lbh589-induced dapk activation triggers apoptosis and … · 2013-09-03 · lbh589-induced dapk...

LBH589-induced DAPK activation triggers apoptosis and autophagy in human colon tumor cells

Die LBH589-induzierte DAPK Aktivierung fördert Apoptose und Autophagie in humanen

Dickdarmkrebszellen

Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

ZUR

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von Muktheshwar Gandesiri

aus Kaleshwaram, Indien

ii

Als Dissertation genehmigt von der Naturwissen- schaftlichen Fakultät der Friedrich-Alexander-Universität

Erlangen-Nürnberg

Tag der mündlichen Prüfung: 08.03.2013 Vorsitzender der Promotionskommission: Prof. Dr. Johannes Barth Erstberichterstatter: Prof. Dr. Andreas Burkovski Zweitberichterstatter: Prof. Dr. Regine Schneider-Stock

ACKNOWLEDGEMENTS

iii

Acknowledgements First and foremost, I would like to thank Prof. Regine Schneider-Stock, Head of the

Experimental Tumor Pathology for giving me the opportunity to do PhD under her

supervision. I would like to thank her for providing excellent guidance during my entire stay

in her lab and for her critical reading of my PhD thesis.

I would like to express my gratitude to Prof. Arndt Hartmann, Director of the institute of

pathology for giving the opportunity to do PhD thesis in his institute.

I would also like to thank Prof. Matthias Ocker and Prof. Olaf Prante for their help and

cooperation in my research work.

I am indebted to Prof. Dr. Andreas Burkovski for agreeing as a first supervisor and also

critical reading of my PhD thesis.

I would like to thank Prof. Dr. Robert Slany and Prof. Dr. Lars Nitschke for generously

agreeing as examiners for my PhD thesis.

I am very much thankful to my colleagues, Jelena Ivanovska, Natalya Benderska, Sabine

Knaup, Chirine El Baba, Adrian Koch for their friendship and help.

I would like to thank my mother, sisters, brothers and other family members for their

continuous support, encouragement and motivation in my life.

Finally, my wife Saritha Chakilam, you deserve a special thank you for all the support, love,

and cooperation.

I dedicate this thesis to my father.

At the end, I would like to thank all those people who helped me to make this thesis possible.

INDEX

iv

ABBREVIATIONS...…………………………………………………………………… viii

ABSTRACT……………………………………………………………………………. xi

ZUSAMMENFASSUNG………………………………………………………………. xiii

1. Introduction……………………………………………………………………… 1

1.1 Chromatin structure………………………………………………………………. 1

1.2 Epigenetics and Cancer…………………………………………………………... 2

1.3 Histone deacetylases (HDAC)……………………………………………………. 2

1.4 Histone acetyl transferases (HAT)……………………………………………….. 3

1.5 DNA Methylation……………………………………………………………….... 3

1.6 DNA methyl transferases (DNMTs)……………………………………………... 4

1.7 Histone deacetylase inhibitors (HDACi) as anti-cancer agents………………….. 4

1.7.1 Histone deacetylase inhibitors (HDACi)……………………………………. 4

1.7.2 LBH589 (Panobinostat)…………………………………………………….. 6

1.8 Death Associated Protein Kinase (DAPK)………………………………..…….. 7

1.8.1 DAPK structure…………………………………………………………….... 7

1.8.2 DAPK function……………………………………………………………… 8

1.9 Programmed cell death pathways……………………………………………….. 9

1.9.1 Apoptosis……………………………………………………………………. 9

1.9.2 Autophagy…………………………………………………………………… 10

2. Aims of the work………………………………………………………………... 14

3. Materials and Methods………………………………………………………… 16

3.1 Materials………………………………………………………………………… 16

3.1.1 Chemicals and Reagents…………………………………………………….. 16

3.1.2 Cell culture………………………………………………………………….. 18

3.1.3 Cell lines…………………………………………………………………….. 18

3.1.4 Buffers and Solutions……………………………………………………….. 18

3.1.5 Antibodies…………………………………………………………………… 20

3.1.6 Drugs and Chemicals……………………………………………………….. 21

3.1.7 Inhibitors…………………………………………………………………….. 21

3.1.8 Kits………………………………………………………………………….. 21

3.1.9 Equipments………………………………………………………………….. 22

3.1.10 Consumables……………………………………………………………….. 23

INDEX

v

3.1.11 Software……………………………………………………………………. 23

3.1.12 Database……………………………………………………………………. 23

3.2 Methods…………………………………………………………………………. 24

3.2.1 Tumor cell lines and Cell culture…………………………………………… 24

3.2.2 Generation of DAPK shRNA stable cell line……………………………….. 24

3.2.3 Generation of DAPK+++ overexpressing cell line…………………………. 24

3.2.4 xCELLigence Real-Time cell assay………………………………………… 25

3.2.5 MTT assay………………………………………………………………….. 25

3.2.6 Crystal violet Assay………………………………………………………… 25

3.2.7 Clonogenic assay…………………………………………………………… 26

3.2.8 Western blotting…………………………………………………………….. 26

3.2.8.1 Determination of protein concentration………………………………. 26

3.2.8.2 Western blot analysis…………………………………………………. 27

3.2.9 Detection of autophagic vacuoles by acridine orange………………………. 28

3.2.10 Detection of autophagosomes by LC3 immunofluorescence staining……. 28

3.2.11 Flow cytometric detection of Apoptosis by Annexin V-FITC labelling….. 28

3.2.12 Flow cytometric analysis of cell cycle distribution……………………….. 28

3.2.13 Immunoprecipitation………………………………………………………. 29

3.2.14 In vitro kinase assay……………………………………………………….. 29

3.2.15 RNA Isolation and cDNA synthesis………………………………………. 29

3.2.16 Real-Time Reverse Transcription-PCR……………………………………. 30

3.2.17 In vivo Experimental Methods…………………………………………….. 30

3.2.17.1 Xenograft model…………………………………………………….. 30

3.2.17.2 Tissue microarray and Immunohistochemistry……………………… 31

3.2.17.3 Assessment of immunohistochemical protein expression…………… 31

3.2.17.4 Preparation of animals for MicroPET Imaging……………………… 31

3.2.17.5 MicroPET Imaging…………………………………………………… 32

3.2.18 Statistical analysis………………………………………………………….. 32

4. Results…………………………………………………………………………….. 33

4.1 LBH589 decreases cell viability in DAPK wt cells…………………………….. 33

4.2 Generation of DAPK knockdown stable cell line (DAPK shRNA) in

DAPK wt cells…………………………………………………………………… 34

INDEX

vi

4.3 Generation of tamoxifen-inducible DAPK overexpression stable cell line

(DAPK+++) in DAPK wt cells………………………………………………. 34

4.4 Effect of Tamoxifen and/or LBH589 on the expression of DAPK

in DAPK +++ cells……………………………………………………………… 35

4.5 LBH589 induces acetylation of histones H3 and H4 in human colon tumor cells.. 36

4.6 Knockdown and overexpression of DAPK in DAPK wt cells did not influence

cell proliferation…………………………………………………………………. 37

4.7 LBH589 inhibits cell proliferation in human colon tumor cells having different

DAPK levels…………………………………………………………………….. 38

4.8 LBH589 reduces the long-term survival in DAPK wt cells having different

DAPK levels…………………………………………..………………………… 39

4.9 LBH589 induces up-regulation and activation of DAPK in human colon cancer

cells in vitro…………………………………………………………………….. 40

4.10 Role of DAPK in LBH589-induced apoptosis………………………………… 42

4.10.1 LBH589 induces cell cycle arrest in human colon tumor cells……………. 42

4.10.2 LBH589 causes formation of apoptotic bodies in a DAPK-independent

manner……………………………………………………………..…….… 43

4.10.3 LBH589 activates caspases and induces apoptosis in a DAPK-independent

manner……………………………………………………..………………. 43

4.10.4 zVAD rescued the cells from LBH589 induced apoptosis in human colon

tumor cells……………………………………………….…………………. 46

4.10.5 LBH589 also induces caspase independent cell death in human colon

tumor cells…………………………………………………………….…… 46

4.10.6 LBH589-induced apoptosis is independent of DAPK kinase activity…….. 47

4.11 Role of DAPK in LBH589-induced autophagy……………………………….. 49

4.11.1 LBH589 induces autophagy in human colon cancer cells in a DAPK

-dependent manner………………………………………………..………… 49

4.11.2 Effect of LBH589 on other autophagy markers…………………………… 51

4.11.3 Inhibition of autophagy decreased acidification of vesicular organelles in

human colon tumor cells…………………………………………………… 51

4.11.4 Inhibition of autophagy causes accumulation of LC3-II and p62 protein…. 52

4.11.5 Crosstalk between LBH589-induced apoptosis and autophagy…………… 53

4.12 DAPK up-regulation or activation is important for the pro-apoptotic effects of

LBH589 in different colorectal cancer cells…………………………………… 55

INDEX

vii

4.13 DAPK up-regulation and activation is a general phenomenon and is correlated

with the mode of cell death after treatment with HDACi……………………… 57

4.14 In vivo colon tumor xenografts………………………………………………… 59

4.14.1 LBH589 suppresses the growth of colon tumor xenografts……………….. 59

4.14.2 LBH589 up-regulates DAPK in colon tumor xenografts………………….. 60

4.14.3 LBH589 reduces proliferation in DAPK wt and DAPK shRNA xenografts.. 60

4.14.4 LBH589 does not suppress growth of HT29 colon tumor xenografts …….. 61

4.14.5 µPET imaging with an 18F-fluoroglycosylated RGD peptide ([18F]FGlc-RGD)

in HT29-xenograft mice…………………………………………………… 62

5. Discussion…………………………………………………………………………. 64

5.1 LBH589 induces the acetylation of core histones H3 and H4………………….. 65

5.2 LBH589 up-regulates tumor suppressor DAPK expression in vitro and in vivo.. 66

5.3 LBH589 reduces the proliferation in vitro and in vivo and causes G2/M cell cycle

arrest in a DAPK-independent manner…………………………………………. 67

5.4 LBH589 causes DNA damage………………………………………………….. 68

5.5 LBH589 induces apoptosis in a DAPK-independent manner………………….. 68

5.6 LBH589 induces autophagy in a DAPK-dependent manner…………………… 70

5.7 Conclusion and outlook………………………………………………………… 73

6. References………………………………………………………………………... 75

7. Curriculum Vitae……………………………………………………………..... 88

7.1 List of Original Research Publications…………………………………………. 88

7.2 Poster/Oral Presentations……………………………………………………….. 89

Abbreviations

viii

Abbreviations AA/Bis-AA Acrylamide/Bis-Acrylamide

Ac-H3 Acetyl-Histone 3

Ac-H4 Acetyl-Histone 4

Apaf-1 Apoptotic protease activating factor 1

APS Ammonium persulfate

ATCC American Type Culture Collection

BCA Bicinchoninic acid

Bcl-2 B-cell lymphoma 2

BSA Bovine serum albumin

cDNA Complementary deoxyribonucleic acid

C.I. Cell Index

CTCL Cutaneous T-cell lymphoma

CT Threshold cycle

DAPI 4’-6 Diamidino-2-phenylindole

DAPK Death-associated protein kinase

DD Death domain

DMEM Dulbecco’s modified Eagle’s medium

dNTP Deoxy nucleotide triphosphate

DNMT DNA methyltransferase

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DTT Dithiothreitol

ECL Enhanced Chemiluminescence

EDTA Ethtylenediamine-tetra-acetic acid

ER Endoplasmic reticulum

FACS Fluorescence-activated cell sorting

FBS Fetal bovine serum

FDA Food and Drug Administration

Abbreviations

ix

Fig Figure

FITC Fluorescein isothiocyanate

h Hour

HAT Histone acetyltransferases

HDAC Histone deacetylases

HDACi Histone deacetylase inhibitors

HEPES N-2-Hydroxyethylpiperazine-N’-2-Ethanesulfonic Acid

HRP Horseradish peroxidase

HT Hydroxy tamoxifen

IFN-γ Ιnterferon-γ

Ig Immunoglobulin

IRS Immunoreactive score

LC3 Microtubule-associated protein 1 light chain 3

LTR long terminal repeats

mAb Monoclonal antibody

min Minute

mRNA messenger RNA

MTT 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium-bromide

PAGE Polyacrylamide gel electrophoresis

PBS Phosphate-buffered saline

PCD Programmed cell death

PCR Polymerase chain reaction

PFA Paraformaldehyde

PI Propidium iodide

p.i. Post-injection

PMSF Phenylmethylsulfonyl fluoride

RNA Ribonucleic acid

ROIs Regions of interest

Abbreviations

x

RPMI Roswell Park Memorial Institute Medium

RT Room temperature

RT-PCR Reverse transcription-polymerase chain reaction

s Second

SAHA Suberoylanilide hydroxamic acid

SDS Sodium dodecyl sulfate

SEM Standard error of the mean

SI Staining intensity

SUV Standard uptake values

TBS Tris-buffered saline

TEMED N,N,N',N'-Tetramethylethylenediamine

TNF Tumor necrosis factor

TMAs Tissue microarrays

TOR Target of rapamycin

TSA Trichostatin A

UAS Upstream activating sequences °C Degree Celsius

kDa Kilodalton

M Molar

MBq Megabecquerels

ml Milliliter

mM Millimolar

MW Molecular weight

ng Nanogram

pmol Picomole

µg Microgram

µl Microliter

µM Micromolar

rpm Revolutions per minute

V Volts

Abstract

xi

Abstract

Histone deacetylase inhibitors (HDACi) are relatively new class of anticancer agents

displaying pleiotropic activities against various cancers. Among HDACi, panobinostat

(LBH589) is currently under extensive investigation both in vitro and in vivo as it shows

promising antitumor activity at low nanomolar concentrations. The identification and

characterization of new targets for LBH589 action would further enhance our understanding

of the molecular mechanisms involved in HDACi therapy. Death associated protein kinase

(DAPK) is a Ca2+/CaM serine/threonine protein kinase and it was identified as a positive

mediator in various cell death pathways including apoptosis and autophagy. It has been

characterised as a tumor suppressor protein whose expression is lost or reduced in several

cancers. Although LBH589-induced signaling pathways have been studied in various cancer

cell lines, the role of the tumor suppressor DAPK in LBH589-induced cytotoxicity has not

been investigated so far.

To study the role of DAPK in LBH589-induced signalling pathways in human colon cancer

cells, stable DAPK knockdown (DAPK shRNA) and DAPK overexpressing (DAPK+++) cell

lines were generated from HCT116 wildtype (wt) colon cancer cells. Cell viability was

evaluated using the crystal violet assay. Long term survival was studied by anchorage

dependent clonogenic assay and proliferation was determined by MTT assay as well as using

xCELLigence real-time cell proliferation method. Apoptosis was assessed by immunoblotting

against caspases-3, 8, 9, PARP cleavage and also by flow cytometric analysis. Autophagy was

assessed by immunoblotting and immunofluorescence against autophagy marker LC3-II,

acridine orange staining and also by p62 protein degradation. DAPK kinase activity was

determined using in vitro kinase assay. DNA-damage was evaluated by detection of pH2AX

levels by immunoblotting. To functionally differentiate between apoptosis and autophagy

corresponding inhibitors were used. The in vivo relevance of our findings was confirmed in a

colon xenograft mouse model.

We showed that the LBH589 significantly inhibited cell proliferation and reduced long-term

survival in all the three cell lines which was independent of DAPK levels. We also showed

that LBH589 up-regulated tumor suppressor DAPK both in vitro and in vivo. DAPK was also

activated after LBH589 treatment as shown by kinase assay and a remarkable decrease in the

levels of autoinhibitory phosphorylation of ser308. Moreover, LBH589 significantly

suppressed the growth of DAPK shRNA and DAPK wt colon tumor xenografts but not colon

tumor xenografts of the apoptosis resistant cell line HT29. Furthermore, LBH589 treatment of

Abstract

xii

human colon tumor cells having different DAPK levels induces apoptosis which was

independent of DAPK levels.

In parallel, LBH589 induced a DAPK-dependent autophagy as assessed by punctuate

accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62

protein. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage

of caspase 3 and PARP but didn’t rescue the cells from LBH589-induced cell death in crystal

violet staining suggesting both caspase-dependent as well as -independent apoptosis

pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-

mediated apoptosis in a DAPK-dependent manner.

In conclusion, LBH589 simultaneously induced hallmarks of apoptosis and autophagy in

colon cancer cells having different DAPK levels by up-regulating and/or activating the tumor

suppressor DAPK. For the first time we show that dephosphorylation of DAPK at ser 308 is

the most important mechanism in DAPK activation by HDACi. Autophagy induction seems

to be rather caused by DAPK protein interactions than by its catalytic activity. In autophagy

deficient cells, DAPK plays an essential role in committing cells to LBH589-induced

apoptosis. Taken together, our study identifies the tumor suppressor DAPK as a novel

molecule which plays an important role in LBH589-induced signalling pathways and further

confirms the high therapeutic potential of LBH589 in the treatment of colon cancer.

ZUSAMMENFASSUNG

xiii

ZUSAMMENFASSUNG

Histondeacetylaseinhibitoren (HDACi) sind relativ neue Anti-Tumor-Substanzen, die eine

pleiotrope Aktivität gegen verschiedene Tumorarten zeigen. Als ein HDACi wird derzeit

besonders die Wirkung von Panobinostat (LBH589) sowohl in vitro als auch in vivo intensiv

geforscht, da diese Substanz viel versprechende Anti-Tumor-Aktivität im nanomolaren

Bereich zeigt. Die Identifizierung und Charakterisierung neuer Targets für LBH589 würde

unser Verständnis von den molekularen Mechanismen, die in eine HDACi-Therapie involviert

sind, verbessern. Die DAPK (Death associated protein kinase) ist eine Ca2+/CaM

Serin/Threonin Proteinkinase und wurde als positiver Mediator in verschiedenen

Zelltodformen wie Apoptose und Autophagie beschrieben. Die DAPK wurde als ein

Tumorsuppressorprotein charakterisiert, dessen Expression in verschiedenen Krebsarten

verloren geht oder reduziert ist. Obwohl LBH589-induzierte Signalwege in verschiedenen

Krebs-Zelllinien untersucht wurden, ist die Rolle der DAPK in der LBH589-induzierten

Zytotoxizität bisher noch nicht verstanden.

