DIFFERENTIAL EFFECT OF SULINDAC ON NORMAL AND MALIGNANT
RETINAL CELLS
by
Arunodoy Sur
A Dissertation submitted to the Faculty of
The Charles E. Schimdt College of Science
In Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
Florida Atlantic University
Boca Raton, FL
August 2013
Copyright by Arunodoy Sur, 2013
ii
ACKNOWLEDGEMENTS
I would like to thank everyone for their contribution in the completion of my PhD
project. First and foremost I would like to thank my PhD. advisor Dr. Janet Blanks for
the guidance and support she had provided me throughout my time in her laboratory. I
am indebted to her for the role she played in my development as a researcher.
I would also like to express my gratitude towards Dr. Howard Prentice who played an
instrumental role in my academic training. Besides providing me with valuable
scientific inputs and guidance in my research he was always there to encourage and
support me, whenever things were not going right.
This research project would not have been possible without the vast background work
on sulindac that has been conducted under the leadership of Dr. Herbert Weissbach over
the past several years. His knowledge of preconditioning agents and their application in
anticancer studies was crucial in the completion of my thesis. It was a privilege to
receive Dr. Weissbach’s guidance and he serves as an inspiration to students like me.
I would also like to express my thanks to Dr. Wen Shen who provided valuable for
insight for my dissertation and for her contribution towards my academic training.
iv
Besides my PhD committee members several others contributed with scientific ideas
and helped me in my research. I am grateful to every member of Dr. Weissbach’s
research group for each of their valuable contribution towards the completion of this
project. I would especially like to thank Dr. Diane Baronas-Lowell for her help with the
retinal cell protection experiments. The cancer research conducted by Dr. Shailaja
Kesaraju and Dr. Kasirajan Ayyanthan provided me with valuable background
information on which the retinoblastoma assays were designed. The information I
gained from the prior research done by Dr. Maria Marchetti, Dr. Ian Moench and Alex
Kreymerman was also essential for designing and conducting experiments associated
with this project and I am grateful to all of these researchers.
I would also like to thank my former lab member Manas Biswal for his help with the
retinal cell protection study and I am also thankful to Dr. Wong for helping me towards
the later stages of my PhD project. I am grateful towards the two undergraduate research
interns, Andreica Ancuta and Daria Iarossevitch, for their contribution in this research
project.
Besides everyone involved with my PhD research I would like to thank Keyla Thamsten,
at the Center for Complex Systems for her role in handling the financial aspects of our
lab and helping us with ordering of laboratory supplies. I also like to express my gratitude
towards the Biology Department of Florida Atlantic University for providing me with the
opportunity of teaching assistantships. It not only offered financial support but also
enabled me to gain valuable teaching experience. With regards to my teaching, I would
v
like to thank our teaching coordinator, Geri Mayer for her guidance and support during
my time as a teaching assistant.
Finally I would like to thank all my family members, especially my mother, Dr. Aparna
Sur, for the affection, support and encouragement she has provided and my father, Late
Dr. Robin Kumar Sur, who served as an inspiration and instilled in me a love for science
at an early age.
During the past few years as a graduate student I have gained valuable academic
knowledge, made several new friends and gathered cherished experiences. I am thankful
to each and every one who have been a part of this journey and made it possible.
vi
ABSTRACT
Author:Arunodoy Sur
Title:
Differential Effect of Sulindac on Normal and Malignant
Retinal Cells
Institution:
Florida Atlantic University
Dissertation Advisor
Dr. Janet C. Blanks
Degree:
Doctor of Philosophy
Year:
2013
Age related macular degeneration (AMD) is the leading cause of blindness
among the elderly population and oxidative stress induced damage of ocular tissues is
thought to play an important role in the etiology of this disease. This current study is
aimed at testing pharmacological preconditioning as a possible protective mechanism
for retinal cells against oxidative stress induced damage. Since the retinal pigment
epithelial (RPE) cells located adjacent to the photoreceptor cells, are believed to be
responsible for the initial pathology in AMD we tested our therapy on this cell type.
Earlier research conducted with lung cells and in a cardiac model with the nonsteroidal
anti-inflammatory drug (NSAID) sulindac (Moench et al., 2009; Marchetti et al., 2009)
indicate that sulindac can protect normal cells from oxidative damage through ischemic
preconditioning (IPC) pathways. This led us to test whether sulindac can also protect
retinal cells against oxidative stress. Our findings indicate that preincuabtion with
vii
sulindac produces significant protection against both tert-butyl hydrogen peroxide
(TBHP) induced chemical stress and UVB light induced photooxidative damage
through a preconditioning mechanism.
The second goal of this project was to use sulindac in combination with known
anticancer drugs against retinoblastoma (Rb) the most common ocular childhood
malignancy, worldwide. Other laboratories have reported that sulindac sensitizes cancer
cells to anticancer agents targeting mitochondria and thus causing enhanced killing of
cancer cells. The anticancer property of sulindac was tested by sensitizing Rb with
sulindac prior to administration of agents that affected mitochondrial function. Our
results indicate that a combination of sulindac and other compounds that induce oxidative
stress enhanced cell death in an Rb cell line.
Findings of this project support previous data that pharmacological
preconditioning agents can be used for the dual purpose of protecting normal retinal
cells against oxidative stress and also sensitize cancer cells to oxidizing agents in the
same organ. Our findings serve as a platform for developing a clinical therapy for
oxidative stress induced disorders as well as possibly provide an alternative to existing
anticancer therapies. Our future goal is to test the efficacy of both protective and
anticancer properties of sulindac in appropriate animal models to establish clinical
significance.
viii
DIFFERENTIAL EFFECT OF SULINDAC, ON NORMAL AND MALIGNANT
RETINAL CELLS
LIST OF FIGURES .......................................................................................................... xii
LIST OF ABBREVIATIONS .......................................................................................... xiv
CHAPTER -1: INTRODUCTION ...................................................................................... 1
Photoreceptor cells .......................................................................................................... 3
Retinal Pigment Epithelium (RPE) ................................................................................. 8
Reactive oxygen species (ROS) .................................................................................... 13
Oxidative stress generated by photooxidative stress ..................................................... 14
Age related macular degeneration (AMD) .................................................................... 18
Preconditioning in the retina ......................................................................................... 23
Cancer............................................................................................................................ 24
Retinoblastoma .............................................................................................................. 25
Treatment of cancer with sulindac alone or in combination with other drugs .............. 28
Peroxisome proliferator-activated receptor (PPAR) ..................................................... 37
CHAPTER-2: MATERIALS AND METHODS .............................................................. 41
Methods for ARPE19 experiments ............................................................................... 41
Cell culture studies using RPE cells .......................................................................... 41
Experiments involving TBHP induced oxidative stress ............................................ 41
Exposure of RPE cells to UVB radiation .................................................................. 42
Blocking of PKC pathways ....................................................................................... 42
ix
Cell viability assays ................................................................................................... 43
Western blotting protocol .......................................................................................... 43
Statistical analysis...................................................................................................... 43
Methods for cancer cell assays ...................................................................................... 44
Y79 cell culture and viability assay ........................................................................... 44
Killing of cancer cells with combination of IPC agents and oxidizing agents .......... 44
Assays to determine mechanism of anticancer properties ......................................... 44
CHAPTER 3: RESULTS .................................................................................................. 46
A. Protection of retinal cells by sulindac .................................................................... 46
Sulindac and sildenafil protect ARPE19 cells from chemical oxidative stress and
UV induced photooxidative stress ............................................................................. 46
Protection by sulindac is independent of COX inhibition ......................................... 48
Activation of PPARα is involved in the protective mechanism of sulindac ............. 49
Involvement of PPARγ in sulindac’s protective role ................................................ 52
Protection by sulindac is dependent on PKC............................................................. 54
PKCε but not PKCδ is involved in the preconditioning pathway ............................. 56
Protection by sulindac is dependent on PKG ............................................................ 58
Role of ROS and the mK(ATP) channels in the protection of RPE cells against
oxidative stress by sulindac ....................................................................................... 60
Preconditioning markers are upregulated in cells incubated with sulindac............... 62
B. Killing of retinoblastoma cells by IPC/drug combinations.................................... 63
Sulindac sensitizes Y79 cells to agents that affect mitochondria .............................. 63
Sildenafil also enhances death in cancer cells exposed to oxidative stress ............... 66
Effect of PPARγ agonists on cancer cells ................................................................. 70
x
Enhanced killing of cancer cells by sulindac is dependent on PKC .......................... 73
Role of PKC isoforms in the anti-cancer effect of sulindac ...................................... 75
ROS generation by our drug combination is required for killing of cancer cells ...... 76
Enhanced killing of cancer cells by pharmacological preconditioning is not
limited to Rb .............................................................................................................. 78
CHAPTER 4: DISCUSSION ............................................................................................ 81
Conclusion................................................................................................................... 102
REFERENCES ............................................................................................................... 105
xi
LIST OF FIGURES
Figure 1 Morphology of the retina .......................................................................................2
Figure 2: The retinal cells ....................................................................................................3
Figure 3 Distribution of photoreceptors across the retina ....................................................4
Figure 4 Diagram of phototransduction ...............................................................................6
Figure 5 RPE and the photoreceptor ....................................................................................7
Figure 6 Stages of development of the RPE and the retina .................................................8
Figure 7 The visual pigment cycle .....................................................................................10
Figure 8 Origin of lipofuscin ............................................................................................11
Figure 9 : Drusen formation in AMD ................................................................................20
Figure 10 Progression of AMD..........................................................................................22
Figure 11 Histopathology of Rb ........................................................................................25
Figure 12: Structure of sulindac . .......................................................................................30
Figure 13 Structure of sildenafil ........................................................................................36
Figure 14 Function of PPARs ............................................................................................39
Figure 15 Chemical structure of the 3 glitazones ..............................................................40
Figure 16 Protective effect of sulindac and sildenafil .......................................................48
Figure 17 Effect of non-NSAID sulindac sulfone against UVB damage ..........................50
Figure 18 Role of PPARα in protection by sulindac ........................................................52
Figure 19 Protection of RPE cells by TZDs ......................................................................54
Figure 20 PKC involvement in preconditioning . ..............................................................56
xii
Figure 21 Role of specific PKC isoforms in the protective pathway ................................58
Figure 22 Involvement of PKG in the preconditioning mechanismB ...............................59
Figure 23 Role of ROS generation and mK(ATP) channels in sulindac protection ..........62
Figure 24 Induction of preconditioning markers ...............................................................63
Figure 25 Effect of sulindac on viability of Y79 cells .......................................................66
Figure 26 Effect of sildenafil on Y79 cells exposed to oxidative stress ............................70
Figure 27 Effect of treating Y79 cells with PPARgamma agonists in combination with
compounds that induce oxidative stress .............................................................................73
Figure 28 Effect of PKC inhibition by chelerythrine on the anti-cancer effect of
sulindac ..............................................................................................................................74
Figure 29 Role of PKC isoforms in the anti-cancer effect of sulindac ..............................75
Figure 30 Involvement of ROS in enhanced killing o cancer cells by preconditioning
agents .................................................................................................................................77
Figure 31 Effect of treating lung cance and skin cancer with IPC/drug combination .......80
Figure 32 Proposed pathway of IPC protection .................................................................91
Figure 33 Pathways showing differential effect of sulindac ..............................................99
xiii
LIST OF ABBREVIATIONS
A549…Lung cancer cell line
AMD….Age related macular degeneration
ARPE19…. Retinal Pigment Epithelial cell line
AREDS….Age related eye disease study
ATP….Adenosine triphosphate
BM….Bruch’s Membrane
COX….Cycloxygenase
DCA….Dichloroacetate
DFMO….Difluoromethylornithine
DOX….Doxorubicin
Hsp27….Heat Shock Protein 27
iNOS…Inducible Nitric Oxide Synthetase
IPC….Ischemic Preconditioning
INL….Inner Nuclear Layer
IPL….Inner Plexiform Layer
Msr….Methionine Sulfoxide Reductase
NSAID…..Non-steroidal anti-inflammatory drug
ONL….Outer Nuclear Layer
OPL…Outer Plexiform Layer
PDE5….Phosphodiesterase-5
xiv
PKC….Protein Kinase C
PKG….Protein Kinase G
PPAR….Peroxisome Proliferator activated receptor
Rb….Retinoblastoma
RGC….Retinal Ganglion Cell
ROS….Reactive Oxygen Species
RPE….Retinal Pigment Epithelium
RXRα….Retinoid-X-Receptor-α
SC225….Squamous Epithelial skin cancer cell line
TBHP….Tert-butyl hydrogen peroxide
TZD…Thiazolinidinedione
UV….Ultraviolet
VEGF….Vascular Endothelial Growth Factor
Y79….Retinoblastoma cell line
xv
CHAPTER -1: INTRODUCTION
The retina: Human vision is a complex mechanism that requires numerous components of
the eye and the brain to work together. The initial step of vision occurs in the retina, a
multilayered light sensitive tissue which lines the posterior surface of the eye. These
multiple internal layers of the retina can be divided into three layers of neuronal cell
bodies and two synaptic layers. The cell bodies of both rod and cone cells are located in
the outer nuclear layer (ONL), while the inner nuclear layer (INL) contains the cell
bodies of the bipolar, horizontal and amacrine cells (Figure 1). Signals generated by the
photoreceptor cells are transmitted to the bipolar cells. Horizontal cells located in the INL
can modify transmission between photoreceptors and bipolar cells (See figure 2).
Synapses between photoreceptors, horizontal cells and bipolar cells are located in the
outer plexiform layer (OPL) (Kaneda, 2013).
The light sensitive photoreceptor cells exist as two types: rods and cones. The inner
segment region of cones are conical in shape while the rods’ inner and outer segments
are very-slender (rod-like). Cones, although much fewer in number, are responsible for
colour vision and acuity.
Retinal ganglion cells (RGCs), located adjacent to the
vitreous, receive the signals processed by the bipolar cells. The inner plexiform layer
serves as the site for synaptic connection between the RGCs and bipolar cells (Kolb et
1
al, 2001). Amacrine cells present in the INL form synaptic connections with RGC
dendrites and bipolar cell terminals (Dowling and Boycott, 1966). Amacrine cells are
involved in the lateral processing of vision and also play an important role in sensing of
contrast, brightness and movement (Kaneda, 2013) Ganglion cells fire action potentials
which propagate down the optic nerve to the brain. RGCs are characterized by having
long axons which bundle together to form the optic nerve which exits the eyeball and
carries visual information to the brain for processing (Dowling. 1987).
Figure 1: Morphology of the retina: The retina is a thin layer of tissue
located at the back of the eye next to the choroid. A single layer of retinal
pigment epithelial (RPE) cells is situated between the choroid and the
photoreceptor outer segment (OS). Inner segment (IS), Outer segment (OS),
Outer nuclear layer (ONL), Outer plexiform layer (OPL), Inner nuclear layer
(INL), Inner plexiform layer (IPL) Ganglion cell layer (GCL). (Adapted from
(Sung and Chuang, 2010).
2
Figure 1 The retinal cells: Photoreceptors line the back of the eye and are of
two types: Rods and (R) Cones (C). Anterior to photoreceptor cells are
several types of neurons: Bipolar cells (B), horizontal cells (H) and amacrine
cells (A), as well as Muller cells (M), the major glial cell in the retina. The
signal produced by light is transmitted from photoreceptors to bipolar cells to
ganglion cells (G), which project the signal through their extended axons
from the eye via the optic nerve to the brain (Adapted from Sung and
Chuang, 2010).
Photoreceptor cells- Photoreceptors in the retina are classified into two types, rods and
cones, based on their characteristic shapes. The rods are responsible for vision in dim
light (scotopic) and function during low light intensity. Cones, on the other hand,
function to produce acuity in bright light (photopic vision) and are responsible for
3
colour vision. The distribution of rods and cones also varies across the retina. The
number of rods increases in the peripheral retina while the cones are concentrated in the
central region called the macula (Figure 3). The density of rods is much greater than
cones throughout most of the retina. The cone density peaks sharply at the foveal center
to a highest concentration of approximately 200,000 cones per square millimeter, and
drops rapidly in the first few degrees (Lewis et al., 2003). The number of cones
gradually decreases towards the retinal periphery and is reduced to 2500 cells/mm2
close to the ora serrata (the edge of the retina) from 6000 cones/mm2 at a distance of
1.5 mm from the foveal center (Jonas et al., 1992).
