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ON THE INFLUENCE OF DIETARY
PHYTOCHEMICALS ON THE SIGNALING
TRANSDUCTION IN HUMAN CELLULAR SYSTEMS REDOX-BALANCE &
ELECTROPHILIC ATTACK:
THE BIDIRECTIONAL FUNCTION OF
SELECTED PHYTOCHEMICALS:
DOCTORAL THESIS
submitted in
fulfilment of the
requirements of the
degree of DOCTOR
OF PHILOSOPHY
(PhD)
Martina C.F. Überall (Naschberger), Mag.rer.nat.
April 2016
Division of Medical Biochemistry
Centre for Chemistry and Biomedicine (CCB)
Medical University of Innsbruck (MUI)
Innrain 80-82, 6020 Innsbruck
i
Eidesstaatliche Erkla rung
“Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation selbständig
angefertigt habe. Die aus fremden Quellen direkt oder indirekt übernommenen
Gedanken sind als solche kenntlich gemacht.
Die Arbeit wurde bisher weder in gleicher noch in ähnlicher Form einer anderen
Prüfungsbehörde vorgelegt und auch noch nicht veröffentlich.“
Statement of Originality
„Herewith, I declare that this work has not been previously submitted for a degree or
diploma in any university.
To the best of my knowledge and belief, the thesis contains no material previously
published or written by another person except where due reference is made in the
thesis itself.”
Natters, am 12.04.2016 ______________________________
Martina C.F. Überall
ii
ACKNOWLEDGEMENTS
Empirical research for this project was performed at the Department for Medical
Biochemistry at the Medical University of Innsbruck and funded to a large extent by the
Austrian Research Agency (FFG), with the following grant: 844686 (KITCHEN
APPLIANCES) Development of new kitchen appliances for healthier cooking, a head
quarter project closely linked to Philips Austria GmbH and Carinthian Tech Research
(CTR). Thanks to both partners, for adding value to my project by directly applying the
outcomes and implementing them in innovative technologies.
Most importantly, I would like to express my gratitude to my supervisor, Florian, for his
guidance, advice, and support throughout my time as a PhD student and beyond. Besides
never growing tired of guiding me intellectually - often packed into entertaining stories -
he gave me space to find my own path and continuously supported and promoted my
personal development. Being my mentor, he did not just teach me about holistic
approaches when it comes to health and disease, but moreover, he set an example for
me of telos (from the Greek τέλος for "purpose", or "goal"), the concept of viewing one’s
own purposes and goals in life, as well as entelecheia (from the Greek ἐντελέχεια), the
particular type of motivation needed for self-determination and inner strength in
directing one’s life and growth in order to become all one is capable of. Also, he and his
wife, Andrea, gave me strength in the occasional tricky situations with their mantra of
tashi delek (from the Tibetan བཀྲ་ཤིས་བདེ་ལེགས).
Also, I would like to thank my former professor and significant colleague from across the
Indian Ocean, Kathryn Tonissen, for her insightful and extremely skilled supervision,
especially towards the end of my thesis. Thanks to her, I originally found my love for
Nrf2 and thioredoxin when working in her lab in beautiful Brisbane, Australia.
As well, I would like to pronounce my appreciation for Gabriele Werner-Felmayer,
who became my mentor not just inside the University, but even outdoors to as far as the
holy land. Fond memories of our trip to Israel in March 2014 will keep fueling my drive
for (bio-)ethically adequate scientific practice and life in general.
Furthermore, I would like to acknowledge all the past and present members of the
‘Überall’ group, the ‘Hengst’ department, my MCBO colleagues and all others with whom
iii
I had the opportunity to work during my PhD studies. In particular, I would like to thank
Lisa Maly-Kindler, for her assistance in Western blotting and sharing approx. 3 m2 of
office space without stepping on each other’s toes; Anto Nogalo, for continuously
cheering me up during coffee breaks and his view on cell culture practices; Hubert
Hackl, for introducing me to the rather complex to grasp, but fascinating, field of
biomedical statistics, thereby actually curing my phobia of large numbers and data sets;
my colleagues Andrea Casari, Kathrin Becker, Peter Gruber, Johanna Gostner and
Johannes Hochleitner for being such great lab members and colleagues, always willing
to lend a hand and providing assistance.
A heartfelt thanks goes to Maria Lerchbaumer, who I met even before my PhD and who
has, probably mostly unaware of the extent, helped me tremendously with her advice
and friendship throughout my PhD. And, huge thanks also belong to my dear
colleagues at the PHT, who gave me balance and support from the other end, bridging
over the obligatory strenuous episodes of my PhD project.
Last but not least, the biggest thanks are directed to my friends and family. Over the last
three years, I have received endless love and support on countless occasions, in words,
hugs and manifold forms of affection. My friends did not grow tired of sending me
cheerful messages from all over the world, no matter if from Iceland (thanks, Birna!) or
the states (Gigo, you rock!) or elsewhere. The ones close-by are real gems: Lisi, Michi,
Maria, Johanna, Susi & Christoph and many others (you know who you are <3) took
turns in kicking my butt (in fact, mostly for working too much) and caressing my soul
(outweighing the formerly mentioned, thankfully). In particular, my family often had to
accept my elegant absences while losing myself in the lab, but instead of complaints and
nagging they gifted me with their unconditional love and understanding. Thank you,
mum Lydia, dad Raimund, granny Emmi, and all beloved family members. I clearly
realize that without this backing up, my PhD could not have happened, and hence this is
not actually a product of my work solely, but rather the product of shared efforts (even
though absolutely neglected in the statement of originality above). Thus, the person to
definitely thank mostly is my husband Simon, who must be relieved about not having to
share me with my cells so much in the future. Thank you Simon, for everything!
iv
ABSTRACT
SIGNIFICANCE ‘Health’ has been proposed as the result of the organism’s ability to cope
with and adapt to stresses from our environment (1). In regard to the adaptive stress
response triggered in cells by electrophiles and oxidants, the transcription factor ‘nuclear
factor erythroid 2-related factor 2’ (Nrf2) has become known as the key molecule, or even a
“master switch” (2). This is highlighted by the fact that the Keap1-Nrf2 pathway orchestrates
more than 600 cytoprotective genes, which regulate cellular detoxification, the elimination of
ROS, xenobiotic metabolism, and drug transport (3). CRITICAL ISSUE While in healthy cells
this mechanism provides a strategy for the cell to “detox” by expressing these protective
enzymes, cancer cells apply the exact same tactic to ensure their survival. This “dark side” of
Nrf2 has often been neglected when discussing the effect of (dietary) antioxidants and their
potential benefit for health (4), which is why until now many promising dietary phytochemicals
have failed as chemopreventives in randomized controlled trials, while others exhibited even
harmful effects (5) (6). AIM To elucidate the question of the influence of anti- and prooxidant
dietary phytochemicals as “friends or foes” on the signaling transduction in a human cellular
cancer model thus became the focus of this thesis. This project to an extent deciphers the
effect of selected dietary phytochemicals on the Nrf2 pathway and on endogenous
antioxidant systems. Most importantly, the doctoral thesis at hand aims to define the
bidirectional – direct and indirect – anti-/prooxidative properties of nutrigenomic activators of
Nrf2 and thereby their potential to activate the thioredoxin detoxification system and heme-
oxygenase-1. METHODS The hepatocyte has been described as “a systemic hub”, because
it engages in the bodily metabolic demand, iron homeostasis and, most prominently,
detoxification processes, which are all redox-regulated (7). Therefore HepG2 cells, a well
characterized and robust liver cancer cell line, were employed, a model which in this field of
research is denoted “state-of-the-art”. The parameters investigated were cell viability with a
resazurin-based assay, anti- and prooxidant effect with cell-based assays using a peroxyl-
radical (AAPH) and a fluorescing indicator (DCF) as well as the reporter cell line HepG2-
ARE-bla™. Moreover, HepG2 cells were subfractioned into their major (and relevant)
compartments – cytoplasm, nuclei, mitochondria – and as such Western blotted to analyze
changes in Nrf2-target protein expression, selecting heme oxygenase-1 (HO-1), thioredoxin-
1 (Trx-1) and thioredoxin reductase-1 (TrxR-1) as candidates. Furthermore, to follow up on
endogenous ROS-production and the cells’ redox states, mitochondrial membrane potential
changes were detected with the confocal microscope and a fluorescing dye (TMRM), as well
as the multi-plate reader utilizing a different indicator stain (m-MPI). To obtain some in vivo
insights also, a Kaplan Maier analysis was performed on two Nrf2 target genes (Trx-1 and
v
TrxR-1) and their influence on survival probability. RESULTS Having established that
quercetin (QUE) acts predominantly as a direct antioxidant by scavenging ROS (the peroxyl-
radical AAPH), sulforaphane (SFN) is proven as a lead substance in protecting the cells from
oxidative stress via Nrf2-dependent modulation of the thioredoxin endogenous redox system,
and at the same time as a weak prooxidant. Epigallocatchin-3-gallate (EGCG) employs both
strategies, as the cell tries to re-establish homeostasis, which proves that these three
substances make a highly interesting match. The influence of SFN, QUE, and EGCG in
combination revealed novel and promising results on the IC50 of these liver cancer cells,
which was lowered significantly (after 24 hrs: 76.36 µM for SFN only; 180.5 µM for EGCG
only; compared to 52.54 µM of SFN when paired with 50 µM EGCG), and thus, EGCG is
shown to aggravate the anti-tumorigenic effect of SFN. Moreover, SFN plus EGCG raised
HO-1 levels significantly (↑ 2.81-fold) as well as TrxR-1 (↑ 1.85-fold) in reduced monomeric
form. Another significant effect of EGCG is demonstrated in its capability to lower Trx-1
levels in HepG2 cells. As shown in the Kaplan Maier analysis, Trx-1 is a protein, which if
overexpressed in cancer patients lowers their survival probability. While revealing synergistic
effects of these three lead substances on Nrf2-target protein expression, one novel and
striking finding is also that TrxR-1, a crucial part of the “redoxisome”, occurs in two sizes
[kDa] depending on the treatment: a monomeric 55 kDa form, which polymerizes upon
oxidative stress and appears clearly visible at a bigger molecular weight of ≈110-120 kDa. As
demonstrated in the paper at hand, this phenomenon is counteracted by QUE, the prime
direct antioxidant tested. Moreover, this thesis presents a dual approach to assessing
mitochondrial membrane potential and shows the effects of SFN, QUE, and EGCG in
qualitative and quantitative analyses, as single compounds and in combinations, which
revealed synergistic, antagonistic, additive, and indifferent effects. Overall, this project
challenges, first of all, the “antioxidant hypothesis”, according to which oxidative stress can
be overcome by dietary intake of antioxidant phytochemicals, and reveals how these can
work either directly as ROS scavengers or indirectly via the Nrf2 pathway – exemplifying their
bidirectional functionality. Secondly, this paper also examines the “oxidant hypothesis”, by
exploring and employing prooxidative modes to lower the survival probability of cancer cells
and thereby yielding significant findings. FUTURE PROSEPCTS Naturally, more detailed
concentration-time-organelle resolved studies as a follow-up to our study are advisable.
Ideally, future research will assess both individual significant markers of cellular status at
biochemical or phenotypical level and next generation –omics sequencing. Some results of
this project promise therapeutic successes, but more pre-clinical tests, in particular under
physiologically true oxygen conditions (known as “physoxia”), are advisable.
vi
LIST OF ABBREVIATIONS
AAPH α,α′-Azodiisobutyramidine
dihydrochloride
ARE/EpRE antioxidant responsive
element/electrophilic
A.d. Aqua destillata, destilled water
Bach1 BTB and CNC homolog 1
CAT catechin
CIN cinnamic acid
CTB cell titer blue®
CUR curcumin
CVD cardio vascular diseases
DCF dichlorofluerescin
DMSO dimethyl sulfoxide
DNTB 5,5’-dithiobis(2-nitrobenzoic)
acid
EGCG epigallocatechin-3-gallate
EtOH ethanol
FDR false discovery rate
GAL gallic acid
ITCs isothiocyanates
Keap1 kelch-like ECH-associated
protein-1
NF-ĸB nuclear factor kappa B
Nrf2 nuclear factor erythroid 2-
related factor 2
NES nuclear export signal
NLS nuclear localization signal
PKC protein kinase C
Prx peroxiredoxins
ROS reactive oxygen species
SEM standard error of mean
SFN sulforaphane
SOP standard operating procedures
Trx thioredoxin
TrxR thioredoxin reductase
QUE quercetin
TABLE OF CONTENTS
1 GENERAL INTRODUCTION .......................................................................................... 1
2 BACKGROUND.............................................................................................................. 3
2.1 Reactive oxygen species (ROS) .............................................................................. 5
2.1.1 ROS & modulation of carcinogenesis ............................................................... 7
2.1.2 ROS & biomedical applications ........................................................................ 9
2.2 Redox systems biology ..........................................................................................10
2.3 The nuclear factor E2-related factor 2 (Nrf2)-pathway ............................................13
2.3.1 Heme oxygenase-1 (HO-1) as a Nrf2-target protein ........................................15
2.4 The thioredoxin system ..........................................................................................16
2.5 Antioxidants............................................................................................................18
2.6 Dietary phytochemicals ..........................................................................................20
2.6.1 Selection & rational behind tested substances ................................................22
2.7 Hormetic concept ...................................................................................................31
2.8 Synergies ...............................................................................................................32
2.9 Research aims .......................................................................................................34
3 RESULTS......................................................................................................................36
3.1 General assessment ..............................................................................................36
3.2 Cell proliferation & viability .....................................................................................38
3.2.1 IC50 calculation based on metabolic activity of HepG2 of single compounds ...39
3.2.2 IC50 calculation based on metabolic activity of HepG2 of multiple compounds 42
3.3 Effects on intracellular ROS-inhibition ....................................................................44
3.4 Effects on intracellular Nrf2-transactivation ............................................................52
3.4.1 Assessment via the CellSensor® ARE-bla HepG2 Cell Line ...........................52
3.4.2 Effect on heme oxygenase-1 (HO-1) protein expression .................................60
3.4.3 Effect on thioredoxin-1 (Trx-1) protein expression ...........................................61
3.4.4 Effect on thioredoxin reductase-1 (TrxR-1) protein expression ........................62
3.5 Effects on intracellular Nrf2 (trans-)location & expression levels.............................65
3.6 Effects on mitochondrial membrane potential .........................................................67
3.7 Trx/TrxR and survival probability in vivo .................................................................72
4 FINAL DISCUSSION .....................................................................................................75
4.1 Summary of the Results & Discussion ....................................................................76
4.2 Cell proliferation & viability .....................................................................................78
4.3 Effects on intracellular ROS-inhibition ....................................................................81
4.4 Effects on intracellular Nrf2-transactivation ............................................................82
4.4.1 Assessment via the CellSensor® ARE-bla HepG2 Cell Line ...........................82
4.4.2 Effect on heme oxygenase-1 (HO-1) protein expression .................................83
4.4.3 Effect on thioredoxin-1 (Trx-1) protein expression ...........................................84
4.4.4 Effect on thioredoxin reductase (TrxR-1) protein expression ...........................85
4.5 Effects on intracellular Nrf2 (trans-)location & expression levels.............................86
4.6 Effects on mitochondrial membrane potential .........................................................87
4.7 FINAL SUMMARY substance-wise ........................................................................89
4.7.1 Sulforaphane (SFN) ........................................................................................90
4.7.2 Epigallocatechin-3-gallate (EGCG) ..................................................................91
4.7.3 Quercetin (QUE) .............................................................................................92
4.7.4 Wrap-up - all three substances in combination ................................................93
4.8 Conclusions............................................................................................................95
4.9 Future Directions .................................................................................................. 102
5 MATERIALS & METHODS .......................................................................................... 105
5.1 Dietary phytochemicals ........................................................................................ 105
5.2 Antibodies ............................................................................................................ 105
5.3 Chemicals, reagents & kits ................................................................................... 107
5.4 Cell culture ........................................................................................................... 108
5.5 Cell proliferation & viability ................................................................................... 112
5.6 Measurement of intracellular ROS-inhibition......................................................... 113
5.7 Assessment of intracellular Nrf2-transactivation ................................................... 114
5.7.1 ARE-GeneBLAzer β-lactamase reporter gene assay .................................... 114
5.8 Assessment of intracellular Nrf2-translocation ...................................................... 115
5.8.1 Subcellular fractionation & Western blot analysis .......................................... 115
5.9 Assessment of mitochondrial membrane potential (MMP) .................................... 118
5.9.1 MMP investigated via confocal microscopy analysis...................................... 118
5.9.2 MMP investigated via fluorescence plate reader ........................................... 119
5.10 Statistical analyses ............................................................................................... 120
6 Works Cited ................................................................................................................. 121
6.1.1 Competing interests & Funding ..................................................................... 140
Lists of figures and tables
Figure 1: Pathologies and diseases caused by oxidative stress. ........................................... 4
Figure 2: Exogenous and endogenous scavengers of ROS................................................... 5
Figure 3: ROS and their atomic specificity ............................................................................. 6
Figure 4: ROS & RES homeostasis and strategies to modulate redox dynamics for potential
therapeutic application – “oxidation therapy” .................................................................. 9
Figure 5: The Redox code - major strategies for mammalian redox homeostasis. ................12
Figure 6: The Nrf2-pathway ..................................................................................................14
Figure 7: The thioredoxin system .........................................................................................17
Figure 8: The bidirectional – A) direct and B) indirect - function of antioxidants. ...................19
Figure 9: Classification of dietary phytochemicals ................................................................24
Figure 10: Sulforaphane (SFN) - C6H11NOS2. .......................................................................24
Figure 11: Quercetin (QUE) - C15H10O7.................................................................................27
Figure 12: Epigallocatechin-3-gallate (EGCG) - C22H18O11 ...................................................29
Figure 13: Theoretical biotransformation pathways for epigallocatechin-3-gallate (EGCG) and
its metabolites. ..............................................................................................................30
Figure 14: Graphical abstract of Workflow/Milestones, stating the research aims .................35
Figure 15: HepG2 cells visualized under the confocal microscope .......................................37
Figure 16: HepG2 cells undergoing cell division visualized under the confocal microscope ..38
Figure 17: Effect of sulforaphane on cell viability ..................................................................39
Figure 18: Effect of quercetin on cell viability ........................................................................39
Figure 19: Effect of epigallocatechin-3-gallate on cell viability ..............................................40
Figure 20: Effect of curcumin on cell viability ........................................................................40
Figure 21: Effect of cinnamic acid on cell viability .................................................................41
Figure 22: Effect of gallic acid on cell viability .......................................................................41
Figure 23: Effect of sulforaphane and epigallocatechin-3-gallate combined on cell viability ..42
Figure 24: Effect of sulforaphane and quercetin combined on cell viability ...........................42
Figure 25: Effect of sulforaphane, epigallocatechin-3-gallate and quercetin combined on cell
viability ..........................................................................................................................43
Figure 26: Effect of sulforaphane, epigallocatechin-3-gallate and quercetin combined on cell
viability.. ........................................................................................................................43
Figure 27: Measurement of intracellular ROS upon treatment with sulforaphane. .................45
Figure 28: Measurement of intracellular ROS upon treatment with quercetin .......................45
Figure 29: Measurement of intracellular ROS upon treatment with epigallocatechin-gallate. 47
Figure 30: Measurement of intracellular ROS upon treatment with curcumin ........................48
Figure 31: Measurement of intracellular ROS upon treatment with cinnamic acid .................49
Figure 32: Measurement of intracellular ROS upon treatment with gallic acid ......................50
Figure 33: Measurement of intracellular ROS upon treatment with catechin .........................51
Figure 34: Activation of antioxidant response element (ARE)-driven β-lactamase reporter
gene expression upon treatment with sulforaphane .......................................................53
Figure 35: Activation of antioxidant response element (ARE)-driven β-lactamase reporter
gene expression upon treatment with quercetin ............................................................54
Figure 36: Activation of antioxidant response element (ARE)-driven β-lactamase reporter
gene expression upon treatment with epigallocatechin-3-gallate ...................................55
Figure 37: Activation of antioxidant response element (ARE)-driven β-lactamase reporter
gene expression upon treatment with curcumin .............................................................56
Figure 38: Activation of antioxidant response element (ARE)-driven β-lactamase reporter
gene expression upon treatment with cinnamic acid ......................................................57
Figure 39: Activation of antioxidant response element (ARE)-driven β-lactamase reporter
gene expression upon treatment with gallic acid ...........................................................58
Figure 40: Activation of antioxidant response element (ARE)-driven β-lactamase reporter
gene expression upon treatment with catechin ..............................................................59
Figure 41: Heme oxygenase-1 (HO-1) protein expression ....................................................61
Figure 42: Thioredoxin-1 (Trx-1) protein expression .............................................................62
Figure 43: Western blot of TrxR-1 staining plus GAPDH as loading control. .........................63
Figure 44: Thioredoxin reductase-1 (TRXR-1) protein expression (I) ....................................64
Figure 45: Thioredoxin reductase-1 (TRXR-1) protein expression (II) ...................................65
Figure 46: Nrf2 protein expression levels..............................................................................66
Figure 47: HepG2 cells, after treatment with selected dietary phytochemicals, visualized
under the confocal microscope ......................................................................................68
Figure 48: Comparison of means the area fraction vs. mean grey values from these fractions
assessed of HepG2 cells, after treatment with selected dietary phytochemicals,
visualized under the confocal microscope .....................................................................69
Figure 49: Changes in mitochondrial membrane potential ....................................................70
Figure 50: Kaplan Meier analysis of the influence of Trx-1 levels in cancer patients .............73
Figure 51: Kaplan Meier analysis of the influence of Trx-1 levels in cancer patients. ............74
Figure 52: The identified hallmarks of cancer – the next generation .....................................77
Figure 53: The “redox code” .................................................................................................89
Figure 54: Chemical structure of quercetin as a role model for the key features of flavonoids
with antioxidant activity ..................................................................................................92
Figure 55: Bifunctional antioxidative capacity, A) direct ROS-scavenging action of dietary
phytochemicals like quercetin, B) indirect antioxidant action via Nrf2 of bioactives like
sulforaphane. ................................................................................................................96
Figure 56: Nrf2-pathway, 1) degradation under normoxia, 2) induction via an electrophilic
attack, leading to the expression of phase II enzymes ...................................................97
Figure 57: CellTiter-Blue™ Cell assay, to assess cell viability. ........................................... 113
Figure 58: ROS-assay, to evaluate the direct antioxidant potential. .................................... 114
Figure 59: ARE-assay, to measure the indirect antioxidant potential. ................................. 115
Figure 60: Mito-assay, to obtain changes in the mitochondrial membrane potential ........... 119
Table 1: Exogenous and endogenous sources of ROS ......................................................... 7
Table 2: Mediators of ROS catabolism .................................................................................10
Table 3: pH-Value assessment for single substances ..........................................................36
Table 4: pH-value assessment for single substances (repeated) and for combinations ........37
Table 5: Values derived from densitometric analysis of Western blots for HO-1. ..................60
Table 6: Values derived from densitometric analysis of Western blots for Trx-1 ...................61
Table 7: Values derived from densitometric analysis of Western blots for TrxR-1 (I).............63
Table 8: Values derived from densitometric analysis of Western blots for TrxR-1 (II). ...........64
Table 9: Values derived from densitometric analysis of Western blots for Nrf2. ....................66
Table 10: Summary of IC50 calculation based on the metabolic activity of HepG2 cells treated
with single compounds. .................................................................................................78
Table 11: Summary of IC50 calculation based on the metabolic activity of HepG2 cells treated
with multiple compounds. ..............................................................................................80
Table 12: Summary of ROS-inhibition values of single substances in HepG2 cells ..............81
Table 13: Summary of ARE-fold induction values of single substances in HepG2 cells ........82
Table 14: Alignments of the sequence of Trx-1 and Trx2 ......................................................85
Table 15: Summary of the main results yielded by the presented study of single substances.
......................................................................................................................................99
Table 16: Summary of the main results yielded by the presented study of substances in
combinations. .............................................................................................................. 100
Table 17: Identification and characterization of antibodies used for Western Blot analysis. 106
Table 18: Identification and source of chemicals, reagents and kits applied. ...................... 107
Table 19: Identification and source of specific cell culture materials and reagents. ............. 109
Table 20: Experimental set up of coverslips for microspial analysis. ................................... 118
1
1 GENERAL INTRODUCTION
Starting my work in the lab of ao.Univ.-Prof. Mag. Dr. Florian Überall in October 2012, I have
been privileged to gain scientific insights and work experience by actively contributing to the
following projects - intellectually and with actual lab work - in the course of my PhD studies:
2013-2016, FFG 844686 (KITCHEN APPLIANCES) Development of new kitchen appliances for
healthier cooking.– MAIN PROJECT, still ongoing
2012-2015, FFG 834169 (VOConCELL) Cellular and molecular risk assessment of volatile organic compounds from wood-based materials on human cell models using a new type of emission and exposure chamber.
2012-2013, FFP 834251 (PHYTORAF I &II) Analysis of bioactive extracts – cascading use of waste from plant harvest and processing.
Three years ago, I started immersing myself in the field of the “Special Biochemistry of
Nutrition”, getting acquainted with various bioactive substances, and learning about their
modes of action in cellular models of liver, lung, prostate, and intestine origin. The
bidirectionality in their activity became our focus, since the cellular redox balance is of utmost
importance in many intracellular signaling events. Moreover, this proved to be a great
starting point in identifying the versatile action of dietary phytochemicals in regard to
assessing the health-modulating capacity that substances from fruits and vegetables have
often been attributed with. Hence, my work, particularly of the first year, contributed to the
establishment of suitable cellular models and experimental set-ups. I strived for the analysis
of the pro- and antioxidant effects of selected phytochemicals enhancing my knowledge as
well as capabilities in regard to the Nrf2-pathway and its key players.