Um die Bedeutung der DAPK in LBH589-induzierten Signalwegen in humanen

Dickdarmkrebszellen zu untersuchen, wurden zunächst eine stabile DAPK-Knockdown

(DAPK shRNA) und eine DAPK überexprimierende (DAPK+++) Zelllinie aus HCT116

Wildtyp (wt) Dickdarmkrebszellen entwickelt. Das Überleben der Zellen wurde mittels

Kristallviolett-Assay evaluiert. Das Langzeitüberleben der Tumorzellen wurde mit dem

Anchorage-dependent clonogenicity Assay und die Proliferation mittels MTT und der

xCELLigence real-time Methode bestimmt. Die Apoptose wurde über Immunblots gegen

Caspase -3, 8, 9, und PARP Spaltung und auch mittels Flow Cytometrie gemessen. Die

Autophagie wurde über Immunblots und Immunfluoreszenz gegen den Autophagiemarker

LC3-II, eine Acridin Orange Färbung und den Nachweis einer p62 Protein Degradation

bestimmt. Die katalytische Aktivität der DAPK wurde über einen in vitro Kinaseassay

gemessen. Die DNA-Schädigung wurde durch Bestimmung der pH2AX-Proteinmengen

mittels Western Bloting evaluiert. Um funktionell zwischen Apoptose und Autophage zu

unterscheiden, wurden entsprechende Inhibitoren verwendet. Die in vivo Relevanz unserer

Ergebnisse wurde in einem Xenotransplantations-Mausmodell bestätigt.

Wir konnten zeigen, dass LBH589 die Zellproliferation signifikant beeinträchtigt und das

Langzeit-Überleben in allen drei Zelllinien unabhängig von der DAPK-Menge reduziert. Wir

konnten nachweisen, dass LBH589 die DAPK Menge sowohl in vitro als auch in vivo erhöht.

Zusätzlich wird die DAPK nach LBH589-Behandlung aktiviert, was über die Kinase-

Untersuchung und über den Nachweis einer bemerkenswerten Reduktion der Menge an auto-

ZUSAMMENFASSUNG

xiv

inhibitorischer Phosphorylierung von ser308 gezeigt werden konnte. In vivo unterdrückt

LBH589 signifikant das Wachstum von DAPK shRNA und DAPK wt Tumorzellen im

Xenotransplantationsmodell während HT29 Tumorzellen eher resistent gegen LBH589 sind.

Überdies induziert eine LBH589-Behandlung von humanen Kolontumorzellen die Apoptose

unabhängig von der Menge und Aktivierung der DAPK. Parallel ist die durch LBH589

induzierte Autophagie DAPK-abhängig. Wir beobachten eine punktuelle Akkumulation von

LC3-II in der Immunfluoreszenz, eine vermehrte Bildung von sauren Organellen sowie eine

Degradation des p62 Proteins. Der Caspaseinhibitor zVAD erhöht die Bildung von

Autophagosomen, reduziert die Spaltung von Caspase 3 und PARP, schützt die Zellen aber

nicht vor dem LBH589-induzierten Zelltod in der Kristallviolett-Färbung, was auf einen

Caspase-abhängigen und auch auf einen Caspase-unabhängigen Apoptose-Weg hindeutet.

Eine Vorbehandlung mit dem Autophagie-Inhibitor Bafilomycin A1 führt zu einer DAPK-

abhängigen Caspase 3-vermittelten Apoptose.

Zusammenfassend lässt sich sagen, dass LBH589 über eine Hochregulation und/oder

Aktivierung des Tumorsupressorproteins DAPK simultan Merkmale von Apoptose und

Autophagie in Dickdarmkrebszellen mit unterschiedlichen DAPK-Mengen induziert. Erstmals

konnten wir zeigen, dass die Dephosphorylierung von DAPK am ser 308 der wichtigste

Mechanismus bei der DAPK-Aktivierung durch einen HDACi ist. Die Autophagieinduktion

scheint eher durch Protein-Interaktionen der DAPK als durch ihre katalytische Aktivität

verursacht zu werden. In durch Autophagie beeinträchtigten Zellen spielt DAPK eine

wesentliche Rolle bei der LBH589-induzierten Apoptose.

Wir haben in unserer Studie den Tumorsuppressor DAPK als neues Molekül im LBH589-

induzierten Signalweg identifiziert und überdies das hohe therapeutische Potential von

LBH589 bei der Behandlung von Dickdarmkrebs bestätigt.

Introduction

1

1. Introduction

1.1 Chromatin structure

The eukaryotic genome is packed into chromatin. The nucleosome is the fundamental unit of

chromatin. Nucleosomes are made up of approximately 146 base pairs of DNA wrapped

around a histone octamer containing two copies each of histones H2A, H2B, H3, and H4

(Horn PJ et al., 2002). Chromatin structure plays an important role in the regulation of gene

expression. Chromatin is a complex structure consisting of DNA, RNA, histones, non-histone

proteins and other chromosomal proteins. Based on the density of the staining of the nucleic

acids chromatin was divided into two domains, euchromatin and heterochromatin.

Euchromatin is transcriptionally active whereas heterochromatin is transcriptionally inactive

(Trojer P et al., 2007; Bannister AJ et al., 2011). Transcriptional regulation in eukaryotes

occurs within a chromatin and is influenced by posttranslational modifications such as

acetylation, phosphorylation, methylation, ubiquitylation, sumolytion, carbonylation and

glycosylation (de Ruijter AJ et al., 2003; Trojer P et al., 2007).

DNA methylation or histone deacetylation closes chromatin whereas histone acetylation or

demethylation of DNA relaxes chromatin leading to transcriptional repression and

transcriptional activation respectively (Figure 1, adopted from Johnstone RW et al., 2002).

Fig. 1. Closed and Open structures of chromatin. (A) Chromatin structure is closed by DNA methylation or histone deacetylation leading to transcriptional repression. (B) The relaxed structure of chromation due to histone acetylation or demethylation of DNA allows transcriptional activation of genes (adopted from Johnstone RW et al., 2002).

Introduction

2

1.2 Epigenetics and Cancer

Epigenetics is defined as heritable changes in gene expression without a change in the DNA

sequence (Egger G et al., 2004; Rodríguez-Paredes M et al., 2011). Epigenetic modifications

such as acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation are

able to influence gene expression without changing the DNA sequence (Gibney ER et al.,

2010; Reik W et al., 2007). Epigenetic modifications seem to play an important role in the

pathogenesis of several diseases and in particular cancer. Furthermore, these modifications

offer hope and the promise to be used as novel biomarkers for early cancer detection,

prediction and prognosis (Hamilton JP et al., 2011). The use of compounds directed towards

epigenetic changes such as histone deacetylase inhibitors (HDACi) and DNA

methyltransferase (DNMT) inhibitors has become a hot topic in the cancer research (Gravina

GL et al., 2010). Several HDACi and DNMT inhibitors are actively investigated both in vitro

and in vivo. These drugs/inhibitors could restore the normal epigenetic landscape in cancer

cells by inhibiting enzymes of the epigenetic machineries. Indeed, to date two DNMT

inhibitors, vidaza and decitabine and also two HDAC inhibitors, vorinostat and romidepsin

have been approved by the US Food and Drug Administration (FDA) for cancer treatment

(Rodríguez-Paredes M et al., 2011).

1.3 Histone Deacetylases (HDACs)

Histone deacetylases (HDACs) are enzymes which catalyze the removal of the acetyl groups

from the histones and cause transcriptional repression (Marks PA and Xu WS et al., 2009).

HDACs are involved in various important cell regulatory pathways and play a role in

regulation of gene expression, cell proliferation, cell migration, cell death, and angiogenesis

(Marks PA and Xu WS et al., 2009). HDACs are often overexpressed in various types of

cancers, and their overexpression is well correlated with poor prognosis (Weichert W et al.,

2008). In addition to deacetylation of histones, HDAC can also regulate non-histone proteins

including p53, E2F, a-tubulin and MyoD, demonstrating the complex function of HDACs

(Hubbert C et al., 2002; Juan LJ et al., 2000).

Based on the phylogenetic analyses and sequence homology, at least 18 human HDACs were

identified and were divided into four classes in eukaryotic cells (Table 1; Balasubramanian S

et al., 2009; Marks PA and Xu WS et al., 2009; Dokmanovic M et al., 2007).

Introduction

3

Table 1: Classification of human HDACs

1.4 Histone acetyl transferases (HAT)

Histone acetyl transferases (HATs) are enzymes that acetylate lysine residues present in the

histone tails by transferring an acetyl group from an acetyl CoA to form ε-N-acetyl lysine.

Histone acetylation occurs post-translationally and is a reversible process (Marmorstein R and

Roth SY et al., 2001; Kuo MH and Allis CD et al., 1998). Acetylation of histones is well

correlated with the relaxed chromatin state which in turn favours accessibility of

transcriptional factors to DNA elements leading to an increase in gene expression (Emanuele

S et al., 2008). Based on the subcellular localization and function HATs can be divided into

two types, Type A and Type B. Type A HATs are located in the nucleus and functioning as

transcriptional co-activators whereas Type B HATs are located in the cytoplasm and are

responsible for chromatin synthesis and assembly (Brownell JE and Allis CD et al., 1996;

Emanuele S et al., 2008). Kuo MH and Allis CD et al. (1998) reviewed that several

transcriptional regulators have been found to possess intrinsic HAT activity including Gcn5p

and homologs, PCAF, p300/CBP, TAFII250 and homologs, and SRC-1 and ACTR. In

addition, only one type B HAT, yeast Hat1p, has been identified and characterized (Kuo MH

and Allis CD et al., 1998).

1.5 DNA Methylation

The DNA Methylation is involved in regulating several cellular processes including

embryonic development, transcription, chromatin structure, X chromosome inactivation,

genomic imprinting, and chromosome stability (Robertson KD et al., 2005). DNA

Methylation is a crucial epigenetic modification usually taking place at the 5′ position of the

cytosine ring within CpG dinucleotides. In human cancer several key genes involved in the

regulation of DNA repair, apoptosis, cell cycle regulation, cell proliferation, and metastasis

HDACs Localization Co-factor Class I

HDACs 1,2,3, and 8 Mainly nucleus Zn+2-dependent

Class IIa HDACs 4,5,7, and 9

Nucleus/Cytoplasm Zn+2-dependent

Class IIb HDACs 6 and 10 Mainly cytoplasm Zn+2-dependent

Class III Sirtuins 1-7 NAD+-dependent

Class IV HDAC 11 Nucleus/Cytoplasm Zn+2-dependent

Introduction

4

are aberrantly methylated resulting in transcriptional repression (Esteller M et al., 2001;

Michie AM et al., 2010). Hypermethylation of CpG islands in the promoter region of gene

results in transcriptional silencing and subsequent loss of protein expression (Hamilton JP et

al., 2011). Indeed, DAPK promoter regions are hypermethylated in several cancer cells

resulting in decreased expression of DAPK (Michie AM et al., 2010; Satoh A et al., 2002).

1.6 DNA methyl transferases (DNMTs)

Methylation is controlled by a family of enzymes known as DNA methyltransferases

(DNMTs). In mammals, to date four DNMTs have been identified: DNMT1, DNMT2,

DNMT3a and DNMT3b (Weber and Schübeler et al., 2007). Blocking DNA methylation by

inhibiting DNMTs led to demethylation of the CpG islands and/or reexpression/reactivation

of tumor suppressor genes, ultimately inhibiting tumor growth. There are several DNA de-

methylating compounds that are approved or under investigation, including 5-azacytidine

(Vidaza), 5-aza-2’-deoxycytidine (decitabine), zebularine, procainamide, and procaine

(Roberts LR and Gores GJ et al., 2005; Hamilton JP et al., 2011).

1.7 Histone deacetylase inhibitors (HDACi) as anti-cancer agents

1.7.1 Histone deacetylase inhibitors (HDACi)

HDACi are emerging as potent anticancer agents for the treatment of solid and

haematological malignancies (Ellis L and Pili R et al., 2010). HDACi inhibit the enzyme

activity of histone deacetylases (HDACs), which remove acetyl groups from both histone and

non-histone cellular proteins. The anti-tumor effects of HDAC inhibition have been explored

in a variety of cancer cell lines, in vivo tumor models, and have been observed in patients

with hematologic and solid tumors (Bolden JE et al., 2006; Shao W et al., 2010; Yoshida M et

al., 1990).

HDACi are a relatively new group of structurally divergent anticancer agents of which two

drugs (Vorinostat and Romidepsin) have been already approved for the therapeutic treatment

and several other drugs are currently in various stages of clinical trials. HDACi are currently

classified according to their chemical structure and can be divided in to four major classes

including hydroxamates, cyclic peptides, aliphatic acids, and benzamides (Table 2; Rikiishi H

et al., 2011; Dickinson M et al., 2010; Dokmanovic M et al., 2007). In general, HDACi are

characterized as class I specific or pan-deacetylase (pan-DAC) inhibitors. The pan-DAC

inhibitors include LBH589 (panobinostat), SAHA (Vorinostat) and TSA (Trichostatin A)

inhibiting classes I, II, and IV HDACs whereas valproic acid and butyrate inhibit Class I, and

Introduction

5

IIa HDACs. Romidepsin (depsipeptide, FK228), Entinostat (MS275, SNDX 275) and

Mocetinostat (MGCD0103) are considered to inhibit Class I HDACs. (Table 2; Figure 2;

Dickinson M et al., 2010; Balasubramanian S et al., 2009).

Table 2: Histone deacetylase inhibitors which are in clinical trials or already approved for the treatment (selected compounds). *Approved by the FDA for the treatment of cutaneous T cell lymphoma.

Most of the HDACi which are currently in clinical trials inhibit multiple classes of HDACs to

varying degrees in three of these classes: Class I, II and IV (Figure 2, Dickinson M et al.,

2010; Balasubramanian S et al., 2009; Marks PA et al., 2004).

Fig. 2. Chemical structures of HDAC inhibitors

HDACi class Compounds HDAC targets Hydroxamates LBH589 (Panobinostat) Classes I, II, and IV

SAHA (Vorinostat)* Classes I, II, and IV Trichostatin A (TSA) Classes I, II, and IV

Cyclic peptide Romidepsin (depsipeptide, FK228)* HDAC 1, 2, 4, and 6

Aliphatic Acids Valproic Acid Classes I, and IIa Butyrate Classes I, and IIa

Benzamides Entinostat (MS275, SNDX 275) HDAC1, 2, 3, and 9 Mocetinostat (MGCD0103) HDAC1, 2, 3, and 11

Introduction

6

The mechanism of action of HDACi is a complex process and mediates cell death through

various signalling pathways. HDAC inhibitors induce their effects in various cell lines and

animal models including histone acetylation, cell cycle arrest, growth arrest (LaBonte MJ et

al., 2009; Di Fazio P et al., 2010; Pettazzoni P et al., 2011), autophagic cell death pathway

(Shao Y et al., 2004), cell death by activating the intrinsic apoptotic pathway (Shao W et al.,

2010), activating the extrinsic apoptotic pathway (Martín-Sánchez E et al., 2011),

simultaneous activation of the intrinsic and extrinsic apoptotic pathways (Rosato RR et al.,

2003), polyploidy, senescence (XU WS et al., 2005; Xu WS et al., 2007), reactive oxygen

species-induced cell death, induction of p21 (Ruefli AA et al., 2001; Rosato RR et al., 2003),

activating tumor suppressor genes such as DAPK (WU J et al., 2010), and inhibiting tumor

growth and proliferation in in vivo models (Shao W et al., 2010). Other drug targets have been

described to be responsible for their anticancer effects which include down-regulation of

NFkB and IRAK1 (LaBonte MJ et al., 2011), proteasomal degradation of the damage sensor

CHEK1 (Brazelle W et al., 2010), induction of the stress protein CHOP (Di Fazio P et al.,

2010; Rao R et al., 2010), or degradation of c-FLIP a negative regulator of death-receptor

induced cell death (Kauh J et al., 2010). The induction of a particular response in transformed

cells seems to be dependent on the type of tumor cell lines, the type of HDACi used, and the

concentration and time of exposure of the HDACi to the cells (Dokmanovic M et al., 2007).

1.7.2 LBH589 (Panobinostat)

The panHDACi LBH589 is emerging as one of the potent anti-cancer therapeutic agent for

various cancers, including colon cancer. LBH589 is a novel HDACi that potently inhibits all

classes of HDACs including I, II and IV at nanomolar concentrations (Atadja P et al., 2009;

Mariadason JM, 2008).

It is known that the LBH589 induces histone acetylation, cell cycle arrest, and apoptosis in a

variety of cancer cell lines (LaBonte MJ et al., 2009; Di Fazio P et al., 2010; Pettazzoni P et

al., 2011). It has the potential to sensitize colon cancer cells to TRAIL-mediated apoptosis

(Lee SC et al., 2011). Ellis et al. in 2009 reported that LBH589 induces autophagy in cells

lacking effective apoptosis machinery. Moreover, Fazzone W et al. (2009), showed a

synergistic interaction between HDACi and 5-FU in colon cancer cells. Different drug targets

have been described to be responsible for its anticancer effects which include down-regulation

of NFkB and IRAK1 (LaBonte MJ et al., 2011), proteasomal degradation of the damage

sensor CHEK1 (Brazelle W et al., 2010), induction of the stress protein CHOP (Di Fazio P et

Introduction

7

al., 2010; Rao R et al., 2010), or degradation of c-FLIP a negative regulator of death-receptor

induced cell death (Kauh J et al., 2010).

In vitro and in vivo studies of Shao W et al. in 2010 demonstrated that LBH589 induced cell

death in cutaneous T-cell lymphoma (CTCL). CTCL cell lines and tumor regression in CTCL

mouse xenografts. In fact, the potent anti-cancer effects of LBH589 in cellular as well as

tumor models of CTCL were corroborating with the activity of LBH589 observed in Phase I

and II clinical trials (Ellis L et al., 2008; Duvic M et al., 2012).

Recently combination of various drugs with HDACi has gained attention in various cancers.

Indeed, Ocio EM et al. (2010) performed experiments in mouse models with triple

combinations of LBH589 to dexamethasone and either bortezomib or lenalidomide. They

found that triple combinations of LBH589 have superior activity compared to single agents or

double combinations in a murine model of multiple myeloma (Ocio EM et al., 2010).

1.8 Death Associated Protein Kinase (DAPK)

1.8.1 DAPK structure

Death associated protein kinase (DAPK, also called as DAPK 1) is a Ca2+/CaM

serine/threonine 160 kDa large protein kinase. The DAPK structure contains various

functional domains, including a kinase domain, a CaM regulatory domain, eight consecutive

ankyrin repeats (AR), two P-loops, a cytoskeletal binding domain and a death domain (DD;

Figure 3). Among multiple structural domains of DAPK, the kinase domain and the death

domain are the most important regions for its pro-apoptotic function (Diess LP et al., 1995;

Cohen O et al., 1997; Lin Y et al., 2010).

Fig. 3. Schematic diagram of multi-domain structure of DAPK. DAPK consists of multi-domain structure, comprising a kinase domain, a CaM regulatory domain, eight consecutive ankyrin repeats, two P-loops, a cytoskeletal binding domain and a death domain. Numbers refer to amino acids (Adopted from Lin Y et al., 2010). To date, five members of the DAP kinase family of related death kinases have been identified.

DAPK is one of the members of a family of related death kinases and shares sequence

homology with other group of kinases that is restricted to the N-terminal kinase domain. The

other four kinases are death associated protein kinase related protein 1/ death associated

Introduction

8

protein kinase 2 (DRP-1/DAPK2), dap like kinase/zipper interacting protein kinase (DlK/ZIP

kinase), DAP kinase related apoptosis-inducing kinases 1 and 2 (DRAK 1 and DRAK 2; Inbal

B et al., 2000; Kawai T et al., 1998; Kogel D et al., 1998). The DAP kinase family members

have distinct subcellular localization (Table 3, adopted from Schneider-stock R, 2005).

