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University of Veterinary Medicine Hannover

Institute for Food Toxicology and Analytical Chemistry

The effect of resveratrol and resveratrol imine analogues on human

tumor cell lines

THESIS

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY

(PhD)

awarded by the University of Veterinary Medicine Hannover

by

Shan Wang

Henan, P.R. China

Hannover, Germany 2016

Supervisor: Prof. Dr. Pablo Steinberg

Supervision Group: Prof. Dr. Pablo Steinberg

Prof. Dr. Hassan Y. Naim

Prof. Dr. Peter Winterhalter

1st Evaluation: Prof. Dr. Pablo Steinberg

Institute for Food Toxicology and Analytical Chemistry,

University of Veterinary Medicine Hannover, Germany

Prof. Dr. Hassan Y. Naim

Department of Physiological Chemistry,

University of Veterinary Medicine Hannover, Germany

Prof. Dr. Peter Winterhalter

Department of Food Chemistry,

Technical University of Braunschweig, Germany

2nd Evaluation: Prof. Dr. Sabine Kulling

Department of Safety and Quality of Fruit and Vegetables,

Max Rubner-Institut, Karlsruhe, Germany

Date of final exam: 06.04.2016

Shan Wang received financial support from the China Scholarship Council (CSC).

To my family

Publications and presentations:

Publications:

Empl, M. T., Albers, M., Wang, S., Steinberg, P. (2015): The resveratrol tetramer r-viniferin

induces a cell cycle arrest followed by apoptosis in the prostate cancer cell line LNCaP.

Phytother Res 29(10):1640-1645.

Oral Presentations:

Wang, S., Empl, M. T., Steinberg, P.: Antiproliferative effect of an imine analogue of resveratrol

on human tumor cells. 23rd

VETPHARM-Symposium. Sep. 12.-13. 2013, Giessen, Germany.

Poster presentation:

Wang, S., Empl, M. T., Steinberg, P.: The antiproliferative effect of imine analogues of resveratrol

on human tumour cells. 11th International PhD Student Symposium, Horizons in Molecular Biology.

September 09.-12. 2013, Göttingen, Germany.

Wang, S., Empl, M. T., Steinberg, P.: Inhibitory effects of resveratrol imine analogues on the

proliferation and cell cycle progression of human tumor cells. Annual Spring Congress of the

European Society of Veterinary Oncology, May 28.-30. 2015, Krakow, Poland.

Contents

Abbreviations ................................................................................................................................................ I

Summary ...................................................................................................................................................... III

Zusammenfassung ...................................................................................................................................... V

1. Introduction ........................................................................................................................................... 1

1.1. Cancer and dietary polyphenols ................................................................................................... 1

1.2. Effect of resveratrol in vitro and in vivo studies ....................................................................... 2

1.3. Resveratrol analogues ..................................................................................................................... 6

1.4. The cell cycle and its regulation .................................................................................................... 7

1.5. The COX-2/PGE2 pathway................................................................................................................ 9

1.6. The aim of study .............................................................................................................................. 14

2. Materials and Methods ...................................................................................................................... 15

2.1. Materials............................................................................................................................................. 15

2.2. Methods ............................................................................................................................................. 16

3. Results ................................................................................................................................................. 23

3.1. Growth inhibitory effect of the test substances ...................................................................... 23

3.2. Cell cycle distribution analysis .................................................................................................... 34

3.3. Effect of resveratrol and compound 5 on the COX-2/PGE2-pathway ................................. 46

3.4. Metabolic stability of compound 5 .............................................................................................. 48

4. Discussion .......................................................................................................................................... 49

4.1. Effect of resveratrol and IRAs on the proliferation of human tumor cell lines ............... 49

4.2. Effect of resveratrol and compound 5 on cell cycle distribution ........................................ 50

4.3. Effect of resveratrol and compound 5 on the COX-2/PGE2-pathway ................................. 54

4.4. Metabolism of compound 5 .......................................................................................................... 54

5. References .......................................................................................................................................... 57

6. Annex ................................................................................................................................................... 73

7. Acknowledgments ............................................................................................................................. 77

Abbreviation

I

Abbreviations

AA Arachidonic acid

ANOVA Analysis of variance

Apc Adenomatous polyposis coli

APS Ammonium persulfate

BSA Bovine serum albumin

CDK Cyclin-dependent kinase

COX Cyclooxygenase

dH2O Distilled water

DMEM Dulbecco's Modified Eagle Medium

DMSO Dimethyl sulfoxide

DMSZ German Collection of Microorganisms and Cell Culture

DNA Deoxyribonucleic acid

DTT Dithiothreitol

ECL Electrochemiluminescence

EDTA Ethylenediaminetetraacetic acid

EtOH Ethanol

FasL Fas ligand

FBS Fetal Bovine Serum

HEPES-buffer 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid buffer

HRP Horseradish peroxidase

IC50 Half maximal inhibitory concentration

IgG Immunoglobulin G

LC-MS Liquid chromatography-mass spectrometry

LC-UV Liquid chromatography-ultraviolet detection

M Molar

mA Milliampere

mM Milimolar

NF-kB Nuclear factor kB

NSAIDs Non-steroidal anti-inflammatory drugs

P/S Penicilin/Streptomycin

PBS Phosphate-buffered saline

Abbreviation

II

PGG Prostaglandin G

PGH Prostaglandin H

PGE2 Prostaglandin E2

PGs Prostaglandins

PI Propidium iodide

PMSF Phenylmethylsulfonyl fluoride

RCF Relative centrifugal force

RNA Ribonucleic acid

RNAse A Ribonuclease A

RPMI-1640 Medium Roswell Park Memorial Institute Medium

SD Standard deviation

SDS Sodium dodecyl sulfate

SRB Sulforhodamine B

T/E Trypsin/EDTA

TEMED Tetramethylethylenediamine

TRIS Tris-(hydroxymethyl)-aminomethane

V Volt

μm Micrometer

Summary

III

Summary

The effect of resveratrol and resveratrol imine analogues on human tumor cell

lines

Shan Wang

Dietary polyphenols such as resveratrol, which are commonly present in fruits and

vegetables, have been shown to possess cancer chemopreventive effects in vitro.

Resveratrol has been described as a cancer chemopreventive agent due to its growth

inhibiting activity in several different tumor cell lines. However, its chemopreventive

effect in vivo is a matter of debate, predominantly because of its low bioavailability.

Therefore, resveratrol analogues with potentially stronger health-promoting effects

have attracted the interest of the scientific community. Newly synthesized resveratrol

imine analogues (IRAs) have been reported as inducing stronger antioxidative effects

than resveratrol itself, but information regarding their potential anticarcinogenic

activities is still very limited. Therefore, the biological properties of five IRAs on

different human tumor cell lines were investigated.

The antiproliferative activity of five IRAs was first evaluated in HCT-116p53wt cells. The

results show that only compound 5 exerted a stronger antiproliferative effect than

resveratrol, whereas the other compounds were either not or only slightly cytotoxic.

Thus, only compound 5 was chosen as a candidate for studies to determine its ability

to inhibit the growth of various cancer cell lines, to modulate COX-2 protein

expression and to analyze its metabolic stability.

In agreement with previous studies, resveratrol led to a growth inhibition in cancer cell

lines, however, the effect was cell type-dependent. In this respect, compound 5

showed a similar behavior. Flow cytometric analyses revealed that both compounds

induce alterations in the cell cycle, thus indicating that the growth inhibitory effect of

resveratrol and compound 5 is associated with a cell cycle dysregulation. Western blot

Summary

IV

analyses confirmed the down-regulation of COX-2 protein expression after resveratrol

treatment in the HCA-7 cell line. In contrast, compound 5 induced the expression of

that protein. Further analyses by LC-MS revealed that resveratrol as well as

compound 5 both inhibited PGE2 production. Finally, the metabolic stability of

compound 5 was investigated by an in vitro glucuronidation assay, which revealed

that this IRA is less conjugated than resveratrol by rat liver microsomes.

In conclusion, the effects of compound 5 on human tumor cells is similar to that of

resveratrol, but the bioavailability of compound 5 is higher, mainly owing to the fact

that it is conjugated to a lesser extent than resveratrol itself.

Zusammenfassung

V

Zusammenfassung

Die Wirkung von Resveratrol und Resveratrol-Imin-Analoga auf humane

Tumorzelllinien

Shan Wang

In der Ernährung vorkommende Polyhenole wie Resveratrol, welche allgemein in

Früchten und Gemüse vorhanden sind, weisen in vitro antikanzerogene

Eigenschaften. Resveratol wurde als krebshemmende Substanz beschrieben, da sie

das Wachstum einer Reihe von Tumorzelllinien hemmt. Trotzdem ist seine

krebspräventive Wirkung in vivo sehr beschränkt, vorwiegend auf Grund seiner

geringen Bioverfügbarkeit. Deshalb hat die Suche nach Resveratrol-Analoga mit einer

potenziell stärkeren gesundheitsfördernden Wirkung als Resveratrol das

wissenschaftliche Interesse geweckt. In jüngster Zeit synthetisierte

Resveratrol-Imin-Analoga (IRAs) sollen eine stärkere antioxidative Wirkung im

Vergleich zu Resveratrol aufweisen; ihre antikanzerogene Aktivität ist bis heute aber

kaum untersucht worden. Aus diesemGrund wurden die biologischen Wirkungen von

fünf IRAs in verschiedenen Humantumorzelllinien untersucht.

In einem ersten Schritt wurde die antiproliferative Wirkung von fünf IRAs in

HCT-116p53wt-Zellen getestet. Die Ergebnisse zeigen, dass nur die Substanz 5 eine

stärkere Wirkung als Resveratrol aufwies, während die anderen Substanzen gar

keine oder nur eine geringfügige Zytotoxizität zeigten. Somit wurde nur die Substanz

5 bezüglich einer hemmenden Wirkung auf das Wachstum von Tumorzelllinien, der

Regulation der Expression von COX-2 und dessen metabolischen Stabilität weiter

untersucht.

