Natural Vitamin E, α-Tocotrienol, as a Neuroprotectant
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of The Ohio State University
By
Han-A Park, M.S.
Graduate Program in Nutrition
The Ohio State University
2010
Dissertation Committee:
Dr. Chandan K. Sen, Adviser
Dr. Savita Khanna
Dr. Sashwati Roy
Dr. Narasimham Parinandi
Copyright by
Han-A Park
2010
ABSTRACT
Natural vitamin E exists as the well known tocopherols and poorly studied tocotrienols. α-Tocotrienol (TCT) represents the most potent neuroprotective form of vitamin E. This work addresses a novel molecular mechanism by which α-TCT may be protective against stroke. Reduced glutathione (GSH) is a low molecular weight intracellular thiol in all aerobic cells. Under conditions of oxidative stress such as during stroke, large amounts of GSH are rapidly oxidized to glutathione disulfide (GSSG). The current study demonstrates that elevation of intracellular GSSG concentration may trigger neural cell death via a 12-lipoxygenase (12-Lox) dependent mechanism. Furthermore, this work elucidates that α-TCT may improve GSSG clearance in cells subjected to
oxidative stress via upregulaton of multidrug resistance-associated protein 1
(MRP1) in the stroke-affected brain.
Objective : This dissertation addresses three specific aims: (i) determine the
role of intracellular GSSG in neural cell death; (ii) characterize the
significance of MRP1 as a GSSG efflux pathway in experimental stroke; and
(iii) determine the MRP1-dependent neuroprotective property of α-TCT
against stroke
ii
Experimental approach and results : To elucidate the specific significance of
GSSG in neural cell death, intracellular GSSG was specifically elevated by single cell microinjection. Control HT4 neural cells were microinjected with either the corresponding reduced form GSH or the vehicle (PBS). GSSG, but not GSH, caused cell death at pathophysiologically relevant concentrations.
GSSG-induced death of the neural cells was protected in the presence of 12-
Lox inhibitor or α-TCT. GSSG-dependent glutathionylation of 12-Lox emerged a critical player in neural cell death. Next, to test whether impaired cellular clearance of GSSG aggravates stroke-induced brain injury in vivo, middle cerebral artery occlusion (MCAO) was performed in MRP1-/- mice. Larger stroke-induced lesion in MRP-/- mice recognized a protective role of MRP1. In vitro, protection against glutamate-induced neurotoxicity by α-TCT was
attenuated under conditions of MRP1 knockdown suggesting a role of MRP1
in α-TCT-dependent neuroprotection. In vivo studies demonstrated that oral
supplementation of α-TCT protected brain against stroke-induced injury.
MRP1 expression was elevated in the stroke affected tissue of α-TCT- supplemented mice. Efforts to elucidate the underlying mechanism identified
MRP1 as a target of miR-199a-5p. In α-TCT supplemented mice, miR-199a-
iii 5p was downregulated in the stroke-affected tissue. This work recognizes
MRP1 as a protective factor against stroke.
Conclusions: Work in this dissertation adds a new dimension to the current understanding of the molecular bases of α-TCT neuroprotection by identifying
MRP1 as a α-TCT-sensitive target and by unveiling the general prospect that oral α-TCT may regulate microRNA expression in stroke-affected brain tissue.
iv DEDICATION
This dissertation is dedicated to my grandmother ( 최행이),
my parents ( 박종주; 정윤봉),
my brother and his wife ( 박건호; 최수미).
v ACKNOWLEDGEMENTS
My sincere gratitude goes to my mentor Dr. Chandan K. Sen for his guidance
and passion for research. I also especially want to thank Dr. Savita Khanna,
Dr. Sashwati Roy and Dr. Narasimham Parinandi for their support during the
dissertation process.
Learning how to work in a team was a great education in a lab. I appreciate support of all the members of the Sen team (arranged alphabetically):
Sami Albeiroti, Maurea Al-Khouri, Harshesh Amin, Pavan Ananth, Ali Azad,
Jaideep Banerjee, Ashley Bath, Corey Beals, Samantha Bellamy, Vineet
Bhasker, Sabyasachi Biswas, Sarah Carothers, Kyle Caution, Soma Chaki,
Yuk Chen Chan, Sang-Yong Choi, Eric Collard, Jacqueline Corry, Amitava
Das, Bhakti Deshpande, Ryan Dickerson, Jason Driggs, Haytham Elgharably,
Omar Ezziddin, Huiqing Fang, Drew Franklin, Muna Findley, Melissa Fox,
Kasturi Ganesh, Kirthana Ganeshan, Reza Ghoorkhanian, Surya Gnyawali,
Urmila Gnyawali, Gayle M. Gordillo, Sudip Gupta, Justin Harper, Mallory
Heigel, Jonathan Holmes, Syed-Rehan Hussain, Omar Hussian, Daniel Iscru,
vi Vijay Jayanti, Courtney Kauh, Suchin Khanna, Natalia Kubicki, Donald Kuhn,
Shivani Kwatra, Suguna Lonchin, Dashayini Mahalingam, Marc Mathias,
Mariah Maurer, Molly McCoy, Pankaj Mishra, Marcus Mithoefer, Chitrangada
Das Mukhopadhyay, Christopher Myers, Kishore Nallu, Paul Nolan, Navdeep
Ojha, Darshan Patel, Virenkumar Patel, Yojan Patel, Laura Peterson, Tina
Phillips, George Pryor III, Nagi Reddy Putluri, Kofi Quaye, Jared Radtke,
Faraz Rahman, Rashmeet Reen, Sandy Rees, Cameron Rink, Trent Rink,
Richard Schugart, Claire Seguin, Rebecca Schnitt, Hiral Shah, Shrenik Shah,
Zubin Shah, Yingli Shang, Shani Shilo, Hannah Siegle, Nathan Schomaker,
Steven Solomon, Arun Tewari, Roy Thompson, Rishi Verma, Vivek Yedavalli, and W. Taylor Williams.
vii VITA
1993 – 1996…………………… Jinju Girls’ High School, South Korea
1996 – 2001…………………… B.S. Food and Nutrition,
Sookmyung Women’s University, South Korea
2001 – 2003…………………… M.S. Food and Nutrition,
Sookmyung Women’s University, South Korea
2004 – present……………….. OSU Nutrition Ph.D program,
The Ohio State University, Columbus, OH
PUBLICATIONS
Park HA , Kubicki N, Gnyawali S, Chan YC, Roy S, Khanna S, and Sen CK
(2011) Natural Vitamin E α-Tocotrienol Protects Against Ischemic Stroke by
Induction of Multidrug Resistance-Associated Protein 1. Submitted
Park HA , Roy S, Khanna S, and Sen CK (2010) Method in Redox Signaling,
Chapter 10: Current Technologies in Single-Cell Microinjection and
Application to Study Signal Transduction. Mary Ann Liebert, Inc. p71-77
viii Gordillo G, Fang H, Park HA , and Roy S (2010) Nox-4 dependent nuclear
H2O2 drives DNA oxidation resulting in 8-OHdG as urinary biomarker and hemangioendothelioma formation. Antioxid Redox Signal. 15:12(8) 933-43
Park HA , Khanna S, Rink C, Gnyawali S, Roy S, and Sen CK (2009)
Glutathione disulfide induces neural cell death via a 12-lipoxygenase pathway.
Cell Death and Differentiation. 16(8):1167-79
Khanna S, Park HA , Sen CK, Golakoti T, Sengupta K, Venkateswarlu S, and
Roy S (2009) Neuroprotective and anti-inflammatory properties of a novel demethylated curcuminoid. Antioxid Redox Signal .11: 449-468
Khanna S, Roy S, Park HA , and Sen CK (2007) Regulation of c-Src activity in glutamate-induced neurodegeration. J. Biol. Chem. 282: 23482-90
FIELDS OF STUDY
Major Field : Human Nutrition
Emphasis : Signal Transduction in Neural Cell Death, Stroke
ix TABLE OF CONTENTS
Abstract…………………………………………………………………………... ii
Dedication……………………………………………………………………….. v
Acknowledgments ……………………………………………………………… vi
Vita………………………………………………………………………………... viii
List of Tables…………………………………………………………………….. xii
List of Figures…………………………………………………………………… xiii
Doctorate of Philosophy ……………………………………………………….. 1
CHAPTERS
1. GSSG as a new trigger for neural cell death
1.1 Introduction……………………………………………………….…. 8
1.2 Materials and Methods…………………………………………….. 14
1.3 Results and Discussion……………………………………………. 25
1.4 Conclusions…………………………………………………………. 41
1.5 Tables……………………………………………………………...… 42
1.6 Figures……………………………………………………….………. 43
2. Multidrug resistance-associated protein 1 in experimental stroke
x 2.1 Introduction………………………………………………………….. 70
2.2 Materials and Methods…………………………………………….. 75
2.3 Results and Discussion……………………………………………. 84
2.4 Conclusions…………………………………………………………. 92
2.5 Tables…………………………………………………………...…… 93
2.6 Figures…………………………………………………………….…. 95
3. Regulation of multidrug resistance-associated protein 1 by α-
tocotrienol in stroke
3.1 Introduction………………………………………………………….. 111
3.2 Materials and Methods…………………………………………….. 120
3.3 Results and Discussion……………………………………………. 125
3.4 Conclusions…………………………………………………………. 130
3.5 Tables…………………………………………………………….….. 131
3.6 Figures……………………………………………………………..… 132
4. Summary and Closing Thoughts…………………………………………… 143
List of References………………………………………………………….…… 147
xi LIST OF TABLES
Table 1.1 List of glutathionylated proteins in publications……………..….. 42
Table 2.1 Summary of MRP family……………………………………...…... 93
Table 2.2 Substrates of MRP1……………………………………………..... 94
Table 3.1 Tocopherol and tocotrienol in edible plants parts …………...… 131
xii LIST OF FIGURES
1.1 Chemical structure of GSH and GSSG………………………...……... 43
1.2 Mechanism of glutaredoxin and thioredoxin reaction..…….………… 44
1.3 Microinjection workstation…………………………………………….… 45
1.4 Micro-capillary preparation………………………………...………….... 46
1.5 Microinjected GSH failed to rescue, while GSSG potentiated
glutamate-induced death of HT4 neural cells……………………..… 47
1.6 Image of a HT4 neural cell taken by atomic force microscopy ….…. 48
1.7 Dose-dependent GSSG toxicity……………………………………...… 49
1.8 Intracellular GSH and GSSG levels in response to BSO treatment.. 50
1.9 Dose-dependent GSSG toxicity following BSO treatment .……...…. 51
1.10 Cytosolic injection of GSSG compromised mitochondrial
membrane potential…………………………………….……………… 52
1.11 The γ-Glutamyl cycle….…………………………………………..….... 53
1.12 GSSG-induced cell death attenuated by 12-Lox inhibitors ……….. 54
1.13 Arachidonic acid-induced cell death attenuated by 12-Lox
inhibitors………………………………………………………………… 55
1.14 Bischloroethylnitrosourea-induced cellular GSSG accumulation…. 56
1.15 Bischloroethylnitrosourea-induced cell death attenuated by 12-
xiii Lox inhibitors …………………………………………………………... 57
1.16 Bischloroethylnitrosourea-induced cell death aggravated in the
presence of MRP1 inhibitor …………….…………………………….. 58
1.17 12-Lox activity in response to GSSG………………………..……….. 59
1.18 Protein glutathionylation in response to GSSG …………………….. 60
1.19 Protein glutathionylation in response to GSSG under reducing
conditions………………………………………………………..……… 61
1.20 GSS-12-Lox formation in response to GSSG…………….…………. 62
1.21 Glutaredoxin 1 transfection in HT4 neural cells…………………….. 63
1.22 Glutaredoxin 1 reversed glutathionylation in HT4 neural cells……. 64
1.23 Glutaredoxin 1 protected cells against glutamate challenge………. 65
1.24 Glutaredoxin 1 reversed glutathionylation of 12-Lox in HT4 neural
cells……………………………………………………………………… 66
1.25 Glutaredoxin 1 microinjection protected HT4 neural cells against
GSSG as well as arachidonic acid challenge………..………...…… 67
1.26 MCAO-induced infarct hemisphere contained elevated level of
GSSG……………………………………………………………………. 68
1.27 12-Lox-deficient mice resistant to GSSG-induced brain injury….… 69
2.1 Topology of MRPs.…………………………………………………….… 95
2.2 Induction of MRP1 in response to glutamate challenge…...……...… 96
xiv 2.3 MRP1 knockdown attenuated the neuroprotective effects of α-TCT. 97
2.4 MRP1 activity was increased by glutamate challenge in HT4 neural
cells……………………………………………………..………...……..… 98
2.5 MCAO-induced brain injury was aggravated in MRP1 deficient
mice………………………………………………………..…….…...…… 99
2.6 MCAO-induced MRP1 protein expression.………………………..….. 100
2.7 MCAO-induced GSSG accumulation in MRP1 deficient mice……… 101
2.8 Increased abundance of MRP1-positive cells in infarct hemisphere
of FVB..………………………………………………………………....… 102
2.9 Increased level of neurodegeneration in infarct hemisphere of
MRP1 deficient mice………………………………………………..…… 103
2.10 Alignment between miR-199a-5p and MRP1……………………….. 104
2.11 miR199a-5p levels in response to miR199a-5p mimic or inhibitor
delivery in HT4 neural cells……………………………………………... 105
2.12 miR199a-5p silenced MRP1 gene in HT4 neural cells…………….. 106
2.13 miR199a-5p downregulated MRP1 protein expression…….…...…. 107
2.14 miR-199a-5p directly targets MRP1……………………………….…. 108
2.15 miR-199a-5p mimic delivered HT4 cells were vulnerable to
glutamate-induced toxicity…………….…..………….……………….… 109
2.16 Neuroprotective effect of miR-199a-5p inhibitor abrogated in the
xv presence of MK571 in HT4 neural cells……………………...... …….. 110
3.1 Chemical structure of vitamin E...……………………………………… 132
3.2 Classification of stroke………………………………………………….. 133
3.3 Design of oral α-TCT supplementation………………………………... 134
3.4 Brain vitamin E level after oral α-TCT supplementation…………...... 135
3.5 Oral α-TCT supplementation protected against MCAO-induced
brain injury ……..…………………………..……………………….….… 136
3.6 Total RNA collection using LMPC…………………………………...… 137
3.7 Oral α-TCT supplementation upregulated MRP1 gene expression
specifically at the MCAO-affected site of the brain…………………… 138
3.8 Oral α-TCT supplementation upregulated MRP1 protein expression
specifically at the MCAO-affected site of the brain………………...… 139
3.9 Histologic evaluation of MRP1 expression in α-TCT supplemented
mice………………………………………………………..…………..….. 140
3.10 Oral α-TCT supplementation attenuated MCAO-induced
neurodegeneration………………………………………...... 141
3.11 Oral α-TCT supplementation attenuated MCAO-induced lipid
peroxidation………………………………………………...……..……… 142
xvi CHAPTER 0
DOCTORATE OF PHILOSOPHY
Introduction
The Doctor of Philosophy degree, abbreviated PhD or Ph.D., is the highest academic qualification in the United States’ educational system. The number of recipients of the Doctor of Philosophy degree has steadily increased in the United States over the past 150 years. According to the
United States Department of Education, Institute of Education Science, more than 60,000 doctoral degrees were earned from 2007 to 2008.
Biomedical sciences and public health, of which nutrition is an integral part, have garnered ever increasing academic interest during recent decades.
Growth of patients with special needs, increased social attention to health services, and individual interests have all worked in concert to increase demand for professionals in health science. Furthermore, these fields are
1 becoming more highly specialized and in most cases now require a postgraduate or professional degree. As a result, almost 10% of students who hold a bachelor’s degree in health science go on to gain a doctorate. This number is double those who pursue literature doctorates and 10% higher than business majors. Notably, the number of female scholars who hold a PhD has dramatically increased in the last 5 years.
History of PhD
July 25 th 1861 marks a monumental date in the academic history of the
US. Three recipients (Arthur W. Wright in physics, James M. Whiton in classics, and Eugene Schuyler in philosophy) received the first PhD degrees conferred in the US, awarded by Yale University.
Although the true origin of the PhD is not clear, the precursor of our current system of academic degrees appears to have started in European universities in the middle ages. The word doctorate originates from the Latin docere , which means ‘to teach’. The doctorate degree served as a license to
teach at medieval universities in Europe, but the term doctor was used to
2 label Christian authorities who taught or interpreted the bible. The University of Bologna and University of Paris are among the oldest institutions to confer the degree of doctor. However, the modern curriculum of study for PhD was developed in German universities and from there introduced into other countries, including the United States. The Department of Philosophy and
Arts of Yale University was the first to adopt the German system and produced the first PhDs in United States. Later, the PhD was awarded by the
University of Pennsylvania in 1871, Cornell in 1872, and Harvard in 1873.
Personal Philosophy of PhD
Procedure: I consider this the most personal chapter of my dissertation, based on my experiences and thoughts. I define PhD as the establishment of the process of thought . Then, I must answer ‘what is the process of thought ?’ I consider the following the four major steps: the process of understanding the problem, the process of finding the solution, the process
of action, and finally, the process of evaluation . My PhD years were a
countless repetition of these four steps, and now I have come to the point that
I understand and can establish my own way to accomplish these four steps.
3 Karl Marx is well known as a communist and sociologist. The title of his PhD thesis was The Difference Between the Democritean and Epicurean
Philosophy of Nature. Obviously, his impact on the field of ancient Greek philosophy is minor compared to his impact on 19 th century world history.
However, I believe the practice during his PhD education of studying and evaluating Greek philosophy trained and informed his process of thought and eventually allowed him to establish his own completely new theory of political economy. As Umberto Eco mentioned in his manual Come si fa una tesi di laurea ; 논문 잘쓰는 방법 (Korean edition; translated to ‘How to write a
thesis’) in 1977, the process of experience and thought while composing a
dissertation may be more important than the actual theme of the thesis.
Therefore, I see the dissertation as the first indicator of a person who has
established their own procedure of thought.
Communication: The next step is to address personal goals after
receiving a PhD. Interestingly, when I was asked to add this chapter, several
non-science works I read more than ten years ago came to my mind. 점, 선,
면 (Korean edition: translated to ‘Point and Line to Plane’) written by Wassily
Kandinsky is a landmark book in the plastic theory of art. Its main purpose is
4 to define the geometrical elements as indicated by the title. However, several definitions used in this book were intriguing to me. One of these terms was direction . Kandinsky introduced two directions in art. The first direction is from artist to art, and the second is from art to the observer. Although Kandinsky considered the relationship between artist, art and observer in a subjective manner, it was a revelation for me to consider this multiplicity of views as a consumer of art. Understanding the point of view of the artist, interpreting the path of the material surface of art to the observer, and finally assessing the connection or disconnection between these two are critical to evaluate art.
Since reading Point and Line to Plane, I have started to understand art as an object with multiple viewpoints; my own perspective, that of the art itself, and finally the viewpoint of the artist. The reason why I mention Kandinsky’s theory in my dissertation is to make the supposition that his formula is also in accordance with scientific investigation. Let’s redefine his critical viewpoints as from nature to the scientist and from the scientist to the general public. We now face exactly the same issues previously described; the explanation of the event in nature, the interpretation of scientist’s viewpoint, and finally the scientist’s communication with the general public.
5 Philosophy: What I tried to learn from PhD training was using science as a tool to approaching an understanding of philosophy . I expect to employ this approach to examining and appreciating everything that I value in life.
Admiration for the ‘Renaissance Man’ might be old fashioned in our modern day society which strives for specificity and accuracy, but I believe the separate fields made distinct in our current society all become one in the end. Looking to the past, Art, Science and Philosophy were so interwoven as to be almost indistinguishable. Artists used to be scientists, and scientists used to be musicians, and so on. It is not difficult to find people across the path of human history who understood his or her role as an integrator rather than a specialist. Consider Pythagoras, Leonardo Da Vinci, Johann Carl
Friedrich Gauss, and Rabindranath Tagore. In my case, art and music played a large role in my PhD training, and I believe that earning a PhD opens another path to enjoy something I have always appreciated.
I believe that artists, scientists and philosophers share the same qualities. They require keen eyes to observe what nature provides and a creative and limber mind capable of explaining nature’s workings in their own
6 words. As a scientist, I will not consider myself an inventor but an observer of nature, and I will constantly try to find the thread between values that I love.
I consider this dissertation as adding another chapter to my existing philosophy, defining my own process of thought, and as a humble start of my communication. As Eco advised at the end of his book using Bernardus
Carnotensis and Isaac Newton’s famous quote, a young scholar may be small, but they are as dwarfs standing on the shoulders of giants .
7 CHAPTER 1
GSSG as a new trigger for neural cell death
1.1 Introduction
1.1.1 Glutathione disulfide
Free glutathione, a tripeptide with the sequence γ-Glu-Cys-Gly, exists
either in a reduced form with a free thiol group (GSH) or in an oxidized form
with a disulfide between two identical molecules (GSSG) (Fig. 1.1). GSH is
a ubiquitous low molecular weight intracellular thiol present in all aerobic
cells in millimolar concentrations. The sulfhydryl (-SH) group supports the
reducing properties of GSH by way of a thiol-exchange system (-SH to -S-S-
), making GSH one of the most abundant and powerful intracellular
antioxidants. Besides scavenging free radicals and reactive oxygen species,
GSH detoxifies tissues by conjugating with various electrophiles including
xenobiotics. In addition, GSH serves as a major reservoir of cysteine for
cellular protein synthesis.
8
Under basal conditions, GSSG represents 1% of the total GSH in the cell 2. Under conditions of oxidant insult, GSH is rapidly oxidized to GSSG.
Thus, an elevated GSSG/GSH ratio is often used as a marker for oxidative stress 3. Cellular GSSG may be recycled to GSH in the presence of reductases such as NADPH-dependent GSSG reductase. Excessive GSSG, as generated during sudden oxidant insult, is pumped out of the cell by a
ATP-dependent process underscoring the urgent need of the cell to protect itself from a GSSG surge 4-5. In most studies, GSSG is dealt with as a byproduct of GSH metabolism. Because cellular GSH concentration is expected to be in the range of 1-5 mM, millimolar concentrations of GSSG are expected in cells under conditions of oxidant insult. However, knowledge about the potential biological significance of GSSG per se is limited. While excessive oxidant insult causes necrotic cell death, a more moderate challenge triggers secondary responses in the cell that culminate in cell death. Elevation in cellular GSSG levels represents one such rapid cellular response to moderate oxidant insult. In this study, we sought to examine whether elevated cellular GSSG levels may directly influence cell death. Addressing this question would require that cellular GSSG elevation be isolated from all other biological causative factors. Thus, we adopted the
9 microinjection approach to raise cellular GSSG or GSH as control to
investigate the significance of GSSG on cell death. To test the significance
of our findings in vitro , GSSG was stereotaxically injected to the brain in vivo
and MRI was performed to quantify tissue lesion.
1.1.2 S-Glutathionylation
S-Glutathionylation is the reversible formation of mixed disulfides
between protein cysteine and glutathione. This is the thiol/disulfide
exchange mechanism which occurs when an oxidative insult changes the
GSH/GSSG ratio and induces GSSG to bind to protein thiols. Oxidative
stress is known to trigger the incorporation of the glutathione moiety into
target protein. Both ROS 6 and RNS 7 are responsible to induce S-
glutathionylation. Therefore ROS/RNS production by pathologies associated
with inflammation, neurotoxicity and ischemia may induce this post
translational modification in vivo . S-Glutathionylation has been implicated in
the buffering of oxidative stress and protection against cysteine residues by
regulation of target protein or enzyme activity. S-Glutathionylation is
reported as inhibitory modification in phosphofructokinase 8, NF κB9, creatine
kinase 10 , and actin 11 while it activates microsomal glutathione-S-
transferase 12 , HIV-1 protease-Cys67 13 , and matrix metalloproteinase14 .
10 Regardless contrasting effects of glutathionylation in different protein, it appears to change function of the modified protein. Thus, reversible protein-
SSG formation is critical to be recognized as a potential regulatory mechanism.
