DIPLOMARBEIT

Titel der Diplomarbeit

„Statin induced effects on MYCN in Neuroblastoma cell lines“

verfasst von

Tschu-Jie Liu

angestrebter akademischer Grad

Magister der Naturwissenschaften (Mag.rer.nat.)

Wien, 2013

Studienkennzahl lt. Studienblatt: A441

Studienrichtung lt. Studienblatt: Diplomstudium Genetik - Mikrobiologie

Betreuerin / Betreuer: Univ.-Prof. Mag. Dr. Pavel Kovarik

Für meine liebe Familie

4

Danksagung

Danksagung

An dieser Stelle möchte ich alldiejenigen Personen danken, die mich beim Verfassen dieser

Arbeit unterstützt haben:

Ich möchte Univ.-Prof. Mag. Dr. Pavel Kovarik danken, der meine Diplomarbeit betreut hat.

Ein großes Dankeschön geht an Prof. Dr. Martin Hohenegger, der mir das Arbeiten in einer der

besten, nettesten Arbeitsgruppe ermöglicht hat und mich schonend in die Welt der Wissenschaft

und des wissenschaftlichen Arbeitens eingeführt hat.

Auch Mag. Bihter Atil möchte ich ein riesiges Dankeschön für ihre technischen,

wissenschaftlichen und Labor-organisatorischen Hilfestellungen aussprechen.

Weiters möchte ich Mag. Christine Wasinger und Murtaza Kulaksiz danken, die stets ihre

wertvollen Erfahrungen mit mir geteilt haben und mich mit diversen technischen Feinheiten

unter die Arme gegriffen haben.

Der größte Dank gebührt meiner lieben Familie, die trotz allen schwierigen Zeiten während

meines Studiums immer an mich geglaubt und mich vorangetrieben haben:

Meinem Vater danke ich herzlichst für all die motivierenden Zusprüchen, sowie etwaige

finanzielle Unterstützung.

Bei meiner Schwester und ihrem Mann Marcus bedanke ich mich besonders für ihre weisen,

praktischen Ratschläge, sowie für die tollen Zeiten in Wien, Hamburg und Zürich.

Den allergrößten Dank bin ich meiner Mutter verpflichtet, die trotz allen schwierigen

Lebenssituationen mir immer auf die verschiedenste Art und Weise unermüdlich geholfen hat.

Vielen Dank Euch allen!

5

Table of Contents

1 Table of Contents

Danksagung ...................................................................................... 4

1 Table of Contents ........................................................................ 5

2 Zusammenfassung ...................................................................... 8

3 Abstract ....................................................................................... 9

4 Abbreviations ............................................................................ 10

5 Introduction .............................................................................. 14

5.1 Neuroblastoma ................................................................................................... 14

5.1.1 Definition ...................................................................................................... 14

5.1.2 Staging .......................................................................................................... 15

5.1.3 Risk groups and prognosis ............................................................................. 16

5.1.4 Treatments .................................................................................................... 17

5.2 MYC proto-oncogene family ............................................................................. 19

5.2.1 MYC/c-Myc .................................................................................................. 20

5.2.2 MYCL/L-myc ............................................................................................... 22

5.2.3 MYCN/N-myc .............................................................................................. 22

5.3 Statins ................................................................................................................. 27

5.3.1 Definition ...................................................................................................... 27

5.3.2 Types of statins ............................................................................................. 28

5.3.3 Anti-cancer effects of statins ......................................................................... 30

6 Aims ........................................................................................... 35

7 Materials ................................................................................... 36

7.1 Antibodies ........................................................................................................... 36

7.2 Cell lines ............................................................................................................. 37

7.3 Drugs .................................................................................................................. 37

7.4 Equipment .......................................................................................................... 38

7.5 Growth Media .................................................................................................... 39

7.6 Protease-Inhibitors............................................................................................. 40

7.7 Tools ................................................................................................................... 40

7.8 Other substances ................................................................................................ 41

8 Methods ..................................................................................... 43

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Table of Contents

8.1.1 Cell culture treatments ................................................................................... 43

8.2 Cell culture techniques....................................................................................... 43

8.2.1 Cell culture maintenance and conditions ........................................................ 43

8.2.2 Splitting, cell counting and cell seeding ......................................................... 43

8.2.3 Microscopy and picture acquisition ............................................................... 44

8.3 Protein methods ................................................................................................. 45

8.3.1 Cell lysis in IP-Buffer.................................................................................... 45

8.3.2 Nuclear extraction method ............................................................................. 45

8.3.3 Protein concentration determination .............................................................. 47 8.3.3.1 Bio-Rad protein concentration determination ..................................................................47 8.3.3.2 BCA protein concentration determination ........................................................................47

8.3.4 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) .. 48

8.3.5 Western Blot ................................................................................................. 51 8.3.5.1 Wet Blotting ....................................................................................................................51 8.3.5.2 Semi-Dry Blotting ...........................................................................................................52 8.3.5.3 Ponceau staining and blocking ........................................................................................52 8.3.5.4 Incubation with primary/secondary antibody ...................................................................53 8.3.5.5 Detection by enhanced chemiluminescence ......................................................................53 8.3.5.6 Stripping of nitrocellulose membrane ..............................................................................54 8.3.5.7 Quantification of Western Blot ........................................................................................55

8.4 Flow cytometry ................................................................................................... 55

8.4.1 Fixation of cells ............................................................................................. 55

8.4.2 Flow cytometric analysis ............................................................................... 56

8.5 Senescence-associated β-Galactosidase Assay ................................................... 58

8.6 Statistical analysis .............................................................................................. 59

9 Results ....................................................................................... 60

9.1 N-myc protein expression in SH-SY5Y, Kelly and IMR-32 cells treated with simvastatin and mevalonate ...................................................................... 60

9.2 N-myc protein expression in Kelly and IMR-32 cells treated with increasing simvastatin concentration ................................................................ 64

9.3 Altered N-myc expression in Kelly and IMR-32 cells treated with DMSO ..... 66

9.4 Altered N-myc protein expression in IMR-32 and Kelly cells treated with simvastatin and mevalonate analysed by flow cytometry ................................. 69

9.5 N-myc protein expression changes in Kelly and IMR-32 cells after long-term simvastatin treatment................................................................................ 71

9.6 N-myc in nuclear extracts .................................................................................. 71

9.7 Expressional changes of small G-proteins of the Rac-family by simvastatin treatment in IMR-32 and Kelly cells ............................................. 73

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Table of Contents

9.8 Alterations of Aurora Kinase A and Max protein expression in IMR-32 and Kelly cells treated with simvastatin ............................................................ 76

9.9 Apoptosis induction by simvastatin treatment in IMR-32 and Kelly cells....... 78

10 Discussion .................................................................................. 83

10.1 N-myc over-expression is confirmed in human Kelly and IMR-32 cells .......... 83

10.2 Simvastatin is able to downregulate N-myc level in Kelly, but not in IMR-32 cells ....................................................................................................... 84

10.3 Simvastatin is able to reduce the expressional level of selected small GTPases .............................................................................................................. 85

10.4 Simvastatin treatment may cause functional alterations of N-myc .................. 86

10.5 Simvastatin treatment induces apoptosis .......................................................... 87

11 Concluding Remarks ................................................................ 89

12 List of Figures ........................................................................... 90

13 List of Tables ............................................................................. 92

14 References ................................................................................. 93

15 Curriculum Vitae.....................................................................107

8

Zusammenfassung

2 Zusammenfassung Der Transkriptionsfaktor N-myc (V-myc related myelocytomatosis viral-related oncogene,

neuroblastoma derived [avian]) ist ein etablierter biologischer und klinischer Marker für

Neuroblastoma, eine der häufigsten, pädiatrischen Tumorerkrankung. Eine Amplifizierung

dessen kodierenden Gens MYCN kommt bei 40 % schwerwiegender Erkrankung vor und steht

mit aggressivem Tumorwachstum, fortgeschrittenem Krebsstadium und schlechten Prognosen in

Verbindung. Unterdrückung der N-myc Expression könnte eine neue Form der Therapie

darstellen. Tatsächlich haben in vitro Experimente gezeigt, dass die Herunterregulierung des N-

mycs mit 5-Hydroxyurea mit Seneszenz in menschlichen Neuroblastom Zelllinien einhergeht.

Statine sind HMG-CoA Reduktase Inhibitoren und verhindern somit die Synthese von

Isoprenoiden und Cholesterol. Diese Medikamente werden daher weitläufig für Behandlung von

Hypercholesterinämie eingesetzt. Des Weiteren wurde Statinen auch eine

proliferationshemmende Wirkung auf Tumorzellen in vitro und in vivo nachgewiesen.

In dieser Arbeit wurden menschliche Neuroblastoma Zelllinien mit Amplifikation in MYCN

untersucht, um klarzustellen, ob Statine eine Wirkung auf N-myc Expression haben. Erstens

wurde die erhöhte Expression von N-myc in menschlichen IMR-32 und Kelly Neuroblastom

Zelllinien nachgewiesen, wohingegen N-myc unter denselben Bedingungen in der MYCN-nicht-

amplifizierten Neuroblastom Zelllinie SH-SY5Y kaum nachzuweisen war. Zweitens führte das

prototypische Statin „Simvastatin“ zu einer konzentrationsabhängigen Abnahme von N-myc in

Kelly Zellen, wobei ein gegenteiliger Effekt bei IMR-32 Zellen erzielt wurde. Interessanterweise

wurde auch die Menge von cdc42, einem kleinen G-Protein, mittels Simvastatin-Behandlung bei

beiden MYCN-amplifizierten Neuroblastom Zelllinien reduziert. Drittens, bei einem

Schwellenwert von 1 µM Simvastatin wurde auch Aurora Kinase A in diesen Zellen signifikant

reduziert. Ein ähnlicher Effekt wurde bei Max – einem Interaktionspartner von N-myc – nicht

beobachtet.

Wir schließen daraus, dass Simvastatin N-myc Expression bei bestimmten, menschlichen

Neuroblastom Zelllinien unterdrücken kann, was eine neue Form der Therapie darstellen könnte.

9

Abstract

3 Abstract The transcription factor N-myc (V-myc related myelocytomatosis viral-related oncogene,

neuroblastoma derived [avian]) is considered the best-established biological and clinical marker

for neuroblastoma, which is a common childhood tumour. Amplification of its encoding gene

MYCN occurs in 40 % of high-risk tumours and is associated with aggressive tumour growth,

advanced clinical stage and poor prognosis. The inhibition of N-myc expression might represent

a therapeutic option. Indeed, in vitro experiments have successfully described N-myc down-

regulation with 5-hydoxyurea, simultaneously inducing senescence in human neuroblastoma cell

lines.

Statins inhibit the HMG-CoA reductase and thereby prevent the synthesis of isoprenoids and

cholesterol. These drugs are therefore widely used to treat hypercholesterolemia. Emerging

evidences show that statins also possess anti-proliferating effects on tumour cells in vitro and in

vivo.

Here, human neuroblastoma cells with amplified MYCN were investigated to answer the

question whether statins have an impact on N-myc. First, the amplified expression of N-myc in

human IMR-32 and Kelly neuroblastoma cell lines could be confirmed, whereas N-myc was

hardly detectable in the human, MYCN-non-amplified SH-SY5Y neuroblastoma cell line under

the same conditions. Second, the prototypical statin, simvastatin, induced a concentration and

time dependent down-regulation of N-myc in Kelly cells, while the opposite effect was triggered

in IMR-32 cells. Interestingly, the small G-protein cdc42 was down-regulated by simvastatin

exposure in both MYCN amplified neuroblastoma cell lines, Kelly and IMR-32 cells. Third, a

threshold concentration of 1 µM simvastatin was also sufficient to significantly down-regulate

Aurora kinase A in these cells. The N-myc interaction partner Max was not altered in a similar

manner by simvastatin application.

Thus, we conclude that simvastatin is able to reduce amplified N-myc levels in certain human

neuroblastoma cell lines, which may give head to new therapeutical approaches.

10

Abbreviations

4 Abbreviations

5-HU 5-Hydroxyurea

ABC ATP binding cassette

Akt Protein kinase B

ALK anaplastic lymphoma kinase

ARF ADP-ribosylation factor

AURKA Aurora Kinase A gene

BARD1 BRCA1-associated RING domain protein 1

BCA Bicinchoninic Acid

BCL B-cell lymphoma

BDNF Brain Derived Neurotrophic Factor

BET Bromodomain and Extra-Terminal

BSA Bovine Serum Albumin

bHLH basic Helix loop Helix

cdc Cell division control

cdk Cyclin dependent kinase

cm centimeter

CO2 Carbon dioxide

COG Children’s Oncology Group

Cu Copper

DMEM Dulbecco’s Modified Eagle Medium

DMSO Dimethyl sulfoxide

dmin double minute

DNA Desoxyribonucleic Acid

dox doxorubicin

DPYSL Dihydropyrimidinase-like

EDTA Ethylenediaminetetraacetic acid

ERK Extracellular-signal Regulated Kinase

FBS Fetal Bovine Serum

Fbxw7 F-box and WD repeat domain containing 7

FSC Forward Scatter

GAP GTPase-activating Protein

11

Abbreviations

GCN5 General Control of amino acid synthesis yeast homolog like 2

GD2 Ganglioside G2

GDI Guanine Nucleotide Dissociation Inhibitors

GDP Guanosine Diphosphate

GEF Guanine Nucleotide Exchange Factors

GM-CSF Granulocyte-Macrophage-Colony Stimulating Factor

Gsk3β Glycogen synthase kinase 3 β

GTP Guanosine Triphosphate

GTPase Guanosine Triphosphate hydrolase

H2O Water

HCl Hydrochloric acid

HDAC Histone Deacetylase

HMG-CoA 3-hydroxy-3-methyl-glutaryl-Coenzyme A

HRP Horseradish Perxoxidase

HSR homogeneous staining regions

INRG International Neuroblastoma Risk Group

INSS International Neuroblastoma Staging System

IP Immunoprecipitation

K2HPO4 Dipotassium phosphate

K3[Fe(CN)6] Potassium ferricyanide

K4[Fe(CN)6] Potassium ferrocyanide

kb Kilobase-pairs

KCl Potassium Chloride

KH2PO4 Monopotassium phosphate

kDa Kilo Dalton

Lap2β Lamina-associated polypeptide 2 β

LDL Low Density Lipoprotein

Leu leucine

mA milliampere

MAP3-K12 Mitogen-activated protein kinase kinase kinase 12

MAX MYC associated factor X

MCM Minichromosome Maintenance Complex

MDM2 Mouse double minute 2 homolog

12

Abbreviations

MDR Multiple Drug Resistance

MEF2D Myocyte-specific enhancer factor 2D

MeOH Methanol

mev Mevalonate

mg milligram

MgCl2 Magnesium chloride

miRNA Micro Ribonucleic Acid

MKP Mitogen-activated protein Kinase Phosphatase

mL milliliter

µL microliter

mm millimeter

µM micromolar

MQ MilliQ water

mRNA Messenger Ribonucleic Acid

MRP Multidrug Resistance Protein

MYC V-myc avian myelocytomatosis viral oncogene homolog

MYCL V-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog

MYCN V-myelocytomatosis viral related oncogene, Neuroblastoma derived [avian]

NaCl Sodium chloride

NaH2PO4 Monosodium phosphate

Na2HPO4 Disodium phosphate

NaN3 Sodium azide

NaOH Sodium hydroxide

NBPF23 Neuroblastoma Breakpoint Family, Member 23

nM nanomolar

nm nanometer

NP-40 Nonidet-P40 (octyl phenoxypolyethoxylethanol)

PAGE Polyacrylamide gel electrophoresis

PARP Poly ADP Ribose Polymerase

PBS Potassium Buffered Saline

Penc/Strep Penicillin/Streptomycin

PHOX2B Paired-like homeobox 2b

PI3K Phosphatidylinositide 3 kinase

13

Abbreviations

p21WAF1/CP1 cyclin-dependent kinase inhibitor 1/CDK-interacting protein 1

p27KIP1 Cyclin-dependent kinase inhibitor 1B

qPCR quantitative Polymerase Chain Reaction

Rac Ras-related C3 botulinum toxin substrate

Ras Rat Sarcoma

Rho Ras homologue

RPMI Roswell Park Memorial Institute medium

SSC Side Scatter

SCF Skp, Cullin, F-box containing ubiquitin ligase complex

SCLC Small Cell lung Cancer

SD Standard Deviation

SDS Sodium Dodecyl Sulfate

sim Simvastatin

SIOPEN International Society of Pediatric Oncology Europe Neuroblastoma Group

SIRT1 Sirtuin-1

TBP TATA Box binding Protein

TBS Tris-Buffered Saline

TBS-T Tris-Buffered Saline with Tween-20

Temed Tetramethylethylendiamin

TERT Telomerase Reversse Transcriptase

TG2 Transglutaminase-2

Tris Tris-(hydroxymethyl)-aminomethan

TrkA Neurotrophic Tyrosine Kinase Receptor Type 1

TrkB Neurotrophic Tyrosine Kinase Receptor, type 2

TRRAP Transformation/Transcription Associated Protein

V Volt

vin Vincristine

v/v Volume/volume %

v/w Volume/mass %

W Watt

x g Gravity

X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactropyranosid

14

Introduction

5 Introduction

5.1 Neuroblastoma

5.1.1 Definition

Neuroblastoma is an extracranial, solid tumour of the sympathetic nervous system that occurs in

patients from one to fifteen years of age; it is considered the most common pediatric tumour as it

accounts for 8 % of all pediatric tumours (Bell et al., 2010; Maris, 2010). The median age of

diagnosis of the general population is 1.5 years (Brodeur, 2003; Maris, 2010). The tumours can

be found anywhere in the peripheral nervous system, however, the majority arise from the

adrenal medulla and paraspinal ganglia. Metastasis can be formed in regional lymph nodes,

bones and bone marrow, in the liver (Figure 1). The symptoms depend on the location of the

tumour: Tumour formation in the upper chest may cause Horner’s syndrome. Tumour in the

paraspinal ganglia may lead to compression and paralysis (Maris, 2010). Neuroblastoma tumour

cells originate from neuronal precursor cells during the embryonic development called

neurulation. The tumorigenic characteristic is based upon the inability of neuronal precursor cells

to cease the cell cycle and to differentiate (Tonini et al., 2012).

