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’ Chemopreventative Effects
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, 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.
Table of Content
Description
I.
Title page
II.
Student Demographics
i
III.
Table of Content
ii
IV.
Abstract
1
V.
Chapters
1. Introduction
2
2. Review of Literature
4
3. Presentation of Research (Novel Stilbene-based Compound Demonstrates an
Anti-migratory Effect Against Glioblastoma Cells.)
4. Conclusion
14
VI.
References
16
VII.
Appendix A
Abstract
Glioblastoma multiforme (GBM), the most common type of brain tumor, is highly aggressive
and malignant. The migratory nature of these tumor cells inside a delicate organ such as 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, its limited capacity to repair
itself, 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. It is found in plants, specifically grape skins,
blueberries, and peanuts1. Resveratrol is a stilbenoid and has become known as a cancer
preventative that can cross the blood brain barrier2. 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 anti-tumor effect on brain tumor cell lines.
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 month 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.
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. 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. 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 3. Temozolomide (TMZ), for example, targets rapidly dividing cells. While TMZ is
effective in cause 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 cells, 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, or 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
decrease inflammation. Some have even been found to be useful in treating and preventing
cancer. One advantage to some of these compounds is that therapeutic levels can be achieved
through diet, or consumption of foods. These 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 1. 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 isoforms4. Stilbenes are synthesized by plants under
conditions of stress5.
It 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 barrier2.
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 anti-tumor effect on brain tumor cell lines.
2. Review of Literature
Cell cycle inhibition
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 cells can
still grow into another mass. Therefore preventing proliferation in GBM is fundamental. Gao, et
al.
6
found that Resveratrol prevents proliferation in GBM cells through histone modifications.
This occurs by preventing the ubiquitination, or 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 GBM7. Leone, et al. 7 reported that cells accumulated in the S phase at doses under
80 µM of Resveratrol. They also found that Resveratrol induces DNA double stranded breaks,
leading to expression H2AX phosphorylation7. H2AX are proteins that hold and repair double
stranded breaks. Leone, et al. 7 believe that Resveratrol can also inhibit topoisomerase IIa, which
is found at replication forks, and is necessary for survival of growing cells. This is known as a
TOPO poison, and kills the cells by eliminating the activities of topoisomerase.
Gagliano, et al. 2 also reported that, especially after 72 hours of Resveratrol treatment, in
a dose dependent manner, there is reduction in proliferation.
Resveratrol as an antioxidant
Because the brain requires large amounts of oxygen to operate, reactive oxygen species
(ROS) occur readily. In combination with that fact that the brain does not have many antioxidant
compounds to combat ROS, it is highly vulnerable to oxidative damage8.
Too much ROS
activity can lead to protein, lipid and DNA oxidation, the latter causing damage to synthesized
protein function8. DNA damage that cannot be repaired has been linked to carcinogenesis 8. One
study, done by Quincozes-Santos, et al. 8, looked at Resveratrol’s DNA protective capabilities.
They oxidative assault that occurred after one hour incubation in Resveratrol resulted in nearly
complete protection of DNA from H2O28. Quincozes-Santos, et al.
8
also mentioned that at
concentrations greater than 250µM, for periods longer than 12 hours, Resveratrol itself causes
DNA damage.
Resveratrol as an anti-inflammatory drug
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 factors9. 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
potential9. 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.
Resveratrol decreases MMP-2 and SPARC protein levels
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 tumors2. This is especially true with MMP-2,
which is capable of breaking down collagen type IV and laminin2. Another protein that may
play a role in invasion of surrounding tissue is Secreted Protein Acidic and Rich in Cysteine
(SPARC). SPARC has anti-adhesive properties, may help the tumor cells survive stressful
conditions of the brain, and activates MMP-22. Gagliano, et al. 2 found that at both 48 hours and
72 hours of Resveratrol treatment there is a reduction in MMP-2 and a reduction of SPARC
levels also seen at 24 hours. This was achieved at 1 µM, the concentration that was found in
mouse plasma after being administered red wine.
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 way. 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. 10showed that the occurrence of autophagy was decreased, therefore, increasing the amount of
apoptosis. They believe that this occurs because reactive oxygen species (ROS) generation
signals for the cell to begin autophagy. By blocking ROS and its downstream effects, which Lin,
et al. 10found Resveratrol to do, they believe that apoptosis is increased in GBM.