In Übereinstimmung mit vorangegangenen Untersuchungen führte Resveratrol zu

einer Hemmung des Wachstums verschiedener Humantumorzelllinien, die jedoch

vom Zelltyp abhängig war. Diesbezüglich zeigte auch die Substanz 5 ein ähnliches

Verhalten. Durchflusszytometrische Untersuchungen bestätigten, dass beide

Zusammenfassung

VI

Substanzen zu Veränderungen im Zellzyklus führten, was darauf hinweist, dass die

hemmende Wirkung von Resveratrol und Substanz 5 in Verbindung mit der

Dysregulation des Zellzyklus steht. Eine Western-Blot-Analyse bestätigte die

Herunterregulation der COX-2-Proteinexpression nach Behandlung mit Resveratrol in

HCA-7 Zellen. Im Gegensatz dazu, induzierte die Substanz 5 die Expression dieses

Proteins. Weiterhin zeigt eine LC-MS-Analyse, dass sowohl Resveratrol als auch die

Substanz 5 die PGE2-Produktion unterdrücken können. Letztendlich wurde mittels

eines Glucuronidierungs-Assays die metabolische Stabilität der Substanz 5 in vitro

untersucht. Dabei wurde nachgewiesen, dass dieses IRA in einem geringeren

Umfang als Resveratrol durch Rattenleber-Mikrosome glucuronidiert wird.

Zusammenfassend lässt sich sagen, dass die Wirkung der Substanz 5 und

Resveratrol auf menschliche Tumorzelllinien ähnlich ist, dass aber die

Bioverfügbarkeit der Substanz 5 höher ist, da sie in geringerem Maße konjugiert wird.

Introduction

1

1. Introduction

1.1. Cancer and dietary polyphenols

Cancer is still a main health problem and one of the major reasons for mortality

worldwide (ZENG et al. 2013). There are over 100 different types of cancer, including

lung, prostate, colon, stomach, and breast cancer. An estimate of cancer incidence,

mortality and prevalence by the World Health Organization (WHO) showed that in

2012 there were 14.1 million new cancer cases, 8.2 million cancer deaths and 32.6

million people who are living with cancer (FERLAY et al. 2015; FITZMAURICE et al.

2015).

The causes of cancer are complex. 90-95 % of cancer cases can be attributed to

environmental factors, whereas the remaining 5-10 % is due to genetic factors

(ANAND et al. 2008). Environmental factors include diet, smoking, obesity, alcohol,

low amounts of physical exercise, as well as stress, radiation and environmental

pollutants. Although the inherited factors are not modifiable, some environmental

factors such as diet, tobacco consumption and physical inactivity can be controlled

(DIVISI et al. 2006). Therefore, cancer can be considered, at least partially, a

preventable disease, and much of the cancer burden could be lowered by a lifestyle

change.

In this context, a “good” dietary consumption and regular physical exercise may be

important modifiable ways for reducing cancer incidence (BOFFETTA et al. 2010;

DAVIES et al. 2011; YUSOF et al. 2012). A healthy diet on the one hand means

consuming food mostly based on plants (such as fruits, vegetables and products

gained from them), while on the other hand ingesting less red meat and salt (DAVIES

et al. 2011). It has been shown that fruits and vegetables present in the diet have

potentially preventable effects on cancer (DIVISI et al. 2006). Various substances in

these food products have been identified (STEINMETZ et al. 1996; MANACH et al.

2004). For example, flavones in fruits, quercetin in plant products and resveratrol in

Introduction

2

plants were intensely studied due to their potential beneficial health effects in humans

(D'ARCHIVIO et al. 2010).

Some studies have indicated that dietary polyphenols commonly found in vegetables

and fruits may play a role in the prevention of diseases such as cancer and

cardiovascular disorders (SCALBERT et al. 2000; SCALBERT et al. 2005).

Resveratrol is one of most studied compounds because of its cardioprotective,

antiinflammatory and antioxidative effects in animals and human cell lines (POULSEN

et al. 2013; RAEDERSTORFF et al. 2013; GAMBINI et al. 2015). However, preventive

effects in animals and human cell lines are difficult to extrapolate to humans, and

results from a few clinical studies proved not to be reproducible (ARTS et al. 2001;

ARTS et al. 2002; SCALBERT et al. 2005).

1.2. Effect of resveratrol in vitro and in vivo studies

1.2.1. Background on resveratrol

Resveratrol (3,5,4’-trihydroxystilbene, Figure 1.1) is a polyphenol found in many

plants and plant products such as grapes, peanuts and red wine (BHAT et al. 2001b).

Its content is 50-100 mg per gram grape skin and 0.2 mg/l to 7.7 mg/l in red wine

(CELOTTI et al. 1996). After the discovery of the so-called “French Paradox”, the

potential health-promoting properties of resveratrol have attracted a lot of attention.

The “French Paradox” describes the phenomenon that French people take up high

amounts of saturated fat but show a low incidence of coronary heart diseases (CHD)

when compared to other countries, and this has been partly attributed to the high wine

consumption in France (FERRIERES 2004). In addition to its antiinflammatory,

antioxidative and cardioprotective properties, resveratrol also could suppress cancer

progression.

JANG et al. (1997) found out that resveratrol induces a cancer-preventive effect,

which is associated with the three major steps of carcinogenesis. In that study,

resveratrol induced quinone reductase (phase II enzyme) activity, which is associated

Introduction

3

with the inhibition of tumor initiation, inhibited the cyclooxygenase and

hydroperoxidase activities, which correlates with tumor promotion, and inhibited

preneoplastic lesions in 7,12-dimethylbenz[a]anthracene (DMBA)-treated mouse

mammary glands. This finding attracted much scientific interest regarding the

anticarcinogenic activity of resveratrol.

Figure 1.1: The structure of trans-resveratrol (JANG et al. 1997)

1.2.2. In vitro studies

Resveratrol has been shown to inhibit the proliferation of various tumor cell lines, for

example cells originating from colon, prostate, breast, head and neck, liver, lung,

stomach, pancreatic and muscle cancers (HSIEH et al. 1999; ATTEN et al. 2001;

MAHYAR-ROEMER et al. 2001; MATSUOKA et al. 2001; SERRERO et al. 2001; JOE

et al. 2002; KUWAJERWALA et al. 2002; POZO-GUISADO et al. 2002; NARAYANAN

et al. 2003; POUSSIER et al. 2005; BUJANDA et al. 2006; KIM et al. 2006; LIN et al.

2008; CUI et al. 2010; ZHOU et al. 2011; LIU et al. 2012; FOUAD et al. 2013).

Exposure to 25 μM resveratrol caused a 70 % growth inhibition in Caco-2 cells, which

was due to the accumulation of cells at the S/G2 phase transition of the cell cycle

(SCHNEIDER et al. 2000). Moreover, the growth of androgen-responsive and

androgen-nonresponsive cancer cells was inhibited by this substance (HSIEH et al.

1999; KUWAJERWALA et al. 2002). In another colon cancer cell line, resveratrol

inhibited proliferation, which was associated with a decrease of the cyclin D1/CDK4

protein complex and an increase of cyclin E and cyclin A levels (WOLTER et al. 2001).

In addition to the suppression of cellular proliferation, resveratrol can induce apoptosis

Introduction

4

via different pathways. It has been shown to induce apoptosis through the activation

of the mitochondrial apoptosis pathway in a p53-independent manner in HCT-116

cells (MAHYAR-ROEMER et al. 2001). DELMAS et al. (2003) reported that

resveratrol-induced apoptosis in SW-480 cells was associated with the activation of

Bax and Bak proteins. Resveratrol also induced apoptosis in HL-60 and T47D cells,

which was specifically dependent on Fas signaling (CLEMENT et al. 1998). This

pathway also seems to be the predominantly activated one in HCT-116 cells; in this

context. MAHYAR-ROEMER et al. (2001) described that resveratrol induced

apoptosis through the intrinsic apoptotic pathway (by accumulation of p21 and BAX).

Resveratrol-induced apoptosis is associated with the activation of p53 (SHE et al.

2002). p53 is a tumor suppressor gene and plays an important role in apoptosis.

Normally, it is inactive (through binding to Mdm2), and (in most cases) only activated

in response to oncogenic stress/DNA damage (through the ATM/ATR pathway) in

order to induce cell cycle arrest and apoptosis (MAXIMOV et al. 2008).

The inhibitory activity of resveratrol on cyclooxygenase-2 (COX-2) has been

suggested to be a major factor contributing to its anti-cancer activity (JANG et al.

1997). Research studies indicated that the incubation of HCA-7 cells with resveratrol

for 24-96 h suppressed COX-2 protein expression and prostaglandin E2 (PGE2)

production. COX-2 expression was shown to be controlled by NF-kappaB, and COX-2

expression also inhibits the nuclear translocation of NF-kB, thereby suggesting a dual

role for COX-2 (POLIGONE et al. 2001). Additional studies support the view that

resveratrol suppresses DMBA-induced mammary carcinogenesis, which is associated

with the suppression of DMBA-induced NF-kappaB activation and COX-2 expression

(BANERJEE et al. 2002).

1.2.3. In vivo studies

Besides its effects in vitro, resveratrol also has been investigated regarding its activity

in vivo. LI et al. (2002) demonstrated that resveratrol could decrease the size and

number of tumors in DMBA-treated rats. Moreover, it was shown that resveratrol was

Introduction

5

able to reduce the formation of preneoplastic ductal lesions and inhibit mammary

tumorigenesis (BHAT et al. 2001a). In another study, it was suggested that resveratrol

inhibits PMA-promoted mouse skin tumors and that tumorigenesis in mouse skin was

associated with reactive oxygen species-related pathways (JANG et al. 1998).

TESSITORE et al. (2000) investigated the effect of resveratrol on azoxymethane

(AOM)-induced colon carcinogenesis in male F344 rats for 100 days, and the results

showed that resveratrol decreased the formation of colonic aberrant crypt foci by 40 %

and their multiplicity by 50 %.

The number of clinical studies performed with resveratrol in humans is still small.