Figure
3
Distribution
of
photoreceptors
across
the
retina:
Photoreceptors are not evenly distributed throughout the retina. The
peripheral region has a higher concentration of rods while cones are
primarily located at the central region in the fovea (Figure adapted from
Osterberg, 1935).
As a result of this regional variation in photoreceptor distribution the fovea is better at
high resolution (acuity). In the central macular region, photoreceptors send information
4
directly to relatively fewer ganglion cells which further enables this region to achieve
higher visual acuity. The peripheral retina is better equipped for vision in dim light
because it has a much higher concentration of rods, the photoreceptors adapted to vision
in low light. Rod density reaches a peak of about 150,000 rods/mm2 near the retinal
periphery, at a distance of about 5mm from the fovea (Jonas et al., 1992). The
morphology of the photoreceptor cells can be divided into four regions. The outer
segments (OS) are composed of discs of folded double membranes which contain
molecules of visual pigments. Next to the OS is the inner segment which serves as the
site for assembly of opsin molecules. The mitochondria, ribosomes and membranes are
located in the inner segment. The cell body contains the nucleus of the cell and finally
there is the synaptic terminal where photoreceptor cells contact synaptic processes from
bipolar and horizontal cells.
The process by which photoreceptors are stimulated by light and initiate the visual
sensory response is termed phototransduction. The process of phototransduction is
dependent upon changes in membrane potential of the photoreceptors. When they are
not being stimulated by light, rods and cones are in a depolarized state. Conversely,
photoreceptors become hyperpolarized in response to light. Depolarization of
photoreceptors is caused by an increase in concentration of cGMP which opens ion
channels (Miller, 1982). The positive ions entering a photoreceptor cell lower its
electrochemical gradient and keep it in a depolarized state in the absence of light.
Hyperpolarization is triggered by absorption of light by the photopigments present in
the membranous disks of the photoreceptor outer segments. The absorption of photons
results in activation of rhodopsin molecules in the disc membrane of the outer segments
5
to form activated rhodopsin (denoted as R*) (Lamb and Pugh, 2006). Activated
rhodopsin initiates catalysis of GTP to GDP resulting in the formation of Gα-GTP, also
known as activated G-protein. In the following step activated G-protein dissociates from
R* and binds to the γ-subunit of PDE leading to its activation (Leskov et al., 2000).
This activated PDE subsequently causes the hydrolysis of cyclic GMP (cGMP).
Reduction in cGMP concentration allows Na+ channels to close and thereby generating
the cell’s electrical response, a reduction in the circulating current and a consequent
hyperpolarization (Kawamura, 1993) (Figure 4).
Figure 4: Diagram of phototransduction. This figure depicts the molecules
involved in the series of steps involved in the process of phototransduction.
a) Absorption of light by rhodopsin results in b) activation of transducin,
which releases GDP and binds to GTP and activates phosphodiesterase
(PDE). c) PDE facilitates the conversion of cGMP to GMP causing a drop in
cGMP concentration preventing the opening of cGMP-gated ion channels. d)
6
Transducin causes hydrolysis of GTP which allows phosphate (P) to be
released (reviewed in Blumer. 2004).
Photoreceptor cells play such a key role in the visual process any damage to them
impairs the physiological function of vision and may eventually lead to blindness. The
health of photoreceptor cells is intimately linked with the function of the epithelial cell
layer termed the retinal pigment epithelium (RPE), a monolayer of epithelial cells
located between the retina and choroid (Figure 5). While the neural retina is formed
from the inner layer of optic cup the outer layer gives rise to the adjacent RPE (Figure
6).
A
B
Figure 5: RPE and the photoreceptor: A) An electromicrograph showing
a rod outer segment (ROS) surrounded by the apical process of an RPE
cell. B) The stages of disc shedding by photoreceptor outer segement and
its phagocytosis by the RPE (Figure from Saari, 2000).
7
Retinal Pigment Epithelium (RPE)- Cells of the RPE are hexagonal in shape but they
appear cuboidal when viewed in cross section. They are connected by tight junctions and
arranged in a monolayer. The pigment responsible for the colour and hence the name of
the RPE layer is melanin, which is concentrated as cytoplasmic granules called
melanosomes in the apical end of the cells (Figure 8). This dark pigment absorbs stray
light and reduces scattering in the eye (Strauss, 2005).The second most abundant pigment
found in the RPE is lipofuscin, which is thought to arise from outer segments of
photoreceptors which are phagocytosed by the RPE (reviewed by Kennedy et al., 1995).
Some lipofuscin is present at childhood but it gradually accumulates with age and is
associated with age-onset ocular disorders (Delori et al., 2001)
Figure 6 Stages of development of the RPE and the retina: Stage a)
Thickening and invagination of the ectoderm and the underlying
neuroepithelium. Stage b) Neural retina is formed from the inner layer of
optic cup while the outer layer develops into RPE. Stage c) Three distinct
8
cellular layers of the retina (photoreceptors, interneurons and RGC) are
formed adjacent to the RPE monolayer (From Ali and Sowden, 2011).
The RPE cells are involved in various key roles that are crucial to the survival and
function of the retina. Their RPE cells transport ions, water, and metabolic end products
from the subretinal space to the retina. It also performs delivery of nutrients from blood
vessels in the choroid and maintain the retinal cycle (for review see Strauss, 2005). RPE
cells are essential for maintaining the retinal cycle since the photoreceptors themselves
are not capable of reisomerizing the all-trans-retinal back into all-cis-retinal after photon
absorption. To maintain the visual cycle, the RPE layer receives all-trans-retinal from the
photoreceptors, reisomerizes it back to all-cis-retinal and then transports it back to the
photoreceptor cells (Figure 7) (Baehr et al., 2003).
9
Occurs in photoreceptor cells
Occurs in RPE cells
Figure 7: The visual pigment cycle: Retinal alternates between 11-cisretinal and 11-trans-retinal. RPE is the site of reisomerization of the visual
pigment (Figure from Saari, 2000).
Besides its role in the reisomerization of visual pigment, the RPE layer is also responsible
for other functions essential for survival of the adjacent photoreceptor cells. These
functions include, transporting glucose from the blood into the retina (Bergersen et al.,
1999, Ban and Rizzolo, 2000), secretion of growth factors (Jablonski et al., 2000; Walsh
et al., 2001), production of DHA (Bazan et al., 1994), transport of water, and movement
of ions between the retina and the choroid (Hughes and Takahira, 1998; Hamann, 2002;
Maminishkis et al., 2002).
Photoreceptors are constantly exposed to bright light which leads to generation of
photo-induced harmful products and accumulation of damaged proteins and lipids.
(Beatty et al, 2000). To ensure proper function, the photoreceptors go through a
constant process of shedding and renewing their outer segments. One of the most
10
important roles of the RPE is phagocytosis and digestion of light-damaged
photoreceptor outer segments (Figure 5B). After digesting outer segments the RPE
recycles retinal and DHA back to the photoreceptors to aid in their function (Bok et al.,
2002). Phagocytosis of photoreceptor outer segments leads to the buildup of oxidative
stress burden in the RPE and contributes to ROS-induced RPE cell death (Kennedy et
al., 1995) which is believed to be an underlying cause of AMD.
Rod Outer segment
B
A
Figure 8 : A) Origin of lipofuscin The phagocyosis of outer segments of
rods and their incomplete digestion gives rise to lipofuscin particles which
accumulate in the RPE and act as a source of ROS. B) A transmission
electron micrograph of RPE layer next to the Bruch’s membrane (B). The
lipofuscin granules (L), melanosomes (M) and phagosomes (P) are evident
within the RPE cell. (Figures from Kennedy et al., 1995).
As evident from the various roles of the RPE, any damage to this layer drastically
affects the health and survival of other retinal cells. High levels of oxygen delivered to
11
the RPE by the choriocapillaris, coupled with the intense focus of light on this area,
create the perfect microenvironment for production of lethal amounts of ROS making
the RPE layer highly vulnerable to ROS damage (Mettu et al., 2012). In order to prevent
ROS damage, the RPE cells possess antioxidant enzymes such as superoxide dismutase
(SOD) and catalase (Newsome et al., 1990; Tate et al., 1995). Damage to RPE cells,
responsible in part for the development of AMD, can be categorized in three distinct
phases. Initial ROS induced RPE injury results in extrusion of cell membrane "blebs,"
together with decreased activity of matrix metalloproteinases (MMPs) promoting
accumulation of basal laminar deposits (BLD) (Hageman et al. 2001). BLD is a mixture
of collagen and basement membrane proteins deposited between the RPE basal lamina
and the Bruch’s membrane (Sarks et al., 2007). This process leads to progression of
drusen by admixture of blebs into Bruch’s membrane, followed by the formation of new
basement membrane under the RPE to trap these deposits within Bruch’s membrane.
Finally, macrophages are recruited to sites of RPE injury and deposit formation. The
recruitment of certain activated or reparative macrophages, through the release of
inflammatory mediators, growth factors, or other substances, may promote
complications and progression to the late form of AMD (Mettu et al., 2012).
Oxidative damage to RPE cells may be involved in several pathological conditions,
including AMD. The loss of vision in advanced AMD is linked to loss of photoreceptor
cells in the macula. This damage is preceded by damage, dysfunction and death of the
underlying RPE cells (Strauss, 2005). Exposure of RPE cells to elevated levels of ROS
inhibits the ability of these cells to perform vital functions in the maintenance of retinal
health (Bailey and Cassone, 2004)) and can lead to programmed cell death (Kim et al.,
12
2003; Pitha-Rowe et al., 2009). Since one of the major underlying causes of AMD is the
oxidative stress induced by endogenous free radicals and exogenous oxidants,
therapeutic strategies like the one described in this current project aimed at protecting
RPE cells against oxidative damage may be particularly important in retarding AMD.
Reactive oxygen species (ROS) – ROS are produced by cells at very low levels as a
byproduct of several normal mechanisms and its controlled production is critical for
multiple regulatory processes and signal transduction pathways. However, when the
concentration of ROS exceeds the normal cellular level, it leads to serious cellular
damage. ROS is a common term used to include various kinds of partly reduced oxygen
species such as superoxide anions, OH free radicals, H2O2 and singlet oxygen (Jezek et
al., 2004). Reactive nitrogen species are also known to inflict damage owing to its
radical status (Jezek et al., 2004). Among the various cellular components,
mitochondria are the major source and a prime target of ROS. The mitochondrial
electron transport chain (ETC) is considered the primary source of superoxide anion
generation. Other events that have been demonstrated to release ROS include, leukocyte
activity involved in inflammatory responses and phagocytosis (Jezek et al., 2004;
Wiseman, 2006). But the main source of ROS is the mitochondria which only produce
ROS as a byproduct of ATP generation. ETC is an array of four complexes that are
organized based on their redox potential (Moro et al., 2005). ROS production is
associated especially with complex I and complex III of the ETC (Brand et al., 2004)).
During ischemic stress complex III is known to generate superoxide (Han et al., 2003).
ROS production is further elevated under hyperglycemic conditions. Oxidative damage
caused by ROS leads to mitochondrial DNA damage, loss of efficiency of the Electron
13
Transport chain, and oxidation of mitochondrial proteins which in turn leads to further
accumulation of ROS. Since mitochondrial DNA is closer to the site of free radical
generation, they undergo mutations at a faster rate than nuclear DNA. This vicious
cycle of ROS production and resulting damage has been shown to result in loss of
mitochondrial membrane permeability leading to release of cytochrome C in the
cytoplasm. The release of cytochrome C is a two-step process, initiated by release of the
hemoprotein from its binding to cardiolipin in the inner mitochondrial membrane (Ott et
al., 2002). This results in a pool of free cytochrome C in the intermembrane space.
Subsequent permeabilization of the outer mitochondrial membrane releases cytochrome
C into the cytosol where it binds apoptotic peptidase activating factor 1 (APAF1) which
triggers the apoptotic pathway, resulting in cell death. ROS also leads to modification of
phospholipids and cellular protein which results in alteration of membrane permeability
and dysfunction of several cellular proteins (Dhalla et al., 2000).
Certain tissues, which exhibit a very high metabolic activity, tend to be more prone to
damage due to oxidative stress. Since the retina uses oxygen at a rate higher than most
other tissues in the body (Yu and Cringle, 2001), it has an increased susceptibility to
damage induced by oxidative stress arising from elevated glucose and elevated
mitochondrial activity. Indeed, oxidative stress has been implicated in the etiology of
several ocular disorders such as cataract, glaucoma, diabetic retinopathy and AMD.
Oxidative stress generated by photooxidative stress – The eye is constantly exposed
to light making it a prime target of damage from photooxidative stress. Light induced
oxidative mechanisms are known to contribute to aging changes in both RPE as well as
14
photoreceptors cells and they underlie the pathogenesis of AMD. The extent of damage
is determined by multiple factors including intensity, duration and wavelength of the
light rays. Biological factors such as age of the individual, diet and protection rendered
by intracellular antioxidants also play key roles in determining the extent of damage
(Sparrow et al., 2006). An important source of oxidative damage is the photoreactive
pigments that accumulate and constitute the lipofuscin located in RPE cells (Sparrow et
al, 2006; Zhou et al, 2006). Lipofuscin, made up of photosensitive bisretinoid
compounds, is thought to be one of the major sources of ROS responsible for
pathophysiology of AMD (Yoon et al., 2012). Accumulation of lipofuscin in the RPE is
known to increase with age (Sparrow et al, 2006).
Photoecxitation of lipofuscin (A2E) components present in RPE cells leads to
generation of free radical species and A2E photodegradation results in the release of
aldehyde-bearing products, one of which is methylglyoxal (MG), which leads to further
damage (Yoon et al., 2012). The AMD mouse model, lacking the antioxidant enzyme
SOD2, shows increased levels of A2E and iso-A2E as well as elevated levels of other
markers of oxidative damage (Justilien et al., 2007) indicating a correlation between
A2E accumulation and oxidative stress. Previous studies in cultured RPE cells showed
that when levels of A2E reaches levels comparable to that found in the aging human
eye, the RPE cells are more vulnerable to light and oxidative stress (Sparrow et al.,
2006). It is noteworthy that A2E accumulation specifically slows down the
phagolysomal digestion of the outer segments in cell culture studies (Finnemann et al,
2002). A recent study found that mitochondrial dysfunction resulting from increased
accumulation of A2E is a major component of oxidative stress induced damage caused
15
by A2E. Increased levels of A2E led to loss of mitochondrial membrane potential and
decreased oxidative phosphorylation (Vives-Bauza et al, 2008). Another important
finding from this study was that the harmful effects of A2E levels were reversed by
application of antioxidants suggesting that A2E induced damage is due to oxidative
stress (Vives-Bauza et al., 2008). Besides lipofuscin, another product of
photodegradation that affects the retina is MG, a low molecular weight reactive
dicarbonyl that is responsible for advanced glycation endproduct (AGE) modification of
proteins (Thornalley, 2008). It is significant for AMD, since AGE-modified proteins are
detected in drusen deposits that accumulate between RPE cells and Bruch’s membrane
in vivo (Crabb et al, 2002; Curcio et al, 2001). The hypothesis that drusen are formed
by the RPE was first made by Farkas who suggested that drusen represented
incompletely digested outer segment material expelled by the RPE into the extracellular
matrix between the RPE and Brüch's membrane (Farkas et al., 1971). Other researchers
have provided evidence that RPE cells shed portions of their basal plasma membrane
plus cytoplasm into this matrix (Burns and Feeney-Burns, 1980), thereby contributing
further to the process of drusen formation. Photoreceptor outer segments live in a
potentially toxic environment that includes high oxygen hence their phagocytosis and
digestion exposes the RPE cells to further oxidative stress (Dorey et al., 1989). These
conditions are conducive to photooxidative damage and the production of byproducts of
lipid peroxidation such as malondialdehyde, which can cross link proteins (Katz and
Eldred, 1989). Considering the current evidence it is likely that multiple sources,
namely cells of the choroidal vasculature, the RPE, and blood components, contribute to
origin of drusen. In the article by Crabb et al. (Crabb et al., 2002) the authors identified
16
components that could potentially arise from all of these sources. It is well established
that drusen formation is associated with the pathogenesis of AMD (Anderson et al.,
2002). Photoreceptors are modified to transduce light energy but this constant intense
light exposure is also responsible for triggering a series of events that causes strand
breaks in DNA and alterations in the nucleus that eventually results in apoptosis of
these cells (Gordon et al., 2002). As demonstrated in the in vivo studies with light
damaged albino mice, the presence of antioxidants such as ascorbic acid or
dimethylthiourea significantly reduces the damage caused by bright light, thus
providing further evidence of the role of oxidative stress in light induced retinal damage
(Organisciak et al., 1990; Gordon et al., 2002).