2
My overarching goal has always been to translate profound (biomedical) knowledge into
application and to grant others access to it, my motivation in accordance with public health
principles. Therefore, I also endeavored to enhance my skills in the area of health education,
communication and promotion, a set of competences especially convenient for our
headquarter project on kitchen appliances. Together with Philips and CTR, we set ourselves
the goal to offer customers a convenient way for healthy cooking (a concept, which is not
easy to define) by developing several kitchen appliances. From a biomedical perspective, the
output has been more than fruitful.
Plus, another project that has been at the core of my heart also taking a public health
approach, is “Klasse Forschung!”, an initiative of the Cemit Tyrol, supported by the Austrian
Research Agency. Within this project, we invited school classes to our laboratory to fulfil our
educational mandate by teaching the youngsters about natural as well as artificial flavors and
tastes and discussing their effects in our body.
3
2 BACKGROUND
It has been prognosticated that cardiovascular diseases (CVDs) and cancer, the two leading
causes of death worldwide (8), and hence the resulting risk of mortality, will continuously
increase until 2030 (9). Life-style decisions have been shown to considerably influence the
risk factor, which determines the likelihood to develop these diseases. Thus, when it comes
to nutritional behavior, every person’s particular choice matters. For decades, numerous
studies have tackled the question of how specific foods or single compounds might impinge
on this risk.
Fruits and vegetables, as well as their dietary phytochemicals, have become the center of
attention when it comes to beneficial health-modulating capacities. As a direct consequence
of epidemiological studies, which have shown that the intake of fresh produce lowers the risk
for cardiovascular diseases (10-12), type II diabetes (13), and certain cancers, i.e. of the
mouth, the pharynx, the larynx, the esophagus, the stomach, and the lungs, the WHO
recommends eating ≥400 g per day, not counting potatoes or starchy tubers such as
cassava (14). Additionally, the WHO estimates insufficient intake of fruits and vegetables to
be responsible for around 14% of gastrointestinal cancer deaths, about 11% of ischemic
heart disease deaths, and about 9% deaths due to a stroke worldwide (15). As a matter of
fact, the majority of Europeans are not able to reach these recommendations, even though
the increase has also been clearly stated in the European Commission’s White Paper in
Nutrition from 2007 (16), which has led to national nutrition policies such as “5-a-day-
campains” and the “school fruit schemes”.
Thus, in this thesis, “the antioxidant hypothesis”, stating that phytochemicals are known
“antioxidants”, which could “potentially” help to overcome “oxidative stress” as the root for a
number of pathologies, shall be challenged and discussed in depth.
4
The fact is that aerobic life holds an inherent double-edged sword: oxygen. While being the
essential element for life, oxygen can also occur as an intermediate oxygen carrying
metabolite in form of a free radical, with unpaired electrons. Oxygen radicals, collectively
termed “reactive oxygen species” (ROS), are either produced internally: as a normal part of
metabolism, under the circumstance of an inflammation, or usual physical exercising; or, can
be caused by external factors: by cigarette smoke, environmental pollutants, ozone or others
(Table 1). Under physiological/basal conditions, the body’s own antioxidant defense system
is capable of dealing with these free radicals, and balances free ROS with antioxidants by
directly detoxifying and metabolizing them, or by repairing resulting damage when required
(Table 2). Under special circumstances though, “oxidative stress” - the inability to stabilize
this balance - can cause severe damage to an organism and lead to numerous pathologies
(Figure 1) (17).
Figure 1: Pathologies and diseases caused by oxidative stress. (Source: NIST, National Institute of Standards and Technology, from (17))
Controversially, ROS in moderate doses also serve beneficial purposes in the human body.
Mittal and Murad (1977) provided evidence for advantageous use of free radicals, when they
showed that hydroxyl radicals (●OH) stimulate activation of guanylate cyclase and formation
of “second messenger” cyclic guanosine monophosphate (cGMP) (18-19).
5
Hence, “redox biology” really is a delicate life-decisive balance, for which, the body depends
on endogenous as well as exogenous sources to master a highly complex interplay (Figure
2) (20). To elucidate on these mechanisms, this thesis highlights the endogenous
cytoprotective gene expression induced by some representative exogenous dietary
phytochemicals, in particular the thioredoxin system, with the Nrf2-Keap1 system as a
prime molecular target, in a human hepatocarcinoma model.
Figure 2: Exogenous and endogenous scavengers of ROS. (Modified from (20))
2.1 Reactive oxygen species (ROS)
Reactive oxygen species (ROS) have triggered a growing body of evidence pointing towards
them as pivotal influences on the human body’s health. ROS are known to react
preferentially with certain atoms to orchestrate various biological phenomena ranging from
cell homeostasis to cell death. They are mostly endogenously produced, small, reactive
signaling molecules. Alternatively, they may arise from interactions with exogenous sources
such as xenobiotic compounds. Molecular reactions comprise inhibition as well as activation
6
of proteins, mutagenesis of DNA, and the modulation of gene transcription (21). Worst case,
they can cause a cell to undergo malignant transformation, when the signal is too strong, it
lasts too long or arises at the wrong time and place, and thus becomes cytotoxic. So called
“oxidative stress” evokes upon an overwhelming ROS stimulus and an inadequate response
of the cellular antioxidant system. ROS-mediated damage affects nucleic acids, proteins, and
lipids. The classification includes superoxide, hydrogen peroxide, and hydroxyl radicals,
besides singlet oxygen and ozone. Further ROS are the hypochlorous (HOCl), hypobromous
(HOBr), and hypoiodous (HOI) acids, which arise when peroxidases catalyze the oxidation of
halides by hydrogen peroxide (H2O2) as well as important products of the reaction of ROS
with other molecules that hold strong oxidizing potential. Furthermore, ROS at low levels
have the capacity to react reversibly with a limited number of atoms such as e.g. selenium or
sulphur in a subset of cysteine or methionine residues. At higher levels, on the other hand,
ROS are likely to react irreversibly with certain iron and carbon atoms (Figure 3).
Figure 3: ROS and their atomic specificity. Upon reduction of oxygen to water, sequential one-electron
subtractions can produce reactive oxygen intermediates (ROIs), a subset of ROS such as e.g. superoxide, hydrogen peroxide, and hydroxyl radicals, besides singlet oxygen and ozone. Further ROS are the hypochlorous (HOCl), hypobromous (HOBr), and hypoiodous (HOI) acids. (Modified from (21))
7
Scientists in recent years have gained a deeper understanding of the origin and the multiple
targets and actions influenced by ROS in cells. Endogenous sources include seven isoforms
of NADPH oxidases (NOXs), the mitochondrial respiratory chain, the flavoenzyme ERO1 in
the endoplasmic reticulum (ER), xanthine oxidase, lipoxygenase, cyclooxygenase,
cytochrome p450s, a flavin-dependent demethylase, oxidases for polyamines and amino
acids, and nitric oxide synthases. Moreover, haem groups, metal storage proteins or copper
or iron ions can serve to convert O2●- and/or H2O2 to ●OH, to mention just a few examples
(for a complete list, please see Table 1).
Table 1: Exogenous and endogenous sources of ROS. (Modified from (21))
Exogenous sources of ROS Endogenous sources of ROS
Smoke
Air pollutants
Ultraviolet radiation
γ-irradiation
Xenobiotic compounds
NADPH oxidases
Mitochondria
ER flavoenzyme ERO1
Xanthine oxidase
Lipoxygenases
Cyclooxygenases
Cytochrome P450 enzymes
Flavin-dependent demethylase
Polyamine and amino acid oxidases
Nitric oxide synthases
Free iron and copper ions
Haem groups
Metal storage proteins
2.1.1 ROS & modulation of carcinogenesis
Cumulative experimental data indicate that ROS play a major role in the initiation, promotion,
and progression of carcinogenesis, highlighted by the fact that cancer cells show increased
levels of ROS and impairment in their redox regulation. The increased levels are primarily
due to the characteristically elevated and altered oxygen metabolism, a change from
oxidative phosphorylation to glycolysis, and the increased activity of NADPH oxidases (NOX)
8
(22-23). In cancer development, mutagenic and carcinogenic agents like tobacco smoke,
asbestos or N-nitrosamines, have been discovered to trigger this process by acting as pro-
oxidants, triggering these changes and thereby inducing genetic alterations, cellular
proliferation along with resistance to apoptosis, metastasis, and angiogenesis, etc., which is
generally understood as “oxidative damage” (22).
However, more recently it has been discovered that pro-oxidants can also function as
effective agents in the elimination of cancer cells as they enforce intracellular toxic levels. In
this sense, the pro-oxidative capacities of some natural products, i.e. polyphenols such as
quercetin or epigallocatechin-3-gallate, shall be discussed, as they show promising results as
chemotherapeutic adjuvants, not just by increasing ROS, but even more so by enhancing the
cytotoxic activity of cytostatics for cancer cells only, while affecting normal cells only
marginally. Nonetheless, caution has to be taken when using polyphenols in anticancer
therapy, since their effect has been shown to depend on factors such as the applied dose,
the cell type, the time period of exposure as well as environmental conditions. Especially,
since a successful therapy, which selectively targets cancer cells, has to rule out any
antioxidative effects, but instead has to modify redox homeostasis in order to achieve toxic
levels and induce apoptosis as well as cell cycle arrest by “oxidation therapy” (Figure 4).
While it is not yet fully understood why certain pro-oxidants have the capacity to kill cancer
cells selectively, the following findings indicate a direction: firstly, it has been observed that
cancer cells are more susceptible to H2O2 than their corresponding normal cells, as e.g.
ascorbic acid at high doses generated more H2O2 and thereby significantly reduced tumor
progression in mice without toxicity to normal tissue (24). It has also been shown that, they
are capable of producing higher quantities of H2O2 than non-cancerous cells (25). Secondly,
this effect might be due to the elevated levels of transition metals such as e.g. copper,
stimulated by pro-oxidants, which can then generate ROS through Fenton and Fenton-like
reactions (26), as it has been shown that most types of cancer cells over-express e.g.
transferrin receptors or the copper transporter 1 (27).
9
Currently, two innovative criteria for drug development are gaining awareness, the pro-
electrophilic drugs (PED) and the pathologically activated therapeutics (PATs), both of which
become electrophiles and are activated when triggered by oxidative stress which they can
then fight (28).
Therefore, the quest for natural, dietary phytochemicals with inherent electrophilic properties
for cancer prevention, progression or cure, is an emerging strategy in modern drug-targeted
therapies and their “druggability” as pressing as never before. Thus, these indications have
made it even more crucial to investigate the precise mechanisms of action of dietary
phytochemicals (29).
Figure 4: ROS & RES homeostasis and strategies to modulate redox dynamics for potential therapeutic application – “oxidation therapy”. Reactive oxygen species (ROS) and reactive electrophilic species (RES)
levels can vary in normal cells also, but will be regulated via homeostatic mechanisms. However, in cancer cells, just like in ageing cells, these strategies are lost as redox regulation gets impaired. From a therapeutical perspective, cancer cells treated with pro-oxidants should enter the desired apoptosis by increasing the intracellular ROS level, just like cell death theoretically could be avoided for healthy and ageing cells by eliminating ROS with antioxidant strategies. (Modified from (30))
2.1.2 ROS & biomedical applications
As mentioned above, beneficial use of ROS occurs at low and moderate concentrations,
such as intracellular signaling, in particular the modulation of transcription factor activation,
10
and in the defense against infectious agents (31). Therefore, particularly in recent years,
many drugs which apply the functional mechanism of ROS, or reactive electrophilic species
(RES) for that matter, were introduced to the market. They either work by inducing
intracellular ROS production or sensitizing cells to them, diminishing their production or
enhancing their catabolism (Table 2).
Table 2: Mediators of ROS catabolism. (Modified from (21))
Catabolism by endogenous antioxidant
systems
Catabolism by small molecules that react
with ROS non-enzymatically
Superoxide dismutases
Catalases
Glutathione peroxidases
Glutathione reductases
Thioredoxins
Thioredoxin reductases
Methionine sulphoxide reductases
Peroxiredoxins or peroxynitrite reductases
Ascorbate
Pyruvate
Α-ketoglutarate
Oxaloacetate
Hence, many antibiotics eradicate bacteria by enhancing their ROS production, and so do
anti-infectives, such as e.g. clofazimine, and anti-cancer reagents which exercise antibiotic
actions such as e.g. adriamycin and bleomycin. Also, the anti-inflammatory function of statins
is based on decreased ROS production of endothelial cells (19; 21).
2.2 Redox systems biology
Mammalian cells utilize a variety of antioxidants, antioxidant systems, and antioxidant repair
systems not only to prevent oxidative damage, but, furthermore, to ensure the regulation of
essential signaling pathways (32-41). The “redox code”, as portrayed in Figure 5, denotes a
set of reduction-oxidation (redox) biological strategies such as of the nicotinamide adenine
dinucleotide (NAD, NADP), the thiol/disulphide, and other redox systems along with the thiol
11
redox proteome at specific spatiotemporal set points in cellular organization (42). Cysteine
(Cys) and methionine (Met) are the two amino acids which can undergo reversible oxidation
and therefore are known as “sulfur switches” (43). Upon activation of this switch, the
following events are triggered: protein conformation, enzyme activity, transporter activity,
ligand binding to receptors, protein-protein interactions, protein-DNA interactions, protein
trafficking, and protein degradation (44). “Sulfur switches” are understood as “redox control
nodes”, control points – a basic principle in systems biology – which occupy decisive
crossroads within a network of pathways. Some mechanisms have been defined and the
activation of apoptosis signal-regulating kinase (Ask-1) was linked to the oxidation of
thioredoxins (45), while the oxidation of glutaredoxins (GSH/GSSG) was related to the Nrf2-
transactivation, for instance (46). Nrf2, or rather Keap1 as is explained below, is a classical
“redox sensor”, since it is not the ultimate target, but merely a switch upstream of a signaling
cascade. As a third major redox control node, Cys/CySS has been identified (44). Redox
control in the system cell occurs quasi-independently for each cellular compartment, which
allows for temporally and spatially separated regulation of the subcellular redox status.
While a lot of attention is already being paid to reactive oxygen species, reactive
electrophiles are a rather unexplored entity, due to the fact that they are very diverse in their
chemical structure and appearance. They constitute positively charged compounds, which
are inherently attracted and react with other compounds which possess an electron rich
center. Even though they come with a diverse structure inducing numerous biological
activities, electrophiles share the electron-deficient carbon centers, with an electron density
in the carbonyl oxygen of their structure (28). As a consequence they react with nucleophiles,
such as for instance protein thiolsor sulfhydryl groups (-SH), for instance found in reduced
glutathione (GSH). This mechanism could indeed contribute to a decrease in the reductive
capacity of the cell, but their action can also result in the initiation of intracellular signaling
pathways, such as for instance via Nrf2, thereby triggering cytoprotective capacity (47).
12
Figure 5: The Redox code - major strategies for mammalian redox homeostasis. Reduction-oxidation (redox) strategies act via post-translational modification of proteins via
particular target cysteines that have a low acid dissociation constant (pKa), by changing their oxidation states and thereby their function. Oxidative modifications can be reversed, for instance, via the two most prominent antioxidant systems, namely the thioredoxin (Trx)- and the glutathione (GSH)-system. Nrf2 is an essential “control knob” for redox homeostasis, as it potentially induces the expression of these antioxidant enzymes, and thereby regulated imbalances between oxidants and reductants to maintain this homeostasis. (Modified from (41))
13
2.3 The nuclear factor E2-related factor 2 (Nrf2)-pathway
Enhancing the cellular antioxidant capacity by the up-regulation of antioxidant detoxification
genes, and thereby the so-called “phase II detoxification”, is essential in the cellular
adaptation to oxidative stress and the protection of the cell from oxidative damage. In this
process, electrophilic ROS sensing by cysteine residues can provide feedback control to
regulate intracellular ROS levels. Kelch-like ECH-associated protein-1 (Keap1) possesses an
“oxidative/electrophilic interface”, which consists of redox-active cysteine residues (Cys).
Upon oxidative stress, it may form disulfide bonds with nearby cysteines (-S-S-) and thereby
change the protein’s structure and function (48). Therefore, under normal conditions, it
anchors its molecular partner nuclear factor E2-related factor 2 (Nrf2) in the cytoplasm where
Nrf2 eventually becomes ubiquitinated and subject to degradation in the proteasome. But,
upon the oxidization of its cysteines (Cys-151, -273, and -288), Keap1’s conformational
change triggers Nrf2’s release and subsequent translocation into the nucleus. Additionally,
modification of Cys-151 followed by PKCδ phosphorylation of Nrf2’s Ser-40 also results in
the escape from Keap1 and import into the nucleus (49). But, not only Keap1, also Nrf2 has
been found to be regulated via redox mechanisms, as it contains at least two redox-sensitive
Cys residues within its nuclear localization signal (NLS) and nuclear export signal (NES)
sequences. Trx-1 was, for instance, shown to promote nuclear export of Nrf2 via this
“oxidative interface” at its Cys506 in the NES region (50). Plus, Nrf2 can be phosphorylated
by Fyn at Tyr568 in the nucleus, which also results in nuclear export, presumably by
promoting its interaction with the chromosome region maintenance 1 (Crm-1; exportin) (51).
Also further mechanisms determining its activation have been discussed in recent reviews
(52-56). Interestingly, it has also been discovered that de novo synthesis outdoes the rate of
Nrf2 translocation into the nucleus in response to low (12.5 μM) H2O2. This could mean that
there are still undiscovered Keap1-independent H2O2-sensors involved in Nrf2 activation (57)
(58).
14
Once translocated into the nucleus, Nrf2 may bind with a small Maf protein (Maf-F, Maf-G,
and Maf-K) and to the antioxidative (aka electrophilic) response element (ARE/EpRE), hence
causing its activation (59). Nrf2 belongs to the family of the cap ‘n’ collar (CNC) b-zip
transcription factors, and contains a cysteine located in the DNA binding domain (Cys-514),
which serves as a conserved site for Redox factor-1 (Ref1) (60). Thus, under oxidative stress
conditions, a portfolio of “phase II detoxification” target genes are transcribed, which promote
antioxidant detoxification, such as e.g. glutathione S-transferase (GST) (61), NADPH
quinone oxidoreductase-1 (NOQ1) (62), heme oxygenase-1 (HO-1) (63-64), ferritin H (FH)
(65-66), and thioredoxin (32; 52; 67-70). Similarly, the oxidation of another b-zip
transcriptional repressor of ARE/EpRE, the human BTB, and CNC homolog 1 (Bach1)
causes Bach’s translocation to the cytoplasm, hence also triggering the activation of
ARE/EpRE (71). Noteworthy is also the Nrf2’s function as a proto-oncogene when
deregulated, for instance once corrupted by the oncogenes K-Ras, B-Raf, and Myc (72).
Figure 6: The Nrf2-pathway. While under a normal oxidation status, Nrf2 is bound and thus inhibited by Keap1,
the cysteine residues of Keap1 can be subjected to an electrophilic attack, which causes conformational changes and releases Nrf2. Hence, Nrf2 is free to translocate into the nucleus and serve as a transcription factor potentially initiating the expression of at least 500 known target genes, including several key proteins of the Trx- and the GSH-system and others such as heme oxygenase-1 (HO-1).
15
Therefore, Nrf2 has been established as a major regulator of mammalian cells to orchestrate
cellular responses to oxidative and electrophilic stress (52-56; 58). The first reference to Nrf2
in the scientific community appeared in 1994, with around 5 500 subsequently published
papers pronouncing its significance (73-74).
In terms of pathway-inducers, as described by Talalay at el. (75), Nrf2-inducing substances
belong to the subsequently listed chemical classes and quinones: (i) oxidizable diphenols,
phenylenediamines and quinones; (ii) Michael acceptors; (iii) isothiocyanates; (iv)
thiocarbamates; (v) trivalent arsenicals; (vi) dithiocyanates; (vii) hydroperoxides; (viii) vicinal
dimercaptans; (ix) heavy metals; and (x) polynes (4). Nrfs repressors, on the other hand, are
not yet so well-characterized (47), but for instance brusatol has been characterized as such
and found to sensitize chemoresistant cells to cisplatin through increasing the ubiquitination
rate and hence degradation of Nrf2 (76).
2.3.1 Heme oxygenase-1 (HO-1) as a Nrf2-target protein
HO-1 is one of the major enzymes readily induced for the purpose of antioxidant
detoxification and defense (63-64). Its main postulated function is heme degradation, thereby
releasing iron, carbon monoxide, and biliverdin. It responds to various noxious stimuli or
conditions including hyperoxia, hypoxia, pro-inflammatory cytokines, nitric oxide, heavy
metals, UV irradiation, heat-shock, shear-stress, H2O2, thiol-reactive substances, amongst
others (77). Nrf2 is considered to play the most significant role in its endogenously rooted
transcriptional activation, thereby promoting its cytoprotective function (78). Thus, this protein
has been shown to have an essential role in cellular and tissue defenses against oxidative
stress and inflammation, as its overexpression can inhibit pathological developments
including vascular proliferation and chronic transplant rejection (79). More specifically, Nrf2
16
has been proven critical in the anti-inflammatory effects of interleukin-10 and 15-deoxy-delta
12, 14-prostaglandin J2 (80). This is why, in order to strengthen the cellular responsiveness,
HO-1 is considered a critical target gene of Nrf2. Its induction is therefore of biomedical
interest.
In a recent study on Nrf2 target proteins, it has been shown that electrophilic compounds
typically modulate both - TrxR-1 and Nrf2 (41). TrxR-1 is expressed at low submicromolar
concentration in cells, but makes a suitable and easy target at its Sec residues and shows a
rather unique reactivity. As Nrf2 acts as a transcription factor for the key molecules of the Trx
system and TrxR-1 serves a function in Nrf2 activation, the Trx system shall now be
discussed in more detail.
2.4 The thioredoxin system
The thioredoxin system composes a key regulatory system to defend oxidative stress,
similarly to the GSH-dependent enzymes (81-82). It consists of thioredoxin (Trx) existing in
different forms: Trx-1, the main form, present in the cytoplasm (83); Trx2 in the mitochondria
(84); SpTrx mainly expressed in spermatozoa (85); as well as of the enzyme thioredoxin
reductase (TrxR), the main enzyme propelling the whole Trx system, which reduces Trx or
related proteins when oxidized at the expense of NADPH.1 In mammalian organisms, TrxR
also exists in three forms: TrxR-1 in the cytosol, TrxR2 in the mitochondria, and TGR in testis
(32; 86). Thioredoxins are 12 kDa small reductases, with a conserved -CGPC- active site
motif.
1 Unless otherwise indicated, Trx in this thesis refers to Trx-1, the best studied and most ubiquitous
form. The same holds true for TrxR referring to TrxR-1, unless stated otherwise.
17
Figure 7: The thioredoxin system. It consists of isoenzymes of thioredoxin reductase (TrxR) that use NADPH
as electron donor to reduce their main substrates, isoforms of thioredoxin (Trx), and related proteins. It sustains various pathways by providing redox enzymes either with electrons or via protein-protein interactions.
The thioredoxin system comprises a key antioxidant system and as such plays a crucial role
in cell survival. This has been shown by thioredoxin knockout mice, which are embryonically
lethal (87). More specifically, it sustains cell proliferation and viability (88-89), as well as
protein folding and signal transduction (82; 90-91). Its main action is ROS-scavenging,
directly quenching singlet oxygen and hydroxyl radicals and it regulates H2O2 homeostasis
via peroxiredoxins (Prx), also called thioredoxin peroxidases, which utilize thioredoxin as an
immediate electron donor (92-93). The different Prx isoforms (Prx1-6) occurring in diverse
cellular compartments have shown different substrate specificities and reaction mechanisms,
but are all highly reactive with peroxides (recently reviewed in (94-95)). In this context, it
should be mentioned that the Prxs gain awareness as mediators or oxidation states as
means of redox signaling (96-97), as they can for instance over-oxidize Trx in the absence or
inhibition of TrxR-1 (76). Moreover, the Trx system acts indirectly by reducing oxidized
cysteine residues within proteins to regulate their activity (86). For instance, it functions via
the reduction of protein tyrosine phosphatases (PTPs) (98-99).
Thioredoxin as well as thioredoxin reductase are known target genes of the transcription
factor Nrf2, and are up-regulated by Nrf2 under conditions of oxidative stress to re-balance
the cell’s intracellular redox environment. On the downside, elevated levels of Nrf2 have also
been found to be present in many types of cancers (72; 100). Over-expression has been
18
linked to cancer cell growth, metastasis, and resistance to various chemotherapeutic agents.
Therefore, it has become a favored candidate for anticancer therapy (101-102). Regarding
the cell’s extracellular environment, it has to be mentioned, that thioredoxin is also secreted
and then shows chemokine properties (103). Thus, thioredoxin is a key player in many
cellular strategies that involve thiol-redox states and orchestrates the removal of reactive
oxygen and nitrogen species at a high turn-over rate (82). Only recently, interest in TrxR has
increased, when it was identified as a potent regulator of the Nrf2-Keap1 response system,
as the selenoprotein TrxR-1 acts together with Keap1 in sensing cellular stresses and
modulating adequate Nrf2-responses (41). Animal studies and cell culture experiments have
shown that there is a direct causal relationship between TrxR-1 inhibition or deletion and
profound Nrf2 activation.