DRAK1, DRAK2, Zip kinase are nuclear proteins, DAPK1 is associated with the actin

filaments of the cytoskeleton whereas DRP-1/DAPK2 is localized in the cytoplasm (Shani G

et al., 2001; Schneider-stock R et al., 2005). Several lines of evidence indicate that all DAP

kinase family members are ubiquitously expressed and involved in different cell death

pathways (Kögel D et al., 2001).

Table 3:

1.8.2 DAPK function

DAPK is a novel prototype of death-associated serine/threonine protein kinases with multiple

structural domains and plays a role in both proapoptotic and/or antiapoptotic signal

transduction pathways (Cohen O et al., 1997; Deiss LP et al., 1995; Lin Y et al., 2010). The

kinase activity of DAPK has been shown to be crucial for triggering a number of cell death

pathways (Jin Y et al., 2006). Indeed, DAPK has been shown to be involved in cell death

induced by various cytokines and others molecules including TNF-α and FAS (Cohen O et

al., 1999), IFN-γ (Deiss LP et al., 1995), TGF-β (Jang CW et al., 2002), Trichostaton A (Wu J

et al., 2010), sodium butyrate (Shin H et al., 2012; Zhang HT et al., 2007), LBH589

(Gandesiri M et al., 2012) and sodium selenite (Jiang Q et al., 2012). It also mediates the

induction of autophagy in response to oxidative damage (Eisenberg-Lerner A et al., 2012).

The DAPK was characterized as a tumor suppressor because the expression of DAPK is lost

in many human cancer cell lines and tumors and this loss of expression correlates with the

Introduction

9

recurrence and/or metastasis incidence of several human cancers (Raveh and Kimchi et al.,

2001; Bialik and Kimchi et al., 2004). Its is evident from several studies that the loss of

DAPK expression is mainly caused by DNA methylation (Kissil JL et al., 1997) and/or

deregulation of histone acetylation (Satoh A et al., 2002; Toyooka S et al., 2003).

Interestingly, DAPK inactivation is associated with chemotherapy resistance (Eisenberg-

Lerner A and Kimchi A et al., 2012; Ogawa T et al., 2012)

DAPK activity is known to be negatively regulated by auto-phosphorylation of Serine-308

(Jin Y et al., 2006; Shamloo M et al., 2005). However some of the biological effects of DAPK

are likely mediated through protein recruitment in a phosphorylation independent manner (Lin

Y et al., 2010; Chuang YT et al., 2011). There are several well studied phosphorylation sites

on DAPK protein, including, Ser308 autophosphorylation site (Shohat G et al., 2002), ERK-

phosphorylation of DAPK at Ser735 (Chen CH et al., 2005), Src phosphorylation of DAPK at

Y491/492 (Wang WJ et al., 2007), and the phosphorylation at Ser289 by RSK (Anjum R et

al., 2005).

1.9 Programmed cell death pathways

Programmed cell death (PCD) is a basic biological process in multicellular organisms, and

has many important functions that include maintenance of tissue homeostasis, morphogenesis,

and elimination of damaged, harmful or abnormal cells (Sun Y and Peng ZL et al., 2009).

Dysfunction of PCD leads to several diseases in humans, including autoimmunity,

neurodegenerative diseases and importantly to a cancer (Okada H et al., 2004). Based on the

PCD criteria such as morphological alterations, initiating a death signal, and the activation of

caspases, the PCD has been subdivided into three categories: 1) Apoptosis or Type-I cell

death, 2) Autophagy or Type-II cell death, and 3) Necrosis or Type-III cell death (Sun Y and

Peng ZL et al., 2009; Gozuacik D and Kimchi A et al., 2007). The type of PCD occurs

majorly depending on the stimulus and also the cellular context. Under some circumstances,

cells may switch from apoptosis to necrosis or autophagy, and vice versa, inferring cross-talk

between these pathways (Mevorach D et al., 2010; Maiuri MC et al., 2007).

1.9.1 Apoptosis

Apoptosis is one of the forms of programmed cell death (PCD) that may occur in multicellular

organisms to maintain cellular homeostasis. The pathogenesis of many diseases including

cancer is attributed to its malfunctioning (Norbury CJ and Hickson ID et al., 2001; Elmore S

Introduction

10

et al., 2007). The term apoptosis was first described by Kerr et al in 1972 to differentiate a

morphologically distinct form of cell death (Kerr JF et al., 1972).

Apoptosis (Type-I PCD) is the best characterized mode of PCD from necrosis and is executed

by caspase proteases which become activated by either the death receptor or the mitochondrial

pathways. Apoptotic cell death is characterized by chromatin condensation, cell shrinkage,

nuclear fragmentation, and blebbing (Duprez L et al., 2009; Portt L et al., 2010). The process

of apoptosis is controlled by a diverse range of cellular signals which may originate either

extracellularly or intracellularly. Apoptosis can be initiated by two main pathways, 1)

Extrinsic or death receptor pathway, and 2) intrinsic or mitochondrial pathway. Both of these

pathways activate caspases (Cysteine aspartyl-specific caspases) leading to the biochemical

and morphological changes that are characteristic of apoptosis (Igney FH and Krammer PH et

al., 2002). Numerous techniques are available for the detection and quantitation of apoptosis.

Based on the mode of detection, apoptosis assays can be classified into six major categories:

1) Cytomorphological alterations, 2) DNA fragmentation, 3) Detection of caspases, cleaved

substrates, regulators and inhibitors, 4 ) Membrane alterations, 5) Detection of apoptosis in

whole mounts, and 6) Mitochondrial assays (Elmore S et al., 2007). Apoptosis can be

regulated by several pro-apoptotic proteins such as Bid, Bax, Bak, Bim, Bad, HRK, Bmf,

Puma, and DAPK and also by anti-apoptotic proteins such as Bcl-xL, Mcl-1, and Bcl-2 (Fulda

S et al., 2008; Cory S et al., 2003). DAPK participates in both intrinsic and extrinsic apoptotic

pathways (Kögel D et al., 2001). The kinase activity of DAPK has been shown to be crucial

for triggering a number of cell death pathways (Jin Y et al., 2006). It is involved in

endoplasmic reticulum stress-induced autophagy by suppressing integrin activity and

disrupting matrix survival (Gozuacik D et al., 2008; Wang WJ et al., 2002). In several cancer

models it has been reported that HDACi have been shown to activate either intrinsic or

extrinsic apoptotic pathway or both of these apoptotic pathways simultaneously (Rosato RR et

al., 2003).

1.9.2 Autophagy

Autophagy is an evolutionally conserved catabolic process in which cellular organelles such

as mitochondria, ribosomes; the endoplasmic reticulum and bulk proteins are degraded

through the lysosomal system (Klionsky DJ, 2007; Levine B and Kroemer G et al., 2008).

Based on the mechanisms used for the delivery of cargo to lysosomes, at least three different

forms of autophagy have been described in mammalian cells, 1) macroautophagy, 2)

microautophagy, and 3) chaperone mediated autophagy (CMA) (Klionsky DJ and Emr SD et

Introduction

11

al., 2000; Mizushima N et al., 2005). Macroautophagy (hereafter referred to as autophagy) is

another form of programmed cell death (Type II cell death) which is morphologically and

biochemically different from apoptosis (Type I cell death) (Sperandio S et al., 2000).

Autophagy is a dynamic process containing several stages that include induction as well as

formation of the autophagosome, fusion with lysosomes, and finally degradation and

recycling (Figure 4; Elmore S et al., 2007; Kondo Y et al., 2005).

Fig. 4. Schematic overview of autophagy pathway. Autophagy is a multi-step process starts with isolation of double-membrane structures followed by elongation and maturation. LC3 protein is recruited to the membrane leading to the formation of autophagosomes. The autophagosomes then fuse with lysosomes to form autolysomes and the lysosomal hydrolases degrade the sequestered contents for recycling (Adopted from Kondo Y et al., 2005). The process of autophagy begins with formation of the double-membrane layered vesicles

known as autophagosomes which then fuse with lysosomes to form autolysosomes where the

engulfed contents are degraded by lysosomal hydrolases and are recycled (Meijer AJ and

Codogno P et al., 2004; Kim J and Klionsky DJ et al., 2000; Kondo Y et al., 2005).

Introduction

12

Microtubule-associated protein1 light chain 3 (LC3) is commonly used to monitor autophagy.

LC3-II has been used as a marker of autophagy, because it is present on isolated membranes

and autophagosomes and the amount of LC3-II protein clearly correlates with the number of

autophagosomes (Klionsky DJ et al., 2008; Mizushima N et al., 2007). Furthermore,

autophagic flux can be measured either by using Bafilomycin A1, which inhibits

autophagosome-lysosome fusion or by p62 (SQSTM1/ sequestosome 1) protein degradation

(Klionsky DJ et al., 2008; Komatsu M et al., 2007; Bjorkoy G et al., 2005; Yamamoto A et

al., 1998; Mizushima N et al., 2005).

Autophagy occurs virtually in all cells at low basal levels to maintain cellular homeostasis

(Dalby KN et al., 2010; Levine B and Kroemer G et al., 2008). The role of autophagy is to

perform routine housekeeping functions like the elimination of damaged or long lived

organelles, defective proteins, and the removal of intracellular pathogens (Mizushima N et al.,

2008). Activation of the autophagy pathway is necessary for multiple cellular functions,

including survival during starvation, immunity and development (Mizushima N et al., 2008;

Kondo Y et al., 2006). Dysfunction of autophagy is associated with different pathologies,

including cancer, aging, neurodegeneration, heart diseases and microbial infection

(Mizushima N et al., 2008).

The role of autophagy in cellular death pathways is not clearly understood. Moreover,

whether autophagy causes death or protects cells from death is controversial. There are

several key regulators of autophagy that include, the target of rapamycin (TOR), the

eukaryotic initiation factor 2α (eIF2α), the ER-membrane-associated protein, the stress-

activated kinase c-Jun-N-terminal kinase, Erk1/2, ceramide, calcium, BH3-only proteins, and

also tumor suppressors such as DAPK, Beclin 1, PTEN and p53, AMP-activated protein

kinase (AMPK) (Criollo A et al., 2007; Maiuri MC et al., 2007; Meijer AJ and Codogno P et

al., 2006; Rubinsztein DC et al., 2007).

With regard to the close relationship between apoptosis and autophagy, the role of DAPK in

the regulation of these two pathways is particularly interesting. Recently, it has been reported

that DAPK modulates both apoptotic and autophagic programmed cell death in various cancer

cell lines (Cohen O et al., 1997; Wu J et al., 2010; Gozuacik D et al, 2008; Kang C et al,

2010). Inbal B et al. (2002) showed that both DAPK and DRP-1 kinases have a direct

involvement in autophagy and membrane blebbing and further reported that these events

occur in a caspase-independent manner. Some of the other compounds such as sodium

selenite induce the activation of DAPK via protein phosphatase 2A which in turn promotes

autophagy in human leukemia HL60 cells (Jiang Q et al., 2012). It has been demonstrated that

Introduction

13

both vorinostat and butyrate induce autophagic cell death in addition to apoptosis in HeLa

cells (Shao Y et al., 2004). In our own study, we have demonstrated that histone deacetylase

inhibitor LBH589 induces autophagy in human colon tumor cells in a DAPK-dependent

manner (Gandesiri M et al., 2012).

Aims

14

2. Aims of the work

Histone deacetylase inhibitors (HDACi) have recently emerged as potent anticancer agents

against solid and haematological cancers, including colon cancer. LBH589 is currently under

investigation in several preclinical studies and shows promising antitumor activity at low

nanomolar concentrations (Neri P et al., 2012). Its multi-target property appears to be very

attractive. The identification and characterization of new targets for LBH589 action would

further enhance our understanding of the molecular mechanisms involved in HDACi therapy.

Death associated protein kinase (DAPK) has been shown to be a key molecule in various

types of cell death pathways including apoptosis and autophagy. It has been characterized as a

tumor suppressor protein whose expression is greatly reduced and/or lost in a significant

number of human malignancies, including colon cancer. DAPK plays a role in many different

cell death pathways. A direct involvement of DAPK in HDACi-induced cell death was not

studied yet, and questions such as whether DAPK is being activated in this cellular setting

were not addressed as well.

The objective of this dissertation was to study the role of DAPK in LBH589-induced

functions such as apoptosis, autophagy, proliferation, mRNA expression, long-term survival,

and DNA damage in colon cancer cells having different expression levels of DAPK.

The specific aims were:

� To investigate if LBH589 induces up-regulation of tumor suppressor protein DAPK in

human colon cancer cells in vitro and in vivo

� To study the long-term growth inhibitory effect of LBH589 on the colony formation

ability

� To evaluate the effect of LBH589 on cellular viability

� To examine potential DAPK-dependent or–independent pro-apoptotic effects of

LBH589

� To investigate the role of DAPK kinase activity in LBH589-induced death signalling

pathways

� To study if LBH589-induced autophagy is DAPK-dependent or –independent

� To study the influence of apoptosis and autophagy inhibitors on colon cancer cells and

also to assess the existence of cross-talk between LBH589-induced apoptosis and

autophagy

Aims

15

� To investigate if DAPK up-regulation and/or activation is important for the pro-

apoptotic effects of LBH589 in different colorectal cancer cells-HT29 and DLD1

� To assess DAPK up-regulation and activation is a general phenomenon and whether it

is correlated with the mode of cell death after treatment with other HDACi

� To evaluate the anti-tumor activity of LBH589 in colon tumor xenografts

Materials and Methods

16

3. Materials and Methods

3.1 Materials

3.1.1 Chemicals and Reagents

Chemicals Source

Agarose Biozym Scientific GmbH, Hessisch

Oldendorf, Germany

Acrlyamide/Bisacrylamide solution Carl Roth GmbH & Co, Karlsruhe,

Germany

Ammoniumpersulfate (APS) Carl Roth GmbH & Co, Karlsruhe,

Germany

Biotinylated Protein Ladder Cell Signaling, Danvers, MA, USA

Bromophenolblue Serva, Heidelberg, Germany

Bicinchoninic acid-Assay (BCA) Pierce, Rockford, IL, USA

Bovine serum albumin (BSA) Carl Roth GmbH & Co, Karlsruhe,

Germany

4',6-diamino-2-phenylindole (DAPI) Invitrogen-Life Technologies, Darmstadt,

Germany

Chemiluminiscence reagent HRP substrate Millipore Corporation, Schwalbach,

Germany

Dimethyl sulfoxide (DMSO) Pan-Biotech GmbH, Aidenbach, Germany

Di-sodium-hydrogenphosphate Carl Roth GmbH & Co, Karlsruhe,

Germany

DNA standards (1 kb, 100 bp) Invitrogen, Karlsruhe, Germany

Ethanol Carl Roth GmbH & Co, Karlsruhe,

Germany

Ethidiumbromide Carl Roth GmbH & Co, Karlsruhe,

Germany

Ethylene-diamine-tetra-acetic acid (EDTA) Sigma-Aldrich GmbH, München,

Germany

Glycine Carl Roth GmbH & Co, Karlsruhe,

Germany

HEPES Sigma-Aldrich GmbH, München,

Germany


17

Hydrogen chloride (HCl) Merck, Darmstadt, Germany

Methanol Carl Roth GmbH & Co, Karlsruhe,

Germany

Milk powder (Blotting grade) Carl Roth GmbH & Co, Karlsruhe,

Germany

Nonidet P-40 Roche Diagnostics GmbH, Germany

Phenylmethylsulfonyl fluoride (PMSF) Carl Roth GmbH & Co, Karlsruhe,

Germany

Pre-stained Protein Ladder Cell Signaling, Danvers, MA, USA

Ponceau S Sigma-Aldrich GmbH, München,

Germany

Potassium chloride (KCl) Carl Roth GmbH & Co, Karlsruhe,

Germany

Protease inhibitor cocktail Set III Calbiochem, Darmstadt, Germany

Restore TM Western blot Stripping buffer Thermo Scientific, Rockford, IL, USA

Sodium chloride (NaCl) Carl Roth GmbH & Co, Karlsruhe,

Germany

Sodium docecyl sulfate (SDS) Carl Roth GmbH & Co, Karlsruhe,

Germany

Sodium phosphate (Na3PO4) Carl Roth GmbH & Co, Karlsruhe,

Germany

N,N,N’,N’-tetramethylethyldiamine (TEMED) Sigma-Aldrich GmbH, München,

Germany

Tris Carl Roth GmbH & Co, Karlsruhe,

Germany

Triton-X100 Sigma-Aldrich GmbH, München,

Germany

Tween-20 Merck, Darmstadt, Germany

All other chemicals used in this work were obtained with the best analytical quality by the

following companies: Boehringer (Mannheim), Invitrogen (Karlsruhe), Merck (Darmstadt),

Sigma (München), and Carl Roth GmbH & Co (Karlsruhe). All chemicals were stored

according to the manufacturer’s instructions.