Among the various optical wavelengths, UV radiation is known to be one of the most
potent agents in initiating production of ROS in retinal cells. The primary source of UV
exposure is the rays of the sun, which contains radiation across the entire UV spectrum.
The ozone depletion in combination with increase in human lifespan has resulted in a
significant increase in the cumulative lifetime exposure to harmful UVB radiation
(Patton et al., 1999). Research has shown that short-wavelength light exposure to
cultured RPE cells results in release of toxic oxygen radicals from the mitochondria
(Youn et al., 2009). Exposure of cultured RPE cells to UV caused a decrease in
mitochondrial membrane potential that eventually lead to apoptosis of RPE cells in an
ROS mediated pathway. This provides further evidence supporting generation of ROS
by UV irradiation in RPE (Wang et al, 2009). Even though both UVA and UVB reach
the earth’s surface and both are considered deleterious to the eye, UVB is considered
more potent and damaging (Patton et al., 1999). For this reason, the current study has
17
concentrated on designing a protective method targeting protection against UVB
radiation in our attempt to develop a therapy for AMD.
Age related macular degeneration (AMD)- ROS oxidize an array of cellular
constituents including lipids, amino acids and nucleic acids, and these effects of ROS are
implicated in the progression of various ocular disorders including AMD. AMD is the
leading cause of blindness among elderly persons. AMD is caused by multiple factors
including environmental stresses and the risk of developing AMD is known to be
enhanced by genetic influences (Gorin, 2012). AMD pathophysiology results from loss of
RPE cells and its effect on adjacent photoreceptors and choriocapillaries.
It is believed that the pathophysiology of AMD is due to cumulative oxidative damage
to the RPE resulting from an imbalance between generation of ROS and the ability to
destroy and/or protect against ROS damage to macromolecules (Beatty et al, 2000;
Dentchev et al, 2005; Dunaief et al, 2002; Milam et al, 2002). The loss of efficacy of
naturally occurring antioxidant protective mechanisms associated with age contributes
further to the progression of AMD. RPE cells are known to possess a high level of
metabolic activity making these cells sensitive to oxidative stress (Dong et al, 2011).
The Age Related Eye Disease Study (AREDS) conducted by the National Eye Institute
(NEI) in 2001, showed that progression of AMD can be slowed by an antioxidant
cocktail containing beta-carotene, vitamin E and vitamin C, providing further evidence
for association of ROS with AMD. The AREDS study revealed that when treated with
the AREDS formula, the incidence of vision loss in people with high risk of advanced
AMD was reduced by 25 percent (AREDS, 2001). However this treatment with high
18
levels of antioxidant and zinc did not have any significant effect on the progression of
cataracts. Currently the second phase of AREDS study called AREDS2 is ongoing. The
main aim of this study is to refine the formulation of the treatment based on information
from current scientific work. The main changes to the formulation have been the
addition of pigments lutein, zeaxanthin and also fish oil. The role of the fish oil is to
serve as a source of omega-3-fatty acids DHA and EPA which is believed to be
beneficial for ocular health. The goal of AREDS2 is to build upon the findings of
AREDS1 to develop a more efficient formulation for preventing the occurrence or
progression of AMD in the elderly population (AREDS, 2012). The recently declared
results of the AREDS2 study show that inclusion of omega-3 fatty acids DHA and EPA
as supplements did not reduce the risk of advanced AMD in the AREDS2 trial.
However, substituting beta-carotene with lutein/zeaxanthin in the original AREDS
formulation, did help lower the risk of advanced AMD. This effect was most prominent
among current and former smokers, and for people who did not intake adequate amount
of green leafy vegetables in their diet.
According to recent scientific studies approximately 1.7 million Americans currently
suffer from AMD and the number of AMD patients in the United States is estimated to
grow to 3 million by the year 2020 (Friedman et al, 2004). Based upon the pattern of
progression and pathophysiology macular degeneration can be divided into two basic
types of AMD "dry" and "wet". Approximately 85% to 90% of the cases of AMD are
the "dry" (atrophic) type (Seddon et al, 2001). In the dry form of AMD, areas of focal
RPE cell loss develop in the central region termed the macula followed by a loss of the
adjacent photoreceptor cells.
19
In the dry form, areas of degenerating RPE develop near the macula, followed by loss
of overlying photoreceptors. This deterioration of the retina is associated with the
formation of drusen, near the macular region.
RPE
BM
A
B
Figure 9: Drusen formation in AMD: A) A fundus picture showing
development of drusens as dark deposits in the retina is a major feature of
pathophysiology of AMD. B) A section of the retina showing drusen
20
deposited between RPE and Bruch’s membrane (BM) (indicated by asterisks).
(Figure from Webvisionmed.utah.edu).
In the early stages of AMD, patients have only a few small druse and they fail to notice
any symptoms or vision loss. Druse are generally located between the RPE basal lamina
and Bruch’s membrane (Figure 11). Fragmentation and tearing of Bruch’s membrane
along with calcification of the Bruch’s membrane are reported to occur in AMD (Burns
and Feeney-Burns, 1980; Grindle and Marshall, 1978). Another hallmark of AMD
diagnosis is basal laminar deposits which accumulate between RPE basal plasma
membrane and basal lamina (Haimovici et al., 2001; Anderson et al, 2002). Drusen and
other deposits result in morphologic and biochemical damage in the photoreceptors of
the affected regions. Drusen-associated reductions in photoreceptor cell densities have
been reported suggesting that degenerative changes in photoreceptors ultimately lead to
cell death (Johnson et al., 2003). If untreated, there is a progression to one or more large
druse and patients with this condition start experiencing difficulty in performing simple
daily tasks like reading. These phenomena lead to a thinning and drying out of the
macula, causing the macula to lose its function. The resulting blind areas expand at a
slow rate but can eventually progress to cause significant loss of vision.
21
Figure 10 Progression of AMD: The progression of AMD from initial
depositions of drusen (shown by large arrows) to development of
choroidal neovascularization (indicated by smaller black arrows). (Figure
from Mettu et al., 2012).
Acute loss of central vision associated with the more severe “wet” (neovascular) form
of AMD is the result of a phenomenon known as choroidal neovascularization (CNV)
(Figure 12). CNV is characterized by development of new, unstable blood vessels that
originate from the choroid and grow into the macular region (Li et al., 2001). Virtually
any pathologic process that involves the RPE and damages Bruch’s membrane can be
complicated by CNV. The unstable and abnormal vessels leak fluid and disrupt the
sensitive environment of the RPE and photoreceptors (Browning et al., 2004).
Development of CNV in advanced cases of AMD results in retinal edema which may
further disrupt the retina. This causes sudden and irreversible damage to the macula
leading to loss of central vision (Li et al, 2011). Although affecting only 20% of AMDafflicted individuals, the most severe vision loss is associated with the neovascular
form. Neovascular AMD (CNV) is known to be initiated by increased oxidative stress,
22
and hypoxia and inflammation, leading to enhanced expression of vascular endothelial
growth factor (VEGF) (Beatty et al, 2000).
Preconditioning in the retina Despite the deleterious effects of ROS on cellular
function and viability, recent findings indicate that ROS play a critical role in multiple
signaling pathways involved in enzymatic processes and transport channels that are
essential for normal physiological activities. Interestingly, when ROS production is
stimulated at sublethal levels, protective pathways have been shown to be activated in
cardiac and other tissues (Bolli, 2000). This protection afforded by low levels of ROS is
most likely through preconditioning which has been shown in the heart to induce well
characterized prosurvival pathhways at
multiple levels that involve kinase family
members, mitochondrial components, including mitochondrial membrane ion channels,
redox sensitive transcription factors and effectors such as superoxide dismutase and
NOS (Dhalla et al., 2000). These protective pathways have been found to involve
upregulation of Bcl-2 and cause an increase in Bcl-2:BAX ratio (Bartling et al., 1999).
Applying preconditioning in protecting RPE can prove to be an efficient mechanism
since microarray analysis performed on retinal cells exposed to preconditioning agents
were shown to result in changes in gene expression of a number of retinal genes
(Kamphuis et al., 2007); (Thiersch et al., 2008). Preconditioning of cultured ARPE-19
cells by controlled exposure to non-lethal doses of hydrogen peroxide protected them
from subsequent oxidative stress induced by lethal dose of H2O2 (Sharma et al., 2009).
Retinal ischemic damage was shown to be attenuated in ND4 Swiss-webster rats when
the animals were preconditioned by increasing their intraocular pressure for 5 minutes.
23
This preconditioning afforded morphological as well as functional protection against
ischemic insult (Zhu et al., 2002). The mechanism of ischemic preconditioning (IPC) in
the retina is thought to occur through a complex pathway which is believed to involve a
number of downstream signal transduction factors.
Mitochondrial KATP channels
(mK(ATP)), PKC, ROS and NOS are major components of this neuroprotective pathway
(Junk et al., 2002; Roth et al., 2006; Dreixler et al., 2008). A previous cardiac study by
Moench and colleagues showed that sulindac protects normal cells against oxidative
stress through IPC (Moench et al., 2009). In this current study we tested if a similar
preconditioning by sulindac can be used as a therapeutic approach in protecting normal
RPE cells against oxidative stress induced damage. This pharmacological preconditioning
could form a platform for future clinical application in preventing or retarding the
progression of AMD and other oxidative stress induced diseases affecting the human eye.
Besides protecting normal cells against oxidative stress, another property of
pharmacological preconditioning agents that has been recently studied, is their ability to
sensitize cancer cells to compounds that induce oxidative stress. Therefore the second
aim of our project is to test the implement the anticancer property of sulindac in targeting
ocular malignancy.
Cancer – Cancer is another disease affecting millions of people worldwide that has been
linked to mitochondrial dysfunctions and consequent production of ROS. Cancer is not
just one ailment but a combination of multiple diseases. The primary underlying cause of
cancer is the failure of the cellular mechanism that regulate cell division resulting in
unregulated growth and uncontrolled cell division which results in tumor formation. In
24
advanced stages, cancer results in metastasis or invasion of neighbouring healthy tissues.
Types of cancer are broadly classified based on the organ or tissue affected.
Unfortunately, the eye is also affected by this lethal ailment.
Retinoblastoma- Cancer affecting the retina known as retinoblastoma (Rb), is the most
common inherited childhood malignancy, worldwide. This type of tumor usually starts by
affecting the immature or developing retina in children and is detected within the first 3
years of life. The first sign of retinoblastoma is luekocoria, a whitening of the pupil
commonly termed as “Cat’s eye”. Checking for red reflection exhibited by normal eyes
and corneal reflection may also reveal initiation of Rb in children (Balmer and Munier,
2007). Detection with certainty can be achieved by computerized tomography (CT) scan
or Magnetic Resonance Imaging of the head region (MRI).
Figure 11 Histopathology of Rb: The Rb tumor is characterized by
formation of rosettes (indicated by boxes), which appear to have a circular
shape with a hyperchromatic nuclei (Figure from Wippold and Perry,
2006).
25
The incidence of this ocular malignancy ranges around 1 in 15,000 to 20,000 live births.
About 250 to 300 new cases of Rb are reported in the United States annually and 5000
cases are diagnosed worldwide (Sareen et al., 2006). All of the patients affected by Rb
either have a mutation or loss of both the alleles of the Rb tumor-suppressor gene, located
on the 13th chromosome. In its normal form this gene is responsible for coding a
phoshpoprotein (pRb) that plays a decisive role in suppressing tumor formation (Sareen
et al., 2006). The pRb performs this task by preventing certain regulatory proteins from
triggering DNA replication. Thus, upon its mutation leads to unchecked tumor growth.
Based upon the mode of inheritance Rb can be classified as either hereditary or nonhereditary. The genetics of Rb indicates that it follows the “two-hit” model (Knudson,
1971). The first “hit” is generally an inherited mutation while the next “hit”/mutation
causes the loss of the only remaining normal allele. This causes mutation of both Rb
alleles and leads to disruption of the cell cycle (Harbour and Dean, 2000). In the heritable
form, only one of the alleles undergoes mutation in the germline, but the second allele is
either mutated or lost in developing retinal cells (Sareen et al., 2006). Multiple reasons
that lead to loss of heterozygosity include loss of alleles, mitotic recombination, or even
deletion of the entire chromosome 13 (Chintagumpala et al., 2007).
While the children affected with the non-hereditary forms have a singly tumor in one eye
the hereditary form is characterized by multiple tumors in either eye and is considered to
be more deleterious. The phosphopoprotein coded by the Rb gene plays a crucial role in
suppression of tumor and regulation of cell cycle by influencing genes involved in the
transition of G1-S phase (Mendoza-Maldonado et al., 2010). Hence patients affected by
26
malignancy associated with mutation in Rb gene are likely to be more vulnerable to
cancers affecting even non-ocular cancers such as liver, breast and brain.
Several different researchers have attempted to determine the origin of Rb cells. It has
been reported that in human Rb, all the glial, neuronal, RPE, and photoreceptor cell
markers, were found to be present in parts of rosette-forming tumor cells (Figure 11)
(Ohira et al., 1994). Another study discovered that markers of postmitotic cone
precursors are expressed in retinoblastoma cells. The same study also demonstrated that
human cone precursors prominently express MDM2 and N-Myc, and both of these
proteins are also required for the survival and proliferation of retinoblastoma cells. These
findings provide support for a cone precursor origin of retinoblastoma (Xu et al. 2009).
Even though Rb starts in the retina, if untreated it can be fatal as it metastasizes to other
areas. Intracranial metastasis has been associated with almost all fatal cases of Rb while
approximately half of them suffered from distant organ metastasis (Provenzale et al.,
1995; MacKay et al., 1984). The intracranial invasion of the disease happens through the
optic nerve and into the central nervous system and it can also metastasize t distant
organs, hematogenously following the once it had reached the optic vein. Mortality is
almost certain whenever there is extraocular invasion of the tumor (Abramson, 1982).
Owing to the proximity of the eye to the brain, metastatic Rb is highly likely to affect the
brain or other parts of the central nervous system (MacKay et al, 1984). The most
important criteria required to confine Rb within ocular tissues is detecting it early
enough. Unfortunately, Rb in children is rarely detected very early making its
containment more difficult. Another issue associated with the current therapeutic
27
strategies is the aggressive nature of available treatment options. The most aggressive
therapy, enucleation of affected eye, results in loss of vision and facial deformity
(Wippold and Perry, 2006). In very advanced stages, mostly in children affected by the
hereditary form of Rb, aggressive radiotherapy or chemotherapy is used. These kinds of
treatments significantly enhance the risk of development of secondary malignancies such
as melanoma or osteosarcoma, in later stages of the patient’s life. These findings further
emphasizes the need for developing novel, therapeutic methods aimed at prevention or
containment of Rb so that we can preserve visual function and ensure higher rate of
survival among children affected by this ocular malignancy.