Figure 1. Neuroblastoma tumour and metastasis development (Maris, 2010)

15

Introduction

The genetic cause of neuroblastoma may lie within hereditary mutational factors or be

somatically acquired (5 to 15 %) (Maris, 2010). Mutations within ALK and homeobox gene

PHOX2B are responsible for the majority of hereditary neuroblastoma cases. Single nucleotide

mutation in the kinase domain of ALK gene results in constitutive activation of the kinase

(Mossé et al., 2008) and enhances the oncogenic property of MYCN/N-myc by stabilizing it

(Berry et al., 2012). The loss-of-function mutation in the homeobox gene PHOX2B prohibits

normal sympathetic nervous tissue development and differentiation capabilities (Mossé et al.,

2008; Raabe et al., 2008). Somatically acquired – or “spontaneous” - neuroblastoma cases arises

from combinatory interactions between DNA variants in certain putative genes, such as single-

nucleotide-polymorphisms within the genes FLJ22536, BARD1 or NBPF23 (Capasso et al.,

2009; Maris, 2010). Another predisposition locus is the short arm of chromosome 16 (16p12-13)

(Brodeur, 2003).

5.1.2 Staging

The staging of neuroblastoma aims to clarify the distribution of tumours within patient’s body

and is based on physical exams, imaging tests, and biopsies of the main tumour and other tissues

to allow prognosis (cited from the homepage of American Cancer Society). The most commonly

used neuroblastoma staging system since 1990 is the International Neuroblastoma Staging

System (INSS), proposed by Smith et al. (Smith et al., 1989). The stages are described shortly in

Figure 2. Noteworthy, patients are often reported to have a special disease stadium called 4S

stage. Characteristic for this stage is a unique pattern of metastasis formation in the skin and the

liver. However, this special stage is generally associated with good prognosis and spontaneous

regression with nothing than supportive care and observation (Bell et al., 2010; Maris, 2010).

16

Introduction

Figure 2. Neuroblastoma stages classified by INSS (Brodeur et al., 1993)

5.1.3 Risk groups and prognosis

The neuroblastoma is known for its broad spectrum of disease outcome: On the one hand it has a

highly disproportionate mortality and morbidity rate; on the other hand it has the highest rate of

spontaneous regression amongst all pediatric tumours. Older children diagnosed with tumour

stage 4 are prone to suffer from refractory disease, whereas patients diagnosed at lower stages

have a good chance for spontaneous regression (Maris, 2010). Thus, treatment strategies need to

be tailored according to the patient risk.

The development of a risk group classification system may come in handy to allow fast

diagnosis, precise prognosis and righteous application of treatments. However, diverse

classification systems exist within different institutions (i.e. International Society of Pediatric

Oncology Europe Neuroblastoma Group “SIOPEN”, Children’s Oncology Group “COG”)

depending on the respective criteria of choice (i.e. age of patient, surgery risk, histology, MYCN

17

Introduction

amplification, postsurgical staging etc.), resulting in inhomogeneous risk definitions (Cecchetto

et al., 2005; Kushner et al., 2005). To overcome this problem, the International Neuroblastoma

Risk Groups (INRG) Task Force was founded in 2004, wherein research information is pooled

from neuroblastoma research institutions of several countries (North America, Australia, Europe

and Japan) and classified according to 13 prognostic markers, i.e. age of diagnosis, tumour stage

and characteristics, genetic predisposition and genomic aberrations (Cohn et al., 2009). INRG

assigns neuroblastoma cases to 4 classes with regards to survival rate: low to very low risk class

holds a high survival rate (< 75 % to ≤ 85 % and > 85 % respectively), intermediate class

predicts a survival rate of < 50 % to ≤ 75 % and high risk class neuroblastoma cases have a

survival rate of below 50 %. Promising improvements of this classification system is thought to

come along with advances in the research on tumour biology, genetic aberrants, epigenetic

alterations, as well as the development of identification of prognostic transcriptional patterns

typical for neuroblastoma (Maris, 2010). INRG classification system is a valuable step to reach

consensus among international neuroblastoma research groups and to allow evaluation and

validated comparisons of methodology, interpretation and risk-based therapeutic approaches.

Genetic features such as ploidy or structural rearrangements of DNA material in neuroblastoma

are considered reliable tools for disease prognosis. Near-triploid or hyperdiploid karyotypes and

expression of the neurotrophin receptor TrkA are associated with favourable outcome. Patients

with these tumours are mostly less than 1 year old (Kamijo and Nakagawara, 2012). On the

contrary, diploid karyotypes, structural changes such as deletions of 1p or 11q, unbalanced gain

of 17q or amplification of MYCN, coupled with expression of TrkB and its ligand – brain-

derived neurotrophic factor (BDNF) – are associated with unfavourable outcome; concomitantly

these tumours occur in children older than 1 year of age (Brodeur, 2003; Kamijo and

Nakagawara, 2012).

A more recent achievement in prognosis has been reported by non-invasive determination of

MYCN copy number by qPCR in blood plasma (Kojima et al., 2013).

5.1.4 Treatments

Treatments for neuroblastoma are accommodated to the patients according to the risk

classification and staging. Patients are preserved from exhausting therapeutic dosages as much as

possible. Low risk, benign, localized tumours are removed by surgery or may regress without

any intervention but supportive care (Hero et al., 2008). Intermediate risk, localized tumours are

18

Introduction

eradicated either by surgery or carefully dosed cycles of chemotherapy with alternating

combinations of chemotherapeutics such as carboplatin, etoposide, vincristine, doxorubicin and

cyclophosphamide. The numbers of cycles depend on the severity of the disease, such as age of

diagnosis, MYCN amplification and (un-)favourable histo-pathological features of the tumour

(Baker et al., 2010). Treatments of high-risk tumours are more complicated due to the high

probability of relapse. The treatment proceeds through three phases: The first phase (remission

induction) consists of cycles of chemotherapeutic administration such as treatments with

combinations of cisplatin, etoposide, vincristine, doxorubicin, cyclophosphamide and topotecan

(Garaventa et al., 2003; La Quaglia et al., 1994). The response to the induction phase is crucial

for the prognosis of the outcome (Beiske et al., 2009; Schmidt et al., 2008). Induction is followed

by rapid, sequential, tandem myeloablative consolidation therapy (consolidation phase) that aims

to eliminate residual drug resistant tumours, most importantly in the hematopoietic compartment

(Berthold et al., 2005; George et al., 2006; Matthay et al., 1999a; Pritchard et al., 2005).

However the optimal regiment for consolidation therapy still needs to be evaluated. The third

phase further increases the event-free survival rate (maintenance phase). For this purpose,

treatments with 13-cis retinoic acid (isoretinoid) have been proven successful (Matthay et al.,

1999b; Sidell, 1982; Thiele et al., 1985). This can be further improved by an immune-cellular

cytotoxicity treatment: It combines isoretinoid with coadministration of anti-GD2-monoclonal

antibody “ch14.18” (an antibody targeting disialoganglioside “GD2”, which is expressed by

neuroblastoma cells only) and interleukin-12 or granulocyte-macrophage-colony stimulating

factor (GM-CSF) in alternating cycles (Navid et al., 2010).

A common feature of high-risk tumours is the likeliness of a segmental genomic aberration

which the amplification of the oncogene MYCN belongs to. Thus, development of new

therapeutic approaches often targets MYCN to repress its amplification. Most recently, a study

proved a dual treatment strategy with anti-GD2 antibody and an Aurora kinase A inhibitor to be

successful in treatment of neuroblastoma cell lines (Horwacik et al., 2013). Other strategies are

based on development of tumour specific agents (such as metaiodobenzylguanidine), of new

high-dose chemotherapy and stem cell transplantation (Mugishima, 2012).

Recently, a study by Puissant et al. stated that MYCN amplified neuroblastoma cells show

sensitivity and response to treatment with JQ1, a small molecule inhibitor of BET bromodomain

proteins, by directly targeting the expression and down-regulation of MYCN. This epigenetic

19

Introduction

approach shows great potentials of bromodomain inhibitors and has already found its way to be

evaluated in clinical trials (Puissant et al., 2013).

Due to its importance in therapeutic strategies, MYCN and its proto-oncogene family will be

discussed in the following chapters.

5.2 MYC proto-oncogene family

MYC proto-oncogene family comprises of three gene members: MYC, MYCL and MYCN.

These proteins are transcription factors containing a basic-region/helix-loop-helix/leucine-zipper

(BR/HLH/LZ or bHLH-leucine zipper) motif in their C-terminus (hence called bHLH

transcription factors). They all localize to the nucleus and respond to diverse external factors by

hetero-dimerization with other bHLH transcription factors (most prominently with Max) with

their C-terminus and modulate the expression of their target genes by interaction with co-

activators or co-repressors by their N-Terminus (Cole and Nikiforov, 2006; DePinho et al., 1987;

Gherardi et al., 2013a; Kleine-Kohlbrecher et al., 2006). Their binding sites on the DNA are

marked by the consensus sequence CACGTG (E-box MYC sites) or non-canonical DNA

sequences (CA-NN-TG) (Blackwell et al., 1990; Corvetta et al., 2013; Gherardi et al., 2013b).

Oncogenic manifestation of the MYC genes – especially their amplification – is associated with

aggressive tumour phenotypes such as unlimited cell proliferation, prohibited proliferation,

genomic instability, angiogenesis and metastasis formation (Adhikary and Eilers, 2005).

Despite their close relation and structural homology (Figure 3), MYC, MYCN and MYCL are

expressed in distinct patterns during embryogenesis (Zimmerman and Alt, 1990; Zimmerman et

al., 1986); concomitantly, many studies suggest that MYC oncoproteins regulate distinct, but

overlapping set of target genes (Nesbit et al., 1999).

20

Introduction

Figure 3. Structural homologous regions in c-Myc, N-myc and L-myc (Cole and Cowling, 2008a) The N-terminus contains a transactivation domain with conserved regions known as MYC boxes. The C-Terminus

has a bHLH-leucine zipper motif through which these proteins bind to DNA.

5.2.1 MYC/c-Myc

MYC (V-myc avian myelocytomatosis viral oncogene homolog) is located at chromosome 8q24

gene and was initially identified according to its similarity to the v-myc oncogene, which was

reported by studies on oncogenic retroviruses induced myelocytomatosis in the chicken

(Duesberg and Vogt, 1979; Hu et al., 1979; Sheiness and Bishop, 1979). MYC holds a central

role in a broad spectrum of physiological and signalling pathways downstream of many ligand-

membrane receptor complexes (Armelin et al., 1984; Kelly et al., 1983), as well as in

reprogramming to pluripotent stem cell state (Laurenti et al., 2009; Singh and Dalton, 2009;

Takahashi and Yamanaka, 2006). Physiological roles include cell growth, proliferation (DNA

replication), tumorigenesis and tumour maintenance (Arvanitis and Felsher, 2006). On the

molecular aspect MYC is most prominently known as transcription factor that activates the

transcription of a vast number of genes (Dang, 2012). However, MYC also assumes a role as

repressor, best known for its negative regulation in the transcription of Miz-1 activated genes

(Schneider et al., 1997). MYC was also shown to be involved in regulation of cap-dependent

translation (Cole and Cowling, 2008b; Cowling and Cole, 2007).

Interestingly, MYC mRNA bears two translation initiation sites: An N-terminally extended form

of c-Myc is initiated at CUG in exon 1 (p67) and a major, predominant c-Myc protein initiated at

an internal, canonical AUG in exon 2 (p64) (Blackwood et al., 1994a). Both forms localize to the

cell nucleus and have the same short half-life. It was shown that the loss of the alternative CUG

site is not a requirement for tumorigenesis (Blackwood et al., 1994b). Functional differences

21

Introduction

between these two forms are not reported, however, one study confirmed the shorter product to

be involved in stress response (Spotts et al., 1997; Xiao et al., 1998a). A cleavage form of MYC

- “MYC-nick” - is localized to the cytoplasm and is responsible for promoting cell differentiation

(Conacci-Sorrell et al., 2010).

Abnormal MYC transformation events like translocation and – most frequently – amplification

are associated with various types of tumours including Burkitt lymphoma (Dalla-Favera and

Bregni, 1982; Taub et al., 1982), myeloma (Shou et al., 2000), leukaemia (Palomero et al., 2006;

Sharma et al., 2006; Weng et al., 2006) and solid carcinoma (Beroukhim et al., 2010; D’Cruz et

al., 2001). In normal cells, amplification of MYC triggers checkpoints such as ARF or p53

activation. Tumorigenesis may occur in cells bearing mutational impairment of ARF and p53

(Eischen et al., 1999; Zindy et al., 1998). Due to its proto-oncogenic role, its expression is tightly

transcriptionally regulated by motifs found in the proximity of its promoter region (Brooks and

Hurley, 2010; Hurley et al., 2006; Levens, 2010).

In order to bind to the DNA, c-Myc is able to hetero-dimerize with Max via its bHLH domain in

its C-terminus (Fladvad et al., 2005; Liu et al., 2003; McEwan et al., 1996; McMahon et al.,

2000; Nikiforov et al., 2002); remarkably, in certain cell types MYC may also exert its

transcriptional function in the absence of Max (Hopewell and Ziff, 1995). Subsequently, c-Myc

can bind to other transcription factors such as TRRAP, GCN5 and TBP via its N-terminal

domain (Follis et al., 2009; Hu et al., 2005; Mustata et al., 2009; Sauvé et al., 2007).

Therapeutic approaches aim to suppress tumour formation by targeting MYC amplification. It

was shown, that knock-down of MYC may reduce proliferation in tumour cells and trigger

apoptosis (Cappellen et al., 2007; Koh et al., 2011; Wang et al., 2008). Recently, several

alternative phosphorylation sites of MYC have been identified whose mutations render MYC

stronger transforming activity in SH-EP neuroblastoma line. These sites may represent potent

sites for therapeutic attack (Wasylishen et al., 2013).

Recent research focuses on the tumorigenic role of c-Myc in regulation of microRNA (miRNA)

expression (Bui and Mendell, 2010; Chang et al., 2008; Dews et al., 2006; Gao et al., 2009a; Liu

et al., 2013; O’Donnell et al., 2005). It has been shown, that c-Myc and N-myc control an

overlapping set of miRNAs. The role of miRNAs in oncogenesis will be discussed for N-myc in

5.2.3.

