1
The effect of resveratrol on ultraviolet lightinduced skin cell death
George Grady
This thesis is submitted in partial fulfillment
of the requirements of the Research Honors Program
in the Department of Chemistry
Marietta College
Marietta, Ohio
April 25, 2013
2
This Research Honors thesis has been approved for
the Department of Chemistry and
the Honors and Investigative Studies Committee by
____________________________________________________
Faculty thesis advisor
_____________
Date
____________________________________________________
Thesis committee member
_____________
Date
____________________________________________________
Thesis committee member
_____________
Date
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Table of Contents
Item
Page Number
Glossary
4
Abstract
5
Introduction
6-16
Methods & Materials
17-20
Results
21-24
Discussion
25-28
References
29-30
Appendix
31-33
4
Glossary
M624 cells------------------------------------human melanoma skin cells
HaCaT cells-----------------------------------human keratinocyte skin cells
p53---------------------------------------------human tumor suppressor protein
MAPK------------------------------------------protein kinases that regulate cell activities
TGF-β2----------------------------------------secreted cytokine that regulate growth
Survivin---------------------------------------inhibitors of caspase’s and apoptosis
JAK/STAT-------------------------------------proteins that transmit signals into gene regulations
Cytochrome c--------------------------------component of mitochondria and apoptosis pathways
NF-Кβ------------------------------------------protein complex that regulates transcription of DNA
Fas death receptor--------------------------receptor on membrane that leads to apoptosis
TNF--------------------------------------------family of cytokines that can cause apoptosis
Lipid rafts------------------------------------group of lipids in membrane that recruit proteins
Caspase cascade----------------------------proteins that when activated lead to apoptosis
PARP------------------------------------------nuclear protein and an indicator of apoptosis
Bax/Bcl-2------------------------------------pro/anti-apoptotic proteins that compete
Apoptosis-inducing factor---------------intrinsic regulator of apoptosis and caspase cascade
SMAC/Diablo--------------------------------mitochondrial protein involved in apoptosis
EndoG-----------------------------------------mitochondrial enzyme that can cleave DNA
5
Abstract:
Antioxidants are suspected to have anti-cancer and anti-tumor effects due to their
capability to exhaust harmful free radicals in the body. The effects of resveratrol, a naturally
occurring antioxidant, on ultraviolet light-induced skin cell death have only begun to be
investigated. Skin cancer is one of the most common types of cancer prevalent in today’s
society; most cases of skin cancer are caused by overexposure to ultraviolet light irradiation
emitted from the sun. Results show that resveratrol alone had no significant effect on M624
melanoma skin cell and HaCaT normal skin cell viability or cell proliferation, but upon
resveratrol and UVB treatment there is a significant decrease in cell viability in M624 cells.
Upon starvation of M624 cells, resveratrol was shown to help the cells survive and proliferate.
Western blot analysis of PARP and caspase-8 show that resveratrol alone and resveratrol with
UVB irradiation induces apoptosis in M624 cells. Resveratrol should be continued to be
investigated and other therapeutic agents should be tested in the presence of ultraviolet light
due to potential amplified toxicity.
6
Introduction:
Recent research has shown that antioxidants have anti-cancer and anti-tumor effects
due to their ability to exhaust free radicals, which are harmful to the body. Investigation of
therapeutics used to treat and prevent skin cancer is an important area of research because
skin cancer is one of the most prevalent cancers in today’s society. There are three types of skin
cancer: basal cell carcinoma, squamous cell carcinoma, and melanoma. Melanoma is the
deadliest form of skin cancer, and because of this, human melanoma (M624) skin cells are one
of the cell lines under investigation. The sun produces ultraviolet (UV) rays which are high in
energy and capable of some beneficial effects such as vitamin D synthesis but also can cause
much harm to our bodies. There are three main classes of UV rays: UVA, UVB, and UVC. UVA
rays have wavelengths of 320-400 nm, which are the lowest in energy and capable of
penetrating the Earth’s ozone layer. UVA also has the ability to penetrate the skin deep into the
Figure 1. UV light spectra. UV rays have shorter wavelengths than visible light rays, which
makes them higher in energy and give them potential to do damage to our skin.26
7
dermis layer and cause repairable DNA damage. UVB has wavelengths of 290-320 nm, which
are higher in energy and only partially able to penetrate the Earth’s ozone layer. UVB only
penetrates into the epidermis, the outermost skin layer, but causes non-repairable DNA
damage which can lead to skin cell death or cancer. UVC has wavelengths of 100-290 nm, which
are the highest in energy but unable to penetrate Earth’s ozone layer. An overview of the
ultraviolet light spectra can be found in Figure 1. For this investigation, UVB light was used due
to its properties which enable UVB irradiation to cause skin cell death or cancer formation.