Resveratrol was shown to be absorbed from grape juice and to reduce the risk of

atherosclerosis (PACEASCIAK et al. 1996). The absorption of resveratrol in humans

is high, but its bioavailability is low (WALLE et al. 2004). A pharmacokinetics

investigation in healthy adult subjects showed that the mean peak concentration of

resveratrol in plasma was low after oral administration, thus suggesting that high-dose

resveratrol consumption in humans cannot induce the same chemopreventive effect

as in vitro (BOOCOCK et al. 2007a). Moreover, after the oral consumption of

resveratrol, it was mainly present as glucuronide and sulfate conjugates in serum and

urine (GOLDBERG et al. 2003).

Due to the structure of resveratrol, it is easily metabolized to sulfates and

glucuronides in the intestine and liver (TOME-CARNEIRO et al. 2013). Extensive

metabolism of resveratrol is an important factor responsible for its low bioavailability.

KUHNLE et al. (2000) showed in an isolated rat small intestine model that only small

amounts of resveratrol remained unmetabolized and only small amounts of

resveratrol were absorbed across the enterocytes of the jejunum and ileum. However,

the low amount of bioavailable resveratrol might still exert a cancer-preventive effect

due to its accumulation in certain tissues and to the resveratrol metabolites (WALLE et

al. 2004; WALLE 2011). For example, the potential biological properties of

dihydroresveratrol (a metabolite of resveratrol) might be important to enhance the

Introduction

6

activity of resveratrol (KAGEURA et al. 2001; STIVALA et al. 2001;

ROTCHES-RIBALTA et al. 2012).

1.3. Resveratrol analogues

Since bioavailability limits the activity of resveratrol, research has focused on the

search or synthesis of new resveratrol analogues, with the aim of finding optimized

and more potent substances regarding growth-inhibition activity and metabolic

stability (SALE et al. 2005). Metabolites of resveratrol hydroxylated at the aromatic

rings demonstrated a higher COX-2 inhibition rate than celecoxib (a selective COX-2

inhibitor) (MURIAS et al. 2004). The anti-tumor activity of resveratrol triacetate was

also reported to inhibit cell growth and lead to a cell cycle arrest in human

adenocarcinoma colon cells (MAREL et al. 2008). r-Viniferin (a resveratrol tetramer)

was found to inhibit the growth of LNCaP cells and to induce a cell cycle arrest in the

G1 phase, which was accompanied by a higher apoptotic activity than resveratrol

(EMPL et al. 2015). The synthetic resveratrol analog 3,4,5,4'-tetramethoxystilbene

(DMU-212) was reported to exert a stronger anti-proliferative activity than resveratrol

in human colon cancer cells (SALE et al. 2004).

Imine resveratrol analogues (IRAs, Figure 1.2) are a series of synthetic polyphenols,

in which the C=C double bond has been replaced by a C=N bond in addition to

several modifications at the aromatic rings (LU et al. 2012). IRAs are easy to

synthesize and have a higher antioxidative activity than resveratrol. SERSEN et al.

(2009) found out that these analogues have the potential to scavenge DPPH radicals.

The replacement of the C=C double bond in the resveratrol molecule does not lead to

a loss of its antioxidant effects (LU et al. 2012; KOTORA et al. 2016). In addition, IRAs

also were shown to exert neuroprotective effects (LI et al. 2014). The synthesized

resveratrol analogues possess an antiinflammatory potential by significantly reducing

the activity of NF-kB (GOBEC et al. 2015). The anticarcinogenic effect of the

synthesized analogues was shown to be stronger than that of resveratrol. In this

context, it has been shown that they can inhibit human breast cancer cell growth by

Introduction

7

inhibiting the activation of protooncogenes by an estrogen receptor-dependent

pathway (RONGHE et al. 2014). However, studies regarding the biological effects of

IRAs are still scarce.

Figure 1.2: The structure of IRAs (LU et al. 2012)

1.4. The cell cycle and its regulation

The cell cycle is a cyclic procedure leading from the end of one cell division to the end

of the next cell division and in which a series of events end up in DNA replication

(VERMEULEN et al. 2003). The correct cell division is essential for the development

of tissues, and cell cycle control is important for an appropriate repair when a DNA

damage has occurred during this process (LODISH et al. 2008).

The cell cycle is divided in four different phases (Figure 3). Before entering the G1

phase, cells can remain in a resting stage (G0 phase), which is a phase of

non-proliferation. Cell division starts when the cells enter the G1 phase. In this phase,

cells prepare DNA synthesis and chromosome replication, i.e. prepare for the S phase.

Introduction

8

DNA replication occurs during the S phase, resulting in a doubled DNA content. In the

G2 phase, cells prepare for cell division. The subsequent process of mitosis, also

called the M (mitotic) phase, is the phase in which the nucleus and cytoplasm are

divided to yield two daughter cells. These can then re-enter the cell cycle or enter the

resting phase (This paragraph is summarized from LODISH et al. (2008)).

In each phase, there are a number of different cellular proteins that regulate the

transition between the G1, S, G2 and M phases. They consist of the cyclin subunit and

the cyclin-dependent kinase (CDK) subunit. Cyclins were first discovered in 1982

(EVANS et al. 1983; PINES 1991). They are synthesized during the cell cycle and

degraded in the M phase. CDKs can be activated by cyclins and form a cyclin-CDK

complex, which is important to ensure an accurate cell division (LEES 1995;

ARELLANO et al. 1997).

The cyclin-CDK complexes in each phase are different (VERMEULEN et al. 2003).

CDK 2 is important when cells enter the S and G2 phase. For example, the cyclin

E/A-CDK 2 complex can help the cells enter the S phase from the G1 phase, and the

cyclin A-CDK 2 complex is active between the S and G2 phases. Moreover, the cyclin

D-CDK 4/6 complex assists the cell through the G1 phase and the cyclin A/B-CDK 1

complex helps in the progression of the cells into the M phase (GALDERISI et al.

2003; SATYANARAYANA et al. 2009). For the correct cell division and progression of

the cell cycle, a strict control is essential. Checkpoints assure the proper order of cell

cycle events, regulate the progress of the cell cycle, and arrest the cell cycle in case

there is (DNA) damage that needs time to be repaired (HARTWELL et al. 1989;

ELLEDGE 1996).

Introduction

9

Figure 1.3: Overview of the cell cycle (LODISH et al. 2008)

Unrestrained cell proliferation could cause cancer (VERMEULEN et al. 2003). The

mutation of some genes may lead to cell cycle dysregulation, which in turn may lead

to tumor formation (CHAMPERIS TSANIRAS et al. 2014). EASTON et al. (1998)

described an alteration of CDK4 and CDK6 genes, which leads to the loss of

p16INK4a gene binding in neuroblastoma cells. p53 is a tumor suppressor gene; its

reduced expression is related to an increased risk of tumor formation

(VENKATACHALAM et al. 1998). A G1 phase arrest can be induced by p53; It acts by

enhancing the expression of the cyclin-dependent kinase inhibitor 1 (p21CIP), which in

turn inhibits CDKs 2, 4 and 6 (Figure 3) (SHAW 1996).

1.5. The COX-2/PGE2 pathway

Cyclooxygenase (COX) is an enzyme which is involved in prostaglandin synthesis

(VANE et al. 1998). There are two forms of COX. COX-1 was first cloned from sheep

in 1988 (DEWITT et al. 1988; YOKOYAMA et al. 1988), and is known to maintain the

normal lining of the gastrointestinal system and present in most tissues (KARGMAN

Introduction

10

et al. 1996; JACKSON et al. 2000). In contrast to that, COX-2 is mainly expressed in

sites of inflammation and undetectable in many normal tissues (ONOE et al. 1996;

SEIBERT et al. 1997; JACKSON et al. 2000). COX-2 is mainly expressed in response

to growth factors, cytokines or tumor promoters (DEWITT et al. 1993; EBERHART et

al. 1994; ONOE et al. 1996; RISTIMAKI et al. 1997).

The antitumor activity and mechanisms action of non-steroidal anti-inflammatory

drugs (NSAIDs) have been analyzed in a number of in vitro and in vivo studies

(KELLER et al. 2003). The mode of action of most NSAIDs is based on the inhibition

of COX, which in turn leads to a reduced production of prostaglandins (VANE 1971;

ABRAMSON et al. 1989). Both COX-2 and its main product PGE2, play a key role in

the development of cancer. COX-2 expression is upregulated in human colonic

tumors, and PGE2 has been demonstrated to promote tumor cell proliferation

(KANAOKA et al. 2007; NAKANISHI et al. 2013). DI POPOLO et al. (2000) showed

that the use of aspirin or other NSAIDs lowers the risk of colorectal cancer, and these

findings support the view that the COX-2/PGE2 pathway plays an important role in

cancer development (KELLER et al. 2003).

1.5.1. COX-2

COX-2 is an enzyme whose biosynthesis can be induced by diverse growth factors as

well as cytokines, and it is known that COX-2-expression is closely associated with

inflammatory mediators and signs of inflammation (BAKHLE et al. 1996; RUMZHUM

et al. 2015). Because of the fact that NSAIDs also block COX-1, which is constitutively

expressed in normal gastrointestinal tissue, intake of high NSAID doses may cause

gastrointestinal and ulcerative diseases (TRAVERSA et al. 1995; MALFERTHEINER

et al. 2009). Selective COX-2 inhibitors (“coxibs”) have the advantage, when

compared to regular NSAIDs, that they only target COX-2, but clinical data have

shown that COX-2 inhibitors can lead to a high incidence of cardiovascular diseases

(FUNK et al. 2007).

The link between chronic inflammation and cancer has been confirmed in a number of

Introduction

11

studies, and inflammation might play a role in tumor development, including tumor

initiation, promotion and proliferation (SUGANUMA et al. 1999; COUSSENS et al.

2002; HUSSAIN et al. 2007). COX-2, which is normally induced in inflammation,

appears to be directly implicated in tumor development. Overexpression of COX-2

has also been demonstrated in colorectal and breast cancer cells (CAO et al. 2002;

SECCHIERO et al. 2005). It has been suggested that COX-2 could increase mutated

cell growth and affect cell death (CHAN et al. 2007; SOBOLEWSKI et al. 2010).

COX-2 overexpression is related to the production of PGE2, which is known to inhibit

cell growth and tumor invasion.