Glutaredoxin (GRx), also known as thiol transferase, is an oxidoreductase mostly found in cytosol (GRx1) and mitochondria (GRx2).
GRx closely resembles thioredoxin (TRx), but GRx acts GSH-dependent manner (Fig. 1.2). GRx coupled with glutathione reductase and glutathione to maintain its own redox status. GRx appears to be responsible for deglutathionylation. S-Glutathionylation is important regulatory mechanism due to post translational modification of protein thiol. GRx is able to reverse glutathionylation by breaking mixed disulfide bonds. Protein glutathionylation occurs under condition of oxidative stress due to GSSG accumulation. This post translational modification is responsible to change structure and activity of numerous proteins such as caspase 3, c-Jun, creatine kinase, GAPDH, and beta-actin. GRx is selectively break disulfide bonds (Protein-SSG, not Protein-S-S-Cys) in glutathionylated proteins to contribute thiol homeostasis (Table 1.1).
11 1.1.3 Microinjection as novel tool to study redox molecules
Historically, a number of delivery methods including cDNA transfection,
viral infection, and electroporation have been used to introduce exogenous
substances such as functional proteins, peptides, DNA, RNA, or drugs into
live biological cells. Although these methods have several advantages, they
also suffer from some key limitations. In some cases, reagents that stress or
kill the cells need to be used. Efficiency of delivery is a frequent concern. In
addition, the exact time and dose of delivery is difficult to track on a single-
cell basis. For example in the case of transfection of an expression vector
with the goal to deliver protein in the cytosol of cells, the temporal kinetics of
vector delivery and performance of the protein expression machinery remain
uncertain and may vary from one situation to the other. Thus, such
transfection approaches may not be suitable for experiments where delivery
of the protein is necessary at a given time point of the signaling cascade.
Such timed studies are helpful in delineating the temporal sequence of a
signaling cascade.
Microinjection refers to the process of using a micro needle to insert
substances at a microscopic or borderline macroscopic level into a single
living cell. It is a simple mechanical process in which an extremely fine
12 micro needle penetrates the cell membrane and sometimes the nuclear envelope and releases its contents. Microinjection is normally performed under a specialized optical microscope setup called a micromanipulator. As such, microinjection represents a powerful tool that can directly and instantly deliver small molecules into compartments of a single cell. The approach allows for fine adjustments of pressure, volume, and duration of each injection depending on the target cell type or cell compartment (Fig. 1.3). It is also possible to inject the same cell multiple times without disturbing neighboring cells. The efficiency of introducing biological materials into a cell with microinjection is much higher compared to other delivery methods.
Limitations of substance to be microinjected are minimal enabling delivery of nucleic acid, small molecules, proteins as well as blocking antibodies.
Microinjection does not require transfection reagents or viral vectors.
Furthermore, this method only requires pico- to femtoliter volumes of the substance being introduced into the cell. For experiments seeking to temporally dissect the sequel of signaling events in a given cascade, microinjection represents a unique powerful tool. In addition, the capability of introducing substance into specific compartments of the cell, e.g. cytosol versus nuclear, helps address signaling events at the level of subcellular compartments 15-19 .
13 1.2 Materials and Methods
1.2.1 Cell culture
Mouse hippocampal HT4 neural cells were grown in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal calf serum, 100
units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in humidified
20-24 atmosphere of 95% air and 5% CO 2 as described previously .
Glutamate treatment : Immediately before experiments, the culture
medium was replaced with fresh medium supplemented with serum and
antibiotics. Glutamate (10mM, Sigma-Aldrich, St. Louis, MO) was added to
the medium as an aqueous solution. No change in the medium pH was
observed in response to the addition of glutamate 21,23 .
L-Buthionine-sulfoximine (BSO) treatment : Before experiments, the
culture medium was replaced as described above, and freshly prepared
BSO (50µM, Sigma-Aldrich) in sterile PBS was added to the medium as
described in relevant legends.
1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment: Before
experiment, the culture medium was replaced as described above, and
BCNU (50µM, Sigma-Aldrich) prepared in ethanol was added to the medium.
Respective controls were treated with an equal volume of ethanol.
14 α-Tocotrienol (TCT) and 5,6,7,-trihydroxyflavone (BL-15) treatment: A
stock solution of α-TCT (Carotech Inc, Malaysia) was prepared in ethanol,
and BL-15 (Enzo Life Sciences, Plymouth Meeting, PA), was prepared in
dimethyl sulfoxide (DMSO). Before experiments, culture medium was
replaced with fresh medium supplemented with serum and antibiotics, and
α-TCT (1µM) or BL-15 (2.5µM) was added to the culture dishes as
described in relevant legends.
1.2.2 Microinjection
The mouse hippocampal HT4 cells (0.1 10 6 / plate) were grown on
35mm plates 24h before microinjection. Microinjection was performed using
a micromanipulator Femtojet B 5247 and Injectman NI 2 (Eppendorf,
Hamburg, Germany) with 80hPa of pressure and 0.2s duration. The
compensation pressure during injection was 40hPa. The glass micropipettes
were made from GD-1 glass capillaries (Narishige, Japan) by using a
Narishige PC-10 Puller with heater set at 52.7 °C (Fig. 1. 4).
Cells were injected with GSSG, GSH, sham (PBS) or reagents
described in the relevant legends. Live or dead cells were counted at 24h
after microinjection. Stock solutions of GSSG or GSH were freshly prepared
in sterile PBS, and co-injected with QDot streptavidin conjugate with the
15 emission maximum near 605nm (Invitrogen Corporation, Carlsbad, CA).
QDot streptavidin conjugate was used as a fluorescent marker to localize
the injection site. Digital images were collected using a specialized phase
contrast as well as a fluorescent Zeiss Axiovert 200M microscope
(Göttingen, Germany) suited for imaging cells grown in routine culture plates.
The sample stage was maintained at 37 °C, and the sample gas
environment was maintained exactly as in the culture incubator.
1.2.3 Reduced and oxidized glutathione assay
GSH and GSSG were detected simultaneously in HT4 cells and mice
brain tissues using an HPLC coulometric electrode array detector
(CoulArray Detector, model 5600 with 12 channels; ESA Inc., Chelmsford,
MA, USA) as described previously 21-22,25 . The CoulArray detector employs
multiple channels set at specific redox potentials. Data were collected using
channels set at 600, 700, and 800mV. The samples were snap-frozen and
stored in liquid nitrogen until HPLC assay. Sample preparation, composition
of the mobile phase, and specification of the column used were as
previously reported 23,25 .
16 1.2.4 Measurement of mitochondrial membrane potential
Mitochondrial membrane ∆ψ was measured using the fluorescent
lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM,
Invitrogen), which accumulates within mitochondria in a potential dependent
manner 26 . Following 24h of seeding, HT4 cells were injected with 500
attomole GSSG or sham (PBS). Dextran alexa fluor 488 (Invitrogen) was
co-injected, and used as a fluorescent marker. After 2h incubation, cells
were resuspended in Hanks’ balanced salt solution and stained with 8nM
TMRM and 0.5 µl/ml plasma membrane potential indicator (PMPI, Molecular
Devices Corp., Sunnyvale, CA) for 30 min at 37 °C in the dark. The cells
were washed with PBS, and digital images of stained live cells were
collected using a Zeiss Axiovert 200M microscope 27-29 .
1.2.5 Cell viability
The viability of cells in culture was assessed by measuring leakage of
lactate dehydrogenase (LDH) from cells into media 24h following glutamate
treatment using an in vitro toxicology assay kit from Sigma Chemical Co. (St.
Louis, MO, USA). The protocol has been described in detail in a previous
report 30 . In brief, LDH leakage was determined using the following
equation : % total LDH leaked = (LDH activity in the cell culture medium /
17 total LDH activity ) × 100 20-22 . Total LDH activity represents the sum of LDH
activities in the cell monolayer, detached cells, and the cell culture medium.
Survival of HT4 cells was also quantified by using a calcein acetoxymethyl
ester (AM) assay 31-32 . Briefly, HT4 cells (40,000/well) were seeded in 12-
well plates. After 2h of incubation, cells were treated with either 2.5 µM BL-
15 or 1µM α-TCT. After 6h incubation, cells were treated with 50µM BCNU.
Following 12h incubation, media was removed from each well, and 5µM
calcein AM in sterile PBS was added. After incubation at 37 °C for 1h,
fluorescence was measured by using the fluorescence multi-well plate
reader Cyto Fluor TM II (PerSeptive Biosystems) with the excitation
wavelength at 485nm and the emission wavelength of 530nm.
1.2.6 Determination of 12-lipoxygenase (Lox) activity
The in vitro activity of 12-Lox was assayed using a standard
spectrophotometric method measuring the increase in the formation of
conjugated dienes from the substrate arachidonic acid as described 33 , with
minor modifications as specified below. To ensure greater solubility of
arachidonic acid and to minimize the use of ethanol in the assay medium,
the potassium salt of arachidonic acid was freshly prepared by mixing
arachidonic acid with 0.1M KOH (1:1). The final assay mixture (total volume
18 of 1ml) contained 10 µmol/L of arachidonic acid (10 µl from 1mmol/L stock)
and 2 units of 12-Lox (porcine leukocyte enzyme, Caymen Chemical, Ann
Arbor, MI) in 100 mmol/L Tris-HCl buffer (pH 7.4). The mixture was then
gently mixed, reaction was started by adding the enzyme, and absorbance
of the reaction mixture was measured at 234 nm (as an index of formation
of conjugated dienes) using a Shimadzu model UV-2401PC
spectrophotometer. The activity of 12-Lox was calculated from the
absorbance values as n mole/min using the ε of 2.52 X 10 4 mol/L -1 and
normalized as % control 22 .
1.2.7 12-Lox overexpression
Following 24h of seeding (0.5 10 6 / well), HT4 cells were transfected
with plasmid pcDNA3.1 + 12-Lox (ResGen; Invitrogen Corporation,
Carlsbad, CA) or empty pcDNA 3.1 containing V5 epitope tag using
Lipofectamine TM LTX Reagent (Invitrogen Corporation, Carlsbad, CA). Cells
were maintained in regular culture conditions for 48h to allow for protein
expression, and cells or cell lysates were treated as described in the
respective figure legends.
19 1.2.8 Immunoprecipitation and immunoblots
HT4 cells (0.5 10 6 / well) were seeded in 6-well plates for
immunoprecipitation as previously described 20 . Cells were transfected with
pcDNA3.1 + 12-Lox or empty pcDNA 3.1 containing V5 epitope tag as
described above. During harvest, the cells were washed with ice-cold
phosphate-buffered saline (pH 7.4) and lysed with 0.2ml lysis buffer (Cell
signaling Technology, Inc, Danvers, MA). Protein concentration was
determined using the BCA protein assay kit (Pierce Biotechnology). Cells
were treated as described in the respective figure legends, and then cell
lysates (500 µg) were incubated with 20 µl immunoprecipitating antibody
overnight at 4 °C (V5 antibody affinity purified agarose immobilized
conjugate, Bethyl Laboratories, Inc, Montgomery, TX). Immunoprecipitated
complexes were washed four times with lysis buffer (centrifugation at 1000
× g at 4 °C for 5 minutes), and boiled for 30 min under non-reducing
conditions. Next, equal volumes of samples were loaded onto SDS-PAGE
gel and probed with anti-V5 antibody (1: 5000 dilution, Invitrogen).
1.2.9 GSSG-induced glutathionylation
HT4 cell lysates (20 µg) or 12-Lox from porcine leukocytes (30 µg) were
incubated with 10mM GSSG or the same volume of PBS at 37 °C. After 1h
20 incubation, samples were either processed for immunoprecipitation or
subjected to SDS-PAGE under non-reducing conditions. Samples were
quickly centrifuged, resuspended with sample buffer lacking thiol reductant,
and boiled for 30min. After quick centrifugation, samples were loaded to
10% SDS-PAGE, and transferred to PVDF. After transfer, membranes were
blocked using 10% nonfat milk overnight at 4 °C, and incubated with anti-
glutathione monoclonal antibody (1:500 dilution, ViroGen Corporation,
Watertown, MA), or anti-12-Lox polyclonal antibody (1:1000 dilution,
Cayman chemical, Ann Arbor, MI) for 2.5h. Membranes were washed three
times. Next, blots were incubated with secondary antibody (1: 2000 dilution,
Amersham anti-mouse IgG horseradish peroxidase linked whole antibody,
GE Healthcare), or (1: 3000 dilution, Amersham anti-rabbit IgG horseradish
peroxidase linked whole antibody, GE Healthcare) for 1h, and visualized by
enhanced chemiluminescence Western blotting detection reagent (GE
Healthcare).
1.2.10 Adenoviral expression of glutaredoxin 1
The mouse hippocampal HT4 cells (0.5 10 6 / well) were grown on 6-
well plates for 24 h, and infected with specific multiplicities of infection (m.o.i.
100, 200, 500, 1000, and 2000) of adenoviral vector containing the
21 glutaredoxin 1 cDNA construct (Ad-GRx1, a gift from Dr. J.J. Mieyal, Case
Western Reserve University, Cleveland, OH, U.S.A.) or ad-LacZ (control) in
750 µl of serum-free DMEM for 4h. Cells were cultured for 72h in
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Transfected
cells or cell lysates were treated as described in relevant figure legends.
After protein extraction, protein concentrations were determined using BCA
protein reagents. Samples (30 µg / lane) were separated on 12% SDS-
PAGE and probed with anti-glutaredoxin 1 (1:1000 dilution, R&D systems,
Inc, Minneapolis, MN). To evaluate the loading efficiency, membranes were
re-probed with anti-GAPDH.
1.2.11 Immunocytochemistry
HT4 cells (0.5 10 6 / well) were seeded in 35mm plates for 24h, and
transfected with ad-GRx1 as described above. Cells were washed with PBS
three times and then fixed in 10% buffered formalin for 20 min. Next, the
cells were by permeabilization using 0.1% Triton X-100/PBS for 15 min.
After washing, cells were incubated with 10% goat serum (Vector
Laboratories, Burlingame, CA) for 1h at room temperature, then incubated
with GRx-1 antibody (1:100, in 10% goat serum) overnight at 4 °C. After
22 incubation with primary antibody, cells were washed with PBS three times
and incubated with an Alexa-flour rabbit (green) for 1h at room temperature.
After incubation with 4’,6’-diamino-2-phenylindole (1:10,000) for 2 min, cells
were mounted in gelmount (aqueous mount, Vector Laboratories) for
microscopic imaging as described previously 20 .
1.2.12 Stereotaxic injection
12-Lox knockout (B6.129S2-Alox15 tm/Fun ) and corresponding
background C57BL6/J mice were obtained from Jackson Laboratory. Young
(8 to 10 week of age; 24-27g) male mice were anesthetized with isoflurane
in oxygen-enriched air delivered through a facemask. The mouse was
secured by ear bars, the skull was exposed, and injected with 5 µl of GSSG
stock (100 mM) in sterile PBS into the cortex using a Hamilton RN syringe
with a stainless steel needle (Hamilton Company, Reno, Nevada). The
location of injection site (coordinates: -0.5mm anterior, 3.5mm lateral, and
1.0mm ventral to bregma) was measured by Benchmark TM Stereotaxic
Digital (myNeurolab.com, St.Louis, MO). To minimize pressure-induced
damage, GSSG was injected at a rate of 0.2 µl/min. All animal protocols
were approved by the Institutional Laboratory Animal Care and Use
Committee (ILACUC) of the Ohio State University, Columbus, Ohio.
23
1.2.13 Data analysis
Data are reported as mean + SD of at least three independent
experiments. Comparisons of mean were tested using Student’s t-test or
one-way ANOVA with Tukey’s test. P < 0.05 was considered statistically
significant.
Summary of Methods • Single cell microinjection • Measurement of mitochondrial potential • HPLC : reduced and oxidized glutathione assay • Cell viability assay • 12-Lox activity assay • Immunoprecipitation and immunoblot • GSSG-induced glutathionylation • Overexpression of 12-Lox / GRx1 • Immunocytochemistry • Stereotaxic injection of GSSG
24 1.3 Results and Discussion
1.3.1 Glutathione disulfide induced neural cell death
Increased extracellular glutamate depletes intracellular GSH 23 . To test
the significance of this loss of cellular GSH during glutamate-induced loss of
HT4 cell viability, GSH was replenished in glutamate-treated cells by
microinjection. Previously we have reported that 4h of glutamate treatment
markedly depletes the cellular GSH pool 23-24 . Thus, 4h glutamate treatment
was performed in these experiments. After 4h of treatment, glutamate was
withdrawn and cell were microinjected with GSH. Because the antioxidant
properties of GSH were being tested, GSSG was selected as control. As
control for GSH injection, GSSG injection was performed. GSH
microinjection did not significantly rescue cells from glutamate-induced
death. This observation was consistent with our previous indirect
observation that glutathione depletion is not critically important in causing
cell death because we were previously able to afford complete protection by
the α-TCT form of natural vitamin E under conditions where glutamate-
induced glutathione loss remained unaffected 23 . Of striking interest,
however, was the observation that the control cells microinjected with
25 GSSG were all lost to death. GSSG microinjection proved to be potently cytotoxic (Fig. 1.5).
This serendipitous observation led us to examine the threshold of intracellular GSSG concentration ([GSSG]i) that triggers cell death of HT4 neural cells. Based on atomic force microscopy measurements (Fig. 1.6), we determined that the mean HT4 cell volume is in the order of 1 pl (not shown). Microinjection of graded amounts of GSSG was performed to identify the threshold concentration of GSSG that trigger cell death. It was noted that injection of 500 attomoles of GSSG which is equivalent to 0.5 mM of [GSSG]i was the threshold at which GSSG caused death of HT4 cell under standard culture conditions (Fig. 1.7).
Under conditions of glutamate challenge or other insult leading to cell death, cellular GSH levels are known to sharply fall 23 . Therefore, we chose to lower GSH levels in cells by arresting GSH synthesis using BSO. BSO sharply increased GSSG levels (Fig. 1.8A) and depleted cellular GSH pool by 80% (Fig. 1.8B) similar to the magnitude noted during glutamate challenge 23 . Under such GSH-depleted conditions, HT4 cells were noted to become more sensitive to GSSG-induced cell death. In such GSH-deficient
26 cells, the threshold for GSSG-induced lethality was lowered by twenty-fold such that microinjection of 25 attomoles (corresponding to 0.025 mM) of
GSSG caused cytotoxicity (Fig. 1.9). These observations underscore the heightened significance of GSSG as an inducer of cell death under conditions of GSH depletion. Characterization of GSSG-induced death of
HT4 neural cells was started by testing the involvement of mitochondrial dysfunction as is commonly associated with cell death. Cytosolic injection of
GSSG was observed to selectively compromise mitochondrial membrane potential while not affecting plasma membrane potential (Fig. 1.10).
In mammals, the intracellular synthesis of glutathione and its utilization is described by the γ-glutamyl cycle, a concept that was put forth almost four decades ago by Dr. Alton Meister34 . Reduced glutathione represents the centerpiece of the γ-glutamyl cycle (Fig. 1.11), involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission. While Dr Alton Meist er 1 1922-1995 GSH may form numerous adducts in the human
27 body 35 , the most abundant GSH derivative is represented by GSSG.
Oxidation of intracellular GSH by oxidants such as hydrogen peroxide and organic peroxides generally leads to the formation of GSSG. GSH can be further oxidized to the sulfenic, sulfinic, and sulfonic acid derivatives via successive two-electron oxidations of the thiol group.
GSSG is mostly viewed as a byproduct of GSH generated following reaction of GSH with an oxidizing species. As a result, GSSG/GSH ratio in the tissue emerged as a frequently used biochemical measure of oxidative stress 36 . Advances in the concept of redox signaling, and redefining of oxidative stress in that light 10 , has led to rethinking of the significance of
GSSG in the cell 37 . It is now widely acknowledged that changes in the cellular reduced/oxidized glutathione ratio trigger signal transduction mechanisms influencing cell survival. GSSG is capable of causing protein
S-glutathionylation or reversible formation of protein mixed disulfides
(protein-SSG). Post-translational reversible S-glutathionylation is known to regulate signal transduction as well as activities of several redox sensitive thiol-proteins 38 .
28 Studies with exogenous non-permeable GSSG have demonstrated that extracellular GSSG may trigger apoptosis by a redox-mediated p38 mitogen-activated protein kinase pathway 39 . While this addresses the significance of extracellular GSSG, the specific properties of intracellular
GSSG remain under veil. GSH is oxidized to GSSG within the cell and pumped to the extracellular compartment 4-5. Intracellular compartment being the primary site of GSSG generation, the significance of this disulfide within the cell becomes an important issue to address. Studies examining the significance of intracellular GSSG are complicated by the lack of a specific approach that would only elevate intracellular GSSG levels. For example, exposure of cells to pathogen related chemicals or to direct oxidant insult does elevate cellular GSSG but activates numerous other aspects of cell signaling 40 . While the study of GSSG driven reactions in a cell-free system is relatively straightforward in approach, the in vivo significance of such findings remains questionable 41 . This work presents first evidence from the use of a microinjection approach to study the significance of GSSG within the cell. Previously, we have utilized this approach to differentially study the cytosolic and nuclear compartments of HT4 cells as well as of primary cortical neurons 22 . The approach is powerful in instantly and selectively introducing agents into specific compartments of the cell. Results of this
29 study provide the first evidence demonstrating that the specific elevation of
GSSG within the cell may cause cell death. Our observation demonstrating loss of mitochondrial membrane potential without affecting cell membrane integrity argues in favor of an apoptotic fate. Disorders of the central nervous system are frequently associated with concomitant glutathione depletion and oxidation 42-43 . Our observation that cells with compromised
GSH levels are substantially more sensitive to GSSG-induced death leads to the notion that GSSG may play a role in cell death under conditions of disease and aging. We note that in GSH-sufficient cells (experimental) 0.5 mM GSSG is lethal. In GSH-deficient cells, a condition that mimics oxidative stress situation, the threshold of lethality sharply goes down by 20 fold to
0.025 mM GSSG. Our estimates show that in HT4 neural cells, total GSH content is in the range of 13 mM (not shown). This is consistent with the literature reporting that under basal conditions, cellular GSH levels is in the tune of 10 mM 34,44 . In response to oxidant insult, GSSG levels sharply go up and may represent up to 50% of the total GSH in the cell 42,45 . Oxidative stress depletes cellular reducing equivalents such NADPH 46 compromising
GSSG reductase function.
30 Summary of Observations • Elevated intracellular GSSG (0.5mM) triggers neural cell death. • Under the condition of GSH depletion, GSSG-induced lethality is lowered by 20 fold.
1.3.2 Glutathione disulfide induced cell death via 12-Lox pathway
Previously, we had reported that GSH-depletion in glutamate-
challenged HT4 neural cells leads to the activation of 12-Lox which is
central in executing glutamate-induced neural cell death. Inhibitors of 12-
Lox, including BL-15 and α-TCT, prevent glutamate-induced HT4 cell death
21-23,28 . We therefore sought to examine whether GSSG-induced death of
HT4 neural cells is mediated by 12-Lox. Both inhibitors of 12-Lox, BL-15 as
well as α-TCT, significantly protected against GSSG-induced loss of cell
viability suggesting the involvement of 12-Lox in this death pathway (Fig.
1.12). Next, we utilized the advantages of the microinjection approach to
test the significance of 12-Lox and its substrate arachidonic acid in the
death of GSH-deficient HT4 cells. While depletion of cellular GSH reserves
by arresting GSH synthesis using BSO does not cause cell death, such
GSH-deficient cells are known to be highly sensitive to extracellular
arachidonic acid treatment 21 . Consistently, in this study we observed that
31 microinjection of small amounts of free arachidonic acid to GSH-deficient
HT4 cells caused cell death. Both BL-15 as well as α-TCT protected against such death suggesting the involvement of 12-Lox in intracellular free arachidonic acid induced cell death. Supporting this conclusion is our observation that direct administration of active 12-Lox into GSH-deficient cells causes cell death in a BL-15 and α-TCT sensitive manner (Fig. 1.13).