22

Introduction

5.2.2 MYCL/L-myc

MYCL (MYCL v-myc avian myelocytomatosis viral oncogene lung carcinoma derived

homolog) is not as well studied as MYCN and MYC. It has been mapped to chromosome 1p34.2

(Nau et al., 1985a). Its product L-myc is expressed in the developing kidney, new-born lung and

in both proliferative and differentiative compartments of the brain and neural tube (Hatton et al.,

1996). MYCL is found to be frequently amplified in human small cell lung cancer (SCLC) cells

(Nau et al., 1985b). A more recent study linked a single nucleotide polymorphism in the second

intron of MYCL (rs3134613) to enhanced susceptibility to diffuse-gastric cancer and

differentiation of gastric cancer (Chen et al., 2010).

5.2.3 MYCN/N-myc

MYCN (V-myc myelocytomatosis viral related oncogene, neuroblastoma derived [avian]) is

found on the distal short arm of chromosome 2p24 and encodes for the protein/transcription

factor N-myc (Brodeur et al., 1984a; Schwab et al., 1983). The human N-myc consists of 464

amino acids and has a molecular weight of about 60 to 63 kDa. N-myc shares wide structural

homologies with c-Myc, comprising of 3 exons, where the first is not translated and exons 2 and

3 encode for the protein product (Kohl et al., 1986).

MYCN is reported to be amplified in several tumours such as gliomablastoma (Garson et al.,

1985; Stenger et al., 1991), lung tumour (Nau et al., 1985b; Wong et al., 1986),

rhabdomyosarcoma (Driman et al., 1994; Williamson et al., 2005), Wilm’s tumor (Nisen et al.,

1986), and retinoblastoma (Doz et al., 1996; Lee et al., 1984). However, MYCN is most notably

an established biological and clinical marker for neuroblastoma and its amplification is highly

associated with a poor outcome (Brodeur et al., 1984b). The N-myc amplification is found in

approximately 25 % of all neuroblatoma cases and in 40 % of high risk neuroblastoma tumours

(Bell et al., 2010). It is preferentially amplified from the paternal allele (Cheng et al., 1993).

MYCN amplification may occur extra-chromosomally as double minutes by uneven segregation

during mitosis or intra-chromosomally as homogenous staining regions by integration of

amplified DNA into chromosomes (Brodeur, 2003). Amplification results in both higher MYCN

transcript (mRNA) steady-state level (100 times than non-amplified) and high protein level

(Brodeur, 2003). However, high protein level may also derive from increased protein stability

(Otto et al., 2009a). The amplification may provide selective advantages to the cells, such as the

23

Introduction

progression through the G1 phase of the cell cycle (Otto et al., 2009b), but also results in

physiological changes such as dependence on glucose and glutamine as nutrients (Qing et al.,

2012a). MYCN amplification is considered an intrinsic, biological feature in a subset of

neuroblastomas and tumours without MYCN amplification at time of diagnosis rarely if ever will

acquire this abnormality (Brodeur, 2003).

It was reported that other gene amplifications mainly occur with MYCN amplification, such as

allelic gain or amplification of 1p, 4q, 6p, 7q, 11q, 12q, 17q and 18q (Brodeur, 2003).

Like MYC, MYCN expression is tightly regulated on epigenetic, transcriptional and translational

level. On translational level, N-myc’s activity is dependent on the phosphorylation status of

Serine 62 (S62) and Threonine 58 (T58) located in the Mycbox I domain at the N-terminus.

Activation is carried out by S62 phosphorylation by Cyclin B/Cdk 1. S62-phoshorylated N-myc

is a target for Gsk3β. It phosphorylates N-myc at T58 which subsequently leads to Ubiquitin-

ligase dependent degradation of N-myc by SCFFbw7-complex (Otto et al., 2009b). Degradation

can be counteracted by PI3K/Akt pathway, which inhibits Gsk3β activity, or by Aurora Kinase A

whose function in promoting N-myc stability is proposed to be Ubiquitin chain synthesis at non-

Lysin 48 (K48) locations such as at K63 and K11 (Kim et al., 2007a). Ubiquitination at K48 do

not support degradation and thus is thought to represent a competitive mechanism opposed to

ubiquitination at K48 carried out by SCFFbw7-complex (Kim et al., 2007b; Otto et al., 2009b).

The role of MYCN stabilization and degradation in promoting cell cycle or differentiation is

depicted in Figure 4.

24

Introduction

Figure 4. Stabilization and degradation of N-myc in cell cycle progression and differentiation (Otto et al., 2009b)

Another study reported the role of histone deacetylase SIRT1 in MYCN associated oncogenesis

of neuroblastoma. SIRT1 was found to bind to N-myc at Mycbox I and to form a transcriptional

repression complex at the promoter of MKP3, thus inhibiting its expression. MKP3 is required

for ERK dependent N-myc phosphorylation/activation and subsequent degradation. Hence,

SIRT1 acts in a negative feedback loop to prevent N-myc degradation and promotes its stability

and oncogenesis (Marshall et al., 2011).

Over the past years, many downstream targets of N-myc have been uncovered. N-myc leads to

expression of oncogenes, most notably genes involved in cell cycle (MCM7), tumour

suppression (p53, TG2, MDM2) or cell survival (TERT, MRP1) (Figure 5) (Bell et al., 2010)

N-myc also shows activity of gene repression, which is most prominently achieved by

interaction with co-repressors such as Sp1 and Miz-1 at promoters of target genes and

recruitment of histone deacetylase HDAC1 (Iraci et al., 2011; Liu et al., 2007).

A more recent finding reported that, among genes which are negatively regulated by N-myc, the

dihydropyrimidinase-like protein DPYSL3 has been proven to be targeted by N-myc and its up-

regulation is associated with favourable outcome (Tan et al., 2013).

25

Introduction

Figure 5. A selection of downstream targets of N-myc (Bell et al., 2010) Over the last years, emerging evidences show a regulative role of N-myc in transcription of

microRNAs (miRNAs). MicroRNAs are the most widely studied non-coding RNAs which are

involved in negative regulation of protein expression in cells (Bartel, 2009). Important for

cancerogenesis, miRNAs hold a regulative function in many cellular processes such as

differentiation, cell fate decisions, signal transduction and apoptosis. Deregulated miRNA

expression has been associated with a broad range of diseases, including cancer. Hence, cancer

related miRNAs are termed “oncomirs” (Buechner and Einvik, 2012a).

One of the best-characterized groups of oncomirs regulated by N-myc belongs to the mir-17-92

cluster (Figure 6). Their functions involve repression of tumour suppressors (for example p21)

(Fontana et al., 2008a), proteins of apoptosis (for example BIM) (Fontana et al., 2008b) or

proteins involved in differentiation (like BCL2, MEF2D or MAP3-K12) (Beveridge et al., 2009).

26

Introduction

Figure 6. The mir-17-92 cluster and its proposed functions in oncogenesis (Buechner and Einvik, 2012b)

Interestingly, besides its role inducing miRNA expression, N-myc was also confirmed to be a

repressor of miRNA expression, especially in MYCN-amplified neuroblastoma tumours (Bray et

al., 2009; Chen and Stallings, 2007; Mestdagh et al., 2009; Schulte et al., 2010; Shohet et al.,

2011). It was suggested that dysregulation of Dicer and Drosha, which are key enzymes in the

smiRNA-processing pathway, are responsible or miRNA downregulation (Lin et al., 2010).

Moreover, N-myc is also targeted by miRNAs. The most prominent MYCN-targeting miRNA is

mir34a. It is located at chromosome 1p36, a region frequently deleted in MYCN-amplified

neuroblastoma tumours (Maris et al., 2007). Other miRNAs found to target N-myc are mir-34c,

mir-449, mir-19, mir-29, mir101 and let7/mir-202 (Buechner and Einvik, 2012b). They were all

found to interact with the 3’UTR of MYCN-transcript (Buechner et al., 2011).

27

Introduction

5.3 Statins

5.3.1 Definition

The chemical structure of statins consists of two components: A hydrophobic ring system and a

dihydroxyheptanoic acid segment covalently attached to it. Statins are 3-hydroxy-3-methyl-

glutaryl-CoA (HMG-CoA) reductase inhibitors and can thereby block the mevalonate pathway

(Figure 7).

Figure 7. Statins are negative regulators of the mevalonate pathway (Gazzerro et al., 2012) Statins are 3-hydroxy-3-methyl glutaryl CoA (HMG-CoA) reductase inhibitors and block the conversion of HMG-

CoA to mevalonate. This blockage depletes cells from cholesterol and other isoprenoids. The underlying mechanism is the dose-dependent, reversible competition of the

dihydroxyheptanoic acid segment (the pharmacophore) with the HMG-CoA for the binding site

within the HMG-CoA reductase enzyme (Figure 8). The ring system keeps the statin close to the

28

Introduction

enzyme and prevents its competitive displacement by the endogenous substrate HMG-CoA

(Gazzerro et al., 2012).

Dihydroxyheptanoic acid segment of statins

HMG-CoA

Figure 8. Pharmacological efficiency of statins is based on the similarity of the pharmacophore moiety of statins and HMG-CoA

R = ring moiety of statins; chemical structures are taken from wikipedia

The blockade of the mevalonate pathway results in depletion of cellular cholesterol, which cells

compensate by triggering the up-regulation of LDL receptors (particularly in the liver cells) and

uptake of low-density lipoprotein (LDL-) cholesterol from the circulation. Consequently, plasma

LDL-cholesterol levels decline and cardiovascular events are prevented (Taylor et al., 2011).

Pharmacokinetic profiles, plasma concentrations of statins and side-effects are stated to be

dependent on various factors such as ethnicity, age and sex, food intake, the type of statin, drug-

drug interactions, and concomitant diseases - especially hepatic or renal diseases which implicate

requirement for careful dosage administration (Gazzerro et al., 2012). Nonetheless, statins are

considered to be generally well tolerated (Hindler et al., 2006) and are widely used to manage

and prevent cardiovascular and coronary heart diseases.

5.3.2 Types of statins

Statins differ in their chemical structures, their solubility (lipophilicity and hydrophilicity) and

systemic bioavailability, their kinetic profile, the rate of metabolism and their ability to form

active and inactive metabolites after their hydrolysis in liver (Gazzerro et al., 2012). These

characteristics are dependent on the substituents of the ring moiety.

29

Introduction

Statins are divided in to two types according to their origins: Type 1 statins are natural or fungal-

derived (lovastatin, simvastatin and pravastatin) and type 2 statins are synthetic statins

(fluvastatin, cerivastatin, atorvastatin and rosuvastatin) (Figure 9).

Type 1 statins

lovastatin simvastatin pravastatin

Type 2 statins

fluvastatin cerivastatin atorvastatin rosuvastatin

Figure 9. Examples for type 1 and type 2 statins The pharmacophore moiety is depicted in red, the ring systems in black; chemical structures are modified from the

website of Molecular Anatomy Project (http://maptest.rutgers.edu/drupal/?q=node/39) Lovastatin, simvastatin, atorvastatin and fluvastatin are lipophilic, whereas pravastatin and

rosuvastatin are hydrophilic. The lipophilic property is crucial for pharmacokinetic effect of the

statin, because the liver is the target organ and the lipophilicty allows them to passively penetrate

cells of the extrahepatic tissues. Concomitantly, the high lipophilicity is accompanied by low

systemic bioavailability which results in a more pronounced (Gazzerro et al., 2012). On the

30

Introduction

contrary, hydrophilic statins are not able to encompass other tissues or enter hepatocytes by

passive diffusion, and thus requires active carrier uptake by hepatocytes (Hamelin and Turgeon,

1998; Nezasa et al., 2003), which results in higher hepatoselectivity. Another difference relates

to their form of administration: All statins are given orally in their active hydroxy acid forms,

except for lovastatin and simvastatin, which are applied as lactone pro-drugs which are then

hydrolysed to the active hydroxyl acid form (Corsini et al., 1995).

The different types and characteristics of statins enable huge variability in their modes of action,

which may contribute to their pleiotropic properties. These effects are discussed below, with a

special focus on the anti-cancer effects.

5.3.3 Anti-cancer effects of statins

From large clinical trials it became obvious that statins exert actions beyond the LDL-cholesterol

lowering effects, which are called pleiotropic effects (Pasterkamp and Van Lammeren, 2010).

This includes in particular endothelial (angiogenic and thrombotic), immune suppressive or

modulatory and anti-inflammatory actions, which have been issues of extensive clinical studies

and whose underlying mechanisms have been largely uncovered (exemplified in Figure 10)

(Gazzerro et al., 2012). In this regard, statins exhibit enormous therapeutic potentials from which

treatments for various non-cardiovascular-related diseases – including osteoporosis, dementia,

multiple sclerosis and autoimmune diseases – can benefit.

Another interesting aspect of statin is its controversial cancer promoting and inhibiting effect. On

the one hand, statins may cause tumour progression or metastasis formation due to their pro-

angiogenic effect. However, on the other hand, numerous studies in the last years have shown

promising therapeutic possibilities of statins, as they were shown to exert anti-proliferative

actions in various cancer types, including breast cancer (Campbell et al., 2006; Denoyelle et al.,

2003; Koyuturk et al., 2007; Rao et al., 1998), colon carcinoma (Agarwal et al., 1999),

glioblastoma (Jiang et al., 2004), leukaemia (Cafforio et al., 2005; Dimitroulakos et al., 2000;

Sassano et al., 2007), melanoma (Cafforio et al., 2005; Collisson et al., 2003), pancreatic cancer

(Kusama et al., 2001), prostate cancer (Marcelli et al., 1998), thyroid cancer (Bifulco, 2008;

Zhong et al., 2005).

31

Introduction

Figure 10. Examples of pleiotropic effects of statins and their mode of actions (Mason, 2003) Statins are known to promote various endothelial, immune responsive and anti-inflammatory modulations.

The anti-cancer effects of statins are strongly associated with their ability to regulate small

GTPases and hence GTPase-mediated signalling pathways: Mevalonate pathway inhibition by

statins reduces the production of geranylgeranylgeranyl- and farnesylpyrophosphate which are

essential for activation for small GTPases of the Rho and Ras family, respectively (Brandes,

2005; Visvikis et al., 2010). Geranylgeranylation and farnesylation are post-translational

isoprenylation steps for GTPases, allowing their translocation from cytoplasm to the cell

membrane for anchorage and subsequent activation to its GTP bound form by release of guanine

nucleotide dissociation inhibitors (GDIs) and GDP-GTP exchange by guanine nucleotide

exchange factors (GEFs) (Liao and Laufs, 2005; Visvikis et al., 2010). This process is

exemplified for Rho GTPases activation in Figure 11.

32

Introduction

Figure 11. Regulation of GTPase by isoprenylation (Liao and Laufs, 2005) Geranylgeranylation of Rho GTPases is required for their translocation to the membrane and subsequent GEF

mediated activation. Statins reduce the synthesis of geranylgeranyl-pyrophosphate and interfere with this process. Active Rho proteins are inactivated by GAP mediated GTP hydrolysis in order to return to its GDP bound state. GDI = guanine nucleotide dissociation inhibitors; GEF = guanine nucleotide exchange factors; GAP = GTPase-

activating proteins.

Thus, statins inhibit the activation of small GTPases and their signalling pathways. Rho and Ras

GTPases are key players in co-ordinating the organization of actin cytoskeleton downstream of

cell-surface signalling receptors (Nobes and Hall, 1995; Ridley and Hall, 1992; Ridley et al.,

1992). Consequently, they hold important roles in cytoskeleton dependent processes, such as

progression of cell division, cell migration, co-ordination of cell adhesion and cohesion of cells

in endothelial and epithelial tissues, phagocytosis, cellular defences, regulation of transcription,

cell differentiation, survival and cell death (Visvikis et al., 2010).

With regards to their cell proliferation promoting functions, deregulated activation of members

of the Rho GTPase family (for example: RhoA, cdc42, Rac) and/or of the Ras GTPase family

(prominent oncogenic members are N-Ras, K-Ras, H-Ras) may promote cancer development and

33

Introduction

progression (Downward, 2003; Ridley, 2001), which therefore can be counteracted by

administration of statins (Bifulco, 2008). Indeed, statins induced inhibition of

geranylgeranylation or farnesylation is shown to reduce cell proliferation (Bifulco, 2008; Wang

et al., 2003; Zhong et al., 2005) involving G1-S cell cycle arrest (Declue et al., 1991; Hirai et al.,

1997; Rao et al., 1998) and induce apoptosis by up-regulation and activation of proteins of

programmed cell death (i.e. caspases 3, 7, 8 and 9) (Cafforio et al., 2005; Marcelli et al., 1998;

Wang et al., 2000). Figure 12 summarizes these anti-tumoric mechanisms.