Studies have shown that NSAIDs and selective COX-2 inhibitors may reduce the risk

of cancer. Celecoxib significantly reduced tumor size (to about 17 %) and multiplicity

(to about 29 %) in C57BL/6J (Min/+) male mice (the adenomatous Apc mutant Min

mouse model), and its adenoma preventing effect is thought to be based on the

inhibition of the conversion of arachidonic acid to prostaglandins (JACOBY et al.

2000). Similarly, celecoxib has shown tumor preventive effects regarding breast

cancer development in in vitro and in vivo experiments (DAI et al. 2012), and induced

apoptosis in human prostate cancer cells (HSU et al. 2000).

COX-2 is a tumor therapy target for many compounds. Polyphenols could reduce

tumor progression through inhibition of COX-2. Quercetin metabolites can

down-regulate COX-2 expression in human colon cells, which may be the basis of its

chemopreventive effect (MIENE et al. 2011). The anti-tumor activity of resveratrol can

be attributed in part to the inhibition of COX-2 protein expression (SALE et al. 2005).

Green tea polyphenols have been shown to exert an inhibitory effect on COX-2 in

colon carcinogenesis (PENG et al. 2006).

1.5.2. PGE2

Prostaglandin E2 (PGE2) was discovered by Bunting in 1976 (BUNTING et al. 1976). It

is a member of the prostanoid family, is synthesized from arachidonic acid (AA) and

plays a predominant role in inflammation as well as cancer development and

Introduction

12

promotion (PARK et al. 2006; NAKANISHI et al. 2013). PGE2 has diversity important

regulatory functions, including the regulation of body temperature, inflammation,

glucose metabolism, cell growth and other physiological activities (MURAKAMI et al.

2004). It is known that PGE2 protects the gut mucosa and inhibits gastric acid

secretion (BRZOZOWSKI et al. 2005; DEY et al. 2006).

PGE2 has also been described as a cancer promoting agent. The mechanism of

action is based on the binding of PGE2 to its cognate receptors (EP1-4), which results

in an increase of cell proliferation, apoptosis inhibition, cell invasion, and immune

response suppression (WANG et al. 2006). The main role of PGE2 in tumor

progression has been attributed to the activated EP receptor, subsequently triggering

different signaling pathways. PGE2 promoted the motility and invasiveness of human

colorectal carcinoma cells (SHENG et al. 2001). Administration of PGE2 to male F344

rats could increase the incidence and multiplicity of colon adenomas (KAWAMORI et

al. 2003). In vitro and in vivo data support the tumor-promoting effect of PGE2.

However, PGE2 also has tumor inhibiting effects. WILSON et al. (2000) reported that

treatment of Min/+ mice with a stable PGE2 analogue (16,16-dimethyl-PGE2) for 8

weeks clearly decreased the number and size of tumors (10-70%) in the whole

intestine, which suggests that PGE2 perhaps has the ability to inhibit tumor

development in some cases.

Introduction

13

Figure 1.4: The COX-2/PGE2 pathway (SOBOLEWSKI et al. 2010). The COX-2

enzyme is responsible for catalyzing arachidonic acid to prostaglandin G2 (PGG2),

and subsequently to prostaglandin H2 (PGH2). Then, PGH2 is converted to different

prostaglandins, which mediate their effects through different receptors

(SOBOLEWSKI et al. 2010).

Introduction

14

1.6. The aim of study

Since the bioavailability of resveratrol is limited in vivo by its fast metabolism, and the

chemopreventive activity of newly generated resveratrol analogues has not been

elucidated, we investigated the biological properties of five IRAs on human tumor cell

lines and compared them with those of resveratrol.

The aims of study were the following:

1. To examine the anti-proliferative activity of IRAs on human colorectal carcinoma as

well as other tumor cell lines and compare it to that of resveratrol.

2. To investigate the effects of resveratrol and the most potent IRA(s) on the cell

cycle distribution of the above-mentioned cell lines.

3. To investigate the effects resveratrol and the most potent IRA(s) on COX-2

expression and activity

4. To investigate the metabolic stability of the most potent IRA(s)

Materials and methods

15

2. Materials and Methods

2.1. Materials

2.1.1. Chemicals

Dimethyl sulfoxide (DMSO), ethanol, trichloroacetic acid (TCA), acetic acid,

phenylmethylsulfonyl fluoride (PMSF), urea, sodium dodecyl sulfate (SDS),

tris-(hydroxymethyl)-aminomethane (Tris), glycine, ammonium persulfate (APS),

nocodazole, powdered milk, methanol, NaCl, glucose, isopropanol, and

acrylamide-bisacrylamide solution (37.5:1) were purchased from Carl Roth (Karlsruhe,

Germany), while ribonuclease A (RNase A), sulforhodamine B (SRB), propidium

iodide (PI) solution, protease inhibitor cocktail, dithiothreitol (DTT), Ponceau S,

tetramethylethylenediamine (TEMED), goat anti-rabbit IgG-HRP, and resveratrol were

acquired from Sigma-Aldrich (Taufkirchen, Germany). Cell culture reagents such as

fetal bovine serum (FBS), sodium bicarbonate (NaHCO3) solution,

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer, non-essential

amino acids (NEA) solution, trypsin/EDTA (T/E), trypan blue solution and all cell

culture media were purchased from Biochrom (Berlin, Germany). The resveratrol

imine analogues (IRAs, purity ≥ 99 %) were kindly provided by the research group of

Prof. Dr. Y. Pan (Zhejiang University, Hangzhou, P.R. of China). In terms of an easy

identification, we named them compound 1, 2, 3, 4 and 5 (Figure 1.2). The exact

composition of all buffers used in the present study can be found in the annex.

2.1.2. Other materials

Ionic detergent compatibility reagent (IDCR) Thermo Scientific

Pierce 660nm protein assay reagent Thermo Scientific

GAPDH antibody Santa Cruz

Biotinylated protein ladder Santa Cruz

COX-2 antibody Cell Signaling


16

Goat anti-mouse IgG-HRP Cell Signaling

peqGOLD protein-marker IV PEQLAB Biotechnologie

Bromophenol blue sodium salt AppliChem

Tween® 20 Prolabo

ECL solution (HRP-substrate) Millipore

2.1.3. Cell lines

HCT-116p53wt Colorectal carcinoma DMSZ

HCT-116p53-/- Colorectal carcinoma Provided by Prof. Dr. Regina

Schneider-Stock (Institute of

Pathology，University of Erlangen,

Erlangen, Germany)

A-431 Epidermoid carcinoma ATCC

Caco-2 Adenocarcinoma DMSZ

LNCaP Prostate carcinoma DMSZ

HCA-7 Adenocarcinoma ECACC

2.2. Methods

2.2.1. Cell culture

HCT-116p53wt cells were grown in RPMI 1640 medium supplemented with 10 % (v/v)

FBS, 2 mM L-glutamine and 100 IU/mL penicillin/100 μg/mL streptomycin. A-431,

HCA-7 and HCT-116p53-/- cells were grown in Dulbecco’s Modified Eagle Medium

(DMEM) supplemented with 10 % (v/v) FBS, 2 mM L-glutamine and 100 IU/mL

penicillin/100 μg/mL streptomycin. LNCaP cells were grown in RPMI 1640 medium

supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 IU/mL penicillin/100 μg/mL

streptomycin, 0.15 % NaHCO3, 1 mM sodium pyruvate, 10 mM HEPES, and 4.5 g/L

glucose. Caco-2 cells were grown in DMEM supplemented with 10% (v/v) FBS, 2 mM

L-glutamine, 100 IU/mL penicillin/100 μg/mL streptomycin and 1% (v/v) NEA.


17

All cell lines were incubated at 37 °C in an atmosphere with 5 % CO2 and 95 %

relative humidity. To count the cells, a hemocytometer and the trypan blue exclusion

method were used (STROBER 2001). Resveratrol and the five resveratrol imine

analogues were dissolved in DMSO, aliquoted in 0.5 ml centrifuge tubes and stored at

-20 °C until use.

2.2.2. Cell proliferation measurements

The proliferation of cells was determined by the SRB assay (SKEHAN et al. 1990) as

previously described (VICHAI et al. 2006), but with slight alterations. Briefly, 1000 (in

the case of the cell lines HCT-116, A-431, Caco-2, HCA-7) or 3000 cells (in the case

of the cell line LNCaP) per well were seeded into 96-well plates (TPP®, Trasadingen,

Switzerland). Then, the cells were treated with the solvent control (0.1 % DMSO) and

increasing concentrations of resveratrol as well as the resveratrol imine analogues (1,

5, 10, 20, 40, 60, 80, 100 µM) and incubated for 48, 72 or 120 h. Thereafter, cellular

proteins were fixed by the addition of 50 % TCA solution. Subsequently, 70 μl of a

0.4 % (w/v) SRB solution were added to each well, and the plates rinsed five times

with 1 % acetic acid after an incubation time of 55 min. Finally, 100 μl of a 10 mM Tris

buffer were added to each well and the absorption measured at 510 nm in a plate

reader (infinite® M200; Tecan, Crailsheim, Germany).


18

2.2.3. Flow cytometry

Cell cycle analyses were performed with slight changes as previously described

(EMPL et al. 2015). In short, 1.5 x 106 cells were seeded in 10 cm cell culture dishes

(TPP) and incubated for 24 hours. Culture medium was then replaced by 10 ml fresh

FBS-free medium for another 24 h. Then, the cells were exposed to 0.1 % DMSO

(solvent control), the test substances (resveratrol and compound 5; 10, 40, 80 μM) or

10 μM nocodazole (positive control) for 24 and 48 h. In a next step, the supernatants

were collected in 50 ml tubes and the cells detached by the addition of a T/E solution.

Both the supernatant and the cells were then combined in the same tube.

Subsequently, the cells were centrifuged and 1 x 106 cells (in 300 µl cold PBS)

transferred drop by drop to a new 1.5 ml centrifuge tube containing 700 μl of ethanol.

Samples were then stored at 4 °C until analysis. After staining with PI, the DNA

content was measured using BD Accuri® C6 cytometer (BD Biosciences, Heidelberg,

Germany) controlled by the CFlow Plus software (version 1.0.264.15; BD Biosciences,

Heidelberg, Germany). 20,000 events were gated and the flow rate was set to

14 μL/min for each sample. Finally, the acquired data was analyzed with FlowJo

(version 7.6.5; FlowJo, Ashland, OR, USA).