Next, we sought to test whether endogenous GSSG may kill cells by a
12-Lox dependent mechanism. BCNU, a well-characterized specific GSSG reductase inhibitor, was chosen as a pharmacological tool to increase cellular GSSG:GSH ratio (Fig. 1.14)47-48 . Indeed, BCNU-induced elevation of cellular GSSG caused cell death. BCNU-induced cell death was significantly lowered by BL-15 as well as α-TCT suggesting the involvement of 12-Lox (Fig. 1.15). Inhibition of GSSG efflux by MK-571, a specific inhibitor for multidrug resistance associated protein-1 (MRP1) 42,49 , significantly increased loss of HT4 viability (Fig. 1.16). Consistent observations indicating that GSSG may induce cell death by a 12-Lox dependent mechanism led us to investigate the relationship between GSSG and 12-Lox. Using a standard assay to measure 12-Lox activity it was noted
32 that the presence of GSSG in the reaction mixture significantly increases the catalytic function of 12-Lox (Fig. 1.17).
Glutamate toxicity is a major contributor to pathological cell death within the nervous system and is known to be mediated by reactive oxygen species and GSH loss 50-51 . Glutamate-induced death of neural cells is known to be associated with GSH loss and oxidation 23-24 . Our previous studies have identified 12-Lox as a key mediator of glutamate-induced neural cell death 21 . We and others have reported that 12-Lox deficient mice are protected against stroke dependent injury to the brain 21,52 . GSH depletion causes neural degeneration by activating the 12-Lox pathway 53-54 .
Observations in this study suggest a direct influence of GSSG on 12-
Lox activation. Arachidonic acid is converted into several more polar products in addition to 12-l-hydroperoxyeicosa-5,8,10,14-tetraenoic acid
(12-HPETE) and 12-l-hydroxyeicosa-5,8,10,14-tetraenoic acid (12-HETE) by 12-Lox. Previously it has been demonstrated that the presence of 0.5-1.5 mM GSH in the reaction mixture prevents the formation of the more polar products and produces 12-HETE as the only metabolite from arachidonic acid by the 12-Lox pathway. It was therefore concluded that 12-HPETE
33 peroxidase in the 12-Lox pathway is a GSH-dependent peroxidase and the more polar products might be formed from the non-enzymatic breakdown of the primary 12-Lox product of 12-HPETE, owing to insufficient capability of the subsequent peroxidase system to completely reduce 12-HPETE to 12-
HETE 55 . In a cell system, disulfides are known to be able to act as biological oxidants that oxidize the zinc-thiolate clusters in metallothionein with concomitant zinc release 56 . Intracellular zinc release is known to cause 12-
Lox activation and neurotoxicity 57 . Studies with BSO-treated GSH-deficient cells highlighted the significance of microinjected free arachidonic acid in neurotoxicity. These observations are consistent with the literature reporting a central role of the free arachidonic acid mobilizing enzyme phospholipase
A2 in neurotoxicity 58 .
The GSSG reductase inhibitor BCNU, clinically known as carmustine, is a proven chemotherapeutic agent 59 . Cytotoxicity is a widely recognized side-effect of BCNU 60 . Because BCNU treatment is associated with GSSG accumulation in cells 61 , the significance of GSSG in BCNU-induced cytotoxicity is of interest. Observations of this study support that both GSSG as well as BCNU-induced cell death follow the same 12-Lox dependent path suggesting the possibility that BCNU may cause cell death via GSSG.
34
Affirmation of this hypothesis would warrant examining the significance of pro-GSSG strategies for cancer therapy. Major forms of cancer therapy including radiation therapy as well as chemotherapy rely on oxygen- centered free radicals for their action. Thus, these interventions cause overt oxidative insult associated with elevated levels of cellular and tissue
GSSG 62-63 . GSSG generated within the cell is pumped out of the cell perhaps to avert cytotoxicity caused by GSSG accumulation. Approaches to selectively block the GSSG efflux mechanisms in cancer cells might be useful for cancer therapy. Findings of this study support that BSO sensitizes cells to GSSG-induced death. Indeed, BSO has been founds to be an useful adjunct for both radiation 64 as well as chemotherapy 65 . Also, inhibition of
GSSG efflux by inhibition of MRP1 enhanced BCNU-induced cytotoxicity suggesting that GSSG extrusion play a role in neural sensitivity to GSSG.
Summary of Observations • GSSG-induced neural cell death is mediated by 12-Lox • BCNU-induced neural cell death is mediated by 12-Lox • GSSG induces 12-Lox activity
35 1.3.3 GSSG-12-Lox formation in response to GSSG
GSSG was noted to cause marked S-glutathionylation of proteins in
HT4 cells (Fig. 1.18). Control cells did show baseline glutathionylation levels
but they are not noticed in Fig. 1.18 because film exposure time was
minimized to obtain a good resolution blot of the GSSG treated cells. Signal
detected as protein glutathionylation by immunoblots was reversed under
reducing conditions demonstrating specificity of the antibody used (Fig.
1.19). To determine whether 12-Lox is subject to GSSG-induced S-
glutathionylation HT4 cells were transfected with vectors expressing V5-
tagged 12-Lox. Cell lysates from such cells, containing V5-tagged 12-Lox,
were incubated with GSSG. Interestingly, the formation of GSS-12-Lox was
noted (Fig. 1.20A). Thus, GSSG is capable of S-glutathionylating 12-Lox.
These findings were consistent with our observation that GSSG is able to S-
glutathionylate pure 12-Lox obtained commercially (Fig. 1.20B).
GRx1 is the most specific and efficient deglutathionylating enzyme in
the cytoplasm of mammalian cells 38 . To test the overall significance of S-
glutathionylation on glutamate-induced cell death, HT4 cells were infected
with an adenoviral vector expressing GRx1. The gene delivery process was
successful in markedly increasing cellular GRx1 expression (Fig. 1.21).
36 Globally, GRx1 overexpression lowered the empirical abundance of S- glutathionylated proteins in the cell indicating elevated catalytic function of
GRx1 in the cells (Fig. 1.22). Overexpression of GRx1 significantly protected HT4 neural cells against glutamate challenge. These findings suggest the involvement of S-glutathionylation reactions in glutamate- induced cell death (Fig. 1.23). To expand on the mechanism by which GRx1 may have protected the cells against glutamate-insult, S-glutathionylation of
V5-tagged 12-Lox was examined in HT4 cells. Glutamate increased 12-Lox glutathionylation which was prevented in GRx1 over-expressing cells (Fig.
1.24). These findings were consistent with results from studies employing the microinjection approach demonstrating that cytosolic delivery GRx1 significantly rescued HT4 neural cells against GSSG or arachidonic acid- induced cytotoxicity (Fig. 1.25). These observations collectively support the notion glutamate induces S-glutathionylation of 12-Lox in HT4 neural cells.
GRx1 protected against glutamate-induced 12-Lox glutathionylation as well as cell death.
S-Glutathionylated proteins (PSSG) can result from thiol/disulfide exchange between protein thiols (PSH) and GSSG 3. Protein S- glutathionylation, the reversible binding of glutathione to low-pKa cysteinyl
37 residues in PSH, is involved in the redox regulation of protein function.
Several enzymes are known to undergo this post translational modification.
Importantly, whether glutathionylation inhibits or augments protein function may vary depending on the individual case (Table 1.1). For example, glutathionylation inhibits phosphofructokinase 8, NF κB66 , glyceraldehydes-3- phosphate dehydrogenase 67 , protein kinase C-α68 , creatine kinase 10 , as well as actin 11 . In contrast, glutathionylation-dependent gain of protein function has been reported for microsomal glutathione S-transferase 12 , HIV-1 protease-Cys67 13 , and matrix metalloproteinases 14 . Furthermore, specific electron transport proteins of the mitochondria are sensitive to S- glutathionylation 69-70 . Consistently, we noted that GSSG induced cell death was associated with loss of mitochondrial membrane potential. The presence of multiple cysteine residues in 12-Lox makes it susceptible to S- glutathionylation. Results of this study provide first evidence demonstrating that 12-Lox may be glutathionylated in glutamate challenged neural cells.
The finding that both GRx1 expression as well as delivery may protect cells against glutamate-induced neurotoxicity suggests that glutamate-induced glutathionylation is implicated in the cell death pathway. GSSG is a functionally active byproduct of GSH metabolism that, under appropriate conditions, may trigger cell death. S-glutathionylation seems to be a
38 mechanism that favors 12-Lox but not the classical caspase-3 dependent 71
death pathway.
Summary of Observations • GSSG glutathionylates 12-Lox • GRx1 deglutathionylates 12-Lox • GRx1 is neuroprotective
1.3.4 Glutathione disulfide-induced brain injury in 12-Lox deficiency mouse in vivo
The estimated concentration of GSH in the brain is 1.9 ± 0.37 mM 72 .
Consistent with the literature 73 , resting GSH levels in the mouse brain tissue
was noted to be 2.8 µmol per gram wet weight (not shown). Acute ischemic
stroke, as illustrated in Fig. 1. 26A, resulted in over 7-fold increase in GSSG
levels of the affected brain (Fig. 1. 26B). We have previously reported that
12-Lox deficient mice are protected against brain injury caused by acute
ischemic stroke 22 . To test whether GSSG is indeed capable of causing brain
lesion in vivo , GSSG was stereotaxically injected into the cortex. Evidence
of clear infarction was noted. 12-Lox deficient mice were significantly
protected against GSSG-induced lesion of the cortex in vivo (Fig. 1. 27).
39 These results indicate that under specific conditions GSSG may cause brain lesion in vivo via a 12-Lox dependent pathway.
Three-fourth of all stroke in humans occur in distributions of the middle cerebral artery 74 . Therefore, MCAO represents a common approach to study stroke in small as well as large animals 22 . Using a MCAO approach we were able to obtain just over 16% infarction of the ipsilateral hemisphere as assessed by MRI. Stroke caused 8-fold increased in GSSG levels in the affected brain tissue. This is consistent with the known incidence of oxidative stress in the stroke affected brain 75 . Our observation that the stereotaxic injection of GSSG to the brain may cause lesion by a 12-Lox sensitive mechanism leads to question the significance of GSSG in numerous brain pathologies commonly associated with elevated levels of
GSSG in the brain 76-77 .
Summary of Observations • GSSG accumulates in the stroke-affected brain • Elevated GSSG causes injury to the brain tissue • 12-Lox deficient mice are resistant to GSSG-induced injury
40 1.4 Conclusion
Taken together, this work presents first evidence demonstrating that
intracellular GSSG may trigger cell death. GSSG cytotoxicity is substantially
enhanced under conditions of compromised cellular GSH levels as
observed during a wide variety of disease conditions as well as aging 77-78 .
BSO-assisted glutathione lowering approaches are known to be effective to
facilitate both chemo- as well as radiation- therapies. Furthermore, BCNU-
dependent arrest of GSSG reductase activity leads to elevation of cellular
GSSG and has chemotherapeutic functions. Findings of this study lead to
question the significance of GSSG in such processes. From the standpoint
of novel therapeutic approaches, strategies directed at improving or
arresting cellular GSSG clearance may be effective in minimizing oxidative
stress related tissue injury or potentiating the killing of tumor cells,
respectively.
41 1.5 Tables
Protein Stimulus Reference
β-Actin GSSG, GSH 79 Aldose reductase GSNO, GSSG 80-81 Ca2+/CaM (calmodulin)-dependent diamide 82 kinase Caspase-3 NO 83 Creatine kinase GSSG 10 ER ryanodine receptor ischemia 84 67,85-87 GAPDH GSSG, GSNO, H 2O2, diamide 88 Glutathione S-transferase GSSG, H 2O2 Hemoglobin GSSG 89 HIV-1 protease GSSG 13,90 H-Ras GSNO 91 c-Jun NO, GSNO 92 12-Lipoxygenase GSSG, glutamate 93 94 Metallothionein GSNO, H 2O2, diamide Nuclear factor-1 Diamide 9 95 Nuclear factor-κB H2O2 96 P53 H2O2, diamide, cisplatin Protein disulfide isomerase (PDI) NO 97 68 Protein kinase C diamide 98 Protein tyrosine phosphatase 1B H2O2 99 Stromal interaction molecule 1 (STIM1) H2O2
Table 1.1 List of glutathionylated proteins in publications
42 1.6 Figures
Figure 1.1 Chemical structure of GSH and GSSG . The reduced form, GSH is a tripeptide with glutamate, cysteine, and glycine. The oxidized dimer,
GSSG is formed by a disulfide bond between two GSHs.
43
Figure 1.2 Mechanism of glutaredoxin and thioredoxin reaction.
Glutaredoxin (GRx) catalyses the reduction of disulfide bonds in
glutathionylated proteins. During the reaction, cysteine active sites of GRx are
converted to a disulfide. Oxidized Grx is reduced by GRx reductase while
thioredoxin (TRx) is reduced by TRx reductase.
44
Figure 1.3 Microinjection workstation . Zeiss Axiovert 200M microscope and Eppendorf Injectman N12 rest on a TMC vibration-free table. The
Eppendorf micromanipulator Femtojet B 5247 is connected with a glass capillary. The stage is maintained at 37°C by Tempcontrol-37. Digital images are collected and analyzed using AxioVision software. Microinjection was performed into HT4 neural cell with 80 hPa of pressure and 0.2 s duration.
The compensation pressure during injection was 40 hPa. QDot streptavidin conjugate was used as a fluorescent marker to localize the injection site. Qdot is delivered to the cytosolic or nuclear compartment of HT4 neural cells.
45
Figure 1.4 Micro-capillary preparation . The glass microinjection needles are made from GD-1 glass capillaries (Narishige, Japan) by using a Narishige
PC-10 Puller with heater set at 52.7°C and weight set at 250g. This setting creates supple capillary with < 0.5 µm tip diameter. Size and Shape of needles
are customized by adjusting weight and heating value.
46
Figure 1.5 Microinjected GSH failed to rescue, while GSSG potentiated glutamate-induced death of HT4 neural cells . PBS ( A), 500 attomole GSH
(B), or GSSG ( C) was injected into the cytoplasm after 4h glutamate challenge. After 24h incubation, some (15-20%) HT4 cells injected with PBS
(D) or GSH ( E) remained alive, while all GSSG ( F) injected cells were dead suggesting GSSG toxicity (G). attomoles injected [micromolar]i, Bar=20 µm, n=3, Results are mean ± SD, *p < 0.05 compared with PBS injected cells.
47
Figure 1.6 Image of a HT4 neural cell taken by atomic force microscopy .
HT4 neural cells were fixed with glutaraldehyde, then horizontal, vertical, and surface distances of five specific spots were measured and used for determination of single cell volume (n=3).
48
Figure 1.7 Dose-dependent GSSG toxicity . GSH (500 attomoles) or GSSG
(0, 12.5, 25, 50, 250, and 500 attomoles) was microinjected into the cytoplasm of HT4 cells for 24h. attomoles injected [micromolar]i, n=3, Results are mean ± SD, *p < 0.05 compared with PBS injected cells.
49
Figure 1.8 Intracellular GSH and GSSG levels in response to BSO
treatment . Cellular GSSG/total glutathione ( A) was increased, and absolute value of GSH ( µmol/ mg protein) was decreased after 50 µM BSO treatment
(B). n=3, Results are mean ± SD, *p < 0.05 compared with control.
50
Figure 1.9 Dose-dependent GSSG toxicity following BSO treatment .
GSSG (0, 1.25, 2.5, 5 and 25 attomoles) was injected into the cytoplasm of
HT4 cells pre-treated with 50 µM BSO for 12h. attomoles injected
[micromolar]i, n=3, Results are mean ± SD, *p < 0.05 compared with PBS
injected cells.
51
Figure 1.10 Cytosolic injection of GSSG compromised mitochondrial membrane potential. HT4 cells were injected with either PBS ( A, Dextran alexa fluor 488; B, 8nM TMRM; C, 0.5 µl/ml PMPI; D, merged image) or 500
attomole GSSG ( E, Dextran alexa fluor 488; F, 8nM TMRM; G, 0.5 µl/ml
PMPI; H, merged image). I, GSSG injected cells showed lower mitochondrial membrane potential ( F) compared to sham injected cells ( B) while plasma membrane potential was not affected ( C and G). attomoles injected
[micromolar]i, Bar=30 µm, n=3, Results are mean ± SD, *p < 0.05 compared with corresponding control.
52
Figure 1.11 The γγγ-Glutamyl cycle . Summary of glutathione metabolism modified from Meister et al (1983) Glutathione 100 .
53
Figure 1.12 GSSG-induced cell death attenuated by 12-Lox inhibitors.
HT4 cells were injected with PBS ( A), 500 attomole GSH ( B), or 500 attomole
GSSG ( C to E). 2.5 µM BL-15 ( D) and 1 µM α-TCT ( E) were added to culture medium prior to cytoplasmic injection with GSSG. After 24h incubation, cells injected with PBS ( F) and GSH ( G) were alive, while GSSG ( H) injected cells were dead. Treatment of BL-15 ( I) and α-TCT ( J) protected neural cells against GSSG challenge (K). attomoles injected [micromolar]i, Bar=20 µm, n=3, Results are mean ± SD, *p < 0.05 compared with PBS or GSH injected cells. †p < 0.05 compared with GSSG injected cells.
54
Figure 1.13 Arachidonic acid-induced cell death attenuated by 12-Lox inhibitors . Cultured HT4 cells were treated with 50 µM BSO for 12h, then
either 0.15 attomole arachidonic acid or 5 ×10 -8 units 12-Lox were injected.
After 24h incubation, cells injected with arachidonic acid lost cell viability while
BL-15 or α-TCT treated cells were protected. Similarly, cells injected with 12-
Lox lost cell viability while BL-15 and α-TCT treated cells were protected.
attomoles injected [micromolar]i, n=3, Results are mean ± SD, *p < 0.05 compared with PBS injected cells. †p < 0.05 compared with arachidonic acid or 12-Lox without treatment of 12-Lox inhibitors.
55
Figure 1.14 Bischloroethylnitrosourea-induced cellular GSSG
accumulation . Bischloroethylnitrosourea (BCNU, 50 µM) treatment increased cellular GSSG over time. n=3, Results are mean ± SD, §p < 0.05 compared with control.
56
Figure 1.15 Bischloroethylnitrosourea-induced cell death attenuated by
12-Lox inhibitors . HT4 cells were treated with ethanol ( A), 50 µM 1,3-Bis (2- chloroethyl)-1-nitrosourea; BCNU ( B), 50 µM BCNU and 2.5 µM BL-15 ( C), or
50 µM BCNU and 1 µM TCT ( D). After 12h of BCNU treatment, live cells
were visualized using calcein-AM ( A-D); cell viability was also assayed using
a calcein AM based cell viability kit ( E). Bar=100 µm. n=3, Results are mean ±
SD, *p < 0.05 compared with control. †p < 0.05 compared with BCNU treated cells.
57
Figure 1.16 Bischloroethylnitrosourea-induced cell death aggravated presence of MRP1 inhibitor. MK571 (20 µM), a pharmacological inhibitor of
MRP1, was added to HT4 cell culture medium for 6h prior to 50 µM BCNU treatment. After 12h of adding BCNU into cell culture medium, loss of cell viability was assessed by measuring leakage of LDH. MK571 treated HT4 cells were more vulnerable to BCNU-induced loss of cell viability. n=3,
Results are mean ± SD, §p < 0.05 compared with control. †p < 0.05 compared with BCNU treated cells.
58
Figure 1.17 12-Lox activity in response to GSSG . The in vitro activity of 12-
Lox was assayed by using a standard spectrophotometric method. The final
assay mixture contained 10 µmol/L of arachidonic acid and 2 units of 12-Lox.
To determine the effects of GSSG, 10 µM GSSG or GSH were incubated with
this mixture as shown. The absorbance of the reaction mixture was measured
at 234 nm as an index of formation of conjugated dienes. GSSG increased
12-Lox activity by increasing conjugated diene (reaction product) formation.
n=3, Results are mean ± SD, *p < 0.05 compared with GSH injected cells or control.
59
Figure 1.18 Protein glutathionylation in response to GSSG. HT4 cell
lysates (20µg) were incubated with GSSG (10 mM) for 1h and subjected to
SDS-PAGE and immunoblotting for the detection of formation of protein-
glutathione mixed disulfides (glutathionylation). Cells incubated with GSSG
were rich in glutathionylated proteins.
60
Figure 1.19 Protein glutathionylation in response to GSSG under
reducing conditions. HT4 cell lysates (20µg) were incubated with GSSG (10
mM) for 1h and subjected to SDS-PAGE under either reducing or non-
reducing condition for the detection of formation of protein-glutathione mixed
disulfides (glutathionylation). Cells incubated with GSSG under non-reducing
condition were rich in glutathionylated proteins. Samples processed under
reducing condition were lacked in glutathionylated proteins.
61
Figure 1.20 GSS-12-Lox formation in response to GSSG . A, After transfection with 12-Lox containing V5 epitope, HT4 cell lysates were incubated with 10mM GSSG for 1h and cell lysates (500µg) were subjected to immunoprecipitation (IP) with V5 antibody. IP were subjected to SDS-PAGE and immunoblotting for the detection of formation of protein-glutathione mixed disulfides. Cell lysates incubated with GSSG increased the formation of GSS-
12-Lox. B, Porcine leukocytes 12-Lox (30µg) was incubated with 10mM
GSSG for 1h. Western blot was used for detection of GSS-12-Lox formation.
12-Lox incubated with GSSG increased formation of glutathionylation. n=3,
Results are mean ± SD, * p < 0.05 compared with control.
62
Figure 1.21 Glutaredoxin 1 transfection in HT4 neural cells . After 24h of
seeding, HT4 cells were transfected with adenoviral vector containing the
glutaredoxin 1 (ad-GRx1) cDNA construct with dose-dependent manner. ( A,
Western blot; B-C, immunocytochemistry, blue-DAPI stained nuclei; green-
GRx1 protein; B, cells transfected with 2000 m.o.i ad-LacZ; C, cells tranfected
with 2000 m.o.i ad-GRx1). Bar=50 µm.
63
Figure 1.22 Glutaredoxin 1 reversed glutathionylation in HT4 neural cells.
After transfection with ad-LacZ or ad-GRx1 (2000 m.o.i), HT4 cell lysates
(30µg) were subjected to SDS-PAGE and immunoblotting for the detection of formation of glutathionylation. HT4 cells transfected with ad-GRx1 expressed lower formation of glutathionylation.
64
Figure 1.23 Glutaredoxin 1 protected cells against glutamate challenge.
HT4 cells were transfected ad-LacZ or ad-GRx1. After 72h of transfection, cells were re-split and incubated for 24h. Cells were challenged with or without 10mM glutamate for 24h, and viability of HT4 cells were assessed by measuring leakage of LDH. Cells overexpressing GRx1 were more resistant to glutamate-induced loss of cell viability. n=3, Results are mean ± SD, * p <
0.05 compared with LacZ transfected control, †p < 0.05 compared with LacZ transfected- glutamate treated cells.
65
Figure 1.24 Glutaredoxin 1 reversed glutathionylation of 12-Lox in HT4
neural cells , After transfection with 12-Lox containing V5 epitope, HT4 cells were challenged with 10mM glutamate for 8h. Cell lysates (500µg) were subjected to IP with V5 antibody. IP were subjected to SDS-PAGE and immunoblotting for the detection of formation of glutathionylation. Cells treated with glutamate expressed higher formation of GSS-12-Lox. However, in GRx1 transfected cells glutamate-induced 12-Lox glutathionylation was blunted. n=3, Results are mean ± SD, * p < 0.05 compared with corresponding control.
66
Figure 1.25 Glutaredoxin 1 microinjection protected HT4 neural cells against GSSG as well as arachidonic acid challenge . Cultured HT4 cells were either injected 500 attomole GSSG ( A and B) or 0.15 attomole arachidonic acid ( C and D), and co-injected with 2 attomole Grx1 ( B and D).
After 24h incubation, cells injected with GSSG ( E) lost cell viability, while
GRx1 co-injected cells ( F) were significantly protected. Cells injected with arachidonic acid ( G) lost cell viability, while GRx1 co-injected cells ( H) were protected ( I). attomoles injected [micromolar]i, Bar=20 µm, n=3, Results are mean ± SD, * p < 0.05 compared with corresponding control.