2.5 Antioxidants
The first hype started with an article by Tappel and Zalkin published in Nature 1960 (104),
describing the protective effect of antioxidants like glutathione and vitamin E. The human
antioxidant defense network is complex and reflects human evolution. Generally, the network
can be classified 1) according to the mode of action: into enzymatic antioxidants and non-
enzymatic oxidants; 2) based on the source: into exogenous and endogenous; and 3)
depending on solubility: into hydrophobic and hydrophilic. Originally, Halliwell and Gutteridge
(1995), characterized antioxidants as “any substance that, when present at a low
concentration compared with that of an oxidized substrate, significantly delays or inhibits
oxidation of that substrate” (105). Later on, they defined them as “any substance that delays,
prevents or removes oxidative damage to a target molecule” (106). Khlebnikov at al. (2007)
described antioxidants as “any substance that directly scavenges ROS or indirectly acts to
up-regulate antioxidant defenses or inhibit ROS production” (107). Hence, the term
“antioxidants” implies either that a compound A) quenches radicals directly or B) augments
the endogenous antioxidant capacity by up-regulating the expression of cytoprotective,
detoxifying, and antioxidant genes. The latter is done via Nrf2 and the regulation of phase II
19
detoxifying enzymes, as will be elaborated in more detail, since so far this mechanism has
often been ignored when discussing antioxidants.
Figure 8: The bidirectional – A) direct and B) indirect - function of antioxidants. The diagram presents the
inert meaning of an antioxidant, which can either quench radicals directly, or augments the endogenous antioxidant capacity by up-regulating the expression of cytoprotective, detoxifying, and antioxidant genes via Nrf2 and the regulation of phase II detoxifying enzymes.
Antioxidant action can occur as particularized subsequently:
- as preventive oxidants, by inhibiting free radical oxidation reactions;
- as chain breakers, by interrupting the diffusion of the autoxidation chain reaction;
- as singlet oxygen quenchers; by synergizing with other antioxidants (e.g. vitamin E
and polyphenols);
- as reducing agents;
- as metal chelators by converting metal pro-oxidants into stable products (mostly iron
and copper derivatives);
- as inhibitors of pro-oxidative enzymes (108-110).
20
In the course of action numerous new radicals can occur. Prominent in vivo examples
include the urate radical (UrH●-), the ascorbyl radical (Asc●-), the vitamin E radical (VE●), and
phenoxyl radicals (Phl●) (111).
Thus, while the dominant share of the “total antioxidant capacity” of human cells and tissues
is due to endogenously-synthesized antioxidant molecules such as reduced glutathione
(GSH), peroxiredoxins, and superoxide dismutase (Table 2), the role of diet-derived
antioxidants has not yet been fully explored. Clearly, one significant effect is that some are
capable of activating transcription of endogenously-synthesized antioxidant molecules of
phase II detoxification, by inducing Nrf2. Hence, antioxidant induction of Nrf2/ARE-mediated
cytoprotective gene expression serves as a very important mechanism of antioxidant
protection (112). In fact, the transcriptional gene regulation of dietary bioactives may indeed
be more important in vivo than their ascribed antioxidant capacity (31).
2.6 Dietary phytochemicals
Scientists estimate that there are more than 5 000 different phytochemicals in our food (113).
They comprise phenolic antioxidants, vitamins, and other naturally occurring phytochemicals
sharing one characteristic: they eliminate the excess of oxygen metabolites and thereby
counteract chronic diseases, as found in several epidemiological investigations. Thus, they
play a key role in the delicate equilibrium between oxidation and antioxidation in biological
systems (114). Some are promising chemopreventive agents and known to protect against
neurogenerative, cardiovascular, and renal diseases (115). They either function via direct
interactions with carcinogens and/or coordination of phase I and/or phase II enzymes.
Generally, phytochemicals are defined as bioactive non-nutrient plant compounds consumed
by dietary intake of fruits and vegetables that have been linked to risk-reduction concerning
major chronic diseases (116). They do so by protecting cellular systems from oxidative stress
and thus from damage (117-119). However, the literature reflects a divergence, when it
comes to in vitro versus in vivo effects. While all in vitro models have their limits,
21
epidemiological studies of the past were critically investigated and a number of inherent
errors were identified (113). Definitely the most prominent amongst them is the
administration of purified compounds as supplements in high doses. Studies have shown
that isolated compounds show a different behavior (e.g. loss of bioactivity or bioavailability) in
the human body, compared to their occurrence in a complex mixture as present in naturally
occurring whole foods (120-122). Therefore, it has to be highlighted that the health-promoting
effect has only clearly been attributed to these naturally occurring mixtures (113; 123-124).
The putative beneficial pharmacological effects, as well as potential toxicity of any dietary-
phytochemical-rich foods or herbal extracts are dependent on their bioavailability subsequent
to oral intake. The issue of bioavailability and bioaccessibility has been hovering over this
field of research and has become the ammunition of many critics. Since an in vivo detection
of liberation, absorption, distribution, metabolism, and excretion (LADME) in human beings is
tricky and cost intensive, many in vitro assays have been developed, non without limitation,
but all with advantages and disadvantages. For instance, Bouayed, Hoffmann and Bohn
(2011) have developed an in vitro simulation of gastro-intestinal digestion and found that
polyphenol release was mainly achieved during the gastric phase (approx. 65% of phenolics
and flavonoids) (125). But, regardless of the up-take, in vivo bioactivity may already have
started in the gastrointestinal tract (GIT).
Thus, for decades, scientists have tried to track down the health-modulating effects that
antioxidants might have on the human body. As a result, most agree that antioxidants are
beneficial and play a significant role in the human homeostasis. As a consensus, low doses
of antioxidants may be favorable, while high doses might even disrupt this delicate balance.
As stated by Devasagayam et al. (2004), the fact that in vitro effects have failed to be shown
on an in vivo level, this should not discourage, but rather stimulate further research (126).
While most investigations discuss the controversies of exogenous antioxidants in terms of
both established and non-established health effects when it comes to type, dosage, and
matrix, recently the discovery of their potential pro-oxidative nature has caused an uproar.
22
The pro-oxidative characteristics have been shown to occur under certain conditions, such
as high doses or the presence of metal ions (127). Quercetin, epigallocatechin-3-gallate, and
gallic acid are amongst the known pro-oxidants at high doses. For instance, quercetin above
50 µM can potentiate superoxide radical (O2●-) in isolated mitochondria and cultured cells
(128). Moreover, it was found that green tea produces H2O2 in the mouth cavity (129). In fact,
the antioxidant function of green tea is due to its capacity to induce ROS-formation, so that
endogenous antioxidant systems are activated. This is supported by the finding that at least
40 genes can be activated by hydrogen peroxide in mammalian cells (130). As pro-oxidants
they can act as messengers to trigger transcription regulators such as the nuclear factor
kappa B (NF-ĸB), a key regulator in inflammation (131), or Nrf2 (112). Also curcumin (CUR)
has been shown to efficiently kill tumour cells, while leaving normal cells largely unaffected
(132-133). Thus, these dietary agents are becoming highly interesting candidates for
“oxidative therapy”, especially when they act selectively, either as anti- or pro-oxidants,
depending on their cellular target. Moreover, there is hope that when co-administered they
reduce drug resistance and prevent some of the deleterious effects of the anticancer therapy
on normal cells (134) (chapter: ROS & modulation of carcinogenesis).
2.6.1 Selection & rational behind tested substances
For the purpose of this project, a smart selection of a few dietary phytochemicals based on a
scientific rational had to be made. Firstly, the selection was based on the most current
literature and the reported promising candidates, since it was not our intention to discover
new substances but rather validate our model and do our analyses with established
bioactives. Secondly, a scan of their physico-chemical properties revealed e.g. whether we
would have to exclude them due to pan assay interference (PAIN). It describes compounds
(PAINS) that show functionalities across a range of assay platforms and against a range of
proteins. For instance, some source metal chelation, chemical aggregation, or have an inert
fluorescence. They are therefore completely unsuitable for assays assessing this quality.
23
Also, having elaborated on the LADME-principle and bioavailability, the compounds chosen
are all conform with Lipinski’s rule of five. Compounds matching Lipinski’s criteria are likely
membrane permeable and readily absorbed by the body. The benchmarks predict drug-
likeness and specify that compounds should have:
- a weight of less than 500 g/mol;
- a logP (the logarithm of the partition coefficient between water and 1-octanol) of less
than 5 (lipophilicity);
- less than five groups in the molecule that can donate hydrogen atoms; and
- less than ten groups that can accept hydrogen atoms (135).
Only epigallocatechn-3-gallate exceeds the molecular weight a little, but can still be
considered bioavailable as has been shown in in vivo studies (mentioned below). Thirdly, the
selected substances on purpose stem from different groups. Hence, sulforaphane (SFN) was
chosen as a representative of organosulfur compounds. From the large group of phenolics,
quercetin (QUE) was picked to represent flavonols, and epigallocatechin-3-gallate (EGCG)
selected as one of the flavanols (catechins).
In general, dietary phytochemicals may be classified into carotenoids, phenolics, alkaloids,
nitrogen-containing compounds, and organosulfur compounds. They commonly determine
color, flavor, and aroma of many vegetables and fruits, either repelling predators or attracting
pollinators of those plants.
24
Figure 9: Classification of dietary phytochemicals. (Modified from (124))
2.6.1.1 Sulforaphane
Figure 10: Sulforaphane (SFN) - C6H11NOS2, which occurs in e.g. broccoli. A) Picture of broccoli [Work by PDPics (136)]; B) Skeletal chemical structure of SFN [Work by Klaus Hoffmeier (137)]; C) Ball-and-stick model of the SFN molecule [Work by Ben Mills (138)]; all pictures from public domains.
From the group of organosulfur compounds, sulforaphane is of particular interest, as its Nrf2-
activating potential evokes its ascribed chemopreventive capacity. It is classified within the
isothiocyanate group of organosulfurs and obtained naturally from cruciferous vegetables
25
such as broccoli, Brussels sprouts or cabbages. The thioglucoside glucoraphanin is a
predominant glucosinolate within these vegetables and upon hydrolization (catalyzed by
myrosinase) gets converted into sulforaphane. While usually myrosinase is stored in a
different compartment than glucoraphanine, it becomes a part of a plant’s defense response.
When the plant is wounded, it gets released to activate cytoprotective mechanisms (i.e.
sulforaphane then activates Nrf2). The metabolic fate of glucosinolates has been
investigated and dithiocarbamates have been determined quantitatively in human plasma,
serum, erythrocytes, and urine. Data showed, for instance, that single doses of 200 µmol of
isothiocynates were absorbed rapidly and reached peak concentrations of 0.943-2-27 µmol/l
already 1 hr after consumption (139). The same group (2002) was able to calculate a half-life
of 1.77 +/- 0.13 hrs. Up to now, a number of clinical trials have examined the bioavailability of
sulforaphane and have found the sulforaphane-N-acetyl cysteine (SF-NAC) to be an
appropriate marker detectable in urine (140). Moreover, it can be measured indirectly by
detecting 1,3-benzodithiole-2-thione (detectable at 365 nm), a product of its stoichiochemical
reaction with 1,2-benzenedithiol (i.e. cyclocondensation reaction) (141). Myrosinase, the
crucial factor in sulforaphane availability, is known to be heat-inactivated. Therefore, any
cooking process involving higher temperature diminishes the chance of sulforaphane up-
take, and the hydrolization of glucoraphanine hence relies on the gut’s microflora. One
solution for this problem suggested by Cramer and Jeffery (2011) would be the additional
consumption of myrosinase-rich sources, which results in early and more complete
absorption (140).
Polyphenols are biosynthesized via the shikimic acid pathway as well as the polyacetates
pathway and cover a very heterogeneous group of secondary metabolites. Polyphenolic
compounds compose the major share of secondary plant metabolites and also of dietary
antioxidants (125). They are most abundant in fruits and vegetables and occur in
concentrations of up to several 100 mg per 100 g (114). Defined chemically, they possess
26
one or more aromatic rings with one or more hydroxyl groups and may be categorized into
phenolic acids, flavonoids, stilbenes, coumarins, and tannins. In food science, they have
often been suggested as essential parts of functional foods, due to their health-promoting
reputation.
From the group of phenolics, flavonoids account for approximately two thirds, and more
than 5 000 have been distinguished so far (142-145). Structurally defined, they consist of two
aromatic benzene rings (A and B rings) linked by 3 carbons that are usually in an oxygenated
heterocyclic pyran or pyrone ring (ring C), which makes differentiation possible between
flavonols (e.g. quercetin), flavones (e.g. luteolin), flavanols (e.g. epigallocatechin-3-gallate),
flavanones (e.g. naringenin), anthocyanidins, and isoflavonoids (e.g. genistein). They occur
in nature as conjugates in glycosylated or esterified form, and, to a smaller extend, also as
aglycones.
Various activities are ascribed to them, such as cytoprotective, antibacterial, antiviral, anti-
aging, anti-inflammatory, antiallergenic, antimutagenic, vasodilatory, anxiolytic,
antidepressant, and cognitive enhancing effects (146-147).
27
2.6.1.2 Quercetin
Figure 11: Quercetin (QUE) - C15H10O7, which occurs in e.g. onion. A) Picture of onions [Work by Costanzimarco (148)]; B) Skeletal chemical structure of QUE [Work by Yikratuul (149)]; C) Ball-and-stick model of the QUE molecule [Work by Jynto (150)]; all pictures from public domains.
Quercetin is a naturally-occurring dietary flavonol, highly abundant in onions (284-
486 mg/kg), apples (15 mg/kg), tea infusions (10-25 mg/l), red wine (4-16 mg/l), as well as
other fruits and vegetables (151-152).
Since most flavonoids enter the body as hydrophilic glycosides, and quercetin has a
relatively high molecular weight, its absorption in the small intestine was once thought to be
precluded. Moreover, it was believed that intestinal hydrolases do not affect quercetin and
hence, its uptake was originally found to take place in the large intestine, supported by the
glycosidases from the microflora there, which releases the aglycone from its sugar (153).
More recently a study with human ileostomy volunteers proved that quercetin glycosides can
be taken up in the small intestine and even that this absorption of 52% outperforms the one
of the aglycone which lies at only 24% (154). Its absorption has been further explained by
either immediate deglycosylation (155) or carrier-mediated transport (156). Normal quercetin
plasma concentrations are in the nanomolar range, but upon supplementation can increase
to the high nanomolar or even low micromolar range (157).
28
Several biological and pharmacological functions have been ascribed to quercetin.
Regarding its health-modulating properties in humans, Knekt et al. (2002) showed that
people with higher intake have a lowered mortality risk when suffering from a myocardial
infarction (158). It has been shown to act antivirally, by inhibiting the replication of viruses, as
well by deterring the ability of DNA and RNA polymerases and reverse transcriptases. Also, it
enhances the function of interferon and tumor necrosis factor, suggesting carcinostatic
activity. Moreover, its effect on platelet aggregation, LDL oxidation, and vasodilatation attests
to interrupt the pathophysiology of atherosclerotic plaque formation (159).
In vitro, oxidative degradation of quercetin may occur and lead to the formation of an
intermediate free radical ortho-semiquinone, which can ultimately be converted to an ortho-
quinone. Hence, ROS, such as superoxide and hydrogen peroxide, are produced which
qualifies quercetin as a pro-oxidative substance at high-dose levels (160).
In human plasma, in vivo, quercetin metabolites have been identified in the plasma about
1.5 hrs after the consumption of foods rich in flavonoid glycosides (e.g. onion), such as
quercetin-3-glucuronide, 3′-methyl-quercetin-3-glucuronide, and quercetin-3′-sulfate, with
substitutions in the B and/or C ring respectively (161). These compounds are assumed to
possess differing biological activity profiles. While following a single quercetin-rich meal the
plasma levels have shown to be rather low, the consumption of 114 mg quercetin from
onions on 7 consecutive days led to plasma levels of 453 ng/ml (162). And, since the half-
lives of quercetin metabolites are rather high – i.e. 11 to 28 hrs, repeated supplementation
can result in considerable plasma levels (157). Moreover, it has been established that the
food matrix containing quercetin plays a significant role (163). Although toxic effects of
quercetin have been shown in vitro, most recent reviews have concluded that orally
administered quercetin is unlikely to cause any adverse effects (164).
29
2.6.1.3 Epigallocatechin-3-gallate
Figure 12: Epigallocatechin-3-gallate (EGCG) - C22H18O11, which occurs in e.g. green tea. A) Picture of green tea [Work by Peggy_Marco (165)]; B) Skeletal chemical structure of EGCG [Work by Su-No-G (166)]; C) Space-filling model of the EGCG molecule [Work by Jynto (167)]; all pictures from public domains.
Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea (Camellia
sinensis). Green and oolong teas on average contain 30 to 130 mg per cup (237 ml),
whereas black teas only hold 0 to 70 mg (168). The dietary phytochemical is comprised of
the ester of epigallocatechin and gallic acid. Thus, it has a trihydroxyl group at carbons 3’, 4’,
and 5’ on the B ring and a gallate moiety esterified at carbon 3 on the C ring.
Catechin levels measured in human plasma peaked 2 to 4 hrs post consumption (169). The
highest concentration of individual catechins in the human body measured was slightly
higher than 1 µM after a single dose. The average peak for EGCG was found to be 1.3 µmol/l
after administration of 1.5 mmol, and after 24 hrs it had returned to baseline (170). Another
study administering a single dose of 1.75 mmol (800 mg) of EGCG solely found an average
of 0.96 μmol/l, compared to 0.82 μmol/l after a single dose of a green tea catechin mixture
containing the same amount of EGCG (171). Another source states that in humans, the
maximal pharmacological concentration is typically ≈1 µmol/l (172). Thus, it appears that
EGCG may be less bioavailable to the human body, but the specificities of its
30
pharmacokinetics require further investigation. Additionally, catechin esterase activity has
been attributed to saliva, indicating that EGCG could be degalloylated in the mouth and
esophagus. Also, catechins which are not absorbed in the small intestine reach the large
intestine and are presumed to be metabolized by colonic bacteria there. Conjugated
catechins are also excreted in the bile, and hence can be absorbed thereafter (173).
Figure 13: Theoretical biotransformation pathways for epigallocatechin-3-gallate (EGCG) and its metabolites. (Adapted from Yang et al. (173)) Abbreviations: COMT: Catechol-O-methyl transferase, PAP:
Adenosine 3’,5’-bisphosphate, PAPS: 3’-Phosphoadenosine 5’-phosphosulfate, SAH: A-Adenosyl-L-homocysteine, SAM: S-Adenosyl-L-methionine, SULT: Sulfotransferase, UDP: Uridine diphosphate, UDP-GA: UDP-glucuronic acid, UGT: UDP-glucuronosyl transferase.
Regarding pharmacological properties attributed to EGCG, beneficial as well as harmful
effects have been defined. A decisive factor regarding health-modulation has been shown to
be the concentration, whether EGCG is administered in physiological or pharmacological
(non-nutritional) doses, as well as the cellular environment. Depending on its cellular target, it
can act either as anti- or prooxidant, as has been explained above. This is a specialty of very
few polyphenols. EGCG is also one of the few substances for which even pharmacological
31
effects on mental health have been discovered such as e.g. anti-anxiety and antidepressant
activities (125). Notwithstanding, the main focus has been to show that it exerts preventive
effects against chronic diseases such as CVDs, particularly atherosclerosis and coronary
heart disease, partially due to its potential to scavenge free radicals (174). Moreover, it has
been found to inhibit carcinogenesis in animal models of the skin, mammary glands, liver,
esophagus, colon, stomach, lung, small intestine, prostate, and bladder (175-173). Moreover,
Ellinger et al. (2011) have composed a comprehensive review of controlled interventional
studies on the consumption of green tea (176). They concluded that beneficial effects, such
as e.g. reduced lipid/protein peroxidation, and the oxidation of LDL in particular, or the
protection against DNA damage, are more likely to occur in individuals with increased
oxidative stress (due to smoking, benzene exposure or exhaustive exercise) when compared
to “healthy” individuals.
On a molecular level, green tea polyphenol extracts have been shown to significantly
increase ARE-mediated reporter gene activity in transiently transfected HepG2 cells in
correlation with the activation of the MAPK pathway (177), and also in stably transfected
HepG2 cells (24), at a concentration of 25 µM. More specifically, EGCG showed potent
activation of all three MAPKs (ERK, JNK, and p38) at doses of 25 to 50 µM.
Finally, phenolic acids, which can be subdivided into hydrobenzoic acids (e.g. gallic acid,
GAL) and hydroxycinnamic acids (e.g. p-coumaric acid), are important representatives of
phenolics and usually occur in bound form.
2.7 Hormetic concept
In toxicology studies, the dogma that still governs is what was already recognized by
Paracelsus in 1538: “poison is in everything, and no thing is without poison; the dosage
makes it either a poison or a remedy.” Herewith he declared that the effect of any toxic
32
chemicals depends on the dosage (178). The fact that the dose can create a much more
sophisticated effect was voiced more than a century ago by Hugo Schulz. The Arndt-Schulz
law expresses that low, intermediate, and high doses of the same substance can have
different effects in a biological system, and, hence, at low dose a compound can have
beneficial and stimulatory effects, while acting lethal at a high dose (179). This biphasic dose
response is nowadays understood as hormesis. In biology and medicine, according to
literature, the hormetic effect is considered an adaptive response of single cells and an
organism to an intermediary environmental stressor (180). Established examples comprise
ischemic preconditioning, exercise, dietary energy restriction, and exposures to low doses of
certain phytochemicals. The involved cellular signaling pathways and molecular mechanisms
of such hormetic responses have to a certain extent been illuminated. Besides enzymes
such as kinases and deacetylases, transcription factors such as Nrf2 and NF-κB have also
been suggested. Nrf2 turns into a key example, since upon its activation the cellular
machinery of cytoprotection is started. The knowledge of this trigger has become a powerful
strategy for the prevention and treatment of various pathologies. Its value can be understood
by the rise of about 10 citations per year in the 1980s to nearly 6 000 in 2013 in the
biomedical community, as well as its inclusion in textbooks of pharmacology and toxicology
(181).
2.8 Synergies
One prominent hypothesis speculating on the reason for the health benefit of fruit and
vegetables places synergies and interactions of bioactive compounds at the heart of the
discussion. Hence, recent interest has focused not on single compounds, but rather on the
whole fruit and vegetable, following up on additive or synergistic actions of complex mixtures
of phytochemicals and nutrients (31; 116).
Research has shown that bioactive substances play a concerted role in influencing health.
Therefore, unless a clear deficiency can be named the root of a certain disease, the attention
33
on single nutrients cannot significantly advance public health dietary advice. This is
particularly true when talking about actual food, which is always composed of a non-random
assortment of molecules, and has proven significant for the respective organism in the
course of evolution. Having highlighted the multifarious mechanisms of action of antioxidants,
one can imagine how broad their effect would be when taken up in combination. Of course
the question arises which combinations might be especially effective. Consequently, for food-
based studies, it might seem short-sighted to assess single nutrients and draw conclusions,
which might lead to an oversimplification of a complex metabolic scenario. However,
investigating the physiological and biochemical functions of single bioactives provides a
useful starting point for further assessment. The aspiration to gain deeper insights into the
relationship between food, health, and disease fuels further research.
In a healthy human body, ROS-levels are hence controlled by a series of antioxidant defense
mechanisms either by dietary derived or endogenously synthesized compounds. Thereby,
they are metabolized and reduced to allow useful functions, but not completely eradicated.
Hence, high doses of exogenous antioxidants may disrupt this delicate balance and a
healthy biological system (182). In this regard, it has been shown that a typical vegetarian
diet contains 20 times less quercetin than a single dose of the average supplement sold on
the market (183).
Accordingly, the dose really becomes the key code to well programmed modes of action, and
one way to exert balance control is via intracellular signaling pathways, which in turn are
audited by feedback loops.
34
2.9 Research aims
Dietary phytochemicals in their function as “antioxidants” in recent years have become
known for their health-modulating capability. In fact however, they have been discussed to be
health-promoting, as well as health-compromising in this regard. Particularly in the evolution
of cancer, the multiples roles of ROS and thus the antioxidants’ counteractivity have yet to be
fully explored. An aspect almost completely neglected poses the synergistic effects of co-
administered substances – from food as a matrix, or as dietary supplements, which must
hence be elucidated further. Clearly, antioxidants in general have been shown to play a
major role in the redox homeostasis within cells. Since an imbalance in the redox status
promotes not just cancer, but various pathologies, knowledge about this balancing act has to
be increased. Such an imbalance has been shown to often directly correlate with derailed
Nrf2-homeostasis (17; 184).
Therefore, the aim of this study was to investigate how extracellular dietary
phytochemicals bidirectionally impact the signaling along the Nrf2 pathway and the
expression of its target genes, which comprise the endogenous thioredoxin system as
well as heme oxygenase-1, in a human hepatocellular carcinoma model.
Consequently, first, the antioxidant – or, for that matter, also prooxidant – effect of
selected dietary phytochemicals was systematically addressed in an optimized
cellular model. Then, the control over the Nrf2 master redox switch was elucidated on,
before assessing the influence of the dietary phytochemicals on the Nrf2 pathway and
the chosen target genes.
35
Figure 14: Graphical abstract of Workflow/Milestones, stating the research aims.
36
3 RESULTS
In assessing the mode by which dietary antioxidants control the redox balancing strategies of
a cell, and how they interact with the endogenous antioxidant systems in place, the study has
yielded the following results.2
3.1 General assessment
When working with extractable phytochemicals, it is of utmost importance to guarantee a
stable matrix and control the pH-value as well as temperature. Thus, the study protocol
included multiple pH-value measurements before any other analysis to assure consistency,
while the state-of-the-art laboratory environment provided for constant temperature and air
pressure.