2.2.4. Protein extraction and Western blotting

2.2.4.1. Sample preparation

1.6 x 106 cells/10 ml medium were seeded in 10-cm cell culture dishes and left to

attach for 24 h. Then, the cells were exposed to fresh medium containing the control

(0.1 % DMSO) and the test substances (resveratrol and compound 5 [1, 50, 100 μM])

for 24 h. In order to collect the cellular protein, the cell culture dishes were placed on

ice, the adherent cells were scrapped off the dishes with a plastic spatula (TPP), and

the supernatant transferred to 50 ml tubes. Remainders in the dishes were washed

down twice with 5 ml cold PBS and transferred to the same 50 ml tubes. The mixtures

were then centrifuged at 4 °C and 12,000 rcf for 15 min. Thereafter, the supernatant


19

was aspirated, while the cell pellet was resuspended in 1 mL cold PBS and

transferred to a new 1.5 mL centrifuge tube. Then, the tubes were centrifuged at 4 °C

and 4,400 rcf for 10 minutes and the samples stored at -80 °C overnight.

Subsequently, the samples were lysed with the cell lysis buffer (100-200 μL) by

incubating each sample on ice for 60 min accompanied by vortexing intervals every

10 min as well as a 10 s sonication step. Finally, the samples were stored at -80 °C

until protein quantitation.

2.2.4.2. Determination of protein concentration

The protein concentration of all samples was measured by using the Pierce 660 nm

protein assay kit according to the manufacturer’s instructions.

2.2.4.3. Western blotting

For electrophoresis (SDS-PAGE), a 10 % gel was prepared. 20 μg protein of each

sample were transferred to a 1.5 ml centrifuge tube together with 5 x and 2 x loading

buffer to reach a final sample volume of 30 μl/lane. Additionally, 8 μl of protein marker

and 10 μl of biotinylated protein ladder were added to 1.5 ml centrifuge tubes. The

tubes were then shaken and shortly centrifuged, and the proteins were denaturized by

boiling at 95 °C for 5 min. In a next step, 20 μg of total protein were loaded onto each

well of the gel, along with the protein markers/ladders. The separation was firstly

performed for 30 min at 30 mA, before the current was increased to 50 mA for about

1 h until the proteins were separated. The transfer onto nitrocellulose blotting

membranes (GE Healthcare Life Science, UK) was done under “wet” conditions.

Firstly, the gel was taken off the glass and placed in cold transfer buffer for 10 min.

The membrane and filter paper were soaked in cold transfer buffer before being used.

Then, the gel and membrane were tightly “sandwiched” between two sponges and

three filter papers and the proteins transferred onto the membrane at 30 V for 12-16 h

using a Mini trans-blot® cell system (Bio-Rad Laboratories GmbH, München,

Germany). After the transfer, the membrane was stained with Ponceau S solution to


20

check the transfer quality and to enable cutting into two pieces, which was accurately

performed at 50 kDa. Then, both membrane pieces were rinsed three times with

washing buffer until the Ponceau S solution was completely removed. Subsequently,

the membrane was blocked in 5 % milk blocking buffer at room temperature for 2 h

under shaking. Thereafter, the primary antibody (Table 2.1) was diluted in blocking

buffer and bathed with the membrane overnight at 4 °C, before being washed with

washing buffer 3 times for 5 min. The HRP-conjugated secondary antibody was

prepared with blocking buffer and contained an HRP-linked anti-biotin antibody (Table

2.2). Then, an incubation with the HRP-conjugated secondary antibody for 1 h at

room temperature was performed, the membrane rinsed three times for 5 min with

washing buffer and ECL substrate applied as recommended by the manufacturer.

Images of the blots were finally obtained by using an Imager (INTAS, Göttingen,

Germany).

Table 2.1 Primary Antibody

Protein binding Species Company Dilution Clone

COX-2 Rabbit Cell Signaling 1:1000 Polyclonal

GAPDH Mouse Santa Cruz 1:2000 Polyclonal


21

Table 2.2 Secondary Antibody

Protein binding Species Company Dilution Clone

Rabbit-IgG Goat Sigma-Aldrich 1:1000 Polyclonal

Mouse-IgG Goat Santa Cruz 1:10,000 Polyclonal

Biotin Goat Cell Signaling 1:1000 Polyclonal

2.2.5. LC−UV and LC-MS Analysis

LC−UV and LC-MS analyses were performed by Michael Krohn and Ina Willenberg as

previously described (WILLENBERG et al. 2012; WILLENBERG et al. 2015b)

2.2.6. Glucuronidation Assays

The glucuronidation assay was carried out by Michael Krohn as described

(WILLENBERG et al. 2012)

2.2.7. Statistical analysis

For data analysis and IC50-value calculation, GraphPad Prism version 6 (GraphPad

Software Inc., San Diego, CA, USA) was used. A p value ≤ 0.05 was considered to be

statistically significant.


22

Results

23

3. Results

3.1. Growth inhibitory effect of the test substances

3.1.1. Effect of resveratrol and IRAs on colon cancer cell proliferation

The growth inhibitory activity of resveratrol and five IRAs was first investigated in the

human colorectal carcinoma cell line (HCT-116p53wt). Cells were exposed to the test

substances at concentrations of 1-100 μM for 48, 72 and 120 h. As shown in Figure

3.1, the properties of the compounds differ.

While resveratrol and compound 5 showed a strong inhibition of cellular growth, the

other compounds had no such pronounced effects on HCT-116p53wt cells. The

inhibitory activity of resveratrol is thereby dependent on time and dose. The cell

growth inhibition by resveratrol started at a concentration of 20 μM after 48 h and

reached a value of 92 % after 120 h of treatment with 100 μM (Fig. 3.1 F). The effect

of compound 5 is also time as well as dose-dependent and much stronger than

resveratrol, as the earliest significant growth inhibition effects were already seen at a

concentration of 1 μM after 72 h of incubation. Maximum growth inhibition (95 %) was

observed at a concentration of 100 μM after 120 h of treatment (Fig. 3.1 E).

In general, the other four compounds showed no marked cytotoxic activity towards the

above-mentioned colon cancer cell line. The cells still survived when exposed to

compound 1 and compound 3 (Fig. 3.1 A and C), while compound 2 and compound 4

had a slight inhibitory effect on cell growth (22 and 19 % at concentrations of 60 μM

and 80 μM after 120 h of incubation, respectively) (Fig. 3.1 B and D).

Results

24

Figure 3.1: Inhibitory effect of resveratrol (F) and IRAs (A-E) on the proliferation of HCT-116p53wt cells. Results are presented as

mean ± standand deviation (SD) of five independent experiments. ▪: p ≤ 0.05; #: p ≤ 0.01; +: p ≤ 0.001; *: p ≤ 0.0001

Results

25

3.1.2. Effect of resveratrol and compound 5 on the proliferation of other tumor

cell lines

After determining the most potent IRA in the previous step, we analysed the

growth-inhibiting effects of resveratrol and compound 5 on other cancer cell lines (the

p53-deficient colorectal carcinoma cell line HCT-116p53-/-, the epidermoid carcinoma

cell line A-431, the prostate carcinoma cell line LNCaP, the colorectal

adenocarcinoma cell line Caco-2, and the colonic adenocarcinoma cell line HCA-7).

Generally, compound 5 and resveratrol also induced a strong growth-inhibiting effect

in the above-mentioned cell lines. This effect was cell line-dependent. In HCT-116p53-/-

cells, after 48 h of treatment, 20 μM resveratrol was able to significantly suppress cell

growth (Fig. 3.2 A). After 120 h, the cell count decreased to about 13 % of the control

value when the cells were treated with the highest concentration (100 μM). Compound

5 also inhibited cellular proliferation, a marked inhibition starting at 10 μM after 72 h of

incubation and a 90 % maximal inhibition reached when the cells were exposed to a

concentration of 100 μM after 120 h (Fig. 3.2 B).

Results

26

Figure 3.2: Inhibitory effect of resveratrol (A) and compound 5 (B) on the proliferation

of HCT-116p53-/- cells. Results are presented as mean ± SD of five independent

experiments. #: p ≤ 0.01; *: p ≤ 0.0001

Results

27

In the A-431 cells, both resveratrol and compound 5 were able to inhibit the cell

growth in a time- and dose-dependent manner (Fig. 3.3 A and B). After 72 h of

incubation, resveratrol and compound 5 reduced cell number significantly, starting at 5

and 10 μM, respectively. The maximum inhibition rates were approximately 90 % for

both test compounds (100 μM resveratrol and compound 5).


of A-431 cells. Results are presented as mean ± SD of five independent experiments.

#: p ≤ 0.01; +: p ≤ 0.001; *: p ≤ 0.0001

Results

28

As in A-431 cells, the growth-inhibiting activity of compound 5 is similar to that of

resveratrol in LNCaP cells, both substances starting to reduce cell proliferation at a

low concentration (5 μM) after 72 h of treatment. 100 μM resveratrol and compound 5

caused a 69 % growth inhibition (Figure 3.4 A and B).


of LNCaP cells. Results are presented as mean ± SD of five independent experiments.

#: p ≤ 0.01; +: p ≤ 0.001; *: p ≤ 0.0001

Results

29

The same can be also said for Caco-2 cells: A treatment of the cells with 10 µM of

both compounds already led to a significant suppression of cell growth. After treating

the cells with 100 µM resveratrol and compound 5 for 120 h, the percentage of the

remaining cells was 12 % and 15 % of the control value, respectively (Figure 3.5 A

and B).


of Caco-2 cells. Results are presented as mean ± SD of five independent experiments.

#: p ≤ 0.01; +: p ≤ 0.001; *: p ≤ 0.0001

Results

30

While resveratrol and compound 5 also led to a time- and dose-dependent growth

inhibition in HCA-7 cells, this cell line was more resistant to the cytotoxic effects

emanating from the test compounds. This assumption is supported by the fact that the

growth inhibition reached values of 72 and 68 % when the cells were treated with

100 μM resveratrol and compound 5. Interestingly, resveratrol was more cytotoxic

than compound 5, as shown by the fact that first significant effects were observed with

10 μM resveratrol and 20 μM compound 5 (Figure 3.6 A and B).


of HCA-7 cells. Results are presented as mean ± SD of six independent experiments.