18

3.1.2 Cell culture

Ingredients Source

RPMI 1640 PAA Laboratories, Pasching, Astria

FCS PAN Biotech, Aidenbach, Germany

Pencillin PAN Biotech, Aidenbach, Germany

Streptomycin PAN Biotech, Aidenbach, Germany

Trypsin-EDTA PAN Biotech, Aidenbach, Germany

3.1.3 Cell lines

Tumor cell lines Source

HCT116 wt ATCC, Manassas, VA, USA

DAPK shRNA Generated in our lab by Gandesiri M

DAPK +++ Generated in our lab by Ivanovska J

HT29 ATCC, Manassas, VA, USA

DLD-1 ATCC, Manassas, VA, USA

3.1.4 Buffers and Solutions

Protein lysis buffer

4 M Urea

0.5 % SDS

62.5 mM Tris

pH adjusted to 6.8

PMSF and Protease inhibitors were added freshly before lysing the cell pellets

RIPA lysis buffer

150mM NaCl

0.5% Desoxycholat,

0.1% SDS

1% NP40

50mM Tris

pH adjusted to 8

PMSF and Protease inhibitors were added freshly before cell lysis


19

Loading buffer (2X)

4 M Urea

2% SDS

62.5 mM Tris

Loading buffer (5X)

4 M Urea

10% SDS

62.5 mM Tris

20 % DTT

Tris buffered saline (TBS)

20 mM Tris

500 mM NaCl

pH adjusted to 7.4

TBS-Tween (TBS-T)

TBS-1X

0.05% Tween-20

Stacking gel buffer

1.875 M Tris

0.5% SDS

pH adjusted to 6.8

Separating gel buffer

1.875 M Tris

1% SDS

pH adjusted to 8.8

SDS- Electrophoresis buffer (5X)

1.92 M Glycine

230 mM Tris

1% SDS


20

pH adjusted to 8.3

Blotting buffer

26.4 mM Tris

192 mM Glycine

16% Methanol were added freshly before performing blotting

Milk blocking solution

5% nonfat dry milk dissolved in TBS-T buffer

Bovine serum albumin (BSA) blocking solution

5% BSA dissolved in TBS-T buffer

Ponceau S staining solution (100 ml)

0.5 g Ponceau S

1 ml glacial acetic acid

Freezing medium

80 % RPMI complete medium

10% FBS

10% DMSO

3.1.5 Antibodies

Antibodies Source

Ac-H3 Active Motif, Rixensart, Belgium

Ac-H4 Active motif, Rixensart, Belgium

DAPK BD Transduction Laboratories, NY, USA

pDAPKSer308 Sigma-Aldrich, Missouri, USA

LC3 Nanotools, Teningen, Germany

pH2AX Millipore corporation, Billerica, USA

Beclin 1 Cell Signaling, Danvers, MA, USA

Atg7 Cell Signaling, Danvers, MA, USA

p62 Cell Signaling, Danvers, MA, USA

Caspase 3 Cell Signaling, Danvers, MA, USA


21



PARP Cell Signaling, Danvers, MA, USA

Goat anti-mouse Thermo Scientific, Rockford, IL, USA

Goat anti-rabbit Thermo Scientific, Rockford, IL, USA

β-actin Sigma-Aldrich, Missouri, USA

GAPDH Abnova, Walnut, CA, USA

3.1.6 Drugs and Chemicals

Drugs Source

Acridine orange Sigma-Aldrich, St. Louis, MO, USA

3-(4,5-dimethylthiazol-2-yl)

-2,5-diphenyltetrazolium bromide (MTT) Sigma-Aldrich GmbH, München,

Germany

LBH589 (Panobinostat) Novartis, Basel, Switzerland

Vorinostat (SAHA) Santa Cruz Biotechnology, CA, USA

Trichostatin A (TSA) Sigma-Aldrich, St. Louis, MO, USA

3.1.7 Inhibitors

Inhibitors Source

Bafilomycin A1 Sigma-Aldrich, St. Louis, MO, USA

zVAD-FMK R&D systems, Wiesbaden, Germany

DAPK inhibitor MolPort, Riga, Lativa

3.1.8 Kits

Kits Source

Annexin-V-FLOUS Roche Diagnostics GmbH, Germany

Bio-Rad Dc Protein Assay Bio-Rad Laboratories, Hercules, CA

Dynabeads Protein G and A Kit Invitrogen, Darmstadt, Germany

Immobilon western chemiluminesce Millipore Corporation, USA

RNA isolation kit Qiagen GmbH, Hilden, Germany

QuantiFast SYBR Green PCR kit Qiagen GmbH, Hilden, Germany


22

3.1.9 Equipments

Instrument Source

Blotting Chamber Bio-rad, München,Germany

Centrifuges Thermo Fischer Scientific, Erlangen,

Germany

CO2 Incubators Heraeus, Rodenbach, Germany

FACS Becton Dickinson, San Jose, CA, USA

Fluorescence Microscope Nikon, Tokyo, Japan

Fridge and Freezers Thermo Fischer Scientific, Erlangen,

Germany

Gel electrophoresis system (Western blotting) Bio-rad, München, Germany

Heating block comfort Eppendorf, Hamburg, Germany

Ice Machine Horst Zimmermann GmbH,

Nürnberg, Germany

Laminar flow hoods Heraeus, Hanau, Germany

Liquid nitrogen tank MVE, New Prague, MN, Wokingham, UK

Luminescence imaging system GeneGnome, Syngene, UK

Magnetic stirrer with hot plate Heidolph, Schwabach, Germany

Micropipettes Eppendorf, Hamburg, Germany

Microscope Nikon, Tokyo, Japan

NanoDrop Peqlab, Erlangen, Germany

pH meter Schott, Mainz, Germany

Real-Time RT-PCR Bio-rad, München, Germany

Rocking Platforms VWR International GmbH, Darmstadt,

Germany

Rotator VWR International GmbH, Darmstadt,

Germany

Spectrophotometer PerkinElmer, Rodgau, Germany

Sterile work bench Heraeus, Hanau, Germany

Sonicater G. Heinemann, Schwabach Gmund,

Germany

Thermomixer Eppendorf, Hamburg, Germany

UV-Transilluminator Decon DC Scientific, Germany


23

Vortexer Carl Roth GmbH&Co, Karlsruhe,

Germany

Water bath Memmert GmbH & CO KG, Schwabach,

Germany

3.1.10 Consumables

Consumables Source

5 ml FACS tubes BD Biosciences, Belgium

Filter-paper, nitrocellulose membrane Whatman GmbH, Dassel, Germany

Gloves Peha-soft, Heidenheim, Germany

Pipette tips VWR International GmbH, Darmstadt,

Germany

Reaction tubes (0.5/1.5/2.0 ml) Eppendorf, Hamburg, Germany

Sterile filters (pore size 0.2 µM) Sartorius, Göttingen, Germany

Sterile falcon tubes (15ml and 50ml) Greiner-bio-one, Frickenhausen, Germany

Sterile cell scrapers Corning, NY, USA

Sterile tissue culture plastic flasks Corning, NY, USA

Sterile tissue culture plastic plates Corning, NY, USA

3.1.11 Software

Software Company/Source

Adobe Acrobat X pro Adobe systems Inc., USA

Adobe Photoshop CS5 Adobe systems Inc., USA

Windows XP Professional Microsoft Corporation, USA

Gene Tools Syngene, Cambridge, UK

ImageJ (Version 1.45s) National Institutes of Health, USA

3.1.12 Database

http://www.ncbi.nlm.nih.gov/pubmed


24

3.2 Methods

3.2.1 Tumor cell lines and Cell culture

The human colon cancer cell lines HCT116 wt, HT29, DLD 1 and two generated cell lines

DAPK shRNA, DAPK+++ were maintained in RPMI medium and supplemented with 10%

fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). All cell lines were

maintained at 37°C in a humidified atmosphere and 5% CO2. All cell lines were cultured in

75cm² tissue culture flasks. The medium was changed every 2 to 3 days to remove non-

adherent cells. When cells reached approximately 80% confluence, they were trypsinised

using 1% trypsin-EDTA solution. Thereafter, the cells were used either for further cultivation

or for performing various experiments.

3.2.2 Generation of DAPK shRNA stable cell line

HCT DAPK shRNA stable cell line was generated using DAPK shRNA lentiviral particles

according to the manufacture’s instructions (Santa Cruz). HCT116 cells were transfected with

lentiviral particles containing a set of expression constructs each encoding DAPK specific 19-

25 nt shRNA to knock down DAPK expression. After transduction stably shRNA expressing

cells were isolated via puromycin selection. Transduction efficiency was measured by FACS

and DAPK knock down was verified by PCR and Western blotting.

3.2.3 Generation of DAPK+++ overexpressing cell line

Using a recently described 4-hydroxy tamoxifen (4HT)-inducible lentiviral expression system

(Vince JE et al., 2007; Diessenbacher P et al., 2008), we aimed to permit over-expression of

DAPK in transduced HCT116 cell line in a 4-HT dose-dependent manner. The DAPK+++

overexpressing cell line was generated by Jelena Ivanovska in collaboration with Prof. Martin

Leverkus group.

To generate cells expressing 4-HT-inducible DAPK, HCT cells were transduced with a

lentivirus pF GEV16 Super PGKHygro, which expresses a Gal4 DNA binding domain fused

to a mutant estrogen receptor and GEV16, and a lentivirus pF 5 UAS hs DAPK SV40 Puro,

which expresses DAPK in a Gal4-dependent fashion. In the first vector, the ubiquitin

promoter constitutively drives expression of a GEV16 transcription factor. In the absence of

4HT, GEV16 is retained in the cytoplasm, but in the presence of 4HT it translocate to the

nucleus where the GAL4-DNA binding domain (DBD) directs DNA binding to GAL4

upstream activating sequences (UAS) expressed by the second vector whereby its VP16

transactivation domain induces gene transcription. The GEV16 and 5-UAS constructs are


25

contained within the lentiviral 5´-long terminal repeats (LTR) and 3´-self-inactivating LTR.

To generate lentiviral supernatants, 293T cells were transfected with 3 mg pMD2.G, 5 mg

pMDlg/pRRE, and 2.5 mg pRSV-Rev of the lentiviral packaging vectors (Rubinson DA et al.,

2003) together with the constructs described above. The supernatants were harvested 24 hours

post-transfection, filtered (45 mm filter, Schleicher & Schuell), and concentrated by

centrifugation (19,500 g, 2 hours at 121C). The concentrated virus was added to cells as

described (Diessenbacher P et al., 2008). After 24–48 h hygromycin and puromycin are added

to select cells infected with both viruses.

3.2.4 xCELLigence Real-Time cell assay

Human colon tumor cells having different endogenous DAPK levels were seeded at 7500

cells per well in triplicate in 96X microtiter E-plates for impedance-based real-time cell

analysis using the xCELLigence RTCA system (Roche Molecular Diagnostics, Mannheim,

Germany). Cellular viability and proliferation was measured continuously every 15 min for

72h as described previously (Gloesenkamp CR, 2012). Results are expressed as an arbitrary

unit called Cell Index (C.I.) and are given as mean +/- SD of triplicates.

3.2.5 MTT assay

Cells were seeded in 96-well flat bottom microtiter plates at a density of 7500 cells in 200µl

per well. After treatment, 10 µl of 5mg/ml MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-

diphenyltetrazolium-bromide, Sigma) solution was added to each well and incubated in a

humidified 5% CO2 incubator at 37°C for 2 h. The medium with MTT solution was removed

and 100 µl of DMSO was added and incubated for 15 min at RT. The absorbance of the

solution was read spectrophotometrically at 450 nm with a reference at 650 nm using a

microtitre plate reader (VictorX3).

3.2.6 Crystal violet Assay

DAPK wt cells (7500 cells/well) were seeded in 96-well tissue culture plates and were

incubated for 24 h. Next day, cells were washed once with PBS and stimulated with different

concentrations of LBH589 (0.005, 0.01, 0.05, 0.075 and 0.1 µM) for 24, 48, 72 h. At the end

of incubation time, the cell viability of the LBH589 was evaluated by crystal violet staining.

The optical density was measured at 595 nm using a microplate reader. The average

absorbance values of untreated controls were taken as 100% cell viability.


26

3.2.7 Clonogenic assay

Human colon tumor cells (1000 cells/well) were seeded in a 6 cm tissue culture plate and

incubated for 24 h. After 24 h, cells were washed with PBS and treated with 0.05µM

LBH589. After overnight incubation, the medium containing the drug was replaced by growth

medium and incubated further for 12-14 days. As the colonies became visible, the cells were

fixed with 70% methanol for 20 min. Afterwards colonies were stained with crystal violet for

20 min, washed with water and let them dry. Pictures of stained colonies were made and area

of interest was analyzed by Image-Pro Plus software.

3.2.8 Western blotting

3.2.8.1 Determination of protein concentration

The protein concentration of the cell lysates was determined by using Bio-Rad DC Protein

Assay (BioRad Laboratories, Hercules, CA, USA). The Bio-Rad DC Protein Assay is a

colorimetric protein assay used for estimation of protein concentration. Add 5 µl of standards

and protein lysates into a 96-well plate. A color reagent (mixture containing 20 µl reagent S

and 1 ml reagent A) and 200 µl of Reagent B were added to each well and incubated for 15

minutes at room temperature. For the generation of a standard curve, Bovine Serum Albumin

(BSA, 10 mg/ml) was used. The resulting color was read at 750 nm using a

spectrophotometer.

Table 4: Preparation of protein standards (BSA) to determine protein concentration

Concentration BSA (mg/ml) Water

0 - 100 µl

0.10 1 µl 99 µl

0.25 2.5 µl 97.5 µl

0.50 5 µl 95 µl

1.0 10 µl 90 µl

1.50 15 µl 85 µl

2.0 20 µl 80 µl

3.0 30 µl 70 µl


27

Table 5: Solutions used for casting two Separating and Stacking gels

Ingredient Separating gel 10% Separating gel 12% Stacking gel Water 7 ml 5.895 6.33 ml 30% AA/Bis-AA 4.99 ml 5.895 1.65 ml Separating gel buffer 3 ml 3 ml - Stacking gel buffer - - 2 ml 10% APS 112.5 µl 112.5 µl 75 µl TEMED 12 µl 12 µl 8 µl

3.2.8.2 Western blot analysis

Whole cell lysates were prepared from DAPK wt, DLD-1, HT29, DAPK shRNA, and L27

DAPK +++ tumor cells. Protein concentration of lysates was determined with Bio-Rad Dc

Protein Assay (BioRad Laboratories, Hercules, CA), and 30 µg proteins were loaded onto

10% or 12% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to

nitrocellulose membranes before immunodetection processing with anti-DAPK (BD

Transduction Laboratories, Lexington NY), anti-phosphoDAPKSer308 (Sigma), anti-LC3

(Nanotools, Teningen, Germany), anti-pH2AX (Millipore corporation, Billerica, USA), anti-

Beclin 1, anti-Atg7, anti-p62, anti-caspase 3, anti-PARP (Cell Signaling Technology Inc.),

and with secondary antibodies (anti-mouse or anti-rabbit IgG peroxidase conjugated; Thermo

Scientific, Rockford, IL). Bound antibodies were detected by incubating the blots in

Immobilon western chemiluminescent HRP substrate (Millipore Corporation).

Immunoreactivity was measured as peak intensity using an image capture and analysis system

(GeneGnome, Syngene, UK). Hybridization with anti-β-actin or GAPDH was used to control

equal loading.

Table 6: Primary and respective secondary antibodies used in Western blotting. All antibodies were diluted using either 5% milk powder or 5% BSA.

Primary Antibodies

Species Dilution Secondary Antibodies

Dilution

Ac-H3 Rabbit 1:2000 Goat-anti- rabbit 1:20000 Ac-H4 Rabbit 1:5000 Goat-anti-rabbit 1:20000 Caspase 3 Rabbit 1:1000 Goat-anti- rabbit 1:10000 Caspase 8 Mouse 1:1000 Goat-anti-mouse 1:10000 Caspase 9 Rabbit 1:1000 Goat-anti- rabbit 1:10000 DAPK Mouse 1:250 Goat-anti-mouse 1:10000 pDAPK Mouse 1:250 Goat-anti-mouse 1:10000 LC3 Mouse 1:200 Goat-anti-mouse 1:10000 pH2AX Mouse 1:2000 Goat-anti-mouse 1:10000 Beclin 1 Rabbit 1:1000 Goat-anti-rabbit 1:10000 Atg7 Rabbit 1:1000 Goat-anti-rabbit 1:10000 p62 Rabbit 1:1000 Goat-anti-rabbit 1:10000


28

PARP Rabbit 1:1000 Goat-anti-rabbit 1:10000 β-actin Mouse 1:5000 Goat-anti-mouse 1:20000

3.2.9 Detection of autophagic vacuoles by acridine orange

Cells were plated at a density of 2 X 105 on glass cover slips in six-well plates and incubated

for 24 h. Cells were treated with 0.05µM LBH589 for 24 and 48 h. At the appropriate time

points, cells were incubated with 1 µg/ml acridine orange (Sigma, Germany) for 15 min. Cells

were treated with 3µg/ml bafilomycin A1 for 60 min before the addition of acridine orange to

inhibit the acidification of autophagic vacuoles. The acridine orange was removed and

fluorescent photographs were obtained using an inverted fluorescence microscope (Nikon).

3.2.10 Detection of autophagosomes by LC3 immunofluorescence staining

Cells were plated at a density of 2 X 105 on glass cover slips in six-well plates and incubated

for 24 h. Cells were treated with 0.05µM LBH589 for 24 and 48 h. At the appropriate time

points, cells were fixed with 4% (w/v) paraformaldehyde for 30 min and then made permeable

with methanol at -20 °C for 10 min. The cells were then covered with 10% (v/v) goat serum

for 30 min at room temperature to block non-specific adsorption of antibodies to the cells.

After this procedure, the cells were incubated with primary antibody against LC3 at 4°C

overnight. Cells were then probed with Alexa Fluor 488 goat anti-mouse secondary antibodies

and incubated at room temperature for another 2h. Fluorescent signals were detected using

inverted fluorescence microscope (Nikon).

3.2.11 Flow cytometric detection of Apoptosis by Annexin V-FITC labelling

To detect apoptosis the Annexin-V-FLUOS kit (Roche Diagnostics) was used. DAPK wt,

DAPK shRNA, and L27 DAPK +++ cells were stimulated with LBH589 for 24 and 48 h

under the above mentioned conditions. After washing twice in PBS, 1X106 cells were stained

with 100 µl annexin V staining solution, consisting of 20 µl FITC-conjugated annexin V

reagent (20 µg/ml), 20 µl isotonic propidium iodide (PI, 50 µg/ml), and 1000 µl of 1 M/L

HEPES buffer, for 15 minutes at room temperature. Cells were analyzed on a FACS Calibur

flow cytometer (Becton Dickinson, CA).

3.2.12 Flow cytometric analysis of cell cycle distribution

The distribution of cells in different phases of cell cycle was determined by flow cytometric

analysis of DNA content. DAPK wt, DAPK shRNA, and L27 DAPK +++ cells were

stimulated with LBH589 for 24 and 48 h. After indicated times, both floated and adherent


29

cells were harvested, washed twice with PBS, and stained with PI solution (50 µg/ml PI, 0.1%

Sodium Citrate, 0.1% Triton X-100 in PBS). Distribution of cell cycle phases with different

DNA contents was determined using a flow cytometer (Becton Dickinson, CA, USA).

Analysis of cell cycle distribution and the percentage of cells in the G0/G1, S, and G2/M

phase of the cell cycle were determined using the software CellQuest Pro (BD).

3.2.13 Immunoprecipitation

Immunoprecipitation was performed according the manufacturer’s instructions (Invitrogen,

Karlsruhe, Germany). Protein G magnetic Dynabeads were coated with DAPK antibody

(1:250 dilution) for 2 hours with rotation at RT. Supernatants were removed using a magnetic

stand and Dynabeads-antibody complex was washed with 200 µl Antibody Binding and

Washing Buffer. 900 µg of protein lysate was added to the Dynabeads-antibody complex and

gently resuspend by pipetting. The Dynabeads-antibody-antigen complex was incubated

overnight at 4°C with rotation. The Dynabeads-antibody-antigen complex was washed 3 times

each with 200 µl washing buffer. Immunoprecipitates were eluted in 20 µl elution buffer.

Eluted immunoprecipitates were resuspended with SDS reducing loading buffer and

incubated 5 min at 95°C. The proteins were separated by 10% SDS-PAGE and analysis was

performed by western blotting.