According to the World Cancer Report in 2004, skin cancer made up 30% of all newly diagnosed
cancers and UV irradiation causes approximately 90% of skin cancers.1
There are two types of cell death, programmed healthy cell death, apoptosis, or
uncontrolled unhealthy cell death, necrosis. Apoptosis was first introduced by Kerr et al. and
the term has been used to describe cells that change morphology and undergo alterations in
which components, usually confined to the interior of the cell, are expressed on the outer
surface.2,3 Also, during apoptosis the nucleus undergoes changes in which the DNA and
chromatin become condensed, are cleaved into fragments, and packaged into compartments to
be phagocytosed by phagocytes or neighboring cells.4,5 These cells undergoing apoptosis are
sequestered and phagocytosed quickly enough to prevent content spilling and inflammation
occurrence and hence healthy cell death.5 Necrosis on the other hand, is a rapid lysis of cells
that causes much inflammation and damage to the surrounding area.5 This is very unhealthy
and may cause damage to other, neighboring healthy cells due to nearby cell rupturing and
release of intracellular contents. An overview of these two cell death pathways can be seen in
Figure 2. When investigating therapeutic potential of compounds, compounds that attack
8
Figure 2. Apoptosis versus Necrosis. Apoptosis (right pathway) is programmed healthy cell death,
whereas necrosis (left pathway) is uncontrolled unhealthy cell death that causes damage.27
cancerous cells and kill them through an apoptotic pathway, instead of a necrotic pathway, are
very beneficial and the ones that retain our focus in the laboratory.
The antioxidant investigated, resveratrol (shown in Figure 3), is a naturally occurring
derivative of stilbene found in grapes, berries, peanuts, and red wine with a high reducing
capacity and ability to chelate metal ions.6,7 The compound emerged in cancer research
Figure 3. Resveratrol (Left) vs. Stilbene (Right)
9
because it was the compound thought to be responsible for the “French Paradox” in which red
wine, which is relatively high in resveratrol content, acted as a chemotherapeutic agent and
aided people of France to have relatively low risk of coronary disease.1 Resveratrol belongs to a
group of naturally occurring plant products termed, polyphenolic phytoalexins, which may
have significant photoprotective effects on the skin preventing melanoma and non-melanoma
cancer formation by relieving oxidative stress, dysregulation of cell signaling, and DNA damage
caused by UVB light.7 Resveratrol has been most widely known for its cardioprotective and
antiaging effects, but has also been shown to have both chemopreventative and therapeutic
effects on melanoma carcinogenesis along with low toxicity and limited side effects in the
body.8,17
Numerous studies have been conducted using the phytoalexin, resveratrol, on a variety
Figure 4. Overview of extrinsic, intrinsic, and cell survival pathways. Many of the pathways
altered, or affected, by resveratrol can be seen here.28
10
of different cancerous cell lines and animal systems to determine, or research, the different
pathways and effects it may have on the cells. Figure 4 shows some of the pathways currently
being researched that resveratrol may act upon in cancer cells: extrinsic, intrinsic, and cell
survival pathways. George et al. has shown that resveratrol induces apoptosis in mouse skin
tumors via cell cycle arrest, activation of tumor suppressor protein p53 activity, and alteration
of apoptosis-related proteins such as the inhibition of MAPKs cell signaling proteins.9 Another
recent study looked into resveratrol’s effect on the tumor transforming growth factor (TGF-β2)
in mouse tumors, which was found to be downregulated along with other related regulatory
proteins upon resveratrol treatment when compared to the control.10 Resveratrol has been
shown to possibly slow tumor development by the downregulation of various cell cycle proteins
and increase apoptotic pathway signaling to combat tumors and tumor formation.6 It had been
suggested that resveratrol had significantly reduced tumor incidence and delayed the onset of
tumorigenesis in UVB irradiated mice by downregulating the Survivin protein and mRNA levels,
which are highly overexpressed in cancers.1 These studies demonstrate how resveratrol may act
through a variety of different pathways and affect a wide variety of cell components to induce
apoptotic cell death. Other in vivo studies have been conducted using resveratrol for rodent
trials and preclinical trials which will be discussed later.