Figure 12. Anti-cancer mechanisms of statins (Chan et al., 2003) Statins can cause up-regulation of cell cycle inhibitors p21WAF1/CIP1 and p27KIP1 to induce G1-S cell cycle arrest.

Furthermore, down-regulation of geranylgeranyl protein level results in apoptosis induction and RhoA inactivation and subsequently in anti-invasive effects

For instance, induced apoptosis by lovastatin was shown in human anaplastic thyroid carcinoma

cells (Wang et al., 2003; Zhong et al., 2005) and in K-ras-transformed thyroid cells (Laezza et

al., 2008). Cerivastatin inhibits proliferation and invasion of breast cancer cells (Denoyelle et al.,

2003). Simvastatin is found to induce apoptosis in breast cancer cells (Koyuturk et al., 2007).

Statins are also found to inhibit ATP binding cassette (ABC)-transporter in human

rhabdomyosarcoma and neuroblastoma cells (Sieczkowski et al., 2010; Werner et al., 2013),

which would help to overcome the extrusion of co-administrated chemotherapeutics via ABC-

34

Introduction

transporters, importantly ABCB1 (P-glycoprotein, MDR1). This may have clinical relevance of

statins in adjuvant chemotherapy. For example, 1 µM simvastatin increases intracellular

doxorubicin concentration, which translates into enhanced apoptosis in human neuroblastoma

and rhabdomyosarcoma cells (Sieczkowski et al., 2010).

In 2011, Cao et al. reported atorvastatin can successfully lower the activation of MYC by

interfering with its phosphorylation. Phosphorylation stabilizes c-Myc and thereby prevents its

Ubiquitin-dependent degradation by proteasome. Importantly, in a transgenic model of MYC-

induced hepatocellular carcinoma, atorvastatin again inhibited c-Myc phosphorylation, tumour

initiation and growth (Cao et al., 2011).

Based on this publication, it was the aim of this master thesis to investigate the effects of

simvastatin on MYCN-amplified neuroblastoma cells and to uncover its potential mechanisms.

35

Aims

6 Aims

The aims of this study are …

… to confirm high N-myc level in various human neuroblastoma cells

…to clarify, whether simvastatin has an effect on N-myc expression

… to provide mechanistical insights into alterations of N-myc activity regulation upon

simvastatin treatment

… and to characterize phenotypic and morphological changes of the human

neuroblastoma cells upon treatment with simvastatin

36

Materials

7 Materials

7.1 Antibodies

Primary Antibodies

Anti-α-Tubulin Sigma Aldrich, Anti-α-Tubulin Antibody, monoclonal, T6074, 50 kDa, mouse; Missouri, USA

Anti-Aurora kinase A Cell signaling, Anti-Aurora A Antibody, monoclonal, #4718, 48 kDa, rabbit; Cambridge, UK

Anti-β-Actin Sigma Aldrich, Anti-Actin Antibody, monoclonal, #A4700, 40 kDa, mouse; Missouri, USA

Anti-β-Actin Sigma Aldrich, Anti-Actin Antibody, polyclonal, #A5060, 40 kDa, rabbit; Missouri, USA

Anti-cdc42 Cell Signaling, Anti-cdc42 (11A11) Antibody, monoclonal, #2466, 21 kDa, rabbit; Cambridge, UK

Anti-Lap2β Kindly provided by Prof. Dr. Roland Foisner, 51 kDa, mouse; Max Perutz Laboratories; Vienna, Austria

Anti-Max Abcam, Anti-Max Antibody, monoclonal, 73C5a, 18/34 kDa, mouse; Cambridge, UK

Anti-N-myc Cell Signaling, Anti-N-Myc Antibody, polyclonal, #9405, 62 kDa, rabbit; Cambridge, UK

Anti-N-myc Abcam, Anti-N-myc Antibody [NCM II 100] - CHIP Grade, monoclonal, ab16898, mouse; Cambridge, UK

Anti-PARP Cell Signaling, Anti-PARP Antibody, polyclonal, #9542, 116/89/24 kDa, rabbit; Cambridge, UK

Anti-Rac1/2/3 Cell Signaling, Anti-Rac1/2/3 Antibody, polyclonal, #2465, 21 kDa, rabbit; Cambridge, UK

Anti-RhoA Cell Signaling, Anti-RhoA Antibody, monoclonal, #67B9, 21 kDa, 21 kDa, rabbit; Cambridge, UK

Anti-Mouse IgG Fluorescence Controls BD Bioscience, Mouse IgG2a Pure, monoclonal, # 349050, mouse; New Jersey; USA

37

Materials

Secondary Antibodies

Anti-Rabbit Cell Signaling, Anti-rabbit IgG HRP-linked Antibody, #7074, goat; Cambridge, UK

Anti-Mouse Cell Signaling, Anti-mouse IgG HRP-linked Antibody, #7076, goat; Cambridge, UK

Anti-Mouse Alexa Fluor 488 nm Invitrogen, Alexa Fluor® 488 Anti-mouse IgG (H+L) Antibody, #A11001, goat; Carlsbad, USA

7.2 Cell lines

Cell lines

Kelly Human neuroblastoma cell line; kindly provided by Children Cancer Research Institute; Vienna, Austria

IMR-32 Human neuroblastoma cell line; kindly provided by Children Cancer Research Institute; Vienna, Austria

SH-SY5Y Human neuroblastoma cell line; kindly provided by Children Cancer Research Institute; Vienna, Austria

7.3 Drugs

Drugs

Simvastatin Merck Millipore : Calbiochem, Simvastatin (MK-733), CAS 79902-63-9; Massachusetts, USA

Mevalonic Acid Sigma-Aldrich, (±)-Mevalonolactone, ≥ 96.0 %, #69761 Fluka; Missouri, USA

Vincristine Kindly provided by Dr. Christoph Minichsdorfer, AKH; Vienna, Austria

DMSO Roth GmbH; Rotipuran® ≥ 99.8 %, p.a., #4720.4; Karlsruhe, Germany

38

Materials

7.4 Equipment

Centrifuges

BR4i Centrifuge Multi-Function Bench Top Centrifuge BR4i, # 11175603; Jouan, rotor: S40, Swing-out rotor, Radius: 161mm

Eppendorf Centrifuge Eppendorf Centrifuge 5415 R, standard rotor F45-24-11, diameter: 11 mm

Sorvall Ultracentrifuge Sorvall ® RC-5B-Refrigerated Superspeed Centrifuge, DuPont Instruments, Rotor SM-24, radius: 4.34; Delaware, USA

Refrigerators

Automatic Flake Ice-Machine Scotsman ® AF 100; Scotsman Industries, Illinois, USA

Liebherr Premium Refrigerators; Bulle Switzerland

Elektra Bregenz Refrigerators; Vienna, Austria

Forma Freezer 700 Series, Thermo Scientific; Massachusetts, USA

Incubators

Thermomixer comfort, Eppendorf ®, #5355 000.011; Hamburg, Germany

Cell Culture Incubator, Thermo Scientific, Model 381; Massachusetts, USA

37°C Incubator; Binder, KB720; Tuttlingen, Germany

Sonificator

Sonificator, Branson, Bransonic B-220 Ultrasonic Cleaner; Hessen, Germany

Microscope and camera

Light microscope; Nikon Eclipse TS 100; Tokyo, Japan

Nikon Coolpix P5000, Nikon Adapter Ring, UR-E20 and microscope lens tube adaptor; Tokyo Japan

Analyzing equipments and softwares

FACS Canto II V96100032; New Jersey, USA

BD FACS DIVA Software, Version 6.1.2 (build 2008 09 23 06 01), Firmware Version 1.47 (BD FACS Canto

39

Materials

II) CST Version 1.1.2, PLA Version 1.1.2; New Jersey, USA

Flowing Software 2.5.0, by Perttu Terho, Turku Centre for Biotechnology, University of Turku, Finland

Neubauer Chamber Cell Counting, Celeromics, Grenoble, France)

ImageJ 1.43u, Wayne Rasband, National Institutes of Health, USA, Java 1.6.0_01 (32-bit); Maryland USA

Spectrophotometer, Hitachi, UV-VIS spectrophotometer U-2001 ultraviolet visible photometer, Tokyo, Japan

SigmaPlot for Windows, version 10.0, build 10.0.0.54; Systat Software ®, Inc., 2006; California, USA

Victor 3 V Multilabel Readers, PerkinElmer, Inc.; Massachusetts, USA

SDS PAGE and Western Blot equipments

SDS PAGE Bio-Rad Mini-PROTEAN® Tetra Cell, catalogue number 165-8000 and 165-8001; California, USA

Western Blot Bio-Rad Mini Trans-Blot® Electrophoretic Transfer Cell; California, USA

Semi-dry Western Blot apperture Bio-Rad Trans-Blot® SD Semi-Dry Electrophoretic Tranfer Cell, 170-3940; California, USA

Computer controlled Power Supply Bio-Rad Model 3000xi computer controlled Power Supply, 165-0554; California, USA

Power Supply Bio-Rad PAC 3000 Power Supply, 165-5056; California, USA

7.5 Growth Media

Growth Media

RPMI 1640 Sigma-Aldrich, RPMI-1640 Medium, with L-

glutamine and sodium bicarbonate, liquid, sterile-filtered, R8785; Missouri, USA

DMEM/Ham’s F12

Sigma-Aldrich, Dulbecco’s Modified Eagle’s Medium - high glucose, with 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, without sodium pyruvate, liquid, sterile-filtered, D5796; Missouri, USA

40

Materials

7.6 Protease-Inhibitors

Protease inhibitors

Aprotinin Bayer, Trasylol®; Leverkusen, Germany

Leupeptin Roth, Leupeptin Hemisulfat, CN33.1; Karlsruhe, Germany

Pefablock Roth, Pefabloc®, A154.1; Karlsruhe, Germany

7.7 Tools

Pipetting and measurement tools

96 well microtiter plate Sigma-Aldrich, Nunc-ImmunoTM MicroWellTM 96 well solid plates; Missouri, USA

GELoader tips PEQLAB, GelLoader; Bayern, Germany

Micro pipettes Gilson, Pipetman® (2 µL, 20 µL, 200 µL, 100 µL and 5000 µL); Wisconsin, USA

Plastic pipettes Sarstedt, Serological pipettes (2 mL, 5 mL and 10 mL); Nordrhein-Westfalen, Germany

Pipette tips Greiner bio-one, Micro-Pipette Tips (20 µL, 200 µL, 1000 µL); Kremsmuenster, Austria

Cell culture containers and tools

T75 cell culture flask Greiner Bio One, CELLSTAR®, T-75 Flask, Tissue

Culture Treated, 250 mL, w/ Filter Cap, # 658175; Kremsmuenster, Austria

Micro tubes Sarstedt, Micro tubes (1.5 mL and 2 mL); Nordrhein-Westfalen, Germany

10 cm dish SPL Life Science, Cell culture Dish 20100; Gyeonggi-do, Korea

Falcon tubes Sarstedt, tubes with conical base (50 mL and 15 mL); Nordrhein-Westfalen, Germany

41

Materials

7.8 Other substances

Other substances

Acrylamide : N,N-methylenebisacrylamide Fisher Scientific, Acrylamide: Bis-Acrylamide 37.5:1 (40% Solution/Electrophoresis); Massachusetts, USA

Ammoniumpersulfate Roth, 9592.3; Karlsruhe, Germany

Bicinchoninic Acid Protein Assay Kit Sigma-Aldrich, B9643 (Solution A) and C2284 (Solution B); Missouri, USA

Bio-Rad Protein dye Bio-Rad, Protein Assay Dye Reagent Concentrate, #500-0006; California, USA

Bovine Serum Albumin Roth, 8076.4; Karslruhe, Germany

Citric Acid H2O Sigma-Aldrich, 5949-29-1, C-1907; Missouri, USA

EDTA Roth, 8043.2; Karlsruhe, Germany

FBS Sigma-Aldrich, F7524; Missouri, USA

Glycerolphosphate Sigma-Aldrich, G6251; Missouri, USA

Glycin Roth, 3908.2; Karlsruhe, Germany

KCl Sigma-Aldrich:Fluka, 60132; Missouri, USA

K3[Fe(CN)6] Merck, 0119893; Darmstadt, Germany

K4[Fe(CN)6] Merck, 4984; Darmstadt, Germany

K2HPO4 Merck, 5099; Darmstadt, Germany

KH2PO4 Merck, 4873; Darmstadt, Germany

MeOH Roth, 8388.5; Karlsruhe, Germany

MgCl2 Sigma-Aldrich:Fluka, 63064; Missouri, USA

NaCl Roth, 3957.2; Karlsruhe, Germany

NaH2PO4*H2O Merck, 6346; Darmstadt Germany

Na2HPO4*2H2O Sigma-Aldrich:Fluka, 71643; Missouri, USA

NaN3 Sigma-Aldrich, 438456; Missouri, USA

NP-40 Sigma-Aldrich:Fluka, 74385; Missouri, USA

Paraformaldehyd Sigma-Aldrich, P6148; Missouri, USA

Penicillin/Streptomycin GE-Healthcare:PAA, Penicillin/Streptomycin (100

42

Materials

x), P11-010; Buckinghamshire, UK

Ponceau S Roth, C.I.27195; Karlsruhe, Germany

rpm Rounds per minute

SDS Roth, 2326.2; Karlsruhe, Germany

Temed Sigma-Aldrich, T9281; Missouri, USA

Tris Roth, 0188.1; Karlsruhe, Germany

Triton X-100 Sigma-Aldrich:Fluka, 93420; Missouri, USA

Trypan Blue Sigma-Aldrich, T8154; Missouri, USA

Trypsin Sigma-Aldrich, T3924; Missouri, USA

Tween-20 Roth, Tween® 20; Karlsruhe, Germany

X-Gal Sigma-Aldrich, 5-bromo-4-chloro-3-indolyl-β-D-galactropyranosid; B9146; Missouri, USA

43

Methods

8 Methods

8.1 Cell culture techniques

8.1.1 Cell culture treatments

Cell culture treatments were carried out either by pipetting drugs directly to the cells covered in

media or by preparing a dilution row of media supplemented with the required drug

concentration and pipetting the media to the cells.

8.1.2 Cell culture maintenance and conditions

Cells were kept in T75 flasks or plates of different sizes dependent on the experimental

procedure. Kelly and IMR-32 cell lines were grown in RPMI 1640, while SH-SY5Y cells were

grown in DMEM/Ham’s F12.Both media were supplemented with 10 % Fetal Bovine Serum and

1 % Penicillin/Streptomycin. Of note, RPMI 1640 should contain L-glutamine as the MYCN-

overexpressing lines (Kelly and IMR-32) are glutamine dependent. This is due to the fact that

cells with oncogenic amplification of MYC or MYCN are associated with increased

glutaminolysis and thus are dependent on glutamine supplementation (Gao et al., 2009b; Qing et

al., 2012b; Wise et al., 2008).

The cells were kept in the cell culture incubator at 5 % CO2 and adjusted to 37°C. These settings

will be referred to as “cell culture condition” in all subsequent chapters.

8.1.3 Splitting, cell counting and cell seeding

Cells were washed in Potassium Buffered Saline (PBS, Table 1) solution and collected by

trypsinisation and centrifugation in the cell culture centrifuge (220 x g, 5 minutes at room

temperature). Trypsin and PBS were removed and the cell pellet was resuspended thoroughly in

the appropriate growth media.

44

Methods

PBS 137 mM NaCl (80 g)

10.14 mM Na2HPO4 (14.4 g)

2.7 mM KCl (2 g) 1.8 mM KH2PO4 (2.4 g)

∑ 10 L MQ pH= 7.4

Table 1. PBS recipe

An aliquot of the resuspended cells was stained with trypan blue and vital cells (not stained)

were counted using the Neubauer Chamber Cell Counting method (more technical information is

provided in an online technical note “Neubauer Chamber Cell Counting” from Celeromics,

Grenoble, France. The counting result gives the absolute number of cells per mL. Dependent on

the experiment and the amount of cells required, an appropriate number (see Figure 13) of cells

is seeded in tissue flasks or plates and grown under cell culture conditions.

Figure 13. Useful numbers for cell culture (provided by Invitrogen)

8.1.4 Microscopy and picture acquisition

Cell cultures were examined by light microscopy on a daily basis. Images were taken by

attaching the Nikon P5000 camera to the ocular via the adapter ring and the microscope lens tube

adaptor.