67
Figure 1.26 MCAO-induced infarct hemisphere contained elevated level
of GSSG . A, Transient focal cerebral ischemia was induced in 8 to 10 week
old C57BL6/J mice by middle cerebral artery occlusion for 90 min. Brain
infarction was detected by T2-weighted MRI images at 24h after reperfusion.
B, HPLC coulometric electrode array detector was used to detect GSSG and
GSH level in the infarct tissue, and stroke-induced injured hemisphere contained elevated level of GSSG. n=3, Results are mean ± SD, *p < 0.05 compared with non-infarct hemisphere.
68
Figure 1.27 12-Lox-deficient mice resistant to GSSG-induced brain injury .
GSSG was injected to the brain cortex of C57BL6/J (control, n=3) or 12-Lox knock-out (12-Lox -/-, n=3) mice. Two days after injection, T2-weighted MRI
images were collected. WT mice injected with GSSG (A) had clear lesion in
the brain while 12-Lox deficient mice ( B) were significantly resistant to GSSG-
induced brain injury ( C). Solid arrows represent damage caused by GSSG.
Results are mean ± SD, * p < 0.05 compared with non-infarct hemisphere.
69 CHAPTER 2
Multidrug resistance-associated protein 1 in experimental stroke
2.1 Introduction
2.1.1 Multidrug resistance-associated protein 1 (MRP1)
Multidrug resistance-associated protein 1 (MRP1/ ABCC1) is a
member of the ATP-binding cassette (ABC) transporter protein superfamily.
Although many MRP isoforms present specificity of tissue distribution,
MRP1 is ubiquitously detected in most part of body including brain (Table
2.1). ABC superfamily contains membrane-bound, ATP-binding cassette
domains which allow to process translocation of various substrates such as
drugs, drug metabolites, endogenous metabolites, lipids, sterols, proteins
and peptides. ABC superfamily is divided into 7 different subfamilies such
as subfamily A (ABCA), subfamily B (ABCB; MDR; P-glycoprotein),
subfamily C (ABCC; MRP), subfamily D (ABCD), subfamily E (ABCE),
subfamily F (ABCF), and subfamily G (ABCG).
70
ABC subfamily C, MRP, is structurally similar with a multidrug resistance protein (MDR; ABCB). Both MRP and MDR contain N-terminal hydrophobic domain (MSD1) to C-terminal, but MRP contains additional five transmembrane domains (MSD0) and intracellular loops (L0). MRP1, MRP2,
MRP3, MRP6 and MRP7 present three transmembrane domains (MSD0,
MSD1, and MSD2) but MRP4, MRP5, MRP8 and MRP9 are lacking a
MSD0 membrane spinning domain (Fig. 2.1).
Cole et al first cloned cDNA encoding
MRP1 from doxorubicin-selected human small lung cancer cell line (H69AR)101 . MRP1 is found predominantly in heart, small intestine, brain, and skin in human 102-103 , but it expresses very low level in the liver. MRP1 is reported to present similar distribution Dr Susan Cole Queen’s University across different species including rats, dogs, http://www.path.queensu.ca/ mice and cows 104-107 . MRP1 is capable to transport lipophilic anions while substrates of MDR1 are neutral or positively charged compounds. GSSG, glutathione, glucuronate and sulfate conjugate are exported by MRP1
71 (Table 2.2). Thus MRP1 has gained attention in phase II cellular
detoxification and phase III elimination of toxic endogenous metabolites. In
addition, inflammatory mediator leukotriene C 4 (LTC 4) is reported as a
MRP1 substrate with high affinity (Km ≈100nM), so MRP1 appears to play
an important role in immune response 108 . Anticancer drugs such as
anthracycline, etoposide, and vincristine are well studied MRP1 substrates.
Thus, MRP1 has been considered an important target of chemotherapy.
2.1.2 MRP1 in brain
Brain cells such as neurons and astrocytes express various transporter
proteins which are responsible to translocate lipids 109 , peptides, and amino
acids including glutamate 110-111 . Brain cells present both importers (organic
cation transporter and organic anion transporter) and exporters (MRP, MDR,
and BCRP). DeCory et al reported MRP-like transporter in mouse cortical
neurons 112 . Hirrlinger et al also found MRP1 as well as MRP3, MRP4 and
MRP5 expression in embryonic rat neurons 49 . Beside of neurons,
expression of MRP1 in astrocytes was detected by this group. Astrocytes
are reported to release high level of GSSG, thus MRP1 may play a critical
role to maintain glutathione homeostasis in astrocytes.
72 Mechanisms of multidrug resistance and clinical outcomes in response
to manipulation of MRP expression have been extensively studied in the
context of various types of cancers 113-116 . Induction of MRP1 in brain tumors
such as astrocytoma, glioblastoma, and neuroblastoma are also reported.
However, the significance of MRP1 in brain-related pathology other than
cancer is poorly developed. Currently, the limited information available
consistently demonstrates that under pathological conditions known to be
associated with oxidant insult, i.e Alzheimer’s and epilepsy, MRP1
expression is elevated in the brain 117-119 . These studies indicate that MRP1
play a role in dysplastic neuron and reactive astrocytes in such conditions
and suggest MRP1 as a novel therapeutic target for neurodegenerative
disorders.
2.1.3 microRNA
microRNAs (miRs) are family of short single-stranded non-coding
RNAs and powerful regulators of gene expression. A miR is approximately
18-25 ribonucleotides-long with a potential to recognize multiple mRNA
targets guided by sequence complementarity and RNA-binding proteins.
Recent evidence suggests that the number of unique miR genes in human
exceeds 1000 and may be as high as 20,000 120 . miRs are functionally
73 versatile, with the capacity to specifically inhibit translation initiation or elongation as well as induce mRNA destabilization, through predominantly targeting the 3’-untranslated regions (UTR) of mRNA.
miR are transcribed in the nucleus and exported to the cytoplasm, then mature miR that can interact with matching mRNAs by RNA-RNA binding.
This binding with the assistance of the RNA induced silencing complex
(RISC) leads to modes of action, resulting in mRNA degradation or translational inhibition 121 . This mechanism of action is termed as post transcriptional gene regulation (PTGS) 122 .
Transcript of miR gene (pri-miRs) are generated by either RNA polymerase II 123 or RNA polymerase III 124 in nucleus. pri-miRs are shorten to 60nt long hairpin precursors (pre-miR) by ribonuclease Drosha. pri-miRs are moved to cytoplasm by Exportin 5 then cleaved by Dicer. The miR is incorporated into the RNA-induced silencing complex (RISC) where it binds to the 3’UTR of target transcripts and induces either translational silencing or transcriptional degradation .
74 2.2 Material and Method
2.2.1 Small interfering RNA delivery and analysis of genes
HT4 neural cells (0.1 10 6 cells / well in 12-well plate) were seeded in
antibiotic free medium 24h prior to transfection. DharmaFECT TM 1
transfection reagent was used to transfect cells with 100nM siRNA pool
(Dharmacon RNA Technologies, Lafayette, CO) for 72h as described
previously 20,28,125 . For controls, siControl non-targeting siRNA pool (mixture
of 4 siRNA, designed to have ≥ 4 mismatches with the corresponding gene)
was used. HT4 cells were harvested and re-seeded for treatment with
glutamate (Sigma-Aldrich, St, Louis, MO) or α-TCT (Carotech, Malaysia) as
indicated in the respective figure legends. For determination of mRNA
expression after siRNA transfection, total RNA was isolated from cells using
the Absolutely RNA ® Miniprep kit (Stratagene, La Jolla, CA). The abundance
of mRNA for MRP1 was quantified using real time PCR using SYBR green-I
(Applied Biosystems, Forster City, CA).
The following primer sets were used: m_MRP1_F, 5’-GGT CCT GTT
TCC CCC TCT ACT TCT T-3’; m_MRP1_R, 5’-GCA GTG TTG GGC TGA
CCA GTA A-3’; m_GAPDH_F, 5’-ATG ACC ACA GTC CAT GCC ATC ACT-
3’; m_GAPDH_R, 5’-TGT TGA AGT CGC AGG AGA CAA CCT-3’.
75
2.2.2 miRIDIAN miR mimic/ inhibitor delivery
HT4 neural cells were seeded (0.1 10 6 / well, 12 well plate) in
antibiotic free medium for 24h prior to transfection. DharmaFECT TM 1
transfection reagent was used to transfect cells with miRIDIAN mmu-miR-
199a-5p mimic or mmu-miR-199a-5p hairpin inhibitor (Dharmacon RNA
Technologies, Lafayette, CO) as per the manufacturer’s instructions.
miRIDIAN miR mimic or inhibitor negative controls (Dharmacon RNA
Technologies) were used for control transfections. Samples were collected
after 72h of miR mimic/inhibitor delivery for quantification of miR, mRNA and
protein expression as described 126 .
2.2.3 pGL3-MRP1-3’UTR luciferase reporter assay
miRIDIAN mmu-miR-199a-5p mimic or mmu-miR-199a-5p hairpin
inhibitor was delivered to HT4 neural cells followed by transfection with
pGL3-MRP1-3’UTR firefly luciferase expression construct (Signosis,
Sunnyvale, CA) together with renilla luciferase pRL-cmv expression
construct using Lipofectamine TM LTX PLUS TM reagent. Luciferase assay
were performed using the dual-luciferase reporter assay system (Promega,
76 Madison, WI). Firefly luciferase activity was normalized to renilla luciferase
expression for each sample as described 126 .
2.2.4 Quantification of microRNA expression
Total RNA including miR fraction was isolated using miRVana TM
miRNA isolation kit (Ambion, Austin, TX), according to the manufacturer’s
protocol. miR-199a-5p levels were quantified using Taqman Universal
Master Mix (Applied Biosystems, Forster City, CA). miR levels were
quantified with the 2 (-∆∆ CT) relative quantification method using miR-16 as the
house keeping miR 125-128 .
2.2.5 Western blotting
After protein extraction, the protein concentration was determined
using BCA protein reagents. The samples (40-50 µg of protein / lane) were
separated on a 4-12% SDS-polyacrylamide gel electrophoresis as
described 23,93,129 and probed with anti-MRP1 (1:20 dilution, Enzo Life
Sciences, Plymouth Meeting, PA). To evaluate the loading efficiency,
membranes were probed with anti-GAPDH antibody (Sigma-Aldrich, St.
Louis, MO).
77 2.2.6 Cell viability assay
The viability of cells in culture was assessed by measuring leakage of
lactate dehydrogenase (LDH) from cells into media 24h following glutamate
treatment using an in vitro toxicology assay kit from Sigma Chemical Co. (St.
Louis, MO, USA). The protocol has been described in detail in a previous
report 30 . In brief, LDH leakage was determined using the following
equation : % total LDH leaked = (LDH activity in the cell culture medium /
total LDH activity ) × 100 20-22 . Total LDH activity represents the sum of LDH
activities in the cell monolayer, detached cells, and the cell culture medium.
2.2.7 Immunocytochemistry
HT4 neural cells (0.5 10 6 / well) were seeded in 35mm plates for 24h
then treated with or without 10mM glutamate. Cells were washed with PBS
three times and fixed in 10% buffered formalin for 20 min, then underwent
permeabilization using 0.1% Triton X-100/PBS for 15 min. The cells were
washed and incubated with 10% goat serum (Vector Laboratories,
Burlingame, CA) for 1h at room temperature, and incubated with MRP1
antibody (1:50, Abcam, Cambridge, MA) overnight at 4 °C. After incubation
with primary antibody, cells were washed with PBS three times and
incubated with an Alexa-flour 488 (1:200 dilution) for 1h at room
78 temperature. After three washes and incubation with 4’,6’-diamino-2-
phenylindole (1:10,000 dilution) for 2 min, cells were mounted in gelmount
(aqueous mount, Vector Laboratories, Burlingame, CA) for microscopic
imaging as described previously 20,93 .
2.2.8 Calcein clearance assay
Calcein clearance assay was used to measure MRP1 activity in neural
cells against glutamate insult. To improve poor specificity of
pharmacological inhibitors, RNA interference approach was applied to
measure specific MRP1 activity. After 72h of MRP1 siRNA transfection as
described above, HT4 cells were re-seeded and treated with glutamate as
described in relevant legends. After glutamate challenge, calcein-AM (25nM,
Invitrogen, Carlsbad, CA) was loaded to the cells for 15 min at 37°C. HT4
cells were washed with PBS, collected and analyzed using the Accuri C6 TM
(Accuri Cytometers, Ann Arbor, MI) flowcytometer. MRP1 activity was
measured on the basis of intracellular calcein retention 130 .
79 2.2.9 Mouse stroke model
Transient focal cerebral ischemia was induced in 8 weeks old MRP1
deficient (n=20, male, Taconic, Hudson, NY) or background FVB mice
(n=15, male, Taconic, Hudson, NY) by middle cerebral artery occlusion
(MCAO) as previously described 22,93,131-132 .
Occlusion of the right middle cerebral artery was achieved by using the
intraluminal filament insertion technique. Briefly, mice were anesthetized by
inhaling halothan, and 6-0 nylon monofilament was inserted into the internal
carotid artery, via the external carotid artery. Then the filament tip was
positioned for occlusion at a distance of 6mm beyond the internal carotid
artery-pterygopalatine artery bifurcation. We observed that this approach
results in a 70 ± 10% drop in cerebral blood flow as measured by laser
Doppler (DRT4, Moor Instruments). Once the filament was secured, the
incision was sutured and the animal was allowed to recover from anesthesia
in its home cage. After 90min of occlusion, the animal was briefly re-
anesthetized, and reperfusion was initiated via withdrawal of the filament
from MCA. This surgical protocol typically results in a core infarct limited to
the parietal cerebral cortex and caudate putamen of the right hemisphere.
After 48h of reperfusion, T2-weighted image was taken to measure infarct
volume. Mice suffering from surgical complications ( e.g. hemorrhage or
80 death) during MCAO were excluded. Immediately after imaging, tissues
from control and stroke-affected hemispheres of MRP1 (n=12) and FVB
(n=8) were harvested.
2.2.10 Magnetic resonance imaging (MRI)
T2-weighted imaging was performed on stroke-affected mice. Imaging
experiments were carried out in an 11.7T (500 MHz) MR system comprised
of a vertical bore magnet (Bruker Biospin, Ettlingen, Germany) as described
previously by our group133 . High-resolution magnetic resonance imaging
was performed on mice brain. Mice were anesthetized by inhaled isoflurane
via a nose cone. The mouse under MR scan was placed in a MR compatible
animal holder with the proper respiratory sensor unit. The mouse was held
with tape during contingent supply of a mixture of carboxin (95% air and 5%
carbon dioxide) and isoflurane through tubing. A 30 mm birdcage coil was
used which surrounds the mouse brain. The animal was then placed inside
the radio frequency coil (resonator) and finally the whole arrangement was
placed inside the vertical magnet. A spin echo (SE) sequence was used to
acquire T2-weighted MR images from the mouse head on the 11.7-T MRI
system. A spin echo technique with rapid acquisition with relaxation
enhancement (RARE) sequence providing 8 echo train length (ETL) was
81 used with the following parameters: field of view (FOV) = 30×30 mm, acquisition matrix 256×256, repetition time (TR) = 3000 ms, echo time (TE)
= 30 ms, flip angle (FA) = 180 degrees, images in acquisition = 15, resolution = 8.533 pixels/mm, and number of averages 4. Shim currents were initialized by manual adjustments on all linear and higher order field inhomogeneities. After several localizer scans were completed, a T2- weighted spin echo RARE sequence was applied to generate 15 images corresponding to 15 short axis slices.
Post image processing was done using ImageJ software (NIH). For stroke-volume calculation from 2D images, raw MRI images were first converted to digital imaging and communications in medicine (DICOM) format and read into ImageJ software. By delineating both the whole brain and injured part of the brain borders, whole brain area and injury area were calculated. The areas were summed from all short axis slices and the volumes were computed from the area of traced boarders by multiplying slice thickness. A percentage fraction of infarction to whole brain was determined from the volumes calculated as above.
82 2.2.11 Statistics
Data are reported as mean + SD of at least three independent
experiments. Difference in means was tested using Student’s t-test or one-
way ANOVA with Tukey’s test. P < 0.05 was considered statistically
significant.
Summary of Methods • siRNA delivery and analysis of gene • miRIDIAN miR mimic/ inhibitor delivery and quantification • pGL3-MRP1-3’UTR luciferase reporter assay • Western blotting • Cell viability assay • Immunocytochemistry • Calcein clearance assay • Middle cerebral artery occlusion (MCAO) • MRI
83 2.3 Results and Discussion
2.3.1 Induction of MRP1 in response to glutamate challenge of neural cells
In neural cells, glutamate toxicity is associated with oxidant insult
resulting in oxidation of GSH and rapid elevation of intracellular GSSG. We
were therefore led to test the hypothesis that under such conditions of
GSSG loading, GSSG efflux mechanisms such as MRP1 would be
augmented as a defense response in favor of cell survival. We present the
first evidence that extracellular glutamate challenge may induce MRP1 gene
expression in neural cells (Fig. 2.2A). Consistently, MRP1 protein
expression was also augmented in cells challenged with glutamate (Fig.
2.2B-D).
2.3.2 Neuroprotective effect of α-TCT was compromised in neural cells subjected to MRP1 knockdown
Previously we have reported that α-TCT is potently neuroprotective
against a number of insults including GSSG-induced cell death 23,28,93 . To
test whether such neuroprotective property of α-TCT depends on the MRP1
system for cellular GSSG clearance we adopted the RNA interference
approach to knockdown MRP1. The siRNA-based approach lowered MRP1
84 mRNA expression (Fig. 2.3A). Importantly, the ability of α-TCT to protect
neural cells against glutamate challenge after silencing MRP1 was
compromised (Fig. 2.3B). This observation suggests that the MRP1
pathway is functionally active as a part of the overall mechanism by which
α-TCT protects neural cells against glutamate-induced toxicity. Functionally,
MRP1 activity can be measured by the calcein clearance assay. Higher
MRP1 activity results in greater clearance of calcein from preloaded cells.
Exposure of calcein-preloaded cells to glutamate increased calcein
clearance establishing that glutamate challenge upregulates MRP1 function.
Such improved clearance of calcein in glutamate challenged cells was
blunted by MRP1 knockdown demonstrating that MRP1 is indeed a key
contributor to the calcein clearance process (Fig. 2.4).
2.3.3 MCAO-induced brain injury was exacerbated in MRP1 deficient mice
Transient focal cerebral ischemia was induced by MCAO in MRP1
deficient mice (n=20) or corresponding background FVB mice (n=15). Both
FVB and MRP1 deficient mice showed comparably decreased levels of
MCA-area blood flow during occlusion (Fig. 2.5A). Interestingly, MRP1
deficient mice showed a larger hemispherical infarct volume than that noted
in corresponding FVB mice (Fig. 2.5B-D). Increased abundance of MRP1
85 protein was observed in the infarct hemisphere of FVB mice (Fig. 2.6).
MCAO-induced brain injury increased tissue GSSG levels in both MRP1
deficient as well as in background control mice. Notably, MRP1 deficient
mice contained 1.6-fold higher tissue levels of GSSG in the infarct
hemisphere compared to that detected in background mice (Fig. 2.7). These
data indicate that loss of MRP1 function impairs GSSG clearance during
stroke and leads to increased brain injury. Histochemical studies depicted
that increased MRP1 positive cells were primarily localized in the infarct
hemisphere of FVB mice (Fig. 2.8). Both MRP1 deficient as well as
background FVB mice presented positive Fluoro-Jade immunofluorescence
staining in the stroke-affected side supporting the incidence of
neurodegeneration. Importantly, the infarct hemisphere of MRP1 deficient
mice had a more abundant Fluoro-Jade signal indicating a higher level of
neurodegenerative outcome (Fig 2.9).
2.3.4 miR-199a-5p targets MRP1 expression
Given the key functional significance of MRP1 in neural cell death as
well as in stroke-associated brain injury we turned our attention to factors
that regulate the final levels of MRP1 in the cell. To test whether MRP1
expression is subject to post-transcriptional gene silencing by miR, we first
86 performed in silico studies using databases such as Target Scan
(www.targetscan.org), Miranda (www.microrna.org) and PicTar
(www.pictar.org). miR-199a-5p emerged as a major candidate miR which is likely to target MRP1 exhibiting a single conserved binding site to the 3’UTR of MRP1 transcript (Fig. 2.10). Next, we turned towards biological validation of MRP1 as a target of miR-199a-5p.
In neural cells, miR-199a-5p levels were modulated by delivery of mmu-miR-199a-5p mimic or mmu-miR-199a-5p inhibitor (Fig. 2.11). miRIDIAN miR mimic negative control was used as a control. miR-199a-5p silenced MRP1 mRNA (Fig. 2.12) as well as protein (Fig. 2.13). To determine whether MRP1 is a direct target of miR-199a-5p in HT4 neural cells, a fragment of the 3’UTR of MRP1 mRNA containing the putative miR-
199a-5p binding sequence cloned into a firefly luciferase reporter construct was used. This construct was co-transfected with a control renilla luciferase reporter construct into cells along with miR-199a-5p mimic or inhibitor. miR-
199a-5p mimic significantly decreased luciferase activity while miR-199a-5p inhibitor significantly increased the activity of the miR-199a-5p firefly luciferase reporter recognizing MRP1 as a direct target of miR-199a-5p (Fig.
2.14).
87
To test whether alterations in cellular miR-199a-5p levels influence the response to glutamate challenge, cell viability was measured. miR-199a-5p mimic delivered cells were noted to be vulnerable against glutamate challenge (Fig. 2.15A). GSSG levels in such cells were higher (Fig. 2.15B).
These observations indicate that miR-199a-5p may impair GSSG clearance in the face of glutamate insult resulting in augmented cytotoxicity. Cells delivered with miR-199a-5p inhibitor were protected against glutamate challenge better than corresponding control cells. Such beneficial effects were abrogated by the presence of a pharmacological inhibitor of MRP1,
MK571, indicating that the protective effects of miR-199a-5p inhibitor was mediated by elevated MRP1 function (Fig. 2.16). These studies establish that MRP1 serve as a direct target of miR-199a-5p.
This study indicates that MRP1 as an important defense mechanism against oxidative insult and GSSG clearance via MRP1 may be the critical to maintain cell survival after stroke. Although multiple pathways are involved in GSSG clearance, they may not be fully facilitated under specific condition such as stroke. During stroke-related oxidative insult, GSSG is rapidly formed but its redox cycling to GSH is severely impaired. GSSG
88 reductase activity is impaired following stroke 134-136 . Ischemia-reperfusion depletes NADPH impairing all reductase functions dependent on this reducing equivalent 137 . Therefore, a sharp rise in brain tissue GSSG/GSH ratio occurs following stroke 93,138 . In the current chapter we note that such elevation of intracellular GSSG is associated with the induction of MRP1 perhaps as an adaptive survival response. This contention is consistent with previous reports demonstrating upregulation of MRP1 in response to oxidant insult as an adaptive defense response favoring cell survival 49,139 .
Mechanism of multidrug resistance and clinical outcome via MRP1 expression have been intensively studied in various type of cancers including leukemia 116 , bone tumor 115 , breast cancer 114 , and neuroblastoma
113 . However, role of MRP1 in brain pathology other than cancer is still under investigations. Sisodiya et al reported upregulation of MRP1 expression in glia and neuron from human epilepsy and they also observed that induction of MRP1 in dysplastic neurons in cortex 117 . Similarly, Kubota et al reported upregulation of MRP1 in parahippocampal gyrus and microvascular endothelial cells from epileptic patients 119 . Higher expression of MRP1 protein was reported in hippocampal tissue from Alzheimer’s patients compare to age matching control. Although knowledge about the
89 biological significance of MRP1 on stroke is limited, it is clear that MRP1 is responsible to pathological condition of brain.