Some specific apoptotic pathways and proteins have been studied to investigate the
effects of resveratrol. One recent study looked specifically into the apoptotic JAK/STAT pathway
to illustrate the role of mitochondrial membrane potential transition and release of cytochrome
c in resveratrol-induced apoptosis in human epidermoid cells (A431), which can be seen in
Figure 5. It was found that resveratrol had regulated and possibly activated apoptosis via the
11
Figure 5. Proposed mechanism of action by resveratrol in A431 cells (Madan et al., 2008). Figure
shows overview of some of resveratrol’s anti-proliferative and apoptotic effects that contains
part of caspase cascade and PARP proteins which are under investigation in this study.11
caspase cascade activation along with PARP cleavage which are indicators of apoptosis.11
Another study, also on A431 cells, investigated the combinational treatment of UVB light and
resveratrol on the nuclear factor-kappaβ (NF-Кβ), STAT, and survivin proteins. Preeti et al.
found that the combinational treatment inactivated NF-Кβ and survivin, a cell survival protein,
and STAT1, a nuclear translocator and gene regulator.12 Other in vitro studies on a variety of
cell lines have found that resveratrol induces apoptosis through a p53-dependent apoptotic
pathway, protects normal cells from UVA light oxidative stress by downregulating inhibitors of
antioxidant enzyme gene regulators, and by causing cell cycle arrest in S-phase prior to DNA
fragmentation independently of the Fas or TNF pathways.13-15 Most previous studies
12
investigated the levels of the Fas death receptor, but another study investigated the clustering
of these Fas death receptors in lipid rafts to induce the death-inducing signaling complex.16
Researchers found that resveratrol does not affect the expression or amounts of the Fas death
receptor, but rather that resveratrol induced the clustering of the Fas death receptor into the
lipid rafts composed of cholesterol and sphingolipids along with FADD and pro-caspase-8, and
other apoptotic proteins on the surface of the cell membrane, and enabled apoptotic cell death
initiation.16
Much of the focus in recent cancer research has been on the role of the mitochondria in
cancerous cells because of the Warburg effect, and the effects that resveratrol has on the
mitochondria. Over 80 years ago, Otto Warburg, a biochemist, described how cancer cells
consume increasing amounts of glucose, which were used for fermentation rather than
oxidative phosphorylation; this became known as the Warburg effect. Madan et al. found that
resveratrol had stimulated reactive oxygen species (ROS) generation, mitochondrial membrane
depolarization, and altered Bax/Bcl-2 levels which created cytochrome c release, and led to the
caspase cascade and PARP cleavage.11 Another study focused on the intrinsic pathway of
apoptosis through the mitochondria in human retinoblastoma cells. They have shown that
resveratrol creates mitochondrial membrane depolarization possibly through a target protein in
the mitochondria due to the fact that resveratrol also depolarized the membrane in a cell-free
system at low concentration.17 It was also shown that the depolarization and release of
cytochrome c had preceded the activation of the caspase-9 and caspase-3 along with the
release of the apoptotic-factor, SMAC/Diablo, and EndoG proteins into the cytosol.17 Boyer et
al. investigated the effect on mitochondria when normal human keratinocytes (HaCaT) were
13
treated with resveratrol and UVA light. They found that the combination of UVA and resveratrol
caused oxidative stress on the mitochondria, caused depolarization of the membrane, and had
actually created a pore, or opening, in the mitochondrial membrane leading to apoptosis. 18
Although there has been much controversy lately over the scandal at the University of
Connecticut where the director of the cardiovascular research center, Dr. Dipak K. Das, had
allegedly falsified about 145 pieces of data on resveratrol, the compound has still been, and will
be used, due to the overwhelming data gathered detailing resveratrol’s protective and
therapeutic effects coming from other researchers throughout the world. Resveratrol studies
still show great promise and possible use as a therapeutic agent against diseases and cancer,
but it also has some clinical challenges. Resveratrol has poor in vivo bioavailability because it is
quickly metabolized by the intestines and liver, being broken down into other, non-antioxidant
compounds about 30-60 minutes after administration, but researchers are working on ways to
fix this problem.19 One group has attempted to fix this problem by designing cationic and
zwitterionic liposomes in which resveratrol can be carried into the body. They found that the
resveratrol interacted with the cationic liposomes much better, the resveratrol loaded
liposomes were not toxic or did not affect cell viability of stabilized cell lines, and they proposed
that liposomes may be candidates for resveratrol delivery in therapeutic situations. 20 Even
though the circulating levels of resveratrol are fairly low, the low levels have still been efficient