45

Methods

8.2 Protein methods

8.2.1 Cell lysis in IP-Buffer

Cells grown on 10 cm dishes were washed twice with ice-cold PBS and then shock-frozen by

liquid nitrogen. Thawed cell were lysed in approximately 500 µL IP-Buffer (Table 2) already

containing 100 µM of each of the protease inhibitors leupeptin, aprotinin and pefabloc – and

incubated for 10 minutes on ice. The cells were then scratched off the plate, pipetted into a pre-

cooled 1.5 mL tube and again shock-frozen in liquid nitrogen. Rapidly thawed cells were

incubated for 30 minutes on ice and shortly vortexed in ten-minute-intervals. After incubation,

the lysates were sonificated for one minute and centrifuged at 28.000 x g for 30 minutes at 4°C

by the Sorvall RC-3 centrifuge. The supernatant was transferred into a new 1.5 mL tube and

stored at -80°C.

IP-Buffer 25 mM Tris-HCL (pH = 7.5)

150 mM NaCl 10 mM EDTA

0.1% Triton X-100 0,5 % NP-40

10 mM Glycerolphosphate Stored at room temperature

Table 2. IP-Buffer recipe for IP-Buffer protein extraction

8.2.2 Nuclear extraction method

For nuclear extraction four replicates of 10 cm dishes were used, in order to retrieve sufficient

amount of nuclear proteins.

Cells on the dish were washed twice with PBS solution at room temperature and trypsinized at

37°C. The cells were washed off from the plate with ice-cold PBS, transferred into a falcon tube

and centrifuged at 220 x g for 5 minutes at room temperature by the BR4i centrifuge. The

supernatant was removed and the cell pellet was resuspended in PBS and centrifuged at 220 g

46

Methods

with the BR4i Centrifuge for 5 minutes. PBS was then removed and the cell pellet was

resuspended gently in 500 µL buffer A (Table 3) containing 100 µM of each protease inhibitors

– aprotinin, leupeptin and pefabloc. Gentle resuspension of the cell pellet is crucial to avoid

premature breakdown of the nucleus and leakage of nuclear protein into the cytosolic fraction.

The resuspended cell pellet in Buffer A was transferred to a new 1.5 mL tube and allowed to

stand on ice for 10 minutes for extraction of the cytosolic fraction. After incubation the lysate

was centrifuged at 950 x g at 4°C for 10 minutes by the Eppendorf centrifuge. The supernatant

(represents the cytosolic protein fraction) was transferred to a new 1.5 mL tube. Noteworthy,

after centrifugation, the pellet contains large membrane pieces and intact nuclei. Proper removal

of residual cytosolic liquids is crucial to avoid contamination of the nuclear protein fraction with

the cytosolic one. Therefore, transfer of the cytosolic fraction requires GELoader tips that allow

precise removal of remaining cytosolic fraction trapped in-between the pellet by stabbing into

the pellet and removing residual fluids.

The cytosolic fraction was frozen in liquid nitrogen and kept at -80°C in the Forma freezer. The

remaining pellet in the 1.5 mL tube was resuspended in 400 µL of buffer B (Table 3). The pellet

was homogenized thoroughly by repeated uptake in a syringe and injection back to the tube for

30 times. The homogenized lysate was allowed to stand on ice for 30 minutes. The shearing

force of this homogenizing step results in thorough breakdown of the nuclei.

After incubation the lysates were centrifuged at 27.713 x g for 20 minutes at 4°C in the Sorvall

RC-3 centrifuge. The supernatant containing the nuclear extract was transferred into a new 1,5

mL tube and stored at -80°C.

Buffer A Buffer B 10 mM HEPES

1.5 mM MgCl2 10 mM KCl

0.5 mM DTT 0.05 % NP-40

pH = 7.9 Stored at 4°C

5 mM HEPES 1.5 mM MgCl2

0.2 mM EDTA 0.5 mM DTT

26 % glycerol pH = 7.9

Stored at 4°C

Table 3. Buffer A and buffer B recipe for nuclear extraction (provided by Abcam)

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Methods

8.2.3 Protein concentration determination

8.2.3.1 Bio-Rad protein concentration determination

Bio-Rad protein concentration determination was performed by using the Bio-Rad Protein Assay

Dye Reagent Concentrate according to the manufacturer’s recommendation. This method

involves the binding of Coomassie Brillant Blue G-250 dye to proteins. Upon binding, the

compound is converted from a protonated red cationic form (absorption maximum at 470 nm) to

a stable unprotonated blue form (absorption maximum at 595 nm).

Aliquots of the protein suspension (5 µL) were mixed with 200 µL Bio-Rad protein assay dye

reagent in a 1.5 mL tube and brought up to an end volume of 1 mL with MQ water and

thoroughly mixed by vortexing. The optical density of the colorimetric reaction was

photometrically measured at 595 nm with a photometer. The protein concentration is

proportional to the absorption at 595 nm.

8.2.3.2 BCA protein concentration determination

The Bicinchoninic acid (BCA) protein concentration determination was performed according to

the recommendations and technical bulletin of Sigma Aldrich for its Bicinchoninic Acid Protein

Assay Kit. The working solution A and B were also purchased from Sigma Aldrich.

This method is based on the formation of Cu2+-protein complex, followed by the reduction of

Cu2+ to Cu1+ by the amino acids cysteine, cysteine, tryptophan and tyrosine and by the peptide

bond. The level of reduction is proportional to the protein in the sample. BCA then forms a

purple-blue complex with Cu1+ under alkaline conditions, which can be measured at 562 nm or

at a range of 540 nm to 590 nm.

A dilution row of increasing concentrations of Bovine Serum Albumine (BSA) was required as a

protein standard. For this purpose, 3 mg BSA was dissolved in 3 mL MQ and diluted in a range

from 0 to 1 mg/mL. The extraction solution in the absence of proteins was used as blank.

For the measurement, 10 µL of the protein suspension, blank solution or protein standard

dilutions were pipetted in doublets into a 96 well microtiter plate. A 50:1 mixture (200 µL) of

working solution A (containing BCA, sodium carbonate, sodium tartrate and sodium bicarbonate

in 0.1 N NaOH) and solution B (containing 4 % w/v copper II sulphate pentahydrate) was

pipetted to each sample, followed by short mixing. Bubbles should be avoided as they would

disturb the photometric measurement.

48

Methods

The microtiter plate was enclosed with parafilm and incubated at 37°C for 30 minutes. The

measurement was carried out with the plate reader Victor 3 V, using an appropriate filter to

capture the wavelength at 562 nm (544 ± 15 nm). The values generated were exported as Excel

file and analysed off-line with the Sigma Plot software. The protein concentrations were

calculated relative to the BSA protein standard calibrated to linear regression, according to the

formula: y = y0 + ax.

8.2.4 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE)

SDS Gels were cast by using the Mini-PROTEAN® Tetra Cell equipment provided by Bio-Rad.

For the exact assembly of the gel casting cassette see the instruction manual for the catalogue

numbers 165-8000 and 165-8001 provided by Bio-Rad online. Dependent on the desired

volume/µg of proteins used for electrophoresis, a 0.75 mm or 1.00 mm spacer plate can be

chosen to produce a gel of the appropriate thickness.

The prescription for the separating and stacking gels is given in Table 5 and Table 6,

respectively. The components (Table 4) were mixed at room temperature and polymerisation

was initiated by the addition of tetramethylethylendiamin.

After polymerisation of the separating gel 100 % isopropanol was pipetted onto the gel to

provide an even surface. After 30 minutes isopropanol was removed and the stacking gel

solution containing tetramethylethylendiamin was layered onto the separating gel. After casting,

combs were immediately inserted into the still fluid stacking gel mixture. The stacking gel was

then allowed to polymerize at room temperature for 45-60 minutes.

49

Methods

1.5 M Tris-HCl Buffer 10 % SDS 30%-0,8% Acryl Bisacryl

45,43 g Tris

∑250 ml MQ, pH=8.8

10 g SDS

∑ 100 ml MQ

29,211 g acrylamid 0,779 g methylenebisacryl

∑ 100 ml MQ Stored at 4°C Stored at RT Stored at 4°C

0.5 M Tris-HCl Puffer 10 % Ammoniumpersulfat

6,06 g Tris ∑100 ml MQ, pH=6.8

5 g Ammoniumpersulfat ∑ 50 ml MQ

Stored at 4°C Stored at -20°C

Table 4. Compounds for SDS gels

Separating Gel

Compounds 10 % 15 %

Distilled water 0.396 0.229

1.5 M Tris-HCl, pH = 8.8 0.25 0.25

30 % acrylamide/0.8 % N,N-methylenebisacrylamide

0.333 0.5

10 % sodium dodecylsulfate 0.01 0.01

10 % ammonium persulfate 0.01 0.01

Tetramethylethylendiamin 0.001 0.001

Table 5. Preparation of 10 % and 15 % Separating Gel Required amounts are given in % v/v of the final volume.

50

Methods

Stacking Gel

Compounds 5 %

Distilled water 0.562

0.5 M Tris-HCl, pH = 6.8 0.25

30 % acrylamide/0.8 % N,N-methylenebisacrylamide

0.167

10 % sodium dodecylsulfate 0.01

10 % ammonium persulfate 0.01

Tetramethylethylendiamin 0.001

Table 6. Preparation of 5 % Stacking Gel Applied amounts are given in % v/v of the final volume.

Gels are assembled and placed into the Mini-PROTEAN Tetra Cell Electrophoresis Module from

Bio-Rad (for details on assembling see instruction manual). The tank is filled with cold 1 x

Running Buffer (Table 7) according to the manufacturer’s instructions.

Before loading, protein lysates in 1 x SDS Sample buffer were heated to 95°C in Eppendorf

Thermomixer for 3 minutes under continuous shaking. The protein samples were allowed to cool

down at room temperature and then kept on ice until loading procedure. Samples including a

prestained molecular mass standard were loaded with GELoader tips

After loading the lid was placed correctly following the colour coded electrode orientation. The

gels were run by constant voltage of 150 V. SDS PAGE was finished when the migration front

of the bromphenol-dye left the gel. The gels were immediately recovered from the aperture and

prepared for Western Blot transfer.

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Methods

10 x Running Buffer 1 x Running Buffer 250 mM Tris (60.58 g)

1.92 M Glycin (288.26 g)

10 mM EDTA.Na2 (7.44 g) 1% SDS (10 g)

∑ 2 l MQ, pH= 8.3 Stored at room temperature

0.1 % v/v of 10 x Running Puffer

Diluted in cold distilled water Prepared before use, stored at 4°C until usage

Table 7. Running Buffer recipe for SDS PAGE

8.2.5 Western Blot

8.2.5.1 Wet Blotting

The blotting procedure was performed in Mini Trans-Blot® Electrophoretic Transfer Cell

purchased from Bio-Rad. For details on assembling see the Instruction Manual for catalogue

numbers 170-3930, -3935, -3989 and -3836 provided by Bio-Rad online.

After electrophoresis ended, a “sandwich” was formed, consisting of sponge – Whatman paper –

SDS-polyacrylamide gel – nitrocellulose membrane – Whatman paper – sponge, orientated in the

direction from the cathode to the anode, respectively. The sandwich was placed vertically in the

buffer tank, covered in ice cold Transfer Buffer (Table 8) and cooled by an ice block. Blotting

was set to constant 150 volts for 30 minutes.

10 x Transfer Buffer 1 x Transfer Buffer 250 mM Tris (60.58 g)

1.92 M Glycin (288.26 g)

1% SDS (20 g) ∑ 2 L MQ

pH= 8.3

Stored at room temperature

10 x Transfer Buffer (100 ml) MeOH (200 ml)

∑ 1 L MQ Stored at 4°C

Table 8. Transfer Buffer recipe for Wet Blotting

52

Methods

8.2.5.2 Semi-Dry Blotting

Semi-Dry Blotting was carried out with the Trans-Blot® SD Semi-Dry Electrophoretic Transfer

Cell purchased from Bio-Rad. For details on assembling see the Instruction Manual for catalogue

number 170-3940.

After electrophoresis gels were retrieved from between the glass plates and placed inside a gel-

sandwich and incubated in Bjerrum buffer (Table 9) with SDS for approximately 5 minutes.

Nitrocellulose membrane was incubated in Bjerrum buffer with methanol for approximately 5

minutes. A sandwich was assembled, consisting of 7 Whatman papers drenched in methanol –

nitrocellulose membrane – gel – 7 Whatman papers drenched in SDS. The sandwich is placed

horizontally in the Semi-Dry Electrophoretic Transfer Cell, with the Whatman papers drenched

in Bjerrum buffer with methanol. The nitrocellulose membrane should be close to the platinum

anode. The cathode lid was placed carefully onto the Whatman papers drenched in Bjerrum

buffer with SDS. The settings were as follows: limited voltage at 100 V, limited current at 300

mA, limited power at 400 W and constant current at 80 mA per gel. Blotting was carried out for

45 minutes mostly.

Bjerrum Buffer with SDS Bjerrum Buffer with SDS 48 mM Tris (5,815 g)

39 mM Glycin (2,928 g)

1.3 mM SDS (0,374 g)

∑ 1 L MQ

Stored at 4°C

48 mM Tris (5,815 g)

39 mM Glycin (2,928 g)

20 % v/v MeOH (200 mL)

∑ 1 L MQ

Stored at 4°C

Table 9. Bjerrum Buffer recipes for Wet Blotting

8.2.5.3 Ponceau staining and blocking

After blotting the nitrocellulose membrane is retrieved from the sandwich, washed twice with

distilled water in a small box. The membrane was then stained with Ponceau S solution for

approximately 10 minutes. After scanning of the staining, the membrane was washed with TBS-

53

Methods

T until the dye was removed entirely. The membrane was then blocked with 5 % BSA solution

for at least 2 hours at room temperature to reduce unspecific binding sites.

8.2.5.4 Incubation with primary/secondary antibody

After blocking the membrane was washed once with TBS-T and then incubated with specific

primary antibody (1:1.000 in 2 % BSA solution with 0.1 % NaN3) overnight at 4°C shaking, to

bind to the protein of interest on the membrane. The next day, the membrane was washed 3 times

5 minutes with TBS-T and then incubated with secondary antibody coupled to the horseradish

peroxidase (HRP) (1:10.000 in 2 % BSA solution) for at least 1 hour at room temperature

shaking. The secondary antibody binds to the primary antibody on the membrane by targeting its

species-specific site. The membrane was then washed 3 times 15 minutes with TBS-T and 1 time

15 minutes with 1 x TBS.

10 x TBS 1 x TBS TBS-T

0.2 M Tris (24.22 g) 1.5 M NaCl (87.66 g)

∑ 1 L MQ, pH= 7.6 Stored at room temperature

10 x TBS (100 mL)

∑ 1 L MQ Stored at room temperature

10 x TBS (100 mL)

∑ 1 L MQ 1 mL Tween-20

Stored at room temperature

Table 10. TBS and TBS-T recipes

8.2.5.5 Detection by enhanced chemiluminescence

The detection was performed with ECL Prime Detection Reagent from GE Healthcare Life

Sciences according to manufacturer’s recommendations. The membrane was incubated with a

1:1 mixture of solution A (luminol solution) and B (peroxide solution). The HRP of the

secondary antibody will oxidise its substrate luminol in the presence of peroxide or other

enhancers in a reaction called enhanced chemiluminescence (ECL) which provides increased and

stable light emission (Figure 14). This signal was detected by placing a photographic film on the

membrane in the darkroom. The luminescence will darken the film at the sites of the protein of

interest.

54

Methods

Figure 14. Enhanced chemiluminescence (ECL) reaction (GE Healthcare) In the presence of HRP and peroxide, luminol is oxidized to give a dianion (luminol radical anion). The oxygen produced from the hydrogen peroxide (not shown) reacts with the dianion which results in an organic peroxide

(luminol endoperoxide). This product is very unstable and disintegrates to aminophtalate and N2, while electrons go from excited state to the ground state to emit energy as photon.

8.2.5.6 Stripping of nitrocellulose membrane

Mostly, membranes were reused for detection of the loading control protein (β-Actin or α-

Tubulin). In case that the primary antibody of the loading control protein is from the same

species as the primary antibody of the protein of interest, nitrocellulose membrane has to be

stripped to remove the former antibodies. Of note, an undeterminable portion of proteins will be

washed away.