To test the neuroprotective effect of MRP1 in vivo , MRP1 deficient mice were used. MRP1 deficient mice carry a disruption of the MRP1 (also known as ABCC1a and CFTR) gene and lacking functional MRP1 protein in variety of tissues. Wijnholds et al first developed MRP1 targeted mutation mouse model. This group reported that MRP1 deficient mice are fertile but show reduction in transport of glutathione conjugated substrates. In addition, they also observed that impaired inflammatory response from this mouse model 140-141 . Using a MCAO approach in current study, we observed aggravated brain injury in MRP1 deficient mice due to GSSG accumulation.
Our observation that stroke related injury to the brain is exacerbated in
MRP1 deficient mice establishes a key significance of MRP1 on stroke outcomes.
Post-transcriptional gene silencing of MRP1 remains a unexplored area. The current study identified MRP1 as a biologically validated target of miR-199a-5p. This provided a great segue for us to address the significance of miRs in stroke 142-144 . Survey-based studies have identified hundreds of
90 miRs to be sensitive to brain ischemia. Antagomir miR-497 treatment has been recognized for its potential to attenuate injury related to acute ischemic stroke 145 . The miR-200 family and miR-182 has been found to be upregulated in ischemic pre-conditioning 146 . Although the significance of miR-199a-5p in stroke has not been studied, miR-199a-5p was reported to be a hypoxia-sensitive miR 147 . Thus, the hypoxia component of stroke- related ischemia may be responsible for downregulating miR-199a-5p following stroke.
Summary of Observations • MRP1 is neuroprotective • MCAO-induced brain injury is exacerbated in MRP1 deficient mice • miR-199a-5p targets MRP1 silencing
91 2.4 Conclusions
Elevation in cellular GSSG triggers cell death and accumulation of
GSSG is commonly found is injured brain due to stroke. Thus, facilitating
GSSG clearing system may be a powerful strategy to attenuate stroke-related brain injury. Although MRP1 has been studied for more than 20 years, the majority of MRP1 research is focused on decreasing MRP1 activity or expression in cancer cells to increase the efficacy of chemotherapy. In this chapter, we establish MRP1 as a neuroprotective factor under conditions of
GSH oxidation as during oxidant insult associated with a wide range of diseases including stroke. A major addition to this knowledge is represented by the observation that MRP1 is subject to control by miR. This opens up the possibility for miR-based therapies in all settings involving MRP1 including stroke as well as cancer.
92 2.5 Tables
MRP/Gene Substrates Distribution References
MRP1/ ABCC1 Table 2.2 Ubiquitous Table 2.2
MRP2/ ABCC2 Similar to MRP1, bilirubin Liver, kidney, gut 148-151 glucuronide, cisplatin, methotrexate
MRP3/ ABCC3 Glycocholic acid, etoposide, Intestine, kidney, 152-154 methotrexate, bile acid pancreas, prostate
MRP4/ ABCC4 Cyclic nucleotides (cAMP, Prostates, kidney, lungs, 155-158 cGMP), nucleotide analogs pancreas, testis, ovary (PMEA, azidothymidine- monophosphate), prostaglandins, methotrexate
MRP5/ ABCC5 Cyclic nucleotides (cAMP, Skeletal muscle, heart 158-160 cGMP), nucleotide analogs (PMEA, azidothymidine- monophosphate)
MRP6/ ABCC6 Small peptides (BQ123), Kidney, liver 161-163 glutathione conjugates
164-165 MRP7/ ABCC10 LTC 4, estradiol-17 β- Skin, colon, testes, glucuronide, spleen
MRP8/ ABCC11 Cyclic nucleotides, nucleotide Breast, testis, liver, 166 analogs placenta MRP9/ ABCC12 N/A Testes, skeletal muscle, 167 ovary
Table 2.1 Summary of MRP family Information is modified from Dallas et al
(2006) Multidrug Resistance-Associated Proteins: Expression and Function in the Central Nervous System 168
93
Substrates References
GSSG 151,169-170 171-172 Leukotriene C 4 171 Leukotriene D 4 171 Leukotriene E 4 173 Prostaglandin A 1 glutathione conjugate 4-Hydroynoneal glutathione conjugate 174 17 β-Estradiol-17( β-D-glucuronide) 175 Glucuronosyl bilirubin 148 Unconjugated bilirubin 176 Estrone-3-sulfate 177 Daunorubicin, doxorubicin 178-180 Vinblastine, vincristine, etoposide 170,178-179 Methotrexate 150 Calcein-AM, Calcein 181 Folate 182 Flutamide 183 Mercury 184 Arsenite, Arsenate 178,184
Table 2.2 Substrates of MRP1
94 2.6 Figures
Figure 2.1 Topology of MRPs. Schematic depicting the organization of
protein domains is modified from Kruh et al (2003) The MRP family of drug
efflux pumps 185
95
Figure 2.2 Induction of MRP1 in response to glutamate challenge . After
24h of seeding, HT4 neural cells were treated with glutamate (10mM) for 12h.
Glutamate challenge induced the expression of MRP1 mRNA ( A) and protein
(B, Western blot; C-D, immunocytochemistry, blue-DAPI stained nuclei; green-MRP1 protein; C, control; D, cells treated with glutamate). n=3,
Results are mean ± SD, *p < 0.05 compared with control.
96
Figure 2.3 MRP1 knockdown attenuated the neuroprotective effects of α-
TCT . After 72h of control scrambled siRNA or MRP1 siRNA transfection,
MRP1 mRNA expression was significantly down-regulated in MRP1 siRNA transfected cells ( A). B, Cells were re-split 72h after transfection. α-TCT
(1 µM) was added into cell culture medium 6h before glutamate (10mM) treatment for 12h. After 12h of glutamate challenge, loss of cell viability was assessed by measuring leakage of LDH. The ability of α-TCT to protect against glutamate toxicity was significantly compromised under conditions of
MRP1 knockdown. n=3, Results are mean ± SD, *p < 0.05 compared with
corresponding control; §p < 0.05 compared with control siRNA transfected, glutamate treated HT4 neural cells.
97
Figure 2.4 MRP1 activity was increased by glutamate challenge in HT4
neural cells . HT4 neural cells were re-split 72h after control siRNA or MRP1 siRNA transfection. Next, cells were challenged with glutamate (10mM) for 0
(non glutamate), 2h or 4h. Cells were preloaded with calcein-AM (0.025 µM)
for 10 min and intracellular calcein fluorescence was measured. Glutamate-
challenged cells with MRP1 knockdown exhibited loss of functional MRP1 by
retaining more calcein compared to the corresponding control cells. n=3,
Results are mean ± SD, *p < 0.05 compared with corresponding control.
98
Figure 2.5 MCAO-induced brain injury was aggravated in MRP1 deficient mice . Transient focal cerebral ischemia was induced in 8- to 10-week-old
MRP1 knockout (n=20) or background FVB mice (n=15) by middle cerebral
artery occlusion (MCAO). Brain infarction was detected by T2-weighted MRI
imaging at 48h after reperfusion. A, Cortical blood flow was monitored during
surgery. Both FVB and MRP1 deficient mice showed comparably decreased
level of MCA-area blood flow during occlusion. B-D, MRP1 deficient mice
presented larger stroke-induced lesion in the brain compared to
corresponding background mice ( B, FVB; C, MRP1 deficient mice; D, % of
hemispherical infarct volume, FVB, n=8; MRP1 KO, n=12). Results are mean
± SD, †p < 0.05 compared with FVB.
99
Figure 2.6 MCAO-induced MRP1 protein expression . Protein expression of
MRP1 was increased in infarct hemisphere of FVB background mice. n=3,
Results are mean ± SD, *p < 0.05 compared with corresponding contralateral hemisphere.
100
Figure 2.7 MCAO-induced GSSG accumulation in MRP1 deficient mice .
Stroke-induced injured hemisphere contained elevated level of GSSG in both
MRP1 deficient as well as in control FVB mice. MRP1 deficient mice
contained significantly high level of GSSG in stroke-induced hemisphere
compared to FVB background mice. n=4, Results are mean ± SD, *p < 0.05
compared with corresponding contralateral hemisphere; §p < 0.05 compared with infarct hemisphere of FVB.
101
Figure 2.8 Increased abundance of MRP1-positive cells in infarct
hemisphere of FVB. A-I, MRP1 expression was significantly increased at the
infarct site of FVB control mice ( B-I, red-MRP1 protein; blue-DAPI stained nuclei; B and F, non-infarct hemisphere of FVB mice; C and G, infarct hemisphere of FVB mice; D and H, non-infarct hemisphere of MRP1 deficient
mice; E and I, infarct hemisphere of MRP1 deficient mice). Bar=50 µm, n=3,
Results are mean ± SD, *p < 0.05 compared with corresponding contralateral
hemisphere.
102
Figure 2.9 Increased level of neurodegeneration in infarct hemisphere of
MRP1 deficient mice . A, The infarct hemisphere of MRP1 deficient mice presented abundant Fluoro-Jade signals ( B-I, green-FluoroJade positive
neurons; blue-DAPI stained nuclei; B and F, non-infarct hemisphere of FVB
mice; C and G, infarct hemisphere of FVB mice; D and H, non-infarct
hemisphere of MRP1 deficient mice; E and I, infarct hemisphere of MRP1
deficient mice). Bar=50 µm, n=3, Results are mean ± SD, *p < 0.05 compared with corresponding contralateral hemisphere; §p < 0.05 compared with infarct hemisphere of FVB.
103
Figure 2.10 Alignment between miR-199a-5p and MRP1 . The alignment between Mus musculus miR-199a-5p and the 3’UTR of MRP1 is analyzed by target mRNA search from Miranda (www.microRNA.org).
104
Figure 2.11 miR199a-5p levels in response to miR199a-5p mimic or inhibitor delivery in HT4 neural cells . After 72h of miR-199a-5p mimic delivery, miR-199a-5p expression was significantly upregulated ( A) while miR-199a-5p hairpin inhibitor significantly downregulated miR-199a-5p ( B). n=3, Results are mean ± SD, *p < 0.05 compared with control.
105
Figure 2.12 miR199a-5p silenced MRP1 gene in HT4 neural cells . After
72h of miR-199a-5p mimic ( A) or inhibitor ( B) delivery, miR-199a-5p negatively regulated MRP1 mRNA. n=3, Results are mean ± SD, *p < 0.05 compared with control.
106
Figure 2.13 miR199a-5p downregulated MRP1 protein expression . After
72h of miR-199a-5p mimic ( A) or inhibitor (B) delivery, miR-199a-5p negatively regulated MRP1 protein expression. n=3, Results are mean ± SD,
*p < 0.05 compared with control.
107
Figure 2.14 miR199a-5p directly targets MRP1 . To test whether MRP1 is a direct target of miR-199a-5p, cells were transfected with a pGL3-MRP1-
3’UTR firefly luciferase expression construct and co-transfected with control renilla luciferase reporter construct along with miR-199a-5p mimic or inhibitor. miR-199a-5p mimic delivered cells showed lower luciferase activity ( A) while miR-199a-5p hairpin inhibitor delivered cells showed higher luciferase activity
(B). n=3, Results are mean ± SD, *p < 0.05 compared with control.
108
Figure 2.15 miR-199a-5p mimic delivered HT4 cells were vulnerable to glutamate-induced toxicity. A, 72h after either miR mimic negative control or mmu-miR-199a-5p mimic delivery, cells were re-split and treated with
10mM glutamate for 12h. Loss of cell viability was assessed by measuring
LDH leakage. miR-199a-5p mimic delivered cells were vulnerable to glutamate-induced loss of cell viability. B, Intracellular GSSG level was significantly increased in miR-199a-5p delivered cells after 12h of glutamate challenge. n=3, Results are mean ± SD, *p < 0.05 compared with miR mimic negative control delivered control; #p < 0.05 compared with miR-199a-5p mimic delivered control; §p < 0.05 compared with miR mimic negative control delivered, glutamate treated HT4 neural cells.
109
Figure 2.16 Neuroprotective effect of miR-199a-5p inhibitor abrogated in
the presence of MK571 in HT4 neural cells. After 72h of miR-199a-5p
hairpin inhibitor delivery, cells were re-split. MK571 (20 µM) was added 6h before glutamate challenge. After 12h of glutamate treatment, LDH leakage was significantly increased in both control and miR-199a-5p inhibitor delivered cells. Glutamate challenged, miR-199a-5p inhibitor delivered cells showed protection against loss of cell viability compared to corresponding control cells challenged with glutamate. However, the neuroprotective effect of miR-199a-5p inhibitor was abrogated in the presence of MK571. n=3,
Results are mean ± SD, *p < 0.05 compared with miR hairpin inhibitor
negative control delivered control; #p < 0.05 compared with miR-199a-5p hairpin inhibitor delivered control; §p < 0.05 compared with miR hairpin inhibitor negative control delivered, glutamate treated HT4 neural cells.
110 CHAPTER 3
Regulation of multidrug resistance-associated protein 1
by α-tocotrienol in stroke
3.1 Introduction
3.1.1 Vitamin E
Vitamin E is one of the major lipid-soluble, chain-breaking antioxidant
in the body and plays a central role in maintaining neurological structure and
function 186 . Vitamin E is a generic term for the family of eight major
compounds: α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-
tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. Tocochromanol
contains a polar chromanol head group with a long isoprenoid side chain.
Depending on the nature of the isoprenoid chain, tocopherol (TCP; with a
phytyl chain) or tocotrienol (TCT; with a geranylgeranyl chain) can be
distinguished from each other (Fig. 3.1).
111 Eight forms of TCPs and TCTs share close structural homology and possess comparable antioxidant efficacy. Vitamin E family is also reported to present functions independent of their antioxidant properties. α-TCP inhibits protein kinase C, 5-Lox, and phospholipase A2 at the posttranslational level. α-TCP is reported to inhibit cell proliferation, platelet aggregation and monocyte adhesion.
Although TCPs have been widely studied, TCTs have recently become of interest for their anticancer, neuroprotective, and antioxidant effects 21-
23,28,187 . α-TCT supplementation influences the course of carotid atherosclerosis in human and also regulates signal transduction pathways by mechanisms that are independent of its antioxidant properties 187-188 . α-
TCT has been shown to significantly suppress the activity of hydroxy-3- methylglutaryl-coenzyme A reductase, the key regulated enzyme of cholesterol biosynthesis 189 . TCTs inhibit proliferation and tube formation of bovine aortic endothelial cells, suggesting TCTs have a potential to minimize tumor angiogenesis 190 . Supplementation of TCTs shows high protection against free radical ferric nitrilotriacetate induced elevation of bone-reabsorbing cytokines including interleukins 1 and 6 (IL-1 and IL-6).
TCTs differ from TCPs by the presence of three trans double bonds in the
112 hydrocarbon tail. Because of an additional unsaturated side chain, TCTs
possess unique functions that are not shared by TCPs. Nanomolar
concentration, α-TCT, not α-TCP prevents stroke-related
neurodegeneration 23 . The unsaturated side chain of α-TCT allows for more
efficient penetration into tissues that have saturated fatty layers 191 . The test
examining the antioxidant free radial scavenging effects of TCPs and TCTs
revealed that TCTs appear superior due to their better distribution in the
fatty layers of the cell membrane 191 .
3.1.2 History of vitamin E
α-TCP was first introduced as factor X in 1922 described as a fat-
soluble nutrient 192 . In Science, Evans group renamed this compound
tocopherol from Greek words tokos (offspring), phero (to bear), and ol from
alcohol 192 . Then other isoforms of TCP ( β-TCP and γ-TCP in 1937 193 ; δ-
TCP in 1947 194 ) were isolated from vegetable oils. Two decades later, four
isoforms of TCTs were identified 195-196 .
Clinical approaches of vitamin E supplementation on human subjects
started in the late 1930s. Although promising results have been obtained
experimentally, clinical trial outcomes testing the TCP form of natural
113 vitamin E have been disappointing 197-198 . Such failures have led to the
reconsideration of the wisdom to solely invest in the α-TCP form of natural
vitamin E. Attention to other naturally occurring forms of vitamin E such as
γ-TCP as well as the TCT family intensified. Studies on TCTs started only in
the 1980s, six decades after the discovery of vitamin E. The current state of
knowledge presents a compelling case supporting that functionally TCTs
have properties that are not shared by TCPs199-202 .
3.1.3 Vitamin E as a nutrient
Animal including human can not synthesize vitamin E, therefore
adequate dietary intake of vitamin E is crucial to maintain health and normal
development. Green plant tissue contains TCPs. Cereal, salad, cooking oil
and mayonnaise are excellent sources of TCPs as a diet. Unlike TCPs,
TCTs are preferentially found in seeds and fruits. Crude palm oil contains a
high amount of TCTs up to 800mg kg -1. Both TCPs and TCTs are abundant
in grains such as oat, barley rye, and wheat (Table 3.1). TCPs are mainly
present in the germ fraction, whereas the TCTs are present in the pericarp
and endosperm fraction.
114 Both TCPs and TCTs are absorbed in intestinal lumen as a form of
mixed micelle. In enterocytes, chylomicrons are composed with fatty acids,
acylglycerides, and cholesterols, then transported via circulatory system. In
the blood, lipoprotein lipase hydrolyzes chylomicrons to chylomicron
remnants. At this point, TCPs and TCTs are delivered to tissue or form high
density lipoproteins to transfer in circulation.
Although vitamin E deficiency is considered uncommon, fat
malabsorption syndrome, abnormality in TCP transfer protein, pathological
condition in GI track such as Crohn’s disease, and genetic disorder such as
abetalipoproteinemia can cause vitamin E deficiency. Vitamin E toxicity
from natural source is very rare. Although several side effects such as
muscle weakness, fatigue, emotional disturbance and nausea are reported
after high dose of vitamin E supplementation, vitamin E tolerance is
relatively high in adult human.
3.1.4 Neuroprotective effect of α-tocotrienol
Structural difference of TCTs, the presence of three trans double
bonds in the hydrocarbon tail, gives unique characters in this molecules.
Because of additional unsaturated bonds in a side chain, it is more efficient
115 to penetrate into tissue that has saturated fatty layers such as the brain and liver. TCTs also appear superior due to their better distribution in the fatty layers of the cell membranes 191 .
α-TCT-dependent neuroprotection includes a significant antioxidant-independent mechanism has been now established by Sen group 21-
23,28,129,202 . Glutamate-toxicity is a major contributor to neurodegeneration and α-TCT is capable to block glutamate-induced neural cell toxicity. c-Src and the structurally related Dr Chandan K. Sen The Ohio State Univ. members of the Src family are non-receptor tyrosine kinase that reside within the cell associated with cell membranes and appear to transduce signals from transmembrane receptors to the cell interior. c-Src contains SH2 and SH3 domains which play a central role in regulating the catalytic activity of Src protein tyrosine kinase. c-Src is known to regulate several aspects of cellular function including osteoclastic bone resorption and gap junction 203 . The relationship between c-Src activation and cancer progression has been reported 204 .
116 c-Src is highly expressed in the brain. In neuron and astrocyte, c-Src is presented as 15-20 times higher levels than that found in fibroblasts. The specific activity of the c-Src protein from neuronal culture is 6-12 times higher than that from the astrocyte culture, suggesting a key function of this protein in neurons. During oxidative stress such as glutamate-induced neural toxicity, cellular Src tyrosine kinase is activated by SHP-1, a cytoplasmic tyrosine phosphatase. SHP-1 unlocks and activates c-Src by dephosphorylating the inhibitory tyrosine phosphorylation. Activated c-Src phosphorylates 12-Lox and finally induces neurotoxicity. Previously the neuroprotective mechanism of α-TCT is reported by disruption of c-Src pathway 20,205 . Overexpression of catalytically active Src-kinase markedly sensitized the cells to glutamate-induced death. α-TCT treatment completely prevented glutamate-induced death even in active c-Src kinase overexpressing cell, indicating that it either inhibited c-Src kinas activity or regulated one or more events upstream of c-Src kinase activation.
Brain is rich in arachidonic acid (20:4 w-6). Massive amount of arachidonic acids are released from the membranes in response to brain ischemia or trauma 206-207 . During ischemic condition, arachidonic acid is produced via activation of phospholipase A2, then converted to eicosanoids
117 by cyclooxygenase (Cox) or Lox. Among all arachidonic acid pathway, 12-
Lox appears to be the major metabolite of arachidonic acid in the brain as well as cultured neurons 208-209 . Previously, we had reported that GSH- depletion in glutamate-challenged HT4 neural cells leads to the activation of
12-Lox which is central in executing glutamate-induced neural cell death 21-
23,28 . Similarly Wang et al reported that 12-Lox, not Cox or cytochrome P450, is the major pathway on cystine deprivation-induced neuronal death 57 .
12-Lox produces 12(S)-hydroperoxy-eicosatetraenoic (HPETE) acid which is further metabolized into four distinct products: an alcohol, a ketone, or two epoxy alcohols. 12-HPETE is proved to be capable of causing cell death 21 . Our previous study showed that α-TCT directly interacts with the pure 12-Lox enzyme and attenuates 12-Lox induced neural cell death.
Study examining possible docking sites of α-TCT to 12-Lox support the presence of α-TCT binding solvent cavity close to the active site. It is therefore plausible that the binding position of α-TCT prevents access of the natural substrate arachidonic acid to the active site of 12-Lox.
118 3.1.5 Stroke
Stroke is the most common life-threatening neurologic disease, and it
is the third leading cause of death in the United States after heart disease
and cancer. Although stroke is more often disabling than lethal, it was
accounted for 1 out of every 18 deaths in 2006. The American Heart
Association (AHA) reported that ≈ 795,000 people experienced initial ( ≈
610,000) or recurrent stroke ( ≈ 185,000) in 2010. AHA estimated that direct
and indirect cost of stroke is $73.7 billion in 2010 210 .
Stroke is classified into two categories, ischemic and hemorrhagic
stroke (Fig. 3.2). Ischemic stroke is characterized by an interruption of
glucose and oxygen supply due to blocking blood flow to the brain while
hemorrhagic stroke is caused by accumulation of blood within the brain.
Among all the cases of stroke, more than 85% are ischemic stroke, 10% are
hemorrhagic stroke, and less than 5% are caused by other reasons 210 .
The middle cerebral artery (MCA) is the largest of the major branches
of internal carotid artery, and it is the most commonly affected artery in
stroke. The MCA supplies most of the convex surface of the brain. This
artery irrigates almost all of the basal ganglia and capsules including the
119 extreme capsule, claustrum, putamen, the upper parts of the globus pallidus,
parts of the substantia innominata of Reichert, the posterior portion of the
head and all of the body of the caudate nucleus 211 . Focal stroke models
occluding MCA is currently most common in research, because the majority
of human ischemic stroke is caused by an occlusion in the region of MCA.
In this chapter, we applied transient MCAO to exam damage caused by
both ischemia and reperfusion.
3.2 Material and Method
3.2.1 α-Tocotrienol supplementation
C57BL/6 (5 weeks, male, Harlan, Indianapolis, IN) mice were randomly
divided into two groups, control (n=18) and supplemented (n=23) group.
The control group was orally gavaged with vitamin E stripped corn oil with
volume matching the mean volume of the supplement in the test group.
Stock solution of α-TCT supplement solution was prepared in vitamin E-
stripped corn oil. The test group was orally gavaged with α-TCT (Carotech
Inc, Malaysia) in oil (same as placebo) at a dosage of 50 mg/kg body weight
for 13 weeks. Incorporation of orally supplemented vitamin E to the brain is
120 a slow process. Longer supplementation period improves bioavailability of
vitamin E to the brain. Stroke was performed at 20 to 24h after the last
supplementation of α-TCT or corn oil. After 48h of MCAO, T2-weighted
image was taken to measure infarct volume. Mice suffering from surgical
complications ( e.g. hemorrhage or death) during MCAO were excluded.
Immediately after imaging, tissues from control and stroke-affected
hemispheres of control (n=9) and α-TCT fed (n=10) mice were harvested.
Mice were maintained under standard conditions at 22+2°C with 12:12 dark:
light cycles. All animal protocols were approved by the Institutional Animal
Care and Use Committee (IACUC) of the Ohio State University, Columbus,
Ohio.