Treatment of cancer with sulindac alone or in combination with other drugs- One of
the main goals of this current project was to find a suitable treatment for treating the
ocular malignancy mentioned above. As it is evident from the information detailed earlier
in this thesis, ROS and damages to ocular tissues associated with it, is the primary reason
for designing a therapy based on preventing oxidative stress induced damage for the
treatment of AMD. Besides being involved in AMD the mitochondria and oxygen free
radicals also play a key role in malignant cells. This study was designed not only to
protect the retina from ROS but also to test a therapy for increased killing of cancer cells.
To achieve this goal we used the known non-steroidal anti-inflammatory (NSAID)
compound sulindac. The reason for choosing sulindac was that, in addition to its well
established anti-inflammatory activity and more recently discovered role as a
preconditioning agent, sulindac and its metabolites have been also shown to act as
potential anti-cancer agents, by an NSAID independent mechanism. Sulindac was first
28
known as an FDA approved NSAID, which affects prostaglandin production by
inhibiting cyclooxygenases (COX) 1 and 2.
One of the chief reasons for selecting this drug was that it was known to protect against
oxidative stress induced by ischemia/reperfusion in a cardiac system (Moench et al,
2009). Sulindac gained attention when it was discovered that it might act as a substrate
for the reducing, Msr system, which has been shown to be an important cellular
protective molecule against oxidative stress, and may play a role in aging. It has been
shown that the S epimer of sulindac is a substrate for methionine sulfoxide reductase A
(MsrA) (Etienne et al., 2003), which reduces the S epimer of sulindac to sulindac sulfide,
an active COX inhibitor. A recent study has shown that an MsrB like enzyme specifically
targets the R epimer of sulindac (Brunell et al., 2011). The cycle of reduction of sulindac
to sulindac sulfide by the Msr system and its oxidation by free radicals might act as a
catalytic antioxidant mechanism (Figure 12). Therefore, it has been proposed that
sulindac has the potential of serving as both an anti-inflammatory agent and an
antioxidant in the presence of the Msr system.
A
B
29
Figure 12 Structure of sulindac A: The methyl sulfoxide moiety of sulindac can
be reduced to sulindac sulfide or irreversibly oxidized to sulindac sulfone. The
sulfone lacks COX inhibition properties. B: Sulindac is known to act as a substrate
for the reducing enzymes of the Msr family. The R epimer of sulindac is a
substrate for MsrB while MsrA reduces the S epimer (Adapted from Brunell et al.,
2011).
As mentioned above previous studies have shown that sulindac can protect cardiac
myocytes against hypoxia/reoxygenation induced oxidative stress (Moench et al., 2009).
With specific relevance to ocular dysfunction, sulindac has been shown to inhibit aldose
reductase in the lens (Jacobson et al., 1983). With regards to specific hyperglycemic
ocular disorders, sulindac is capable of preventing accumulation of sorbitol in cataract
and neurons incubated in high glucose (Jacobson et al., 1983). Sulindac has also been
shown to protect against retinal capillary basement membrane thickening induced by
diabetic retinopathy (Gardiner et al., 2003). Sulindac has been shown to protect different
kinds of tissues against ischemic insult and oxidative stress. Sulindac was found to
prevent the depletion of GSH in the hippocampus exposed to oxidative stress induced by
quinolinic acid (Dairam et al., 2007). Though the definite mechanism of protection
offered by sulindac has not been elucidated, tissue preconditioning is suggested to be the
most likely possibility. A short, mild ischemic episode has been proven to have a
protective effect against a later, more severe ischemic insult in various types of tissues
including brain and heart (Bolli, 2000; Dawson et al., 2000; Athar et al., 2004). In the
cardiac study by Moench et al, 2009, feeding sulindac to mice protected their cardiac
tissue from high level ischemia. In this cardiac study inhibiton of PKC by chelerythrine
30
resulted in reversal of the protective function of sulindac indicating that PKC plays an
important role in this mechanism. Analysis of cardiac protein of sulindac fed mice
revealed a 3-4 and 6 fold increases in induction of iNOS and Hsp27, respectively. Both of
these proteins are well known to be end-effectors of late stage preconditioning pathway
and PKC is known to be a component of the preconditioning pathway. These findings
provided further evidence supporting the theory that sulindac can function as a
preconditioning agent.
As previously mentioned, sulindac, in addition to its anti-inflammatory activity and
protective property through preconditioning, has been reported to have anti-cancer
property. This anti-cancer property of sulindac has been studied for almost two decades.
In the earlier anti-cancer studies, sulindac was used by itself against multiple cancer cell
lines with promising results. One of the earliest studies demonstrated sulindac was
efficacious for treatment of pre-malignant cells in the colon. Treatment with sulindac
resulted in almost complete removal of polyps in patients suffering from colon and rectal
polyps (Waddell and Loughry, 1983). Other studies also demonstrated the ability of
sulindac to slow the progression of colorectal polyps to colon cancer, as well as its ability
to kill cancer cells of the colon and other tissues (Takemoto, 1998; Guldenschuh et al.,
2001). Other studies on the mechanism of the anti-cancer action of sulindac, when used
by itself, discovered that it, inhibits PDE5 and also activates PKG suggesting those
properties might be involved in its cytotoxic effect on cancer cells (Soh et al., 2008;
Tinsley et al., 2011). Topical application or feeding of sulindac has been shown to
provide protection against UV induced skin damage in hairless SKH-1 mice (Athar et al.,
2004). In this study it was suggested that sulindac protects through a combination of
31
NFkB and COX inhibition. This effect may be related to altered mitochondrial respiration
in cancer cells, first described by Warburg (Warburg, 1956). In another in vitro study
sulindac induced apoptosis in a cancer cell line by binding to a truncated version of
retinoid-X-receptor-α (RXRα) and activating AKT signaling (Zhou et al, 2010). It should
be noted that sulindac’s cancer killing property has been proven to be unrelated to its
ability to inhibit COX, since earlier studies aimed at elucidating the mechanism of
sulindac’s anti-cancer property showed that sulindac sulfone which lacks the COX
inhibitory function was also capable of killing cancer cells (Zhang et al., 2010).
Besides these initial studies where sulindac was used by itself, other researchers
investigated the effect of using sulindac in combination with other compounds. Sulindac
in combination with arsenic trioxide induced apoptosis in a lung cancer cell line (Jin et al,
2008) while another study reported sulindac enhanced the anti-cancer activity of
bortezomib, a proteosome inhibitor (Minami et al., 2005). Sulindac in combination with
another compound has also been used in a clinical study. A study targeting colorectal
adenoma
showed
positive
results
when
treated
with
a
combination
of
difluoromehtylornithine (DFMO), an ornithine decarboxylase inhibitor and sulindac
(Meyskens et al., 2008). It is important to emphasize that all of these compounds used in
combination with sulindac to enhance killing of cancer cells are known to affect
mitochondrial function.
Recent findings from the Weissbach lab indicate that sulindac, used in combination with
compounds that induce oxidative stress on the mitochondria, is highly effective in
enhancing the killing of cancer cells. The protective effect of sulindac seen in normal
32
cells exposed to oxidative stress, was not seen with cancer cells (Marchetti et al, 2009). In
fact it was observed that sulindac sensitizes several cancer cell lines to oxidizing agents,
or compounds that affect mitochondrial function, resulting in cell death by apoptosis
(Marchetti et al, 2009; Resnick et al., 2009; Ayyanathan et al, 2012). This dual role of
sulindac was aptly demonstrated in a study using normal cells from ling and a lung
cancer cell line (A549). Sulindac protected the normal lung cells from oxidative stress
induced by TBHP but enhanced the killing of lung cancer cells in the presence of TBHP
(Marchetti et al., 2009) In another study conducted on a skin cancer cell line, sulindac
sensitized skin cancer cells to killing by H2O2 (Resnick et al., 2009). Findings from
several other studies have proven that sulindac’s ability to sensitize cancer cells is not
limited to only TBHP or H2O2. Sulindac has been used in combination with
dichloroacetate (DCA), TBHP and H2O2 to target multiple cancer cell lines (Marchetti et
al., 2009, Ayyanathan et al., 2012). It was also successful in a clinical study for the
treatment of actinic keratosis, when used in combination with H2O2 (Resnick et al., 2009).
The lung cancer study mentioned earlier provides strong evidence that loss of
mitochondrial membrane potential, as demonstrated by JC-1 staining, is involved in
sulindac’s ability to sensitize cancer cells to TBHP (Marchetti et al., 2009). Findings
from a more recent study using a combination of sulindac and DCA also support the
hypothesis that the mechanism sensitizing cancer cells by sulindac involves production of
ROS and mitochondrial dysfunction (Ayyanathan et al., 2012). The effect of sulindac on
both normal cells and cancer cells is most likely through its effect on the mitochondrial
respiration. This effect may be related to the altered mitochondrial respiration in cancer
cells, which makes malignant cells much more dependent on glycolysis for their ATP
33
production, unlike normal cells where the bulk of ATP is produced through the
mitochondrial oxidative phosphorylation. The metabolic dissimilarity between normal
and malignant cells, is yet another reason for investigating the use of IPC agents for anticancer therapy. Another finding that makes precondition agents ideal drugs against
cancer is that the use of preconditioning agents has not been limited to in vitro studies.
There are clinical studies that support the use of sulindac/drug combinations for cancer
therapy. These include a large study for treating the recurrence of colon polyps and the
other for treating actinic keratosis. Both have been successfully conducted with
sulindac/drug combinations (Meyskens et al, 2008; Resnick et al, 2009) suggesting a
novel therapeutic approach to cancer treatment is possible. Since it is well established
that the mitochondrion is the most likely end effector of IPC protective mechanisms,
these findings suggest that sulindac’s effect on mitochondria might be a crucial factor in
its anti-cancer activity. This idea gained further support when it was reported that
sulindac could function as an IPC agent and provide protection against ischemia
reperfusion induced damage in a cardiac model (Moench et al, 2009).
As mentioned earlier a cancer study using sulindac discovered that inhibition of PDE5 is
involved in inducing apoptosis (Tinsley et al, 2011) This led researchers to investigate if
a known PDE5 inhibitor can substitute sulindac in its anti-cancer role. Indeed, sildenafil,
a known PDE5 inhibitor, also exhibits the dual property of protecting normal cells and
sensitizing cancer cells, in a fashion similar to sulindac (Das et al, 2010). Unpublished
research from the Weissbach lab provides further evidence that sildenafil, can also
enhance killing of cancer cells, when used in combination with agents that affect the
mitochondria in assays with lung cancer (A549) and a tongue derived squamous
34
epithelial cancer (SCC25) cell lines. Sildenafil, most commonly known as the active
ingredient of the erectile dysfunction drug Viagra, functions as phosphodiesterase -5
(PDE-5) inhibitor. Given the wide distribution and involvement of PDE-5 in a variety of
pathways, much research has been devoted to exploring prospective novel applications of
sildenafil. Sildenafil binds to PDE5 and prevents the biotransformation of the second
messenger 3′,5′-cGMP to 5′-GMP, thus increasing intracellular cGMP levels. It has been
reported that high cGMP levels are associated with increased blood flow as a result of
vascular smooth muscle vasodilation (Shi and Tarbell, 2011). Sildenafil also has
a preconditioning-like cardioprotective effect against ischemia/reperfusion injury in the
intact heart (Kukreja et al, 2005). Mechanistic studies suggest that sildenafil exerts
cardioprotection through NO generated from eNOS/iNOS, activation of protein kinase
C/ERK signaling and opening of mitochondrial ATP-sensitive potassium channels (Das
et al, 2010). Additional studies showed sildenafil attenuates cell death resulting from
necrosis and apoptosis, and increases the Bcl2/Bax ratio through NO signaling in adult
cardiomyocytes (Salloum et al, 2003). Emerging new data further suggest that sildenafil
may be used clinically for treatment of pulmonary arterial hypertension and endothelial
dysfunction (Kukreja et al, 2005). The results of a cardiac study in a rabbit model,
provides evidence that sildenafil induces its cardioprotective role by influencing the
opening of mK(ATP) channels (Ockaili et al, 2002). Besides acting as a preconditioning
agent, recent studies have indicated that sildenafil also sensitizes cancer cells to
compounds that cause mitochondrial dysfunction. Sildenafil was reported to enhance the
sensitivity of breast cancer cells to doxorubicin without exacerbating its toxicity to either
bone marrow cells or macrophages (Di et al., 2010). Sildenafil also increased the
35
chemotherapeutic efficacy of doxorubicin in prostate cancer in vivo and ameliorated
cardiac dysfunction (Das et al, 2010). Demonstration of the protective effect against
oxidative stress and prevention/inhibition of cancer using a relatively safe and effective
FDA-approved PDE5 inhibitor such as sildenafil could have an enormous impact on our
understanding of pharmacologic preconditioning and its role as a potential treatment for
AMD and also a novel anti-cancer therapy (Kukreja et al., 2005, Das et al., 2010).
Figure 13 Structure of sildenafil: The chemical structure of sildenafil
which is commonly known as the active ingredient of the drug viagra (Figure
from NCBI Pubchem Compound).
.
Previous studies, suggesting the cancer cytotoxic effect of sulindac, a preconditioning
agent, on multiple cancer cell lines led to our testing of two preconditioning agents,
sulindac and sildenafil. Our goal was to use these two compounds, in combination with
different agents which induced mitochondrial oxidative stress, to produce enhanced
36
killing of cultured Rb cells, Y-79, and also attempt to elucidate the mechanism by which
these compounds function.
Peroxisome proliferator-activated receptor (PPAR)
PPARs are a family of nuclear transcription factors. This family is composed of three
ligand-activated transcription factors, each of which has specific functions, ligand
specificity and tissue distribution. Three isoforms of PPAR have been identified in
mammals: PPAR alpha (PPARα), beta/delta (β) and gamma (γ). PPAR isoforms are
involved in the regulation of transcription of several genes that are involved in a variety
of functions such as proliferation, apoptosis and differentiation in a number of different
cell types. During gene expression PPARs form a heterodimer receptor complex with the
9-cis-retinoic acid receptor (RXR). This complex is associated with a multiprotein corepressor complex which is dissociated upon the binding of a specific ligand or agonist to
the PPAR/RXR complex. As mentioned earlier in the “Introduction”, RXR alpha (RXRα)
has been reported as a possible target of sulindac and also sulindac binds to PPARγ
(Zhou et al., 2010). PPARγ was shown to be involved in sulindac-sulfide mediated
upregulation of p21, in a human prostate cancer cell line (Jarvis et al, 2005). Hence we
are interested in investigating the possible role of PPARs in sulindac’s protection of
normal cells and its ability to sensitize cancer cells to oxidative stress.
All three PPARs are known to be activated by fatty acid derived eicosanoids (See figure
16) (Willson et al, 2000; Rosen and Spiegelman, 2001). Oxidized fatty acids and
oxidized metabolites of linoleic acid, arachidonic acid and docosahexanoic acid have
been shown to act as agonists of PPARγ (Nagy et al, 1998). PPARγ acts as a key
component in lipid metabolism and is highly expressed in adipose tissue (Willson et al,
37
2000). Expression of PPARα is elevated in tissues having high rates of fatty acid
oxidation. Hence tissues such as liver, retina, skeletal muscle and brown fat exhibit
elevated PPARα expression (Braissant et al, 1996; Auboeuf et al, 1997; Lemberger et
al, 1996).
Figure 14 Function of PPARs: The PPARs bind to specific ligands and
interact with RXRs to control expression of target genes. PPARα and
PPARγ are activated by different specific ligands but both of them influence
genes associated with fatty acid metabolism (Figure adapted from Saltiel et
al, 1996).
38
It has been reported in previous studies that both PPARα and PPARγ are linked to
anti-inflammatory, antiangiogenic and anti-oxidant activity (Bordet et al, 2006; Arca et
al, 2007; (Han et al, 2003). These pathways are crucial for the progression of ROS
induced retinal diseases such as AMD. Therefore PPAR isoforms are of interest to
researchers developing therapies for AMD. It has already been demonstrated that
administration of the PPARα agonist, fenofibrate, results in a decrease in VEGF
resulting in inhibition of angiogenesis (Varet et al., 2003). Another aspect of PPARα
that further supports its role in protecting retina against oxidative stress damage is its
IPC activity. Lotz et al, 2011 demonstrated that inhibition of PPARα reverses the
protection offered by ischemic preconditioning (IPC) in a rabbit model of cardiac
ischemia/reperfusion.