Stripping was performed by washing the membrane 3 times 10 minutes with TBS-T at room

temperature shaking. Then the membranes were incubated in mild stripping buffer (Table 11) 2

times 10 minutes at room temperature without shaking. After further washing steps with PBS for

2 times 10 minutes with and with TBS-T for 2 times 10 minutes at room temperature shaking,

the membrane can again be blocked by blocking solution and processed as described in chapter

8.2.5.4.

55

Methods

Mild stripping buffer

Glycin (15 g) SDS (1 g)

Tween-20 (10 mL) ∑ 1 L MQ, pH= 2.2

Stored at 4°C

Table 11. Mild stripping buffer recipe

8.2.5.7 Quantification of Western Blot

The films were scanned with a transmission light scanner and saved as JPEG files. At least two

exposures of the same blot were used for quantification. The quantification of the signals in

JPEG file was performed with ImageJ software. If necessary, the files were adjusted by the

“Invert” tool. The intensity of bands within each sample set was determined by the “Rectangular

Selection” tool of equal area size, set to closely surround the edge of each band and measured in

triplicates. The intensity within this very same area was measured by the “Measure” tool, which

reports the intensity in arbitrary units.

The background substraction, the normalization of all measurements to the corresponding

loading control and the normalization of the treated samples to the control were performed in

Microsoft Excel. The results were depicted as bar diagrams in percentage to the control.

8.3 Flow cytometry

8.3.1 Fixation of cells

Cells were washed with PBS, trypsinized and transferred into a Falcon Tube. The culture was

pelleted by centrifugation at 1.100 rpm for five minutes. The supernatant was aspired and the cell

pellet was resuspended and washed in PBS, transferred into a 1.5 mL tube. The cells were

pelleted by centrifugation in the Eppendorf centrifuge at 3.000 rpm for 5 minutes at 4°C. The

supernatant was aspired and the pellet was resuspended in 80 % methanol and incubated for 5

minutes at room temperature for fixation. The pellet was then washed with PBS by mixing and

56

Methods

centrifugation at 3.000 rpm for 5 minutes at 4°C. The supernatant was aspired, the pellet was

resuspended in 0,1 % Tween/ 1 % NaN3 and incubated for 20 minutes at room temperature for

permeabilization of the cells. The cells were washed with PBS and pelleted by centrifugation as

described and the supernatant was removed. The pellet was incubated the primary antibody in a

final reaction volume of 50 µL of 10 % FBS/1 % NaN3 in PBS and incubated at room

temperature for thirty minutes. The cells were again washed in PBS and pelleted by

centrifugation as in previous steps, the supernatant was removed and the cells were incubated

with the fluorescent secondary antibody (Alexa 488) in a final reaction volume of 50 µL of 10 %

FBS/1 % NaN3 in PBS. After 30 minutes incubation at room temperature the cells were washed

with PBS and pelleted by centrifugation and taken up in an appropriate volume of PBS for flow

cytometry.

8.3.2 Flow cytometric analysis

Flow cytometry is a method to analyse the properties of cells or cell populations. A flow

cytometer consists of a hydrodynamic fluidics system, a laser system, an optical system,

detection system and computing system (Figure 15 A). A sample stream of living or fixated

cells is focused by the hydrodynamic fluidics system in such way, that cells pass the laser beam

one at a time. When a cell passes through the laser, it will refract or scatter light. There are three

types of light signals (Figure 15 B):

Forward Scatter corresponds to the cell size;

Sideward Scatter corresponds to the granularity of the cell; and

Fluorescence signal may be generated by introducing reporter system into the cell;

These signals pass through different angles and filters and are finally recorded by the detection

system, which converts signal intensity into voltage. The level of voltage changes is proportional

to the corresponding property of the cell; for example, large cells produce large amount of

forward scatter, which results in a larger voltage pulse (Figure 15 C). These data can be plotted

on a histogram as a graphical distribution of the corresponding properties of the cell within a

population.

57

Methods

A

B

C

Figure 15. Schematic view of a flow cytometer and its principles of detection (pictures are taken from Invitrogen Homepage “Introduction to Flow Cytometry”)

Description is provided in the text.

In this study, the fluorescence signal of Alexa 488 antibody bound to N-myc antibody was

relevant. Analysis was performed with FACS Canto II and FACS DIVA software from BD

58

Methods

biosciences. The intensity of the Alexa 488 fluorescence was expressed as geometric mean of

each measurement and analysed by Flowing Software.

8.4 Senescence-associated β-Galactosidase Assay

The method is based on a cytochemical assay by using the chromogenic substrate X-Gal (5-

bromo-4-chloro-3-indolyl-β-D-galactropyranosid), which is cleaved by the cellular, lysosomal β-

Galactosidase to yield an insoluble blue compound. Senescent cells express more of β-

Galactosidase, hence its activity can already be detected at a suboptimal pH of 6 (Debacq-

Chainiaux et al., 2009).

Cells grown on 10 cm dishes were washed twice with PBS and fixed with 4 % para-

formaldehyde in 0.9 % NaCl for 15 minutes at room temperature. After removal of the fixation

solution, cells were washed once with PBS and permeabilized with 0.1 % Triton-X for 5 minutes

at room temperature. Cells were then washed with PBS and incubated with freshly prepared

staining solution 40 mM citric acid in sodium phosphate buffer, 150 nM NaCl, 2 mM MgCl2, 5

mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6] and 1 mg/mL X-Gal overnight at 37°C. After removal of

the staining solution, cells were washed once with PBS and observed under the light microscope.

Pictures were taken with a Nikon P5000 camera.

Sodium phosphate buffer Citric acid in sodium phosphate buffer (100 mL) Fixation solution

200 mM NaH2PO4 (in MQ)

200 mM Na2HPO4 (in MQ) pH = 6

Stored at room temperature

100 mM Citric acid (36.85 mL)

Titrating with alcalic Na2HPO4 to pH = 6

∑ 100 mL with sodium phosphate buffer (pH = 6)

Stored at room temperature

0.9 % NaCl2 in MQ

4 % Formaldehyde Frozen at -20°C

Table 12. Compounds for SA-β-Galactosidase assay

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Methods

8.5 Statistical analysis

Statistical analysis of flow cytometry or Western Blot data was performed as One Way analysis

of variance after Kruskal-Willis (ANOVA) with Sigma Plot by and post-hoc analysis with

Dunnett’s test (Multiple Comparisons versus Control group). Statistical significances are

indicated as * = p < 0.05, ** = p < 0.01 and *** = p < 0.001. No indications mean non-

significant value (p > 0.05).

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Results

9 Results

9.1 N-myc protein expression in SH-SY5Y, Kelly and IMR-32 cells treated with simvastatin and mevalonate

N-myc appears as triple band (Figure 16). These bands may correspond to the polypeptides

ranging from 58 kDa to 64 kDa as reported in Mäkela et al. (Mäkelä et al., 1989); according to

this study, the bands result from two distinct translation initiation sites 24 kb apart from the

second exon and their phosphorylation products. For ImageJ quantification, the two close bands

will be referred to as one whole upper band and the single third as lower band.

Figure 16 comprises blots of the same exposure time in order to compare N-myc levels between

the cell lines. N-myc is situated in the predicted molecular size of approximately 60 to 63 kDa.

Optically, N-myc is hardly visible in the SH-SY5Y cells while it is clearly amplified in Kelly

and IMR-32 cells. β-Actin is depicted in the lower lane as loading control (approximately 40

kDa). It is noted that the protein amounts vary within experiments, which, to some extent, is due

to apoptosis and triggered especially by higher statin concentrations like 10 µM. In order to

overcome this problem the intensities of the bands of interest were normalized to the loading

control depicted in Figure 16 B, C and D for SH-SY5Y, Kelly and IMR-32 cells respectively.

61

Results

A

B

62

Results

C

D

Figure 16. N-myc level in Simvastatin and Mevalonate treated cells The SH-SY5Y, IMR-32 and Kelly cell lines were treated with 10 µM simvastatin, in the absence and presence of 1

mM mevalonate or with 1 mM mevalonate alone for 24 hours. Proteins of whole cell lysates were separated by SDS-PAGE and further blotted to detect N-myc and ß-actin (A). Quantification of band intensities was carried out

with the ImageJ software. N-myc intensities were normalized to the loading control ß-Actin (approximately 40 kDa). Bar diagrams for SH-SY5Y (B), Kelly (C) and IMR-32 (D) cells for the upper band and lower band of N-

myc (approximately 60 – 63 kDa) are depicted. Data represent mean ± SD; * = p < 0.05 (n = 4)

Under intensified conditions, in particular longer exposure times N-myc was readily detected

also in SH-SY5Y cells (Figure 17).

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Results

Figure 17. N-myc is also present in SH-SY5Y cells. Similar to Figure 16, whole cell extracts of SH-SY5Y were analysed for N-myc under conditions of longer

exposure.

In SH-SY5Y (Figure 16, B), the N-myc upper band shows a significant reduction to 86.41 % ±

8.51 % and 82.13 % ± 9.39 % by treatment with 10 µM simvastatin and 1 mM mevalonate, and

non-significantly to 97.02 % ± 16.43 % by combinatorial treatment. N-myc lower band level is

non-significantly altered, disposing an increase to 108.34 % ± 12.11 % and 114.20 % ± 27.39 %

by treatment with simvastatin and simvastatin-mevalonate respectively, but a reduction to 85.72

% ± 4.20 % by treatment with mevalonate alone.

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Results

In Kelly cells (Figure 16, C) the level of the N-myc upper bands and lower band are all

significantly reduced by treatment with 10 µM simvastatin to 81.20 % ± 16.31 % and 78.71 % ±

13.02 % respectively; the levels are further reduced by a combinatorial treatment with 10 µM

simvastatin and 1 mM mevalonate to 57.18 % ± 27.39 % and 60.21 % ± 30.60 %. Treatment

with 1 mM mevalonate mesulted in a reduction to 82.32 % ± 9.51 % and 86.70 % ± 8.15 %.

In IMR-32 cells (Figure 16, D), by contrast, the N-myc level is significantly increased by

simvastatin (10 µM) to 152.85 % ± 35.40 % and 175.46 % ± 78.56 % for upper and lower band

respectively, and even further increased to 257.66 % ± 144.71 % and 250.60 % ± 159.67 % by

treatment with simvastatin and mevalonate. Treatment with mevalonate alone increases the level

to 120.32 % ± 4.83 % and 125.38 % ± 34.81 %.

These data suggest that N-myc protein level is sensitive to regulatory changes upon simvastatin

treatment, which seems to be dependent on the cell line.

9.2 N-myc protein expression in Kelly and IMR-32 cells treated with increasing simvastatin concentration

Following the findings of a possible effect by Simvastatin a concentration dependency has to be

postulated for changes in N-myc expression (Figure 18). While N-myc is up-regulated in IMR-

32 cells (Figure 18 B) it is again down-regulated in Kelly cells (Figure 18 C).

In IMR-32 (Figure 18 B), the N-myc amount follows a bell shaped curve corresponding to the

increasing levels of simvastatin (0.1 µM: 123.10 % ± 6.2 % and 137.11 % ± 8.17 % for lower

band; 1 µM: 142.20 % ± 8.58 % and 167.32 % ± 22.99 %; in both cases for upper and lower

band respectively). However, the increase is restricted at 10 µM simvastatin, resulting in 116.00

% ± 6.55 % and 128.05 % ± 9.11 % for upper and lower band, respectively.

In Kelly cells (Figure 18 C), the decrease of N-myc levels is already pronounced by treatments

with 0.1 µM (78.80 % ± 4.88 % and 72.68 % ± 3.05 %) and not further increased with 10 µM

simvastatin (78.88 % ± 0.74 % and 64.48 % ± 3.73 %; upper and lower band, respectively).

These data show that the alterations in N-myc level induced by simvastatin in IMR-32 and Kelly

cells (observed in Figure 16) are consistent and concentration dependent. However, evaluation

of the data provided is semi-quantitative and underlies the technical limitations of the Western

blot technique.

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A

B

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C

Figure 18. Concentration dependency of N-myc levels in IMR-32 and Kelly cells These two MYCN-amplified cell lines were treated with 0.1, 1 and 10 µM simvastatin for 48 hours and analysed

according to Figure 16. Quantifications for N-myc upper and lower band are indicated and presented as mean ± SD for IMR-32 (B) and Kelly (C) cells. * = p < 0.05 (n = 4)

9.3 Altered N-myc expression in Kelly and IMR-32 cells treated with DMSO

Simvastatin is solubilized in DMSO. Therefore, it was considered plausible to include a DMSO

control to address to the question of a possible DMSO effects on N-myc levels. In order to

overcome the limitations of the Western blot technique (i.e. semi-quantitative N-myc signal was

detected by flow cytometric analysis.

One typical Dotplot is shown in Figure 19 A and B, for IMR-32 and Kelly cells respectively.

Both graphs depict cell populations corresponding to the cell morphology according to FSC and

SSC. Gates (blue border) are set to separate cells to be analysed (red dots) from cell debris or

other contaminants that are excluded from analysis (black dots).

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IMR-32 Kelly

A B

C D

Figure 19. Dotplots (A and B) and Histograms (C and D) obtained by flow cytometric analysis of IMR-32 and Kelly cells

Histograms (Figure 19 C and D) show Alexa Fluor 488 signal (referring to N-myc signal) in

logarithmic scale, which is blotted against the cell number. As expected, control cells that are

incubated with N-myc primary antibody and secondary antibody (conjugated with Alexa 488)

showed stronger signals (higher geomean) than the negative control (cells without any antibody)

and the isotype control (cells incubated with IgG2a antibody and secondary antibody conjugated

with Alexa 488). Thus, the flow cytometric analysis method for Kelly and IMR-32 was

evaluated and it was concluded that the N-myc antibody is highly specific.

Kelly and IMR-32 cells were treated with increasing concentrations of DMSO (0.01 %, 0.05 %,

0.1 % and 1 %) for 48 hours (Figure 19). Values resulting from flow cytometric measurements

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are expressed as geometric means (geomean). The geomean value of the control (untreated) is

assumed 100 % and the treatments were normalized to the control.

Figure 20. DMSO effects on N-myc expression in IMR-32 and Kelly cells evaluated by flow cytometry Cells are treated with 0.01 %, 0.05 %, 0.1 % and 1 % DMSO for 48 hours, then fixed, permeabilized and N-myc is detected by flow cytometry. The mean and SD of the normalized data are depicted. measurements were carried out

in triplicates

In IMR-32 cells, the N-myc amount was unchanged by DMSO (97.27 % ± 0.72 % and 93.78 %

± 0.59 %), 100.83 % ± 13.49 %; 110.89 % ± 3.58 %, by 0.01 %, 0.05 %, 0.1 % and 1 % DMSO

respectively). By contrast, in Kelly cells N-myc level increased by increasing concentration of

DMSO (0.01 % DMSO: 118.26 % ± 0.81 %; 0.05 % DMSO: 135.06 % ± 0.71 %; 0.1 % DMSO:

132.61 % ± 2.70 %; 1 % DMSO: 138.78 % ± 6.78 %) (Figure 20).

These findings suggest that DMSO disposes an effect on N-myc protein level that cannot be

neglected. Efforts were put into subsequent experiments to dilute the DMSO content to 0.001 %

with MQ, an empirically determined concentration without compromising the solubility of

Simvastatin in the respective dilutions.

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9.4 Altered N-myc protein expression in IMR-32 and Kelly cells treated with simvastatin and mevalonate analysed by flow cytometry

Flow cytometric analysis of the N-myc expression in simvastatin treated Kelly and IMR-32 cells

confirmed the results obtained with the Western Blot analysis. In Kelly cells (Figure 21 A), N-

myc expression is reduced by simvastatin to 71.67 % ± 1.08 % and 78.67 % ± 53.49 % with 1

µM and 10 µM simvastatin respectively. Co-application with 1 mM mevalonate partially

prevented reduced N-myc levels, indicating HMG-CoA reductase dependent mechanisms to be

involved. However, it is unclear why mevalonate alone reduced the N-myc level to

approximately 50 %. Of note, a HMG-CoA reductase dependent effect is not seen in Western

blots (Figure 16).

In IMR-32 cells (Figure 21 B), N-myc level is enhanced to 176.39 % ± 3.00 % (1 µM

simvastatin) and to 198.43 % ± 15.05 % (10 µM simvastatin) with increasing concentrations of

simvastatin. Again, co-application of mevalonate prevented the effect of simvastatin, indicating a

HMG-CoA reductase dependent mechanism of N-myc upregulation.

Of note, treatments either with simvastatin or mevalonate did not lead to shifts in the distribution

of the cell population with regards to FSC and SSC depicted in histograms (data not shown).