3.2.14 In vitro kinase assay

In vitro kinase assay was performed as described previously with minor modifications

(Bajbouj K et al., 2009). Immunoprecipitation was performed as described above. Dynabeads-

Antibody-Antigen complex was resuspended in washing buffer and supernatant was removed

using magnetic stand. The Dynabeads-Antibody-Antigen complex was resuspended in kinase

buffer (60 mM HEPES, pH 7.5, 3 mM Mncl2, 3 mM Mgcl2, 3 µM sodium orthovanadate, 1.2

mM DTT, 2.5 µg/µl PEG, 2 µg RB-S6P) before the addition of 7.5 µCi [γ32-P] ATP (GE

Healthcare, Amersham Biosciences) and incubated at 25°C for 30 minutes. Samples were

boiled with SDS reducing buffer at 95°C for 5 min and separated using 10% SDS-poly

acrylamide gel. After gel electrophoresis, the proteins were transferred to nitrocellulose

membrane and autoradiographed.

3.2.15 RNA Isolation and cDNA synthesis

Total cellular RNA was extracted using RNeasy Kit (Qiagen, Hilden, Germany). 350 µl of

buffer RLT was added to the cell pellet and mixed. To the lysate, 350 µl of ethanol (70%) was


30

added. The lysate-ethanol mixture was transferred onto an RNeasy spin column and

centrifuged for 15 s at 8000 x g. The buffer RW1 (700 µl) was added and the solution was

centrifuged (15s, 8000 x g). The column was then washed two times with buffer RPE (1st

wash RPE 500 µl, 15 s at 8000 x g; 2nd wash RPE 500 µl, 2 min at 8000 x g). After the final

wash, the column was removed, placed in to a new test tube, and the RNA was subsequently

eluted with 60 µl RNase-free water by centrifugation (1 min at 8000 x g).

cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen) according to the

manufacturer’s instructions. 1 µg of total RNA was incubated with 2 µl gDNA wipe out

buffer for 2 min at 42°C.Test tubes were kept on ice for 2 min. The reverse transcriptase

master mix (6µl) was added and incubated at 42°C for 30 min. In the next step, the samples

were incubated at 95°C for 3 min. The cDNA was either used immediately or stored at -20°C

until further analysis.

3.2.16 Real-Time Reverse Transcription-PCR

The real-time RT-PCR was performed in a final volume of 25 µl using a CFX 96 (Bio-Rad)

and threshold cycle numbers were determined using the Bio-Rad CFX manager software.

DAPK primer sequences were sense 5-CCTTGCAAGACTTCGAA AGGATA-3 and

antisense 5-GATTCCCGAGTGGCCAAA-3. The final reaction mixture contained the

forward and reverse primer at 10 pmol each, PCR was performed under the following

conditions: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s using

QuantiFast SYBR Green PCR Kit (Qiagen). Real-time RT-PCR was performed in duplicate,

and the threshold cycle numbers were averaged. Expression of genes of interest was

normalized to β2-microglobulin and is expressed as arbitrary units.

3.2.17 In vivo Experimental Methods

3.2.17.1 Xenograft model

In vivo experiments were performed as described previously (Di Fazio P et al., 2010). 5.0 X

106 DAPK wt or DAPK shRNA cells suspended in sterile physiologic NaCl solution were

injected into the flank regions of the 6 to 8 week old male NMRI mice (Harlan Winkelmann

GmbH, Germany). Seventeen to twenty seven animals were used in the xenograft studies.

Tumors were measured every day using a calliper square. When tumors were reached a

diameter of 7 mm, animals were treated with LBH589 (10 mg/kg) or physiologic saline

solution for 30 days by daily intraperitoneal injections. Animals were sacrificed by cervical


31

dislocation at the end of the treatment period or when abortion criteria were met. Tumor

xenografts were fixed in 10% phosphate-buffered formalin or snap-frozen in liquid nitrogen.

Ethical approval was granted by the local government authority (Government of Lower

Franconia, Würzburg, Germany) before the beginning of experiments.

3.2.17.2 Tissue microarray and Immunohistochemistry

Tumor tissues were fixed in formalin and embedded in paraffin. The punches were transferred

onto a new paraffin block to form the tissue microarrays (TMAs). Immunohistochemical

studies were performed on 3 µm thick slices using EnVision (Dako) detection system. TMAs

were deparaffinized in xylene for 30 min at 72°C and rehydrated in descending concentrations

of ethanol. Antigen retrieval was done using pressure cooker (120°C, 5 min, 1 mM Tris-

EDTA buffer). Endogenous peroxidases activity was inhibited by incubating the slices for 5

min with blocking solution (Dako). All slices were incubated with primary antibodies (anti-

DAPK (1:200) and anti-ki67 (1:50, Dako) for 30 min at RT, followed by washing with

washing buffer (Dako) and incubation with secondary antibody linked with horseradish

peroxidase (goat-anti-mouse, Dako) at RT for 30 min. Positive immunohistochemical

reactions were detected using DAB+ (Dako) as chromogen substrate. Nuclei were

counterstained with hematoxylin (Dako).

3.2.17.3 Assessment of immunohistochemical protein expression

The TMAs were investigated by two independent reviewers blinded to other data. For DAPK

protein expression, staining intensity and the percentage of positive cells were assessed semi

quantitatively using the following system: Staining intensity (SI) was classified as 0 (no

staining), 1 (weak), 2 (moderate), and 3 (strong); number of positive cells (PC): 0 (0%), 1

(<10%), 2 (10-50%), 3 (51-80%), and 4 (>80%). For DAPK, an immunoreactive score (IRS)

was calculated using the formula [SI * PC = IRS]. Ki-67 staining was estimated by counting

the percentage of positive nuclei in a representative tumor area. An average immunoreactive

score of triplicates was finally estimated for each group.

3.2.17.4 Preparation of animals for MicroPET Imaging

In vivo experiments were performed as described previously (Maschauer S et al., 2010). The

experiment was performed by Simone Maschauer and Bianca Weigel in the laboratory of

Molecular Imaging and Radiochemistry, supervised by Prof. Olaf Prante. All animal

experiments were performed in compliance with the protocols approved by the local Animal


32

Protection Authorities (Regierung Mittelfranken, Germany, No.54-2532.1-15/08). Athymic

nude mice (nu/nu) were obtained from Harlan Winkelmann GmBH (Borchen, Germany) at 9-

11 weeks of age and were kept under standard conditions (12 h light/dark) with food and

water available ad libitum. HT29 cells were harvested and suspended in sterile PBS at a

concentration of 5x107 cells/ml. Viable cells (5x106) in PBS (100 µl) were injected

subcutaneously in back. One to two weeks after inoculation (tumor weight: 400-800 mg), the

mice (about 10-12 weeks old with about 40 g body weight) were used for microPET studies.

3.2.17.5 MicroPET Imaging

MicroPET Imaging experiments were performed as described previously (Maschauer S et al.,

2010). The experiment was performed by Simone Maschauer and Bianca Weigel in the

laboratory of Molecular Imaging and Radiochemistry, supervised by Prof. Olaf Prante. PET

scans and image analysis were performed using a microPET rodent model scanner (Inveon,

Siemens Medical Solutions). About 4-8 MBq of [18F]FGIc-RGD was intravenously injected

into each mouse (n=3) under isoflurane anesthesia (4%). Animals injected with [18F]FGIc-

RGD were subjected to a 10 min static scan starting from 50-60 min post-injection (p.i.). For

each microPET scan, regions of interest (ROIs) were drawn over the tumor on decay-

corrected whole-body images that were gained by MAP iterative image reconstruction. The

radioactivity concentration within the tumor was obtained from the mean value within the

multiple ROIs and then converted to standard uptake values (SUV) by considering the

injected dose and the individual body weight. For receptor-blocking experiments, nude mice

bearing HT29 tumors were scanned (10 min p.i., static) after coinjection with [18F]FGIc-

RGD (4-8 MBq) and c(RGDfV) (12 mg/kg).

3.2.18 Statistical analysis

Statistical analysis was performed using SPSS (SPSS Inc., Chicago, IL, USA). Differences in

cell proliferation and colony forming ability were compared using the one-way analysis of

variance (ANOVA) followed by Tukey’s HSD, Dunnett’s t, and Student-Newman-Keuls post

hoc tests. Significance of in vivo data was calculated using the t-test for independent samples.

P values less than 0.05 were considered statistically significant.

Results

33

4. Results

4.1 LBH589 decreases cell viability in DAPK wt cells

In order to find the optimal concentration of LBH589 in parental cell line HCT116 (DAPK

wt), cells were treated with increasing doses of LBH589 i.e., 0.001, 0.005, 0.01, 0.05, 0.075

and 0.1 µM for 24, 48 and 72h. The effect of LBH589 on cell viability was evaluated by

crystal violet staining.

As shown in Figure 5, LBH589 treatment decreased the cell viability in a time-and dose-

dependent manner in DAPK wt cells. The doses 0.05 µM LBH589 reduced the cell viability

by approximately 50% at 24 h. No significant viability effects were observed with doses

lower than the 0.01 µM LBH589. Therefore, we used 0.05 µM LBH589 for all experiments in

this study, a dose that has been used also by other groups (Kauh J et al., 2010; Fazzone W et

al., 2009).

Fig. 5. Effect of LBH589 on cell viability in DAPK wt cells. The DAPK wt cells (7500 cells/well) were seeded in 96-well cell culture plates. The cells were treated with different concentrations of LBH589 for 24, 48 and 72 h and cell viability was measured by crystal violet assay. Results are expressed as a percentage of the control cells of the corresponding time point. Data are representative of two independent experiments performed in triplicates. *p<0.05 compared with untreated controls of respective time point.

Results

34

4.2 Generation of DAPK knockdown stable cell line (DAPK shRNA) in DAPK wt cells To investigate the role of DAPK in LBH589-induced signalling pathways in human colon

cancer cells, we have generated DAPK knockdown stable cell line using DAPK specific

shRNA lenti viral particles. After transduction stably shRNA expressing cells were isolated

via puromycin selection. Transduction efficiency was measured by FACS and DAPK knock

down was verified by real-time RT-PCR and Western blotting. As shown in Figure 6A,

95.73% transduction efficiency was achieved. Real-time RT-PCR and western blotting

analysis showed that DAPK was down-regulated both at mRNA (Fig. 6B) and protein levels

(Fig. 6C).

A B C Fig. 6. Knockdown of DAPK in DAPK wt cells using DAPK shRNA lentiviral particles. (A) DAPK wt cells were transfected with DAPK shRNA lentiviral particles and transduction efficiency was measured by FACS. (B, C) DAPK knock down was verified by real-time RT-PCR and western blotting. *P ≤ 0.05 for the comparison with HCT wt control cells. (Ctrl= Control, GFP=Green fluorescence protein) 4.3 Generation of tamoxifen-inducible DAPK overexpression stable cell line (DAPK+++) in DAPK wt cells To better understand the DAPK signalling after LBH589 treatment, we have generated an

inducible lentiviral DAPK vector to preclude clonal selection during the selection procedure.

Using a recently described 4-hydroxy tamoxifen (4HT)-inducible lentiviral expression system

(Vince JE et al., 2007; Diessenbacher P et al., 2008), we aimed to permit over-expression of

Results

35

DAPK in transduced HCT116 cell line in a 4-HT dose-dependent manner (Fig. 7). Using the

generated vector we transduced HCT116 cells and were able to over-express DAPK in a dose-

dependent manner. Whereas the empty control vector did not show any induction of DAPK.

In our studies we used a concentration of 25 nM tamoxifen for 6 h and subsequently

stimulated with HDAC inhibitors.

Fig. 7. Tamoxifen-inducible DAPK overexpression system. Dose-dependent up-regulation of DAPK protein expression after 6 h in DAPK Gev (DAPK vector), L27 GEV (empty vector). C: without tamoxifen (tam), 1: 1nM Tam, 2: 10nM Tam, 3: 25nM Tam, 4: 50nM Tam, 5: 100nM Tam. 4.4 Effect of Tamoxifen and/or LBH589 on the expression of DAPK in DAPK +++ cells To verify the suitability of the tamoxifen-inducible overexpression system after LBH589

treatment, we have stimulated the cells either with tamoxifen or LBH589 or in combination

for 24 and 48 h. The expression of DAPK was assessed by real-time RT-PCR and western

blotting. DAPK is known to be negatively regulated by auto-phosphorylation of serine308.

Thus, the dephosphorylation of autoinhibitory serine308 can activate DAPK (Shamloo M et

al., 2005; Jin Y et al., 2006). As shown in figure 8A, although DAPK +++ cells showed a

slight increase in expression of DAPK at mRNA level with tamoxifen or LBH589 treatment

alone (2 to 3-fold) only the combination of tamoxifen and LBH remarkably enhanced mRNA

expression (up to 100-fold) verifying that the inducible lentiviral expression system is

suitable. Furthermore, DAPK +++ cells showed a slight decrease in the pDAPK/DAPK ratio

also with tamoxifen or LBH589 treatment alone (2 to 3-fold). Nevertheless only the

combination of tamoxifen and LBH589 completely activated DAPK with nearly total loss of

the pDAPK inactive form (Fig. 8B).

Results

36

Fig. 8. Influence of Tamoxifen and/or LBH589 on expression of DAPK in DAPK +++ cells. The DAPK +++ cells were stimulated either with tamoxifen (25 nM) or LBH589 (0.05 µM) or in combination for 24 and 48 h. (A) The DAPK mRNA expression was analysed by real time RT-PCR. The mRNA levels were normalized to levels of β2-microglobulin. *p<0.05 compared with untreated controls of respective time point. .*p<0.05 compared with untreated controls of respective time point. #P<0.05 compared with tamoxifen or LBH589 treated cells. (B) DAPK protein was analysed by Western blotting against pDAPK and DAPK. β-actin was used as an equal loading control. The band intensities were quantified by densitometry analysis. 4.5 LBH589 induces acetylation of histones H3 and H4 in human colon tumor cells

Previous studies have showed that HDACi can induce acetylation of histones by inhibiting the

activity of histone deacetylases (Tang YA et al., 2010; Emanuele S et al., 2008; Romanski A

et al., 2004). Therefore, we aimed to investigate the acetylation status of histones H3 and H4

Results

37

using Western blotting after LBH589 treatment for 24, 48 and 72 h. As shown in Figure 9,

treatment with LBH589 induces acetylation of core histones H3 and H4 in all the three cells

lines, suggesting that HDACi induces histone acetylation in human colon cancer cells.

Fig. 9. Effect of LBH589 on acetylation status of histones H3 and H4. The cells having different DAPK status i.e. DAPK shRNA, DAPK wt, and DAPK +++ were treated with 0.05 µM LBH589 for 24, 48, and 72 h. Western blot analysis of acetylation of histones was done using anti-Ac-H3 and anti-Ac-H4 antibodies. Equal loading was controlled by β-actin. The band intensities were quantified by densitometry analysis. In each cell line the control was adjusted to one after normalization to β-actin.

4.6 Knockdown and overexpression of DAPK in DAPK wt cells did not influence cell

proliferation

To study if established two cell lines i.e. DAPK shRNA and DAPK+++ with different DAPK

levels will affect the viability of tumor cells, we used impedance-based xCELLigence system

to monitor dynamic cell proliferation in real-time. The xCELLigence system will measure

changes in cell size, cell viability, cell number, morphology, and cell adhesion in real time

without any disruptions. The cells were seeded in 96X E-plates and continuously monitored

for 72 h. As shown in Figure 10, the three cell lines- DAPK shRNA, DAPK wt and

DAPK+++ showed almost similar pattern of growth kinetics. The data clearly show that all

three cell lines are continuously growing and reach a stable plateau phase without

spontaneously undergoing cell death when reaching confluence. These results suggest that

knockdown and overexpression of DAPK will not influence the growth kinetics of human

colon tumor cells.

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Fig. 10. Real-time monitoring of cell viability in DAPK wt, DAPK shRNA, and DAPK+++ cells. The indicated cell types were seeded at a density of 7500 cells per well in 96X E-plates. The attachment, spreading and proliferation of cells were monitored every 15 minutes until 72 h using the xCELLigence Real-Time Cell-Analyzer. The results are expressed as Normalized Cell Index. 4.7 LBH589 inhibits cell proliferation in human colon tumor cells having different

DAPK levels

To further evaluate the cell growth inhibition activity of LBH589, we performed MTT cell

proliferation assay. Figure 11 shows the effect of LBH589 on the proliferation of human

colon tumor cells having different DAPK levels. LBH589 (0.05 µM) significantly (p<0.05)

reduced the cell proliferation in all the three cell lines at 24, 48, and 72 h. Furthermore,

reduction in cell proliferation levels was independent of DAPK indicating that LBH589

reduced cell proliferation in human colon tumor cells are DAPK-independent. In addition,

DAPK +++ cells showing the most remarkable decrease in cell proliferation when compared

to other two cell lines i.e. DAPK shRNA and DAPK wt.

Fig. 11. Cell proliferation is independent of the DAPK status in human colorectal cancer cells. The indicated cell types were seeded at a density of 7500 cells per well in 96- well plates and treated with LBH589 24, 48 and 72 h. Cell proliferation was determined by the

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MTT cell proliferation assay. Results are expressed as percentages of control cells. Each value is the mean of two separate experiments performed in triplicates. *P<0.05 compared with untreated controls. #P<0.05 for comparison between LBH589 treated cells.

4.8 LBH589 reduces the long-term survival in DAPK wt cells having different DAPK

levels

The effect of LBH589 on the growth of human colon cancer cells was investigated by a

colony formation assay. To further study the long-term growth inhibitory effect of LBH589,

anchorage dependent clonogenic assay was performed in human colon tumor cells having

different DAPK status. Results showed that treatment with LBH589 significantly (p<0.05)

reduced the colony formation ability of all three colon cancer cell lines (Fig. 12). However,

there was no significant difference between DAPK wt and DAPK shRNA cells. Interestingly,

DAPK +++ cells were more sensitive to LBH589, indicating that DAPK potentiates the

inhibitory effect of LBH589 in colony formation. These data suggest that LBH589 might play

a role in colon tumor suppression.

Fig. 12. Long term survival is independent of the DAPK status in human colorectal cancer cells. The indicated cell types were seeded at a density of 500 cells in 5 cm dishes and incubated with LBH589. After overnight incubation, drug-free medium was applied to the samples. Cells were then allowed to form colonies for 12-14 days. Colonies were fixed in methanol, stained with crystal violet, washed with water and photographed. *P<0.05 compared with untreated controls, #P<0.01 for comparison between LBH589 treated cells.

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4.9 LBH589 induces up-regulation and activation of DAPK in human colon cancer cells in vitro To investigate if DAPK is involved in LBH589 action in tumor cells, we assessed the

expression of DAPK at the mRNA and protein levels in cell lines that have different levels of

DAPK i.e. DAPK wt, DAPK shRNA, and DAPK+++. The latter cells were used as an

internal control of unphysiologically high DAPK levels and to clarify the involvement of

DAPK in LBH589 activity. Cells were stimulated with 0.05 µM LBH589 for 24, 48 and 72h.

Although LBH589 significantly (p<0.05) up-regulated the expression of DAPK at the mRNA

level (Fig. 13A) in all three cell lines, the overall levels were approximately 4 and 20-fold

lower in DAPK shRNA cells when compared to DAPK wt and DAPK+++ cells, respectively.