▪: p ≤ 0.05; #: p ≤ 0.01; +: p ≤ 0.001; *: p ≤ 0.0001

Results

31

3.1.3. IC50 calculation

The IC50 values of resveratrol and IRAs in the six above-mentioned tumour cell lines

were computed as described in chapter 2.2.7. In HCT-116p53wt cells, the

growth-inhibitory potency of compound 5 is approximately 50 times higher than that of

resveratrol, with an IC 50 value of 0.59 vs 31.01 μM (Table 3.1). The other four IRAs

showed no noteworthy cytotoxic effect on the growth of HCT-116p53wt cells. In all the

other tumour cell lines tested, resveratrol and compound 5 were able to inhibit cell

growth, but the effect was cell line-dependent. In HCT-116p53-/- cells, the IC50 value of

compound 5 was 2 times lower than that of resveratrol, contrasting the effects in

A-431 cells (Table 3.2). In LNCaP and Caco-2 cells, the IC50 value of both compounds

was quite similar. Moreover, in HCA-7 cells, the inhibitory effect of compound 5 was

2-4 times higher than that in the other tumour cell lines.

Table 3.1: IC50 values of resveratrol and IRAs in HCT-116p53wt cells after 120 h

IC50 (μM)

C 1 C 2 C 3 C 4 C 5 resveratrol

HCT-116p53wt 151.1 303 - 1507 0.59 31.01

The values (μM) represent the mean of five independent experiments. -: IC50 cannot be

calculated.

Results

32

Table 3.2: IC50 values of resveratrol and compound 5 in HCT-116 p53-/-, A-431, LNCaP,

Caco-2 and HCA-7 cells

IC50 (μM)

HCT-116p53-/- A-431 LNCaP Caco-2 HCA-7

R 28.61 9.19 29.55 16.09 33.78

C 5 16.09 15.42 24.88 13.38 51.60

The values (μM) represent the mean of six independent experiments.

Results

33

Results

34

3.2. Cell cycle distribution analysis

The previous chapter showed that resveratrol and compound 5 exerted cell

growth-inhibitory effects on different tumor cell lines, which might be due to alterations

of the cell cycle. Therefore, the effects of resveratrol and compound 5 on the cell cycle

distribution of the above-mentioned cell lines were investigated.

3.2.1. Effect of resveratrol and compound 5 on the cell cycle distribution of

HCT-116p53wt cells

At 10 μM, compound 5 led to a slight decline of the percentage of cells in the S phase,

which was associated with an increased (p≤0.05) population of cells in the G2 phase

(Figure 3.7 A). However, after 48 h no such an effect was observed (Figure 3.7 B).

When cells were grown in the presence of 40 μM compound 5 a significant increase

(p≤0.0001) in the percentage of cells being in the S phase after 24 h was observed

(Figure 3.7 C). After 48 h of incubation, compound 5 seemed to enhance the number

of cells in the G1 phase, but not significantly (Figure 3.7 D). At the highest used

concentration (80 μM), compound 5 caused a significant S phase accumulation only

after 24 h of treatment (Figure 3.7 E). In contrast, resveratrol induced an accumulation

in the S phase at 40 μM after 24 h, strongly associated with a decrease of cells in the

G1 and G2 phase (Figure 3.7 C). At 80 μM (Figure 3.7 E and F), resveratrol led to an

accumulation of cells in the G1 phase after 24 h and in the S phase after 48 h.

Results

35

Figure 3.7: Effect of resveratrol and compound 5 (10 μM 24 h: A; 10 µM 48 h: B;

40 μM 24 h: C; 40 µM 48 h: D; 80 μM 24 h: E; 80 µM 48 h: F) on the cell cycle

distribution of HCT-116p53wt cells. Results are presented as mean ± SD of four

independent experiments. ▪: p ≤ 0.05; *: p ≤ 0.0001.

Results

36


HCT-116p53-/- cells

As can be seen in Figure 3.8 A and B, 10 μM resveratrol led to an accumulation of

cells in the S phase after 24 h (P≤0.01) and 48 h (P≤0.05) of incubation. In contrast,

compound 5 did not cause any alteration of the cell cycle after 24 h of incubation. At

40 μM, a higher percentage of resveratrol-treated cells were arrested in the S phase

after 24 h of treatment, while compound 5 led to a significant arrest after both 24 and

48 h of incubation (Figure 3.8 C and D). At the highest concentration (80 μM) and after

24 h, resveratrol and compound 5 led to an increase of cells in the G1 and S phase

(p≤0.01), respectively, whereas after 48 h resveratrol and compound 5 led to an

accumulation of cells in the S and G2 phase, respectively (Figure 3.8 E and F).

Results

37



distribution of HCT-116p53-/- cells. Results are presented as mean ± SD of four

independent experiments. ▪: p ≤ 0.05; #: p ≤ 0.01; +: p ≤ 0.001; *: p ≤ 0.0001.

Results

38


A-431 cells

As early as after 24 h, a treatment of the cells with 10 μM resveratrol induced a

significant increase of cells in the G1 phase (Figure 3.9 A), while a concentration of

40 μM resveratrol led to an S phase arrest (Figure 3.9 C). However, both compounds

failed to cause any effect at 10 and 40 μM after 48 h of incubation (Figure 3.9 B and

D). When the cells were treated with an 80 μM concentration of the two test

compounds, a high proportion of cells were arrested in the S phase, this effect being

accompanied by a decrease of cells in the G1 phase (Figure 3.9 E and F).

Results

39



distribution of A-431 cells. Results are presented as mean ± SD of four independent

experiments. ▪: p ≤ 0.05; #: p ≤ 0.01; +: p ≤ 0.001; *: p ≤ 0.0001.

Results

40


LNCaP cells

As early as after 24 h, a treatment with 10 μM resveratrol induced a significant

increase of cells in the G1 phase (Figure 3.9 A) and to an S phase arrest after a

treatment with 40 μM (Figure 3.9 C). However, both compounds failed to cause any

effect at 10 and 40 μM after 48 h of incubation (Figure 3.9 B and D). When the cells

were treated with an 80 μM concentration of the two test compounds, a high

proportion of cells were arrested in the S phase, an effect that was accompanied by a

decrease of cells in the G1 phase (Figure 3.9 E and F).

Results

41



distribution of LNCaP cells. Results are presented as mean ± SD of four independent

experiments. #: p ≤ 0.01; *: p ≤ 0.0001.

Results

42


Caco-2 cells

At a rather low concentration of 10 μM, resveratrol and compound 5 failed to lead to

any effect on the cell cycle distribution in Caco-2 cell line (Figure 3.11 A and B). At

40 μM, both test compounds led to a significant arrest of these cells in the S phase

(P≤0.0001; Figure 3.11 C and D). At 80 μM, only compound 5 induced an alteration of

the cell cycle distribution in a time-dependent manner (Figure 3.11 E and F). In

contrast, resveratrol-treated cells were not affected regarding the G1, S or G2 phase

distribution of the cells; instead, an increase of cells in the Sub-G1 fraction was

observed.

Results

43



distribution of Caco-2 cells. Results are presented as mean ± SD of four independent

experiments. +: p ≤ 0.001; *: p ≤ 0.0001

Results

44


HCA-7 cells

Subsequently, the effect of resveratrol and compound 5 in HCA-7 cells was

investigated. As seen in Figure 3.12 A, C and E, compound 5 led to an accumulation

of cells in the G1 phase, which was accompanied by a decrease of the cellular faction

in the S and G2 phase after 24 h. After 48 h, cells were mainly detected in the S phase

when treated with 80 μM compound 5. Resveratrol had no effect on the cell cycle

distribution at a low concentration (Figure 3.12 A and B), and cells were mainly

arrested in the G1 phase at a 40 and 80 μM concentration (Figure 3.12 C, E and F).

Results

45



distribution of HCA-7 cells. Results are presented as mean ± SD of four independent

experiments. +: p ≤ 0.001; *: p ≤ 0.0001.

Results

46

3.3. Effect of resveratrol and compound 5 on the COX-2/PGE2-pathway

3.3.1. Effect of resveratrol and compound 5 on COX-2 protein expression in

HCA-7 cells

In a next step, we investigated the potential regulation of COX-2 protein expression

and the PGE2 levels by resveratrol and compound 5. The COX-2 protein expression in

HCA-7 cells was evaluated by Western blotting, and cells were treated with

resveratrol and compound 5 at concentrations of 1, 50, and 100 µM for 24 h. As

shown in Figure 3.13, treatment with 100 µM resveratrol led to a reduced expression

of COX-2, whereas lower concentrations did not induce such an effect. In contrast,

compound 5 at concentrations of 50 and 100 µM increased the expression of this

protein when compared to DMSO-treated cells (Figure 3.13).

Figure 3.13: Effect of resveratrol and compound 5 on the expression of COX-2 in

HCA-7 cells. Representative images of three independent experiments.

Results

47

3.3.2. Effect of resveratrol and compound 5 on PGE2 production in HCA-7 cells

The data of compound 5-related effects on PGE2-production were kindly provided by

Ina Willenberg, and the results of resveratrol as well as the analysis method have

previously been reported (WILLENBERG et al. 2015a). According to this publication,

PGE2 levels were significantly reduced by a resveratrol treatment (WILLENBERG et

al. 2015a). Treatment with compound 5 (Figure 3.14) also led to a decrease in PGE2

production (Figure 3.14). The maximum inhibition (64 %) thereby occurred at a

concentration of 50 µM. The corresponding IC50 values of resveratrol and compound

5 are shown in Table 3.3. Resveratrol inhibited PGE2 production with an IC50 value of

4.7 µM, while compound 5 showed a stronger inhibitory activity, the IC50 value being

0.42 µM.

Figure 3.14: The effect of compound 5 on PGE2 production in HCA-7 cells. Cells were

treated with compound 5 (0, 1, 10 and 50 µM) for 24 h. Results are shown as the

mean of two independent exprements.