Besides the possible role of PPARα in preconditioning another isoform, PPARγ has
been reported to be neuroprotective and also acts as a component in the mechanism of
certain anti-cancer drugs. As mentioned earlier in the introduction, RXRα which forms
a co-receptor with PPARs has been reported as a possible target of sulindac. Moreover,
PPARγ has also been shown to be involved in sulindac-sulfide mediated upregulation of
p21, in human prostate epithelial cell line (Jarvis et al., 2005). Thiazolidinediones
(TZDs) have also been reported to have anti-cancer properties in recent studies (Shen et
al., 2012). Besides their anti-cancer properties, TZDs also showed cytoprotection
against. In one such study, rosiglitazone, a well-known TZD, provided protection for
retinal cells against oxidative stress by upregulation of superoxide dismutase (SOD) in a
photoreceptor cell line (661W), retinal explants and also in an in vivo light damage
study (Doonan et al., 2009). Another PPARγ ligand, troglitazone, also demonstrated
39
cytoprotective action in cultured RPE cells exposed to oxidative stress induced by
TBHP (Rodrigues et al., 2011)and also protected RGCs from glutamate induced cell
death (Aoun et al., 2003). Hence there is much interest in investigating the possible role
of PPARs in the mechanism of sulindac’s action, both as a preconditioning agent and
also in its anti-cancer properties.
Figure 15: Chemical structure of the 3 glitazones used in this study (Adapted
from Cuffini et al, 2008).
40
CHAPTER-2: MATERIALS AND METHODS
Methods for ARPE19 experiments
Cell culture studies using RPE cells – For cell culture experiments human RPE cell
line ARPE19 (ATCC# CRL 2302) purchased from American Type Culture Collection
(Rockville, MD) was used. Cells were maintained in DMEM F-12 supplemented with
L-glutamine. The culture media also contained 10% fetal bovine serum, 100 units/ml
penicillin and 100ug/ml streptomycin at 37C and 5% CO2. Cells from passages two to
five were treated with either no drug or a range of concentrations of the experimental
drugs prior to exposing them to oxidative stress. Oxidative stress was induced
chemically by adding tert-butyl hydrogen peroxide (TBHP) or by exposing the cells to
UVB light. ARPE19 cells were plated in 96 well plates at a concentration of
10,000cells/well. Change in cellular viability was measured using the MTS assay kit
from Promega (Madison,WI).
Experiments involving TBHP induced oxidative stress – ARPE19 cells were grown
for 24 hours in 96 well plates in DMEM F-12 complete media. Cells were either treated
with no drug or preincubated with the experimental drug for 24 hours. The next day the
cells were exposed to a range of concentrations (175μM to 300μM) of TBHP for 24
hours. On the following day cell viability was measured by the MTS assay
41
(Promega) according to the manufacturer’s protocol and which was measured by
absorbance at 490 nm using a colorimetric microtiter plate reader (SpectraMax Plus384;
Molecular Devices).
Exposure of RPE cells to UVB radiation- For our UV radiation assays the ARPE19
cells were plated in 96 well plates. After 24 hours of incubation with or without our
drug the cells were exposed to UVB light (Ultraspec 2000, Pharmacia Biotech) that
emit wavelengths at a range of 290nm to 370nm. UVB light at intensities of either
800mj/cm2 or 1200mj/cm2 were used for our experiments.
The duration of exposure the two energy levels were determined using the formula :
Hλ= t X Eλ, where Hλ is the energy level (J/cm2) t is the duration of exposure in
seconds and the Eλ is the irradiance (W/cm2) of the UVB source. Irradiance was
measured at 1.3W/cm2, and the exposure times for energy levels of 800mj/cm2 and
1200mj/cm2 were calculated to be 9 minutes 14 seconds and 14minutes 24 seconds,
respectively.
Immediately after the UVB exposure the media was replaced with fresh complete
DMEM F-12 medium and after 24 hours of incubation at 37C and 5% CO2, cellular
viability was measured using the MTS assay.
Blocking of PKC pathways- To investigate the involvement of PKC pathway in the
preconditioning mechanism, the PKC inhibitor chelerythrine (Sigma) was used at a
concentration of 2µM. The inhibitor was added simultaneously with the drug prior to
exposing the cultured cells to oxidative stress.
42
To further analyze which specific isoform of PKC that plays the most prominent role in
the protective mechanism offered by sulindac, specific inhibitors for the two PKC
isoforms, PKCε and PKCδ were used. For PKCε the inhibitor was the peptide V1-V2
and the inhibition of PKC’s δ isoform was brought about by the addition of rottlerin
(Sigma). The inhibitors were added at the same time as the drug prior to exposing the
cells to TBHP. The PKCε isoform blocker, V1-V2 peptide, was added at a final
concentration of 10uM and rottlerin was used at a concentration of 3µM.
Cell viability assays- Cellular viability was determined using the CellTiter 96 Aqueous
One Cell Proliferation Assay from Promega (Madison, WI) as described elsewhere
(Marchetti et al, 2009). This assay contains a tertrazolium salt that is converted to
formazan dye by the activity of mitochondrial dehydrogenases. The change in colour
imparted by this conversion was detected using measuring absorbance at 490nm using a
colorimetric microtiter plate reader (Spectramax Plus 384, Molecular Devices).
Western blotting protocol- Western blotting was performed according to a protocol
described elsewhere (Chan et al, 2008). Western blotting was carried out on proteins
isolated from ARPE19 cells cultured in 60mm dishes with no drug or 200μM sulindac
or a combination of 200μM sulindac and 2μM chelerythrine. Actin and GAPDH was
used as control for standardizing protein concentrations. Two late phase preconditioning
markers, Hsp27 and iNOS were detected with antibodies (Santa Cruz Biotechnology).
Statistical analysis- Unless otherwise mentioned, results of all cell viability
experiments represent the mean of three replicates of a representative experiment. The
43
error bars indicate standard deviations. The means were compared using standard t- test
and P values <0.05 were considered to be statistically significant.
Methods for cancer cell assays
Y79 cell culture and viability assay- For our retinoblastoma assays the retinoblastoma
cell line, Y79 was purchased from ATCC. The cells were grown in RPMI-40 culture
medium and plated on poly-d-lysine coated 96 well plates for our cell viability assays.
For testing the anticancer properties of the drugs, Y79 cells were grown for 24 hours in
96 well plates in RPMI-40 complete media. They were either treated with no drug or
preincubated with a combination of pharmacological preconditioning agents and
chemicals that affect mitochondria function. On the following day cell viability was
measured by the MTS assay (Promega) according to the manufacturer’s protocol and
which was measured by absorbance at 490 nm using a colorimetric microtiter plate
reader (SpectraMax Plus384; Molecular Devices).
Killing of cancer cells with combination of IPC agents and oxidizing agents- Y79
cells were cultured and plated in 96 well plates by following the methods described
earlier in this methods section. The cells were then treated with either sulindac (600µM)
or sildenafil (20µM) in combination with a range of concentrations of different
chemicals that induce oxidative stress on the mitochondria. In this study we used
TBHP, H2O2, DOX, DCA and As2O3 in combination with the preconditioning agents.
Assays to determine mechanism of anticancer properties- To determine the
mechanism responsible for the anticancer role of this IPC agent and drug combination,
various chemicals were employed to inhibit or upregualte different components of a
44
proposed pathway. These chemicals were coincubated for 48 hours with our IPC drug
and oxidizing agent combinations. As described in the experiments with ARPE19 cells
we employed the same specific chemical inhibitors for blocking PKC and its isoforms.
Chelrythrine was used for nonspecific inhibition of PKC while its isoforms, PKC
epsilon and PKC delta were blocked using V1-V2 peptide (10 µM) and rottlerin (3µM),
respectively.
45
CHAPTER 3: RESULTS
A.Protection of retinal cells by sulindac
Sulindac and sildenafil protect ARPE19 cells from chemical oxidative stress and
UV induced photooxidative stress: As described in Methods two types of oxidative
stress were used in these experiments, either exposure of the RPE cells to an oxidizing
agent such as TBHP, or exposure to UV light. In these experiments the RPE cells were
pretreated for 24 hours with varying concentrations of sulindac, sulindac or sildenafil as
shown in the Figures. Figure 16A shows the effect of sulindac in protecting RPE cells
against varying concentrations of TBHP as measured by cell viability, whereas Figure
16B shows the protection of the RPE cells against UV damage by sulindac, using 1200
mjoules of UV radiation (see Methods). As seen in Figure 16A sulindac at
concentrations of 125 and 200 μM afforded essentially complete protection against
TBHP damage between 225-300 µM of TBHP. Figure 16B shows that sulindac, at 500
µM as well as sildenafil (20µM) can provide about 50% protection against 1200mj of
UVB exposure.
46
A
B
47
Figure 16: Protective effect of sulindac and sildenafil A) The effect of
preincubating ARPE19 cells with sulindac prior to exposing them to
chemical oxidative stress induced by TBHP. B) Shows the comparative
effect of two preconditioning agents, sulindac and sildenafil, on cultured
RPE cells exposed to UVB light exposure.
Protection by sulindac is independent of COX inhibition- To determine whether this
protective effect was due to the NSAID activity of sulindac, we used sulindac sulfone, a
metabolite of sulindac that has no NSAID activity. These results are shown in Figure
17. Sulindac sulfone at 200 μM concentration showed complete protection against UV
damage. It should also be noted that sulindac sulfone is not a substrate for the Msr
system which eliminates the possibility that the sulindac protective effect was related to
its being a substrate for the Msr enzymes and functioning as a catalytic anti-oxidant in
an ROS scavenging system (Levine et al, 2000; Weissbach et al, 2005).
48
Figure 17: Effect of non-NSAID sulindac sulfone against UVB damage: For this
assay RPE cells were preincubated with sulindac or sulindac sulfone, the oxidized
metabolite of sulindac, which lacks the NSAID activity.
Activation of PPARα is involved in the protective mechanism of sulindac
As mentioned earlier RXRα acts as an intracellular target for sulindac. Since it is
established that PPARs interact with RXR to form a co-receptor and PPARα is known
to play a role in preconditioning it was reasonable to investigate if PPARα is involved
in the cellular protection afforded by sulindac to ARPE19 cells. Another reason for
looking into the possible role of PPAR is, that as mentioned above, sulindac has been
shown to react with PPARγ in a cancer study (Jarvis et al, 2005). When sulindac was
substituted with the PPAR agonist, fenofibrate (6µM), it offered protection to cultured
ARPE19 cells against oxidative stress induced by both methods, TBHP (Figure 18A) or
UVB light (Figure 18B). To provide further evidence of the involvement of PPARα,
we tested the effect of chemical inhibitor of PPARα on sulindac’s protective ability
against oxidative stress. GW 6471, a PPARα antagonist, reversed the protection of RPE
49
cells against TBHP induced stress by sulindac, suggesting that this receptor is activated
by sulindac (Figure 18C). These results indicate that activation of PPARα might play a
significant role in the protection of RPE cells by sulindac.
A
50
B
51
C
Figure 18 Role of PPARα in protection by sulindac A: Effect of replacing sulindac
with the PPARα agonist, fenofibrate in ARPE19 cells assays with TBHP (A) or with
UVB (B). Figure C shows the effect of inhibiting PPARα with GW 6471, on protection
by sulindac.
Involvement of PPARγ in sulindac’s protective role- Since PPARα was shown to be
involved in the protection of RPE cells by sulindac we also investigated if another PPAR
isoform is also associated with preconditioning. Previous research has reported that PPAγ
agonists exhibited neuroprotective effects. Therefore we tested if TZDs, which are well
established PPARγ agonists, can replicate the protection afforded by the PPARα agonist,
fenofibrate. Three TZDs (rosiglitazone, pioglitazone and troglitazone) were tested in
protecting RPE from either TBHP or UVB light induced oxidative stress. Only
troglitazone demonstrated protection from both kinds of stress, while neither
52
rosiglitazone nor pioglitazone provided any protection against ROS induced damage in
cultured RPE cells (Figure 19A and B). These findings indicate that involvement of
PPARγ is unlikely in sulindac’s protective function and indicate that troglitazone may
have other targets besides PPARγ activation.
A
0.9
Cellular viability (Abs @ 490nm)
0.8
0.7
0.6
Control
0.5
Sul 200 μM
0.4
Trogl 5 μM
0.3
Trogl 10 μM
0.2
0.1
0
0
-0.1
175
200
225
TBHP (μM)
53
250
275
300
B
Figure 19: Protection of RPE cells by TZD: A: cultured RPE cells were
preincubated with two different concentrations of the PPARγ agonist,
troglitazone, prior to exposing the cells to TBHP induced oxidative stress.
B: The comparative effect of three different TZDs on cultured RPE cells
exposed to UVB light of 1200mj intensity.
Protection by sulindac is dependent on PKC- To understand the mechanism of
protection we looked more closely at the possible role of PKC in the protection of RPE
cells by sulindac, as was shown previously in cardiac studies (Moench et al., 2009). As
shown in figure 20, chelerythrine, a broad spectrum PKC inhibitor, significantly
reversed the protective effect of sulindac on cultured RPE cells against TBHP (Figure
20A) and UV damage (Figure 20B), suggesting that one or more isoforms of PKC were
54
involved in the sulindac protective effect. The IPC response in tissues is often initiated
by ROS and/or NO which activates mitochondrial PKCε as part of a complex
mechanism that also involves the opening of the mitochondrial K(ATP) (mK(ATP))
channel and preventing the formation of the mitochondrial permeability transition pore
(MPTP).
A
55
B
Figure 20 PKC involvement in preconditioning A: RPE cells were
coincubated with the PKC inhibitor chelerythrine (2μM) along with sulindac
to elucidate the role of this kinase in the protective pathway. B) The effect of
chelerythrine on protection by sulindac against UVB light is shown in this
figure.
PKCε but not PKCδ is involved in the preconditioning pathway- Based on previous
preconditioning studies it seemed reasonable to look specifically at PKCε. As shown in
56
Figure 21, V1-V2, a peptide inhibitor of PKCε almost completely reversed the
protective effect of sulindac in both the TBHP and UV systems. In contrast, rottlerin, a
PKCδ inhibitor, when used at 3 µM, a concentration reported to inhibit PKCδ, showed
no reversal of the sulindac protection against UV (Figure 21A). However, as will be
shown below, rottlerin inhibits the enhanced killing of cancer cells by sulindac.
A
57
B
Figure 21 Role of specific PKC isoforms in the protective pathway: A) UVB
assay and B) TBHP assay. Where indicated, PKCε was inhibited by the specific
inhibitor peptide V1-V2 (10μM), while PKCδ was inhibited by rottlerin (3μM).
Protection by sulindac is dependent on PKG: Protection produced by preincubation
with both sulindac and sildenafil was significantly reversed when PKG was inhibited
using, Rp-Br-8-PET-cGMPs (Figure 22). This result suggests that PKG is also involved
in the protective mechanism against oxidative stress, and it also serves as further
evidence for a preconditioning mechanism being involved in both sildenafil and
sulindac protection.
58
A
Cellular viability ( Absorbance @
490nm)
1
0.8
0.6
0.4
0.2
0
Control (No
drug, no UV)
No drug
Sulindac
PKG inhibitor S ulindac + PKG
inhibitor
B
0.6
Cellular viability ( @ 490nm)
0.5
0.4
No drug
0.3
Sildenafil
20uM
PKG
inhibitor
Sildfl + PKG
Inhibitor
0.2
0.1
0
0
-0.1
175
200
225
250
275
300
325
TBHP (μM)
Figure 22 Involvement of PKG in the preconditioning mechanism A: Inhibition of
PKG
with
Rp-8-Bromo-PET-cGMPS
reverses
the
protection
against
UVB
photooxidative stress offered by sulindac. B: Effect of Rp-8-Bromo-PET-cGMPS, on
the protection by sildenafil in the TBHP assay.