In addition, there was a gradual increase in DAPK protein levels from DAPK shRNA cells to

DAPK wt to DAPK+++ cells. There was a high level of inhibitory autophosphorylated form

pDAPKSer308 in all three cell line controls. Under LBH589, the pDAPK levels were decreased

over time. The subsequent reduction in pDAPK/DAPK ratio reflects the activation of DAPK

(Fig. 13B). Indeed there was an increase in kinase activity of DAPK after LBH589 treatment

in all the three cell lines. As expected from the highest pDAPK/DAPK ratio in DAPK shRNA

cells they showed the lowest basal level of kinase activity whereas DAPK wt and DAPK +++

cells did not differ in their basal kinase activity. After LBH589 treatment, the kinase activity

was increased in all three cell lines reaching the same levels in DAPK shRNA and DAPK wt

cells and the highest level in DAPK +++ cells (Fig. 13C). Thus these three cell lines will be a

suitable model to study the DAPK-dependent effects of LBH589 in colorectal cancer cells.

In a separate experiment, we used the chemically related hydroxamic acid derivative,

Vorinostat, to generalize the HDACi effect on DAPK. The three cell lines were stimulated

with Vorinostat for 24, 48 and 72 h. Vorinostat induced an up-regulation of DAPK in all three

cell lines with simultaneous activation of DAPK activity which is similar to LBH589 (Fig.

13D).

A

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B

C

D

Fig. 13. LBH589 enhances DAPK mRNA and protein expression in human colon tumor cells. DAPK shRNA, DAPK wt, and DAPK +++ cells were treated with 0.05 µM LBH589 for 24, 48 and 72 h. (A) The mRNA expression was analyzed using real-time RT-PCR. The mRNA levels were normalized to levels of ß2-microglobulin. Results summarized in the bar graphs are representative of two independent experiments, *P<0.05 (when compared with untreated controls). (B) Inactive (pDAPK) and total levels of DAPK were analyzed by Western blotting using antibodies against pDAPK and DAPK. Results are representative of three independent experiments. (C) Determination of DAPK kinase activity in colon cancer cells. The three cell types were treated with LBH589 for 24 and 48 h. The cell lysates were

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subjected to immunoprecipitation (IP) with anti-DAPK and in vitro kinase assay was performed using RB-S6P as the substrate. Equal loading was controlled by Ponceau S staining. (D) DAPK wt cells having different DAPK status were stimulated with 2 µM Vorinostat for indicated times. After the treatment, whole-cell protein lysates were prepared and Western blotting analysis was performed against DAPK and pDAPK. Equal loading was controlled by β-actin. The band intensities were quantified by densitometry analysis. 4.10 Role of DAPK in LBH589-induced apoptosis

4.10.1 LBH589 induces cell cycle arrest in human colon tumor cells

To examine potential DAPK-dependent cell cycle regulatory effects of LBH589, we aimed to

use three human colon cancer cell lines (DAPK wt, DAPK shRNA, and DAPK +++ cells)

having different DAPK status. Each cell line was stimulated with 0.05 µM LBH589 for 24

and 48 h and DNA content was analyzed by flow cytometry after staining with propidium

iodide. After 24h of LBH589 treatment, sub-G1 population that is indicative of cell death was

observed in all the three cell lines. In addition, LBH589 caused a G2/M cell cycle arrest with

decreased G1/S fractions when compared to untreated controls in all three cell lines (Fig. 14).

The results suggest that cells with different DAPK levels might have similar modes of cell

cycle regulation after LBH589 treatment indicating that LBH589-induced cell cycle arrest

was DAPK-independent.

Fig. 14. LBH589 induces G2/M cell cycle arrest in colon cancer cells. DAPK shRNA, DAPK wt, and DAPK +++ cells were treated with 0.05 µM LBH589 for 24 and 48 h. Adherent and non-adherent cells were harvested and stained with propidium iodide. The distributions of cells in different phases of cell cycle were determined using flow cytometry. DNA content analyses were determined using cell Quest software. Data are from two independent experiments.

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4.10.2 LBH589 causes formation of apoptotic bodies in a DAPK-independent manner

In a separate experiment, we took bright field photos of LBH589-treated cells to detect

apoptotic bodies in the three cell lines. As shown in Figure 15, LBH589 induces time-

dependent increase in the number of apoptotic bodies when compared to the respective

controls; however this increase was DAPK-independent. Cells with artificial overexpression

of DAPK (DAPK+++) showed the most prominent apoptotic body formation at 24h. In

conclusion, there were no differences in LBH589 sensitivity between DAPK shRNA and

DAPK wt cells whereas DAPK+++ cells were highly susceptible to LBH589-induced

apoptosis.

Fig. 15. Effect of LBH589 on apoptotic bodies formation. The cells having different DAPK status was treated with 0.05 µM LBH589 for 24 and 48 h. After LBH589 treatment, apoptotic bodies were photographed. Photos (x20) and the arrow indicate apoptotic bodies. 4.10.3 LBH589 activates caspases and induces apoptosis in a DAPK-independent manner Various studies have already shown that HDACi LBH589 induces apoptosis in cancer cell

lines (Fazzone w et al., 2009; Di fazio P et al., 2010). To examine potential DAPK-dependent

pro-apoptotic effects of LBH589, we first analyzed DAPK wt, DAPK shRNA, and DAPK+++

cells by Annexin V staining. Each cell line was stimulated with 0.05 µM LBH589 for 24 and

48 h DAPK+++ cells were most susceptible to LBH589-induced apoptosis whereas no

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difference in LBH589 sensitivity was observed when comparing DAPK shRNA and DAPK

wt cells. The results suggest that cells with different DAPK levels undergo cell death after

LBH589 treatment in a DAPK-independent manner (Fig. 16A).

To address whether LBH589-induced apoptosis is mediated by caspases we investigated the

cleavage of caspase 3, 8, 9 and also PARP. Interestingly, in general LBH589 induced

cleavage of caspase 3, caspase 8, caspase 9 and also PARP in all three cell lines (Fig. 16B). In

addition there was no significant difference in activation of caspase 3, caspase 8, caspase 9

and also PARP cleavage in DAPK wt and DAPK shRNA cells whereas we observed a strong

apoptosis induction in DAPK+++ cells.

In addition, we investigated if apoptosis induction is caused by DAPK-mediated DNA

damage. To detect DNA double strand breaks after LBH589 exposure we studied the levels of

the DNA damage marker, pH2AX by Western Blotting. An increase in pH2AX levels was

detected in all three cell lines (Fig. 16B). In detail, DNA damage was less pronounced in

DAPK+++ cells at 24h. These data suggest that in this cell line the damaged cells are more

efficiently eliminated by apoptosis or that there are other mechanisms protecting the cells

from excessive damage.

To determine if HDACi-induced apoptotic effects are generally DAPK-independent we tested

caspase 3 and PARP activation in our cell system treated with Vorinostat. As expected, we

could not observe DAPK-dependent apoptosis induction between the three cell lines after

Vorinostat treatment suggesting that DAPK does not seem to play a role in apoptosis

induction by HDACi (Fig. 16C).

A

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B

C Fig. 16. LBH589 induces apoptosis in human colon tumor cells. (A) The cells having different DAPK status were treated for 24 and 48 h with or without LBH589, and Annexin V/ propidium iodide analysis was performed by FACS. The data are representative of two independent experiments. (B) DAPK shRNA, DAPK wt, and DAPK +++ cells were treated with 0.05 µM LBH589 for 24, 48 and 72 h. Western blot analysis showing activation of caspase-3, 8, 9, PARP and pH2AX in all 3 cell types after treatment with LBH589. β-actin was used as an equal loading control. (C) The indicated cell types were treated with or without Vorinostat for various time points. Protein levels of Caspase 3 and PARP were detected by Western blotting. The data are representative of two independent experiments.

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4.10.4 zVAD rescued the cells from LBH589 induced apoptosis in human colon tumor cells To provide further evidence that caspases are involved in LBH589-induced apoptosis, we

treated the cells with the general caspase inhibitor zVAD. Cells with different DAPK levels

were pre-stimulated with 40 µM zVAD for 1h and then treated with LBH589 for the indicated

time points. Pre-treatment with zVAD completely abolished LBH589-induced caspase

cleavage. Furthermore, as expected pre-treatment with zVAD also inhibited PARP cleavage

(Fig. 17). A similar effect was observed in all the three cell lines suggesting that LBH589-

induced cell death is mediated by caspase 3. Interestingly additional caspase 3 cleaved bands

were observed after combined LBH589 and zVAD treatment (Fig. 17), suggesting that zVAD

protects caspase 3 p20 from full maturation or that it is protected by effector caspase

inhibitors such as XIAP (Leverkus M et al., 2003).

Fig. 17. Apoptotic cells were rescued by the zVAD. The indicated cell types were preincubated with a general caspase inhibitor, zVAD for 1 h and then stimulated with LBH589 for 24 or 48 h, respectively. Western blotting analysis for caspase 3 and PARP was performed. The data are representative of two independent experiments. 4.10.5 LBH589 also induces caspase independent cell death in human colon tumor cells

To test whether other cell death pathways are also play a role after LBH589 treatment, we

performed crystal violet cell viability assay in DAPK wt parental cell line. The DAPK wt

cells were stimulated either with zVAD or LBH589 or combination for 24 and 48 h.

Interestingly, LBH589 induced apoptosis was not rescued by general caspase inhibitor zVAD

as we did not observe any increase in cell viability (Fig. 18). Therefore we hypothesize that

LBH589 also activates caspase-dependent and -independent cell death mechanisms.

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Fig. 18. Effect of pre-treatment with general capase inhibitor- zVAD on cell viability. The DAPK wt cells (7500 cells/well) were seeded in 96-well cell culture plates. The cells were pre-incubated with zVAD (40 µM) for 1 h and then stimulated with LBH589 (0.05 µM) for 24 and 48 h. The cell viability was measured by crystal violet assay. Results are expressed as percentage of the control cells of the corresponding time point. Data are from two independent experiments performed in triplicates.*p<0.05 compared with untreated controls of respective time point. 4.10.6 LBH589-induced apoptosis is independent of DAPK kinase activity

To further verify the role of DAPK kinase activity in LBH589-induced apoptosis we used a

potent and selective DAPK inhibitor, 2-phenyl-4-(pyridin-3-ylmethylidene)-4,5-dihydro-1,3-

oxazol-5-one for inhibiting its catalytic activity (Okamoto M et al., 2010). First, we measured

the effect of single and combination treatment in the DAPK wt cells using crystal violet assay.

Pre-treatment with DAPK inhibitor did not influence the LBH589 induced caspase 3-

mediated cell death at 24 hours and 48 hours, respectively, when compared to LBH589 alone

(Fig. 19A). Furthermore, as shown in Figure 19B and C, pre-treatment of DAPK wt and

DAPK +++ cells with the DAPK inhibitor did not alter the LBH589-induced caspase 3 and

PARP cleavage. Together, these data suggest that DAPK´s kinase activity is not important for

the observed caspase 3-dependent cell death.

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Fig. 19. Effect of DAPK inhibitor on LBH589-induced apoptosis. (A) The DAPK wt cells (7500 cells/well) were seeded in 96-well cell culture plates. The cells were pre-incubated with DAPK inhibitor (25 µM) for 1 h and then stimulated with LBH589 (0.05 µM) for 24 and 48 h. The cell viability was measured by crystal violet assay. Results are expressed as percentage of the control cells of the corresponding time point. Data are from two independent

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experiments performed in triplicates.*p<0.05 compared with untreated controls of respective time point. (B, C) DAPK wt and DAPK +++ cells were pre-incubated with DAPK inhibitor (25 µM) for 1 h and then stimulated with LBH589 (0.05 µM) for 24 and 48 h. Western blot analysis was performed against caspase 3 and PARP. β-actin was used as an equal loading control. 4.11 Role of DAPK in LBH589-induced autophagy

4.11.1 LBH589 induces autophagy in human colon cancer cells in a DAPK-dependent manner There is an intricate balance between autophagy and apoptotic cell death pathways

(Mizushima N, 2011). To further explain how DAPK regulates LBH589-induced cell death

we studied whether LBH589 induces typical hallmarks of autophagy, a process where DAPK

has been shown to be a key player (Gozuacik D et al., 2008; Inbal B et al., 2002). We used

Western blot analysis to detect expression of autophagy marker LC3.

Western blot analysis with an anti-LC3 antibody revealed that LBH589 treatment caused an

accumulation of autophagosomes in the three colon cancer cell lines with an increased

conversion of cytosolic LC3-I to autophagosomal membrane-bound LC3-II. LC3-II levels

were more pronounced in DAPK+++ cells followed by DAPK wt and DAPK shRNA cells

(Fig. 20A). Overall, these results suggest that LBH589-induced autophagy is potentiated by

DAPK in colon cancer cells.

To study if DAPK-dependent autophagy is a general effect observed with other HDACi we

treated cells with Vorinostat and analysed LC3-II levels. As shown in Figure 20B, stimulation

with Vorinostat induced LC3-II autophagosome formation in all three cell lines in a DAPK-

dependent manner. These data indicate a DAPK-mediated autophagosome formation as a

general effect of HDACi in the treatment of colorectal cancer cells.

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DA

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Furthermore, we used immunofluorescence microscopy analysis to detect LC3-II positive

punctuate staining. As shown in Figure 20, compared with the control cells treatment with

LBH589 caused the punctuate accumulation of LC3-II, representing the new formation of

autophagic vacuoles in the cytoplasm of all three cell lines. The autophagy marker LC3-II

levels were more pronounced in DAPK+++ cells followed by DAPK wt and DAPK shRNA

cells. Furthermore, the LC3-II positive punctuation correlated with DAPK levels, suggesting

that DAPK enhances autophagosome formation. These results are in line with our western

blotting data. Overall, these results suggest that LBH589-induced autophagy is potentiated by

DAPK in colon cancer cells.

Fig. 20. DAPK-dependent autophagy induction after LBH589 treatment. (A, B) The cells were treated either with LBH589 or vorinostat for 24, 48 and 72 h. Protein levels of LC3 were detected by Western blotting. (C-E) The three cell types were seeded on glass coverslips and treated with LBH589 for various time points as indicated. Endogenous LC3 aggregation was detected using immunofluorescence with anti-LC3 antibody. The appearance of punctuated signals of LC3 is a hallmark of autophagy. The picture shows one representative experiment out of two independent experiments.

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Beclin 1

ß-actin

Atg 7

ß-actin

Beclin 1/ß-actin ratio1.0 1.1 0.9 0.9 1.0 0.9 0.9 0.8 1.0 0.8 0.7 0.7

Atg 7/ß-actin ratio1.0 1.2 0.9 1.2 1.0 1.1 1.2 1.3 1.0 1.2 1.1 1.4

DAPK shRNA DAPK wt DAPK+++

Beclin 1

ß-actin

Atg 7

ß-actinß-actin

Beclin 1/ß-actin ratio1.0 1.1 0.9 0.9 1.0 0.9 0.9 0.8 1.0 0.8 0.7 0.7

Atg 7/ß-actin ratio1.0 1.2 0.9 1.2 1.0 1.1 1.2 1.3 1.0 1.2 1.1 1.4 Atg 7/ß-actin ratio1.0 1.2 0.9 1.2 1.0 1.1 1.2 1.3 1.0 1.2 1.1 1.4

DAPK shRNA DAPK wt DAPK+++

4.11.2 Effect of LBH589 on other autophagy markers

In the process of autophagy activation a series of autophagy related genes play an important

role in autophagosome formation (Klionsky DJ et al., 2008). To find out whether other

essential markers of autophagy are induced in response to LBH589 treatment, we analysed the

protein expression of Beclin1 and Atg7. Immunoblotting analysis against Beclin1 and Atg7

revealed nearly no change in protein levels in all three cell lines indicating that these two

molecules might not have a major role in LBH589-induced autophagy (Fig. 21).

Fig. 21. Effect of LBH589 on autophagy related genes Beclin 1 and Atg 7. The indicated cell types were treated with 0.05 µM LBH589 for 24, 48 and 72h. Protein levels of Beclin 1 and Atg 7 were detected by Western blotting. Equal loading was controlled by β-actin. The band intensities were quantified by densitometry analysis. 4.11.3 Inhibition of autophagy decreased acidification of vesicular organelles in human colon tumor cells Previous studies have shown that various chemical inhibitors and also irradiation of cells

caused acidification of vesicular organelles (Paglin S et al., 2001; Newman RA et al., 2007);

however, if DAPK mediates the effects of LBH589 on acidification of vesicular organelles

has not been investigated so far. To test this hypothesis we used Bafilomycin A1, an inhibitor

of autophagy that acts by inhibiting the H+-ATPase responsible for acidification of the

autophagolysosomal vacuoles (Yamamoto A et al., 1998). The formation of acidic vesicular

organelles was determined by acridine orange staining.

As shown in Figure 22, control cells displayed green fluorescence whereas LBH589-treated

cells displayed red fluorescence in all the three cell types at 24 and 48 h. These results

indicate that formation of numerous autophagolysosomal vacuoles after LBH589 treatment in

all the three cell types analysed.

The observed red fluorescence was more pronounced in DAPK+++ cells followed by DAPK

wt and DAPK shRNA cells suggesting that DAPK dependent formation of

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autophagolysosomal vacuoles. On the other hand, pre-treatment with Bafilomycin A1 reduced

the LBH589-induced red fluorescence in all the three cell typtes, indicating prevention of

acidification of vesicular organelles.

Fig. 22. LBH589 induces formation of acidic autophagic vacuoles. (A-C) The cells were treated with LBH589 for 24 or 48 h in the presence or absence of autophagic inhibitor Bafilomycin A1. The formation of acidic vesicular organelles seen by acridine orange staining was observed using inverted fluorescence microscope.

4.11.4 Inhibition of autophagy causes accumulation of LC3-II and p62 protein

LC3-II can accumulate as a result of increased autophagosome formation or impaired

autophagosome-lysosome fusion. To distinguish these two possibilities we next investigated

autophagic flux in human colon cancer cells by assessing the LC3-II turnover in the presence

and absence of Bafilomycin A1. As shown below, treatment with LBH589 caused an increase

in LC3-II levels. Moreover, the increase in LC3-II potentiated over time and correlated with

DAPK levels, suggesting a DAPK-dependent formation of autophagosomes. When compared

with cells treated with Bafilomycin A1 alone, treatment with Bafilomycin A1 and LBH589

caused not only further induction of LC3-I but also a significant increase in conversion of

LC3-I to LC3-II suggesting that the increase in LC3-II was not due to the blockage of

autophagic degradation (Fig. 23).

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The increased autophagic flux was further confirmed by the decrease of p62, an accepted

substrate of autophagy (Li J et al., 2010; Klionsky DJ et al., 2008), whose level decreases

upon autophagy induction and accumulates when autophagy is inhibited. LBH589 treatment

caused a slight decrease of p62 levels in all three cell lines and the LBH589-induced

reduction in p62 levels was prevented by Bafilomycin A1, confirming an autophagy-mediated

p62 degradation. Treatment with Bafilomycin A1 alone resulted in accumulation of p62. This

accumulation was most pronounced in DAPK+++ cells suggesting that the extent of

autophagosome-lysosome fusion is dependent on endogenous DAPK level of a cell. Whereas

in DAPK shRNA and DAPK wt cells LBH589 did not convert the Bafilomycin-induced

increase in p62 levels, in DAPK+++ cells LBH589 decreased p62 levels to the levels seen in

control cells. Thus we suggest that in DAPK+++ cells p62 might lead to tumor suppression

possibly by its own forced degradation (Fig. 23).