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A

B

Figure 21. N-myc level in dependence on increasing simvastatin concentration in Kelly (A) and IMR-32 (B) cells and evaluated by flow cytometry

Cells were treated with 0.1 µM, 1 µM and 10 µM simvastatin alone or combined with 1 mM mevalonate and 1 mM mevalonate alone for 48 hours. Cells were fixed and permeabilized for flow cytometric analysis.

Error bars = SD; * = p < 0.05; measurements were carried out in triplicates

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9.5 N-myc protein expression changes in Kelly and IMR-32 cells after long-term simvastatin treatment

The expression of N-myc was already altered by 0.1 µM simvastatin (Figure 18 and Figure 21).

Therefore, it was hypothesized that upon long-term exposure simvastatin might induce

senescence in accordance to N-myc down-regulation. IMR-32 and Kelly cells were continuously

exposed to 0.01 µM and 0.1 µM simvastatin in the absence and presence of mevalonate. Flow

cytometry was carried out on a weekly basis. The results are summarized in Figure 22. Both

IMR-32 (A) and Kelly (B) cells show strong fluctuations in the expression of N-myc.

Interestingly, HMG-CoA reductase dependency of the effects was not observed (data not

shown).

Taken together, low Simvastatin concentrations (0.01 µM and 0.1 µM) do not show a significant

effect in long-term exposure experiments over 8 weeks.

9.6 N-myc in nuclear extracts

N-myc’s role as a bHLH transcription factor and the presence of a nuclear targeting sequence in

its N-terminus strongly suggest a nuclear localization of the protein. To confirm whether

increasing simvastatin has an effect on the compartmentalisation of N-myc, nuclear fractions

were prepared from IMR-32 and Kelly cells and compared with the cytosolic fraction.

Vincristine was used as a positive control, as it can induce apoptosis similar to statins.

Both the results in IMR-32 and Kelly cells corroborate the effects seen in Western Blots and

flow cytometry (Figure 18 and Figure 21): In IMR-32 N-myc expression was increased in both

nuclear and cytosolic fractions, whereas it was decreased in the nuclear fraction of Kelly cells in

a concentration dependent manner. A clear down-regulation of N-myc was observed in the

vincristine treated cells. Lap2β (53 kDa) is a nuclear laminin associated protein and was used as

a nuclear marker to confirm isolation of the nuclear and cytosolic fraction. As expected, Lap2β

was detectable in the nuclear fraction only. It is unclear, why Lap2β cannot be detected in

nuclear extracts of IMR-32 cells treated with vincristine.

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A

B

Figure 22. N-myc level in dependence on simvastatin and mevalonate long-term treatments in IMR-32 and Kelly

Cells were treated with 0.01 µM ( ) and 0.1 µM ( ) simvastatin for seven weeks (IMR-32; A) and and eight weeks (Kelly; B). N-myc signal in fixed and permeabilized cells were analyzed by flow cytometry on a weekly

basis. Control (untreated; ) was set as 100 % and the treatments were normalized to the control. Data represents the mean +/- SD of a single experiment carried out in triplicates.

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A

B

Figure 23. N-myc level dependence on simvastatin treatment in nuclear and cytosolic fractions of IMR-32 (A) and Kelly (B)

Both lines were subdued to treatment with 1 µM, 10 µM simvastatin and 0.25 µM vincristine for 48 hours. N-myc levels in nuclear and cytosolic protein fractions were visualized by Western Blot. β-Actin was used as loading

control and Lap2β as marker for the nuclear fraction.

9.7 Expressional changes of small G-proteins of the Rac-family by simvastatin treatment in IMR-32 and Kelly cells

Simvastatin is a HMG-CoA reductase inhibitor and blocks the mevalonate pathway. The activity

of small G-proteins is dependent on the mevalonate pathway as they need to be prenylated in

order to become anchored in the cell membrane and to exert their function. It was shown by Cao

et al. (Cao et al., 2011) that c-Myc activation is dependent on phosphorylation by the Rac

GTPase and that treatment with atorvastatin can inhibit the prenylation and anchorage of Rac

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and subsequently the activation of c-Myc. Likewise, this study aims to clarify whether treatment

with simvastatin may influence Rac and two other Rho GTPases, cdc42 and RhoA, which may

also have an impact on the activity of N-myc. The small G proteins are detected in the cytosolic

fraction. Analysis of RhoA is separated into upper band and lower band, referring to the non-

processed (non-prenylated) and processed (prenylated) form respectively (Zhong et al., 2005).

In IMR-32 cells (Figure 24), treatment with 1 µM and 10 µM simvastatin for 48 hours resulted

in decreased protein levels of Rac1/2/3 (A; 63.61 % ± 35.41 % and 56.90 % ± 34.41 %) and

cdc42 (B; 43.74 % ± 29.81 % and 44.95 % ± 14.46 %). However, level of RhoA increased after

the treatment (C; upper band: 113.76 % ± 3.12 % and 118.49 % ± 14.25 %; lower band: 123.97

% ± 5.21 % and 152.32 % ± 32.43 %). Treatment with 0.25 µM vincristine represents a positive

control for apoptosis induction and resulted in a decrease in Rac1/2/3 (A; 27.46 %), in cdc42 (B;

21.08 %) and in RhoA level (C; upper band: 70.92 %; lower band: 96.42 %).

In Kelly cells (Figure 25), the treatment with 1 µM and 10 µM simvastatin for 48 hours resulted

in an elevated level of Rac1/2/3 (A; 163.80 % ± 47.57 % and 172.97 % ± 63.09 %), but

decreased level of cdc42 (B; 52.89 % ± 1.19 % and 65.49 % ± 1.42 %) and of RhoA (C; lower

band: 97.20 % ± 61.41 % and 41.45 % ± 18.36 %). In contrast to IMR-32 cells, 0.25 µM

vincristine leads to elevated levels of Rac1/2/3 (A; 340.64 %) and RhoA lower band (C; 07.07

%), whereas the levels of cdc42 (B; 73.91 %) and RhoA upper band (C; 94.92 %) are reduced,

but still higher in comparison to the simvastatin treated samples.

Taken together, these results suggest a possible regulation of small GTPases by simvastatin

treatment. Of note, both Kelly and IMR-32 cells show a significant down-regulation in cdc42

level by simvastatin treatment.

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IMR-32

A B

C

Figure 24. Protein levels of small GTPases Rac1/2/3, cdc42 and RhoA in dependence on simvastatin and vincristine treatment in cytosolic fractions of IMR-32 cells Cells were treated with 1 µM and 10 µM simvastatin and 0.25 µM vincristine for 48 hours. Rac1/2/3 (A), cdc42 (B) and RhoA (C) levels were detected in the cytosolic protein fraction by Western Blots (all about 21 kDa) and evaluated by ImageJ. Control (untreated) was set to 100 % and the values of the treatments are normalized to the control. Error bars = SD; * = p < 0.05 (n = 4)

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Kelly

A B

C

Figure 25. Protein levels of small GTPases Rac1/2/3, cdc42 and RhoA in dependence on simvastatin and vincristine treatment in cytosolic fractions of Kelly cells Cells were treated with 1 µM and 10 µM simvastatin and 0.25 µM vincristine for 48 hours. Rac1/2/3 (A), cdc42 (B) and RhoA (C) levels were detected in the cytosolic protein fraction by Western Blots (all about 21 kDa) and evaluated by ImageJ. Control (untreated) was set to 100 % and the values of the treatments are normalized to the control. Error bars = SD; * = p < 0.05 (n = 4)

9.8 Alterations of Aurora Kinase A and Max protein expression in IMR-32 and Kelly cells treated with simvastatin

As previous results have confirmed that simvastatin treatment may cause alterations in N-myc

level, it is interesting to know whether these effects are accompanied by changes in a more

functional level of N-myc. To start off, the protein levels of two interaction partners of N-myc -

Aurora kinase A and Max – are evaluated in the nuclear fraction of IMR-32 and Kelly cells

treated with 1 µM and 10 µM simvastatin for 48 hours.

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Aurora kinase A is significantly down-regulated by 1 µM simvastatin (70.70 % ± 22.16 %) and

10 µM simvastatin (41.66 % ± 24.26 %) in a concentration dependent manner in nuclei of IMR-

32 cells (Figure 26 A). The amount of Max is comparable to the control at 1 µM simvastatin

(99.11 % ± 57.46 %), but is significantly reduced with 10 µM simvastatin (51.62 % ± 27.26 %).

Similarly, treatment of the cells with 0.25 µM vincristine reduced Aurora kinase A (72.23 %)

and Max (77.30 %) on protein level.

In Kelly cells (Figure 26 B), Aurora kinase A is strongly reduced (40.60 % ± 4.88 % and 42.62

% ± 7.80 % with 1 µM and 10 µM simvastatin, respectively), whereas the expression of Max is

slightly elevated (124.73 % ± 31.27 % and 106.27 % ± 3.60 %, respectively). Like IMR-32 cells,

Max is reduced by treatment with 0.25 µM vincristine; by contrast Aurora kinase A level is

increased (112.67 %).

These data show that simvastatin treatment affects important interaction partners of N-myc on

protein level which may result in altered N-myc function. This assumption, however, requires

further investigations.

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IMR-32 Kelly

A B

Figure 26. Protein levels of Max and Aurora A in dependence on simvastatin treatment in nuclear fractions of IMR-32 (A) and Kelly (B)

Cells were treated with 1 µM and 10 µM simvastatin and 0.25 µM vincristine for 48 hours. Max and Aurora A levels were detected in the nuclear fraction in Western Blots and quantified by ImageJ. Control (untreated) is assumed 100 %. Values of the treatments are normalized to the control. Error bars = SD; * = p < 0.05 (n = 4)

9.9 Apoptosis induction by simvastatin treatment in IMR-32 and Kelly cells

Both short-term and long-term treatments with 0.01 µM and 0.1 µM simvastatin did not result in

any obvious phenotypical changes or impairment in growth in IMR-32 and Kelly cells (data not

shown). To investigate whether simvastatin has any effect on senescence or apoptosis in these

cells, β-Galactosidase assays and Poly ADP-ribose polymerase (PARP) cleavage were

performed.

Figure 27 shows that none of the cells has entered senescence after long-term treatment of 5

weeks in IMR-32 cells (A) and of 6 weeks in Kelly cells (B), respectively. The positive control

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shown in Figure 28 was performed with the melanoma strain “WM793” (kindly provided by

Christine Wasinger) to confirm that β-Galactosidase assay worked in a proper way, resulting in

blue staining of senescent cells at high seeding density and after treatment with 50 nM

doxorubicin.

Apoptosis was evaluated by PARP-cleavage performed with nuclear extracts from simvastatin

and vincristine treated IMR-32 and Kelly cells. Apoptosis was confirmed by an increase in

PARP cleavage, resulting in an accumulation of the cleavage product at 89 kDa. The gain of

cleavage product is further quantified as the proportion between upper band (116 kDa) and lower

band (89 kDa) of PARP: The lower the ratio the more cleavage occurred. The ratio observed in

the control was again set to 100 % and the ratios of the treatments were normalized to the

control. Both in IMR-32 and in Kelly cells the augmentation in cleavage related to the increasing

concentration of simvastatin treatment (for IMR-32: 85.34 % ± 27.71 % at 1 µM and 72.65 % ±

13.70 % at 10 µM simvastatin; for Kelly: 85.71 % ± 4.71 % at 1 µM and 76.69 % ± 10.52 % at

10 µM simvastatin). The strongest induction of apoptosis was seen by treatment with 0.25 µM

vincristine in both IMR-32 and Kelly cells (26.82 % and 36.14 %, respectively).

Taken together, these data suggest that short-termed treatments with high concentrations of

simvastatin results in induction of apoptosis in IMR-32 and Kelly cells. Against all odds, long-

termed treatments with lower concentrations did not induce senescence in IMR-32 and Kelly

cells.

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(A) IMR-32

CTL 0.01 µM sim 0.1 µM sim

100 µM mev 0.1 µM sim + 100 µM mev 0.001 % DMSO

(B) Kelly

CTL 0.01 µM Sim 0.1 µM Sim

100 µM Mev 0.1 µM Sim + 100 µM Mev 0.001 % DMSO

Figure 27. β-Galactosidase Assay of IMR-32 and Kelly cells Cells were subdued to treatments with 0.01 µM and 0.1 µM simvastatin, 100 µM mevalonate, a combination of 0.1 µM simvastatin and 100 µM mevalonate and 0.001 % DMSO for five weeks. After fixation and permeabilization cells were incubated with staining solution (with X-gal) overnight, washed with PBS and photographed under the

light microscope. Bar = 0.25 µm; CTL = control; sim = simvastatin; mev mevalonate; DMSO = dimethyl sulfoxide

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WM793

100 nM doxorubicin (- staining) CTL (+ staining)

50 nM doxorubicin staining

Figure 28. β-Galactosidase Assay of WM793 Cells were treated with 50 nM and 100 nM doxorubicin for three days. After fixation permeabilization, cells treated with 100 nM doxorubicin were incubated with staining solution without X-gal overnight; cells treated with 50 nM

doxorubicin and untreated cells were incubated with staining solution with X-gal. After staining, cells were washed with PBS and photographed under the light microscope. Bar = 0.25 µm; CTL = control (untreated)

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(A) IMR-32

(B) Kelly

B

Figure 29. Protein levels of PARP in dependence on simvastatin treatment in nuclear fractions of IMR-32 (A) and Kelly (B) cells

Cells were treated with 1 µM and 10 µM simvastatin and 0.25 µM vincristine for 48 hours. The ratio between PARP uncleaved (116 kDa) and PARP cleaved (89 kDa) levels were detected in the nuclear fraction in Western Blots and quantified by ImageJ. Control (untreated) is assumed 100 %. Values of the treatments are normalized to the control.

Error bars = SD; n = 4

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10 Discussion

Up-regulation of N-myc is strongly associated with malignant tumour phenotypes. Many

publications refer to its cancerous roles in mis-regulating the cell cycle and prevention from

apoptotic pathways. Hence the down-regulation of N-myc may represent a novel strategy of

cancer-therapy (Brodeur, 2003). Statins are known for their anti-proliferative roles in various

cancers. As inhibitors of the mevalonate pathway, they deplete the cells from cholesterol

precursors and thus deprive the cells from building blocks for successful proliferation (Demierre

et al., 2005). However, the mechanisms of these effects in diverse cancer types are not clear.

This study provides evidences that simvastatin is capable to reduce N-myc level in human Kelly

neuroblastoma cells, induce apoptosis and also uncovers a potential mechanism of how this can

be accomplished.

10.1 N-myc over-expression is confirmed in human Kelly and IMR-32 cells

MYCN is frequently amplified in neuroblastoma tumours of advanced stages, resulting in protein

levels higher than in non-amplified cells (Brodeur, 1989, 1990). In this work, high N-myc level

is confirmed by comparative Western Blot analysis and flow cytometry data. In Western Blot –

by using the same conditions (same concentrations of proteins and same exposure time of films)

– it was shown that Kelly cells dispose the highest N-myc level, followed by IMR-32 and SH-

SY5Y (Figure 16). This was also confirmed by flow cytometry analysis, as – under the same

settings and conditions – the absolute geomean values of untreated Kelly cells has always been

higher than those of untreated IMR-32 (data not shown). SH-SY5Y cells do not yield MYCN

amplification (Farina et al., 2012; Roy Choudhury et al., 2010) and was used as a control to show

the basal level of N-myc expression.

Nonetheless, these results only confirm N-myc accumulation on protein level and cannot

determine the nature of the over-expression, i.e whether MYCN/N-myc expression is

deregulated on transcriptional or translational level or results from impaired degradation

pathways in Kelly and IMR-32 cells. Of note, MYCN amplifications have already been stated in

IMR-32 and Kelly cells as HSRs (Corvi et al., 1994; Krawczun et al., 1986; Solovei et al., 2000).

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Studies by fluorescence in situ hybridization with labelled probes against MYCN genomic

sequence (Mathew et al., 2001) may visualize the type of MYCN amplification in the cell lines

used in this study.

