
Ph.D. program. One day, I will be a university professor, sharing my love and enthusiasm of
science with young students.
2
Table of Content
Description
I.
Title page
1
II.
Student Demographics
2
III.
Table of Content
3
IV.
Abstract
4
V.
Chapters
1. Introduction
5
2. Review of Literature
7
3. Presentation of Research (Novel Stilbene-based Compound Demonstrates an
Anti-migratory Effect Against Glioblastoma Cells.)
4. Conclusion
10
15
VI.
References
17
VII.
Appendix
19
3
Abstract
Glioblastoma multiforme (GBM), the most common type of brain tumor, is highly
aggressive and malignant. The migratory nature of these tumor cells inside the many folds of the
brain leads to poor prognosis. Despite great advances in other cancer areas, GBM remains one
of the most difficult to treat because of the sensitivity of the brain and the difficulty of getting
drugs across the blood brain barrier to the tumor. Therefore, discovering natural compounds, or
chemicals that produce the same effect while reducing the negative side effects, is a growing
field. Phytochemicals, or chemicals that are found in plants, have become a popular area of
research.
One photochemical discovered to have a variety of therapeutic effects is called
Resveratrol. Resveratrol is a stilbenoid and has become known as a cancer preventative that can
cross the blood brain barrier. This review article will investigate the stilbene Resveratol’s ability
to prevent cancer formation and cancer progression. Then it will discuss a newly synthesized
Resveratrol derivative’s, known as JKS014, anti-tumor effect on brain tumor cell lines. Our
hypothesis is that, based off the structure’s similarity to Resveratrol, JKS014 will have antiproliferative and anti-migratory properties. We found, using MTT and wound healing assays,
that JKS014 strong anti-migratory potential. More research must be done to determine the
mechanism of action.
4
1. Introduction
1.1 Glioblastoma Multiforme
Glioblastoma multiforme (GBM), the most common type of brain tumor, is highly
aggressive and malignant. GBM accounts for about 52% of all primary brain tumors. While it
only appears in 2-3 individuals out of 100,000, prognosis is usually low, with patients only
surviving 3 months without treatment and about 14 months with treatment. Treatments include
surgery, radiation, and chemotherapy, but Temozolomide and Avastin are currently the only
FDA approved therapeutics for brain tumors. Despite great advances in other cancer areas,
GBM remains one of the most difficult to treat because of the sensitivity of the brain, its limited
capacity to repair itself, and the difficulty of getting drugs across the blood brain barrier to the
tumor. Common symptoms are nausea, headaches, and seizures, along with memory loss and
personality changes.
GBM is derived from the glial cells in the brain. Normally, glial cells are known as
supportive tissues because they maintain homeostasis and protect neurons in the brain. Once the
cells become cancerous, they begin dividing rapidly forming a mass. Unfortunately, the cause of
glioblastoma is still unknown and appears to be random. The group most affected by this cancer
is 45 to 70 years old males.
These cells invade the surrounding brain tissue and induce
angiogenesis. The migratory nature of these tumor cells inside a delicate organ, such as the brain,
makes complete removal of the tumor during surgery difficult. Thus, even with radiation and
chemotherapeutics, recurrence is common.
1.2 Photochemicals
Many chemotherapeutic drugs used today have harmful side effects at effective doses
levels 1. Temozolomide (TMZ), for example, targets rapidly dividing cells. While TMZ is
5
effective in causing apoptosis in tumor cells, there are other normal cells in the body that also
divide quickly, such as the lining of the stomach, skin, and hair follicles, that are also affected.
Treatments may have harsh side effects such as difficulty eating and swallowing, inability to
retaining food, and hair loss, making chemotherapy an unpleasant experience.
Therefore,
discovering natural compounds, or chemicals that produce the same effect while reducing the
negative side effects, is a growing field. Phytochemicals, chemicals that are found in plants, have
become a popular area of research. Scientists have discovered many phytochemicals that exhibit
beneficial effects in a variety of areas, such as stroke recovery, diabetes prevention, and
decreased inflammation. Some have even been found to be useful in treating and preventing
cancer. One advantage of these compounds is that therapeutic levels can be achieved through
diet, or consumption of foods. Phytochemicals can be found in a variety of fruits and vegetables.