Results

48

Table 3.3. IC50 values of resveratrol and compound 5 on PGE2 production in HCA-7

cells [data for resveratrol taken from (WILLENBERG et al. 2015a)]

IC50 (μM)

resveratrol compound 5

HCA-7 4.7 0.42

3.4. Metabolic stability of compound 5

The data on the metabolic stability of compound 5 were kindly provided by Michael

Krohn, and the results for resveratrol as well as details regarding the methods used

have previously been reported (WILLENBERG et al. 2012). Compound 5 was

incubated with rat liver microsomes (RLMs) for 40 min, and as shown in Figure 3.15,

34 % of the unconjugated compound 5 was recovered after that time. Therefore, 66 %

of compound 5 was glucuronidated, while resveratrol was almost fully conjugated

[≈99 %; see (WILLENBERG et al. 2012)].

Figure 3.15: Glucuronidation of compound 5 by RLMs. Shown is the percentage of

free compound 5 after 40 min of incubation with RLMs, expressed as the

mean ± standard deviation of three samples.

Discussion

49

4. Discussion

The results can be summarized as follows: 1) Of the five IRA’s tested, only compound

5 had a significant growth-inhibitory effect on HCT-116p53wt cells, which in addition was

stronger than that of resveratrol; 2) Resveratrol and compound 5 suppressed the

growth of five other human tumor cell lines, the effects being however cell

line-dependent; 3) Resveratrol and compound 5 altered the cell cycle distribution in

different human tumor cell lines, again depending on the concentration and the cell

type; 4) The expression of COX-2 was down-regulated by resveratrol, while it was

up-regulated by compound 5, and resveratrol as well as compound 5 inhibited PGE2

production; 5) Compound 5 is less metabolized to glucuronic acid conjugates in RLMs

when compared to resveratrol.

4.1. Effect of resveratrol and IRAs on the proliferation of human tumor cell

lines

Firstly, an investigation of the anti-proliferative activity of resveratrol and five IRAs on

HCT-116p53wt cells were performed, the results showing that only compound 5 had a

far stronger activity than that of resveratrol in these cells. The growth inhibitory effect

of resveratrol on HCT-116p53wt cells reported in the present work is consistent with

previous studies (LU et al. 2012; FOUAD et al. 2013). Resveratrol has a pronounced

growth-inhibitory effect on these cells, but compound 5 exerts even stronger effects

than those of resveratrol, as shown by its approximately 30 times lower IC50 value.

The fact that IC50 value calculation shows that compound 5 has a stronger

growth-inhibitory effect on HCT-116p53wt cells indicates that p53 most probably

mediates growth inhibition in these colon carcinoma cells. p53 is a tumor suppressor

and plays an important role in cell cycle distribution (MAXIMOV et al. 2008). Activation

of p53 can lead to cell cycling arrest in the G1, S and G2 phases, thereby preventing

cell proliferation (LODISH et al. 2008). Therefore, the stronger inhibitory effect of

compound 5 on HCT-116p53wt cells is probably related to the activation of p53.

Discussion

50

LU et al. (2012) suggested, based on the fact that compound 5 is a much more potent

antioxidant agent than resveratrol, that simple chemical alterations of resveratrol at

the B ring with an ortho-OH group might retain or enhance its biological activity.

However, the investigation of the effects of compound 5 on all the other tumor cell

lines in our study supports the view that substitution modification at the aromatic rings

of resveratrol does not notably modify its antiproliferative effects. The IC50 values

thereby indicate that A-431 cells are the most sensitive cell line regarding a treatment

with resveratrol, while compound 5 is the most effective substance in HCT-116 cells.

In the present study, resveratrol exhibited a stronger inhibitory effect in Caco-2 cells

than in the HCT-116 cell line, and this is consistent with a previous study (LIU et al.

2014). Moreover, Caco-2 cells exposed to compound 5 are also more sensitive to

resveratrol than HCT-116 cells expressing or not expressing the p53 gene. The

analysis of the effects of compound 5 showed a comparable antiproliferative effect on

LNCaP cells, which suggests that the growth-inhibitory effects of resveratrol

analogues were similar to those of resveratrol in this prostate cancer cell line, while

resveratrol is more potent than compound 5 in HCA-7 cells. All in all, the effect of

compound 5 is only stronger in HCT-116p53wt cells, which might be due to the

p53-mediated cell cycle arrest or to the activation of cell repair mechanisms.

4.2. Effect of resveratrol and compound 5 on cell cycle distribution

Growth inhibition is closely associated with cell cycle disturbance and apoptosis in

tumor cells (COTTART et al. 2014). Resveratrol has been reported to affect cell

growth via the induction of a cell cycle arrest, even though not consistently in different

cell lines (AGGARWAL et al. 2004; COTTART et al. 2014). WOLTER et al. (2001)

reported that treatment with resveratrol in the 12.5-200 µM dose range after 24 h

resulted in HCT-116 cells accumulating in the G2 or S phase. In the present study,

after 24 h of incubation, resveratrol led to a slight HCT-116p53wt cell number increase

in the G2 phase at 10 µM and to a significant cell cycle arrest in the S phase at 40 µM,

which is consistent with the previously cited study (WOLTER et al. 2001). Compound

Discussion

51

5 also caused a cell cycle arrest, but different from that induced by resveratrol. Cells

were arrested in the G2 phase (40 µM) and the S phase (80 µM) after 24 h of

incubation. The fact that the growth inhibition exerted by compound 5 is more

pronounced in HCT-116p53wt than in HCT-116p53-/- cells is probably due to effects

mediated by p53. p53 is a transcription factor that induces the expression of p21CIP,

which can inhibit the activity of the Cyclin A/CDK2 complex and thus could induce an

S phase arrest (WAWRYK-GAWDA et al. 2014). Moreover, the S phase arrest could

be due to compound 5 inducing DNA damage at 80 µM. No apoptosis in HCT-116p53wt

cells was observed in our study; thus, the indicated cell growth inhibitory may not be

related to apoptotic events. Resveratrol is known to increase the expression of

proapoptotic proteins in a concentration-dependent manner. The maximal

concentration used in the present study was 80 µM, which might be too low when

wanting to induce apoptosis, as shown in previous studies (MAHYAR-ROEMER et al.

2001; LIU et al. 2014).

In HCT-116p53-/- cells, the effect of resveratrol on the cell cycle at high but not at low

concentrations is the same as in the case of HCT-116p53wt cells. MAHYAR-ROEMER

et al. (2001) reported that resveratrol induced apoptosis in HCT-116 cells

independently of p53, but apoptosis was not detected in our study, since no enhanced

accumulation of cells in the Sub-G1 phase was observed. In the present study, the fact

that the same cell cycle arrest was observed in HCT-116p53wt and HCT-116p53-/- cells

after being treated with a high concentration of resveratrol suggests that the

resveratrol-induced cell cycle arrest is not associated with p53. In contrast, the results

obtained with compound 5 support the view that p53 is involved in the cell cycle arrest

observed in HCT-116p53wt cells.

In the present study, the effect of resveratrol and compound 5 on the cell cycle

distribution of A-431 cells was analyzed and shown not to match with results of

previously published studies. AHMAD et al. (2001) reported that a resveratrol

treatment led to a cell cycle arrest in the G1 phase after an incubation of 24 h in the

Discussion

52

1-50 µM dose range, whereas resveratrol induced an accumulation of cells in the G1

phase (10 µM) and G2 phase (40 µM) in the present study. The accumulation at

10 µM might be explained in the same way as previously described (AHMAD et al.

2001), i.e. resveratrol induced WAF/p21 expression, thus inhibiting the formation of

cyclin D-CDK4/6 and cyclin E-CDK2 complexes and resulting in a G1 phase arrest of

the cells. A resveratrol concentration of 40 µM led to a cell cycle arrest in the G2 phase,

which provides time for cells to undergo apoptosis (DIPAOLA 2002; KAJSTURA et al.

2007). This was actually confirmed after incubating the cells with 80 µM resveratrol,

which resulted in a significant increase of cells in the Sub-G1 phase. Compound 5 was

found to have the same effect as resveratrol at high concentrations, but induced a cell

cycle arrest in the S phase at 40 µM, which was accompanied by a significant

decrease of cells in the G1 phase. Therefore, the growth-inhibitory effect of compound

5 on A-431 cells is most probably due to a disturbance of the cell cycle, i.e. the

induction of a cell cycle arrest in the S phase.

The effect of resveratrol on LNCaP cells has been controversially discussed in the

scientific literature. HSIEH et al. (1999) reported that 2.5 µM resveratrol failed to

cause any effect on the cell cycle after 4 days of incubation. KUWAJERWALA et al.

(2002) reported that treatment with 10 and 20 µM resveratrol led to an accumulation

of LNCaP cells in the S phase after 24 h incubation. BENITEZ et al. (2007) showed

that 50 µM resveratrol induced an increase of the cell counts in the S phase, but only

at higher concentrations (100 and 150 µM) did resveratrol lead to an increasing

number of cells in the G1 phase. The present study showed that resveratrol led a

significant G1 phase arrest after incubation with 10-80 µM for 24 h, which is in line with

recent studies (WANG et al. 2010; EMPL et al. 2015). Again, the inconsistent results

indicated by the above-mentioned studies may be due to different incubation times

and cell batches used. As in the case of resveratrol, cells exposed to compound 5

(10-80 µM) accumulated in the G1 phase. Therefore, the compound 5-induced growth

inhibition is most probably due to the induction of a cell cycle arrest in the G1 phase.

Discussion

53

In Caco-2 cells, we found out that concentrations lower than 40 µM resveratrol caused

a significant cellular accumulation in the S phase, which is in agreement with a

previously published study (WOLTER et al. 2001). Since at higher concentrations

(80 µM) resveratrol induced an accumulation of cells in the Sub-G1 phase, this is most

probably due to the induction of apoptotic processes (KAJSTURA et al. 2007). The

resveratrol induced G1/S phase arrest is mediated by a reduction in the formation of

the cyclin D1-CDK4 complex (MUSGROVE et al. 1994). The compound 5-mediated

arrest in the G1 and S phase at 80 µM might be due to the same effect.