in preclinical models despite being metabolized quickly and having low levels in the blood.21
There is also the question of whether or not resveratrol may be able to accumulate in the right
organ or area in high enough levels to be effective; more delivery routes, other than the
liposome delivery method described above, need to be developed for successful resveratrol
14
therapeutics.21 Another problem that a group of researchers had found is that in Bcl-2
overexpressing cells, resveratrol-induced apoptosis is inhibited by a mechanism involving
interference with cytochrome c release and activity of caspase-3 in the induction of apoptosis.22
Lately scientists have been researching resveratrol analogs that may increase its stability
and ability to be used as a chemopreventative or therapeutic agent. There are several different
derivatives of resveratrol with a variety of functional groups that have been synthesized in
order to solve these problems, and some of the derivatives have actually been found to be
more potent.21 One study used resveratrol and three resveratrol derivatives (viniferin, gnetin H,
and sulffruticosol B) and found that the derivatives had lower IC50 values (required less of the
compound to kill 50% of the cells) and also exhibited cytotoxic activity and induced apoptosis
similar to resveratrol.23 A different group of researchers tried to elongate the conjugation in the
resveratrol chain and incorporate different functional groups onto the benzene rings. They tried
to improve the antioxidant activity by altering these two aspects and had shown that one
analog in particular [trans, trans, trans-1,6-dis(4-hydroxyphenyl)-1,3,5-hexatriene] had
significantly increased antioxidant, cytotoxic, and apoptosis-inducing activities when compared
with resveratrol.24 Therefore, if resveratrol is not the answer, it may have led researchers on
the right path to a compound that can be used as a therapeutic agent. Resveratrol, even
though not fully tested as a therapeutic agent, has been used in a variety of botanicals such as
facial moisturizers, antiaging creams, and eye creams.25 The information provided above
describes how resveratrol may be an important compound in everybody’s lives one day, and
how it may have the potential to solve some health problems present in the world today. In this
project, we investigated the effects of resveratrol on UVB light-induced skin cell death and its
15
possible chemopreventative properties against skin cancer. As of currently, there has been no
investigation of the effects of resveratrol and UVB light on human melanoma skin cells (M624).
Upon resveratrol treatment, and possibly UVB exposure, cell viability and type of cell death was
investigated via clonogenic/trypan blue assays and apoptotic protein research, respectively.
Figure 6. Resveratrol analogs synthesized by Tang et al. C3 is the [trans, trans, trans-1,6-dis(4hydroxyphenyl)-1,3,5-hexatriene] that was shown to be more potent than resveratrol.24
16
The two apoptotic proteins investigated were the poly (ADP-ribose) polymerase (PARP) and
caspase-8. PARP mediates base-excision repair on damaged DNA, but is suppressed, via cleaving by
caspase-3, once the cell has started the apoptotic process.11 Therefore, apoptotic cell death can be
monitored via PARP protein cleavage; if PARP is not cleaved during cell death, necrotic cell death has
ensued. The second apoptotic protein, caspase-8, is part of the caspase cascade which is initiated by
the mitochondrial release of cytochrome c which then causes Apaf-1 to bind to pro-caspase-9 when
the cell is undergoing apoptosis.11 Once pro-caspase-9 is activated the caspase cascade is initiated and
the individual caspase proteins get cleaved by the prior caspase, become activated, and then cleave
the next in line ultimately resulting in the cleaving of PARP and induction of apoptosis.
17
Methods and Materials:
Cell Culture
HaCaT and M624 cells were acquired from Dr. Shiyong Wu’s laboratory at Ohio University.
HaCaT and M624 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin). Cell cultures were
sustained on 60 or 100 mm plates at 37°C in a 5% CO2 humidified incubator and grown to 40-90%
confluency for experiments/treatments.
Cell Lysate Preparation
In order to prepare cell lysate samples, after 3 or 20 hours following resveratrol and/or UVB
treatment, media was removed from the cells and cells were washed with cold, 1X Phosphate Buffered
Saline (PBS) three times. Cells were scraped from the plates using a micropipetter tip and suspension
with cells was transferred to a 1.5 mL Eppendorf tube on ice. Suspension was centrifuged and cells
were washed once more with PBS. Cells were then placed in a -20°C freezer for at least two hours,
removed, and washed once more with PBS. TNET lysis buffer (25mM Tris-HCl pH 7.5, 150mM NaCl,
5mM EDTA, and 0.1% Triton X-100) was mixed with 200mM PMSF to a final concentration of 0.5mM
PMSF-TNET solution. The solution containing cells was homogenized with 23 gauge needles and
syringes and centrifuged at low speeds. Supernatant containing proteins was transferred to a new
Eppendorf tube.