Table 3: pH-Value assessment for single substances.
pH-Value assessment I
Stock [µl] Medium [µl]
RESULT pH [-log10(H3O+)]
Medium x 3 000 7.91
Sulforaphane (SFN) 60 2 940 8.05
Quercetin (QUE) 120 2 880 7.95
Epigallocatechin-3-gallate (EGCG)
240 2 760 7.94
Curcumin (CUR) 60 2 940 7.98
Cinnamic acid (CIN) 120 2 880 8
Catechin (CAT) 240 2 760 8.2
Gallic acid (GAL) 240 2 760 7.83
The pH-value analysis showed no dilution diverging for more than 0.37 pH for single
substances and for more than 0.33 pH for combinations of substances respectively, applying
the value obtained for the medium as standard. The difference calculated with =MAX(x:x)-
MIN(x:x), where x denote the column of results.
2 Please find the corresponding statistical analysis explained at the end of each chapter, in which
section the controls used are explicitly mentioned as well.
37
Table 4: pH-value assessment for single substances (repeated) and for combinations of substances.
pH-Value assessment II (measured again, plus combinations)
Stock [µl] Medium [µl] RESULT pH [-log10(H3O+)]
Medium x 3 000 8.22
Sulforaphane 60 2 940 8.19
Quercetin 6 2 994 8.23
Epigallocatechin-3-gallate 50 2 950 8.38
Sulforaphane + Epigallocatechin-3-gallate
60 + 50 2 890 8.36
Quercetin + Epigallocatechin-3-gallate
6 + 50 2 944 8.22
Sulforaphane + Epigallocatechin-3-gallate + Quercetin
60 + 50 + 6 2 884 8.52
Moreover, visual control on a regular basis is essential. Therefore, cells were visually verified
twice a week, which was particularly crucial for the experiments involving confocal
microscopy to assure proper growth before treatment.
Figure 15: HepG2 (human hepatocellular carcinoma) cells visualized under the confocal microscope. A)
transmitted light image, B) nuclei staining with 100 nM Syto16 (green channel, excitation at 488nm⁄ emission at 518 for DNA) incubated at 37°C after 20 minutes. Digital images were taken using an Olympus IX-70 inverted microscope.
38
The event of growth and proliferation frequently became evident at the moment of
investigation under the microscope, and with the staining of the nuclei could be observed
nicely as shown in Figure 16.
Figure 16: HepG2 (human hepatocellular carcinoma) cells undergoing cell division visualized under the confocal microscope. C) nuclei staining with 100 nM Syto16 (green channel, excitation at 488nm⁄ emission at
518 for DNA) incubated at 37°C after 20 minutes, D) nuclei staining, as in C), and staining of glycoproteins with 100 nM WGA AF647 incubated at 37°C after 20 minutes, (both dyes from Thermo Fisher Scientific GmbH, Germany).
3.2 Cell proliferation & viability
To determine the effect of the selected dietary phytochemicals on HepG2’s cell viability and
to define optimal treatment conditions for further cell culture experiments, HepG2 cells were
treated with increasing concentrations of the bioactives solved in A.d. (e.g. EGCG) EtOH
(e.g. CUR) or DMSO (e.g. SFN, QUE, CIN, CAT & GAL). Cell proliferation and viability was
thus calculated in relation to the respective solvent controls (SFN: 0.5% DMSO; QUE: 1%
DMSO; EGCG: 2% A.d.; CUR: 0.5% EtOH; CIN: 1% DMSO), which were also tested during
each test run.
39
3.2.1 IC50 calculation based on metabolic activity of HepG2 of single compounds
Sulforaphane, as shown in Figure 17, deviated the number of viable HepG2 cells dose-
dependently over time, with an IC50 of 76.36 µM 24 hrs post treatment declining to 52.91 µM
after 72 hrs.
IC 5 0 S u lfo ra p h a n e 2 4 h r s
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 76.36
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 S u lfo ra p h a n e 7 2 h r s
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 52.91
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 17: Effect of sulforaphane on cell viability. IC50 calculation based on the metabolic activity of HepG2
cells upon treatment (24-72 hrs) with increasing concentrations [5-100 µM] of sulforaphane solved in DMSO. n=5, calculated with GraphPad Prism.
Quercetin, as shown in Figure 18, revealed a reduction in the number of viable HepG2 cells
dose-dependently over time, with an IC50 of 354.3 µM 24 hrs post treatment declining to an
IC50 of 132.6 µM after 72 hrs.
IC 5 0 Q u e r c e t in 2 4 h r s
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0IC50 354.3
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 Q u e r c e t in 7 2 h r s
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 132.6
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 18: Effect of quercetin on cell viability. IC50 calculation based on the metabolic activity of HepG2 cells
upon treatment (24-72 hrs) with increasing concentrations [10-200 µM] of quercetin solved in DMSO. n=5, calculated with GraphPad Prism.
40
Epigallocatechin-3-gallate, as shown in Figure 19, detectably decreased the number of
viable HepG2 cells dose-dependently over time, with an IC50 of 180.5 µM 24 hrs post
treatment declining to 173.7 µM after 72 hrs.
IC 5 0 E p ig a llo c a te c h in -3 -g a lla te 2 4 h rs
1 .0 1 .5 2 .0 2 .5 3 .0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 180.5
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 E p ig a llo c a te c h in -3 -g a lla te 7 2 h rs
1 .0 1 .5 2 .0 2 .5 3 .0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 173.7
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 19: Effect of epigallocatechin-3-gallate on cell viability. IC50 calculation based on the metabolic activity
of HepG2 cells upon treatment (24-72 hrs) with increasing concentrations [20-400 µM] of epigallocatechin-3-gallate solved in A.d. n=4, calculated with GraphPad Prism.
Curcumin, as shown in Figure 20, led to a decrease in the number of viable HepG2 cells
dose-dependently over time, with an IC50 of 113.1 µM 24 hrs post treatment declining to
68.74 µM after 72 hrs.
IC 5 0 C u rc u m in 2 4 h r s
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 113.1
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 C u rc u m in 7 2 h r s
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 68.74
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 20: Effect of curcumin on cell viability. IC50 calculation based on the metabolic activity of HepG2 cells
upon treatment (24-72 hrs) with increasing concentrations [5-100 µM] of curcumin solved in EtOH. n=4, calculated with GraphPad Prism.
41
Cinnamic acid, as shown in Figure 21, did not decrease the number of viable HepG2 cells
dose-dependently over time. Thus, the IC50 could not be defined. Due to the recommended
maximum of solvent being reached, no higher concentrations were tested.
IC 5 0 C in n a m ic a c id 2 4 h r s
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 C in n a m ic a c id 7 2 h r s
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 21: Effect of cinnamic acid on cell viability. IC50 calculation based on the metabolic activity of HepG2
cells upon treatment (24-72 hrs) with increasing concentrations [10-200 µM] of cinnamic acid solved in DMSO. n=4, calculated with GraphPad Prism.
Gallic acid, as shown in Figure 22, decreased the number of viable HepG2 cells dose-
dependently over time, with an IC50 of 248.5 µM 24 hrs post treatment declining to 242.0 µM
after 72 hrs.
IC 5 0 G a llic a c id 2 4 h rs
1 .0 1 .5 2 .0 2 .5 3 .0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 248.5
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 G a llic a c id 7 2 h rs
1 .0 1 .5 2 .0 2 .5 3 .0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 242.0
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 22: Effect of gallic acid on cell viability. IC50 calculation based on the metabolic activity of HepG2 cells
upon treatment (24-72 hrs) with increasing concentrations [20-400 µM] of gallic acid solved in DMSO. n=3, calculated with GraphPad Prism.
42
3.2.2 IC50 calculation based on metabolic activity of HepG2 of multiple compounds
SFN & EGCG applied in combination, as shown in Figure 23, resulted in a reduction of the
number of viable HepG2 cells dose-dependently over time, with an IC50 of 52.54 µM 24 hrs
post treatment declining to 33.79 µM after 72 hrs.
IC 5 0 S F N + 5 0 µ M E G C G 2 4 h rs
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 52.54
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 S F N + 5 0 µ M E G C G 7 2 h rs
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 33.79
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 23: Effect of sulforaphane and epigallocatechin-3-gallate combined on cell viability. IC50 calculation
based on the metabolic activity of HepG2 cells upon treatment (24-72 hrs) with 50 µM of epigallocatechin-3-gallate solved in A.d. and increasing concentrations [5-100 µM] of sulforaphane solved in DMSO. n=5, calculated with GraphPad Prism.
SFN & QUE in combination, as shown in Figure 24, decreased the number of viable HepG2
cells dose-dependently over time, with an IC50 of 83.02 µM 24 hrs post treatment declining to
44.43 µM after 72 hrs.
IC 5 0 S F N + 1 0 µ M Q U E 2 4 h rs
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 83.02
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 S F N + 1 0 µ M Q U E 7 2 h rs
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 44.43
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 24: Effect of sulforaphane and quercetin combined on cell viability. IC50 calculation based on the
metabolic activity of HepG2 cells upon treatment (24-72 hrs) with 10 µM of Quercetin solved in DMSO and increasing concentrations [5-100 µM] of sulforaphane solved in DMSO. n=3, calculated with GraphPad Prism.
43
SFN, EGCG & QUE in combination I, as shown in Figure 25, decreased the number of
viable HepG2 cells dose-dependently over time, with an IC50 of 65.47 µM 24 hrs post
treatment declining to 28.90 µM after 72 hrs.
IC 5 0 S F N + 5 0 µ M E G C G + 1 0 µ M Q U E 2 4 h rs
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 65.47
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 S F N + 5 0 µ M E G C G + 1 0 µ M Q U E 7 2 h rs
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 28.90
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 25: Effect of sulforaphane, epigallocatechin-3-gallate and quercetin combined on cell viability. IC50
calculation based on the metabolic activity of HepG2 cells upon treatment (24-72 hrs) with 10 µM of quercetin solved in DMSO, with 50 µM of epigallocatechin-3-gallate solved in A.d. and increasing concentrations [5-100 µM] of sulforaphane solved in DMSO. n=4, calculated with GraphPad Prism.
SFN, EGCG & QUE in combination II, as shown in Figure 26, decreased the number of
viable HepG2 cells dose-dependently over time, with an IC50 of 83.02 µM 24 hrs post
treatment declining to 44.43 µM after 72 hrs.
IC 5 0 S F N + 2 0 0 µ M E G C G + 1 0 µ M Q U E 2 4 h rs
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 89.13
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
IC 5 0 S F N + 2 0 0 µ M E G C G + 1 0 µ M Q U E 7 2 h rs
0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50 17.33
L o g [µ M ]
Via
bil
ity
(%
of
co
ntr
ol)
Figure 26: Effect of sulforaphane, epigallocatechin-3-gallate and quercetin combined on cell viability. IC50
calculation based on the metabolic activity of HepG2 cells upon treatment (24-72 hrs) with 10 µM of quercetin solved in DMSO, with 200 µM of epigallocatechin-3-gallate solved in A.d. and increasing concentrations [5-100 µM] of sulforaphane solved in DMSO. n=3, calculated with GraphPad Prism.
44
Statistical analysis: Cell viability was calculated as log (inhibitor) vs. response -- Variable
slope in relation to the solvent control using GraphPad Prism for Windows, Version 6.00
(GraphPad Software, Inc., La Jolla, CA, USA). Thereby the bottom was set to 0 and the top
the highest value in the data set. The confidence interval was set to 95% with 6 degrees of
freedom and the R square-values were strictly controlled. The experiment was repeated in
case they did not show an excellent fit. Graphs show mean values of n=x (where x is
denoted individually for each substance in the figure legend). Independent experiments were
run in duplicates.
3.3 Effects on intracellular ROS-inhibition
To reveal the direct radical scavenging effects, the antioxidant activity of the selected dietary
phytochemicals was analyzed in living HepG2 cells, by measuring their scavenging potential
against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), peroxyl-radical-induced
ROS. The results are expressed as the mean percentages of dichlorofluorescein (DCF)
fluorescence, as a measure of ROS formation, and shown in relation to the AAPH-treated
HBSS control (set to 100%).
45
Sulforaphane, as shown in Figure 27, within a concentration range of 5–75 µM, did not
influence the DCF fluorescence, as a measure of ROS formation, significantly. Hence,
overall, sulforaphane did not show ROS-inhibitory properties in this assay.
Figure 27: Measurement of intracellular ROS upon treatment with sulforaphane. Inhibition of peroxyl-radical-
(AAPH; 600 µM)-induced formation of ROS in HepG2 cells pretreated with increasing concentrations of sulforaphane (5-75 µM). The mean percentages of DCF fluorescence are shown in relation to AAPH-treated cells (set to 100%). The graph presents mean values ± SEM of n=5 independent experiments, calculated with R.
46
Quercetin, as shown in Figure 28, within a concentration range of 10–100 µM, did
significantly influence the DCF fluorescence by inhibiting ROS formation. At a concentration
of 10 µM, a highly significant reduction of AAPH-stimulated ROS levels to 31.8 ± SEM 3.6%
could be observed, compared to HBSS control cells treated with 600 µM AAPH.
Figure 28: Measurement of intracellular ROS upon treatment with quercetin. Inhibition of peroxyl-radical-
(AAPH; 600 µM)-induced formation of ROS in HepG2 cells pretreated with increasing concentrations of quercetin (10-100 µM). The mean percentages of DCF fluorescence are shown in relation to AAPH-treated cells (set to 100%). The graph presents mean values ± SEM of n=4 independent experiments, calculated with R.
47
Epigallocatechin-3-gallate, as shown in Figure 29, within a concentration range of 20–
200 µM, did significantly influence the DCF fluorescence by inhibiting ROS formation. At a
concentration of 20 µM, a highly significant reduction of AAPH-stimulated ROS levels to 54.8
± SEM 2.7% could be observed, compared to HBSS control cells treated with 600 µM AAPH.
Figure 29: Measurement of intracellular ROS upon treatment with epigallocatechin-3-gallate. Inhibition of
peroxyl-radical-(AAPH; 600 µM)-induced formation of ROS in HepG2 cells pretreated with increasing concentrations of epigallocatechin-3-gallate (20-200 µM). The mean percentages of DCF fluorescence are shown in relation to AAPH-treated cells (set to 100%). The graph presents mean values ± SEM of n=4 independent experiments, calculated with R.
48
Curcumin, as shown in Figure 30, within a concentration range of 5–75 µM, did significantly
influence the DCF fluorescence by inhibiting ROS formation. At a concentration of 10 µM, a
highly significant reduction of AAPH-stimulated ROS levels to 76.4 ± SEM 5% could be
observed, compared to HBSS control cells treated with 600 µM AAPH.
Figure 30: Measurement of intracellular ROS upon treatment with curcumin. Inhibition of peroxyl-radical-
(AAPH; 600 µM)-induced formation of ROS in HepG2 cells pretreated with increasing concentrations of curcumin (5-75 µM). The mean percentages of DCF fluorescence are shown in relation to AAPH-treated cells (set to 100%). The graph presents mean values ± SEM of n=5 independent experiments, calculated with R.
49
Cinnamic acid, as shown in Figure 31, within a concentration range of 10–100 µM, did not
influence the DCF fluorescence, as a measure of ROS formation, significantly. Hence,
overall, cinnamic acid did not show ROS-inhibitory properties in this assay.
Figure 31: Measurement of intracellular ROS upon treatment with cinnamic acid. Inhibition of peroxyl-
radical-(AAPH; 600 µM)-induced formation of ROS in HepG2 cells pretreated with increasing concentrations of cinnamic acid (10-100 µM). The mean percentages of DCF fluorescence are shown in relation to AAPH-treated cells (set to 100%). The graph presents mean values ± SEM of n=5 independent experiments, calculated with R.
50
Gallic acid, as shown in Figure 32, within a concentration range of 20–400 µM, did
significantly influence the DCF fluorescence by inhibiting ROS formation. At a concentration
of 100 µM, a highly significant reduction of AAPH-stimulated ROS levels to 83.0 ± 6.0%
could be observed, compared to HBSS control cells treated with 600 µM AAPH.
Figure 32: Measurement of intracellular ROS upon treatment with gallic acid. Inhibition of peroxyl-radical-
(AAPH; 600 µM)-induced formation of ROS in HepG2 cells pretreated with increasing concentrations of gallic acid (20-400 µM). The mean percentages of DCF fluorescence are shown in relation to AAPH-treated cells (set to 100%). The graph presents mean values ± SEM of n=4 independent experiments, calculated with R.
51
Catechin, as shown in Figure 33, within a concentration range of 20–200 µM, catechin did
not influence the DCF fluorescence, as a measure of ROS formation, significantly. Hence,
catechin did not show ROS-inhibitory properties in this assay.
Figure 33: Measurement of intracellular ROS upon treatment with catechin. Inhibition of peroxyl-radical-
(AAPH; 600 µM)-induced formation of ROS in HepG2 cells pretreated with increasing concentrations of catechin (20-200 µM). The mean percentages of DCF fluorescence are shown in relation to AAPH-treated cells (set to 100%). The graph presents mean values ± SEM of n=4 independent experiments, calculated with R.
Statistical analysis: Intracellular ROS-formation was calculated as mean percentages of
DCF fluorescence, in relation to HBSS control cells treated with 600 µM AAPH. As a
positive control quercetin was chosen and applied to all experiments (data not shown), while
a solvent control was tested for 0-hypothesis as well. p-Values were calculated with R,
Version x64 3.2.1. (The R Foundation for Statistical Computing, Vienna, Austria), performing
a post-hoc Dunnett’s test for multiple comparison, after testing for normal distribution with a
Shapiro Wilk test. This holds true for all, except for gallic acid, which required a Wilcox
analysis coupled with false discovery rate correction (FDR). Graphs show mean values ±
SEM of n=x (where x is denoted individually for each substance in the figure legend)
independent experiments run in quadruplicates (*p=<0.05, **p=<0.01, ***p=<0.001,
compared to AAPH-treated cells).
52
3.4 Effects on intracellular Nrf2-transactivation
3.4.1 Assessment via the CellSensor® ARE-bla HepG2 Cell Line
To expose the indirect radical scavenging effects, which by definition refer to the triggering of
the endogenous antioxidant machinery, the selected dietary phytochemicals were analyzed.
Based on the observation that some dietary phytochemicals characterized as antioxidants
up-regulate various genes of the Nrf2-mediated oxidative stress response and the fact that
Nrf2 is a transcriptional activator of ARE-mediated gene expression, the CellSensor® ARE-
bla HepG2 cell system was used to test substance-induced transcriptional activation of ARE-
driven reporter gene expression, namely bacterial β-lactamase. Thus, the first experiment
conducted intends to test the null hypothesis stating that the Nrf2 pathway is unaffected by
dietary phytochemicals, even in small doses.
53
Sulforaphane, as shown in Figure 34, dose-dependently stimulated ARE-driven β-lactamase
expression as HepG2 cells were treated for 15 hrs. Within a concentration range of 5–
100 µM, 5 µM induced ARE-mediated transcriptional activity 3.8 ± SEM 0.2-fold, while the
highest induction was reached at 10 µM with 4.2 ± SEM 0.3-fold. Even though noted as
significant, concentrations higher than 50 µM should be viewed critically, as this is too close
to the determined IC50 of 76.36 µM 24 hrs post treatment.
Figure 34: Activation of antioxidant response element (ARE)-driven β-lactamase reporter gene expression upon treatment with sulforaphane. Cell Sensor® ARE-bla HepG2 cells were treated with increasing
concentrations of sulforaphane (5-100 µM) for 15 hrs. The mean percentages of β-lactamase activity, as a measure of ARE-mediated transcriptional activation is shown in relation to the solvent control (set to 1). The graph presents mean values ± SEM of n=4 independent experiments, ***p<0.001, calculated with R.
54
Quercetin, as shown in Figure 35, dose-dependently stimulated ARE-driven β-lactamase
expression as HepG2 cells were treated for 15 hrs. Within a concentration range of 10–200
µM, 10 µM caused the highest ARE-mediated transcriptional activity at 1.4 ± SEM 0.1-fold,
compared to the DMSO solvent control. Here, the concentrations above appear to be valid,
as the IC50 was determined at 354.3 µM at a much later point in time at 24 hrs post
treatment. The observed repression (≥ 40 µM) shall be addressed in the final discussion.
Figure 35: Activation of antioxidant response element (ARE)-driven β-lactamase reporter gene expression upon treatment with quercetin. Cell Sensor® ARE-bla HepG2 cells were treated with increasing concentrations
of quercetin (10-200 µM) for 15 hrs. The mean percentages of β-lactamase activity, as a measure of ARE-mediated transcriptional activation is shown in relation to the solvent control (set to 1). The graph presents mean values ± SEM of n=5 independent experiments, ***p<0.001, calculated with R.
55
Epigallocatechin-3-gallate, as shown in Figure 36, only at a high dose stimulated ARE-
driven β-lactamase expression as HepG2 cells were treated for 15 hrs. Within a
concentration range of 20–400 µM, 200 µM was the only concentration that induced
significant ARE-mediated transcriptional activity to 1.9 ± 0.2-fold.
Figure 36: Activation of antioxidant response element (ARE)-driven β-lactamase reporter gene expression upon treatment with epigallocatechin-3-gallate. Cell Sensor® ARE-bla HepG2 cells were treated with
increasing concentrations of epigallocatechin-3-gallate (20-400 µM) for 15 hrs. The mean percentages of β-lactamase activity, as a measure of ARE-mediated transcriptional activation is shown in relation to the solvent control (set to 1). The graph presents mean values ± SEM of n=5 independent experiments, ***p<0.001, calculated with R.
56
Curcumin, as shown in Figure 37, dose-dependently stimulated ARE-driven β-lactamase
expression as HepG2 cells were treated 15 hrs. Within a concentration range of 5–100 µM,
30 µM caused the highest fold induction mediated by ARE-transcriptional activity at 3.0
± SEM 0.1-fold.
Figure 37: Activation of antioxidant response element (ARE)-driven β-lactamase reporter gene expression upon treatment with curcumin. Cell Sensor® ARE-bla HepG2 cells were treated with increasing concentrations
of curcumin (5-100 µM) for 15 hrs. The mean percentages of β-lactamase activity, as a measure of ARE-mediated transcriptional activation is shown in relation to the solvent control (set to 1). The graph presents mean values ± SEM of n=5 independent experiments, ***p<0.001, calculated with R.
57
Cinnamic acid, as shown in Figure 38, only at the lowest chosen dose stimulated ARE-
driven β-lactamase expression as HepG2 cells were treated 15 hrs. Within a concentration
range of 10–200 µM, 100 µM caused an induction mediated by ARE-transcriptional activity at
1.15 ± SEM 0.07-fold, which was calculated to be significant, and at 200 µM to 1.2 ± SEM
0.06-fold, which was calculated to be highly significant. It has to be noted though that at
200 µM the percentage of solvent (DMSO) in the solution was as high as 1%, which made a
difference, when looking at the induction level compared to the medium control. So even
though statistically calculated the result appears significant, the medium control proves it
wrong.
Figure 38: Activation of antioxidant response element (ARE)-driven β-lactamase reporter gene expression upon treatment with cinnamic acid. Cell Sensor® ARE-bla HepG2 cells were treated with increasing
concentrations of cinnamic acid (10-200 µM) for 15 hrs. The mean percentages of β-lactamase activity, as a measure of ARE-mediated transcriptional activation is shown in relation to the solvent control (set to 1). The graph presents mean values ± SEM of n=4 independent experiments, calculated with R.
58
Gallic acid, as shown in Figure 39, dose-dependently stimulated ARE-driven β-lactamase
expression as HepG2 cells were treated 15 hrs. Within a concentration range of 10–400 µM,
20 µM, for instance, induced an ARE-mediated transcriptional activity of 1.7 ± SEM 0.1-fold,
while the highest value was measured at 200 µM at 2.0 ± SEM 0.1-fold.
Figure 39: Activation of antioxidant response element (ARE)-driven β-lactamase reporter gene expression upon treatment with gallic acid. Cell Sensor® ARE-bla HepG2 cells were treated with increasing concentrations
of gallic acid (10-400 µM) for 15 hrs. The mean percentages of β-lactamase activity, as a measure of ARE-mediated transcriptional activation is shown in relation to the solvent control (set to 1). The graph presents mean values ± SEM of n=6 independent experiments, **p<0.01 and ***p<0.001, calculated with R.
59
Catechin, as shown in Figure 40, dose-dependently stimulated ARE-driven β-lactamase
expression as HepG2 cells were treated 15 hrs. Within a concentration range of 20–400 µM,
400 µM induced a slight ARE-mediated transcriptional activity of 1.2 ± SEM 0.05-fold, where
again, it has to be noted that this is only compared to the solvent control but not to the
medium control, and therefore the result loses validity.
Figure 40: Activation of antioxidant response element (ARE)-driven β-lactamase reporter gene expression upon treatment with catechin. Cell Sensor® ARE-bla HepG2 cells were treated with increasing concentrations
of catechin (20-400 µM) for 15 hrs. The mean percentages of β-lactamase activity, as a measure of ARE-mediated transcriptional activation is shown in relation to the solvent control (set to 1). The graph presents mean values ± SEM of n=4 independent experiments, calculated with R.
Statistical analysis: Activation of the antioxidant response element (ARE)-driven β-
lactamase reporter gene expression upon treatment was calculated as fold induction, in
relation to the respective solvent control. As a positive control tert-butylhydroquinone
(tBHQ) was applied to all experiments (data not shown), while the solvent controls were
tested for 0-hypothesis as well. p-Values were calculated with R, Version x64 3.2.1.,
performing a post-hoc Dunnett’s test for multiple comparison, after testing for normal
distribution with a Shapiro Wilk test. Graphs show mean values ± SEM of n=x (where x is
denoted individually for each substance in the figure legend) independent experiments run in
quadruplicates (*p=<0.05, **p=<0.01, ***p=<0.001, compared to solvent-treated cells).