Once discovered, synthetic derivatives are created with the intention of enhancing the abilities of
these compounds.
1.3 Resveratrol
One photochemical discovered to have therapeutic effects is called Resveratrol. It is
found in plants, specifically grape skins, blueberries, and peanuts 2. Resveratrol is a stilbenoid,
or stilbene compound, consisting of two aromatic rings attached by a styrene double bond (see
Figure 1). It exists in both cis and trans isoforms3. Stilbenes are synthesized by plants under
conditions of stress4. Resveratrol has been shown to have cardio-protective, anti-diabetic, and
life extending properties.
More importantly, Resveratrol has become known as a cancer
preventative that can cross the blood brain barrier5. This review article will investigate the
stilbene Resveratol’s ability to prevent cancer formation and cancer progression. Then it will
6
discuss a newly synthesized Resveratrol derivative’s (JKS014) anti-tumor effect on brain tumor
cell lines.
2. Review of Literature
Research shows that stilbenes have many beneficial effects both in cancer prevention and
cancer treatment. Some preventative benefits include preventing inflammation, protecting the
DNA and the cell itself. Two papers have compiled a list of known functions of Resveratrol.
One, by Bisht, et al. 3, discusses the anti-tumor formation properties of Resveratrol (see chart 1).
The second, by Weng and Yen
1
informs us of the anti-tumor progression properties of
Resveratrol (see chart 2).
2.1 Chemopreventative properties
2.1.1 Resveratrol as an antioxidant
Because the brain requires large amounts of oxygen to operate, reactive oxygen species
(ROS) occur readily. This, in combination with that fact that the brain does not have many
antioxidant compounds to combat ROS, makes it highly vulnerable to oxidative damage6.
Too
much ROS activity can lead to protein, lipid and DNA oxidation, the latter potentially causing
damage to synthesized protein function6. Improper function of proteins can lead to deregulation
of normal cellular pathways.
DNA damage that cannot be repaired has been linked to
carcinogenesis6. One study, done by Quincozes-Santos, et al. 6, looked at Resveratrol’s DNA
protective capabilities.
The oxidative assault that occurred after one hour incubation in
Resveratrol resulted in nearly complete protection of DNA from H2O26. Quincozes-Santos, et al.
7
6
also mentioned that at concentrations greater than 250µM, for periods longer than 12 hours,
Resveratrol itself causes DNA damage.
2.1.2 Resveratrol as an anti-inflammatory agent
While inflammation in the body is normal, chronic inflammation may cause cancer. NFκB, or nuclear factor-kappa B, has been implicated in regulating inflammatory responses in cells.
It also has functions in the cellular proliferation and cell adhesion pathways.
In normal
conditions, it is inhibited by IκB and held in the cytoplasm in an inactive state. After stimulation
requiring inflammatory response such as mitogens, ultraviolet irradiation, and viral proteins, IκB
becomes phosphorylated and then degraded. This allows NF-κB to enter the nucleus where it
has access to cytokines, cell adhesion molecules and growth factors7. It is this pathway that can
have carcinogenic effects on cells. One of NF-κB’s target genes is Cox-2.
COX-2 upregulation may play a role in the development of cancer, specifically, prolongs
survival, promotes angiogenesis, and leads to changes that enhance metastatic and invasive
potential7. By targeting COX-2, proliferation can be reduced and apoptosis induced in certain
cell lines. Targeting upstream NF-κB and inhibiting its function could also be a strong candidate
for therapeutic drugs. Resveratrol has been shown to have antioxidative and anti-inflammatory
effects in some cell lines.
2.2 Chemotherapeutic properties
2.2.1 Resveratrol as a cell cycle inhibitor
Unregulated cell cycle progression is a hallmark of cancer. Unchecked, tumor cells can
grow to form large masses. Even after removal of over 98% of the tumor, the left over tumor
cells can still grow into another mass.
Therefore, preventing proliferation in GBM is
8
fundamental. Gao, et al.
8
found that Resveratrol prevents proliferation in GBM cells through
histone modifications. This occurs by preventing the ubiquitination, or the signal for destruction,
of histone H2B that helps hold the DNA tightly wound. Without the release of H2B, DNA
polymerase cannot access the DNA, preventing cells from exiting G1 and entering S phase,
ultimately halting the cell cycle in G0.