Bradford Concentration Assays
After cell lysate samples were prepared, standard curves were created from a known stock of
BSA protein in concentrations of 0.100, 0.200, 0.300, 0.400, and 0.500 mg/mL in triplicate. Cell lysate
18
samples were then diluted until within the range of the standard curve and the protein concentration
was determined from substituting into the standard curve linear equation and multiplying by the
dilution factor. The Bradford assay consists of preparing 100 µL aliquots of the samples and mixing with
2 mL of Bradford reagent, which binds to the proteins and can be measured by the absorbance at 595
nm. Absorbance readings were taken on the Genesys Spectronic 20 apparatus.
Clonogenic Assays
Cells were grown and cultured in 6-well plates until they reached about 20-25% confluency.
Plates were either treated with 40 or 60µM resveratrol, irradiated with UVB light, or a combination of
the two, and controls had neither. After resveratrol treatment or UVB irradiation, media in the wells
were replaced with fresh media, and allowed to sit for about a week or until nutrients were exhausted.
After this time, media was then removed; cells were washed with warm PBS; and then a 1:7 mixture of
acetic acid: methanol for 5 minutes, followed by a 0.5% crystal violet for 2 hours, and then plates were
rinsed with tap water and photographed.
Trypan Blue Exclusion Assays
Cells were grown and cultured in 12-well plates until approximately 70% confluent. On treated
plates the first column of cells had 0µM, the second had 20µM, the third had 40µM, and the fourth had
60µM resveratrol treatment. Some plates were introduced to UVB light while others had combined
resveratrol treatment and UV, and others neither. Media from each well containing dead cells was
centrifuged, and the cells attached to the plate were released using Trypsin. The cell pellet from the
media was re-suspended with the cell suspensions from the plate. A cell suspension aliquot was mixed
19
1:1 with Trypan Blue reagent, and counted on a hemacytometer. Blue cells were labeled “dead cells”
and yellow cells were labeled “living cells”.
Resveratrol Treatment
Cells grown to a specific % confluency (depending on the specific test) were treated with 0, 20,
40, and 60 µM resveratrol treatments, which were prepared from a 10 mM resveratrol stock solution
in ethanol. Cells were treated with resveratrol for 24 hours before UVB irradiation. Resveratrol was
mixed with cell media containing DMEM, FBS, and antibiotics in amounts described above.
UVB Irradiation
For trypan blue exclusion and clonogenic assays cells were irradiated with 50 and 15 mJ/cm2
UVB light, respectively. Cells for SDS-PAGE and Western Blotting procedures for apoptotic protein
research were irradiated with 25 mJ/cm2 UVB light. Amounts of ultraviolet light were altered between
assays in order to obtain an approximate IC50 value for each experiment. Cell media containing
resveratrol was removed from the cells during UV irradiation and then replaced with normal media.
Statistical Analysis
In order to determine significance for the trypan blue exclusion assay with UVB irradiation and
resveratrol treatment, a t-test at a 95% confidence level was performed.
20
Immunoblot
12.5% SDS-polyacrylamide gels (41.65% of 30% Acrylamide; 8% Bis-Acrylamide, 26.00% Tris-HCl,
1.00% of 10% SDS, and 31.35% distilled water) were made with a 4% stacking gel. Cell lysate samples
were subjected to gel electrophoresis (SDS-PAGE) in 5X Protein Loading Buffer. SDS-PAGE Running
Buffer, 1X, was used for separation of proteins according to size.
Proteins from SDS-PAGE electrophoresis were electroblotted to a nitrocellulose membrane in
1X Wet Transfer Buffer. Membranes were placed in 1X TBST buffer and probed for β-actin with
monoclonal, mouse anti-β-actin IgG1 primary antibody (Sigma Aldrich: Catalog #: A5441). Goat antimouse IgG (H+L) with conjugated horseradish peroxidase secondary antibody (Thermo Fisher Scientific:
product number 31431) was then introduced to the membrane and allowed to bind to primary
antibody. Other membranes in 1X TBST were also probed for PARP with monoclonal, rabbit anti-PARP
IgG (Thermo Scientific: Catalog # MA5-15031). Secondary antibody, goat anti-rabbit IgG (H+L) with
conjugated horseradish peroxidase secondary antibody (Thermo Scientific: product number 31466)
was then introduced to the membrane. Chemiluminescence detection method was used with
developer and fixer solutions, films, and SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific: product number 34080).
21
Results:
Trypan blue exclusion assays were conducted to test cell viability of both HaCaT and M624 cells
after exposure to resveratrol. Cells were mixed with trypan blue reagent which permeates the cell
membrane of dead cells and stains them blue. Resveratrol treatment of HaCaT cells shown in Figure 7,
Percent of Cells (%)
demonstrates no significant change in cell
100
viability. When the cancerous M624 cells
80
were investigated, there also was no
60
40
Living Cells
20
Dead Cells
significant change in cell viability. The graphs
for the two M624, resveratrol treated trypan
0
0
20
40
60
blue exclusion assays can be seen in Figure 8.