60
Next, to investigate the interaction with the cells’ Nrf2-dependent endogenous cytoprotective
antioxidant machinery, protein levels of selected candidate genes were assessed.
3.4.2 Effect on heme oxygenase-1 (HO-1) protein expression
Following the reporter cell line transactivation assay, the key players involved were checked
on protein level and thereby the results for the three selected candidates sulforaphane
(SFN), quercetin (QUE), and epigallocatechin-3-gallate (EGCG) validated. Moreover, the
aspect of synergy was studied by testing the combinations of these three substances also.
Judging from the previously reported results, only SFN should cause an increase of more
than 2-fold. Hence, heme oxygenase-1 (HO-1) was selected as a known target gene (185)
and the Western blot bands were assessed via densitometric analysis. The hypothesis that
SFN would induce HO-1 turned out to be absolutely true, and, additionally, also the
combinations SFN+QUE, SFN+EGCG, and SFN+QUE+EGCG caused a clear induction and
significant HO-1 levels compared to the medium-treated control.
As shown in Table 5 and Figure 41, expression of HO-1 was substance-dependently induced
after treatment of HepG2 cells for 24 hrs. 10 μM of SFN raised HO-1 levels to as high as
2.78 ± SEM 0.3-fold. All combinations with SFN induced HO-1 expression to at least two, but
SFN + QUE reached the measured maximum at 3.23 ± SEM 0.9-fold, unfortunately though
with a large SEM. Interestingly, QUE did not induce HO-1 on its own. Please refer to the
following table for all other values:
Table 5: Values derived from densitometric analysis of Western blots for HO-1.
Ctrl SFN QUE
EGCG_ Low
EGCG_ High
SFN+QUE SFN+EGCG S+Q+E
HO-1 rel.
prot. lev. 1.000 2.784 1.010 1.141 1.014 3.232 2.806 2.510
SEM 0.000 0.296 0.121 0.284 0.378 0.940 0.547 0.542
P-values 1.000 0.002 0.936 0.641 0.972 0.064 0.021 0.039
61
Figure 41: Heme oxygenase-1 (HO-1) protein expression. Densitometric analysis of HO-1/GAPDH expression
after treatment of HepG2 cells with just medium (CTRL), 10 µM sulforaphane (SFN), 10 µM quercetin (QUE), 50 µM epigallocatechin-3-gallate (EGCG-lo), 200 µM epigallocatechin-3-gallate (EGCG-hi), 10 µM sulforaphane plus 10 µM quercetin SFN+QUE, 10 µM sulforaphane plus 50 µM epigallocatechin-3-gallate SFN+EGCG, OR 10 µM sulforaphane plus 10 µM quercetin plus 50 µM epigallocatechin-3-gallate S+Q+E, for 24 hrs. The graph presents mean values ± SEM of n=6 independent experiments, *p<0.05 and **p<0.01 compared to medium control, calculated with R.
3.4.3 Effect on thioredoxin-1 (Trx-1) protein expression
For thioredoxin-1 (Trx-1) the scientific community does not yet have sufficient data when it
comes to its induction via dietary phytochemicals. As shown in Table 6 and Figure 42, Trx-1
expression appears to be decreased via SFN stimulating the Nrf2 pathway, at least for the
time point assessed – 24 hrs past treatment. QUE shows a trend of increase, even though
the numbers are not significant due to a large SEM. EGCG at a concentration of 50 µM, on
the other hand, lowered the expression level even more than SFN, to 0.53 ± SEM 0.1-fold,
so to about half the endogenous level. Please refer to the following table for all other values:
Table 6: Values derived from densitometric analysis of Western blots for Trx-1.
Ctrl SFN QUE
EGCG_ Low
EGCG_ High
SFN+QUE SFN+EGCG S+Q+E
Trx-1 rel.
prot. lev. 1.000 0.613 1.286 0.528 0.567 1.074 1.087 1.251
SEM 0.000 0.148 0.627 0.100 0.094 0.327 0.352 0.472
P-values 1.000 0.121 0.693 0.042 0.044 0.842 0.827 0.648
62
Figure 42: Thioredoxin-1 (Trx-1) protein expression. Densitometric analysis of TRX-1/GAPDH expression after
treatment of HepG2 cells with just medium (CTRL), 10 µM sulforaphane (SFN), 10 µM quercetin (QUE), 50 µM epigallocatechin-3-gallate (EGCG-lo), 200 µM epigallocatechin-3-gallate (EGCG-hi), 10 µM sulforaphane plus 10 µM quercetin SFN+QUE, 10 µM sulforaphane plus 50 µM epigallocatechin-3-gallate SFN+EGCG, OR 10 µM sulforaphane plus 10 µM quercetin plus 50 µM epigallocatechin-3-gallate S+Q+E, for 24 hrs. The graph presents mean values ± SEM of n=3 independent experiments, *p<0.05 and **p<0.01 compared to medium control, calculated with R.
3.4.4 Effect on thioredoxin reductase-1 (TrxR-1) protein expression
When measuring the thioredoxin reductase-1 (TrxR-1) protein levels, one band was clearly
visible at a higher/bigger molecular weight of ≈110-120 kDa, while another one showed
pronouncedly at a lower/smaller molecular weight of ≈55-65 kDa, when examining the
Western blots, as shown in Figure 43.
63
Figure 43: Western blot of TrxR-1 staining plus GAPDH as loading control. A) PageRuler Protein Ladder for
gel and blot; B) sample blot picture taken with Odyssey infrared technology, with 1 – the ladder, 2 – the cytosolic fraction of untreated HepG2s, 3 – the mitochondrial fraction of untreated HepG2s, 4 – the nuclear fraction of untreated HepG2s; 5 – the cytosolic fraction of HepG2s treated with 10 µM SFN for 24 hrs, 6 – the mitochondrial fraction of HepG2s treated with 10 µM SFN for 24 hrs, 7 – the nuclear fraction of HepG2s treated with 10 µM SFN for 24 hrs; C) same as B), but greyscaled.
This is surprising, because so far the scientific community only has knowledge of a TrxR-1-
encoding transcript for the ≈55 kDa main form, but not for such a large polypeptide. At the
same time, occurrences of such TrxR-1-positive immunoreactive bands have been reported
in reducing SDS-Page analyses of protein lysates. This issue shall be expanded on and
discussed in the next chapter.
Interestingly, the higher band identified, was significantly lowered by QUE treatment, while a
trend was documented for SFN+EGCG as well as SFN+QUE+EGCG. Please refer to the
following table for all other values:
Table 7: Values derived from densitometric analysis of Western blots for TrxR-1_higher band.
Higher band
Ctrl SFN QUE EGCG_
Low EGCG_
High SFN+Q
UE SFN+EGCG
S+Q+E
TrxR-1 rel. prot.l.
1.000 0.934 0.850 0.885 1.057 1.007 0.412 0.590
SEM 0.000 0.124 0.008 0.230 0.209 0.209 0.141 0.181
P-values 1.000 0.646 0.003 0.666 0.812 0.977 0.053 0.152
64
Figure 44: Thioredoxin reductase-1 (TRXR-1) protein expression (higher/bigger band/protein).
Densitometric analysis of TRXR-1/GAPDH expression after treatment of HepG2 cells with just medium (CTRL), 10 µM sulforaphane (SFN), 10 µM quercetin (QUE), 50 µM epigallocatechin-3-gallate (EGCG-lo), 200 µM epigallocatechin-3-gallate (EGCG-hi), 10 µM sulforaphane plus 10 µM quercetin SFN+QUE, 10 µM sulforaphane plus 50 µM epigallocatechin-3-gallate SFN+EGCG, OR 10 µM sulforaphane plus 10 µM quercetin plus 50 µM epigallocatechin-3-gallate S+Q+E, for 24 hrs. The graph presents mean values ± SEM of n=3 independent experiments, *p<0.05 and **p<0.01 compared to medium control, calculated with R.
Analysis of the lower/smaller TrxR-1 protein band revealed that SFN indeed initiated a
significantly higher protein level at 2.06 ± SEM 0.07-fold, as well as a trend for the
combinations, in particular SFN+EGCG, and S+Q+E 2.49 ± SEM 0.25-fold. Please refer to
the following table for all other values:
Table 8: Values derived from densitometric analysis of Western blots for TrxR-1_lower band.
Lower band
Ctrl SFN QUE EGCG_
Low EGCG_
High SFN+QUE SFN+EGCG S+Q+E
TrxR-1 rel. prot.l.
1.000 2.058 1.765 0.584 0.586 1.069 1.845 2.488
SEM 0.000 0.069 0.450 0.298 0.297 0.544 0.864 0.929
P-values 1.000 0.004 0.231 0.297 0.298 0.910 0.431 0.250
65
Figure 45: Thioredoxin reductase-1 (TRXR-1) protein expression (lower/smaller band/protein).
Densitometric analysis of TRXR-1/GAPDH expression after treatment of HepG2 cells with just medium (CTRL), 10 µM sulforaphane (SFN), 10 µM quercetin (QUE), 50 µM epigallocatechin-3-gallate (EGCG-lo), 200 µM epigallocatechin-3-gallate (EGCG-hi), 10 µM sulforaphane plus 10 µM quercetin SFN+QUE, 10 µM sulforaphane plus 50 µM epigallocatechin-3-gallate SFN+EGCG, OR 10 µM sulforaphane plus 10 µM quercetin plus 50 µM epigallocatechin-3-gallate S+Q+E, for 24 hrs. The graph presents mean values ± SEM of n=3 independent experiments, *p<0.05 and **p<0.01 compared to medium control, calculated with R.
3.5 Effects on intracellular Nrf2 (trans-)location & expression levels
The expression levels of Nrf2 as well as its intracellular (trans-)location were assessed upon
treatment with dietary phytochemicals in order to explore the hypothesis that Nrf2 is bound to
cytosolic Keap1 which mediates its degradation via the proteasomal pathway, a bond which
can be disrupted via electrophilic attacks. If the theory holds true, a disruption of the binding
partners should lead to a translocation into the nucleus. Indeed, the percentage of the
nuclear fraction increased from 29.0% (control) to 45.6% when treated with SFN. When
treated with SFN + QUE, the percentage of Nrf2 in the nucleus shifted to as large a portion
as 62.1%. This level was assessed after the cells had been treated for 24 hrs.
Along with the location, it was of course expected for expression levels to increase, which
was also found to hold true, since SFN causes 1.86 ± SEM 0.2-fold induction of Nrf2 protein
levels. Moreover, it was deduced that 50 µM EGCG lowered the expression level for Nrf2
levels significantly to 0.66 ± SEM 0.1-fold, even below endogenous expression. Treatment
66
with any of the combinations including SFN showed a distinct trend of increased Nrf2,
especially with all three substances combined, even EGCG, 1.92 ± SEM 0.3-fold. Notably,
this is a higher-fold induction than was reached with SFN alone. Please refer to the following
table for all other values:
Table 9: Values derived from densitometric analysis of Western blots for Nrf2.
Ctrl SFN QUE
EGCG_ Low
EGCG_ High
SFN+QUE SFN+EGCG S+Q+E
Nrf2 rel.
prot.l. 1.000 1.864 0.900 0.657 0.814 1.507 1.468 1.924
SEM 0.000 0.242 0.086 0.101 0.358 0.249 0.353 0.299
P-values 1.000 0.016 0.299 0.019 0.626 0.098 0.242 0.027
Figure 46: Nrf2 protein expression levels. Densitometric analysis of Nrf2/GAPDH expression after treatment of
HepG2 cells with just medium (CTRL), 10 µM sulforaphane (SFN), 10 µM quercetin (QUE), 50 µM epigallocatechin-3-gallate (EGCG-lo), 200 µM epigallocatechin-3-gallate (EGCG-hi), 10 µM sulforaphane plus 10 µM quercetin SFN+QUE, 10 µM sulforaphane plus 50 µM epigallocatechin-3-gallate SFN+EGCG, OR 10 µM sulforaphane plus 10 µM quercetin plus 50 µM epigallocatechin-3-gallate S+Q+E, for 24 hrs. The graph presents mean values ± SEM of n=6 independent experiments, *p<0.05 and **p<0.01 compared to medium control, calculated with R.
This data, in summary, proves how food-derived nonnutrient molecules can indeed modulate
gene and thereby protein expression. Moreover, it strengthens the lead that suggests Nrf2 as
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a nutrigenomic biomarker, and demonstrates how it orchestrates the expression of key
molecules of the thioredoxin system and heme oxygenase-1 in this particular model, thereby
facilitating detoxification.
Statistical analysis: Protein expression levels of the individual compartments, subsequent
to subcellular fractionation, were calculated by comparing the intensities of the bands
determined with Western blotting following densitometric analysis using the Odyssey infrared
technology. As a standardized loading control GAPDH was applied, which should only
occur in the cytosolic fraction. Hence, if there was GAPDH present in the mitochondria or
nuclei also, these shares were subtracted for any protein assessed. As a positive control
for the nuclei histone 3 (H3) was used and misplaced shares were subtracted from any
subfraction except for the nuclei. As a negative control the corresponding solvents
(DMSO, A.d., and EtOH) were tested for 0-hypothesis as well (data not shown). p-Values
were calculated with R, Version x64 3.2.1. (The R Foundation for Statistical Computing,
Vienna, Austria), performing a post-hoc Dunnett’s test for multiple comparisons. Graphs
show mean values ± SEM of n=x (where x is denoted individually for each substance in the
figure legend), (*p=<0.05, **p=<0.01, ***p=<0.001, compared to medium-treated cells).
3.6 Effects on mitochondrial membrane potential
To analyze the effect of the selected substances on mitochondrial function, which is
commonly assessed via the effect in the mitochondrial membrane potential (MMP, ∆ψm)
whereby a decrease is associated with mitochondrial dysfunction, two approaches were
followed. First, a live cell staining was performed using tetramethylrhodamine methyl ester
(TMRM), to determine a potential reduction in MMP also qualitatively, with the confocal
microscope. Next, an optimized specific dye, the mitochondrial membrane potential indicator
(m-MPI), was applied in a more high-throughput setting (96-well plates) to validating the
observed changes quantitatively. Results of the prior approach are shown subsequently.
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Figure 47: HepG2 cells, after treatment with selected dietary phytochemicals, visualized under the confocal microscope. HepG2 cells, in four independent experiments, were stained with 100 nM of the
mitochondrial dye tetramethylrhodamine, methyl ester, perchlorate (TMRM) for 20 minutes, to indicate changes in the mitochondrial membrane potential ∆ψm. 24 hrs before, the cells, seeded out in a concentration of 60 000 cells per well, had been treated with just medium (Med), 10 µM sulforaphane (SFN), 10 µM quercetin (QUE), 50 µM epigallocatechin-3-gallate (EGCG_lo), 200 µM epigallocatechin-3-gallate (EGCG_hi), 10 µM sulforaphane plus 10 µM quercetin (SFN+QUE), 10 µM sulforaphane plus 50 µM epigallocatechin-3-gallate (SFN+EGCG), OR 10 µM sulforaphane plus 10 µM quercetin plus 50 µM epigallocatechin-3-gallate (S+Q+E) respectively.
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Area fractions, excluding the cell free areas out of the four independent images presented
above (Figure 47), were then analyzed with Image J (Version win64 Fiji Is Just) software and
the mean grey values were calculated for each area assessed and averaged. The means of
each treatment for these four experiments could subsequently be compared (Figure 48).
Figure 48: Comparison of means the area fraction vs. mean grey values from these fractions assessed of HepG2 cells, after treatment with selected dietary phytochemicals, visualized under the confocal microscope. HepG2 cells, in four independent experiments (n=4), were stained with 100 nM of the mitochondrial
dye tetramethylrhodamine, methyl ester, perchlorate (TMRM) for 20 minutes, to indicate changes in the mitochondrial membrane potential ∆ψm. 24 hrs before, the cells, seeded out in a concentration of 60 000 cells per well, had been treated with just medium (Med), 10 µM sulforaphane (SFN), 10 µM quercetin (QUE), 50 µM epigallocatechin-3-gallate (EGCG_lo), 200 µM epigallocatechin-3-gallate (EGCG_hi), 10 µM sulforaphane plus 10 µM quercetin (SFN+QUE), 10 µM sulforaphane plus 50 µM epigallocatechin-3-gallate (SFN+EGCG), OR 10 µM sulforaphane plus 10 µM quercetin plus 50 µM epigallocatechin-3-gallate (S+Q+E) respectively. Statistical analysis was performed with GraphPad Prism.
While each experimental set is somewhat different, statistical analysis, applying ANOVA
followed by Dunnett’s test for multiple comparisons, show some trends, and even some
significant outcomes. One-way ANOVA calculated a p-value of <0,0001 (****), which was
pinpointed by Dunnett’s to ***p=<0.001 comparing the medium control with 200 µM EGCG
and **p=<0.01 comparing medium control with all three substances added (S+Q+E). These
calculations were performed with GraphPad Prism for Windows, Version 6.00 (GraphPad
Software, Inc., La Jolla, CA, USA).
*** ***
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Next, to determine the changes in mitochondrial membrane potential with an additional
method, the m-MPI indicator dye was added to HepG2 cells according to the manufacturer’s
guidelines (186). m-MPI is positively charged and therefore able to enter the mitochondria
where it aggregated and fluoresces red. Upon a decline in mitochondrial potential, the
indicator dye stays in the cytosol in monomeric form and keeps its green fluorescence. Due
to this capability it allows the distinguishing of two populations, the ones with a high
mitochondrial membrane potential (MMP, ∆ψm), observed as high red and low green
fluorescence, and the cells which remain with a low ∆ψm, therefore enabling a determination
of the actual share of the population in percentage. In this assay, carbonyl cyanide 4-
(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial uncoupler, was used as a
positive control (data not shown), as it reliably lowers the ∆ψm.
Figure 49: Changes in mitochondrial membrane potential. HepG2 cells were treated with just medium
(Medium-Control), 1% DMSO (Solvent-Control), 5 µM or 50 µM sulforaphane (SFN), 10 µM or 100 µM quercetin (QUE), or 20 µM or 200 µM epigallocatechin-3-gallate (EGCG) respectively. The mitochondrial membrane potential, as a measure of monomers vs. J-aggregates, is shown in relation to the solvent control (set to 1). The graph presents mean values ± SEM of n=3 independent experiments, ***p<0.001, calculated with GraphPad Prism.
Statistical analysis, applying ANOVA derived a p-value of <0,0001 (****), which was
pinpointed by Dunnett’s to treatment with 10 µM quercetin, when compared to the medium
control These calculations were performed with GraphPad Prism for Windows, Version 6.00
(GraphPad Software, Inc., La Jolla, CA, USA).
****
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Thus, both independent modes of analysis determined quercetin amongst the selected
candidates that influences the mitochondrial membrane potential, either as a trend, or highly
significant, depending on the method applied. This is in line with the results of others that
have also shown that quercetin has significant influence on membranes, increasing their
permeability (128). What this evidence indicates, is that quercetin, just like the mitochondrial
uncoupler FCCP, first of all targets the mitochondria, and secondly thereby acts as an
oxidative stressor when it induces mitochondrial permeability transition (MPT). Indeed, it
supports the measurements that point out that quercetin potentiates O2●- generation via
targeting the mitochondria of cells. Highly striking is also the fact that only 10 µM of quercetin
had this effect, but not the higher dose of 100 µM, which demonstrates how delicate the
equilibrium is and since quercetin is suspected to utilize iron (Fe) and copper (Cu) for
chelation as well as calcium (Ca2+), these effect have, also by others, only been observed at
lower concentrations (128).
The second interesting finding with this data set is definitely the effect that EGCG,
particularly at a high dose (200 µM) at which it induced Nrf2, apparently leads to a significant
decrease in mitochondrial membrane potential. This theory of mitochondrial membrane
potential collapse has been observed by others and also explained with ROS formation
(187). Now, the second prevailing theory is though, that this phenomenon is the results of
some kind of interference with the confocal laser. These two theories which shall be
discussed further in the next chapter (FINAL DISCUSSION).
Obviously, when performing cell culture experiments, the relation to in vivo situations
remains unclear. Thus, as an addition to our in vitro studies, elucidating to a certain extent on
the interplay between extracellular dietary phytochemicals and endogenous signaling via
Nrf2, attention was of course also directed towards the actual consequences of said
mechanisms for an organism, not just a single cell. Hence, the following two chapters
present a bioinformatical analysis, shifting the focus to life outside cell cultures - to real life.
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3.7 Trx/TrxR and survival probability in vivo
Previously, it has been reported that in the various stages of cancer development, Nrf2 gets
hijacked and overexpressed as well as hyperactivated (72; 188). It is such an attractive
target, because of its role as “master switch” in regulating absolutely crucial processes such
as cellular detoxification, the elimination of ROS, xenobiotic metabolism, and drug transport
(2). Hence, it can have severe consequences and this transcription factor a foe in cancer, a
phenomenon explained at the “dark side of Nrf2” (4).
Thus, to estimate the effects of the two target genes of Nrf2 thioredoxin (Trx) and thioredoxin
reductase (TrxR) when over-expressed in cancer patients, a Kaplan Meier analysis was
performed. And indeed, the results show that if there is an overexpression of both – Trx or
TrxR – this lowers the survival probability of cancer patients dramatically, in the four types of
cancer – lung, ovarian, gastric and breast - assessed (Figure 51,Figure 51).
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Figure 50: Kaplan Meier analysis of the influence of Trx-1 levels in cancer patients. A non-parametric
statistic used to estimate the survival function from lifetime data.
Thioredoxin (Trx) and thioredoxin reductase (TrxR) are overexpressed in many aggressive
tumors since they promote their survival by counteracting their typically elevated ROS levels
(41). Thus, many tumor cells show a higher dependency on the Trx system than normal body
cells. Many emerging anticancer drugs therefore aim to target the TrxR (189-190). Cisplatin
constitutes one prominent example of an anti-cancer agent which causes covalent complex
formation of TrxR-1 with either Trx-1 or TRP14 (thioredoxin like protein of 14 kDa), which
most likely contributes to cisplatin-mediated cytotoxicity (191).
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Figure 51: Kaplan Meier analysis of the influence of Trx-1 levels in cancer patients. A non-parametric
statistic used to estimate the survival function from lifetime data.
Recent publications raise the question of how important these two key molecules of every
cell’s endogenous antioxidant machinery are to a completely new sphere. They suggest a
key role for TrxR not only in reducing Trx, but also, in its capability of regulating the Nrf2-
Keap1 stress response system (41).
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4 FINAL DISCUSSION
Following up on the hypothesis that the precondition for “health” is a living organism’s ability
to adapt to environmental and endogenous stresses, HepG2 cells were challenged with
various redox stressors of anti- and prooxidative quality. The hypothesis investigated focused
on The “master switch” of cells in general, and these liver cells in particular, known as
nuclear factor E2-related factor 2. Nrf2, being the key molecule of adaptive stress responses,
is known to facilitate the expression of more than 600 genes, most of which are of a
cytoprotective nature. The main interest hence became how the selected exogenously
occurring substances, which are regularly consumed by humans with their food, and often
even in isolate form and high doses as dietary supplements, interact with the Nrf2 pathway
and affect the chosen Nrf2 target genes on protein level. More specifically, the governing
objective was to identify how dietary phytochemicals control the redox balancing
strategies by defining whether they act as direct antioxidants or as indirect
antioxidants through interaction with the endogenous antioxidant systems in place.
Even though a very simplified model was employed, some of the complexity of a real life
situation was mimicked by adding these compounds as single substances, but also as
multicompound mixtures (as would be the case if ingested in the complex matrix of food). In
the course of this analysis, we encountered novel and promising results by testing not just
the single compounds, but synergies of the selected candidates and their bifunctional effect
on the target genes HO-1, Trx-1, and TrxR-1, mimicking phase II detoxification in the liver. It
needs to be highlighted that the HepG2 cells challenged are from a human hepatocarcinoma
and therefore show most of the characteristics of primary cancer cells. This is a special
situation for working on Nrf2 altogether, because in cancerogenesis this molecule plays a
decisive role due to the fact that Nrf2 has the capacity to initiate the expression of all these
detoxifying and cytoprotective enzymes and proteins, referred to as its “dark side”, since
thereby it can also contribute to chemoresistance during therapy. In fact, evidence is
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accumulating that Keap1 and Nrf2 are frequently mutated and Nrf2 often hyperactivated in
human cancers (47). At the same time, the controversy whether chemopreventive initiatives
should include the increase of vegetables (e.g. “Five-a-Day”, “Savor the Spectrum”, etc.) and
thereby the intake of potentially Nrf2 activating substances, still prevails (5-6). Many
important contributions to the knowledge base on this matter, have concluded that the
protective and health-promoting potential probably depends on the biological background
and the redox status of the cell or the respective organ. Hence, despite the remarkable
progress in the understanding of carcinogenesis as well as on the mechanism of action of
single phytochemicals, crucial elements are yet to be elucidated on. Hence, the focus of the
project presented in this thesis.
4.1 Summary of the Results & Discussion
Along with the certainty that the organism’s redox balance is delicate yet crucial in health and
disease, arises the need for a fundamental understanding of the underlying mechanisms.
While the direct antioxidant effects of substances bears less of a riddle, the scope and
importance of Nrf2 as a transcription factor orchestrating the endogenous antioxidant
machinery still needs to be fully comprehended. In this regard, the pool of target genes
revealed to the scientific community is constantly growing, and with it the knowledge about
their functionality. As it evidently becomes even more crucial to understand this pathway and
its key players, i.e. in order to find ways to trigger the right therapeutic mechanisms at the
right time, testing the relevant substances in a reliable model becomes crucial and this
conviction inspired the study at hand.