Another study shows that Resveratrol induces dose dependent inhibition of the cell cycle
at S phase in GBM9. Leone, et al. 9 reported that cells accumulate in the S phase at doses under
80 µM of Resveratrol. Leone, et al. 9 believe that Resveratrol can also inhibit topoisomerase IIa,
which is found at replication forks, and is necessary for survival of growing cells. They also
found that Resveratrol induces DNA double stranded breaks, leading to expression H2AX
phosphorylation9. This process is not well understood. H2AX are proteins that hold and repair
double stranded breaks. This is known as a TOPO poison, and kills the cells by eliminating the
activities of topoisomerase.
Gagliano, et al. 5 also reported that, especially after 72 hours of Resveratrol treatment, in
a dose dependent manner, there is reduction in proliferation.
2.2.2 Resveratrol as an anti-migratory agent
Matrix metalloproteinases (MMPs), a family of zinc dependent proteases, break down
extracellular matrix components. While not completely understood, increased activity of MMPs
has been correlated with invasiveness of brain tumors5. This is especially true with MMP-2,
which is capable of breaking down collagen type IV and laminin5. Another protein that may
play a role in invasion of surrounding tissue is Secreted Protein Acidic and Rich in Cysteine
9
(SPARC). SPARC having anti-adhesive properties, may help the tumor cells survive stressful
conditions of the brain, and activates MMP-25.
Gagliano, et al. 5 found that at both 48 hours and 72 hours of Resveratrol treatment there
is a reduction in MMP-2 and SPARC levels. A reduction in SPARC levels is also seen at 24
hours. This was achieved at 1 µM, the concentration that was found in mouse plasma after being
administered red wine.
2.2.3 Resveratrol in combination therapy
Although highly effective at killing other tumor cell types, Temozolomide (TMZ) is not
effective at killing GBM. TMZ alone only reduces GBM viability by about 50%10. Lin, et al. 10
showed that the addition of Resveratrol to TMZ enhances the receptivity of GBM cells to TMZ,
causing apoptosis at increased rates in a concentration dependent manner. They believe that this
occurs because reactive oxygen species (ROS) generation signals for the cell to begin autophagy.
Autophagy, or degrading and reusing organelles and proteins found outside of the cell, is a
process that inhibits cell apoptosis. By experimenting with TMZ with the addition of low
quantities of Resveratrol Lin, et al.
10
showed that the occurrence of autophagy was decreased,
therefore, increasing the amount of apoptosis. By blocking ROS and its downstream effects,
which Lin, et al.
10
found Resveratrol to do, they showed that apoptosis is increased, decreasing
cell viability after TMZ treatment in GBM.
3. Presentation of research
3.1 Novel Stilbene Derivative’s Anti-tumor effects in Glioblastoma Multiforme
Since developing new ways to target glioblastoma is such a necessity, our study looked at
the anti-tumor effects of a series of newly synthesized aromatic stilbene-based compounds
10
(JKS001 – JKS014). These compounds are Resveratrol derivatives and retain the stilbene
backbone. The differences are that the side chains have been altered to chemical groups that
have exhibited an enhanced effect in cancer prevention. We hypothesized that these compounds
(4 µM – 400 µM) would be able to prevent both GBM cell proliferation and/or migration based
on results from compounds synthesized with similar chemical properties and structures. None of
these compounds were able to decrease GBM cell proliferation, but one compound, known as
JKS014 (2-[trans-2-(4-bromophenyl)-vinyl]-3-nitrobenzoic acid), was able to successfully
decrease cell migration. JKS014 is a nitrostyrene compound, with two benzene rings bounded to
a double bonded carbon just as Resveratrol (see figure2).
The difference is the side chains, most notably the bromine and nitrite groups. These
groups were added because of their strong anti-tumor properties seen in other compounds. We
did different tests, specifically, wound healing assays, and MTT assays, in an attempt to
elucidate the function of this compound.