Concentration of Resveratrol (µM)
When M624 cells were exposed to a
combinational treatment of resveratrol and
Figure 7. Resveratrol treatment of HaCaT cells. Resveratrol
treatments of 20, 40, and 60 µM were compared to the
control (0 µM). Results are based on triplicate data.
100
80
60
Living Cells
40
Dead Cells
20
0
Percent of Cells (%)
Percent of Cells (%)
100
UVB irradiation, there is a significant
80
60
Living Cells
40
Dead Cells
20
0
0
20
40
60
Concentration of Resveratrol (µM)
0
20
40
60
Concentration of Resveratrol (µM)
Figure 8. Resveratrol treatment of M624 cells for 48 hours (left graph) and 72 hours (right graph).
Resveratrol treatments of 20, 40, and 60 µM were compared to the control (0 µ). Results are based on
triplicate data.
22
increase in cell death between the 60 µM treatment and the control (0 µM), and a moderately
significant increase between the 40 µM treatment and control. This data is shown in Figure 9.
Clonogenic assays were conducted to confirm results from trypan blue exclusion assays on
resveratrol’s effects on cell viability and cellular proliferation. Clonogenic assays were conducted only
on M624 cells in order to investigate resveratrol effects on cancerous cells. The two clonogenic assays
shown in Figure 10 demonstrate how resveratrol affects nutrient deprived M624 cells. M624 cells were
Figure 10 that resveratrol increases cell
viability and cellular proliferation in a dosedependent manner on M624 cells. Also
shown in Figure 10, is the fact that
Percent of Cells (%)
kept in the incubator for some time after they were deprived of nutrients, or starved. It can be seen in
65
60
55
50
Living Cells
45
Dead Cells
40
35
0
resveratrol increases cell viability and
20
40
60
Concentration of Resveratrol (µM)
proliferation in a time dependent manner.
The clonogenic assays conducted for Figure
10 were treated with 25 and 50 µM
Figure 9. Resveratrol and UVB irradiation of M624 cells.
Cells were irradiated with 50 mJ/cm2. Simple t-tests
were conducted for statistical error bars. Results are
based on six sets of data.
resveratrol, but those shown in Figure 11 were treated with 40 and 60 µM resveratrol and cells were
not starved as in the previous set of clonogenic assays. Resveratrol alone caused an insignificant
decrease in cell viability in a dose-dependent manner as shown in Figure 11 (left). Upon resveratrol
treatment and UVB irradiation there is a decrease in cell proliferation compared to cells treated only
with resveratrol as shown in Figure 11 (right vs. left).
23
Figure 10. Clonogenic assays of resveratrol treated M624 cells for 48 hours (left) and 72 hours (right).
These M624 cells were not fixed until all nutrients were deprived and the cells were starved. Resveratrol
treatments of 25 and 50 µM were compared to the control (0 µM).
The effects of resveratrol on apoptosis induction were investigated through apoptotic protein
analysis. The PARP and caspase-8 proteins are indicators of apoptosis once cleaved, which can be
Figure 11. Resveratrol treatment of M624 cells (left) and resveratrol treatment along with UVB
irradiation (right). M624 cells were irradiated with 15 mJ/cm2. Cells were fixed prior to nutrient
depletion and the 40 and 60 µM treatments were compared with the control (0 µM).
investigated by western blotting. A representative blot of PARP and caspase-8 is shown in Figure 12,
along with the control protein, β-actin. For PARP, it can be seen that resveratrol treatment and UVB
irradiation cause similar amounts of PARP expression and cleavage, with the exception of sample UV20. Combinational treatment of resveratrol and UVB irradiation appears to cause slightly less PARP
24
cleavage than resveratrol in UVB treatment alone. Caspase-8 cleavage appears to follow the same
trend as found for PARP with similar amounts of cleavage between cell samples receiving UVB
irradiation and resveratrol treatment, and upon combinational treatment a slight decrease in caspase8 cleavage occurs, with the exception again being UV-20.
Control
UV-3
UV-20
Resv-3
Resv-20
UV+Resv-3
UV+Resv-20
(kDa)
116
89
PARP
62
A
55
43
Caspase-8
10
Β-actin
Lane:
1
2
3
4
5
6
7
Figure 12. Western Blot analysis of PARP, caspase-8, and the control, β-actin. Cells were treated
with 60 µM resveratrol and irradiated with 25 mJ/cm2 of UVB. PARP and caspase-8 blots are
compared to the control β-actin blot.