Clearly, when working on a pharmacological aim like overcoming the “hallmarks of cancer”,
portrayed in Figure 52, it also becomes apparent how robustness and redundancy make any
single compound intervention due to multifactorial developments a “mission impossible”. On
the other hand, this hypothesis nourishes the multicompound approach, strengthening the
argument that a concerted mixture could tackle the problem from multiple angles. And, in
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fact, various traditional medicinal systems, apply combinations of herbs and plant materials
containing multifarious compounds in the treatment of multifactorial diseases (192).
Figure 52: The identified hallmarks of cancer – the next generation (193).
An important number of studies of various pathologies related to oxidative stress support the
idea that the imbalance of redox homeostasis is often associated with improper detoxification
in the liver, thereby contributing to the development of various disorders. After the selection
and validation of HepG2 liver cells as a suitable model for the hypothesis challenged, the
adequate concentrations for cell culture testing had to be determined. This was done by
performing a resazurin reduction assay which is relatively inexpensive, uses a homogeneous
format, and is more sensitive than, for instance, tetrazolium assays (194). Critically
assessing, as a limitation of the CTB assay, it is important to note that it is very specific, as
resazurin is metabolically changed into resorufin, hence this specific substance and
conversion should not be affected by an interference from any other substance applied.
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Furthermore, it is a redox reaction, which implies that when the cells’ general milieu is
altered, then this reaction cannot function properly either.
4.2 Cell proliferation & viability
To assure sufficient proliferation and viability of the cells also upon treatment, the
concentration influencing this parameter had to be identified. When assessing the influence
of the selected substances on HepG2 cells’ metabolic activity, our study has yielded the
following results: all IC50 of the single compounds are summarized in the subsequent Table
10, for direct comparison.
Table 10: Summary of IC50 calculation based on the metabolic activity of HepG2 cells treated with single compounds.
IC50s of single
compounds
24 hrs
48 hrs
72 hrs
Sulforaphane (SFN) 76.36 58.44 52.91
Quercetin (QUE) 354.3 148.3 132.6
Epigallocatechin-3-
gallate (EGCG)
180.5 141.2 173.7
Curcumin (CUR) 113.1 73.31 68.74
Cinnamic acid (CIN) - - -
Gallic acid (GAL) 248.5 248.8 242.0
When analyzing the data, we detected sulforaphane to be the most influential substance in
comparison to the others, while cinnamic acid did not compromise the cells’ state to a level
where the IC50 could have been calculated. Hence, cinnamic acid was not included in the
selection of the three most promising candidates since we aimed for unambiguously
deducible effects. Moreover, we found the cells’ response to quercetin at this high level after
24 hrs, while 48 hrs post exposure the IC50 concentration was about the same as for
epigallocatechin-3-gallate, showing a delay in response. With this data, the IC50 were
empirically established, with the primary aim of avoiding these concentrations in
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subsequently performed assays. In fact, it was aimed at not to exceed the IC20 in each of the
following steps. This is crucial since interference from cellular processes such as apoptosis,
necrosis or any other cell disturbances, had to be avoided. It is important to work on fully
competent cells when investigating cell signaling events of the kind focused on within this
project framework. In order to validate the obtained values, they were compared to the ones
previously reported from colleagues.
For sulforaphane, Melchini et al. performed a WST1 dye reduction assay (with a kit from
Roche Applied Science) and found an IC50 of 24.89 ± 1.53 µM (195). Hu et al. also assessed
the IC50 for SFN in HepG2 cells and determined it at 14.05 µM; one should note though that
this is the value obtained 72 hrs after treatment and MTT was used as a substance (196).
For quercetin, Musonda et al. demonstrated absolutely sub-cytotoxic treatment of HepG2
cells with up to 50 µM of Quercetin (197). For epigallocatechin-3-gallate, colleagues Cao et
al. obtained the IC50 of 24 hrs and 48 hrs at 133.90 mg/l (which, according to calculations,
corresponds to 292.1 µM) and 78.97 mg/l (which, according to calculations, corresponds to
172.3 µM) when testing the substance on their HepG2 clone (198).
Therefore, when looking at some IC50 values from other groups for comparison, these
numbers are agreeable enough with the results of this study, as they are roughly along the
same lines, and frankly, even though it is a cell line, clone specific traits may occur during the
years, in different laboratories being handled by different scientists, and hence some
variation should be deemed fully acceptable.
One of the novelties in the study performed was the selection of the three candidate
substances SFN, QUE and EGCG, which were also tested in groupings so that synergies
and antagonisms could be determined in addition. Having highlighted the complexity of a
food matrix in the introduction, it is of great relevance to test dietary compounds in realistic
combinations. Beneath these experiments lies the logic that when looking at the prospect of
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utilizing phytochemicals as therapeutical agents, the possibilities for combinational therapy
should never be neglected.
Hence, the investigations led to the following data when testing multiple compounds at once,
presented subsequently in Table 11, for better and direct comparison of all IC50s.
Table 11: Summary of IC50 calculation based on the metabolic activity of HepG2 cells treated with multiple compounds.
IC50s of multiple
compounds
24 hrs
48 hrs
72 hrs
Please note, that the values [µM] denote the actual SFN concentration
at which the IC50 was reached.
SFN +50 µM EGCG 52.54 35.85 33.79
SFN +10 µM QUE 83.02 45.43 44.43
SFN +50 µM EGCG
+10 µM QUE 65.47 33.81 28.90
SFN +200 µM EGCG
+10 µM QUE 89.13 23.26 17.33
Striking is of course, that SFN and EGCG lower the IC50 notably when comparing the values
to the single substances even already after 24 hrs: 76.36 µM for SFN only; 180.5 µM for
EGCG only; compared to 52.54 µM of SFN when paired with 50 µM EGCG. It has to be
highlighted here that 50 µM EGCG are not even close to its IC50 concentration of 180.5 µM.
One could interpret that SFN has anti-tumorigenic effects, which can be aggravated in the
presence of EGCG. In fact, these results absolutely strengthen what colleagues have
touched on when they tested 25 µM SFN and 20 µM EGCG as a low-dose combination in
vitro as well as 25 µM SFN and 100 µM EGCG as a high-dose combination on HT-29 human
colon carcinoma cells and found a reduction in cell viability to 70% (high-dose) and 40%
(low-dose) at 48 hrs without significant changes before that. Although not as pronounced as
in the experimental setting utilized here, our colleagues’ results are very similar.
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These in vitro results are fully in line with the in vivo data that shows that sulforaphane on its
own has cytoprotective effects including the reduction of tumors that develop in carcinogen
exposure studies (199). To the best of our knowledge, so far no studies have been
performed where SFN and EGCG were administered together, therefore, this hold great
potential for future pre-clinical and clinical approached.
4.3 Effects on intracellular ROS-inhibition
Reactive oxygen species (ROS) play a major role in various pathologies and carcinogenesis,
when pro-oxidative agents, such as tobacco smoke, cause a redox deregulation within cells.
Direct antioxidants with the possibility of inhibiting ROS-formation are thus of great value in
avoiding this imbalance and deregulation. Hence, with the following assay, exactly this
capacity of the dietary phytochemicals selected was assessed.
Table 12: Summary of ROS-inhibition values of single substances in HepG2 cells, with IC50 values stated for orientation.
ROS-inhibition
(1 hr, post treatment) [%]
IC50 of compounds
(24 hrs, post treatment) [µM]
Sulforaphane (SFN) No inhibitory effect 76.36
Quercetin (QUE) 31.8 ± SEM 3.6% [10 µM] 354.3
Epigallocatechin-3-
gallate (EGCG) 54.8 ± SEM 2.7% [20 µM] 180.5
Curcumin (CUR) 76.4 ± SEM 5% [10 µM] 113.1
Cinnamic acid (CIN) No inhibitory effect -
Gallic acid (GAL) 88.3 ± SEM 3.1% [30 µM] 248.5
AAPH-induced ROS levels were decreased, as expected, by all substances considered
prime examples of a dietary phytochemical with antioxidant capacity due to their structure.
This was the case for all flavonoids, as well as the hydrobenzoic acid – gallic acid.
Surprisingly, cinnamic acid did not act as an antioxidant, nor did it influence the cells’
viability. SFN turned out not to act as a direct antioxidant towards the free radical-generating
azo compound.
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4.4 Effects on intracellular Nrf2-transactivation
The majority of studies around this key molecule identify Nrf2 as a “master redox switch” (55)
and, hence, as an activator of cellular defense mechanisms. The following assay proved to
be appropriate for a fast screening of multiple substances (high throughput) and
concentrations.
4.4.1 Assessment via the CellSensor® ARE-bla HepG2 Cell Line
Table 13: Summary of ARE-fold induction values of single substances in HepG2 cells, with IC50 values stated for orientation.
ARE-fold induction
(15 hrs, post treatment) [fold]
IC50 of compounds
(24 hrs, post treatment) [µM]
Sulforaphane (SFN) 4.2 ± SEM 0.3-fold [10 µM] 76.36
Quercetin (QUE) 1.4 ± SEM 0.1-fold [10 µM] 354.3
Epigallocatechin-3-
gallate (EGCG) 1.9 ± SEM 0.2-fold [200 µM] 180.5
Curcumin (CUR) 3.0 ± SEM 0.1-fold [30 µM] 113.1
Cinnamic acid (CIN) 1.15 ± SEM 0.07-fold [100 µM] -
Gallic acid (GAL) 1.7 ± SEM 0.1-fold [20 µM] 248.5
Catechin (CAT) 1.2 ± SEM 0.05-fold [400 µM] undetermined
While SFN, QUE and EGCG along with CUR and GAL caused an induction at a certain
concentration [indicated in brackets in the “ARE-fold induction”-column], the results of CAT
and CIN have to be ignored, since the solvent control (set to 1) was below the medium
control indicating that the induction is a false positive.
In regard to the observed regression, often also yielding significant results (significant down-
regulation), when applying higher doses of the individual substances, several reasons could
account for this phenomenon. Previously, we monitored that the HepG2 cell line with the
stably integrated β-lactamase reporter gene is not equally as robust as the normal HepG2
clone (data unpublished). Hence, it is possible that in these modified cells cell growth is
inhibited at a lower concentration. This would lead to a lower cell number, for which no
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normalization is integrated in the experimental work flow, except for the solvent control,
which all other values are of course standardized to. The fact that this phenomenon
correlated with the individual toxicities of the substances - signifying that for SFN this effect
can be witnessed at a much lower concentration, whereas for CIN there was no such effect -
supports this hypothesis. Accordingly, for future experiments with this Promega cell sensor
product, normalization of cell numbers should be included.
4.4.2 Effect on heme oxygenase-1 (HO-1) protein expression
HO-1 has been shown to play an essential role in cellular and tissue defenses against
oxidative stress and inflammation, as its overexpression can inhibit pathological
developments including vascular proliferation and chronic transplant rejection (79).
Sulforaphane has already been described as a potent inducer of HO-1 levels before. Also in
our experiments we saw more than double the expression levels when compared to the
control. Results from another group state that the HO-1 protein accumulates time-
dependently until up to 12 hrs after SFN treatment, while the strongest induction occurs
already 4 hrs after adding the compound (200).
Regarding the flavonoids EGCG and QUE, others have shown that Nrf2 and HO-1 levels are
raised, at least when a 50 µM concentration of them was tested on human retinal pigment
epithelial (RPE) cells (more precisely, in ARPE-19 cells) to determine whether specific
dietary and synthetic flavonoids can protect these cells from oxidative-stress–induced death.
Unfortunately, they do not state accurate numbers, which would have been exciting to
compare, but just refer to an up-regulation (201). Another group has also shown the
importance of the uptake, compartmentalization and transport of EGCG in endothelial cells in
calveolae, the plasma vesicle-like microdomains, whereby EGCG has been shown to induce
Nrf2 and HO-1 expression, provided that this mechanism is fully functional (202). In the
experimental set-up chosen for this project, a high dose of EGCG clearly down-regulated
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HO-1 protein expression 24 hrs post treatment. This might indicate an even faster
degradation of Nrf2, which has also been shown for allyl isothiocyanate AITC, indole-3-
carbinol I3C, and parthenolide PLT (200), and is emphasized by the presented result that
Nrf2 levels in general are lowered by such a high dose of EGCG.
4.4.3 Effect on thioredoxin-1 (Trx-1) protein expression
The Trx system plays a significant role in maintaining a reduced environment within cells,
and, as suggested, also extracellularly. This becomes evident since Trx-1 is highly
conserved in many organisms, found ubiquitously in most organs and in most cells. There it
can be found in the cytosol, the nucleus, as well as in secreted form outside cells under
particular circumstances (203). Thus, it is an important part of the “redoxisome”, a significant
regulator of cellular redox homeostasis, and as such essential for cell survival and function.
Comparing our results to those from other groups, Bacon et al. reported on the “Dual Action
of Sulforaphane in the Regulation of Thioredoxin reductase and Thioredoxin in Human
HepG2 and Caco-2 cells” (204) and showed that SFN is an inducer for both, the enzyme as
well as the substrate, in both cell types. Upon treatment with 10 µmol/l SFN in DMSO, these
colleagues showed a 4-fold induction after 8 hrs for TrxR mRNA levels, and a 2-fold increase
for the amount of protein, in human hepatoma HepG2 cells, whereas the induction for Trx
could only be detected for the mRNA (2.9-fold) after 48 hrs, while the protein levels remained
unchanged. This parallels the results presented here also, where no increase in protein
expression upon treatment with SFN 24 hrs later was detected. On the contrary, the trend to
a down-regulation was observed for SFN as well as EGCG in low and higher concentration.
As it has been observed that Trx can occur outside of cells, mostly under oxidative and
inflammatory conditions (205), this might explain why even though the promoter is activated
by Nrf2 and SFN, this increase might remain undetectable in Western blot analysis. In its
function as a circulatory protein, Trx-1 has been characterized as a chemotactic factor,
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attracting monocytes, neutrophils, and T lymphocytes (103). Yet, the precise mechanism of
its export into extracellular space still needs to be clarified (206).
Another aspect to be discussed is that in the control cells, a portion of the Trx-1 appeared to
occur in the mitochondria. Enquiring at the antibody producing company (Santa Cruz
Biotechnology Inc., Heidelberg, Germany) and thoroughly checking at UniProt revealed that
it must have been Trx-1 (Accession # P10599), since Trx2 (Accession # Q99757), which
actually occurs in the mitochondria, has a rather different sequence:
Table 14: Alignments of the sequence of Trx-1 and Trx2. (207)
4.4.4 Effect on thioredoxin reductase (TrxR-1) protein expression
Without its reducing partner TrxR-1, the bulk of Trx remains in its oxidized form, while a
smaller share is recycled via the action of reduced glutaredoxins (Grxs) (41). This has
multiple consequences, as it is unable to function as a reducing agent for other proteins in
that case. Also the thioredoxin interacting protein (Txnip/TBP-2/VDUP1) can only bind to its
reduced version, resulting in further changes in redox-dependent cellular processes, such as
gene expression, signal transduction, cell growth, and apoptosis (203; 206).
As demonstrated in the results section, curiously, when assessing TrxR-1 at the protein level,
two clearly distinguishable bands became apparent. Along with these findings from our
group, recent research has found TrxR-1 to occur as a 55 kDa sized band in SDS-PAGE
analyses and to have a function as a redox sensor itself at its Trp114, which causes
oligomerization and crosslinking upon being triggered by oxidative stress. It has been shown
that between two oxidized Trp114 residues a covalent link can be established leading to
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dimers, tetramers, and higher multimers of dimers (208). This could have been what was
observed here also. Taking this further, we can now report that the treatment with quercetin
significantly lowered the occurrence of the higher band identified. The same trend was
documented for SFN+EGCG as well as SFN+QUE+EGCG. This data suggests that
quercetin down-regulates the expression of oxidized TrxR multimers and, hence, is indeed a
very potent antioxidant, which acts not only directly, but also via Nrf2. The effect that
quercetin inhibits mammalian TrxR-1 has previously been described (190). Moreover, our
data indicates that EGCG along with QUE has an inhibitory effect on TrxR in vitro, which is
fully in line with the results from other groups that have identified thy molecules Cys and Sec
restudies as a potential target side of EGCG (209) (210).
4.5 Effects on intracellular Nrf2 (trans-)location & expression levels
Mammalian cells utilize the transcription factor Nrf2 as a major mechanism to orchestrate
cellular responses to oxidative or electrophilic attacks on the cell (52-56; 58). Upon triggering
of this mechanism, Nrf2 has been shown to translocate into the nucleus in order to bind to
the antioxidant/electrophilic response element and thereby initiate the transcription of phase
II detoxifying enzymes and proteins that can counteract the oxidative insult. Nrf2, for this
change of cellular compartment, contains a nuclear localization sequence (NLS), just like it
possesses a nuclear export sequence also. The transport of Nrf2 outside of the nucleus has
been shown to be initiated by Bach1, but also by Trx-1 (46).
According to results of colleagues, sulforaphane has been found to be a good inducer of Nrf2
translocation into the nucleus (211-212), which was also apparent in the results presented
here. It could be demonstrated to prolong Nrf2’s stability and hence its half-life (to about
75 minutes; assessed in HepG2 cells) (200). Under normoxia, the activity of Nrf2 under these
basal conditions is limited by its short half-life (<10min), due to rapid ubiquitination and
proteasomal destruction (213-214). Quercetin has previously been suggested to induce
nuclear translocation of Nrf2, e.g. in primary cerebellar granule neurons (CGN) of rats (215),
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as well as to stimulate Nrf2 expression, as e.g. demonstrated in HepG2-C8 cells (216).
Epigallocatechin-3-gallate, on the other hand, was observed to lower Nrf2 expression levels,
which might suggest that EGCG actually increases its degradation in the proteasome or
strengthens its bond with Keap1 thereby inhibiting its release or uses another mechanism to
change this pathway.
4.6 Effects on mitochondrial membrane potential
Amongst cellular organelles, the mitochondria are a major site for ROS production (217), as
they are prone to transfer electrons onto oxygen facilitated by the electron transfer chain
(ETC) complexes I-V (7). It has been suggested that mitochondrial-targeted antioxidants are
more promising inhibitors of tumorigenesis than the ones which remain in the cytosol, since
the mitochondria obviously are also one of the major sites for ROS production within the cell
(218). Moreover, cancer has often been described as a mitochondrial metabolic disease
(219). On the premise that typically cell perturbations occur at the subcellular level, this
requires the analysis of cellular stress responses for various organelles, and in particular of
the mitochondria (220), which was hence tackled in this study.
Like in all fluorescence-based applications, precaution has to be taken when working with
compounds as they might have fluorogenic properties. In fact, this holds true for quercetin,
which has shown such capacity at 488 nm excitation and 500-540 nm emission, so in the
green channel (215). Fortunately, this property can be neglected when assessing other
channels, such as in the case of TMRM where analysis was conducted via the red channel,
an excitation at 543 nm and emitting fluorescence collected at ⩾560 nm. This was the main
reason for analyzing the mitochondrial membrane potential (MMP, ∆ψm) with two methods,
instead of taking a single approach.
Quercetin stood out in the analysis staining HepG2 cells with TMRM 24 hrs after treatment.
At a concentration of 10 µM it increased mitochondrial activity evidently, yet insignifically, and
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did so also in combination with sulforaphane. Comparing these results to those of others, a
phenol-based approach to mitochondrial medicine has been highly recommended before, for
instance by De Marchi et al. (128). Along the same lines, they observed in
electrophysiological experiments that quercetin succeeded in inhibiting the mitochondrial
permeability transition pore (MPTP), but only at at low μM levels. At higher (> 10 μM)
concentrations mitochondrial permeability transition(MPT)-inducing effects have been
observed in the presence of Ca2+, and with dense suspensions of mitochondria. This
supports the findings of this study using method two, where cells were stained with m-MPI
and quercetin was the only substance which lowered the ∆ψm. These data indicate that
quercetin changes the membrane and makes it more permeable. An effect along the same
lines, which was proposed by colleagues, has been shown in vivo when resveratrol in
combination with quercetin was pharmacokinetically more bioavailable to the body than on its
own (221).
A second thought-provoking effect was the one discovered for epigallocatechin-3-gallate,
which must have diminished either mitochondrial activity or quenched the fluorescing dye.
Since a staining of the nuclei as well as all cellular glycoproteins was performed as a control,
it became apparent that the latter effect holds true. Just like for the TMRM staining, the dye
applied for the nuclei, the Syto 16 (green channel), was reduced, proving that the effect
occurred in the reaction of the cells to the treatment with EGCG as a substance and the dye
under the influence of laser. In fact, there are hints to be found in literature that the influence
of the laser in the presence of EGCG might cause the production of singlet oxygen, which in
turn alters the fluorochrome due to its high reactivity (222), as colleagues have published that
exposure to UV radiation caused polyphenols to act prooxidatively increasing ROS levels
(223). This is exactly the prooxidative action of polyphenols, which has been proposed to
cause their pro-apoptotic effect in already altered or damaged cells, hence protecting an
organisms’ health (127; 223).
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4.7 FINAL SUMMARY substance-wise
The “redox code” comprises a major strategy for mammalian redox homeostasis. Since
many diseases evolve around an imbalance in this system, it is crucial to understand it well.
This holds true in particular for cancer prevention, carcinogenesis and cancer therapy. The
post-translational modification of proteins, e.g. oxidations, particularly at certain target
cysteines that have a low pKa, is a highly employed redox-strategy which leads to a
functional change in the concerned protein. The oxidative modifications can be inverted via
the two most prominent antioxidant systems, namely the Trx- and the GSH-system. Nrf2 has
been identified as an essential “control knob” for redox homeostasis, as it potently induces
the expression of these antioxidant enzymes and by doing so regulates imbalances between
oxidants and reductants to return the cellular state into an equilibrium (41).
Figure 53: The “redox code”. (Modified from (41))
For the purpose of this thesis, several substances and their capability to influence the Nrf2-
control knob have been investigated. The subsequent paragraphs will shortly summarize the
three most important, investigated compounds and their effects on the Nrf2 pathway:
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4.7.1 Sulforaphane (SFN)
In conclusion, sulforaphane belongs to the organosulfur compounds which can be obtained
through a diet rich in cruciferous vegetables (such as broccoli, cabbages, cauliflower and
Brussels sprouts) and is taken up as glucoraphanine. Myrosinase from the gastro-intestinal-
tract converts the precursor glucoraphanine into SFN when it is released from plant cells,
e.g. by mechanic destruction while chewing.
In the results presented in this thesis, SFN has provoked the lowest IC50 of all substances
tested (76.36 µM), did not inhibit ROS-formation (-), but instead induced β-lactamase
expression as a sign of inducing the translocation of Nrf2 (***), which could be nicely shown
with the reporter cell line, but also via Western blotting (↑ 1.6-fold Nrf2 expression).
Additional information was revealed via subcellular fractionation and protein analysis, which
showed an increased share of the Nrf2 protein in the nucleus (↑ from 27.1% (control) to
48.8%). Regarding Nrf2 target proteins, SFN increased HO-1 expression (↑ 2.36-fold),
lowered Trx expression (↓ 0.61-fold), and increased TrxR-1 expression (↑ 2.1-fold) in its
monomeric form.
Hence, SFN does not exhibit inherent properties of a “direct” antioxidant, as in fact it is a
weak electrophilic prooxidant (224). Yet, it has significant cytoprotective potential, since it is a
potent inducer of the endogenous antioxidant machinery, by targeting e.g. the thioredoxin
system. On top of it all, it has good bioavailability, which makes it a strong candidate for
therapeutic use as a dietary phytochemical (200).
Its precise mechanism of targeting the Nrf2-pathway is thought to involve the modification of
critical cysteine residues of Keap1 thereby stabilizing Nrf2 and enabling its translocation into
the nucleus to facilitate as a transcription factor there. Research has shown that a
dithiocarbamate functional group is formed between SFN’s isothiocyanate and Keap1’s
sulfhydryl nucleophiles, making this adduct kinetically labile (225).
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In chemoprevention, it has been identified as a potent activator of apoptosis, e.g. via the
increase in Bax upon JNK-mediated Bcl-2 inhibition, which triggers cytochrome c release
from the mitochondria (226). Currently there are at least eighteen registered clinical studies,
pronouncing the fact that sulforaphane is a highly promising candidate for druggability (209).
The outcomes of this project strongly recommend combining SFN with EGCG in order to
yield even more promising results.
4.7.2 Epigallocatechin-3-gallate (EGCG)
In summary, EGCG (along with other catechins mainly consumed with green tea) has been
shown to have a highly peculiar bioactivity profile, as it can exercise both antioxidant and
prooxidant effects (127).
Indeed, our results show both effects clearly, as it was identified as a potent inhibitor of ROS-
formation (***), but at the high concentration of 200 µM also activated Nrf2 and triggered β-
lactamase expression (***). At a low dose, it actually lowered Nrf2 protein expression in
HepG2 cells, as well as Trx-1 (↓ 0.53-fold, low dose) and TrxR-1 monomers (↓ 0.58-fold, low
dose; ↓ 0.59-fold, high dose).
Inducing ROS due to the formation of superoxide during its oxidation in the presence of
oxygen and redox active transition metals, such as a labile aroxil radical, revealed EGCG’s
property as a pro-oxidant and phytotoxin (227-229). Controversial opinions prevail whether
polyphenol-initiated ROS-production is the root or the cause of the triggered apoptosis and
cell cycle arrest, but it is clear that it plays a key role (230). Another hypothesis discussed is
the Warburg effect, as it states that the high level of glycolysis leads to an acidification and
thereby changes the pH of cells which affects the DNA structure and exposes the chromatin-
bound copper. Hence, the chromatin-bound copper becomes available to be attacked by pro-
oxidants like resveratrol and EGCG (231).