59
Role of ROS and the mK(ATP) channels in the protection of RPE cells against
oxidative stress by sulindac
As mentioned previously a key component in triggering the IPC pathways is the
generation of ROS by pharmacological preconditioning agents. In order to confirm the
role of ROS we coincubated cultured ARPE19 cells with the ROS scavenger, tiron
along with sulindac, prior to exposure to TBHP. The presence of tiron caused
essentially complete reversal of sulindac’s protection (Figure 23A) thus providing
further evidence in favor of the involvement of increased ROS levels in this protective
mechanism. Previous studies have also shown that the closure of MPTP, is crucial for
the survival of cells and the mK(ATP) channels function to keep the MPTP closed. To
test the role of the mK(ATP) channels in this system RPE cells were incubated with
sulindac and 5-hydroxydecanoic acid (5-HD), a chemical inhibitor of mK(ATP)
channels. As shown in Figure 23B, the presence of 5-HD caused almost complete
reversal of sulindac’s protective role, confirming the involvement of the mitochondrial
membrane channels. All of these data support the hypothesis that sulindac is protecting
the RPE cells by initiating an ischemic preconditioning response.
60
A
B
Figure 23 Role of ROS generation and mK(ATP) channels in sulindac
protection: A) Effect of tiron (1μM or 2μM) in inhibiting sulindac
61
protection. B) Effect of 5-HD (50μM or 75μM) in inhibiting the sulindac
effect.
Preconditioning markers are upregulated in cells incubated with sulindac- In the
previous cardiac study it was also shown that two late stage preconditioning markers,
iNOS and Hsp27 were induced by sulindac (Moench et al., 2009). As shown in Figure
24A and 24B there was significant induction of iNOS and Hsp27 in RPE cells
pretreated for 48 hours with sulindac, as compared with control, supporting the view
that sulindac is protecting by initiating an ischemic preconditioning response. The use
of the PKC inhibitor chelerythrine resulted in downregulation of both of the
preconditioning markers, indicating that this upregulation is dependent on PKC.
B
A
62
Figure 24 Induction of preconditioning markers: Western blotting
results (n=3) of proteins from cultured ARPE19 cells that were incubated
with either sulindac alone or coincubated with sulindac and a PKC
inhibitor, chelerythrine. Specific antibodies were used to detect the two
proteins, A) iNOS and B) Hsp27.
B. Killing of retinoblastoma cells by IPC/drug combinations
As mentioned above multiple previous studies have shown that besides protecting
normal cells by acting as a preconditioning agent, sulindac is also capable of enhancing
the death of various cancer cell lines when used in combination with agents that affect
mitochondrial function. So, the second aim of this project was to determine if sulindac
is capable of sensitizing a Rb cell line, Y79, to compounds that induce oxidative stress
in the mitochondria and if another preconditioning agent, sildenafil, can substitute for
sulindac. Our in vitro assays with Y79 cells also attempted to elucidate the mechanism
by which sulindac and other preconditioning agents enhance killing of cancer cells.
Sulindac sensitizes Y79 cells to agents that affect mitochondria
In the initial experiments Y79 was incubated with sulindac in combination with TBHP
according to the procedures described in the Methods section. As shown in Figure 25A
the combined treatment with sulindac and TBHP showed significant enhancement in the
loss of viability of cancer cells compared to cells that were treated with the oxidizing
agent alone. Preincubation with sulindac was also tested in Y79 cells exposed to H2O2.
Y79 cells were preincubated with sulindac for 48 hours and then exposed to
concentrations of H2O2 ranging from 250M to 1mM for 2 hours. As demonstrated in
63
Figure 25B the combination of sulindac and the oxidizing agent produced much higher
cell death as compared to treatment with H2O2 alone. Figure 27C shows the result of
coincubating Y79 with a combination of sulindac and DOX (50nm to 300nm) in 96 well
plates for 48 hours. Similar results were obtained in experiments using Y79 cells when
DOX was substituted with arsenic trioxide (As2O3) or dichloroacetate (DCA) and used
in combination with preconditioning agents. These cell viability assays were performed
using a combination of an IPC agent and oxidizing agents show that sulindac is capable
of producing significant enhancement in the death of Y79 cells when used in
combination with different agents that affect mitochondrial function.
A
64
B
65
C
Figure 25 Effect of sulindac on viability of Y79 cells: Sulindac (600μM)
was used in combination with other compounds that can induce oxidative
stress in the mitochondria. In these assays cultured Y79 cells were incubated
for 48 hours with sulindac in combination with one of the following A)
TBHP; B) H2O2; C) DOX) following which cell viability was measured.
Sildenafil also enhances death in cancer cells exposed to oxidative stress- Since
sulindac acts as a preconditioning agent and can also sensitize Y79 cells to agents that
affect the mitochondria, the next step was to investigate if another preconditioning
agent can replace sulindac in our system. As mentioned previously, findings from recent
studies indicate that sildenafil can also enhance cancer killing when used in
66
combination with drugs that affect mitochondrial function, but can protect normal cells
against oxidative stress. As shown above sildenafil protected RPE cells against both
TBHP and UV induced oxidative stress. Y79 cells were treated with sildenafil (in place
of sulindac) in combination with multiple agents that affect mitochondrial function, as
shown in figures 26A-C. In this study we used sildenafil at a concentration of 20M in
combination with either, DCA, DOX, and As2O3 for a period of 48 hours following the
procedures described in the “Methods” section. Results from our experiments with Y79
cells provide evidence that enhancement in cancer killing achieved by these dual drug
treatments is comparable to what we saw when using sulindac. This finding suggests
that sensitizing cancer cells to oxidative stress inducing compounds is not unique to
sulindac and it can be replaced by another preconditioning agent.
67
A
0.3
Cellular Viability
0.25
0.2
Only DCA
0.15
Sil +DCA
0.1
Sul + DCA
0.05
0
0
4
8
12
16
DCA (μM)
68
20
24
B
69
C
Figure 26: Effect of sildenafil and sulindac on Y79 cells exposed to
oxidative stress: Y79 cells were treated for 48 hours with combination of
sildenafil (20μM) or sulindac (400μM) and A) DCA, B) DOX, C) As2O3.
Effect of PPARγ agonists on cancer cells
As mentioned earlier in this dissertation, previous studies have shown that TZDs exhibit
anti-cancer properties. We wanted to investigate whether PPARγ plays any role in the
killing of cancer cells by sulindac. To test the hypothesis we wanted to see if known
PPARγ ligands could substitute for sulindac in its cancer killing role. In our assays with
Y79 cells we treated the cells with three different TZDs, rosiglitazone, pioglitazone and
70
troglitazone in combination with agents that affect the mitochondria and as seen with
sulindac. The procedures were similar to our assays described with sulindac. As shown in
Figure 27A and 27B the enhanced killing of the cancer cells by two of the TZDs,
pioglitazone and rosiglitazone were comparable to the anti-cancer effects seen with
sulindac and sildenafil. The PPARγ agonist rosiglitazone (RGL) enhances the death of
Y79 cells in presence of As2O3 (Figure 27A) and another PPARγ agonist pioglitazone
when used in combination with TBHP also produces increased killing of cultured Y79
cells (Figure 27B). However, troglitazone, did not produce any enhanced cancer killing in
the presence of an oxidizing agent (data not shown). Since it is well know that
troglitazone acts as an agonist of PPARγ and TZDs can have other cellular targets, these
findings led us to believe that targets other than PPARγ are responsible for the cancer
killing properties of pioglitazone and rosiglitazone, and it is unlikely that PPARγ is
involved in the mechanism by which preconditioning agents sensitize cancer cells to
oxidative stress.
71
A
72
B
Figure 27: Effect of treating Y79 cells with PPARγ agonists in
combination with compounds that induce oxidative stress: Y79 cells
were coincubated with TZDs and compounds that induce oxidative stress on
the mitochondria. A) rosiglitazone or sulindac in combination with As2O3
and B) pioglitazone with TBHP.
Enhanced killing of cancer cells by sulindac is dependent on PKC
As reported earlier in our protection studies with RPE cells, in this current study as well
as in previous protection studies in the cardiac model (Moench et al., 2009) sulindac’s
73
cytoprotective role is dependent on PKC. We wanted to study if the protection and
cancer killing mechanisms involve a similar pathway. Consequently, we looked into the
role of PKC by studying the effect of a chemical inhibitor of PKC, chelerythrine, along
with sulindac. Cultured Y79 cells exposed to DOX, were coincubated with sulindac and
chelerythrine for 48 hours following which, cellular viability was determined (Figure
28). The results show that chelerythrine significantly reverses the enhanced killing of
Y79 cells produced by the combination of sulindac and DOX. The involvement of one
of the PKC isoforms in the mechanism of killing by sulindac is evident from these
results.
Figure 28 Effect of PKC inhibition by chelerythrine on the anti-cancer
effect of sulindac: This graph shows the effect of adding the PKC inhibitor
chelerythrine (2μM) in presence of sulindac and DOX combination in our
assay with the Y79 cell line.
74
Role of PKC isoforms in the anti-cancer effect of sulindac- An obvious next step in
elucidating the mechanism of cancer killing was to investigate which specific PKC
isoform is associated with the anti-cancer role of sulindac. Findings from previous
research indicated that among the different PKC isoforms, PKCδ is most likely to be
involved in apoptotic pathways (Brodie and Blumberg, 2003; Steinberg, 2004) while
the ε isoform is more likely to be associated with the protective function of sulindac.
Our results show that inhibition of PKCδ by the specific inhibitor rottlerin resulted in
reversal of the killing effect of sulindac and DOX (Figure 29). However specific
inhibition of the PKCε isoform with V1-V2 yielded no significant effect (Figure 29).
This is in agreement with unpublished research work from the Weissbach laboratory,
performed on other cell lines which showed, PKCδ inhibition can reverse killing by
other sulindac drug combination treatments. Moreover as shown above in our protection
assays with RPE cells the inhibition of PKCδ did not have any significant effect on
protection whereas inhibiting the ε isoform reversed protection (Figure 21). This
indicates that these PKC isoforms are involved in two distinct pathways.
75
Figure 29 Role of PKC isoforms in the anti-cancer effect of sulindac: To
determine which specific PKC isoform is important for the enhanced killing
effect of preconditioning agents in cancer assays, two specific chemical
inhibitors were used. 3μM rottlerin was used to inhibit PKCδ while the
peptide V1-V2 (10μM) was used to inhibit PKCε isoform.
ROS generation by our drug combination is required for killing of cancer cells: As
shown above in the RPE protection data, the generation of ROS by preconditioning
agents is a crucial component of the protective mechanism of sulindac. We looked into
the possible role of ROS in the killing of Y79 cells. Moreover, it has been shown in
previous research that ROS generation and the loss of mitochondrial membrane
potential induced by this increased ROS, has an important role in the cytotoxic effect of
sulindac against malignant cell lines. So in this study the ROS scavenger N76
acetylcysteine (NAC) was used to determine the role of ROS in sulindac’s effect on
Y79 cells. We coincubated the Y79 cells with NAC and sulindac for 48 hours along
with our oxidizing agent, DOX. Measurement of cell viability indicated that the ability
of sulindac to sensitize Y79 cells to agents that induced oxidative stress in the
mitochondria is reduced in the presence of NAC. These data are in accordance with
findings from prior studies and provide further evidence that ROS generation is
involved in the enhanced killing of cancer cells using sulindac/drug combinations.
Figure 30 Involvement of ROS in enhanced killing of cancer cells by
preconditioning agents: To investigate the role of ROS in the anti-cancer
function of sulindac in our Y79 assay the ROS scavenger NAC (2mM) was
added along with sulindac during the 48 hours incubation of the cancer
cells.
77
Enhanced killing of cancer cells by pharmacological preconditioning is not limited
to Rb
The enhancement of killing of malignant cells by a combination of preconditioning
agent and a compound that affects mitochondrial function was not limited to cultured
Rb cells. As seen in Figure 31, either sulindac or sildenafil markedly enhanced the
killing of two other cancer cell lines in combination with different compounds that
cause oxidative damage. These studies were done in collaboration with Dr Ayyanathan
and Dr. Kesaraju from Dr Weissbach’s laboratory. In most cases sulindac was more
effective than sildenafil in enhancing the killing of different cancer cell lines. Figure 31
shows that treating with preconditioning agents, sulindac and sildenafil, sensitize lung
cancer (A549) and squamous cancer (SCC25) cell lines to compounds that induce
oxidative stress. These findings demonstrate that sensitization of cancer cells to
oxidative stress by preconditioning agents is not restricted to Rb cells. This provides
evidence that this dual drug therapy can be adapted to target multiple malignant cells
and thus adds further therapeutic value to this mode of anti-cancer treatment.
78
A
Effect of IPC agents on lung cancer cell line (A549)
B
Effect of IPC agent on lung cancer cell line (A549)
79
C
Effect on lung skin cancer cell line (SC225)
Figure 31: Effect of treating lung cancer cell line with IPC/drug combination: A)
and B) The effect of IPC/cancer drug combination on a lung cancer cell line (A549). C)
Effect of combination of sulindac and DCA on skin cancer cell line (SC225) (The
studies with A549 and SC225 were done in Dr. Weissbach’s laboratory. The A549
studies were done by Dr. Kesaraju and Dr. Ayyanathan did the assay with SC225 cell
line).
80
CHAPTER 4: DISCUSSION
It is apparent that there is significant difference between the metabolism of a cancer cell
and a normal cell and this can be utilized to design an anti-cancer chemotherapeutic
mechanism which is not only efficient in targeting malignant cells but also highly
selective. Results of experiments described here show that preconditioning agents offer
protection in normal retinal cells while they have a cytotoxic effect on Rb cell lines,
when used in combination with compounds that effect the mitochondria.
As mentioned previously in this study, AMD is one of the leading causes of blindness
and ROS induced damage is a key reason for the damage inflicted on the retina in
AMD. The two types of retinal cells that are crucial to the health of the retina and are
highly vulnerable to oxidative stress insult are the RPE and photoreceptor cells.
Degeneration of these two cell types are the hallmark of pathology in AMD.
We employed pharmacological preconditioning to protect cultured RPE cells (ARPE19) against chemical oxidative stress induced by incubation with a range of TBHP
concentrations and photooxidative stress induced by UVB irradiation. As described in
the results section preincubation with sulindac as well as sildenafil showed significant
reduction in loss of cell viability in both types of oxidative stress. These findings are in
81
accordance with earlier studies where both of these drugs protected normal cells against
ischemic stress (Moench et al., 2009; Das et al., 2010). Sulindac displayed protection
against ischemic insult in a cardiac model both in vitro and ex vivo (Moench et al.,
2009). Sildenafil was also shown to have cardioprotective properties in multiple studies
including an in vivo study conducted on a rabbit model (Das et al., 2010).
After the initial experiments with sulindac and sildenafil, it was evident that both the
drugs were protecting retinal cells against oxidative stress. The logical next step was to
perform experiments aimed at understanding the underlying mechanism by which they
were protecting. The most likely possibility was that sulindac is protecting through its
well established COX inhibition property. However, the results of our RPE study
showed that sulindac’s protective property was independent of NSAID property. In an
earlier cardiac study the protection of cardiac cells was also proven to be independent of
sulindac’s NSAID, property. In this current study, sulindac sulfone, the oxidized
metabolite of sulindac, which lacks the COX inhibition property, was able to protect
cultured ARPE19 cells against UVB irradiation. Moreover, sildenafil which is not an
NSAID, also protected the cells against UV light induced photooxidative stress as well
as chemical oxidative stress induced by TBHP.