3.2 Materials
Materials for these experiments included two cell lines, A172wt and U251wk, cultured in
DMEM and 10% fetal bovine serum, and the JKS014 compound, obtained from Dr. Franks in
the North Carolina A&T State University Chemistry Department. We also used MTT assay and
western blotting kits, along with various immunoproteins. We used DMSO as a vehicle control,
and various known cell cycle inhibiting compounds.
3.3 Methods
3.3.1 MTT assay
11
Using MTT assays, we tested the potential anti-proliferative effect of JKS014 (see charts
3 and 4). JKS014 was tested on two cell lines, A172wt and U251wk, at concentrations of 4 µM,
40 µM, and 400 µM. Cells were plated at 10,000 cells into 96 well plates, with four wells per
treatment, except minus and plus serums which had six wells each. The next morning, once the
cells had adhered to the plate, cells were washed three times with minus serum to remove any
growth factor from the plus serum the cells had been plated in. Treatments of minus serum, plus
serum, DMSO vehicle control, and the three concentrations of JKS014 were applied for 48 hours.
Three other treatments of known cell cycle inhibitors LY, UO, and a combination of LY and UO,
remained in minus serum until 14 to 16 hours before MTT treatment began. On the second day
of treatment, 10 µL of MTT was added to each well for four hours. Then 100 µL of Solution C,
was added to remove the coloring in the wells. After an hour to an hour and a half, the 96 well
plate was read, measuring absorbance at 562 nm. High absorbance meant that proliferation rates
were high, and low meant that little proliferation was occurring.
Averages and standard
deviations were taken and graphed.
3.3.2 Wound healing assay
To measure the anti-migratory capability of JKS014, we did wound healing, or scratch
test assays. Again, in two cell lines, A172wt and U251wk, we plated a 24 well plate with cells at
about 100,000 cells per well. The next morning, or once the cells had the opportunity to adhere,
the cells were washed twice with minus serum to remove the growth factor they had been plated
with from the wells. Then a 200 µl pipet tip was scratched down the monolayer of tissue cells.
A third wash was conducted to remove loose cells from the wells. Treatments were placed,
minus serum, plus serum, DMSO control, and JKS014 at 400 µM. Pictures were taken at 0
hours, 16-18 hours, and 24 hours. The distance of the gap was measured three times per well at
12
both 0 and 24 hours. Then the average was taken, and the final value was divided by the initial
value and multiplied by 100. This number was then subtracted from 100% and showed the
percent closure of each treatment. A high percent closure indicated increased migration, while
low percent closure indicated little migration.
In U2251wk, we tested the wound healing capabilities at different concentrations. All
steps above were identical except that the treatments were minus serum, plus serum, and two
different concentrations of JKS014, 40 µM and 400 µM.
3.3.3 Western blotting
After treatment for 24 hours, cells were lysed in an attempt to determine the migratory
pathway JKS014 interacted with. Treatments included, minus serum, minus serum with DMSO,
and minus serum with JKS014, and in plus serum, plus serum, plus serum with DMSO, and plus
serum with JKS014. Thus there were six treatments. The lysates were pipetted into a 10% to
12% gel at about 25 µg/µl, and ran for 45 minutes to an hour. The proteins in the gel were then
transferred over to a PVDF membrane for an hour and ten minutes. The membrane was then
blocked in 5% milk, and immunoprotein treatments were left on overnight. Treatments included
AKT, MAPK, and cofilin, all common proteins cells use in migration. The next morning, the
membrane was washed three times with PBS tween for ten minutes each, and secondary
treatments were placed, which were anti-mouse and anti-rabbit antibodies. This treatment lasted
an hour. Then PBS tween was again used to wash off the treatment, three times for ten minutes.
A substrate was used to activate the fluorescent particle, and x-ray film was used to determine
expression levels.
Dark bands indicate increased amounts of protein expression, but not
necessarily protein activation.
13
3.4 Results
Our MTT assay showed that there was no inhibition of cell proliferation (see graphs 1
and 2). At the highest concentration, 400 µM, we observed apoptosis, indicating that JKS014 is
cytotoxic to the cells at this concentration. In contrast, we saw that JKS014 does have antimigratory capabilities at 400µM (see graphs 3 and 4). In U251wk cell lines, at 40µM and
400µM, the inhibition of migration occurs in a concentration dependent manner (see graph 5).