3.2.2 High performance liquid chromatography (HPLC)-electrochemical detection
Vitamin E analyses of α-TCT or corn oil fed mice brains were
performed using a HPLC-coulometric electrode array detector (Coularray
Detector - model 5600 with 12 channels; ESA Inc., Chelmsford, MA). This
system enables the simultaneous detection of TCTs and TCPs in the same
run as described by us previously 23,212 . Glutathione measurement was
performed using an HPLC system coupled with a electrochemical
121 coulometric detector as described 21-22,25 . The CoulArray detector employs
multiple channels set at specific redox potentials. Data were collected using
channels set at 600, 700, and 800mV. The samples were snap-frozen and
stored in liquid nitrogen until HPLC assay. Sample preparation, composition
of the mobile phase, and specification of the column used were as
described previously 23,25 .
3.2.3 mRNA expression assay from laser captured microdissected brain sample
Laser microdissection and pressure catapulting (LMPC) was
performed using the microlaser system from PALM Microlaser Technologies
AG (Bernreid, Germany). Briefly, Mice were euthanized immediately after
MRI imaging. Next, necropsy was performed to isolate the brain. Coronal
slices were collected using a mouse brain matrix. Sections were rinsed in
PBS, embedded in OCT compound (Sakura Finetek, Japan) and frozen at -
80°C. OCT-embedded in slices were subsequently cut in 12 µm thick
sections on a Leica CM 3050 S cryostat (Leica Microsystems, Wetzlar,
Germany). The sections were placed on polyethylene napthalate membrane
glass slides (PALM Microlaser Technologies AG, Bernreid, Germany),
which had been RNA Zap (Ambion, Austin, TX) and UV treated, for cutting
122 and catapulting as described by our group recently 20,213 . Settings used for
laser cutting were UV-Energy of 70-80 and UV-Focus of 70. Matched area
(2 10 6 µm2) of non-infarct and infarct area was captured into 25 µl of RNA
extraction buffer. The total RNA was isolated using PicoPure RNA Isolation
kit (Arcturus, Sunnyvale, CA). Quantification of MCAO-induced MRP1
mRNA expression in mouse brain was performed by real-time PCR using
SYBR Green-I. Relative gene expression was standardized to 18s rRNA. To
determine miR199a-5p expression in MCAO-induced mouse brain, miRNA
fraction was isolated using miRVana TM miRNA isolation kit (Ambion, Austin,
TX) as described above.
3.2.4 Histology
OCT-embedded frozen brain was sectioned in 12 µm thickness and
mounted onto slides for histologic determinations. This section was stained
with rabbit polyclonal antibody to MRP1 (1: 200, Abbiotec, San Diego, CA),
0.0001% Fluoro-Jade® C (Millipore, Billerica, MA) or 4-hydroxynonenal
(1:1000, Enzo Life Sciences, Plymouth Meeting, PA) . Tissue sections were
analyzed by fluorescence microscopy (Axiovert 200M, Zeiss, Göttingen,
Germany) and images were captured using Axiovert v4.6 software (Zeiss)
28,214 .
123
3.2.5 Statistics
Data are reported as mean + SD of at least three independent
experiments. Difference in means was tested using Student’s t-test or one-
way ANOVA with Tukey’s test. P < 0.05 was considered statistically
significant.
Summary of Methods • α-TCT supplementation • Middle cerebral artery occlusion (MCAO) • MRI • HPLC : Vitamin E, GSH, and GSSG analyses • Laser microdissection pressure catapulting (LMPC) • miR, mRNA analysis from LMPC samples • Western blotting • Immunohistochemistry
124 3.3 Results and Discussion
3.3.1 MCAO-induced brain injury was attenuated in α-TCT supplemented
mice
To test the neuroprotective effects of α-TCT against stroke in vivo ,
randomly divided mice were gavaged with either vitamin E stripped corn oil
or α-TCT (50mg/ kg body weight) for 13 weeks. MCAO was performed one
day after the last supplementation. MCAO affected brain tissues were
collected 48h after reperfusion (Fig. 3.3). α-TCT supplementation
significantly increased brain α-TCT level without changing α-TCP levels (Fig.
3.4). MCAO limited blood flow in α-TCT supplemented as well as control
groups comparably (Fig. 3.5A). However, MCAO-induced hemispherical
infarct volume was significantly attenuated in α-TCT supplemented group
(Fig. 3.5B-D).
3.3.2 Oral α-TCT supplementation protected brain via MRP1 upregulation
To test whether the neuroprotective properties of α-TCT against stroke
in vivo was dependent on the ability of α-TCT to regulate MRP1 expression
in the brain, tissue was collected from infarct as well as contralateral non-
infarct sites using a laser microdissection and pressure catapulting (LMPC)
125 system (Fig. 3.6). Notably, α-TCT supplementation significantly increased mRNA expression of MRP1 at the infarct site. Furthermore, α-TCT supplemented brain had lower miR-199a-5p levels in the infarct hemisphere
(Fig. 3.7A-B). Infarct hemisphere of α-TCT supplemented group significantly increased MRP1 protein (Fig 3.8A). In vitro studies generated consistent data demonstrating significant increased MRP1 protein expression in α-
TCT-treated, glutamate-challenged cells (Fig. 3.8B). Immunohistochemical studies demonstrated a higher abundance of MRP1 positive cells in the brain infarct site of α-TCT-supplemented mice subjected to stroke (Fig. 3.9).
Fluoro-Jade immunofluorescence staining demonstrated that α-TCT supplementation attenuated stroke-induced neurodegeneration (Fig. 3.10).
To test the level of lipid peroxidation, an oxidative stress byproduct commonly associated with stroke, 4-hydroxynonenal (4-HNE) staining was performed. Following stroke, elevated levels of 4-HNE-positive cells were found in the infarct brain tissue of both control as well as α-TCT supplemented mice. Consistent with other observations indicating a beneficial influence of α-TCT supplementation, the level of 4-HNE-positive cells was lower in the infarct hemisphere of α-TCT supplemented mice (Fig.
3.11).
126 Following a number of failed clinical trials testing the conventional form of vitamin E, α-TCP 198,215 , interest in naturally occurring forms of vitamin E such as γ-TCP as well as the TCT family is sharply on the rise. Meta- analyses of clinical trials testing the efficacy of vitamin E in human health suffer from a blind spot because they fail to recognize that α-TCP, the only form of vitamin E tested in such trials, represent only a fraction of the natural vitamin E family 198,215 . Because TCPs and TCTs have unique functional properties it is important to limit title claims to the specific form of vitamin E studied.
The first of such unique properties discovered was the observation that
TCT, but not TCP, is a potent inhibitor of HMG-CoA reductase and therefore hypocholesterolemic 189,216 . In 2000, our laboratory presented the first evidence that at nanomolar concentrations, α-TCT is potently neuroprotective. This is the most potent among all known functions of the vitamin E family 217 . The observation has now been time tested for a decade leading to major mechanistic insights that have identified inhibition of inducible c-Src as well as 12-Lox activity as key pathways responsible for the neuroprotective effects of α-TCT. The neuroprotective actions of α-TCT
127 have been validated in the stroke setting in vivo 20,22,205 as well as in a number of neurotoxicity systems tested by several laboratories 218-219 .
Stroke-associated ischemic insult is known to compromise ATP production as well as deplete the reducing equivalent pool of the brain tissue 220-222 . This, in turn, compromises the function of ATP-dependent ion pumps as well as the ability of the brain to reduce oxidation products generated as a consequence of oxidant insult. As a result, on one hand K + efflux and Ca 2+ influx are increased due to membrane depolarization. On the other hand, excess GSSG builds up within the cell. Both elevated intracellular Ca 2+ as well as GSSG are directly implicated in cell death signaling in a manner that elevated GSSG is associated with Ca 2+ - dependent neurotoxicity 223-226 . Our previous work highlights the critical significance of elevated and trapped cellular GSSG in executing neural cell death 93 . Thus, cellular GSSG clearing mechanisms play a key role in protecting neural cells under conditions of oxidant insult.
Neuroprotective as well as hypocholesterolemic properties of α-TCT make it a good candidate for nutrition-based intervention in people at high risk for stroke. α-TCT is safe and commonly used in the diet of far-eastern
128 countries like Malaysia and Singapore. Our observation that the potent neuroprotective properties of α-TCT may be attenuated under conditions of
MRP1 knockdown identifies MRP1 as a functionally important target of α-
TCT in the context of neuroprotection. Major insight on the mechanism of action of α-TCT was gained with the observation that at the infarct site, α-
TCT was effective at downregulating miR-199a-5p which in turn upregulated
MRP1 expression. Although MRP1 has been studied for more than 20 years, the majority of MRP1 research has focused on limiting MRP1 activity or expression in cancer cells to increase the efficacy of chemotherapy. This work establishes MRP1 as a neuroprotective factor under conditions of GSH oxidation as during oxidant insult associated with stroke. A major addition to this knowledge is represented by the observation that MRP1 is subject to control by miR. This opens up the possibility for miR-based therapies aiming at MRP1 not only in the context of stroke but also for cancer. Finally, this study adds a new dimension to our understanding of the neuroprotective functions of α-TCT by identifying MRP1 as a novel target and by unveiling the general prospect that α-TCT may regulate miR expression in a disease setting.
129 Summary of Observations • Orally supplemented α-TCT induced MRP1 at the stroke site • Orally supplemented α-TCT protected against stroke in mice
3.4 Conclusions
MRP1 expression was elevated in the stroke affected tissue of α-TCT- supplemented mice. Efforts to elucidate the underlying mechanism identified
MRP1 as a target of miR-199a-5p. In α-TCT supplemented mice, miR-199a-
5p was downregulated in the stroke affected tissue. This work recognizes
MRP1 as a protective factor against stroke. Furthermore, findings of this study adds a new dimension to the current understanding of the molecular bases of α-TCT neuroprotection by identifying MRP1 as a α-TCT-sensitive target and by unveiling the general prospect that oral α-TCT may regulate microRNA expression in stroke-affected brain tissue.
130 Table 3.5
α-TCP β-TCP γ-TCP δ-TCP α-TCT β-TCT γ-TCT δ-TCT
Palm 89 - 18 - 128 - 323 72
Soybean 100 8 1021 421 - - - -
Maize 282 54 1034 54 49 8 161 6
Sunflower 670 27 11 1 - - - -
Rapeseed 202 65 490 9 - - - -
Table 3.1 Tocopherol and tocotrienol in edible plants parts (mg/kg). Data from Gunstone et al 1994 The Lipid handbook 227 .
131 3.6 Figures
Figure 3.1 Chemical structure of vitamin E
132
Figure 3.2 Classification of stroke. Modified from NINDS stroke data bank and Amarenco et al Classification of stroke subtypes 228 .
133
Figure 3.3 Design of oral α-TCT supplementation . C57BL/6/ mice were randomly divided into two groups, and orally gavaged with vitamin E-stripped corn oil (n=18) or 50mg α-TCT per kg body weight (n=23) for 13 weeks.
MCAO was performed 24h after the last supplementation and reperfusion was initiated 90 min after occlusion. T2 weighted MRI imaging was performed
48h after reperfusion, and brain sections were harvested .
134
Figure 3.4 Brain vitamin E level after oral α-TCT supplementation. Oral
supplementation of α-TCT significantly increased α-TCT level in the mouse brain ( A) without affecting α-TCP level (B). n=5, Results are mean ± SD, *p <
0.05 compared with corn oil fed mice.
135
Figure 3.5 Oral α-TCT supplementation protected against MCAO-induced
brain injury . A, Reduction in the MCA-area blood flow in corn oil fed and α-
TCT fed mice during occlusion was found to be comparable (corn oil, n=9; α-
TCT, n=10). B-D, MRI images were used to determine infarct size as a percentage of the hemispherical infarct volume. Oral supplementation of α-
TCT significantly reduced MCAO-induced brain injury ( B, corn oil
supplemented mice; C, α-TCT supplemented mice; D, % of hemispherical infarct volume, corn oil, n=9; α-TCT, n=10). Results are mean ± SD, *p < 0.05
compared with corn oil fed mice.
136
Figure 3.6 Total RNA collection using LMPC . After 13 weeks of α-TCT or
corn oil supplementation, MCAO was induced. After 48h of reperfusion,
brains were harvested and embedded in OCT. OCT-embedded slices were
cut in 12 µm thick coronal section. miR and mRNA were collected from contralateral non infarct or stroke hemisphere using a LMPC system ( A, control non-infarct hemisphere; B, infarct hemisphere).
137
Figure 3.7 Oral α-TCT supplementation upregulated MRP1 gene expression specifically at the MCAO-affected site of the brain . A, miR-
199a-5p expression was significantly down regulated in the infarct hemisphere of α-TCT fed mice brains. B, MRP1 mRNA level was significantly
elevated in the infarct hemisphere of α-TCT fed mice. n=3, Results are mean
± SD, *p < 0.05 compared with corresponding control.
138
Figure 3.8 Oral α-TCT supplementation upregulated MRP1 protein
expression specifically at the MCAO-affected site of the brain . A, MRP1 protein expression was increased in the infarct hemisphere of both α-TCT fed
as well as corn oil fed mice. B, to test our finding in vitro , HT4 neural cells were treated either α-TCT (1 µM), glutamate (10mM) or a combination of both.
After 24h of seeding, α-TCT was added into cell culture medium 9h prior to glutamate treatment for 12h. Cell exposed to α-TCT prior to glutamate
challenge expressed significantly higher level of MRP1 protein. n=3, Results
are mean ± SD, *p < 0.05 compared with corresponding control; §p < 0.05
compared with infarct hemisphere of corn oil fed mice.
139
Figure 3.9 Histologic evaluation of MRP1 expression in α-TCT
supplemented mice . A, The number of MRP1 positive cells were significantly upregulated in the infarct hemisphere of α-TCT supplemented
mice ( B-I, red-MRP1 protein; blue-DAPI stained nuclei; B and F, non-infarct
hemisphere of corn oil fed mice; C and G, infarct hemisphere of corn oil fed
mice; D and H, non-infarct hemisphere of α-TCT fed mice; E and I, infarct
hemisphere of α-TCT fed mice). Bar=50 µm, n=3, Results are mean ± SD, *p
< 0.05 compared with corresponding contralateral hemisphere; §p < 0.05 compared with infarct hemisphere of corn oil fed mice.
140
Figure 3.10 Oral α-TCT supplementation attenuated MCAO-induced
neurodegeneration . A, Fluoro-Jade positive neurons were fewer in the
infarct hemisphere of α-TCT fed group compared to infarct site of corn oil fed
control group ( B-I, green-FluoroJade; blue-DAPI stained nuclei; B and F, non- infarct hemisphere of corn oil fed mice; C and G, infarct hemisphere of corn
oil fed mice; D and H, non-infarct hemisphere of α-TCT fed mice; E and I, infarct hemisphere of α-TCT fed mice). Bar=50 µm, n=3, Results are mean ±
SD, *p < 0.05 compared with corresponding contralateral hemisphere; §p <
0.05 compared with infarct hemisphere of corn oil fed mice.
141
Figure 3.11 Oral α-TCT supplementation attenuated MCAO-induced lipid peroxidation . A, The abundance of 4-HNE-positive cells was lower in infarct hemisphere of α-TCT supplemented mice ( B-I, 4-HNE; B and F, non-infarct
hemisphere of corn oil fed mice; C and G, infarct hemisphere of corn oil fed
mice; D and H, non-infarct hemisphere of α-TCT fed mice; E and I, infarct
hemisphere of α-TCT fed mice). Bar =20 µm. n=3, Results are mean ± SD, *p
< 0.05 compared with corresponding contralateral hemisphere; §p < 0.05 compared with infarct hemisphere of corn oil fed mice.
142 4. Summary and Closing Thoughts
In many parts the Far East Asia, including Korea, food is understood
as a necessary component for harmony in the human body. Thus food
consumption and health has been considered indivisible for thousands of
years.
의식동원 (醫食同源 : translated to ‘ Food and Medicine are the same at
the root ’) is the fundamental philosophy in Korean cuisine. Traditionally,
Korean medicine derives from food sources. The book Dongui Bogam
(동의보감 : 東醫寶鑑), published in 1613, is recognized as a UNESCO’s
Memory of the World documentary heritage. It is the one of the first official medical books in Korea, written by the royal physician Jun Heo. Dongui
Bogam contains five chapters: internal medicine, external medicine, miscellaneous diseases, remedies, and acupuncture. Jun Heo devoted the majority of his chapter remedies to explaining nutritional therapy and the proper use of food resources, from staples such as rice and garlic to exotics such as deer antlers, to cure disease.
143 Although food history and human history are indivisible, the concept of nutritional intervention on the natural history of disease is relatively new in western culture. The entry nutrition first appeared in The Oxford English dictionary in the 15 th century, but this word was not widely used until the19 th
century. A definition of nutrition in the scientific community first appeared in
1931, when Graham Lusk wrote in The Science of Nutrition : “Nutrition may
be defined as the sum of the processes concerned in the growth,
maintenance and repair of the living body as a whole or of its constituent
parts. ” Since then, the importance of nutritional intervention in the treatment
of malnutrition and management of metabolic syndromes such as diabetes
has slowly gained attention.
Still, nutritional intervention is considered secondary to surgery or
pharmacological therapy in the treatment of disease in western culture. The
philosophy of western medicine is at the root of this trend. In western
medicine, the disease condition is wholly separated from the healthy or
normal condition. Therefore, the treatment of disease is thought to require
‘change ’ from the diseased condition. In eastern thought, disease is
considered an ‘imbalance ’ of the natural condition and treatment is aimed at
re-adapting the body to the imbalance. To destroy completely the source of
144 disease may seem the more appealing approach in western medical practice, but this approach has the potential to ignore communication and harmony in the human body as a whole.
In stroke management, aspirin is used to prevent platelet aggregation, thus preventing clot formation and the source of ischemia leading the neurological damage. Despite being one of the most commonly used, readily available, and ‘safe’ drugs available, aspirin has innumerable side effects not limited to gastrointestinal bleeding and ulceration. The neuroprotective and hypocholesterolemic properties of α-TCT make it a good candidate for
nutrition-based intervention in populations at high risk for stroke. α-TCT is safe and commonly supplemented in the diet of far-eastern countries like
Malaysia and Singapore. This study confirms that orally supplemented natural
vitamin E, α-TCT, can attenuate stroke-induced brain damage, including
neurodegeneration and lipid peroxidation .
This dissertation explores a natural, non-invasive, and effective approach to managing stroke; using an Eastern approach and providing a
Western explanation. Nutrition has been struggling to carve out its own identity between the medical and culinary arts. Studies of nutritional
145 intervention on disease must be continued. As mentioned in this dissertation ’s chapter on philosophy, nutrition is an underutilized and
underrecognized answer, provided directly from nature, for the medical
challenges we are facing today.
146 LIST OF REFERENCES
1. Kresge N, Simoni, Robert D., Hill Robert L. The Chemistry of Glutathione:
the Work of Alton Meister. J Biol Chem. 2007;28238:e30.
2. Lenton KJ, Therriault H, Wagner JR. Analysis of glutathione and
glutathione disulfide in whole cells and mitochondria by postcolumn
derivatization high-performance liquid chromatography with ortho-
phthalaldehyde. Anal Biochem. 1999;2741:125-30.
3. Rossi R, Dalle-Donne I, Milzani A, Giustarini D. Oxidized forms of
glutathione in peripheral blood as biomarkers of oxidative stress. Clin
Chem. 2006;527:1406-14.
4. Sen CK, Rahkila P, Hanninen O. Glutathione metabolism in skeletal
muscle derived cells of the L6 line. Acta Physiol Scand. 1993;1481:21-
6.
5. Srivastava SK, Beutler E. The transport of oxidized glutathione from human
erythrocytes. J Biol Chem. 1969;2441:9-16.
6. Aslund F, Beckwith J. Bridge over troubled waters: sensing stress by
disulfide bond formation. Cell. 1999;966:751-3.
7. Arnelle DR, Stamler JS. NO+, NO, and NO- donation by S-nitrosothiols:
implications for regulation of physiological functions by S-nitrosylation
147 and acceleration of disulfide formation. Arch Biochem Biophys.
1995;3182:279-85.
8. Mieyal JJ, Starke DW, Gravina SA, Dothey C, Chung JS. Thioltransferase
in human red blood cells: purification and properties. Biochemistry.
1991;3025:6088-97.
9. Bandyopadhyay S, Starke DW, Mieyal JJ, Gronostajski RM.
Thioltransferase (glutaredoxin) reactivates the DNA-binding activity of
oxidation-inactivated nuclear factor I. J Biol Chem. 1998;2731:392-7.
10. Reddy S, Jones AD, Cross CE, Wong PS, Van Der Vliet A. Inactivation of
creatine kinase by S-glutathionylation of the active-site cysteine
residue. Biochem J. 2000;347 Pt 3:821-7.
11. Wang J, Boja ES, Tan W, Tekle E, Fales HM, English S, Mieyal JJ, Chock
PB. Reversible glutathionylation regulates actin polymerization in A431
cells. J Biol Chem. 2001;27651:47763-6.
12. Dafre AL, Sies H, Akerboom T. Protein S-thiolation and regulation of
microsomal glutathione transferase activity by the glutathione redox
couple. Arch Biochem Biophys. 1996;3322:288-94.
13. Davis DA, Newcomb FM, Starke DW, Ott DE, Mieyal JJ, Yarchoan R.
Thioltransferase (glutaredoxin) is detected within HIV-1 and can
148 regulate the activity of glutathionylated HIV-1 protease in vitro. J Biol
Chem. 1997;27241:25935-40.
14. Okamoto T, Akaike T, Sawa T, Miyamoto Y, van der Vliet A, Maeda H.
Activation of matrix metalloproteinases by peroxynitrite-induced protein
S-glutathiolation via disulfide S-oxide formation. J Biol Chem.
2001;27631:29596-602.
15. Lacal P, and Feramisco. Microinjection. 1999.
16. Lappe-Siefke C, Maas C, Kneussel M. Microinjection into cultured
hippocampal neurons: a straightforward approach for controlled
cellular delivery of nucleic acids, peptides and antibodies. J Neurosci
Methods. 2008;1751:88-95.
17. Zhang Y, Yu LC. Microinjection as a tool of mechanical delivery. Curr
Opin Biotechnol. 2008.
18. Zhang Y, Yu LC. Single-cell microinjection technology in cell biology.
Bioessays. 2008;306:606-10.
19. King R. Gene delivery to mammalian cells by microinjection. Methods Mol
Biol. 2004;245:167-74.
20. Khanna S, Roy S, Park HA, Sen CK. Regulation of c-Src activity in
glutamate-induced neurodegeneration. J Biol Chem.
2007;28232:23482-90.
149 21. Khanna S, Roy S, Ryu H, Bahadduri P, Swaan PW, Ratan RR, Sen CK.
Molecular basis of vitamin E action: tocotrienol modulates 12-
lipoxygenase, a key mediator of glutamate-induced neurodegeneration.
J Biol Chem. 2003;27844:43508-15.
22. Khanna S, Roy S, Slivka A, Craft TK, Chaki S, Rink C, Notestine MA,
DeVries AC, Parinandi NL, Sen CK. Neuroprotective properties of the
natural vitamin E alpha-tocotrienol. Stroke. 2005;3610:2258-64.
23. Sen CK, Khanna S, Roy S, Packer L. Molecular basis of vitamin E action.
Tocotrienol potently inhibits glutamate-induced pp60(c-Src) kinase
activation and death of HT4 neuronal cells. J Biol Chem.
2000;27517:13049-55.
24. Tirosh O, Sen CK, Roy S, Packer L. Cellular and mitochondrial changes
in glutamate-induced HT4 neuronal cell death. Neuroscience.
2000;973:531-41.
25. Sen CK, Khanna S, Babior BM, Hunt TK, Ellison EC, Roy S. Oxidant-
induced vascular endothelial growth factor expression in human
keratinocytes and cutaneous wound healing. J Biol Chem.
2002;27736:33284-90.
26. Reid AB, Kurten RC, McCullough SS, Brock RW, Hinson JA. Mechanisms
of acetaminophen-induced hepatotoxicity: role of oxidative stress and
150 mitochondrial permeability transition in freshly isolated mouse
hepatocytes. J Pharmacol Exp Ther. 2005;3122:509-16.
