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))

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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-

106

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).

109

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

111

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.

112

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

113

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.

114

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).

115

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

Metabolize Enzymes and Phase III Drug Transporters. Biopharm Drug Dispos. 2009; 30(7): p.

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140

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