Thus far western blotting has shown no difference in expression levels of proteins involved in
the AKT or MAPK pathways. Neither is there a change in expression levels of cofilin or actin.
3.5 Discussion
After discovering that JKS014 strongly knocked down migration, we did western blots in
an attempted to determine the mechanism it affected. We tested common known migration
pathways such as the AKT, MAPK, pathways, but found no significant change in expression
levels. We also looked at actin and cofilin protein levels, but found no change.
Since the levels of expression levels did not appear to be altered, we thought JKS014 may
be altering the localization of certain proteins involved directly in movement, such as actin and
cofilin. Actin is the microtubule unit that connects with itself to form long chains. These chains
push against the cytoplasm, causing it to extend in one area. Eventually, the rest of the cell
follows the projection and movement is achieved. Cofilin is a protein that helps actin bind to
itself and create the chains. Logically, these proteins are found at the site of migration, or the
front of migration.
If JKS014 was indeed altering where these proteins were located or
inhibiting the formation of actin, we expected to see individual actin subunits or these proteins in
other parts of the cell, instead of around the edge of the cytoplasm. Using immunoflorescence,
14
we determined that under JKS014 conditions, there seemed to be no change in the location of
either actin or cofilin. JKS014 has great potential when used in combination therapy. It could be
considered along with an anti-proliferative compound. Future studies are still needed.
3.6 Conclusion
Even with all the advances in other types of cancers, treating glioblastoma multiforme
continues to be a difficult task. This is because of the delicate nature of the brain, the very little
ability to repair damages and because of its selective protective barrier, preventing drugs from
reaching their targets. After the surgical removal of the majority of a brain tumor, cells that are
left behind seem to become even more aggressive and invasive.
Phytochemicals are becoming more important in the fight against cancer. One such is
Resveratrol.
Resveratrol has many anti-tumor properties including anti-inflammation,
antioxidative, anti-proliferation and anti-migration.
Derivatives of Resveratrol are being
synthesized to enhance these properties.
Thus, a compound that can prevent migration is highly desirable, even if it cannot also
inhibit growth. JKS014, a newly synthesized stilbene derivative, has this capability to inhibit
migration. Potentially, it could be a useful tool in treating cancer, especially when used in
combination with a compound that blocks proliferation.
More testing should be done to
determine the full extent of JKS014’s chemopreventative properties.
The full chemopreventitive capability of JKS014 is not well understood, but research
involving similar compounds indicates many potential beneficial effects of this compound.
These may include anti-inflammatiory, cytoprotective, and DNA protective mechanisms useful
15
in preventing cancer. Also, JKS014 has strong potential to have anti-migration, anti-invasion,
anti-angiogenic, and anti-adhesion properties that would be beneficial in treating cancer.
1-20
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Appendix
Figure 1. Depicts the compound Resveratrol.
Figure 2. Depicts the compound JKS014.
19
Chart 1. Depicts the compound Resveratrol’s cancer preventing properties as documented by
Bisht, et al. 3.
20
Chart 1. Depicts the compound Resveratrol’s anti-cancer progression properties as
documented by Weng and Yen 1.
Graph 1. Depicts a MTT assay in A172wk cell lines. Treatments are lane one: minus serum, lane
two: plus serum, lane three: DMSO, lane four: JKS014 400 µM, lane five: JKS014 40 µM, lane six:
JKS014 4 µM, lane eight: UO, lane nine: LY.
21
Graph 2. Depicts a MTT assay in U251wk cell lines. Treatments are lane one: minus serum, lane
two: plus serum, lane three: DMSO, lane four: JKS014 400 µM, lane five: JKS014 40 µM, lane six:
JKS014 4 µM, lane eight: UO, lane nine: LY.
22
Graph 3. Depicts a wound healing assay in A172wt cell lines. Treatments are lane one: plus
serum, lane two: minus serum, lane three: JKS014 400 µM.
Graph 4. Depicts a wound healing assay in U251wk cell lines. Treatments are lane one: plus
serum, lane two: minus serum, lane three: JKS014 400 µM.
23
Graph 5. Depicts a wound healing assay in U251wk cell lines. Treatments are lane one: plus
serum, lane two: minus serum, lane three: JKS014 40 µM, lane four: JKS014 400 µM.
24