Numerous signaling pathways have been implicated in various pharmacological
protective mechanisms against oxidative stress that are linked to fluctuations in ROS
levels. As mentioned earlier the mitochondria play a key role in loss of retinal cell
viability under high ROS levels (Beatty et al., 2000). Findings from previous studies
strongly indicated that ROS acts as an initiator or mediator in the protective mechanism
82
(Bolli, 2000). Hence one obvious possibility is that sulindac protected the retinal cells
by inducing preconditioning against oxidative damage. Previous studies showed that
sulindac is an ischemic preconditioning agent that can protect cardiac tissue against
ischemia/reperfusion damage (Moench et al., 2009). Sildenafil has also been shown to
protect cardiac tissue against ischemic stress through a preconditioning pathway
(Kukreja et al., 2005). It seemed reasonable to investigate whether both sulindac and
sildenafil are acting as preconditioning agents in our experiments with retinal cells
which are known to have a strong IPC response.
One of the key elements in the preconditioning protective pathway is believed to be the
protein kinase, PKC. The importance of PKC in preconditioning was first highlighted
by Baxter et al (Baxter et al., 1995) in cardiac ischemia experiments performed in a
rabbit model. The results of this study showed that application of chelerythrine, a
general inhibitor of PKG isoforms, inhibited the infarct-sparing effects of
preconditioning. More specifically, preconditioning by sulindac, has been linked to
involvement of PKC in the cardiac study performed by Moench et al (Moench et al.,
2009). In this earlier study, it was shown that sulindac protected cardiac myocytes
against hypoxia/reoxygenation and feeding sulindac to mice can reduce infarct size in a
Langendorff model. The protection against infarct size was abrogated when mice were
fed chelerythrine along with sulindac, indicating an involvement of PKC. In our current
study we also saw a similar reversal of the protective effect, in both the UV assay and
oxidative stress assays using TBHP, when chelerythrine and sulindac were used
together (Figure 15). This provides further evidence for involvement of PKC in our
protective mechanism.
83
To further analyze the role of PKC we investigated the specific part played by key PKC
isoforms in our therapy. Protein kinase C consists of a family of ten isoforms, which
have been divided into 3 groups. The classical isoforms (α, βI, βII, and γ) which require
phospholipids and calcium for activation, the novel isoforms (δ, ε, θ, and η,) which are
not reliant on calcium for activation and the atypical isoforms (ζ, and ι/λ) which can be
activated in the absence of diacylglycerol and calcium (Dempsey et al., 2000). While
these PKC’s have been documented as key kinases in many cellular functions, the novel
isoforms, ε and δ, have been most documented in protective and apoptotic roles in
cancer and normal cells, respectively. The preconditioning response is most often
initiated by ROS and/or NO which activates mitochondrial PKCε as part of a complex
mechanism that also involves the opening of the mK(ATP) channel and prevention of
formation of the mitochondrial permeability transition pore (Bolli, 2000). Based on
previous studies it seemed reasonable to look specifically at PKCε. We employed the
specific PKCε blocker V1-V2 peptide to selectively inhibit the ε isoform while rottlerin
was applied to inhibit PKCδ. Results of our assays indicate that in oxidative stress by
both UVB and TBHP the inhibition of PKCε resulted in reversal of the protective effect
of sulindac (Figure 22). However specific inhibition of PKCδ with rottlerin showed no
significant alteration in the protective influence of sulindac was observed (Figure 22A).
These findings are in accordance with earlier studies which suggest that PKCδ is pro
apoptotic while the PKCε facilitates cellular survival.
As mentioned previously sulindac has been reported to interact with RXR in a cancer
study (Zhou et al., 2010) and more importantly, PPARγ has been shown to be involved
in sulindac sulfide mediated upregulation of p21 in study on human prostate cancer cell
84
line (Jarvis et al., 2005). At this stage, it seemed logical to look into PPAR since RXR
and PPAR are known to function as a co-receptor. Based on the findings of previous
studies investigating the role of different PPAR isoforms in preconditioning we decided
to specifically look into the role of PPARα. The findings from our assays involving the
PPARα agonist fenofibrate, and reversal of sulindac’s protection by a PPARα
antagonist, provide evidence in support of the involvement of PPARα in the protection
of RPE cells by sulindac. We also investigated the possible involvement of the PPARγ
isoform in protection of RPE cells. For this purpose we investigated the protective
ability of three different TZDs, pioglitazone, rosilglitazone and troglitazone, all of
which are well established PPARγ agonists, against both TBHP and UVB light in
ARPE19 cells. Among these three, only troglitazone protected against both TBHP and
UVB damage while the two other agonists failed to protect against either forms of
oxidative stress. Moreover, sulindac’s protective ability against TBHP, was not
significantly reduced when it was coincubated with a PPARγ antagonist (data not
shown). Since the mechanism seems to be independent of PPARγ it is fair to assume
that some target of glitazones other than PPARγ is most likely involved in this
mechanism and this target is most likely associated with mitochondrial function.
Among the various cellular targets of TZDs, the mitochondrial protein mitoNEET
seems to be an ideal candidate owing to its vital role in mitochondrial respiration
(Paddock et al., 2007). Besides mitoNEET PPARγ agonists are also known to inhibit
mitochondrial pyruvate carrier (MPC) (Divakaruni et al., 2013)and this might be
involved in TZDs role in protecting RPE cells against oxidative stress induced damage.
85
These findings strongly indicate that only PPARα is involved in the IPC mechanism
triggered by sulindac. This activation of PPARα by sulindac in RPE cells is novel and is
an important finding since earlier studies have reported a role of PPAR in various age
onset diseases including AMD (Varet et al., 2003). Different isoforms of PPAR have
been also linked to the progression of Alzheimer’s and Parkinson’s disease (Perl, 2000;
Forno, 1996). Since one of the aims of this current study is to develop a therapy for
AMD, the property of PPAR, which adds further clinical value to our findings is its
ability to influence various components, such as, VEGF, docosahexaenoic acid (DHA)
and matrix metalloproteinase (MMP), which participate in the progression of AMD
(Varet et al., 2003). Protection of normal cells by IPC through PPARα activation, as
demonstrated here in RPE cells, has been reported earlier in other organs such as liver
and heart (Massip-Salcedo et al., 2008; Lotz et al., 2011).
PPARα agonist
inhibited MAPK expression following ischemia-reperfuison and this in turn inhibited
adiponectin accumulation in steatotic livers and adiponectin-worsening effects on
oxidative stress and hepatic injury (Massip-Salcedo et al., 2008) The therapeutic value
of IPC through PPARα activation has also been displayed in a cardiac study on New
Zealand white rabbits where it was reported that IPC against myocardial infarction is
mediated by activation of PPARα (Lotz et al., 2011). The same study also reported an
increase in mRNA levels of iNOS associated with activation of PPARα and IPC. The
findings of our current study in combination with the results reported previously by
other researchers, provides confirmation that activation of PPARα is essential for the
IPC triggered by sulindac and this finding can have therapeutic value with respect to
AMD.
86
Another component reported to play an important role in the preconditioning pathways
is protein kinase G (PKG). In a cardiac study, bradykinin, a known preconditioning
agent, offered cardioprotection in rat hearts against ischemia-reperfusion injury (Pasdois
et al., 2007). This cardioprotecctive ability of the drug was lost when a chemical
inhibitor of PKG (KT-5823) was administered along with it (Pasdois et al., 2007),
indicating the necessity of PKG in IPC pathways. In a different cardiac study PKG was
reported to induce the opening of mK(ATP) channels, which are known to be involved
in IPC (Costa et al., 2005). The findings from this study also indicated that PKG plays a
pivotal role serving as the terminal cystosolic component in the IPC protective
mechanism and conveys the protective signal to the mitochondrial membrane from the
cytosol, by a pathway that involves PKCε (Costa et al, 2005). Since the mitochondria
are believed to be the end effector of the IPC pathway these results further highlight the
significance of PKG and PKCε in IPC mechanism. So it was logical to investigate if
this activation of PKG might also play a role in the pharmacological preconditioning
offered by sulindac that protects ARPE19 cells against oxidative stress induced death.
In order to determine the involvement of PKG we used a chemical inhibitor of PKG
along with sulindac in our assay with UVB light. As shown in figure 22A, the PKG
inhibitor, Rp-8-Br-PET-cGMPS, prevented the protection against photooxidative stress
from UVB irradiation seen with sulindac. This finding adds further confirmation to our
hypothesis that suggests involvement of preconditioning pathways are involved in the
protective mechanism. Our data suggesting both PKC and PKG are involved in the
protective mechanism of sulindac are also consistent with a preconditioning
mechanism. But these two biomolecules function as upstream markers for several other
87
pathways. So, to add more credence to our hypothesis of preconditioning we felt it was
imperative to look for up-regulation of the levels of downstream markers of late phase
preconditioning.
iNOS is known to be a major contributor to late myocardial ischemic preconditioning,
and targeted deletion of the iNOS gene has been shown to prevent the preconditioning
induced by a range of stimuli including ischemia (Moench et al., 2009) adenosine
agonists (Zhao and Kukreja, 2002), and exercise in a mouse model (Guo et al., 1999). In
a study performed in the rat retina, two pharmacological iNOS inhibitors,
aminoguanidine and L-N6-(1-iminoethyl) lysine, inhibited protection achieved through
ischemic preconditioning (Sakamoto et al., 2006). Analyses of both mRNA and proteins
isolated from retina of rats exposed to retinal ischemia showed an increase in expression
of Hsp27 in both neuronal and non-neuronal retinal cells (Li et al., 2003). In RGC-5
cells treated with CoCl2, as well as in the retina of animals fed CoCl2, there is induction
of Hsp27 alongside increased HIF-1α levels suggesting a significant role of Hsp27 in
protection of the retina through ischemic preconditioning (Whitlock et al., 2005).
The protective effect we have seen in this study combined with the assays showing the
involvement of PKC are consistent with our conclusion that sulindac is acting as a
pharmacological preconditioning agent. As reported in the results section western
blotting of retinal proteins revealed that ARPE19 cells incubated with sulindac had
significant upregulated levels of both Hsp27 and iNOS relative to ARPE19 cells that
were not treated with the drug (Figure 25). Moreover the levels of both proteins were
reduced in cells that were co-incubated with sulindac and the PKC inhibitor
88
chelerythrine. This provides further evidence in support of our suggested hypothesis
that protection offered by sulindac is dependent on preconditioning mechanisms.
It is evident from the cardiac studies showing the protection against ischemic stress by
the preconditioning agent sildenafil, that PKG is involved in the pathways through
which sildenafil preconditions cardiac tissues, since sildenafil raises the levels of cGMP
in cells (Hassan and Ketat, 2005). Activation of PKG has also been associated with the
anti-cancer activity of sulindac sulfide. To determine the possible role of PKG in
sulindac’s protective efficacy we used a chemical inhibitor of PKG in our assays with
ARPE19 cells. The results of these experiments reveal that inhibition of PKG by Rp-Br8-PET- cGMPS produces partial reversal of sulindac’s protection against oxidative
stress. These findings suggest that activation of PKG might be necessary for
preconditioning of retinal cells by sulindac.
The mitochondria are considered to be the main source of endogenous ROS and it is
also one of the main targets of oxidative stress induced cytotoxicity. Since sulindac is
known to affect cancer cells by inducing production of ROS in the mitochondria when
used in combination with oxidizing agents (Marchetti et al., 2009) and ROS is known to
play a role in inducing preconditioning , it is believed that even in this role as a
pharmacological preconditioning agent of retinal cells, sulindac has some influence on
the mitochondrial ROS levels. Elevated ROS level might be the underlying mechanism
by which downstream PKC activation is triggered in sulindac treated RPE cells. As
demonstrated by our cultured ARPE19 experiments with the ROS scavenger, tiron, the
presence of an ROS scavenger decreases sulindac’s ability to protect RPE cells against
89
TBHP induced oxidative stress (Figure 24A). Previous studies have suggested that
generation of ROS plays an important role in protection provided by pharmacological
preconditioning agents against oxidative stress (Kukreja, 2001). Multiple studies have
reported that the opening of mK(ATP) channels plays an essential role in the protecting
provided by preconditioning (Ettaiche et al., 2001; Ockaili et al., 2002). In a rat model
of retinal ischemia induced by increasing intraocular pressure, ischemic preconditioning
was mimicked by injecting the animals with cromakalim, a known mK(ATP) channel
opener. The results indicate that mK(ATP) channels are essential in enhancing the
resistance of retinal cells against ischemic insult (Ettaiche et al., 2001). In its protective
role seen in the RPE in vitro studies, sulindac might be affecting the mK(ATP) channels
which are believed to play a major role in ischemic preconditioning. It is well
established that opening of the mK(ATP) channels affect the mitochondrial
permeability transition pores (MPTP) and prevents them from opening. Keeping the
MPTPS closed prevents release of cytochrome c into the cytoplasm and thus prevents
cell death. In order to establish the role of mK(ATP) channels in the protective pathway
initiated by sulindac we coincubated cultured RPE cells with a mK(ATP) channel
blocker 5-HD along with sulindac prior to exposing the cells to TBHP. When compared
to cells preincubated with sulindac alone the presence of 5-HD resulted in a major loss
of cellular viability. The results of our experiments with 5-HD combined with data
reported previously by others suggest that sulindac prevents the opening of the MPTP
through its influence on the mK(ATP) channels and this might be a key end effector of
the IPC protective pathway (as shown in figure 31).
90
Figure 32 Proposed pathway of IPC protective pathway: PKC and iNOS
activity play important roles in preconditioning with the mitochondrial
membrane being the final target (Adapted from book chapter by Prentice
and Weissbach, 2012).
The results of this current study provide substantial evidence that application of
sulindac as a preconditioning agent can have therapeutic value in protecting RPE cells
against initiation and progression of AMD. Since there is compelling evidence that
oxidative stress is one of the major factors in macular damage in AMD (Beatty et al.,
2000) a therapy that is dependent on preconditioning and also possibly affects the
mitochondria will be ideal for protecting metabolically highly active tissues, such as the
retina, against ROS induced damage.
Besides the protective role of sulindac against oxidative stress in retinal cells, there may
be another important role for sulindac that could also be possibly related to its role as an
91
IPC agent. Sulindac and its metabolites, including sulindac sulfide and sulindac sulfone,
were shown to have anti-cancer activity more than 15 years ago and have been
intensively studied as anti-cancer agents (Waddell and Loughry, 1983). A unique more
recent observation relates to the difference in how cancer and normal cells react to
sulindac and oxidative damage. It was found that the protective effect of sulindac, seen in
earlier studies using normal lung and cardiac cells exposed to oxidative stress, was not
seen when cancer cells were treated in a similar manner (Marchetti et al., 2009). It was
observed that sulindac, at concentrations that are not toxic to cancer cells, sensitizes
cancer cells to oxidizing agents, resulting in enhanced cell death by apoptosis (Marchetti
et al., 2009, Ayyanathan et al., 2012). Indeed, there were several other reports of killing
of cancer cells using sulindac in combination with agents that affect mitochondrial
respiration resulting in the production of ROS (Minami et al., 2005; Meyskens et al.,
2008; Jin et al., 2008). These cell culture experiments appear to have clinical relevance.
Two clinical studies have been reported using sulindac/drug combinations suggesting a
novel therapeutic approach for cancer treatment. In a small proof of concept study, the
combination of sulindac and H2O2 was used to treat actinic keratoses, a precancerous skin
lesion (Resnick et al., 2009). The anti-cancer efficacy of sulindac used in combination
with a compound that affects mitochondrial function was also tested in a 3 year long
clinical study on patients who had colon polyps surgically removed. The findings of this
study showed that the combination of sulindac and DFMO an ornithine decarboxylase
inhibitor, resulted in a >80% reduction in both the recurrence of the polyps and also
reduced the appearance of colon carcinoma (Meyskens et al., 2008). Here we investigated
if sulindac can enhance killing of the retinoblastoma cell line, Y79, when used in
92
combination with chemicals that affect the mitochondria. In our study we found that both
sulindac and sildenafil were able to sensitize Y79 cells to multiple chemicals including
DOX, DCA, As2O3 and TBHP.