27. Duchen MR, Surin A, Jacobson J. Imaging mitochondrial function in intact
cells. Methods Enzymol. 2003;361:353-89.
28. Khanna S, Roy S, Parinandi NL, Maurer M, Sen CK. Characterization of
the potent neuroprotective properties of the natural vitamin E alpha-
tocotrienol. J Neurochem. 2006;985:1474-86.
29. Nicholls DG. Simultaneous monitoring of ionophore- and inhibitor-
mediated plasma and mitochondrial membrane potential changes in
cultured neurons. J Biol Chem. 2006;28121:14864-74.
30. Han D, Sen CK, Roy S, Kobayashi MS, Tritschler HJ, Packer L.
Protection against glutamate-induced cytotoxicity in C6 glial cells by
thiol antioxidants. Am J Physiol. 1997;2735 Pt 2:R1771-8.
31. De Clerck LS, Bridts CH, Mertens AM, Moens MM, Stevens WJ. Use of
fluorescent dyes in the determination of adherence of human
leucocytes to endothelial cells and the effect of fluorochromes on
cellular function. J Immunol Methods. 1994;1721:115-24.
32. Van Damme P, Van Hoecke A, Lambrechts D, Vanacker P, Bogaert E,
van Swieten J, Carmeliet P, Van Den Bosch L, Robberecht W.
151 Progranulin functions as a neurotrophic factor to regulate neurite
outgrowth and enhance neuronal survival. J Cell Biol. 2008;1811:37-41.
33. Beierschmitt WP, McNeish JD, Griffiths RJ, Nagahisa A, Nakane M,
Amacher DE. Induction of hepatic microsomal drug-metabolizing
enzymes by inhibitors of 5-lipoxygenase (5-LO): studies in rats and 5-
LO knockout mice. Toxicol Sci. 2001;631:15-21.
34. Orlowski M, Meister A. The gamma-glutamyl cycle: a possible transport
system for amino acids. Proc Natl Acad Sci U S A. 1970;673:1248-55.
35. Blair IA. Endogenous glutathione adducts. Curr Drug Metab. 2006;78:853-
72.
36. Schafer FQ, Buettner GR. Redox environment of the cell as viewed
through the redox state of the glutathione disulfide/glutathione couple.
Free Radic Biol Med. 2001;3011:1191-212.
37. Circu ML, Aw TY. Glutathione and apoptosis. Free Radic Res.
2008;428:689-706.
38. Mieyal JJ, Gallogly MM, Qanungo S, Sabens EA, Shelton MD. Molecular
mechanisms and clinical implications of reversible protein S-
glutathionylation. Antioxid Redox Signal. 2008;1011:1941-88.
152 39. Filomeni G, Rotilio G, Ciriolo MR. Glutathione disulfide induces apoptosis
in U937 cells by a redox-mediated p38 MAP kinase pathway. Faseb J.
2003;171:64-6.
40. Kirsch JD, Yi AK, Spitz DR, Krieg AM. Accumulation of glutathione
disulfide mediates NF-kappaB activation during immune stimulation
with CpG DNA. Antisense Nucleic Acid Drug Dev. 2002;125:327-40.
41. den Hartog GJ, Haenen GR, Vegt E, van der Vijgh WJ, Bast A. Efficacy of
HOCl scavenging by sulfur-containing compounds: antioxidant activity
of glutathione disulfide? Biol Chem. 2002;3833-4:709-13.
42. Minich T, Riemer J, Schulz JB, Wielinga P, Wijnholds J, Dringen R. The
multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of
glutathione and glutathione disulfide from brain astrocytes. J
Neurochem. 2006;972:373-84.
43. Schulz JB, Lindenau J, Seyfried J, Dichgans J. Glutathione, oxidative
stress and neurodegeneration. Eur J Biochem. 2000;26716:4904-11.
44. Shaw CA. Glutathione in the nervous system . Washington, DC: Taylor &
Francis, 1998.
45. Pias EK, Aw TY. Early redox imbalance mediates hydroperoxide-induced
apoptosis in mitotic competent undifferentiated PC-12 cells. Cell Death
Differ. 2002;99:1007-16.
153 46. Chen L, Feng P, Li S, Long D, Cheng J, Lu Y, Zhou D. Effect of hypoxia-
inducible factor-1alpha silencing on the sensitivity of human brain
glioma cells to doxorubicin and etoposide. Neurochem Res.
2009;345:984-90.
47. Galter D, Mihm S, Droge W. Distinct effects of glutathione disulphide on
the nuclear transcription factor kappa B and the activator protein-1. Eur
J Biochem. 1994;2212:639-48.
48. Haddad JJ, Safieh-Garabedian B, Saade NE, Lauterbach R. Inhibition of
glutathione-related enzymes augments LPS-mediated cytokine
biosynthesis: involvement of an IkappaB/NF-kappaB-sensitive pathway
in the alveolar epithelium. Int Immunopharmacol. 2002;211:1567-83.
49. Hirrlinger J, Konig J, Keppler D, Lindenau J, Schulz JB, Dringen R. The
multidrug resistance protein MRP1 mediates the release of glutathione
disulfide from rat astrocytes during oxidative stress. J Neurochem.
2001;762:627-36.
50. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and
neurodegenerative disorders. Science. 1993;2625134:689-95.
51. Herrera F, Martin V, Garcia-Santos G, Rodriguez-Blanco J, Antolin I,
Rodriguez C. Melatonin prevents glutamate-induced oxytosis in the
154 HT22 mouse hippocampal cell line through an antioxidant effect
specifically targeting mitochondria. J Neurochem. 2007;1003:736-46.
52. van Leyen K, Kim HY, Lee SR, Jin G, Arai K, Lo EH. Baicalein and 12/15-
lipoxygenase in the ischemic brain. Stroke. 2006;3712:3014-8.
53. Canals S, Casarejos MJ, de Bernardo S, Rodriguez-Martin E, Mena MA.
Nitric oxide triggers the toxicity due to glutathione depletion in midbrain
cultures through 12-lipoxygenase. J Biol Chem. 2003;27824:21542-9.
54. Li Y, Maher P, Schubert D. A role for 12-lipoxygenase in nerve cell death
caused by glutathione depletion. Neuron. 1997;192:453-63.
55. Chang WC, Nakao J, Orimo H, Murota S. Effects of reduced glutathione
on the 12-lipoxygenase pathways in rat platelets. Biochem J.
1982;2023:771-6.
56. Maret W. Cellular zinc and redox states converge in the
metallothionein/thionein pair. J Nutr. 2003;1335 Suppl 1:1460S-2S.
57. Wang H, Li J, Follett PL, Zhang Y, Cotanche DA, Jensen FE, Volpe JJ,
Rosenberg PA. 12-Lipoxygenase plays a key role in cell death caused
by glutathione depletion and arachidonic acid in rat oligodendrocytes.
Eur J Neurosci. 2004;208:2049-58.
155 58. Muralikrishna Adibhatla R, Hatcher JF. Phospholipase A2, reactive
oxygen species, and lipid peroxidation in cerebral ischemia. Free
Radic Biol Med. 2006;403:376-87.
59. Chang SM, Prados MD, Yung WK, Fine H, Junck L, Greenberg H, Robins
HI, Mehta M, Fink KL, Jaeckle KA, Kuhn J, Hess K, Schold C. Phase II
study of neoadjuvant 1, 3-bis (2-chloroethyl)-1-nitrosourea and
temozolomide for newly diagnosed anaplastic glioma: a North
American Brain Tumor Consortium Trial. Cancer. 2004;1008:1712-6.
60. Doroshenko N, Doroshenko P. Ion dependence of cytotoxicity of
carmustine against PC12 cells. Eur J Pharmacol. 2003;4763:185-91.
61. Evers R, Kool M, Smith AJ, van Deemter L, de Haas M, Borst P. Inhibitory
effect of the reversal agents V-104, GF120918 and Pluronic L61 on
MDR1 Pgp-, MRP1- and MRP2-mediated transport. Br J Cancer.
2000;833:366-74.
62. Navarro J, Obrador E, Pellicer JA, Aseni M, Vina J, Estrela JM. Blood
glutathione as an index of radiation-induced oxidative stress in mice
and humans. Free Radic Biol Med. 1997;227:1203-9.
63. Mkoji GM, Smith JM, Prichard RK. Glutathione redox state, lipid peroxide
levels, and activities of glutathione enzymes in oltipraz-treated adult
Schistosoma mansoni. Biochem Pharmacol. 1989;3823:4307-13.
156 64. Leung SW, Mitchell JB, al-Nabulsi I, Friedman N, Newsome J, Belldegrun
A, Kasid U. Effect of L-buthionine sulfoximine on the radiation
response of human renal carcinoma cell lines. Cancer. 1993;717:2276-
85.
65. Mistry P, Harrap KR. Historical aspects of glutathione and cancer
chemotherapy. Pharmacol Ther. 1991;491-2:125-32.
66. Pineda-Molina E, Klatt P, Vazquez J, Marina A, Garcia de Lacoba M,
Perez-Sala D, Lamas S. Glutathionylation of the p50 subunit of NF-
kappaB: a mechanism for redox-induced inhibition of DNA binding.
Biochemistry. 2001;4047:14134-42.
67. Mohr S, Hallak H, de Boitte A, Lapetina EG, Brune B. Nitric oxide-induced
S-glutathionylation and inactivation of glyceraldehyde-3-phosphate
dehydrogenase. J Biol Chem. 1999;27414:9427-30.
68. Ward NE, Stewart JR, Ioannides CG, O'Brian CA. Oxidant-induced S-
glutathiolation inactivates protein kinase C-alpha (PKC-alpha): a
potential mechanism of PKC isozyme regulation. Biochemistry.
2000;3933:10319-29.
69. Hsiao G, Lee JJ, Chen YC, Lin JH, Shen MY, Lin KH, Chou DS, Sheu JR.
Neuroprotective effects of PMC, a potent alpha-tocopherol derivative,
157 in brain ischemia-reperfusion: reduced neutrophil activation and anti-
oxidant actions. Biochem Pharmacol. 2007;735:682-93.
70. Hurd TR, Costa NJ, Dahm CC, Beer SM, Brown SE, Filipovska A, Murphy
MP. Glutathionylation of mitochondrial proteins. Antioxid Redox Signal.
2005;77-8:999-1010.
71. Mastroianni M, Watanabe K, White TB, Zhuang F, Vernon J, Matsuura M,
Wallingford J, Lambowitz AM. Group II intron-based gene targeting
reactions in eukaryotes. PLoS ONE. 2008;39:e3121.
72. Satoh T, Yoshioka Y. Contribution of reduced and oxidized glutathione to
signals detected by magnetic resonance spectroscopy as indicators of
local brain redox state. Neurosci Res. 2006;551:34-9.
73. Ghizoni DM, Pavanati KC, Arent AM, Machado C, Faria MS, Pinto CM,
Gasparotto OC, Goncalves S, Dafre AL. Alterations in glutathione
levels of brain structures caused by acute restraint stress and by nitric
oxide synthase inhibition but not by intraspecific agonistic interaction.
Behav Brain Res. 2006;1661:71-7.
74. Castillo M. Neuroradiology companion: methods, guidelines and imaging
fundamentals . Philadelphia: Lippincott Williams & Wilkins, 2006.
75. Cherubini A, Ruggiero C, Polidori MC, Mecocci P. Potential markers of
oxidative stress in stroke. Free Radic Biol Med. 2005;397:841-52.
158 76. Hiramatsu M, Mori A. Reduced and oxidized glutathione in brain and
convulsions. Neurochem Res. 1981;63:301-6.
77. Adams JD, Jr., Klaidman LK, Odunze IN, Shen HC, Miller CA. Alzheimer's
and Parkinson's disease. Brain levels of glutathione, glutathione
disulfide, and vitamin E. Mol Chem Neuropathol. 1991;143:213-26.
78. Maher P. The effects of stress and aging on glutathione metabolism.
Ageing Res Rev. 2005;42:288-314.
79. Johansson M, Lundberg M. Glutathionylation of beta-actin via a cysteinyl
sulfenic acid intermediary. BMC Biochem. 2007;8:26.
80. Cappiello M, Voltarelli M, Cecconi I, Vilardo PG, Dal Monte M, Marini I,
Del Corso A, Wilson DK, Quiocho FA, Petrash JM, Mura U. Specifically
targeted modification of human aldose reductase by physiological
disulfides. J Biol Chem. 1996;27152:33539-44.
81. Chandra A, Srivastava S, Petrash JM, Bhatnagar A, Srivastava SK.
Modification of aldose reductase by S-nitrosoglutathione. Biochemistry.
1997;3650:15801-9.
82. Kambe T, Song T, Takata T, Hatano N, Miyamoto Y, Nozaki N, Naito Y,
Tokumitsu H, Watanabe Y. Inactivation of Ca2+/calmodulin-dependent
protein kinase I by S-glutathionylation of the active-site cysteine
residue. FEBS Lett. 2010;58411:2478-84.
159 83. Zech B, Wilm M, van Eldik R, Brune B. Mass spectrometric analysis of
nitric oxide-modified caspase-3. J Biol Chem. 1999;27430:20931-6.
84. Bull R, Finkelstein JP, Galvez J, Sanchez G, Donoso P, Behrens MI,
Hidalgo C. Ischemia enhances activation by Ca2+ and redox
modification of ryanodine receptor channels from rat brain cortex. J
Neurosci. 2008;2838:9463-72.
85. Grant CM, Quinn KA, Dawes IW. Differential protein S-thiolation of
glyceraldehyde-3-phosphate dehydrogenase isoenzymes influences
sensitivity to oxidative stress. Mol Cell Biol. 1999;194:2650-6.
86. Klatt P, Pineda Molina E, Perez-Sala D, Lamas S. Novel application of S-
nitrosoglutathione-Sepharose to identify proteins that are potential
targets for S-nitrosoglutathione-induced mixed-disulphide formation.
Biochem J. 2000;349Pt 2:567-78.
87. Lind C, Gerdes R, Schuppe-Koistinen I, Cotgreave IA. Studies on the
mechanism of oxidative modification of human glyceraldehyde-3-
phosphate dehydrogenase by glutathione: catalysis by glutaredoxin.
Biochem Biophys Res Commun. 1998;2472:481-6.
88. Sies H, Dafre AL, Ji Y, Akerboom TP. Protein S-thiolation and redox
regulation of membrane-bound glutathione transferase. Chem Biol
Interact. 1998;111-112:177-85.
160 89. Regazzoni L, Panusa A, Yeum KJ, Carini M, Aldini G. Hemoglobin
glutathionylation can occur through cysteine sulfenic acid intermediate:
electrospray ionization LTQ-Orbitrap hybrid mass spectrometry studies.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009;87728:3456-61.
90. Davis DA, Dorsey K, Wingfield PT, Stahl SJ, Kaufman J, Fales HM,
Levine RL. Regulation of HIV-1 protease activity through cysteine
modification. Biochemistry. 1996;357:2482-8.
91. Ji Y, Akerboom TP, Sies H, Thomas JA. S-nitrosylation and S-
glutathiolation of protein sulfhydryls by S-nitroso glutathione. Arch
Biochem Biophys. 1999;3621:67-78.
92. Klatt P, Molina EP, Lamas S. Nitric oxide inhibits c-Jun DNA binding by
specifically targeted S-glutathionylation. J Biol Chem.
1999;27422:15857-64.
93. Park HA, Khanna S, Rink C, Gnyawali S, Roy S, Sen CK. Glutathione
disulfide induces neural cell death via a 12-lipoxygenase pathway. Cell
Death Differ. 2009;168:1167-79.
94. Casadei M, Persichini T, Polticelli F, Musci G, Colasanti M. S-
glutathionylation of metallothioneins by nitrosative/oxidative stress. Exp
Gerontol. 2008;435:415-22.
161 95. Reynaert NL, van der Vliet A, Guala AS, McGovern T, Hristova M,
Pantano C, Heintz NH, Heim J, Ho YS, Matthews DE, Wouters EF,
Janssen-Heininger YM. Dynamic redox control of NF-kappaB through
glutaredoxin-regulated S-glutathionylation of inhibitory kappaB kinase
beta. Proc Natl Acad Sci U S A. 2006;10335:13086-91.
96. Yusuf MA, Chuang T, Bhat GJ, Srivenugopal KS. Cys-141
glutathionylation of human p53: Studies using specific polyclonal
antibodies in cancer samples and cell lines. Free Radic Biol Med.
2010;495:908-17.
97. Townsend DM, Manevich Y, He L, Xiong Y, Bowers RR, Jr., Hutchens S,
Tew KD. Nitrosative stress-induced s-glutathionylation of protein
disulfide isomerase leads to activation of the unfolded protein response.
Cancer Res. 2009;6919:7626-34.
98. Rinna A, Torres M, Forman HJ. Stimulation of the alveolar macrophage
respiratory burst by ADP causes selective glutathionylation of protein
tyrosine phosphatase 1B. Free Radic Biol Med. 2006;411:86-91.
99. Hawkins BJ, Irrinki KM, Mallilankaraman K, Lien YC, Wang Y,
Bhanumathy CD, Subbiah R, Ritchie MF, Soboloff J, Baba Y, Kurosaki
T, Joseph SK, Gill DL, Madesh M. S-glutathionylation activates STIM1
and alters mitochondrial homeostasis. J Cell Biol. 2010;1903:391-405.
162 100. Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983;52:711-
60.
101. Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC,
Stewart AJ, Kurz EU, Duncan AM, Deeley RG. Overexpression of a
transporter gene in a multidrug-resistant human lung cancer cell line.
Science. 1992;2585088:1650-4.
102. Flens MJ, Zaman GJ, van der Valk P, Izquierdo MA, Schroeijers AB,
Scheffer GL, van der Groep P, de Haas M, Meijer CJ, Scheper RJ.
Tissue distribution of the multidrug resistance protein. Am J Pathol.
1996;1484:1237-47.
103. Rao VV, Dahlheimer JL, Bardgett ME, Snyder AZ, Finch RA, Sartorelli
AC, Piwnica-Worms D. Choroid plexus epithelial expression of MDR1
P glycoprotein and multidrug resistance-associated protein contribute
to the blood-cerebrospinal-fluid drug-permeability barrier. Proc Natl
Acad Sci U S A. 1999;967:3900-5.
104. Stride BD, Valdimarsson G, Gerlach JH, Wilson GM, Cole SP, Deeley
RG. Structure and expression of the messenger RNA encoding the
murine multidrug resistance protein, an ATP-binding cassette
transporter. Mol Pharmacol. 1996;496:962-71.
163 105. Conrad S, Viertelhaus A, Orzechowski A, Hoogstraate J, Gjellan K,
Schrenk D, Kauffmann HM. Sequencing and tissue distribution of the
canine MRP2 gene compared with MRP1 and MDR1. Toxicology.
2001;1562-3:81-91.
106. Taguchi Y, Saeki K, Komano T. Functional analysis of MRP1 cloned
from bovine. FEBS Lett. 2002;5211-3:211-3.
107. Nunoya K, Grant CE, Zhang D, Cole SP, Deeley RG. Molecular cloning
and pharmacological characterization of rat multidrug resistance
protein 1 (mrp1). Drug Metab Dispos. 2003;318:1016-26.
108. Haimeur A, Conseil G, Deeley RG, Cole SP. The MRP-related and
BCRP/ABCG2 multidrug resistance proteins: biology, substrate
specificity and regulation. Curr Drug Metab. 2004;51:21-53.
109. Schmitz G, Kaminski WE. ABCA2: a candidate regulator of neural
transmembrane lipid transport. Cell Mol Life Sci. 2002;598:1285-95.
110. Kanai Y, Hediger MA. The glutamate and neutral amino acid transporter
family: physiological and pharmacological implications. Eur J
Pharmacol. 2003;4791-3:237-47.
111. Mackenzie B, Erickson JD. Sodium-coupled neutral amino acid (System
N/A) transporters of the SLC38 gene family. Pflugers Arch.
2004;4475:784-95.
164 112. DeCory HH, Piech-Dumas KM, Sheu SS, Federoff HJ, Anders MW.
Efflux of glutathione conjugate of monochlorobimane from striatal and
cortical neurons. Drug Metab Dispos. 2001;2910:1256-62.
113. Norris MD, Bordow SB, Marshall GM, Haber PS, Cohn SL, Haber M.
Expression of the gene for multidrug-resistance-associated protein and
outcome in patients with neuroblastoma. N Engl J Med.
1996;3344:231-8.
114. Beck J, Bohnet B, Brugger D, Bader P, Dietl J, Scheper RJ, Kandolf R,
Liu C, Niethammer D, Gekeler V. Multiple gene expression analysis
reveals distinct differences between G2 and G3 stage breast cancers,
and correlations of PKC eta with MDR1, MRP and LRP gene
expression. Br J Cancer. 1998;771:87-91.
115. Tu C, Tian Y, Pei F. [Expression of multidrug resistance-associated
protein 1 in osteosarcoma and its relationship with clinicopathologic
characteristics]. Sichuan Da Xue Xue Bao Yi Xue Ban. 2003;344:684-7.
116. Plasschaert SL, de Bont ES, Boezen M, vander Kolk DM, Daenen SM,
Faber KN, Kamps WA, de Vries EG, Vellenga E. Expression of
multidrug resistance-associated proteins predicts prognosis in
childhood and adult acute lymphoblastic leukemia. Clin Cancer Res.
2005;1124 Pt 1:8661-8.
165 117. Sisodiya SM, Lin WR, Harding BN, Squier MV, Thom M. Drug resistance
in epilepsy: expression of drug resistance proteins in common causes
of refractory epilepsy. Brain. 2002;125Pt 1:22-31.
118. Sultana R, Butterfield DA. Oxidatively modified GST and MRP1 in
Alzheimer's disease brain: implications for accumulation of reactive
lipid peroxidation products. Neurochem Res. 2004;2912:2215-20.
119. Kubota H, Ishihara H, Langmann T, Schmitz G, Stieger B, Wieser HG,
Yonekawa Y, Frei K. Distribution and functional activity of P-
glycoprotein and multidrug resistance-associated proteins in human
brain microvascular endothelial cells in hippocampal sclerosis.
Epilepsy Res. 2006;683:213-28.
120. Perera RJ, Ray A. MicroRNAs in the search for understanding human
diseases. BioDrugs. 2007;212:97-104.
121. Tang G. siRNA and miRNA: an insight into RISCs. Trends Biochem Sci.
2005;302:106-14.
122. Ying S-Y. Current perspectives in microRNAs (miRNA). [Dordrecht]:
Springer, 2008:vii, 464 p.
123. Davison TS, Johnson CD, Andruss BF. Analyzing micro-RNA expression
using microarrays. Methods Enzymol. 2006;411:14-34.
166 124. Aravin AA, Hannon GJ, Brennecke J. The Piwi-piRNA pathway provides
an adaptive defense in the transposon arms race. Science.
2007;3185851:761-4.
125. Shilo S, Roy S, Khanna S, Sen CK. Evidence for the involvement of
miRNA in redox regulated angiogenic response of human
microvascular endothelial cells. Arterioscler Thromb Vasc Biol.
2008;283:471-7.
126. Roy S, Khanna S, Hussain SR, Biswas S, Azad A, Rink C, Gnyawali S,
Shilo S, Nuovo GJ, Sen CK. MicroRNA expression in response to
murine myocardial infarction: miR-21 regulates fibroblast
metalloprotease-2 via phosphatase and tensin homologue. Cardiovasc
Res. 2009;821:21-9.
127. Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor growth.
Oncogene. 2007;2619:2799-803.
128. Biswas S, Roy S, Banerjee J, Hussain SR, Khanna S,
Meenakshisundaram G, Kuppusamy P, Friedman A, Sen CK. Hypoxia
inducible microRNA 210 attenuates keratinocyte proliferation and
impairs closure in a murine model of ischemic wounds. Proc Natl Acad
Sci U S A. 2010;10715:6976-81.
167 129. Khanna S, Parinandi NL, Kotha SR, Roy S, Rink C, Bibus D, Sen CK.