25
Discussion:
The frequency of melanoma has been increasing during recent years and is expected to keep
rising; and although melanoma is treatable in its very early stages, it is one of the most lethal and
incurable cancers once systemic with very low survival rates.8 Scientists are focused on researching and
understanding the altered pathways involved in tumor progression and chemoresistance.8 The
presented research is interesting because no other researchers to this point have investigated the
effects of resveratrol and UVB irradiation on human melanoma cells (M624 cells). Results from trypan
blue exclusion assays show that resveratrol treatment of normal human keratinocytes (HaCaT cells)
and M624 cells causes no significant change in cell viability, which suggests that resveratrol alone is
nontoxic to both cell lines. In contrast, upon combination of UVB irradiation and resveratrol treatment
of M624 cells there is a significant increase in cell death when compared to the control group of cells.
These results support the conclusions from previous research which has shown that resveratrol
sensitized HaCaT cells to UVA-induced apoptosis and cell death, but to a much greater extent
(approximately a 75% increase).18 Together, these results show that melanoma cells are much more
resistant than HaCaT cells because our research utilized higher energy and more potent UVB irradiation
and similar amounts of resveratrol, but we only saw approximately an 8% increase in cell death.
Some of the most interesting results occurred with the clonogenic assays using starved, or
nutrient-deprived, M624 cells upon resveratrol treatment. Our results show a time and dose
dependent increase in cell viability and cellular proliferation in M624 resveratrol-treated cells. The
control group of cells without resveratrol died in both trials, but cells treated with resveratrol had
survived despite starvation and depletion of nutrients. This may possibly be due to the instability of
resveratrol over long periods of time: over time resveratrol may have been broken down into different
26
metabolites, which are one of the problems that resveratrol has in the body. If broken down, whether
from the melanoma cells or UVB irradiation, then the metabolites could have possibly been substituted
as a nutrient in the ever-adapting melanoma cells. Another possibility could be that resveratrol may
have altered some pathway that allows cancer cells to utilize nutrients more efficiently or survive
harsher conditions. Although the Warburg effect states that cancer cells require more glucose and
energy than normal cells, this piece of data does not support that theory, but rather shows a lessened
Warburg effect: when melanoma cells are treated with resveratrol, glucose dependence is decreased.
Clonogenic assays on resveratrol-treated M624 cells without starvation showed that resveratrol
alone caused no significant change in cell proliferation in contrast to the nutrient deprived cells. These
results support results from the trypan blue exclusion assay showing that resveratrol alone does not
affect cell viability. The clonogenic assays of resveratrol-treated and UVB-irradiated cells show no
significant change in cell proliferation. Interestingly, a significant decrease in cell viability upon UVB
irradiation and resveratrol treatment is observed in the trypan blue assay results but not in the
clonogenic assays. This may be due to the differences in analysis and methodology between the two
assays. In trypan blue exclusion assays cells are directly counted on a hemacytometer following
resveratrol and/or UVB treatment and calculated amounts can be recorded, whereas the clonogenic
assays are based on colorimetric intensity of live cells some number of days following treatment. Also,
trypan blue exclusion assays research cell viability right after treatment, whereas clonogenic assays
start with a smaller amount of cells and don’t analyze the cells until about a week after treatment; this
makes clonogenic assays more of a cellular proliferation assay than a cell viability test.
Western blots for both the PARP and caspase-8 proteins were performed in order to determine
if M624 cells underwent apoptosis upon UVB irradiation and/or resveratrol treatment. It can be seen
27
that there is no difference in PARP expression (116 and 62 kDa) or PARP cleavage (89 kDa) between the
control and UV-3 samples. There is less PARP cleavage in the UV+Resv-3 and UV+Resv-20 samples
compared to Resv-3 and Resv-20 samples. Also shown is more cleavage in the Resv-3 and Resv-20
samples than any other sample, and the least amount of PARP cleavage was found in the UV-20
sample. The three samples with a decreased amount of PARP cleavage may have experienced this
decrease due to the differences in the amounts of control protein, β-actin, and, therefore, overall
protein amounts in these samples, which can be seen by the very small amount of β-actin in those
three samples compared with the others. Interestingly, there was a very faint band for the 62 kDa
PARP protein in the UV-20 sample that is not present in either of the UV+Resv samples despite having
similar protein concentrations. It appears that UVB irradiation and resveratrol treatment combined
may cause the PARP protein of size 62 kDa to be downregulated within the cells.