Novel Stilbene Derivative’s Anti-tumor effects in Glioblastoma
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
(JKS001 – JKS014). These compounds are Resveratrol derivatives and retain the stilbene
backbone. The difference is 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 figure______). 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.
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 from
the North Carolina A&T State University Chemistry Department. We also used MTT assay and
western blotting kits, along with various immunoproteins. We also used DMSO as a vehicle
control, and various known cell cycle inhibiting compounds.
Methods
MTT assay
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. 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 were 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 litter proliferation was occurring.
Averages and standard
deviations were taken and graphed.
Wound healing assay
To measure the anti-migratory capability of JKS014 we did a wound healing, or scratch
test assay. Again, in two cell lines, A172wt and U251wk, we plated a 24well 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
both 0 and 24 hours. Then the average was taken, and the 24 hour value was divided by the 0
hour value and multiplied by 100. This number was then subtracted from 100% and showed the
percent closure of each treatment.
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.
Western blotting
After treatment for 24 hours, cells were lysed in an attempt to determine the migratory
pathway JKS014 interacted with. Treatments included, in minus serum, minus serum, DMSO,
and JKS014, and in plus serum, Plus serum, DMSO, and 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.
Results
Our MTT assay showed that there was no inhibition of cell proliferation. 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 anti-migratory capabilities at both
40µM and 400µM. In U251wk cell lines, the inhibition of migration occurs in a concentration
dependent manner. 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.
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,
we determined that under JKS014 conditions, there seemed to be no change in the location of
either actin or cofilin.
Conclusion
Even with all the advances in other types of cancers, treating glioblastoma multiforme
continues to be a difficult task. This is because the brain is a sensitive organ with 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. 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.
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
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.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Goswami SK, Das DK. Resveratrol and chemoprevention. Cancer Letters 2009;284(1):16.
Gagliano N, Moscheni C, Torri C, Magnani I, Bertelli AA, Gioia M. Effect of resveratrol
on matrix metalloproteinase-2 (MMP-2) and Secreted Protein Acidic and Rich in
Cysteine (SPARC) on human cultured glioblastoma cells. Biomedicine &
Pharmacotherapy 2005;59(7):359-364.
Weng C-J, Yen G-C. Chemopreventive effects of dietary phytochemicals against cancer
invasion and metastasis: Phenolic acids, monophenol, polyphenol, and their derivatives.
Cancer Treatment Reviews 2012;38(1):76-87.
Bisht K, Wagner K-H, Bulmer AC. Curcumin, resveratrol and flavonoids as antiinflammatory, cyto- and DNA-protective dietary compounds. Toxicology
2010;278(1):88-100.
Goodwin PH, Hsiang T, Erickson L. A comparison of stilbene and chalcone synthases
including a new stilbene synthase gene from Vitis riparia cv. Gloire de Montpellier. Plant
Science 2000;151(1):1-8.
Gao Z, Xu MS, Barnett TL, Xu CW. Resveratrol induces cellular senescence with
attenuated mono-ubiquitination of histone H2B in glioma cells. Biochemical and
Biophysical Research Communications 2011;407(2):271-276.
Leone S, Cornetta T, Basso E, Cozzi R. Resveratrol induces DNA double-strand breaks
through human topoisomerase II interaction. Cancer Letters 2010;295(2):167-172.
Quincozes-Santos A, Andreazza AC, Nardin P, Funchal C, Gonçalves C-A, Gottfried C.
Resveratrol attenuates oxidative-induced DNA damage in C6 Glioma cells.
NeuroToxicology 2007;28(4):886-891.
Surh Y-J, Chun K-S, Cha H-H, Han SS, Keum Y-S, Park K-K, Lee SS. Molecular
mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals:
down-regulation of COX-2 and iNOS through suppression of NF-κB activation. Mutation
Research/Fundamental and Molecular Mechanisms of Mutagenesis 2001;480–
481(0):243-268.
Lin C-J, Lee C-C, Shih Y-L, Lin T-Y, Wang S-H, Lin Y-F, Shih C-M. Resveratrol
enhances the therapeutic effect of temozolomide against malignant glioma in vitro and in
vivo by inhibiting autophagy. Free Radical Biology and Medicine 2012;52(2):377-391.