Nanomolar vitamin E alpha-tocotrienol inhibits glutamate-induced
activation of phospholipase A2 and causes neuroprotection. J
Neurochem. 2010;1125:1249-60.
130. van de Ven R, de Jong MC, Reurs AW, Schoonderwoerd AJ, Jansen G,
Hooijberg JH, Scheffer GL, de Gruijl TD, Scheper RJ. Dendritic cells
require multidrug resistance protein 1 (ABCC1) transporter activity for
differentiation. J Immunol. 2006;1769:5191-8.
131. DeVries AC, Joh HD, Bernard O, Hattori K, Hurn PD, Traystman RJ,
Alkayed NJ. Social stress exacerbates stroke outcome by suppressing
Bcl-2 expression. Proc Natl Acad Sci U S A. 2001;9820:11824-8.
132. Li X, Blizzard KK, Zeng Z, DeVries AC, Hurn PD, McCullough LD.
Chronic behavioral testing after focal ischemia in the mouse: functional
recovery and the effects of gender. Exp Neurol. 2004;1871:94-104.
133. Ojha N, Roy S, Radtke J, Simonetti O, Gnyawali S, Zweier JL,
Kuppusamy P, Sen CK. Characterization of the structural and
functional changes in the myocardium following focal ischemia-
reperfusion injury. Am J Physiol Heart Circ Physiol. 2008;2946:H2435-
43.
168 134. Adibhatla RM, Hatcher JF, Dempsey RJ. Effects of citicoline on
phospholipid and glutathione levels in transient cerebral ischemia.
Stroke. 2001;3210:2376-81.
135. Aygul R, Kotan D, Demirbas F, Ulvi H, Deniz O. Plasma oxidants and
antioxidants in acute ischaemic stroke. J Int Med Res. 2006;344:413-8.
136. Nazam Ansari M, Bhandari U, Islam F, Tripathi CD. Evaluation of
antioxidant and neuroprotective effect of ethanolic extract of Embelia
ribes Burm in focal cerebral ischemia/reperfusion-induced oxidative
stress in rats. Fundam Clin Pharmacol. 2008;223:305-14.
137. Kim J, Kim KY, Jang HS, Yoshida T, Tsuchiya K, Nitta K, Park JW,
Bonventre JV, Park KM. Role of cytosolic NADP+-dependent isocitrate
dehydrogenase in ischemia-reperfusion injury in mouse kidney. Am J
Physiol Renal Physiol. 2009;2963:F622-33.
138. Anderson MF, Sims NR. The effects of focal ischemia and reperfusion
on the glutathione content of mitochondria from rat brain subregions. J
Neurochem. 2002;813:541-9.
139. Jungsuwadee P, Cole MP, Sultana R, Joshi G, Tangpong J, Butterfield
DA, St Clair DK, Vore M. Increase in Mrp1 expression and 4-hydroxy-
2-nonenal adduction in heart tissue of Adriamycin-treated C57BL/6
mice. Mol Cancer Ther. 2006;511:2851-60.
169 140. Wijnholds J, Evers R, van Leusden MR, Mol CA, Zaman GJ, Mayer U,
Beijnen JH, van der Valk M, Krimpenfort P, Borst P. Increased
sensitivity to anticancer drugs and decreased inflammatory response in
mice lacking the multidrug resistance-associated protein. Nat Med.
1997;311:1275-9.
141. Wijnholds J, Scheffer GL, van der Valk M, van der Valk P, Beijnen JH,
Scheper RJ, Borst P. Multidrug resistance protein 1 protects the
oropharyngeal mucosal layer and the testicular tubules against drug-
induced damage. J Exp Med. 1998;1885:797-808.
142. Jeyaseelan K, Lim KY, Armugam A. MicroRNA expression in the blood
and brain of rats subjected to transient focal ischemia by middle
cerebral artery occlusion. Stroke. 2008;393:959-66.
143. Dharap A, Bowen K, Place R, Li LC, Vemuganti R. Transient focal
ischemia induces extensive temporal changes in rat cerebral
microRNAome. J Cereb Blood Flow Metab. 2009;294:675-87.
144. Liu DZ, Tian Y, Ander BP, Xu H, Stamova BS, Zhan X, Turner RJ,
Jickling G, Sharp FR. Brain and blood microRNA expression profiling
of ischemic stroke, intracerebral hemorrhage, and kainate seizures. J
Cereb Blood Flow Metab. 2010;301:92-101.
170 145. Yin KJ, Deng Z, Huang H, Hamblin M, Xie C, Zhang J, Chen YE. miR-
497 regulates neuronal death in mouse brain after transient focal
cerebral ischemia. Neurobiol Dis. 2010;381:17-26.
146. Lee ST, Chu K, Jung KH, Yoon HJ, Jeon D, Kang KM, Park KH, Bae EK,
Kim M, Lee SK, Roh JK. MicroRNAs induced during ischemic
preconditioning. Stroke. 2010;418:1646-51.
147. Rane S, He M, Sayed D, Vashistha H, Malhotra A, Sadoshima J, Vatner
DE, Vatner SF, Abdellatif M. Downregulation of miR-199a derepresses
hypoxia-inducible factor-1alpha and Sirtuin 1 and recapitulates hypoxia
preconditioning in cardiac myocytes. Circ Res. 2009;1047:879-86.
148. Jedlitschky G, Leier I, Buchholz U, Hummel-Eisenbeiss J, Burchell B,
Keppler D. ATP-dependent transport of bilirubin glucuronides by the
multidrug resistance protein MRP1 and its hepatocyte canalicular
isoform MRP2. Biochem J. 1997;327 ( Pt 1):305-10.
149. Kawabe T, Chen ZS, Wada M, Uchiumi T, Ono M, Akiyama S, Kuwano
M. Enhanced transport of anticancer agents and leukotriene C4 by the
human canalicular multispecific organic anion transporter
(cMOAT/MRP2). FEBS Lett. 1999;4562:327-31.
150. Hooijberg JH, Broxterman HJ, Kool M, Assaraf YG, Peters GJ,
Noordhuis P, Scheper RJ, Borst P, Pinedo HM, Jansen G. Antifolate
171 resistance mediated by the multidrug resistance proteins MRP1 and
MRP2. Cancer Res. 1999;5911:2532-5.
151. Suzuki H, Sugiyama Y. Excretion of GSSG and glutathione conjugates
mediated by MRP1 and cMOAT/MRP2. Semin Liver Dis.
1998;184:359-76.
152. Hirohashi T, Suzuki H, Takikawa H, Sugiyama Y. ATP-dependent
transport of bile salts by rat multidrug resistance-associated protein 3
(Mrp3). J Biol Chem. 2000;2754:2905-10.
153. Zelcer N, Saeki T, Reid G, Beijnen JH, Borst P. Characterization of drug
transport by the human multidrug resistance protein 3 (ABCC3). J Biol
Chem. 2001;27649:46400-7.
154. Meier PJ, Stieger B. Bile salt transporters. Annu Rev Physiol.
2002;64:635-61.
155. Schuetz JD, Connelly MC, Sun D, Paibir SG, Flynn PM, Srinivas RV,
Kumar A, Fridland A. MRP4: A previously unidentified factor in
resistance to nucleoside-based antiviral drugs. Nat Med.
1999;59:1048-51.
156. Wielinga PR, van der Heijden I, Reid G, Beijnen JH, Wijnholds J, Borst P.
Characterization of the MRP4- and MRP5-mediated transport of cyclic
nucleotides from intact cells. J Biol Chem. 2003;27820:17664-71.
172 157. Reid G, Wielinga P, Zelcer N, van der Heijden I, Kuil A, de Haas M,
Wijnholds J, Borst P. The human multidrug resistance protein MRP4
functions as a prostaglandin efflux transporter and is inhibited by
nonsteroidal antiinflammatory drugs. Proc Natl Acad Sci U S A.
2003;10016:9244-9.
158. Reid G, Wielinga P, Zelcer N, De Haas M, Van Deemter L, Wijnholds J,
Balzarini J, Borst P. Characterization of the transport of nucleoside
analog drugs by the human multidrug resistance proteins MRP4 and
MRP5. Mol Pharmacol. 2003;635:1094-103.
159. Jedlitschky G, Burchell B, Keppler D. The multidrug resistance protein 5
functions as an ATP-dependent export pump for cyclic nucleotides. J
Biol Chem. 2000;27539:30069-74.
160. Wijnholds J, Mol CA, van Deemter L, de Haas M, Scheffer GL, Baas F,
Beijnen JH, Scheper RJ, Hatse S, De Clercq E, Balzarini J, Borst P.
Multidrug-resistance protein 5 is a multispecific organic anion
transporter able to transport nucleotide analogs. Proc Natl Acad Sci U
S A. 2000;9713:7476-81.
161. Madon J, Hagenbuch B, Landmann L, Meier PJ, Stieger B. Transport
function and hepatocellular localization of mrp6 in rat liver. Mol
Pharmacol. 2000;573:634-41.
173 162. Belinsky MG, Chen ZS, Shchaveleva I, Zeng H, Kruh GD.
Characterization of the drug resistance and transport properties of
multidrug resistance protein 6 (MRP6, ABCC6). Cancer Res.
2002;6221:6172-7.
163. Ilias A, Urban Z, Seidl TL, Le Saux O, Sinko E, Boyd CD, Sarkadi B,
Varadi A. Loss of ATP-dependent transport activity in pseudoxanthoma
elasticum-associated mutants of human ABCC6 (MRP6). J Biol Chem.
2002;27719:16860-7.
164. Hopper-Borge E, Chen ZS, Shchaveleva I, Belinsky MG, Kruh GD.
Analysis of the drug resistance profile of multidrug resistance protein 7
(ABCC10): resistance to docetaxel. Cancer Res. 2004;6414:4927-30.
165. Chen ZS, Hopper-Borge E, Belinsky MG, Shchaveleva I, Kotova E, Kruh
GD. Characterization of the transport properties of human multidrug
resistance protein 7 (MRP7, ABCC10). Mol Pharmacol. 2003;632:351-
8.
166. Guo Y, Kotova E, Chen ZS, Lee K, Hopper-Borge E, Belinsky MG, Kruh
GD. MRP8, ATP-binding cassette C11 (ABCC11), is a cyclic
nucleotide efflux pump and a resistance factor for fluoropyrimidines
2',3'-dideoxycytidine and 9'-(2'-phosphonylmethoxyethyl)adenine. J
Biol Chem. 2003;27832:29509-14.
174 167. Bera TK, Iavarone C, Kumar V, Lee S, Lee B, Pastan I. MRP9, an
unusual truncated member of the ABC transporter superfamily, is
highly expressed in breast cancer. Proc Natl Acad Sci U S A.
2002;9910:6997-7002.
168. Dallas S, Miller DS, Bendayan R. Multidrug resistance-associated
proteins: expression and function in the central nervous system.
Pharmacol Rev. 2006;582:140-61.
169. Leier I, Jedlitschky G, Buchholz U, Center M, Cole SP, Deeley RG,
Keppler D. ATP-dependent glutathione disulphide transport mediated
by the MRP gene-encoded conjugate export pump. Biochem J.
1996;314 ( Pt 2):433-7.
170. Loe DW, Deeley RG, Cole SP. Characterization of vincristine transport
by the M(r) 190,000 multidrug resistance protein (MRP): evidence for
cotransport with reduced glutathione. Cancer Res. 1998;5822:5130-6.
171. Leier I, Jedlitschky G, Buchholz U, Cole SP, Deeley RG, Keppler D. The
MRP gene encodes an ATP-dependent export pump for leukotriene C4
and structurally related conjugates. J Biol Chem. 1994;26945:27807-
10.
175 172. Jedlitschky G, Leier I, Buchholz U, Center M, Keppler D. ATP-dependent
transport of glutathione S-conjugates by the multidrug resistance-
associated protein. Cancer Res. 1994;5418:4833-6.
173. Evers R, Cnubben NH, Wijnholds J, van Deemter L, van Bladeren PJ,
Borst P. Transport of glutathione prostaglandin A conjugates by the
multidrug resistance protein 1. FEBS Lett. 1997;4191:112-6.
174. Renes J, de Vries EE, Hooiveld GJ, Krikken I, Jansen PL, Muller M.
Multidrug resistance protein MRP1 protects against the toxicity of the
major lipid peroxidation product 4-hydroxynonenal. Biochem J.
2000;350 Pt 2:555-61.
175. Loe DW, Almquist KC, Cole SP, Deeley RG. ATP-dependent 17 beta-
estradiol 17-(beta-D-glucuronide) transport by multidrug resistance
protein (MRP). Inhibition by cholestatic steroids. J Biol Chem.
1996;27116:9683-9.
176. Rigato I, Pascolo L, Fernetti C, Ostrow JD, Tiribelli C. The human
multidrug-resistance-associated protein MRP1 mediates ATP-
dependent transport of unconjugated bilirubin. Biochem J. 2004;383Pt
2:335-41.
176 177. Qian YM, Song WC, Cui H, Cole SP, Deeley RG. Glutathione stimulates
sulfated estrogen transport by multidrug resistance protein 1. J Biol
Chem. 2001;2769:6404-11.
178. Cole SP, Sparks KE, Fraser K, Loe DW, Grant CE, Wilson GM, Deeley
RG. Pharmacological characterization of multidrug resistant MRP-
transfected human tumor cells. Cancer Res. 1994;5422:5902-10.
179. Grant CE, Valdimarsson G, Hipfner DR, Almquist KC, Cole SP, Deeley
RG. Overexpression of multidrug resistance-associated protein (MRP)
increases resistance to natural product drugs. Cancer Res.
1994;542:357-61.
180. Renes J, de Vries EG, Nienhuis EF, Jansen PL, Muller M. ATP- and
glutathione-dependent transport of chemotherapeutic drugs by the
multidrug resistance protein MRP1. Br J Pharmacol. 1999;1263:681-8.
181. Hollo Z, Homolya L, Hegedus T, Sarkadi B. Transport properties of the
multidrug resistance-associated protein (MRP) in human tumour cells.
FEBS Lett. 1996;3831-2:99-104.
182. Assaraf YG, Rothem L, Hooijberg JH, Stark M, Ifergan I, Kathmann I,
Dijkmans BA, Peters GJ, Jansen G. Loss of multidrug resistance
protein 1 expression and folate efflux activity results in a highly
177 concentrative folate transport in human leukemia cells. J Biol Chem.
2003;2789:6680-6.
183. Grzywacz MJ, Yang JM, Hait WN. Effect of the multidrug resistance
protein on the transport of the antiandrogen flutamide. Cancer Res.
2003;6310:2492-8.
184. Vernhet L, Allain N, Bardiau C, Anger JP, Fardel O. Differential
sensitivities of MRP1-overexpressing lung tumor cells to cytotoxic
metals. Toxicology. 2000;1422:127-34.
185. Kruh GD, Belinsky MG. The MRP family of drug efflux pumps. Oncogene.
2003;2247:7537-52.
186. Muller DP, Goss-Sampson MA. Neurochemical, neurophysiological, and
neuropathological studies in vitamin E deficiency. Crit Rev Neurobiol.
1990;53:239-63.
187. Azzi A, Boscoboinik D, Marilley D, Ozer NK, Stauble B, Tasinato A.
Vitamin E: a sensor and an information transducer of the cell oxidation
state. Am J Clin Nutr. 1995;626 Suppl:1337S-46S.
188. Boscoboinik DO, Chatelain E, Bartoli GM, Stauble B, Azzi A. Inhibition of
protein kinase C activity and vascular smooth muscle cell growth by d-
alpha-tocopherol. Biochim Biophys Acta. 1994;12243:418-26.
178 189. Pearce BC, Parker RA, Deason ME, Dischino DD, Gillespie E, Qureshi
AA, Volk K, Wright JJ. Inhibitors of cholesterol biosynthesis. 2.
Hypocholesterolemic and antioxidant activities of benzopyran and
tetrahydronaphthalene analogues of the tocotrienols. J Med Chem.
1994;374:526-41.
190. Inokuchi H, Hirokane H, Tsuzuki T, Nakagawa K, Igarashi M, Miyazawa
T. Anti-angiogenic activity of tocotrienol. Biosci Biotechnol Biochem.
2003;677:1623-7.
191. Suzuki YJ, Tsuchiya M, Wassall SR, Choo YM, Govil G, Kagan VE,
Packer L. Structural and dynamic membrane properties of alpha-
tocopherol and alpha-tocotrienol: implication to the molecular
mechanism of their antioxidant potency. Biochemistry.
1993;3240:10692-9.
192. Evans HM, Bishop KS. On the Existence of a Hitherto Unrecognized
Dietary Factor Essential for Reproduction. Science. 1922;561458:650-
51.
193. Emerson OHE, G. A. Mohammad, A. Evans, H. M. THE CHEMISTRY
OF VITAMIN E: TOCOPHEROLS FROM VARIOUS SOURCES J Biol
Chem. 1937;122:99.
179 194. Stern MH, Robeson CD, et al. delta-Tocopherol; isolation from soybean
oil and properties. J Am Chem Soc. 1947;694:869-74.
195. Pennock JF, Hemming FW, Kerr JD. A reassessment of tocopherol in
chemistry. Biochem Biophys Res Commun. 1964;175:542-8.
196. Whittle KJ, Dunphy PJ, Pennock JF. The isolation and properties of
delta-tocotrienol from Hevea latex. Biochem J. 1966;1001:138-45.
197. Yusuf S, Dagenais G, Pogue J, Bosch J, Sleight P. Vitamin E
supplementation and cardiovascular events in high-risk patients. The
Heart Outcomes Prevention Evaluation Study Investigators. N Engl J
Med. 2000;3423:154-60.
198. Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ,
Guallar E. Meta-analysis: high-dosage vitamin E supplementation may
increase all-cause mortality. Ann Intern Med. 2005;1421:37-46.
199. Theriault A, Chao JT, Wang Q, Gapor A, Adeli K. Tocotrienol: a review
of its therapeutic potential. Clin Biochem. 1999;325:309-19.
200. Packer L, Weber SU, Rimbach G. Molecular aspects of alpha-tocotrienol
antioxidant action and cell signalling. J Nutr. 2001;1312:369S-73S.
201. Schaffer S, Muller WE, Eckert GP. Tocotrienols: constitutional effects in
aging and disease. J Nutr. 2005;1352:151-4.
180 202. Sen CK, Khanna S, Roy S. Tocotrienols: Vitamin E beyond tocopherols.
Life Sci. 2006;7818:2088-98.
203. Miyazaki T, Tanaka S, Sanjay A, Baron R. The role of c-Src kinase in the
regulation of osteoclast function. Mod Rheumatol. 2006;162:68-74.
204. Ishizawar R, Parsons SJ. c-Src and cooperating partners in human
cancer. Cancer Cell. 2004;63:209-14.
205. Khanna S, Venojarvi M, Roy S, Sen CK. Glutamate-induced c-Src
activation in neuronal cells. Methods Enzymol. 2002;352:191-8.
206. Bazan NG, Jr. Effects of ischemia and electroconvulsive shock on free
fatty acid pool in the brain. Biochim Biophys Acta. 1970;2181:1-10.
207. Bazan NG. Free arachidonic acid and other lipids in the nervous system
during early ischemia and after electroshock. Adv Exp Med Biol.
1976;72:317-35.
208. Adesuyi SA, Cockrell CS, Gamache DA, Ellis EF. Lipoxygenase
metabolism of arachidonic acid in brain. J Neurochem. 1985;453:770-6.
209. Ishizaki Y, Murota S. Arachidonic acid metabolism in cultured astrocytes:
presence of 12-lipoxygenase activity in the intact cells. Neurosci Lett.
1991;1312:149-52.
210. Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone
G, Ferguson TB, Ford E, Furie K, Gillespie C, Go A, Greenlund K,
181 Haase N, Hailpern S, Ho PM, Howard V, Kissela B, Kittner S, Lackland
D, Lisabeth L, Marelli A, McDermott MM, Meigs J, Mozaffarian D,
Mussolino M, Nichol G, Roger VL, Rosamond W, Sacco R, Sorlie P,
Thom T, Wasserthiel-Smoller S, Wong ND, Wylie-Rosett J. Heart
disease and stroke statistics--2010 update: a report from the American
Heart Association. Circulation. 2010;1217:e46-e215.
211. Barnett HJM, Furlan AJ, Garcia JH, Ho K-L, Case Western Reserve
University. School of Medicine. Stroke : pathophysiology, diagnosis
and management . 3rd ed. New York ; Edinburgh: Churchill Livingstone,
1998.
212. Roy S, Lado BH, Khanna S, Sen CK. Vitamin E sensitive genes in the
developing rat fetal brain: a high-density oligonucleotide microarray
analysis. FEBS Lett. 2002;5301-3:17-23.
213. Kuhn DE, Roy S, Radtke J, Gupta S, Sen CK. Laser microdissection and
pressure-catapulting technique to study gene expression in the
reoxygenated myocardium. Am J Physiol Heart Circ Physiol.
2006;2906:H2625-32.
214. Rink C, Roy S, Khan M, Ananth P, Kuppusamy P, Sen CK, Khanna S.
Oxygen-sensitive outcomes and gene expression in acute ischemic
stroke. J Cereb Blood Flow Metab. 2010;307:1275-87.
182 215. Schurks M, Glynn RJ, Rist PM, Tzourio C, Kurth T. Effects of vitamin E
on stroke subtypes: meta-analysis of randomised controlled trials. BMJ.
2010;341:c5702.
216. Pearce BC, Parker RA, Deason ME, Qureshi AA, Wright JJ.
Hypocholesterolemic activity of synthetic and natural tocotrienols. J
Med Chem. 1992;3520:3595-606.
217. Sen CK. Cellular thiols and redox-regulated signal transduction. Curr
Top Cell Regul. 2000;36:1-30.
218. Osakada F, Hashino A, Kume T, Katsuki H, Kaneko S, Akaike A. Alpha-
tocotrienol provides the most potent neuroprotection among vitamin E
analogs on cultured striatal neurons. Neuropharmacology.
2004;476:904-15.
219. Mishima K, Tanaka T, Pu F, Egashira N, Iwasaki K, Hidaka R,
Matsunaga K, Takata J, Karube Y, Fujiwara M. Vitamin E isoforms
alpha-tocotrienol and gamma-tocopherol prevent cerebral infarction in
mice. Neurosci Lett. 2003;3371:56-60.
220. Erecinska M, Silver IA. Ions and energy in mammalian brain. Prog
Neurobiol. 1994;431:37-71.
183 221. Onteniente B, Rasika S, Benchoua A, Guegan C. Molecular pathways in
cerebral ischemia: cues to novel therapeutic strategies. Mol Neurobiol.
2003;271:33-72.
222. Doyle KP, Simon RP, Stenzel-Poore MP. Mechanisms of ischemic brain
damage. Neuropharmacology. 2008;553:310-8.
223. Schanne FA, Kane AB, Young EE, Farber JL. Calcium dependence of
toxic cell death: a final common pathway. Science. 1979;2064419:700-
2.
224. Simon RP, Griffiths T, Evans MC, Swan JH, Meldrum BS. Calcium
overload in selectively vulnerable neurons of the hippocampus during
and after ischemia: an electron microscopy study in the rat. J Cereb
Blood Flow Metab. 1984;43:350-61.
225. Leslie SW, Brown LM, Trent RD, Lee YH, Morris JL, Jones TW, Randall
PK, Lau SS, Monks TJ. Stimulation of N-methyl-D-aspartate receptor-
mediated calcium entry into dissociated neurons by reduced and
oxidized glutathione. Mol Pharmacol. 1992;412:308-14.
226. Lee M, Cho T, Jantaratnotai N, Wang YT, McGeer E, McGeer PL.
Depletion of GSH in glial cells induces neurotoxicity: relevance to
aging and degenerative neurological diseases. FASEB J.
2010;247:2533-45.
184 227. Gunstone FD, Harwood JL, Padley FB. The Lipid handbook . 2nd ed.
London ; New York: Chapman and Hall, 1994.
228. Amarenco P, Bogousslavsky J, Caplan LR, Donnan GA, Hennerici MG.
Classification of stroke subtypes. Cerebrovasc Dis. 2009;275:493-501.
185