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In mice, an impressive number of genes have been shown to be regulated by EGCG. This
was demonstrated Nrf2-dependently by employing an Affymetrix mouse genome 430 2.0
array, which in sum comprised most chemopreventive effects determined (232).
4.7.3 Quercetin (QUE)
Polyphenols, such as quercetin, can be considered a prime example of a dietary
phytochemical with antioxidant capacity due to the presence of a catechol group (3’,4’-
dihydroxy) in the B ring; a double bond between carbon 2 and carbon 3 of the C ring
conjugated with a keto group at position 4; as well as the presence of a hydroxyl group as a
substituent in the position 3 of ring C and position 5 of the A ring (230).
Figure 54: Chemical structure of quercetin as a role model for the key features of flavonoids with antioxidant activity.
In our experiments, QUE has proven to be a potent ROS-formation inhibitor (***), as well as
an inducer of the Nrf2 pathway. It did not limit SFN from inducing HO-1 levels, but raised HO-
1 expression further (↑ 3.36-fold, in combination), while lowering TrxR-1 multimeric levels (↓
0.85-fold), strengthening our data on its function as an antioxidant, because the two oxidized
Trp114 residues form a covalent link and hence multimers of dimers.
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4.7.4 Wrap-up - all three substances in combination
While all compounds worked with in the beginning of this project showed highly interesting
profiles, sulforaphane, quercetin and epigallocatechin-3-gallate crystallized as lead
substances for further investigations after the primary analysis stage. They were denoted as
such, because of their rather diverse backgrounds from different groups of secondary plant
metabolites, their distinct pharmacokinetic profiles, and in particular because our hypothesis
was that they would tackle the redox situation of a cell, also in regard to Nrf2, from multiple
angles and hence would provide a thrilling platform to draw conclusions from. This approach
proved fruitful, and the first highlight of the substances in combination presented itself in the
finding that the IC50 of SFN can be lowered dramatically when EGCG was added to the cells
(after 24 hrs: 76.36 µM for SFN only; 180.5 µM for EGCG only; compared to 52.54 µM of
SFN when paired with 50 µM EGCG). Thus, EGCG was shown to aggravate the anti-
tumorigenic effect of SFN. Moreover, SFN plus EGCG raised HO-1 levels significantly (↑
2.81-fold) as well as TrxR-1 (↑ 1.85-fold) in reduced monomeric form. Also SFN plus QUE
raised the level of HO-1, and so did all three in combination (↑ 2.51-fold). Another interesting
discovery was that even though EGCG lowered Nrf2 levels significantly (↓ 0.66-fold, low
dose), this antagonistic effect compared to SFN alone (↑ 1.86-fold) was overcome by cells
treated with all three compounds, where again, Nrf2 levels were found increased (↑ 1.92-fold,
EGCG in low dose), compared to medium treated control cells. Moreover, the mitochondrial
membrane potential revealed yet another interesting result with a significant decrease when
all three substances were added together (***).
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All in all, our study demonstrated that the dietary phytochemicals sulforaphane (SFN),
quercetin (QUE), and epigallocatechin-3-gallate (EGCG) showed diverse nutrigenomical
behaviors alone, but even more so in combination, leading to conclusions which, in our eyes,
have the potential to be taken further. Of course, the obtained data is valid in vitro only and
thus, great care should be taken when extrapolating conclusions from cell culture studies to
dietary in vivo situations. However, it is undeniable that the bioactivities encountered are
indicating meaningful trends, calling for further pharmacokinetic studies and trials. The
results clearly imply that these substances, potentially even in combination, might be
important in the protection against oxidative stress mediated, chronic, degenerative
diseases.
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4.8 Conclusions
This thesis elucidates on how dietary phytochemicals can function as either antioxidants or
prooxidants and therefore influence health by impacting on the cells’ redox status. Thereby
the modes by which these substances control the redox balancing strategies of a cell via
Nrf2 were investigated and it was assessed how they interact with the endogenous
thioredoxin (antioxidant) systems in place.
Within the workflow and research milestones decided on, the following aims were achieved:
Selection and validation of dietary phytochemicals.
Analysis of the effects of the selected dietary phytochemicals on the cell’s viability by
assessing their metabolism in the HepG2 model. The IC50 values were determined
and concentrations for further experiments were chosen accordingly, so that
apoptotic effects did not influence the outcomes.
Furthermore, the precise modes of action of the potential “antioxidants” were
investigated by determining their direct ROS-scavenging capacity, compared to their
indirect Nrf2-transactivating potential in the HepG2 model.
Also, the Nrf2-transactivation mechanism of the selected dietary antioxidants was
analyzed by determining the potential translocation of Nrf2 into the nucleus in HepG2
cells.
To assess the expression level of heme oxygenase-1, thioredoxin-1, and thioredoxin
reductase-1 as target genes of the Nrf2-pathway, cells were first subcellularly
fractioned before the individual compartments were analyzed with the Western
blotting technique.
Moreover, the potential changes in the mitochondrial potential upon treatment with
these dietary phytochemicals in HepG2 cells were measured.
These results agree with previous studies that describe bioactives using different
experimental approaches. Most importantly, this study highlights and validates the previously
suggested hypothesis of the bifunctional mode of action of dietary phytochemicals in their
function as antioxidants (233). This aspect is visualized in the following diagram (Figure 55),
depicting sulforaphane and quercetin as representatives for each conserved function. Please
note, that quercetin is on the direct side, since it has a much stronger effect as such, while it
only slightly induced Nrf2 in HepG2 cells in our study.
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Figure 55: Bifunctional antioxidative capacity, A) direct ROS-scavenging action of dietary phytochemicals like quercetin, B) indirect antioxidant action via Nrf2 of bioactives like sulforaphane.
Moreover, this project accurately highlighted the mechanism of the electrophilic attack,
responsible for the dissociation of Nrf2 from Keap1. Sulforaphane, as our prime example,
acts as an electrophilic Nrf2 activator, as it modifies residues in the IVR region of the Keap1
dimer, thereby disrupting the bond between the DLG motif of Nrf2’s Neh2 domain and
Keap1’s ETGE motif, loosening the hinge and latch mechanism , and thus, triggering Nrf2’s
translocation into the nucleus (47). This way, Nrf2 escapes its proteasomal degradation and
translocates into the nucleus in order to trigger phase II protein expression.
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Figure 56: Nrf2-pathway, 1) degradation under normoxia, 2) induction via an electrophilic attack, leading to the expression of phase II enzymes.
Hence, this report strengthens aspects of the “antioxidant hypothesis”, stating that dietary
compounds might be able to influence an organisms’ “oxidative stress level”, as it implies
interplay not only in a direct mode of action, but also indirectly via facilitating and inducing the
Nrf2 pathway. This subsequently leads to the adaptive stress response exercised by a
battery of phase II enzymes with cytoprotective (antioxidant) properties. Henceforth, when it
comes to a number of pathologies where oxidative stress might be considered the root,
bioactive components in food have two ways to influence this status, proving the public
health strategies of prevention, e.g. 5-a-day-campains, on the right track.
Isothiocyanates, when added to cell culture, are able to up-regulate the phase II
detoxification enzymes, supporting the suggestion that in vivo they might enhance clearance
of chemical carcinogens and thus block chemical carcinogenesis. On the down-side, an
excessive phase II metabolism may, of course, also relate to a drug’s fast degradation.
Even though the limited bioavailability of dietary phytochemicals and especially polyphenols
such as EGCG is often criticized, it has for instance been measured that drinking green tea
results in concentrations of about 50 µM EGCG in the saliva, which has been shown to
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protect salivary glands from the harmful effects of γ-irradiation and chemotherapy with cis-
platinum(II) diamine dichloride (CDDP) (234).
The findings of the study presented should also be a reminder that all compounds are toxic
and that a safe, tolerable upper level needs to be determined, particularly when bioactive
components are isolated from whole foods and provided as a supplement. Hence, it was also
elucidated on how the controversy of the effects of natural compounds arose, and a major
reason is that there is a substantial difference between the effects of a combination of
compounds consumed/administered as a whole and the influences single compounds have,
due to additive, synergistic and/or antagonistic effects. This projects nicely observed this
phenomenon.
Last, but not least, in recent times, the concept of prooxidants as a means of fighting cancer
has triggered the interest of many scientists who are exploring the “oxidation hypothesis”
and its possible therapeutic measures. Prominent members of the scientific community, such
as Jim Watson, have directed their research efforts towards the investigation of the concept
of “oxidation therapy”. This form of therapy is based on the observation that drugs as well as
dietary agents (such as EGCG, resveratrol, curcumin, paclitaxel, etc.) generate hydrogen
peroxide, which can kill cancer cells, but at the same time affect healthy cells only marginally
(132; 223; 235).This study adds to this knowledge base by showing the prooxidative effect of
EGCG under the influence of the laser utilized in confocal microscopy, and thus highlighting
on this side of dietary phytochemicals in their modes of influencing Nrf2 also.
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Table 15: Summary of the main results yielded by the presented study of single substances.
Results summarized – in numbers Results summarized – simplified
Substance tested
IC50 (24 hrs)
[µM] ROS ARE Substance
tested
IC50 (24 hrs)
[µM] ROS ARE
Sulforaphane (SFN)
76.36 No inhibitory
effect
4.2 ± SEM 0.3-
fold [10 µM]
Sulforaphane (SFN)
<100 µM X ***
Quercetin (QUE) 354.3 31.8 ± SEM
3.6% [10 µM]
1.4 ± SEM 0.1-
fold [10 µM] Quercetin (QUE) >100 µM *** ***
Epigallocatechin-3-gallate (EGCG)
180.5 54.8 ± SEM
2.7% [20 µM]
1.9 ± SEM 0.2-
fold [200 µM]
Epigallocatechin-3-gallate (EGCG)
>100 µM *** X / [***]
Curcumin (CUR) 113.1 76.4 ± SEM
5% [10 µM]
3.0 ± SEM 0.1-
fold [30 µM] Curcumin (CUR) >100 µM *** ***
Cinnamic acid (CIN)
No inhibitory
effect
No inhibitory
effect
1.15 ± SEM
0.07-fold [100
µM]
Cinnamic acid (CIN)
X X X / * - ***
Gallic acid (GAL) 248.5 88.3 ± SEM
3.1% [30 µM]
1.7 ± SEM 0.1-
fold [20 µM] Gallic acid (GAL) >100 µM * - *** ** - ***
Catechin (CAT) undetermined X
1.2 ± SEM
0.05-fold [400
µM]
Catechin (CAT) undetermined X X / * - ***
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Table 16: Summary of the main results yielded by the presented study of substances in combinations.
Results summarized - of combinations
Substance tested
IC50
(24 hrs)
SFN [µM]
IC50
(72 hrs)
SFN [µM]
Protein expression levels Mitochondrial membrane potential
(MMP) HO-1 Trx-1 TrxR-1 Nrf2
Sulforaphane (SFN)
76.36 52.91 ↑ ** ↑ **low
band ↑ *
Quercetin (QUE) 354.3 132.6 ↓ **high
band ↑
Epigallocatechin-3-gallate (EGCG)
180.5 173.7 ↓ * ↓ *low
conc. ↓ *high conc.
SFN + 50 µM EGCG 52.54 33.79 ↑ ** ↑ low band
SFN + 10 µM QUE 83.02 44.43 ↑ ↑
SFN + 50 µM EGCG + 10 µM QUE
65.47 28.90 ↑ * ↑ low band ↑ * ↓ *
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In this sense, the data presented revealed that some substances - most prominently
sulforaphane - cause a biphasic dose-response effect, showing toxicity at higher
concentrations, but with a strong capacity to activate adaptive cellular responses pathways at
lower doses due to its electrophilic nature. In general, it can be concluded that the effects
observed in all cases analyzed depend on factors such as the applied dose, the time period
of exposure as well as environmental conditions, and most certainly also on the cell type. On
the other side of the scale, the beneficial use of cells’ of ROS occurs at low and moderate
concentrations, causing threatening effects when in excess. Hence, the bottom line highlights
the very delicate balance, which depends on individual – cells’ and organism’s - needs and
supply.
Thus, while many studies conducted on dietary phytochemicals have merely highlighted that
their biological activities are a direct consequence of their antioxidative properties, emerging
findings (including ours) suggest that the health benefits attributed to the bioactive
constituents of fruits and vegetables are rather due to the electrophilic activation of adaptive
cellular stress response pathways than to acute cellular responses, or, maybe even due to
the capacity to utilize both - bifunctionally.
Along these lines, this thesis highlights the endogenous cytoprotective gene
expression induced by some representative exogenous dietary phytochemicals with
the Nrf2-Keap1 system as a prime molecular target investigated.
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4.9 Future Directions
Naturally, more detailed concentration-time-organelle resolved studies assessing both,
individual significant markers of cellular status at biochemical or phenotypical level, as well
as next generation –omics sequencing, are advisable as a follow-up to our study. For any
similar subsequent study, the parent compound in each model system should be analyzed
along with its in vivo occurring metabolites, an aspect neglected on purpose in the performed
experiments, but is certainly relevant and interesting to explore. Moreover, we propose the
following enhancement as a result to our experiences.
Furthermore, improvement of the “liver model” could be reached by growing HepG2 cells in
3D cultures, which has been shown to improve their morphological differentiation and
enhance their metabolic capacity (236). Co-cultures with other liver-relevant cells would
make the model even more reliable and a better predictor, especially for the influence of a
compound on liver toxicity (220). At the end of the day, the limitations for in vitro
methodologies are of course a given, just like for any technique, but a strategy to address
issues such as population dynamics, since there is no “standard human”, as well as patient
susceptibility could definitely be to employ human stem cell-derived (iPSC) differentiated
hepatocyte models (237). Additionally, advances in material science and bio-engineering
reveal promising indications that in the near future microfluidic devices could be employed,
which represent multi-organ systems in vitro (238). This would of course lead to an advanced
similarity to in vivo mechanisms (239). Most importantly, the purpose of the respective study
has to define the necessary adaptations of the model, just like for our cause the applied
mode has proven valid.
Also, since toxicological studies commonly utilize the Nrf2-pathway, we propose an
enhancement in this respect and suggest the additional evaluation of sulfiredoxin-1 (Srxn1),
because it could be established as a more selective target-gene than most others (240).
Heme oxygenase-1 should be included in any study also though, because others have
shown that is an excellent biomarker for Nrf2 activation, even in vivo, as it is increased in
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acute and chronic renal disease (241-242). Moreover, for analysis in the field of toxicology, it
would be highly thrilling to take further approaches in elucidating the mechanisms of the
debated oxidative response element (ORE). Since there appears to be a certain threshold of
the adaptive stress response, which results in a completely different reply when met, it would
be highly valuable to find out more about how this can be triggered and if there may be a link
to the ORE (243). Obviously, this would be particularly interesting for the therapeutic context.
For future directions concerning the field of nutrigenomics, a number of desired approaches
come to mind. For instance, it would be highly interesting to determine the influence of
dietary phytochemicals under exact physiological conditions, in particular appropriate oxygen
levels as present in the gastro-intestinal system. To date, assays reported in literature have
been performed under normal atmospheric oxygen conditions (normoxic), but this is not
representative of the low (less than 2 %) oxygen levels present in the healthy intestine (the
so-called “physoxia”) (244). Understanding the complex cellular responses of these
compounds at physiologically-relevant oxygen levels could ultimately enable and enhance
the preparation of foods that provide appropriate levels of bioactive nutrients to complement
our lifestyles. This would have implications for both the food industry and the huge industrial
branch producing nutritional supplements. Supplements are believed to all demonstrate
sufficient nutrigenomic capability to modify biochemical and physiological risk factors of
major diseases, which requires valid testing.
Regarding future investigation in cancerogenomics and other diseases, more scientific efforts
and attention should be channeled towards the role of Trx and TrxR and their interplay with
the inflammasome. As explained above, inflammation is a process which leads to the
pathogenesis of various diseases such as cancer, autoimmune diseases, and diabetes
(203). Along these lines, atherosclerosis is one disease highly influenced by the
inflammasome, which can potentially be prevented or therapeutically treated with dietary
phytochemicals, because it has been shown that flavonoids, such as EGCG, can protect
against vascular endothelial inflammation via HO-1 (202). Recent research also highlights
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the Nrf2-HO-1 axis as a promising target for anticancer treatment, particularly in combination
with conventional antineoplastic approaches in the battle again cancer (245). Moreover, Nrf2
plays a decisive role in maintaining quiescence, survival, and stress resistance of cancer
stem cells, which are considered responsible for anticancer drug resistance and tumor
relapse after therapy (246). Similarly, Nrf2 has become a major target for the treatment of
inflammatory bowel disease (IBD), again, because it mediates the NLRP3 inflammasome
(247).
Furthermore, recent efforts in our laboratory have been directed towards measuring the
impact of particulate matter and its interplay with the Nrf2 pathway. This is of significance,
since chronic obstructive pulmonary disease (COPD) is a major and increasing global health
problem which is said to become the third leading cause of death worldwide by 2020. COPD
has also been associated to increased inflammation caused by elevated levels of ROS and
carbonyls. Thus, in the future it would be highly relevant and exciting to develop this
approach further.
While the complex underlying mechanisms of redox regulation are being explored step by
step, there is yet much to learn until we fully comprehend the redox networks and their
governing principles under defined physiological and pathological conditions. Each and every
step will reveal more of the scientific base to understand these biological system’s strategies
in health and disease. One thing is for sure: in the molecular logic of life, the redox code
provides a critical complement to the genetic code, the epigenetic code, and the histone
code.
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5 MATERIALS & METHODS
5.1 Dietary phytochemicals
Epigallocatechin-3-gallate (Cat.No.: 397-05-09, Batch#HWI00686-1) and Quercetin
dihydrate (Cat.No.: 0020-05-95, Batch#HWI00580) were obtained from HWI Analytik
GmbH, Rülzheim, Germany.
L-Sulforaphane (S6317), Tert-butylhydrochinone (112941, Lot#MKBN5279V), Curcumin
(C1386), (+)-catechin hydrate (C1251), Gallic acid (G7384, Lot#100MO258V) Trans-
cinnamic acid (C80857), and tert-Butylhydrochinone (4109BE-148, Lot#MKBN5279V)
were purchased from Sigma-Aldrich Handels Gmbh Vienna, Austria.
To ensure physic-chemical stability the pH-values of every substance investigated (i.e.
dietary phytochemicals) in solution was determined with a pH-meter 691 (from Metrohm
Inula GmbH, Vienna, Austria) and only used for treatment of cells if the value did not
significantly diverge from pure medium.
The dietary phytochemicals were solubilized with either DMSO or EtOH and stored as stocks
of 20 000 or 10 000 µM respectively at -20°C.
5.2 Antibodies
For the Western Blotting technique applied adequate antibodies had to be selected carefully.
Therefore, the following primary antibodies were chosen and acquired from Abcam Plc.,
Cambridge, England: anti-Keap1 [ab150654], anti-Nrf2 (H-300) [ab62352], anti-HO-1
[EP1391Y], and anti-Nrf2 (phospho S40) [EP1809Y]; and the subsequently listed ones from
Santa Cruz Biotechnology Inc., Heidelberg, Germany: anti-Trx (A-5) [sc-166393] and
anti-TrxR (B-2) [sc-28321]. (For more information please see Table 17.) The housekeeping
gene GAPDH was used as a loading control and the following product was applied: anti-
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GAPDH Mouse mAb (6C5) [CB1001], originally from Calbiochem, but purchased from Merck
GesmbH, Vienna, Austria.
Table 17: Identification and characterization of antibodies used for Western Blot analysis.
Primary Antibody against
Description Dilution Nr. Lot # Protein size
Keap1 Mouse, monoclonal, IgG1 raised against aa
380-624 of human Keap1
1:2 000 ab150654 [1F10B6]
GR118806-7 70 kDa
Nrf2 Rabbit, monoclonal, IgG, raised against aa
550 to the C-terminus of human Nrf2
1:2 000 ab62352 [EP1808Y]
GR107472-6 68 kDa
Nrf2p (phosphoS40)
Rabbit, monoclonal, IgG,
raised against phosphor-peptides
corresponding to residues near
serine 40 of Nrf2
1:2 000 ab76026 [EP1809Y]
YJ032305CS2 68 kDa
HO-1 Rabbit, monoclonal, IgG, raised against a peptide near the
C-terminus of human HO-1
1:2 000 ab52947 [EP1391Y]
YJ071709CS 33 kDa
Trx Mouse, monoclonal, IgG1, raised against aa
1-105 (=full length) of human
Trx-1
1:500 A-5, sc-166393
G1510 12 kDa
TrxR Mouse, monoclonal, IgG2a, raised
against aa 71-340 of human
TrxR-1
1:500 B-2, sc-28321
C1813 55 kDa
GAPDH Mouse, monoclonal, mAb
(6C5), Loading control!
1:10 000 CB1001 (6C5)
2626460 38 kDa
GAPDH (1st lot)
Mouse, monoclonal, mAb
(6C5), Loading control!
1:7 000 CB1001 D00155303 38 kDa
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Secondary Antibody against
Description Dilution Cat.Nr. Lot # Company
Anti-Rabbit Alexa Fluor®680, IgG, red,
raised in goat
1:10 000 A21109 37505A Molecular probes, Life
Technologies
Anti-Mouse IRDye® 800CW, green,
raised in donkey
1:10 000 926-32210
C30109-03 LI-COR Biosciences,
Westburg B.V., Leusden, The Netherlands
Anti-Rabbit
IRDye® 800CW, green, raised in goat
1:10 000 926-32211
C30829-02 LI-COR Biosciences,
Westburg B.V., Leusden, The Netherlands
5.3 Chemicals, reagents & kits
Please note that the following list is not meant to be exhaustive, since many compounds are
just routinely used in every laboratory and therefore focus was placed on the most important
and specific chemicals, reagents and kits (Table 18).
Table 18: Identification and source of chemicals, reagents and kits applied.
Product Supplier
Auranofin, Cat.No.: A6733
Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany; Lot#74M4728V
Bradford reagent, dye reagent concentrate, Cat.No.: 500-0006
Bio-Rad Laboratories GmbH, Munich, Germany
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), Cat.No.: C2920
Sigma-Aldrich GmbH, Vienna, Austria;
Celllytic M, Cat.No.: C2978 Sigma-Aldrich GmbH, Vienna, Austria; Lot#25M4084V
CellTiter-Blue™ Cell Viability Assay, Cat.No.: G8081
Promega GmbH, Mannheim, Germany; Lot#24M4003V
Dimethyl sulfoxide hybrid-max sterile (DMSO), Cat.No.: D2650
Sigma-Aldrich GmbH, Vienna, Austria; Lot#RNBC8967
Hank’s Balanced Salt Solution, Cat.No.: H8264
Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany; Lot#RNBD4326
NuPage® Transfer Buffer (20x), Cat.No.: NP0006
Life Technologies, Carlsbad, CA, USA; Lot#1676227
NuPage® MES SDS Running Buffer (20x), Cat.No.: NP0002
Life Technologies, Carlsbad, CA, USA; Lot#1540344
NuPage® 4-12% BT Gel, 1.0 mm, 10 well, Cat.No.: NP0321 Box
Life Technologies Austria, Vienna, Austria; Lot#14043071
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Product Supplier
Odyssey® Blocking Buffer, Cat.No.: 927-40000
LI-COR Biosciences, Westburg B.V., Leusden, The Netherlands; Lot#T2382
PageRuler™ Plus, prestained protein ladder, Cat.No.: 26620
Thermo Fisher Scientific Austria GmbH, Vienna, Austria; Lot#00146407
Phosphatase Inhibitor Cocktail 2, aqueous, Cat.No.: P5726
Sigma-Aldrich GmbH, Vienna, Austria
SYTO® 16, green fluorescent nucleic acid dye, Cat.No.: S7578
Thermo Fisher Scientific GmbH, Darmstadt, Germany
Tetramethylrhodamine, methyl ester, perchlorate, fluorescent dye, Cat.No.: T668
Molecular Probes™, Thermo Fisher Scientific GmbH, Darmstadt, Germany
Thioredoxin (3mg/ml) Kind gift of Kathryn Tonissen, Griffith University
WGA AF647, wheat germ agglutinin, Alexa Fluor® 647 Conjugate
Molecular Probes™, Thermo Fisher Scientific GmbH, Darmstadt, Germany
Kits
Product Supplier
ARE-Assay Kit contents: Beta-Lactamase Loading Solutions, Cat.No.: K1085 Solution D, Cat.No.: K1156
Life Technologies, Madison, WI, USA; 1st - Lot#1389734, 2nd – Lot#1419368, 3rd – 1495733, 4th - Lot#1495733 Lot#1226636B
Cell fractionation Kit, Cat.No.: ab109719
Abcam Plc., Cambridge, England; Lot#GR166157-5, H0387
Mitochondrial membrane potential indicator Kit, Cat.No.: CB-80600-010
Codex BioSolutions, Inc., Gaithersburg, MD, USA
Thioredoxin reductase assay Kit, Cat.No.: CS0170
Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany; Lot#25M4046V
5.4 Cell culture
All experiments involving cell culture techniques were performed according to the
international state-of-the-art SOPs, also valid at the Medical University of Innsbruck, and
specified for instance in (248-249). Moreover, besides the special in-house trainings, an
advanced training course was also undertaken at the Technical University of Dresden (TU
Dresden).
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Table 19: Identification and source of specific cell culture materials and reagents.