Fig. 23. LBH589 causes p62 protein degradation. Cells were pre-stimulated with Bafilomycin A1 for 1 h and then stimulated with LBH589 for 24 and 48 h. Western blotting was performed against LC3 and p62 protein. Bafilomycin A1 increased the accumulation of autophagosomes and p62 protein. All data are representative of two independent experiments. 4.11.5 Crosstalk between LBH589-induced apoptosis and autophagy

In the next series of experiments we aimed to find out if DAPK-dependent autophagy is

responsible for the observed differences in apoptosis induction after LBH589 treatment. For

this the existence of a cross-talk between LBH589-induced apoptosis, autophagy and the role

of DAPK was tested.

At first, we inhibited caspase 3-dependent apoptosis by zVAD and studied autophagosome

formation by Western blotting for LC3-II. In general, combined treatment with zVAD

reinforced the LBH589-induced increase in LC3-II levels in all three cell lines (Fig. 24A)

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independent of the DAPK status suggesting that the less efficient elimination of damaged

tumor cells by apoptosis caused a general increase in autophagosome formation. Moreover,

the data strongly suggest that LBH589-induced autophagy is not an epiphenomenon of

LBH589-induced apoptosis.

Next, we analyzed whether inhibition of autophagy by Bafilomycin A1 influences LBH589-

induced apoptosis. First, we measured the effect of single and combination treatment in the

DAPK wt cells using crystal violet assay. Pre-treatment with Bafilomycin A1 further

increased the rate of apoptotic cells in comparison to LBH589 alone (Fig. 24B). In parallel,

we analyzed the activation of caspase 3 and PARP in all three cell lines. Interestingly, pre-

treatment with Bafilomycin A1 alone caused an increase in active caspase 3 levels in a time-

dependent manner in all three cell lines. PARP cleavage was more prominently induced in

DAPK+++ cells. The enhanced caspase cleavage at 24 h and 48 h was strongly DAPK-

dependent (Fig. 24C). Complete PARP cleavage at 48 h was observed only in DAPK+++

cells. These data show that the induction of autophagy by DAPK partly protects cells from

LBH589-induced apoptosis suggesting a pro-survival role for DAPK.

A

B

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C

Fig. 24. Crosstalk between autophagy and apoptosis. (A) The three cell types were treated with LBH589 in the presence or absence of zVAD for various time points as indicated. The cross inhibition and/or activation were analysed by Western blotting against LC3. (B) The DAPK wt cells (7500 cells/well) were seeded in 96-well cell culture plates. The cells were pre-incubated with Bafilomycin A1 (3 nM) for 1 h and then stimulated with LBH589 (0.05 µM) for 24 h. The cell viability was measured by crystal violet assay. Results are expressed as percentage of the control cells. Data are representative of two independent experiments performed in triplicates. *p<0.05 compared with untreated controls. #P<0.05 compared with LBH589 treated cells. (C) The three cell types were treated with LBH589 in the presence or absence of Bafilomycin A1 for various time points as indicated. The cross inhibition and/or activation were analysed by Western blotting against Caspase 3 and PARP. 4.12 DAPK up-regulation or activation is important for the pro-apoptotic effects of

LBH589 in different colorectal cancer cells

Next we aimed to test if an increase of DAPK levels or its activation is common in colorectal

cancer cells after LBH589 treatment. Investigating two other colorectal cancer cell lines, the

apoptosis-sensitive cell line DLD1 and the apoptosis-resistant cell line HT29, we found an up-

regulation of the DAPK level only in HT29 cells. However the inactive pDAPKSer308 levels

also increased in these cells suggesting an overall lack of DAPK activation for unknown

reasons. In contrast the DAPK level did not change in DLD1 cells whereas the inactive

pDAPKSer308 levels decreased over time indicating DAPK activation in this cell line (Fig.

25A). According to these data a different level of LBH589-induced apoptosis in these two cell

lines was postulated. Indeed, LBH589 induced caspase-3 as well as PARP cleavage in DLD1

but not in HT29 cells, further supporting that DAPK activity might determine the sensitivity

to LBH589 (Fig. 25B).

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To find out whether LBH589 induces autophagy in these two colorectal cancer cell lines, we

detected LC3-II levels. As expected, LBH589 did not increase LC3-II levels as determined by

Western blotting (Fig. 25C). This might be due to the moderate endogenous DAPK levels in

DLD1 cells and on the other hand the lack of DAPK activation in HT29 cells.

Taken together our data suggest that HT29 cells seem to be resistant to LBH589 and do not

show any apoptotic or autophagic responses due to an overall lack of DAPK activation.

D

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Fig. 25. Pro-apoptotic effects of LBH589 might depend on DAPK induction in other colon tumor cell lines. (A-C) DLD1 and HT29 cells were treated with 0.05 µM LBH589 for 24, 48 and 72 h. Whole-cell protein lysates were prepared and Western Blot analysis was performed using the indicated antibodies. (D) Response of DLD1 and HT29 cells and role of DAPK in LBH589-induced pro-apoptotic effects. The symbols indicate unaltered (=), increased ( ↑ ), or decreased ( ↓ ) protein levels of DAPK, pDAPKSer308, caspase 3/ PARP cleavage and LC3-II, respectively. 4.13 DAPK up-regulation and activation is a general phenomenon and is correlated with

the mode of cell death after treatment with HDACi

Although it has been reported that HDACi such as butyrate did not increase DAPK mRNA

levels in HCT116 wt cells (Wilson AJ et al., 2010) DAPK protein levels have not been

investigated so far. In a separate experiment, we thus used two chemically related hydroxamic

acid derivatives, Trichostatin A (TSA) and vorinostat to address the role of DAPK for their

anti-tumor effects. The human colon tumor cells having different DAPK levels i.e. DAPK wt,

DAPK shRNA, and DAPK+++ cells were stimulated either with TSA or Vorinostat for 24, 48

and 72 h. As expected, TSA and vorinostat induced an up-regulation of DAPK in all three cell

lines (Fig. 26). Whereas vorinostat also induced activation of DAPK as determined by the

simultaneous loss of inactive pDAPKSer308, TSA did not reduce pDAPKSer308 levels except for

the early 24h time point. Both drugs induced the highest induction of DAPK and the most

pronounced DAPK activation in DAPK+++ cells, suggesting that the activation of DAPK has

an important role in mediating the pro-apoptotic response of TSA and vorinostat (Fig. 26A,

B).

To correlate these findings with the pro-apoptotic effects of TSA and vorinostat, we

performed immunoblot analysis for caspase 3 and PARP. Stimulation with TSA induced

cleavage of caspase 3 and PARP in all the three cell lines with the most noticeable effects

seen in DAPK+++ cells. Interestingly caspase 3 cleavage was also very pronounced at 24 h in

DAPKsh cells, the time point where inactive DAPKSer308 was undetectable. Of note there

were no differences in apoptosis induction between the three cell lines after vorinostat

treatment suggesting that DAPK does not seems to play a role in apoptosis (Fig. 26C, D).

To study if the effects of TSA and vorinostat on autophagy are mediated by DAPK, we

performed immunoblotting analysis of LC3-II. As shown in Figure 26, stimulation either with

TSA or vorinostat induces LC3-II autophagosome formation in all three cell lines with a more

prominent effect in DAPK+++ cells. These data indicate a DAPK-mediated autophagosome

formation as a general effect of HDACi in the treatment of colorectal cancer cells and reflects

that DAPK seems to be involved in the immediate early stress response to HDACi-induced

DNA-damage.

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Fig. 26. The anti-tumor effect of HDACi depends on DAPK up-regulation and activation. (A-F) DAPK wt cells having different DAPK status were stimulated with either 0.66 µM TSA or 2 µM Vorinostat for indicated times. After the treatment, whole-cell protein

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lysates were prepared and Western blotting analysis was performed against DAPK, pDAPKSer308, caspase 3/ PARP cleavage and LC3-II. 4.14 In vivo colon tumor xenografts

4.14.1 LBH589 suppresses the growth of colon tumor xenografts

To further evaluate the anti-tumor activity of LBH589 in vivo, DAPK wt and DAPK shRNA

colon tumor xenografts were generated in nude mice and treated by daily intraperitoneal

injections of 10mg/kg LBH589 for 30 days. Tumor size of each animal was determined daily

by measurement using a calliper square. At the end of LBH589 treatment (i.e. 30 days) all

animals were sacrificed by cervical dislocation and tissue samples were collected. In general,

LBH589 significantly suppressed the growth of both colon xenograft mouse models

irrespective of DAPK levels when compared to control animals. As shown in Figure 27, a

significant difference (p<0.05) of tumor growth became apparent from day 13 and 15 in

DAPK shRNA and DAPK wt colon xenograft mice respectively. We did not observe any

significant differences in animal weight in all animal groups during the whole experimental

set up.

Fig. 27. Influence of LBH589 on tumor growth in DAPK wt and DAPK shRNA xenografts. Animals with DAPK wt and DAPK shRNA tumors were injected with daily i.p. injections of 10 mg/kg LBH589 or vehicle control for 30 days. Between seventeen to twenty seven animals per group were used in the xenograft studies. The data was normalized against control animals and set the value 1.0 at the start of LBH589 treatment (day 1). Tumor size in

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C LBHDAPK wt

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the treatment group was expressed relative to control animals. * P < 0.05 compared with untreated animals.

4.14.2 LBH589 up-regulates DAPK in colon tumor xenografts

To further corroborate our findings in vivo we aimed to investigate DAPK protein levels in

mice xenograft tissues by immunohistochemistry. For this at the end of the treatment, tumor

samples were collected and fixed in 10% phosphate-buffered formalin or snap-frozen in liquid

nitrogen. As expected, the total DAPK protein level of control animals was significantly

lower in the DAPK shRNA cells than in DAPK wt cells (Fig. 28). In addition, LBH589

increased DAPK protein levels in the cytoplasm of tumor cells of both DAPK wt and DAPK

shRNA xenografts but to a higher extent in DAPK wt cells. These results are in line with our

in vitro observations where LBH589 showed significant up-regulation of DAPK. Moreover,

immunohistochemical analysis of the tissues revealed a high number of necrotic cells with

loss of cell integrity and nuclei in both cell types in LBH589 treated mice.

Fig. 28. Influence of LBH589 on DAPK expression in nude mice xenografts. Immunohistochemical analysis of DAPK protein expression in DAPK wt and DAPK shRNA mice xenograft tissues. DAPK antibody showed up-regulation of the DAPK levels in DAPK wt and DAPK shRNA mice xenograft tissues. Arrows indicate extensive necrosis after LBH589 treatment. Magnification: X200; insets Magnification: X400. * P < 0.05 compared with untreated animals.

4.14.3 LBH589 reduces proliferation in DAPK wt and DAPK shRNA xenografts

In order to further confirm anti-proliferative function of LBH589 in vivo, we have performed

immunohistochemical analysis using Ki-67 antibody. At the end of the treatment, all animals

were sacrificed by cervical dislocation and tumor samples were collected. The collected

tumors were fixed in 10% phosphate-buffered formalin or snap-frozen in liquid nitrogen.

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Immunohistochemical analysis showed that Ki67 score is significantly (p<0.05) decreased in

the nuclei in both colon tumor xenografts upon LBH589 treatment when compared to

respective controls (Fig. 29).These results suggest that LBH589 significantly suppresses

proliferation in vivo, too and depicts its anti-tumorigenic function. Similar to in vivo growth

data, the reduction in proliferation was also independent of DAPK levels.

Fig. 29. Influence of LBH589 on the expression of ki-67. Immunohistochemical analysis of Ki-67 in DAPK wt and DAPK shRNA xenograft tumor tissues. Ki-67 antibody showed a significant reduction in proliferation in LBH589 treated animals compared to respective controls. Magnification: X200. * P < 0.05 compared with untreated controls. 4.14.4 LBH589 does not suppress growth of HT29 colon tumor xenografts

To evaluate the anti-tumor activity of LBH589 in vivo, we used another human colon tumor

cell line HT29 which has mutant form of p53 gene. The HT29 colon tumor xenografts were

generated in nude mice and treated daily with intraperitoneal injections of 10 mg/kg LBH589

for 22 days. As shown in Figure 30, LBH589 treatment did not suppress the growth of HT29

colon xenografts when compared to untreated control xenograft mice, indicating that HT29

xenografts are resistant to LBH589 treatment. These results are in line with our in vitro

findings; where HT29 colon tumor cells are less sensitive to LBH589 when compared to

DAPK wt colon tumor cells.

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Fig. 30. Influence of LBH589 on tumor growth in nude mice. Animals with HT29 tumors were injected with daily i.p. injections of 10 mg/kg LBH589 or vehicle control for 22 days. Twelve animals per group were used in the xenograft studies. At the end of LBH589 treatment (i.e. 22 days) all animals were sacrificed by cervical dislocation and tissue samples were collected. The data were normalized against vehicle treated animals and set the value 1.0 at the start of LBH589 treatment. Tumor size in the treatment group was expressed relative to control animals. 4.14.5 µPET imaging with an 18F-fluoroglycosylated RGD peptide ([18F]FGlc-RGD) in

HT29-xenograft mice

We used [18F]FGlc-RGD for µPET imaging of αvβ3 integrin expression in HT29 xenografts to

assess the response of histone deacetylase inhibitor-LBH589 on tumor-angiogenesis. The

HT29 bearing animals were untreated or treated with 10 mg/kg LBH589 for 11 days. Static

µPET images with [18F]FGIc-RGD were acquired on day 0, day 4 and day 11 upon LBH589

treatment. As shown in Figure 31, interestingly the tumor size is continuously increasing from

day 0 to day 11 after LBH589 treatment, indicating that there is no effect of LBH on tumor-

angiogenesis in vivo. These results further confirm the resistance of HT-29 xenografts to

LBH589 treatment (Fig. 30).

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Fig. 31. Small-animal PET images of HT29 xenografts injected with [18F]FGlc-RGD after administration of 10 mg/kg LBH589 (images are derived from data at 45-60 min post injection). Representative [18F]FGlc-RGD images on day 0, day 4 and day 11 of HT29 xenografts. The corresponding blocking experiment proved the specific binding of [18F]FGlc-RGD in vivo by competition with cRGDfV (10 mg/Kg).

Discussion

64

5. Discussion

In this study, we characterized the role of tumor suppressor protein DAPK in LBH589-

induced cytotoxicity using HCT116 colorectal tumor cells having different endogenous

DAPK levels i.e. HCT116 wt, DAPK shRNA (DAPK knockdown cell line), and DAPK +++

(DAPK overexpressing cell line). Tumor suppressor proteins, such as DAPK are crucial for

the correct execution of cell death under stress conditions. The novel multi-target HDAC

inhibitor LBH589 has been studied in various cancers, including colon cancer but a direct

involvement of DAPK was not studied yet and whether DAPK is being activated in this

cellular setting was not addressed as well.

In this study, we showed that LBH589 induces DAPK-mediated cell death pathways i.e.,

apoptosis and autophagy in human colon tumor cells having different levels of DAPK i.e.

DAPK wt, DAPK shRNA, and DAPK+++.

We demonstrate that LBH589

• Induces up-regulation and activation of DAPK in vitro

• Inhibits cell proliferation in a DAPK-independent manner and reduces the long-term

survival in human colon tumor cells

• Induces apoptosis in a DAPK-independent manner

• Induces autophagy in a DAPK-dependent manner

• In vivo: Reduces proliferation, suppresses colon tumor xenografts growth and up-

regulates DAPK in DAPK wt and DAPK shRNA colon tumor xenografts

We also show that

• The inhibition of autophagy decreased acidification of vesicular organelles, causes

accumulation of LC3-II and p62 proteins

• There exists a crosstalk between LBH589-induced apoptosis and autophagy

• DAPK up-regulation or activation is important for the pro-apoptotic effects of

LBH589 in different colorectal cancer cells i.e., HT29 and DLD1

• DAPK up-regulation and activation is a general phenomenon and is correlated with

the mode of cell death after treatment with other HDACi such asTrichostatin A and

Vorinostat

One of the interesting observations in this study was that two histone deacetylase inhibitors,

LBH589 and vorinostat induced almost similar effects in colon tumor cells having different

DAPK levels i.e. up-regulation and activation of DAPK, induction of apoptosis as well as

autophagy.

Discussion

65

In the present study, we have established first a suitable model to study the role of DAPK in

histone deacetylase inbhibitor LBH589 induced signaling pathways in human colon tumor

cells. We have demonstrated that shRNA knockdown of DAPK in the human colon tumor cell

line-HCT116 proved to be highly effective. As demonstrated by real-time RT-PCR and

Western blotting, we achieved 76% reduction of DAPK gene expression, which was sufficient

to study the multiple roles of DAPK in HCT116 cells after LBH589 exposure. In this regard

Gozuacik D et al. (2008) studied the function of DAPK in mouse embryonic fibroblasts

isolated from DAPK knockout mice. They reported that knockout of DAPK decreased both

apoptosis and autophagy and also led to the protection from endoplasmic reticulum-induced

cell death.

In addition, we demonstrated that tamoxifen-inducible overexpression of DAPK system in the

human colon tumor cell line-HCT116 also proved to be highly effective. As demonstrated by

real-time RT-PCR and Western blotting, tamoxifen alone only slightly enhanced mRNA

expression and slightly decreased pDAPK/DAPK ratio when compared to LBH589. However,

only combination showed a remarkable increase in the DAPK levels. These cells were used as

an internal control of unphysiologically high DAPK levels, and to clarify the involvement of

DAPK in LBH589 activity. Likewise, to study the role of DAPK in membrane blebbing or

autophagy, Inbal B et al. (2002) used in their study constitutively active constructs of DAPK

and DRP-1 (DAPK ∆CaM and DRP-1 ∆73, respectively). They showed that the activated

forms of DAPK and DRP-1 induced both membrane blebbing and autophagy and they also

claimed that these two kinases are necessary for cell death.

The two engineered cell lines, DAPK shRNA and DAPK +++ along with parental cell line i.e.

HCT116 wt (DAPK wt) showed an increase in kinase activity of DAPK when compared to

respective controls. The shRNA-mediated silencing strategy and tamoxifen-inducible

overexpression of DAPK system did not influence the normal cell growth and cell

proliferation when compared to parental cell line DAPK wt. Thus these three cell lines are a

suitable model to study the role of DAPK after LBH589 treatment in colorectal cancer cells.