The caspase-8 protein, which is activated once cleaved just like the PARP protein, is another
indicator of apoptosis. Our results indicate that resveratrol alone causes the most caspase-8 activation
(both Resv-3 and Resv-20 samples). UVB irradiation alone caused some caspase-8 activation only after
3 hours (UV-3), but not after 20 hours (UV-20). The UV-20 sample may not have any caspase activation
because the cells are undergoing necrosis. One theory as to what may be occurring with the UV-20
samples is that cells initially start to undergo apoptosis right after UVB irradiation, but after a certain
amount of time apoptotic proteins are degraded and the cell ends up undergoing necrosis instead of
apoptosis. The UV+Resv-3 and UV+Resv-20 samples cause some caspase-8 cleavage, but not as much
as in the two resveratrol only samples, the UV-3 sample, or the control. This may be due to the small
amount of protein in those two samples when compared to the others (except UV-20), seen though
comparison of amounts of β-actin protein present in each sample. The caspase-8 results appear to
28
show that resveratrol induces apoptosis through the cleavage of caspase-8 more than during
combinational treatment of resveratrol and UVB irradiation.
Overall, our research indicates that resveratrol alone has very little effect on the viability of
M624 cells even though it induces more cells to undergo apoptosis. Resveratrol helps M624 cells
survive upon nutrient depletion, but upon combinational UVB irradiation and resveratrol treatment
causes a significant increase in cell death and induces apoptosis. Effects of resveratrol should continue
to be investigated for different cancer and disease models in order to determine its potential as a
chemotherapeutic agent. Also, other potential therapeutic agents for melanoma should be tested in
the presence of UVB and/or UVA irradiation due to the potential for amplified toxicity or protective
effects of a topical application of the agent in the presence of sunlight.
Acknowledgements:
I would like to thank Marietta College and the Investigative Studies Program along with the
Honors Thesis committee. I would like to especially thank Dr. George, Dr. Egolf, Dr. Howald, and Dr.
Brown of the Honors Thesis committee. I am grateful to Dr. Spilatro for allowing us to utilize some
Biology facilities. I would like to thank Ryan Turnewitsch and Gretchen Miller for their assistance with
protein analysis. Also, I would like to thank Dr. Shiyong Wu (Ohio University) for the supply of the cells.
29
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31
Appendix:
Other PARP Western Blots:
Control
UV-3
UV-20
Resv-3
Resv-20
UV+Resv-3
UV+Resv-20
(kDa)
116
PARP
89
62
PARP
116
89
62
Lane:
1
2
3
4
Solution Recipes:
0.5mM PMSF-TNET Lysis Buffer:
25mM Tris-HCl, pH 7.5
150mM NaCl
5mM EDTA
0.1% Triton X-100
0.5mM PMSF from 200mM stock
100 mL 12.5% SDS-polyacrylamide Solution:
41.65 mL of 30% acrylamide; 8% bis-acrylamide
26.00 mL of 1.5 M Tris-HCl, pH 8.8
1.00 mL 10% SDS
31.35 mL dH2O
12.5% SDS-polyacrylamide Separating Gel Preparation:
5.00 mL of 12.5% SDS-polyacrylamide Solution
5
6
7
32
16.7 µL of 10% APS
3.3 µL of TEMED
101 mL4%-SDS-polyacrylamide Solution:
13.0 mL of 30% acrylamide; 8% bis-acrylamide
25.0 mL of 0.5 M Tris-HCl, pH 6.8
1.0 mL 10% SDS
62.0 mL dH2O
4% SDS-polyacrylamide Stacking Gel Preparation:
2.5 mL of 4% SDS-polyacrylamide solution
12.5 µL 10% APS
2.5 µL TEMED
10 mL 5X PLB:
5.0 mL 0.5M Tris-HCl, pH 6.8
1.0 g SDS(s)
50 mg bromophenol blue
5.0 mL glycerol
350 µL β-mercaptoethanol
1 L of 5X SDS Running Buffer:
15 g Tris-HCl
72 g Glycerin
5 g SDS
***All dissolved in water and brought up to 1 L total
1 L of 5X Wet Transfer Buffer:
15.15 g Tris (THAM)
72 g Glycine
1.875 g SDS
***To make final 1X wet transfer buffer mix the following:
200 mL 5X Wet Transfer Buffer stock
200 mL Methanol
600 mL dH2O
33
1 L 10X TBST Buffer:
80 g NaCl
20 g KCl
30 g Tris-Base (THAM)
500 µL Tween-20
pH to 7.4 with 6M HCl
dilute up to 1 L with dH2O