The findings from our assays showing the protection of ARPE19 cells against UV and
TBHP induced stress indicates that PKC plays an important role in the protective
pathway initiated by pharmacological preconditioning agents. To understand the
involvement of PKC in the enhancement of killing of cancer cells we initially, used the
non-specific PKC inhibitor, chelerythrine, in combination with sulindac and oxidizing
agents in our in vitro Rb cell assays. This compound caused significant reversal of the
killing effect of sulindac in cultured Y79 cells. The results of these experiments clearly
indicate that PKC is also involved in the sulindac enhanced killing effect. The next
logical step in our study was to determine which of the PKC isoforms plays the most
crucial role in the cytotoxicity induced by sulindac in tumor cell lines. Therefore, as a
follow up investigation to the chelerythrine experiments, where classical and novel
isoforms were inhibited, we tested both the PKCδ inhibitor, rottlerin and the peptide
V1-V2, an inhibitor of PKCε. The results of these experiments confirmed our
hypothesis that sulindac does require PKCδ for the enhanced killing of cancer cells with
oxidative stress, since inhibiting PKCδ with rottlerin reverses the cancer killing effect of
sulindac by up to 75% (Figure 28). However inhibition of PKCε using the specific
inhibitor V1-V2 peptide did not produce any noticeable reversal of the killing. From
these results it can be concluded that PKCδ is a major regulator of cell death by
sulindac and oxidative stress treatment in cancer cells. This is in agreement with prior
research in an animal model of skin cancer, that demonstrated PKCδ over-expression
93
inhibited tumor development (Reddig et al., 1999). In multiple previous cancer studies
it has been reported that PKCδ activation was found to be apoptotic in lung, colon,
prostate, and gastric cancers (Nakagawa et al., 2005; Persaud et al., 2005; Cerda et al.,
2001; Tanaka et al., 2003). The findings from the experiments with the non-specific
PKC inhibitor, chelerythrine or the specific isoform inhibitors, rottlerin and V1-V2
peptide, indicate PKC plays an important role in determining the effect of
preconditioning agents on both malignant cells and normal cells and these drugs cause
the up-regulation of different PKC isoforms in these two different types of cells. The
results suggest that in cancer cells sulindac favors the activation of PKCδ, whereas in
cultured ARPE19 cells it confers protection by elevating the PKCε isoform. Since
PKCδ, is known to be anti-apoptotic and the ε isoform has been proven to facilitate
survival this difference in the effect of sulindac on PKC isoforms might provide an
answer to how pharmacological preconditioning agents can be detrimental to cancer
cells, yet protective in normal cells subjected to oxidative stress. Another significant
difference in the role of protein kinases, in the anti-cancer and protective properties of
pharmacological preconditioning agents, is the part played by PKG. While PKG
inhibition by Rp-Br-PET-cGMPS lead to reversal of protection, the use of the same
chemical inhibitor of PKG in our cancer killing assays did not produce any noticeable
change in sulindac’s anti-cancer property (data not shown).
We believe the mechanism by which sulindac sensitizes cancer cells to agents that
affect mitochondrial respiration has similarities with the IPC mechanism by which
preconditioning agents protect normal cells against oxidative stress. One key parallel
between the effect of pharmacological preconditioning agents on both normal retinal
94
cells and various cancer cells, including Rb, is the involvement of the mitochondria.
Considering the findings of previous cancer studies, we believe that the cancer killing
effect of sulindac exhibited in cultured Y79 and other cancer cell lines may be
associated with the Warburg effect. Warburg, more than 60 years ago, showed that
cancer cells obtain less energy from respiration compared to normal cells, even in the
presence of sufficient oxygen, and suggested that there was a defect in mitochondrial
respiration in cancer cells (Warburg, 1956). As a result, any added strain on respiration
in cancer cells, such as seen when glycolysis is inhibited, or pyruvic dehydrogenase
activity is up-regulated is toxic to cancer cells, but not normal cells (Warburg, 1956). It
may not be a coincidence that sildenafil, another IPC agent, has also been shown to
enhance the killing of prostate cancer cells when used in combination with DOX. In
these studies sildenafil also protected the heart against damage due to DOX (Das et al.,
2010). Even in this current study we saw a similar effect of sildenafil on Rb cells
exposed to agents that influence the mitochondria. Thus, there may be some connection
between the ability of IPC agents to protect normal cells against oxidative damage and
their ability to sensitize cancer cells to agents that affect mitochondrial respiration.
Findings of this current study as well as earlier reports strongly suggest that sulindac’s
influence on both normal and cancer cells is dependent on its effect on the
mitochondria. In an earlier cancer study by Marchetti et al, it was shown using JC-1
staining that sulindac preincubation induces loss of mitochondrial membrane potential
in cancer cells (Marchetti et al., 2009). As reported earlier in the results section the
cancer killing effect of sulindac was successfully replicated by TZDs compounds,
pioglitazone and rosiglitazone, both of which act as PPARγ agonists. However they
95
appear to be acting by a PPARγ independent pathway. Since inducing mitochondrial
dysfunction and loss of mitochondrial membrane potential are believed to be essential
for the cancer killing mechanism of sulindac it seemed likely that a mitochondrial target
of TZDs is involved in their cancer killing effect. One such possible target may be the
mitochondrial membrane protein mitoNEET which has been shown to be a target of
pioglitazone and is believed to play an important role in redox sensitive signaling in the
mitochondria (Wiley et al., 2007; Paddock et al., 2007). It has previously been reported
that upregulation of MitoNEET in epithelial cancer cells leads to formation of oxidative
phosphorylation protein complexes and increases tumor growth by about 3 fold (Salem
et al., 2012). At this stage the role of mitoNEET is speculative and more detailed
investigation is required to establish if it is involved in this dual role of sulindac.
It is evident from the results of the experiments described in this project that sulindac
has drastically different effects on normal cells and tumor cells, especially when either
type of cells are under the influence of chemicals that affect mitochondrial function.
However the mechanism by which sulindac or sildenfail enhance cancer cell death and
protection in normal cells share some key components. Findings described by earlier
researchers combined with our observations in this current study indicate that two
components that play decisive roles in both normal and cancer cells are the influence
the drugs exert on the kinase PKC and the mitochondrial membrane potential. The
findings from the experiments with the non-specific PKC inhibitor, chelerythrine or the
specific isoform inhibitors, rottlerin and V1-V2 pepetide, seem to indicate PKC plays an
important role in how preconditioning agents affect both malignant cells and normal
cells. Preconditioning agents cause the upregulation of different PKC isoforms in these
96
two different types of cells. The findings of this current study with retinal cells suggest
that in cancer cells sulindac favors the activation of PKCδ, whereas in cultured ARPE19
cells it confers protection by elevating the PKCε isoform. Since PKCδ, is known to be
pro-apoptotic (Brodie and Blumberg, 2003); (Steinberg, 2004)and the PKCε isoform is
known to facilitate survival, this differential effect of sulindac on the PKC isoforms
might provide an answer to how pharmacological preconditioning agents can be
detrimental to cancer cells, yet protective to normal cells subjected to oxidative stress.
Another key reason for the difference in effect of pharmacological preconditioning
agents, such as sulindac on Rb cells and normal retinal cells can be attributed to the
disparity in respiratory metabolism between these two types of cells. Since the
protection offered by the preconditioning is dependent on the mitochondria, this
organelle plays a much more crucial role in the respiratory metabolism of healthy cells,
and the protection by sulindac from oxidative stress induced damage. On the other hand
in cancer cells, being largely reliant on glycolysis, the same preconditioning agents
induce a cytotoxic effect when exposed to ROS. This property might prove to be
clinically significant. Currently used Rb therapies either use strong chemotherapeutic
agents such as carboplatin, topotecan or vincristine to achieve chemoreduction of
hyperproliferating tumor cells or they use local consolidation of tumors in the form of
radiation or cryotherapy. Recently cancer researchers have started showing interest in
developing cancer therapies that utilize the abnormal microenvironment of the tumor
(Houston et al., 2011). One unique feature of tumor microenvironmet that differentiates
it from normal tissues is localized hypoxic conditions. Most currently existing cancer
treatments are designed to selectively target hyperproliferating cell populations, hence
97
tumor cells that survive under localized hypoxic regions often prove to be resistant to
these chemotherapy and radiation (Boutrid et al., 2008) Cells growing in these hypoxic
regions have altered respiratory metabolism, mostly dependent on glycolytic pathways
for ATP production, and they exhibit a significantly slower metabolism for energy
preservation. Therefore, these cells may not respond to conventional treatments such as
chemotherapy, which are mostly designed to target the rapidly proliferating cells
(Maschek et al., 2004); (Boutrid et al., 2008)h. In a recent study a combination therapy
using antiangiogenic and glycolytic inhibitors achieved significant tumor control in Rb
mouse model study. It was demonstrated that hypoxic cells can be killed in the
LHBETATAG retinal tumor by using the glycolytic inhibitor 2-deoxy-d-glucose (2-DG)
(Houston et al., 2011). This novel idea further highlights the importance of using an
anti-cancer therapy such as the one using pharmacological preconditioning agents
described in this dissertation, which utilizes the dependence on anaerobic respiration
exhibited by most malignant cells, including Rb.
Based on the findings of this current study and multiple previous studies it is possible to
state that IPC agents, including sulindac, have drastically opposite effects on normal
and malignant cells in the presence of oxidative stress (Figure 33). As mentioned above
in both these cases the influence on the mitochondria is crucial and understanding this
will be the key to elucidating the mechanism of IPC agent/drug actions. So far what is
known about these pathways suggests that in normal cells these IPC agents induce
protection by initiating the cell’s own endogenous protective pathway, known as
preconditioning. However, in the case of cancer cells, chiefly because of their altered
cellular respiration, there is no induction of the IPC response. On the contrary they are
98
sensitized and made more vulnerable to oxidative stress induced cell death. This unique
dual property of sulindac and other IPC agents can act as a platform for treating various
diseases and warrants further investigation.
Figure 33: Pathways showing the differential effect of sulindac (and other IPC agents) on
normal and cancer cells exposed to oxidative stress (Adapted from PhD dissertation of
Ian Moench and reprinted with permission from author).
99
Future Research Aims
The study described here is a relatively novel approach where we have successfully
utilized an IPC agent that affects mitochondrial respiration, to protect normal cells
against oxidative damage, but sensitize malignant cells to anti-cancer drugs to enhance
the killing of the cancer cells. However this therapeutic procedure requires further
investigation and more work needs to be done in order to establish with certainty that
these therapeutic methods possess significant clinical importance and they merit use in
clinical trials. The obvious next step is to undertake an in vivo study using a mammalian
model of our target diseases, AMD and retinoblastoma.
In this study we have showed that a preconditioning agent can protect retinal cells in
vitro and our next goal is to test if sulindac can confer morphological protection to the
retina in an animal model of light damage that replicates AMD. To test this hypothesis
we will be utilizing an established mouse model of AMD where BALB/c mice are
exposed to conditions that induce photooxidative stress. Photooxidative stress will be
induced by exposing dark adapted experimental BALB/c mice to bright white light of
around 5000 to 6000 lux for a range of time durations of up to 24 hours. Comparative
histological analysis of the retina from control and experimental animals will be
conducted to evaluate if pretreatment with sulindac prior to exposure to our light
damage paradigm results in protection of photoreceptors against photooxidative stress
induced loss of viability. For further confirmation of sulindac’s protective efficacy
western blotting of retinal total protein will be performed to detect changes in the levels
of rhodopsin. To determine if the mechanism of protection is through preconditioning
100
we will perform western blotting to determine if there is up-regulation of the levels of
known markers of preconditioning such as Hsp27 and iNOS. Besides understanding the
mechanism another chief goal of designing in vivo study will be aimed at testing
multiple modes of application and dosages to determine the ones most appropriate for
clinical purposes. We plan on utilizing intravitreal injections, topical application and
oral administration using a range of doses.
With regards to the cancer study our future plan is to test our combination therapy on a
genetic mouse model that spontaneously develops Rb. Our drug combinations will be
tested in an in vivo model of retinoblastoma using the well characterized Chx10-Cre; Rb
lox/lox
; p107 -/-; p53 lox/lox mouse model that has been shown to accurately recapitulate the
human disease and to be appropriate for evaluating anti-cancer therapies over the long
term. This mouse model will be used over an 18 week period and analyzed according to
established procedures (Nemeth et al., 2011). This model is suitable for testing drugs at
clinically relevant doses as well as monitoring tumor regression in response to
chemotherapy. The untreated mice have been reported to develop retinoblastoma at a
high rate (122 out of 129; 95%, (Nemeth et al., 2011) and 79% (97 out of 122) were
reported to develop bilateral disease. The administration protocols we plan on following
will be similar to that currently followed in clinical trials involving the established Rb
drugs, topotecan and carboplatin. The administration protocol of our drug combination
will consist of a combination of subconjunctival administration and systemic
administration at different stages of the treatment. The efficacy of our therapy will be
determined by monitoring changes in retinoblastoma size with ultrasound and with
ocular histopathology performed at the termination of the experiments to determine
101
reduction in tumor size. Eyes will be removed and also assayed for levels of sulindac
and sulindac metabolites as described previously (Brunell et al., 2011).
Conclusion
Considering the findings of this study we can conclude that the use of pharmacological
preconditioning agents for therapy has significant clinical potential. One crucial positive
quality of drugs such as sulindac is their ability to perform a dual role. As displayed in
the findings of this project and several previous ones sulindac offers protection to
normal cells against oxidative stress induced damage as well as display anti-cancer
property when used in combination with chemicals that influence the mitochondrial
function. The protection offered by preconditioning is not only efficient but also
believed to be better since it uses the body’s own protective mechanism rather than
depending on external agents. Preconditioning is believed to be an evolutionary
adaptation that is triggered under stress of sublethal dose to prepare for a more severe
onslaught. With regards to existing therapies for AMD, there is no efficient preventive
drug that is capable of stopping the progression of AMD before it has caused significant
damage. Currently available therapies aim at stopping the progression of CNV and thus
only come into play once the disease has progressed into the more severe “wet” form.
We believe that the preconditioning therapy described in this dissertation offers a
unique opportunity of prevention of dry AMD prior to the stage where it has progressed
to a more severe stage. Besides preventing ROS induced damage sulindac activates
PPARα. In recent studies using known PPARα agonist such as fenofibrate, researchers
have reported inhibition of CNV in animal models as activation of PPARα results in
102
downregulation of VEGF (Varet et al., 2003). Since sulindac replicated the PPARα
activation of fenofibrate in our ARPE19 assays, it suggests that sulindac will also have
a similar inhibitory effect on VEGF and subsequently CNV in the “wet” form of AMD.
Since IPC/mito drug combinations exploits the metabolic differences between normal and
cancer cells it may have important therapeutic value. This therapeutic method utilizes the
difference in respiratory metabolism between normal and cancer cells. Even though there
are several therapies of RB that have displayed significant success in permanently curing
the tumor, most of these existing methods are highly aggressive. The most commonly
used Rb treatments are chemotherapy using strong chemical agents or radiation therapy.
Both of these procedures have been associated with causing secondary tumors in other
organs in the latter stages of the patient’s life. In several cases patients have to resort to
enucleation of the affected eye and might become completely blind after a few years.
These significant drawbacks of existing Rb therapies demand the development of an Rb
therapy, such as the one described in this project, which is less aggressive and does not
pose the threat of initiating secondary malignancies.
The outcome of this study has the potential for a novel therapy against both oxidative
stress in devastating eye diseases, such as AMD, as well as provide a complementary
approach to systemic chemotherapy, the standard of care for retinoblastoma. There has
been a previous research article that described using an IPC agent, sildenafil, for the dual
purpose of killing prostate cancer while providing protection to normal cardiac cells (Das
et al., 2010). However the research project described in this dissertation is novel in that, it
shows for the first time that an IPC agent protects normal cells from oxidative stress, but
103
sensitizes tumor cells to killing by an oxidizing agent, in the same organ. We believe
these results could have significant therapeutic value. It can be concluded that IPC agents
switch on a protective preconditioning response in normal cells, but in the case of cancer
cells IPC agents do not induce a preconditioning pathway, but sensitizes them to agents
that disrupts mitochondrial function.
104
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