Cell culture materials & reagents
Product Supplier
Blasticidin S-hydrochlorid, Cat.No.: N15205
Sigma-Aldrich GmbH, Vienna, Austria; Lot#BCBN1781V
Cell Culture Dish, 100x20 mm, Cat.No.: 353003
Corning B.V., Amsterdam, Netherlands; Lot#3310550
CellSensor® ARE-bla HepG2 Cell Line, Cat.No.: K1633
Invitrogen™ by Thermo Fisher Scientific Austria GmbH, Vienna, Austria; Lot# 1450643
FBS, dialyzed, Cat.No.: 26400044
Life Technologies, Madison, WI, USA; Lot#1259687
FBS, normal, Cat.No.: A15-101
PAA Laboratories GmbH, Pasching, Austria; Lot#A10109-2400
75cm2-Flask, canted vented, Cat.No.: 353136
Corning B.V., Amsterdam, Netherlands; Lot#3149111
96-well Plate, BLK, CFB, W/LIT, S, IN 39269097, Cat.No.: 3603
Corning B.V., Amsterdam, Netherlands; Lot#314023
96-well Plate, black, clear bottom, Cat.No.: COS3603
Szabo-Scandic GmbH, Vienna, Austria; Lot#25514016
24-well Plate, TCT, PS, W/LIT, S, IN 39269097, Cat.No.: 3526
Corning B.V., Amsterdam, Netherlands; Lot#1014030
6-well Plate, TC, F-Btm, W/LIT, PS50cs, Cat.No.: 353046
Corning B.V., Amsterdam, Netherlands; Lot#3221534
1 µ-Slide 8 well ibiTreat coverslip, for high resolution, tissue culture treated
ibidi GmbH, Martinsried, Germany; Lot#150504/4
Dish 100x20mm TC, Cat.No.: 12648010
Life Technologies Austria, Vienna, Austria; Lot#1645610
HepG2 cells, DSMZ, Cat.No.: ACC 180
Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany
Hepes Buffer (1M) ), Cat.No.: H0887
Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany; Lot#RNBC6920
Minimum Essential Medium Non-Essential Amino Acids (MEM NEAA, 100x), Cat.No.: 11140-035
Gibco by Life Technologies Austria, Vienna, Austria; Lot#1379988
Recovery™ Cell Culture Freezing Medium, Cat.No.: 12648-010
Gibco by Life Technologies Austria, Vienna, Austria; Lot#1645610
RPMI medium, Cat.No.: R8758
Sigma-Aldrich Chemie GmbH, Steinheim, Germany; Lot#RNBC4836
Trypsin Express, Cat.No.: 12604013
Life Technologies Austria, Vienna, Austria; Lot#1459816
Trypsin EDTA, Cat.No.: L11-004
PAA Laboratories GmbH, Pasching, Austria, Lot#L00413-1446
µ-Slide 8 well ibiTreat, 1.5 polymer coverslip, tissue culture treated, sterilized, Cat.No.: 80826
Ibidi, Martinsried, Germany; Lot#150504/4
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For the set of experiments presented in this thesis, the two cell lines shortly presented below
were used. They have been characterized as a suitable in vitro model for the study of
polarized human hepatocytes.
HepG2 – This cell line originated from the liver tissue of a 15-year-old Argentine male, with a
hepatocellular carcinoma, isolated in 1975. When differentiated, these cells grow adherently,
show an epithelial morphology as well as a robust formation of apical and basolateral cell
surface domains (250). HepG2 cells have been characterized as a suitable in vitro model
system, mimicking primary human hepatocytes, for the studies of liver metabolism and
toxicity of xenobiotics, due to their intact and inducible endogenous expression of phase I
and phase II enzymes (as was reviewed in (251)). Limitations of this cellular model are for
instance that
(i) the actual expression levels of enzymes can vary and particularly of phase I it
has been found to be lower than in humans;
(ii) genetic polymorphisms occur in humans, but not in cell lines, hence, also not
in HepG2 cells which makes it less representative as a model;
(iii) after the distribution of the clones from ATCC or any other cell line distributor,
they are treated (slightly) differently in different laboratories (e.g. different
medium), thus, this might for instance cause variability in sensitivities.
(reviewed e.g. in (252)).
Therefore, strictly following good laboratory practice (GLP), comparison between different
clones should be preceded by genetic characterization of each clone (e.g. by assessing short
tandem repeats (STR) as well as establishing a DNA fingerprint), and extrapolation to the
human body should be avoided. Nevertheless, it remains a suitable model of choice for
chemical risk assessment. HepG2 cells are used to study liver diseases (253), mechanisms
of action of drugs (254), as well as gene expression and transcription (255). In general,
human cell lines have been shown to be good in vitro models and sensitive tools for high-
throughput toxicity screening. Furthermore, they have the potential to reduce the use of
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animals in toxicological testing (256-257). Moreover, when studying cancer-related aspects,
recent studies have pointed out the profound advantages of working with a 3D model of
HepG2 cells (258). In 3D cultures, they also express relatively high levels of phase I and
phase II metabolizing enzymes (which is comprehensively reviewed in (259)).
The human hepatocellular carcinoma cell line HepG2 (DSMZ, Germany) was cultured in
RPMI-1640 medium (Sigma-Aldrich, Germany) supplemented with 10% (v/v) heat inactivated
fetal bovine serum (FBS) (Gibco, Germany) at 37°C in a humidified atmosphere with 5%
CO2.
HepG2-ARE-bla – This name depicts a CellSensor® system (Invitrogen, Germany) and
describes modified HepG2 cells which contain a stably integrated bacterial β-lactamase
reporter gene under the control of the Antioxidant Response Element (ARE) (pLenti-
bsd/ARE-bla Vector), which can be induced through the corresponding endogenous
transcription factor Nrf2. Moreover, the valid clone can be selected by supplementing the
medium with 5 μg/ml of Blasticidin (Sigma-Aldrich, Austria). Passaging the cell line requires a
special growth medium, RPMI-1640 (Sigma-Aldrich, Germany) supplemented with 10% (v/v)
heat inactivated and dialyzed FBS (Life Technologies, USA) as well as 0.1 mM non-essential
amino acids (MEM NEAA) (Gibco by Life Technologies, Austria) and 25 mM Hepes (pH 7.3)
(Sigma-Aldrich, Germany). The cell line was treated according to the manufacturer’s
guidelines and the results were compared to the Validation & Assay Performance Summary.
This reporter gene cell line was used to investigate the transcriptional activation of ARE-
mediated gene expression mediated by Nrf2 upon treatment.
During the experiments, the cultures were all maintained in a medium free of antibiotics.
Periodically, the cells were tested for mycoplasms. Fortunately these tests always resulted in
a negative outcome.
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The actual protocols composed and followed for the different cell lines as well as for this
assay were the following:
The sub-culturing procedure was followed as recommended by the ATCC (American Type
Culture Collection). This procedure can be summarized in the following steps:
1. Remove and discard culture medium. 2. Briefly rinse the cell layer with PBS, to remove all traces of serum that contains
trypsin inhibitor, and add 0.25% (w/v) Trypsin 0.53 mM EDTA solution. 3. Add 1 ml of TrypsinEDTA solution to flask, facilitate dispersal by placing them in the
incubator, and observe cells periodically until cell layer is dispersed (usually within 5 to 15 minutes).
4. Add 4 ml of complete growth medium and aspirate cells by gently pipetting up and down.
5. Add appropriate aliquots of the cell suspension to new culture vessels (2x105 cells/ml for HepG2; 1x105 cells/ml for HepG2-ARE-bla).
6. Incubate cultures at 37°C.
A sub-cultivation ratio of 1:4 to 1:6 as recommended by the ATCC was kept by renewing the
medium twice per week.
5.5 Cell proliferation & viability
To measure the viability of HepG2 cells we used the CellTiter-Blue™Cell assay (Promega,
Germany), which provides a fluorometric method using the indicator dye resazurin to
estimate the number of viable cells. The underlying principle of this assay relies on the fact
that living intact cells are metabolically active and able to convert the redox dye resazurin
into the fluorescent end product resorufin, as shown in Figure 57. Therefore, HepG2 cells
(2x104/well) were seeded into 96-well plates, cultured for 24 hrs and then either left untreated
or treated with a solvent (EtOH or DMSO respectively) or increasing doses of dietary
phytochemicals (SFN: 5-75 µM, CUR: 5-100 µM; QUE: 10-100 µM, CIN: 10-200 µM; EGCG:
20-200 µM, CAT: 20-200 µM, GAL: 20-400 µM) for 72 hrs. Thereafter, 10% (v/v) CellTiter-
Blue™ reagent was added. After 2 hrs of incubation the fluorescence was determined at
544 nm excitation / 590 nm emission using a Fluoroskan Ascent FL plate-reader (Thermo
Labsystems, USA), determining the exact percentage of metabolically active and hence
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viable cells. The half maximal (50% inhibitory) concentration (IC50) was then calculated using
the original concept of Chou and Talalay by using the CalcuSyn software (Biosoft, UK) (260).
Later, these values were recalculated and validated by using GraphPad Prism for Windows,
Version 6.00 (GraphPad Software, Inc., La Jolla, CA, USA), which was also used to generate
the figures.
Figure 57: CellTiter-Blue™ Cell assay, to assess cell viability.
5.6 Measurement of intracellular ROS-inhibition
The fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA) (Sigma-Aldrich, Austria)
was used as a substrate to monitor the intracellular accumulation of ROS in HepG2 cells, as
described previously (261). DCFH-DA diffuses through cell membranes and is hydrolyzed by
intracellular esterases to non-fluorescent 2',7'-dichlorofluorescin (DCFH), which is
subsequently trapped within the cell. In the presence of ROS, DCFH is rapidly oxidized to
highly fluorescent 2'-7'- dichlorofluorescein (DCF). The intensity is proportional to the amount
of intracellular ROS (Figure 58, side A) (262-263). Antioxidants, which scavenge the applied
AAPH, hence diminish the fluorescent signal (Figure 58, side B). 10 μM quercetin (Sigma-
Aldrich, Austria) turned out to be a very potent antioxidant and, thus, was established as a
positive control.
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Figure 58: ROS-assay, to evaluate the direct antioxidant potential.
5.7 Assessment of intracellular Nrf2-transactivation
5.7.1 ARE-GeneBLAzer β-lactamase reporter gene assay
2.9x104 ARE-bla HepG2 cells/well were plated into a 96-well plate. 7 hrs after seeding, the
cells were either left untreated or treated with the dietary phytochemicals (SFN, CUR: 5-
100 µM; QUE, CIN: 10-200 µM; EGCG, CAT, GAL: 20-400 µM), or tBHQ (50 μM) as a
positive control or a solvent (EtOH or DMSO respectively) as a negative control. 15 hrs after
treatment, cells were loaded with LiveBLAzer™-FRET B/G substrate CCF4-AM (Invitrogen,
Austria) for 2 hrs, according to the manufacturer’s protocol. The β-lactamase expression was
determined by enzyme-mediated cleavage of the fluorescence resonance transfer (FRET)
substrate (264). Fluorescence emissions (414/460 nm and 414/538 nm) were measured with
a Fluoroskan Ascent FL plate-reader (Thermo Labsystems, USA). The response ratios were
calculated as the mean fold induction of β-lactamase activity relative to the solvent control
(set to 1).
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Figure 59: ARE-assay, to measure the indirect antioxidant potential.
5.8 Assessment of intracellular Nrf2-translocation
5.8.1 Subcellular fractionation & Western blot analysis
Cells were harvested, lysed, and subfractioned 24 hrs after being treated using the Cell
Fractionation Kit (Abcam, England) according to the manufacturer’s protocol. After Bradford
determination, equal amounts of proteins of lysed cells were then separated by SDS-PAGE
on 4-12 % Bis-Tris NuPAGE gradient gels (Invitrogen, Germany) and transferred to a PVDF
Immobilon-FL membrane (Millipore, Germany). Nonspecific binding sites on the membranes
were blocked in Odyssey blocking buffer (LI-COR Biosciences, Germany) and after
incubation with the adequate primary and secondary antibodies imaged with an Odyssey
infrared imaging system (LI-COR Biosciences, Germany) at a scan intensity of 5 and scan
resolution of 169 μm.
Example for an actual protocol composed and followed for whole cell extract preparation:
WESTERN BLOTTING
A) WHOLE CELL EXTRACT PREPARATION/ HARVEST CELLS
wash cells 3 times with ice cold PBS
add 150-200 µl Lysis buffer with supplemented protease inhibitors (for 6-well)
harvest cells with a cell scraper and transfer into eppi
116
crack cells by 3 cycles of freeze (liquid nitrogen for 30’’) and thaw (25°C, shaking) =>
shake for 30’ in the cold room
shock freeze and store at -20°C OR continue with Bradford to measure protein
concentration
B) MEASURING PROTEIN CONCENTRATION/ BRADFORD
(if proteins were stored put them on ice immediately, otherwise keep them on ice)
prepare Bradford reagent 1:4 with A.d. (5 ml + 20 ml) and rinse through 2 sterile filters
put 200 µl into each well of a 96-well plate
in triplicates, add the right amount of BSA-Standard (0-7 µl) or 1 µl of sample
incubate for 5’ at RT, then measure
calculate for the desired amount of protein, mix with A.d. and 5xLaemmli buffer and
freeze at -20°C or continue with loading it onto a gel
C) GEL PREPARATION
if not using a ready-made one, use the recipe of Life technologies (see Appendix)
D) GEL ELECTROPHORESIS
if using the Bolt Mini Gel Tank: place the base on a flat surface, and snap the
electrophoresis tank into the base; place the cassette clamps; fill the chamber with
400 ml running buffer, just above the level of the electrode;
remove the tape (if pre-casted) and place the gel inside => remove the comb and
rinse wells with 1x running buffer
load protein marker ladder (i.e. PageRuler Plus Prestained Protein Ladder); thaw it at
room temperature, mix thoroughly, and load 4 µl per well in a 0.75 to 1 mm thick mini-
gel; return ladder to freezer (-20°C);
heat the samples for 5’ to 95°C, spin them down to, and load them onto the gel
run the gel at 80 V to start with, increase to 140 V or 160 V after 15’
(IMPORTANT: Only valid for NuPage Bis-Tris 4-12% gels and MES)
run gel until LBS marker reaches the bottom of the gel
E) BLOTTING
discard the running buffer, disassemble the gel, and remove the separation gel using
the spacer
place the gel, the sponges (3 thin, 3 thick ones), and the 2 whatman papers in blotting
(transfer) buffer and soak them for 15’
write date, protein & name on the membrane, then activate it in methanol for 15’’, then
2’ in A.d., then 5’ in blotting (transfer) buffer
assemble the “sandwich”:
- black side of cassette
- sponges – 1 thick, 1 thin
- whatman paper
- gel
- membrane (Odyssey)
- whatman paper => role out air bubbles!
- sponges – 2 thin,1 thick
clamp all together tightly after ensuring no air bubbles have formed between the gel
and the membrane => role out properly!
assemble blotting chamber
fill the chamber with cool blotting (transfer) buffer and the outside with cool A.d.
blot at 300 mA for 1.5 hrs (90’)
117
after disassembly, wet the membrane for several minutes in PBS, before transferring
it into the Odyssey blocking buffer
store the blotting (transfer) buffer for the next Western Blot
F) DETECTION OF THE PROTEIN
block the membrane for 1 hr at RT (or overnight at 4°C, reuse buffer up to 3 times)
incubate for 1 hrs with the primary antibody (diluted in Odyssey blocking buffer)
wash 4-5 times for 5’ in PBS 0.1% Tween
incubate for 30’ with the secondary antibody (diluted in Odyssey blocking buffer),
protected from light
wash 4-5 times for 5’ in PBS 0.1% Tween, protected from light
keep membrane in PBS and SCAN with the Odyssey scanner, which detects the
chemiluminescence emanating from the membrane, transforming the infrared
fluorescence signal into a digital image for rapid analysis
afterwards the membrane may be kept in PBS (also overnight) and a second antibody
may be investigated
G) BUFFERS AND REAGENTS
Bradford reagent, Biorad 500-0006
- use 1:4 dilution e.g. 5 ml Bradford + 20 ml A.d.
- always filter before use!
BSA standard: Stock: 10 mg/ml; Working solution: 0.4 µg/µl
- use 1:25 dilution e.g. 6 µl BSA + 144 µl Lysis buffer
Measured at 977 and 595 nm with a PowerWavex (BioTek Instrument, Inc., from
Szabo-Scandic GmbH & Co KG, Vienna, Austria)
Running Buffer, MES SDS Running Buffer (20x),REF:B0002, LOT:1368831 , Life
Technologies
- for 400 ml use 20 ml Buffer + 380 ml A.d.
Transfer Buffer (20x), REF:BT0006, LOT:1351330, Life Technologies
- for 400 ml use 20 ml Buffer + 380 ml A.d. + 40 ml MeOH
H) Further information and resources
http://www.youtube.com/watch?v=yy8f39XCQgs - Invitrogen NuPage® Novex® Gel
System
http://www.youtube.com/watch?v=uTY96pBj26o - How to perform a traditional wet
protein transfer using the XCell SureLock
PageRuler Plus Prestained Protein Ladder (Thermo Scientific)
118
5.9 Assessment of mitochondrial membrane potential (MMP)
5.9.1 MMP investigated via confocal microscopy analysis
60 000 HepG2 cells per well in 200 µl medium were grown in special cell culture treated 1 µ-
Slide 8-well coverslips (ibiTreat, ibidi GmbH, Germany) for 24 hrs. At that point they were
treated respectively for another 24 hrs. After staining the cells with 100 nM TMRM (red
channel), 100 nM Syto16 (green channel) and 100 nM WGA AF647 (blue channel) for 30
minutes at 37°C they were then imaged by fluorescence microscopy. The images were
obtained with an Olympus IX-70 inverted microscope (Olympus America, Melville, NY, USA)
with an Olympus 40x water immersion objective and an Olympus U-RFL-T Mercury-vapor
lamp. Since the primary aim was to analyses the influence of the selected dietary
phytochemicals on the mitochondrial membrane potential, the TMRM staining was analyzed
and quantified in detail, while the other two – Syto 16 and WGA AF647 (both purchased from
Thermo Fisher Scientific, Inc., Germany; see Chemicals, reagents & kits), staining the nuclei
and the general structure of the cell by visualizing its glycoproteins, served as controls.
TMRM (Thermo Fisher Scientific, Inc., Germany) is known as tetramethylrhodamine, methyl
ester, perchlorate and under the molecular formula: C25H25ClN2O7 exhibiting a molecular
weight of 500.93 g/mol. When assessing mitochondrial activity it is a valid and useful tool, as
the cell-permeant, cationic, red-orange fluorescent dye is readily sequestered by active
mitochondria (186). Subsequently, mean grey values were deduced with Image J (Version
win64 Fiji Is Just) software (265), subtracting the background first and then assessing the
mean of all regions of interest (ROI) applying the ROI manager.
Table 20: Experimental set up of coverslips for microspial analysis.
Well 1: untreated Well 2: 10 µM SFN Well 3: 10 µM QUE Well 4: 50 µM EGCG
Well 5: 200 µM EGCG
Well 6: 10 µM SFN + 10 µM QUE
Well 7: 10 µM SFN + 50 µM EGCG
Well 8: 10 µM S + 10 µM Q + 50 µM E
119
5.9.2 MMP investigated via fluorescence plate reader
Based on the principles published (266) and the recommendation supplied by the
manufacturer (Codex BioSolutions Inc., MD, USA) of the mitochondrial dye, the following
parameters were established and optimized for our purposes. Cells were seeded out in 96
well plates at a concentration of 3.2x105 cells/ml, 100 µl/well, the day before the experiment
and incubated at 37°C, 5% CO2. On day 2, the medium was replaced by 50 µl of fresh
culture medium and 50 µl of 2x dye-loading solution was added to each well, before
incubating the plate for 30 minutes at 37°C, 5% CO2. Thereafter, the cells were washed with
m-MPI assay buffer and diluted to a reaction volume 75 µl, cells were either left untreated
(+25 µl HBSS) or treated with 25 µl of (4x) a solvent (EtOH or DMSO respectively) or
increasing doses of the test compounds. The addition was followed by immediate analysis
with the fluorescence plate reader (Infinite® F200 PRO Multimode Reader, Tecan Trading
AG, Switzerland). While filter 1 (excitation 515 nm, emission 538 nm) detected the monomer
form, filter 2 (excitation 544 nm, emission 590 nm) measured the J-aggregated form, thus,
the ratio was used to quantify changes in the mitochondrial membrane potential. Carbonyl
cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was used as a control. Hence, this
dual kinetic assay, with on-line compounds addition, was performed 7 times, every 5 minutes
(for 35 minutes in total).
Figure 60: Mito-assay, to obtain changes in the mitochondrial membrane potential.
120
5.10 Statistical analyses
Since the statistical analysis varied for each assay, please consult the corresponding
RESULTS chapter for a more specific statement. Unless stated otherwise, the mean ±
standard error of mean (SEM) values was calculated to summarize and visualize all
measurements. The significance of the difference among mean values was then determined
adequately (e.g. with ANOVA followed by Dunnett’s multiple comparisons test). Statistical
significance was considered at a p value of ≤ 0.05. All statistical analyses were conducted as
denoted correspondingly in the results section using one of the following applications and
tools:
- IBM SPSS Statistics for Windows, Version 21.0 (IBM Corp., Armonk, NY, USA),
or
- GraphPad Prism for Windows, Version 6.00 (GraphPad Software, Inc., La Jolla,
CA, USA), as well as
- R, Version x64 3.2.1. (The R Foundation for Statistical Computing, Vienna, Austria),
and
- Image J, Version win64 Fiji Is Just (265).
121
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2. Shen G, Kong AN. Nrf2 Plays an Important Role in Coordinated Regulation of Phase II Drug
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6.1.1 Competing interests & Funding
The authors declare that they have no competing interests.
This work was supported by the Austrian Research Promotion Agency (FFG, project number
840590). Nevertheless, the content of this thesis does not necessarily reflect the views or
policies of the funding sources.
Curriculum vitae
Mag.
Martina
ÜBERALL
(Naschberger)
Research Associate & PhD-Candidate
Department for Medical Biochemistry,
Medical University of Innsbruck, Austria
Educational details
2012 – 2016 PhD-Program - Molecular Cell Biology
Department for Medical Biochemistry,
Medical University of Innsbruck, Austria
Title of PhD Thesis: “Redox-Balance & Electrophilic Attack – The Bidirectional Function of Dietary Phytochemicals.”
2011 – 2012 Advanced Teaching Traineeship
Pädagogische Hochschule Tirol, Austria
2005 – 2011 Master in Biology & English - Teacher’s Diploma
University of Innsbruck, Austria
Thesis at the Institute for Biomedical Aging,
Austrian Academy of Sciences (ÖAW)
Title of Diploma Thesis: “Expression of Immunoregulatory Proteins after CMV Infection”
2009 – 2009 Scientific Project {Grant}
School of Biomolecular and Physical Sciences,
Griffith University, Brisbane, Australia
Title of Scientific Project: “Analysis of the Thioredoxin Promoter in Response to Oxidative Stress”
2003 –2005 International Baccalaureate Diploma {Grant}
Lester B. Pearson College, Victoria, Canada
1997 – 2003 High School - Wirtschaftskundliches Realgymnasium
der Ursulinen, Innsbruck, Austria
Professional career
Since 2015
Since 2012
Lecturer at the Management Center Innsbruck (MCI)
Research Associate & PhD-Candidate
Department for Medical Biochemistry,
Medical University Innsbruck, Austria
Main Project: FFG840590 HQ, Philips GmbH and CTR
Competencies: Project management, Grant applications, Scientific research, consulting & translation for industry
Since 2011 Lecturer at the Pädagogische Hochschule Tirol
Physiology
Neurobiology (sensory system) & Immunology
Nutritional ecology
Inter-disciplinary course, focusing on nutrition and:
health, the environment, society & the economy
2011 – 2012 Teacher Trainee, Pädagogische Hochschule Tirol, Austria
2010 – 2012 Editorial Staff, Magazine of the Educational Faculty (BIWI),
University of Innsbruck, Austria
2009 – 2010 Scientific employee & Diploma student
Institute for Biomedical Aging,
Austrian Academy of Sciences (ÖAW)
Since 2005 Apprenticeships
BIOCRATES – Lifesciences AG
Intern marketing department
Tiroler Sparkasse AG
Executive assistant
Oncological Unit at the University Hospital
Miscellaneous responsibilities
Trainings & Workshops (a selection)
11/2015 Project Management, Pentalog Unternehmensberatung, Austria
10/2015 Summer School of Excellence, TU Dresden, Germany {Grant}
10/2015 European Health Forum Gastein 2015, Austria {Grant}
10/2014 European Health Forum Gastein 2014, Austria {Grant}
09/2014 Scientific writing, Medical University Innsbruck, Austria
07/2014 Health Communication & Health Promotion (Summer School)
at the Maastricht University, Netherlands
06/2014 Person-Centered Care, Primary Health Care {Grant}
European Commission, DG Sanco, Brussels, Belgium
09/2013 The Digital Future of our Health Care System {Grant}
European Commission, DG Connect, Brussels, Belgium
10/2013 European Health Forum Gastein 2013, Austria {Grant}
08/2013 Health Care & Social Systems (Summer School) {Grant}
European Forum Alpbach, Austria
2011 – 2012 Diploma in Experiential Pedagogy
College for Social Pedagogy, Stams, Austria
08/2005 NLP-Practitioner
Metaforum, Balatonfüred, Hungary
Community & Voluntary activities
Since 2009 Member of the Society for Biology, Biological Sciences
& Biomedicine, VBio, Munich, Germany
Since 2003 Member of the Austrian United World Colleges Network