10.2 Simvastatin is able to downregulate N-myc level in Kelly, but not in IMR-32 cells

This study shows that short-termed treatments with 0.1, 1 and 10 µM simvastatin are able to

reduce N-myc level in Kelly cells, but causes an opposite effect in IMR-32. Several Western

Blots and flow cytometry experiments confirmed the effects of simvastatin and showed a distinct

dosage dependency (Figure 18, Figure 21 and Figure 23). Remarkably, simvastatin seems to

cause a shift of N-myc from the nucleus to the cytosol in Kelly cells (Figure 23), as its level is

up-regulated in the cytosol upon treatment with 10 µM simvastatin. A possible explanation could

be the enhanced transport of N-myc out of nucleus, followed by proteasomal degradation in the

cytosol; however it has been proposed that ubiquitin-dependent proteasome degradation of N-

myc occurs in the nucleus (Bonvini et al., 1998). Further experiments will include N-myc by

using labelled N-myc antibody which is able to detect ubiquitinated N-myc should answer this

question. In addition, reporter constructs and detection by confocal laser scanning microscopy to

confirm the enhanced presence of N-myc in the cytosolic compartment and help to investigate

nuclear and cytosolic shuttling of N-myc after simvastatin treatment. Furthermore, it will be

possible to perform co-localization assays with organelle markers, hence assigning the N-myc

signal to a specific compartment in order to clarify the role of N-myc in the cytosol.

The effect of mevalonate remains rather unclear, as it seems to act in a synergistic way with

simvastatin to enhance its action in Kelly cells, but shows an opposing effect upon co-treatment

with increasing dosage of simvastatin in both Kelly and IMR-32 (Figure 21 A and B). This

cannot be explained by the effects of DMSO on Kelly cells, as it augments N-myc in flow

cytometry experiments (Figure 20). Nevertheless, many studies already confirmed DMSO as a

differentiation inducing agent (Morley and Whitfield, 1993). The effect of DMSO on Kelly and

IMR-32 cells remains to be clarified.

Long-term treatments with low concentrations (0.01 and 0.1 µM) of simvastatin showed strong

fluctuations of N-myc levels in both Kelly and IMR-32 cells, which makes an interpretation

rather difficult. An explanation for these fluctuations may lie within the experimental procedure.

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For instance, it was confirmed by a control experiment that the fluorescence signal (N-myc)

measured by flow cytometry is strongly influenced by the ratio between the concentration of

antibody and the cell number (data not shown). For future flow cytometry experiments, it is

strongly recommended to perform cell counting and equalization of cell number in all samples

before incubation with the primary antibody or at least using an excess of the same to rule out

the biases caused by cell number variation.

10.3 Simvastatin is able to reduce the expressional level of selected small GTPases

The study by Cao et al. (Cao et al., 2011) confirmed the anti-proliferative effect of statins by

showing that treatment with 10 µM atorvastatin for 96 hours successfully inhibited proliferation

of hepatocellular carcinoma in vitro and in vivo. c-Myc activation is dependent on its

phosphorylation by Rac GTPase. The authors further uncovered the mechanism underlying this

effect by showing that atorvastatin treatment causes inactivation of c-Myc. Furthermore,

atorvastatin inhibits and interferes with the mevalonate pathway, which causes prohibited

prenylation of the small GTPase Rac. This results in impaired function of Rac and hence may be

the cause for the decreased phosphorylation and activation of c-Myc (Cao et al., 2011). Here

likewise, it was hypothesized that GTPases also represent upstream-regulators of N-myc, since

N-myc – analogously to c-Myc – needs to be phosphorylated on S62 to become activated (Otto

et al., 2009b). Hence, it is important to clarify whether simvastatin may indirectly affect N-myc

activity by regulation of GTPases.

All GTPases of the RhoA family – including RhoA, cdc42 and Rac1/2/3 - need to be

geranylgeranylated or farnesylated on the conserved cysteine residue in the CAAX-box at the C-

terminus in order to become anchored in the cell membrane to exert its function (Visvikis et al.,

2010). Here, due to the usage of detergents in a concentration exceeding the critical micelle

concentration (0.05 % NP-40), it was assumed that GTPases lose their anchorage at the cell

membrane and therefore are concentrated in the cytosolic fraction obtained after the cell lysis.

However, as a membrane protein fraction has not been extracted separately from the cytosolic

fraction, it is not possible to give any hint on the cellular compartmentalisation and hence on the

activity of these GTPases. The reduction of Rac1/2/3 and cdc42 level in IMR-32, as well as of

cdc42 and RhoA in Kelly cells by simvastatin treatment may be due to changes in expressional

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Discussion

level by an unknown mechanism, or due to a higher rate of degradation. Analogously, the higher

RhoA level in simvastatin treated IMR-32 cells as well as the higher Rac1/2/3 level in

simvastatin treated Kelly cells, may indicate higher expression or impaired degradation.

Likewise, no clear evidences of changes of prenylation status can be obtained by analysis of

RhoA upper (non-processed) and lower band (processed), as both react in a similar way to

simvastatin treatment.

In particular, the overall reduced level of cdc42 and RhoA in Kelly cells upon simvastatin

treatment may ultimately lead to reduced activation of N-myc (or c-Myc) and cause functional

alterations. As N-myc can only be targeted for degradation upon its activation, (Otto et al.,

2009b) the reduced level of Rac and cdc42 in IMR-32 cells may account for insufficient

activation, inefficient degradation and accumulation of N-myc. However, this explanation cannot

be applied on Kelly cells, as its N-myc level is reduced, which suggests that other regulatory

mechanisms that differ between IMR-32 and Kelly cells exist.

Future experiments will aim to clarify whether the protein level of small GTPases in the

membrane fraction may be influenced by simvastatin treatment. Furthermore, a specific antibody

targeting N-myc phosphorylated at S62 will come in handy to observe N-myc activation upon

treatments.

10.4 Simvastatin treatment may cause functional alterations of N-myc

The result that N-myc expression is affected by simvastatin treatment in two separate

experimental setups prompts the investigation of the effects of simvastatin on a more functional

level. For this purpose, two prominent interaction partners of N-myc were chosen for analysis.

Aurora kinase A, encoded by the proto-oncogene AURKA, is a serine/threonine protein kinase

involved in cell cycle regulation, chromosome segregation and mitotic spindle apparatus

formation (Crane et al., 2004; Meraldi et al., 2002; Sen et al., 1997; [CSL STYLE ERROR:

reference with no printed form.]; Xiao et al., 1998b). It co-localizes with N-myc at G2 to M

transition and is highly involved in the stability of N-myc as it may prohibit the ubiquitin

dependent degradation of N-myc (Otto et al., 2009b). The strong reduction in Aurora kinase A

expression observed by treatments with already 1 µM simvastatin in both IMR-32 and Kelly

cells may indicate that N-myc stability is impaired, which at least in Kelly cells contributes to the

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Discussion

down-regulation seen in Western Blots and flow cytometry experiments Figure 17 C, Figure 18

C and Figure 21 A). Nonetheless, the increase of N-myc expression in IMR-32 cells cannot be

explained by the reduced Aurora kinase A level.

The other interaction partner of N-myc, Max, belongs to the bHLH-zip transcription factors. Its

protein level is found to be increased in Kelly cells and slightly decreased in IMR-32 cells upon

treatment with simvastatin. As N-myc binding to the DNA requires heterodimerization with

Max, these results provide an insight on how simvastatin treatment alters the ability of N-myc to

bind to the promoters of its target genes and regulate their expression. This finding underlines

the importance to further elucidate the effects of simvastatin on the functional level of N-myc. A

reporter assay consisting of a promoter region of an N-myc target gene fused to the luciferase

gene is one example to address to this question. This may show differences in N-myc regulated

expression of target genes that may be of importance for tumour growth and progression.

10.5 Simvastatin treatment induces apoptosis

Statins are able to activate caspases and induce apoptosis (Cafforio et al., 2005; Marcelli et al.,

1998; Sieczkowski et al., 2010; Wang et al., 2000; Werner et al., 2013). PARP (Poly ADP-ribose

polymerase) is a family of nuclear proteins that are involved in DNA repair and may induce

apoptosis through different ways: It can deplete cells from ATP to repair damaged DNA (Satoh

and Lindahl, 1992), or it can stimulate mitochondria to release AIF (Yu et al., 2006) or it can be

inactivated by cleavage by caspases (Cohen, 1997; Lazebnik et al., 1994a; Nicholson et al.,

1995a; Tewari et al., 1995). In the latter case, PARP is cleaved at the aspartic acid 214 and

glycine 215, producing 24 kDa and 89 kDa fragments (Lazebnik et al., 1994b; Nicholson et al.,

1995b) of the protein and prohibited from exerting its function to rescue the DNA. Hence,

cleaved PARP is a marker for caspase activity and subsequently apoptosis (Oliver et al., 1998).

As shown here, short-term treatment with 1 and 10 µM simvastatin was able to induce PARP

cleavage in IMR-32 and Kelly cells, confirming the anti-proliferative effect of statins.

It was shown that treatment with 5-Hydroxyurea (5-HU) is able to reduce MYCN copies in

MYCN-amplified neuroblastoma cells and induce senescence. The underlying mechanism is the

formation of micronuclei with which the cell can discard DNA material such as amplified

MYCN copies (Narath et al., 2007). It was also shown that both dmins and HSRs can be

88

Discussion

expulsed from neuroblastoma cells after treatment with hydroxyurea or other cytostatics

(Prochazka et al., 2010).

Based on these reports, Kelly and IMR-32 cells are tested to show whether long-term treatments

with low concentrations of simvastatin may induce senescence. Surprisingly, both MYCN-

amplified cell lines treated for over 7 weeks did not show any impairment in growth or

phenotypical changes typical for senescence such as increased cell or nucleus size, formation of

dendrites or enhanced granularity (Dimri et al., 1995). Concomitantly, senescence-associated β-

Galactosidase activity was confirmed only in the doxorubicin treated WM793 melanoma cells

(Silini et al., 2010), which were used as a positive control, but not in simvastatin treated Kelly

and IMR-32 cells.

89

Concluding Remarks

11 Concluding Remarks This master thesis shows that simvastatin is able to reduce N-myc level in the human

neuroblastoma cell line Kelly. It further confirms the apoptosis inducing effect of simvastatin in

both Kelly and IMR-32 cell lines. Moreover, the results provide hints on functional and

regulatory alterations of N-myc upon simvastatin treatment and highlight the complexity of

underlying mechanisms and pathways that differ between both neuroblastoma cell lines. This

study gives rise to new investigations and represents a basis for future projects on statins

therapeutic potential in neuroblastoma.

90

List of Figures

12 List of Figures Figure 1. Neuroblastoma tumour and metastasis development (Maris, 2010) ..........................................................14 Figure 2. Neuroblastoma stages classified by INSS (Brodeur et al., 1993) ..............................................................16 Figure 3. Structural homologous regions in c-Myc, N-myc and L-myc (Cole and Cowling, 2008a) .........................20 Figure 4. Stabilization and degradation of N-myc in cell cycle progression and differentiation (Otto et al., 2009b) .24 Figure 5. A selection of downstream targets of N-myc (Bell et al., 2010) ...............................................................25 Figure 6. The mir-17-92 cluster and its proposed functions in oncogenesis (Buechner and Einvik, 2012b) ..............26 Figure 7. Statins are negative regulators of the mevalonate pathway (Gazzerro et al., 2012) ...................................27 Figure 8. Pharmacological efficiency of statins is based on the similarity of the pharmacophore moiety of statins and

HMG-CoA ......................................................................................................................................28 Figure 9. Examples for type 1 and type 2 statins ....................................................................................................29 Figure 10. Examples of pleiotropic effects of statins and their mode of actions (Mason, 2003) ...............................31 Figure 11. Regulation of GTPase by isoprenylation (Liao and Laufs, 2005) ...........................................................32 Figure 12. Anti-cancer mechanisms of statins (Chan et al., 2003) ...........................................................................33 Figure 13. Useful numbers for cell culture (provided by Invitrogen) ......................................................................44 Figure 14. Enhanced chemiluminescence (ECL) reaction (GE Healthcare) .............................................................54 Figure 15. Schematic view of a flow cytometer and its principles of detection (pictures are taken from Invitrogen

Homepage “Introduction to Flow Cytometry”) .................................................................................57 Figure 16. N-myc level in Simvastatin and Mevalonate treated cells ......................................................................62 Figure 17. N-myc is also present in SH-SY5Y cells. ..............................................................................................63 Figure 18. Concentration dependency of N-myc levels in IMR-32 and Kelly cells ..................................................66 Figure 19. Dotplots (A and B) and Histograms (C and D) obtained by flow cytometric analysis of IMR-32 and Kelly

cells ................................................................................................................................................67 Figure 20. DMSO effects on N-myc expression in IMR-32 and Kelly cells evaluated by flow cytometry ................68 Figure 21. N-myc level in dependence on increasing simvastatin concentration in Kelly (A) and IMR-32 (B) cells

and evaluated by flow cytometry .....................................................................................................70 Figure 22. N-myc level in dependence on simvastatin and mevalonate long-term treatments in IMR-32 and Kelly .72 Figure 23. N-myc level dependence on simvastatin treatment in nuclear and cytosolic fractions of IMR-32 (A) and

Kelly (B) .........................................................................................................................................73 Figure 24. Protein levels of small GTPases Rac1/2/3, cdc42 and RhoA in dependence on simvastatin and vincristine

treatment in cytosolic fractions of IMR-32 cells ...............................................................................75 Figure 25. Protein levels of small GTPases Rac1/2/3, cdc42 and RhoA in dependence on simvastatin and vincristine

treatment in cytosolic fractions of Kelly cells ...................................................................................76 Figure 26. Protein levels of Max and Aurora A in dependence on simvastatin treatment in nuclear fractions of IMR-

32 (A) and Kelly (B)........................................................................................................................78 Figure 27. β-Galactosidase Assay of IMR-32 and Kelly cells .................................................................................80 Figure 28. β-Galactosidase Assay of WM793 ........................................................................................................81

91

List of Figures

Figure 29. Protein levels of PARP in dependence on simvastatin treatment in nuclear fractions of IMR-32 (A) and

Kelly (B) cells .................................................................................................................................82

92

List of Tables

13 List of Tables Table 1. PBS recipe ..............................................................................................................................................44 Table 2. IP-Buffer recipe for IP-Buffer protein extraction ......................................................................................45 Table 3. Buffer A and buffer B recipe for nuclear extraction (provided by Abcam) ................................................46 Table 4. Compounds for SDS gels .........................................................................................................................49 Table 5. Preparation of 10 % and 15 % Separating Gel ..........................................................................................49 Table 6. Preparation of 5 % Stacking Gel ..............................................................................................................50 Table 7. Running Buffer recipe for SDS PAGE .....................................................................................................51 Table 8. Transfer Buffer recipe for Wet Blotting....................................................................................................51 Table 9. Bjerrum Buffer recipes for Wet Blotting ..................................................................................................52 Table 10. TBS and TBS-T recipes .........................................................................................................................53 Table 11. Mild stripping buffer recipe ...................................................................................................................55 Table 12. Compounds for SA-β-Galactosidase assay .............................................................................................58

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Weblinks:

Molecular Anatomy Project http://maptest.rutgers.edu/drupal/?q=node/39

Wikipedia http://www.wikipedia.org/

Ich habe mich bemüht, sämtliche Inhaber der Bildrechte ausfindig zu machen und ihre Zustimmung zur Verwendung der Bilder in dieser Arbeit eingeholt. Sollte dennoch eine Urheberrechtsverletzung bekannt werden, ersuche ich um Meldung bei mir.

107

Curriculum Vitae

15 Curriculum Vitae

Personal Data First name Tschu-Jie

Last name Liu Marital status Single Military duty Exempted Place of birth Taipeh/Taiwan Nationality Austrian

E-Mail tschujie@yahoo.de

Education

December 2012 - today Diploma thesis in the group of Prof. Martin Hohenegger at Institut für Pharmakologie (Vienna, Austria); “Statin induced effects on MYCN in human neuroblastoma lines”

July to August 2012 Practical course at Fraunhofer Institute for Toxicology and Experimental Medicine (Hannover, Germany); training in “ex vivo lung function assessments in precision cut lung slices”

Since Oct. 2011 Diploma study at University of Vienna, field of study: Immunology

Feb. 2007 to Jun. 2011 Diploma study at University of Vienna, field of study: Gene- and Biotechnology Research Project at the Max F. Perutz Laboratories (MFPL) for 2 years; Dr. Bohr-Gasse 9, 1030, Vienna

Oct. 2004 to Jan. 2007 Diploma study at University of Vienna, field of study: Biology

Sep. 1996 to Jun. 2004 School education at BRG Rainergasse 39, 1050 Vienna

Tschu-Jie Liu

Vienna, 27.09.2013