In the present study, resveratrol induced an arrest of HCA-7 cells in the G1 (24 h) and

S phase (48h), which is consistent with a previously published study (SALE et al.

2005). Compound 5 mainly caused a cell accumulation in the G1 phase at

concentrations of 40 and 80 µM. One explanation for this effect may be the reduction

of cyclin D, CDK4 and 6 by compound 5, thereby causing a G1 phase arrest

(MUSGROVE et al. 1994). Another explanation could be that compound 5 leads to the

activation of the peroxisome proliferator-activated receptor gamma (PPAR-γ), which is

associated with G1 phase arrest in human colorectal tumor cell lines (BROCKMAN et

al. 1998; KRISHNAN et al. 2007). SMITH et al. (2000) reported that NSAIDs may

inhibit the proliferation of HCA-7 cells and lead to a cell cycle arrest by activation of

PPAR-γ.

All in all, the effect of compound 5 on the cell cycle is similar to that of resveratrol in

the tested human cancer cell lines, since both compounds interfered with cell cycle

distribution. Nevertheless, the data regarding resveratrol shown herein is not in

complete agreement with previously published studies. This might partly be due to

different cell culture media, protocol use, time points or test substance concentrations

used. Depending on the cell type, these effects of compound 5 might be mediated

through cyclins and CDK’s, cyclin-dependent kinase inhibitors or nuclear receptors.

However, the mechanism underlying the observed cell cycle arrest(s) would need

further investigation.

Discussion

54

4.3. Effect of resveratrol and compound 5 on the COX-2/PGE2-pathway

COX-2 and PGE2 have been described as tumor promoters in cancer progression

(GREENHOUGH et al. 2009). Studies have demonstrated that COX-2 and PGE2

might support tumor progression by increasing tumor growth and invasiveness

(WANG et al. 2005; SOBOLEWSKI et al. 2010). Coxibs and other NSAIDs were found

to exert a positive cancer chemopreventive effect (FISCHER et al. 2011). The

mechanism by which most NSAIDs reduce cancer progression involves the

COX-2/PGE2 pathway (CHA et al. 2007; FISCHER et al. 2011). Therefore, it was

important to evaluate the effect of compound 5 on the COX-2/PGE2 pathway. In our

study, HCA-7 cells were used as a model for investigating this matter, since they

overexpress COX-2 (SHENG et al. 1997).

Our results show that resveratrol can inhibit COX-2 protein expression in HCA-7 cells,

which is consistent with a previous study (SALE et al. 2005). COX-2 expression was

only slightly inhibited by resveratrol after a 24 h-incubation. In contrast, compound 5

dramatically enhanced COX-2 expression at a concentration of 50 and 100 µM. The

levels of PGE2 were also analyzed in the present study, compound 5 thereby

exhibiting a stronger effect on the PGE2 levels. Resveratrol is known to be a COX

inhibitor. SUBBARAMAIAH et al. (1998) showed that resveratrol inhibited COX-2

transcription and activity in phorbol ester-treated human mammary epithelial cells.

The mechanism underlying the up-regulation of COX-2 expression is not clear. The

transcription factor NF-kB may be involved in the control of the transcription of this

gene, and NF-kB activation by compound 5 probably contributes to the increase in

COX-2 expression (LI et al. 2011). PGE2 also positively regulates the expression of

COX-2 (BENOIT et al. 2004; VICHAI et al. 2005) .Therefore, it is possible that the

treatment with compound 5 inhibited PGE2 production, and the decreased PGE2

levels might have led to a compensatory up-regulation of the transcription of the

COX-2 gene to increase the amount of COX-2 protein.

4.4. Metabolism of compound 5

Discussion

55

Resveratrol has been shown to inhibit the growth of a broad panel of tumor cells in

vitro (COTTART et al. 2014; GAMBINI et al. 2015). However, studies have

demonstrated that the bioavailability of resveratrol in rats and humans is low

(ANDLAUER et al. 2000; WALLE et al. 2004). Resveratrol is readily absorbed, but is

then rapidly metabolized (GOLDBERG et al. 2003; WALLE et al. 2004). The

concentration of unconjugated resveratrol in vivo is too low to induce the same effects

as those observed in vitro (BOOCOCK et al. 2007a; BOOCOCK et al. 2007b).

However, the bioavailability of resveratrol could be enhanced by the accumulation of

resveratrol and its metabollites in different tissues after absorption of the mother

compound (WALLE 2011). Therefore, it made sense to evaluate the metabolism of

compound 5.

Recent studies have demonstrated that the metabolic stability of resveratrol and

related polyphenols can be determined by an in vitro glucuronidation assay (SCHEBB

et al. 2012; WILLENBERG et al. 2012). Therefore, in our study, we investigated the

metabolic stability of compound 5 by using the same assay and compared it with that

of resveratrol. Compound 5 was shown to be conjugated by RLMs after 40 min

incubation. About 66% of compound 5 was found to be conjugated, a conjugation rate

that is about one third lower than that of resveratrol (99%). The comparison suggests

that the stability of compound 5 is higher than that of resveratrol, but compound 5 is

still metabolized to a great extent. Moreover, the absorption rate of compound 5 is not

known, this may represent a limitation when wanting assess its effects in vivo. The

metabolic stability of compound 5 in humans also needs to be further investigated.

Discussion

56

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Annex

73

6. Annex

Solutions in materials

1 % acetic acid solution solution

20 ml acetic acid acetic acid

500 ml ddH2O

0.4% SRB solution

2 g Sulforhodamine B

500 ml 1 % acetic acid solution

50 % TCA solution

2.5 g TCA

500 ml ddH2O

Tris buffer (pH 10.5)

1.2114 g Tris

1000 ml ddH2O

PBS (pH 7.4)

0.4 g KCl

0.2 g KH2PO4

8 g NaCl

1.43 g Na2HPO4 • 2H2O

1000 ml ddH2O

RNase A solution

1 mg RNase

1 ml ddH2O

Protein lysis buffer (pH 6.8)

12 g Urea

Annex

74

0.25g SDS

0.4 g Tris

50 ml ddH2O

Separating gel buffer (pH 8.8)

56.5 g Tris

2 g SDS

200 ml ddH2O

Stacking gel buffer (pH 6.8)

56.5 g Tris

1 g SDS

200 ml ddH2O

5 X protein loading buffer

2.4024 g Urea

1 g SDS

0.0756 g Tris

0.02% Bromophenol blue

10 ml ddH2O, before using add 20 % DTT

2 X protein loading buffer

0.2 g SDS

0.0756 g Tris

0.02% Bromophenol blue

10 ml ddH2O

10 X Running buffer (pH 8.3)

30.3 g Tris

10 g SDS

144 g Glycin

1000 ml ddH2O

Annex

75

1 X Running buffer

100 ml 10 X Running buffer

900 ml ddH2O

10 X Transfer buffer (pH 8.2-8.4)

30.39 g Tris

144 g Glycin

840 ml ddH2O

1 X Transfer buffer (pH 8.3)

84 ml 10 X Transfer buffer

756 ml ddH2O

160 ml Methanol

1 M Tris solution (pH 7.5)

24.2 g Tris

200 ml ddH2O

5 M NaCl solution

146.1 g NaCl

500 ml ddH2O

1 X Washing buffer

10 ml 1 M Tris solution

30 ml 5 M NaCl solution

0.5 ml Tween® 20

1000 ml ddH2O

Blocking buffer

5 mg milk powder

10 ml 1 X Washing buffer

10% Acrylamide separating gel

Annex

76

7 ml ddH2O

4.99 ml 30 % Acrylamide/Bis-Acrylamid (37.5:1)

3 ml Separating gel buffer

112.5 μl 10 % Ammonium Persulfate (APS)

12 μl Tetramethylethylenediamine (TEMED)

Stacking gel

6.33 ml ddH2O

1.65 ml 30 % Acrylamide/Bis-Acrylamid (37.5:1)

2 ml Separating gel buffer

75 μl 10 % Ammonium Persulfate (APS)

8 μl Tetramethylethylenediamine (TEMED)

Acknowledgments

77

7. Acknowledgments

Firstly, I would like to thank my supervisor Prof. Dr. Pablo Steinberg for providing the

opportunity to work in his great team. Prof. Dr. Pablo Steinberg has always been nice

and patient to help and answer all my questions. I am very lucky to be guided by him.

Secondly, I want to express my deepest gratitude to Dr. Michael Empl for his open and

patient attitude and for giving me numberless valuable comments. He is a very

warm-hearted person, and he helped me a lot in my laboratory work and in my life. I

would not have succeeded in my PhD project without his guidance and support.

I also would like to thank the members of my supervisor group, Prof. Dr. Hassan Y.

Naim and Prof. Dr. Peter Winterhalter. Thanks a lot for their enthusiastic comments

and assistance in all my presentations and meetings.

Thanks a lot to Prof. Dr. Y. Pan for kindly providing the substances used in my PhD

program. Also many thanks to Dr. Ina Willenberg and Michael Krohn for their kind

contribution during my PhD project.

I would like to thank the Department of Immunobiology and Department of

Microbiology for their assistance and permission to work with their laboratory

equipment.

Many thanks to my colleagues Ulrike Oberjatzas, Dr. Maria Brauneis, Dr. Petra Nicken,

Dr. Stephanie Vogel, Janine Döhring, Bettina Seeger, Nicole Brauer and Julia

Hausmann for always helping me with my work. It has really been nice to spend a

wonderful part of my lab life together with them.

I want to give my countless thanks to my friends Dr. Laura Bartel and Dr. Ping-Ping

Tsai for their encouragement and caring during the past years. They encouraged me

to be more self-confident in my work and took care of me a lot at the beginning of my

life in Hannover.

Acknowledgments

78

Also thanks a lot to my Chinese friends Ning Zhang, Lin Li and Wei Wang for their nice

help and advices in my daily life. It is a very precious memory for the time we spent

together abroad.

Special thanks to my family, my mother and my brothers. Thanks a lot for your support

and for giving me the freedom to realize my dream.

Finally, I want to thanks China Scholarship Council (CSC) for financial support during

my study in Germany.

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