5.1 LBH589 induces the acetylation of core histones H3 and H4

Acetylation and deacetylation of histones play an important role in the regulation of gene

expression (Lehrmann H et al., 2002; Xu WS et al., 2007). HDACi are known to induce the

histone acetylation by inhibiting the activity of histone deacetylases (HDAC). Previous

studies have demonstrated that HDACi can induce acetylation of histones in various cancers

(Tang YA et al., 2010; Emanuele S et al., 2008; Romanski A et al., 2004) and this was also

Discussion

66

confirmed by our work, in which the results of Western blot analysis showed an increase in

the acetylation levels of both core histones H3 and H4 in all the three cell lines analyzed.

These results suggest that HDACi induces gene expression by enhancing the acetylation of

histones in human colon tumor cells having different DAPK levels. These results are expected

as LBH589 is a histone deacetylase inhibitor which induces the histone acetylation by

inhibiting the activity of histone deacetylases.

5.2 LBH589 up-regulates tumor suppressor DAPK expression in vitro and in vivo

Several studies have demonstrated that tumor suppressor DAPK expression is lost in many

cancers, including nasopharyngeal carcinomas, pituitary adenomas, lymphomas, colorectal

and gastric cancers. Loss of DAPK is mainly because of aberrant methylation and/or histone

deacetylation and is associated with poor prognosis (Satoh A et al., 2002; Katzenellenbogen

RA et al., 1999; Simpson DJ et al., 2002; Dong SM et al., 2001). To date it is not known

whether inhibition of histone deacetylases can influence the expression of DAPK in human

colon tumor cells. To clarify this issue, we used LBH589 to assess if it can influence the

DAPK expression in human colon tumor cells having different DAPK levels. In this study, we

have demonstrated that LBH589 up-regulates and/or activates DAPK in all the three cell

lines. In addition, we have also demonstrated that LBH589 up-regulates DAPK in an in vivo

colon xenograft mouse model. Our findings demonstrated that another HDACi vorinostat also

influenced the expression of DAPK in human colon tumor cells, suggesting that histone

acetylation might be reinforcing the transcription of DAPK. Zhang X et al. (2006) reported

that HDACi TSA increased the chemosensitivity of anticancer drugs 5-FU, PTX, or SN38 in

gastric cancer cell lines by up-regulating the expression of DAPK1, DAPK2, p21 and p53.

WU J et al. (2010) also reported that treatment with HDACi TSA induces apoptosis in lung

cancer cell lines and enhances sensitivity of cells to cisplatin by up-regulating the DAPK. In

addition, two other groups also reported that HDACi sodium butyrate-induced DAPK

expression led to apoptosis by reducing the FAK protein level in Raji cells and also in human

gastric cancer cells (Zhang HT et al., 2007; Shin H et al., 2012). Overall, we conclude that our

results are consistent with the previous reports that HDACi activates DAPK in various

cancers and activated DAPK induces cell death pathways.

DAPK is known to be negatively regulated by auto-phosphorylation of serine308. The

dephosphorylation of serine308 on DAPK can activate DAPK and induces several signaling

pathways (Shamloo M et al., 2005; Jin Y et al., 2006). In our study, we have shown that

pDAPK levels were decreased and DAPK levels were increased with subsequent reduction in

Discussion

67

pDAPK/DAPK ratio after LBH589 treatment suggesting an increase in kinase activity of

DAPK. Furthermore, our kinase assay studies demonstrated that there was a general increase

in kinase activity of DAPK in all three cell lines reaching the same levels in DAPK shRNA

and DAPK wt cells and the highest level in DAPK +++ cells after LBH589 treatment. From

this we suggest that autophagy seems to be rather independent of kinase activity. Our results

showed that LBH589 is simultaneously able to induce apoptosis and autophagy in DAPK-

independent or -dependent manner, respectively. Thus the histone deacetylase inhibitors

might be a potential chemotherapeutic drug for the treatment of colorectal cancer.

5.3 LBH589 reduces the proliferation in vitro and in vivo and causes G2/M cell cycle

arrest in a DAPK-independent manner

Several studies have already demonstrated that HDACi such as LBH589, SAHA, TSA, and

phenylbutyrate induce the expression of the cell cycle kinase inhibitor p21WAF1 in various

transformed cells (Prystowsky MB et al., 2009; Richon VM et al., 1996; Xiao H et al., 1999;

DiGiuseppe JA et al., 1999). In this regard Ocker M and Schneider-stock R et al in 2007

reviewed that HDAC inhibitors induce the expression of p21WAF1 via increased histone

acetylation around the p21WAF1 promoter and/or the Sp1 sites on the p21WAF1 promoter

releasing the repressor HDACi from its binding. They also reported that expression of

p21WAF1 may play a critical role in various cellular signaling pathways such as cell

differentiation, apoptosis and cell division.

In this study, we have examined the effects of LBH589 on proliferation and acetylation of

histones in human colon tumor cells having different DAPK levels. In vitro, using different

molecular techniques we showed that treatment with LBH589 significantly decreases cell

proliferation in parental cell line HCT116 wt cells as well as in the two engineered cell lines,

DAPK shRNA and DAPK +++ cells in a DAPK-independent manner. In addition, we also

demonstrated significant decreases in cell proliferation rate in HCT116 wt and DAPK shRNA

colon xenografts in a DAPK-independent manner after LBH589 treatment when compared to

control mice. To our knowledge, this is the first study showing that LBH589 reduces human

colon tumor cell proliferation in a DAPK-independent manner both in vitro and in vivo.

Cell cycle arrest either at G1 or G2/M phase in response to HDACi has been reported for

various cancers (Fazzone W et al., 2009; Bolden JE et al., 2006; Munster PN et al., 2001), and

this was also confirmed by our own study, in which the results of flow cytometry analysis

showed cell cycle arrest in the G2/M phase which was DAPK-independent (Gandesiri M et

al., 2012).

Discussion

68

In our study, we used xCELLigence system to monitor cell proliferation in real-time in the

three cell lines-DAPK shRNA, DAPK wt and DAPK+++. The xCELLigence system

measures several parameters like changes in cell size, cell viability, cell number, morphology,

and cell adhesion continuously. In accordance to this, we showed very similar growth

characteristics reaching a stable plateau phase without spontaneously undergoing cell death.

5.4 LBH589 causes DNA damage

Induction of double strand breaks (DSBs) is one of the common outcomes after exposure to

ionising radiation or drugs/cytotoxic agents. The phosphorylation of H2AX (pH2AX or γ-

H2AX) is considered as a marker for DSBs (Kim YB et al., 2012; Redon CE et al., 2010). As

expected, we observed that LBH589 induced the phosphorylation of H2AX in all the three

cell lines in a DAPK-independent manner. This observation is in line with previous studies

showing that HDACi TSA (Zhang F et al., 2009) and LBH589 (Scuto A et al., 2008)

phosphorylate H2AX as an early cellular response to DNA damage. However, DNA damage

was less evident in DAPK+++ cells despite of pronounced apoptosis. The reason might be

that under conditions of high DAPK activity, damaged cells are more efficiently eliminated

by autophagy-driven cell death or that autophagy seems to protect the cells from severe

damage altogether.

5.5 LBH589 induces apoptosis in a DAPK-independent manner

HDACi have recently emerged as potent anticancer agents for both hematologic and solid

cancers. Strikingly, these inhibitors showed promising anti-tumor activity both in vitro and in

vivo (Lee MJ et al., 2008; Wu J et al., 2010). Previous studies have demonstrated that

LBH589 induces apoptosis in various cancer cell lines, including colon cancer (Atadja P et

al., 2009; Hague A et al., 1993). In the last decade the tumor suppressor DAPK has been

linked with regulation of apoptosis and autophagy (Bialik S and Kimchi A et al., 2006).

DAPK is a calcium/calmodulin regulated cytoskeleton-associated serine/threonine kinase and

many of the DAPK substrates are cytoskeleton-associated proteins (Lin Y et al., 2010).

DAPK interacts with different MAPKs such as ERK (Chen CH et al., 2005) or p38 (Bajbouj

K et al., 2009) in response to inflammatory apoptotic stimuli. Otherwise DAPK

phosphorylation by RSK and Src is considered to rather protect from cell death (Anjum R et

al., 2005; Wang WJ et al., 2007). Thus, DAPK can serve as a mediator between death-

inducing and pro-survival signals (Michie AM et al., 2010).

Discussion

69

The role of DAPK in LBH589-induced cytotoxicity has not yet been studied. For the first

time we show that dephosphorylation of DAPK at ser 308 is the most important mechanism in

DAPK activation by HDACi. However, LBH589-induced apoptosis seems to be independent

of DAPK levels. Although the kinase activity of DAPK has been shown to be crucial for

DAPK mediated cell death pathways, so far it is not known whether HDACi LBH589-

induced cell death also requires DAPK kinase activity in human colon tumor cells. To verify

this we used a recently described DAPK inhibitor in our study. By using structure based

virtual screening, Okamoto M et al. (2010) recently identified this novel potent and selective

DAPK inhibitor i.e. (4Z)-2-phenyl-4-(pyridin-3-ylmethylidene)-4,5-dihydro-1,3-oxazol-5-

one. This compound showed excellent selectivity for inhibition of DAPK catalytic activity.

To our knowledge, it was used for the first time in our lab and it has not been used elsewhere

in the world. Using this DAPK inhibitor, we demonstrated that pre-treatment of DAPK wt and

DAPK +++ cells did not alter the LBH589-induced caspase 3 and PARP cleavage suggesting

that DAPK´s kinase activity is not important for the observed caspase 3-dependent cell death.

This is in full agreement with our findings that DAPK sh RNA cells and DAPK wt cells have

the same level of catalytic activity suggesting that catalytic activity of DAPK plays only a

minor role for caspase 3-dependent apoptosis induction.

Several studies have already reported that HT-29 human colon tumor cell line was highly

resistant when compared to other human colon tumor cell lines after treating with variety of

apoptosis inducing drugs. Na YS et al. (2011) recently reported that the HDACi PXD101 or

SB-38, the active form of irinotecan, showed dose-dependly anti-proliferative effects on both

human colon tumor cell lines HCT116 and HT-29. However they showed that HCT116 cells

are more sensitive than HT-29 cell line. Fazzone W et al. (2009) reported that in human colon

tumor cell lines HCT116 and HT29 treatment with HDACi vorinostat and LBH589, caused

apoptosis in both cell lines but again HT29 cells were less susceptible to both HDACi. In

another study, we have shown that Thymoquinone (TQ), a product of black seed, induces

apoptosis in human colon cancer cell line DLD-1 but not in HT-29 cells (El-Najjar N et al.,

2009). In the current study, we showed that LBH589 did not activate apoptotic markers such

as caspase-3, PARP and autophagy marker LC3‑II in HT29 colon cancer cells. These results

suggest that HT29 cells are resistant to induction of apoptosis or autophagy by LBH589. One

possible reason might be that HT-29 cells show an increase in DAPK as well as inactive

pDAPK levels suggesting the lack of activation of DAPK. Another possible explanation

might be that after LBH589 treatment the phosphatase (eg: protein phosphates 2A, Widau RC

Discussion

70

et al., 2010) that dephosphorylates Ser308 is not available or inhibited due to some unknown

reasons. The later hypothetical explanation needs to be further validated.

To further strengthen these interesting findings, we performed in vivo experiments in HT-29

mouse colon xenografts. We demonstrated that LBH589 did not suppress the growth of HT29

colon xenografts when compared to untreated control xenograft mice. To further support our

hypothesis that HT29 cells are resistant, we performed µPET imaging of αvβ3 integrin

expression in HT29 xenografts to assess the effect of LBH589 on tumor-angiogenesis. Our

results suggested that there is no effect of LBH on tumor angiogenesis in vivo. Altogether we

demonstrated that HT-29 cells are resistant to LBH589 both in vitro and in vivo. This is in

agreement with LaBonte MJ et al. in 2009 who analyzed the gene expression profiles in these

two cell lines after treatment with the two HDAC inhibitors, SAHA and LBH589. They found

significant cell line specific alterations on genes involved in apoptosis, mitosis, angiogenesis,

and DNA replication. These different cell line specific effects most probably explain the

differential sensitivities of HCT116 wt and HT29 cell lines to both HDAC inhibitors. Overall,

the results from our group and also from others confirm that HT-29 cells are generally

resistant to various types of stimuli. Further studies are necessary to unravel exact molecular

mechanisms responsible for resistance of human colon tumor cell line HT-29 after HDACi

treatment.

5.6 LBH589 induces autophagy in a DAPK-dependent manner

Autophagy is either a protective or survival mechanism in response to intracellular stress such

as radiotherapy and chemotherapy or a mechanism of cell death under certain conditions

especially in response to treatments that trigger caspase-independent autophagy (Gozuacik D

and Kimchi A et al., 2007). Recently, Lin Y et al. (2010) summarized DAPK interacting

proteins, the different methods to detect these interactions, and the responsible DAPK domain

mediating these interactions. Microtubule-associated protein 1B (MAP1B), Beclin 1, and

tuberous sclerosis 2 (TSC 2) are the proteins that interact with DAPK and might play a role in

autophagy. For Beclin 1 the binding region on DAPK was not identified whereas for MAP1B

and TSC 2 the binding is mediated through the kinase domain and death domain, respectively.

In this regard, Harrison B et al. (2008) showed that DAPK interacts with MAP1B and LC3

protein resulting in the stimulation of DAPK-dependent membrane blebbing and autophagy.

It has been reported that DAPK phosphorylates the autophagy-associated molecule Beclin 1,

thus reducing the BH3-pocket interaction between Beclin 1 and its inhibitor Bcl-2, finally

promoting autophagy (Zalckvar E et al., 2009). However, down-regulation of Bcl-2 triggers

autophagy, but does not promote apoptosis in leukemic cells (Saeki K et al., 2000). The two

Discussion

71

important autophagy related genes are Beclin 1 and Atg 7, which play an important role in the

regulation of autophagy. Autophgy is a multi-step process and Beclin 1 is involved in the

early stage of autophagosome formation. Recently, it has been described that two distinct

types of autophagy-canonical and non-canonical autophagy pathways exist in cancer cells

(Komatsu M et al., 2007; Scarlatti F et al., 2008; Miracco C et al., 2010). In our study,

LBH589 treatment did not change the activity of autophagy-related proteins Beclin1 and Atg7

in all three cell lines. This might be due to the fact that LBH589-DAPK pathway is a non-

canonical autophagy pathway. Further studies are required to investigate that interesting

notion.

Lin Y et al. (2010) reviewed that DAPK can act either as a pro-survival factor or promotes

apoptosis in autophagy signalling pathways. Autophagy related genes such as LC3

(microtubule-associated protein 1 light chain 3) play a critical role in mammalian autophagy

and are widely used to monitor autophagy. LC3 is processed in to soluble LC3-I (MW, 18

kDa) and a lipidated LC3‑II (MW, 16 kDa), which is present on only autophagosomes. Thus,

LC3‑II levels correlate with the number/amount of autophagosomes and are considered as a

classical autophagy marker (Mizushima N et al., 2007; Klinosky DJ et al., 2008; Miracco C et

al., 2010). Previous studies have reported that blockage of autophagy with Bafilomycin A1

led to an accumulation of autophagosomal structures (Yamamoto A et al., 1998). Here we

show that Bafilomycin A1 prevented the acidification of lysosomes and caused accumulation

of LC3-II protein.

If autophagy mediates the cytotoxicity we would expect that Bafilomycin A1 co-treatment

would lead to an attenuated cell death. We analysed PARP, which detects and signals

downstream of DNA strand breaks and subsequently activates DNA repair programs or cell

death pathways. It is activated at an intermediate stage of apoptosis and is then cleaved and

inactivated at a late stage by apoptotic proteases, such as caspase 3. In our system, an increase

in PARP cleavage was noted by combination treatment in all three cell lines and complete

cleavage occurred in DAPK+++ cells further indicating that DAPK-driven autophagy might

initially be triggered to protect the cells by sequestering and degrading damaged organelles.

Thus, inhibition of autophagy sensitized the tumor cells to treatment-induced apoptosis and

DAPK may be responsible to initiate apoptosis if the capacity for autophagy is insufficient.

This indicates that under autophagy inhibition DAPK could act to switch between autophagy

and apoptosis.

p62 is known to be a selective substrate of the autophagic machinery and impaired autophagy

is accompanied by the accumulation of p62. In this study, Bafilomycin A1 treatment

Discussion

72

increased the level of p62 in a DAPK-dependent manner suggesting a role for DAPK in

autophagic flux induced by LBH589. Bafilomycin A1 failed to block the LBH589-induced

p62 degradation in the DAPK+++ cells perhaps due to their artificially high DAPK levels.

Alternatively, a proteasomal degradation of p62 after combination treatment cannot be

excluded (Myeku N et al., 2011). Furthermore we showed that Bafilomycin A1 inhibited

lysosomal hydrolases by decreasing the acidification of lysosomal compartment. These data

suggest that the vacuolar type H+-ATPase is essential for the regulation of the late stage of

macroautophagy after LBH589 treatment. Consistent with other studies, our reports showed

that a commonly synthetic peptide inhibitor of caspases, zVAD, slightly enhanced LC3-II

levels in all three cell lines irrespective of the DAPK expression and activity. The reason

could be that zVAD inhibits both apoptosis and autophagic flux by preventing lysosomal

degradation of autophagosome contents (Wu YT et al., 2008; Yu L et al., 2004). Our data

show that caspase 3 cleavage in LBH589-treated cells was recovered upon zVAD treatment,

yet there was no rescue of death in crystal violet assay. This suggests both caspase-dependent

and caspase-independent apoptotic mechanisms are at play after LBH589 treatment. We have

recently shown in liver cancer cells that LBH589 up-regulated CHOP, a marker of the

unfolded protein response and endoplasmatic reticulum (ER) stress (Di Fazio P et al., 2010).

There is evidence that ER stress can facilitate autophagosome formation and thus triggers

autophagy (Cheng Y et al., 2011). ER-stress-induced autophagy is important for clearing

polyubiquitinylated protein aggregates and consequently protects against cell death. In this

respect, it has been shown that treatment of neuroblastoma cells with ER stressors induced

autophagosome formation and autophagy-inhibited cells demonstrated an increased

vulnerability to ER stress and increased caspase activity (Ogata M et al., 2006). In our study,

there was a clear increase in PARP cleavage when autophagy was blocked as a sign of

augmented apoptosis induction supporting the initiation of cytoprotective autophagy in a

DAPK-dependent manner. Gozuacik D et al. (2008) have been reported that DAPK is

important in integrating autophagy and apoptosis induced by ER stress. They emphasize the

fact that there are always multiple executers securing redundancy in signalling pathways. This

finally results in a robust and fine-tuned cellular stress response and DAPK´s regulatory role

could be rather upstream of these signalling cascades. Furthermore, we observed that

LBH589-induced autophagy was rather dependent on total protein levels than on catalytic

activity of DAPK. We did not find significant difference in catalytic activity between DAPK

shRNA and DAPK wt cells after LBH589 treatment. Therefore, we assume that autophagy

induction is predominantly caused by DAPK protein interactions than by its catalytic activity.

Discussion

73

Under autophagy deficient conditions, DAPK plays a role in sensitizing human colon tumor

cells to LBH589-induced apoptosis.

A schematic model of DAPK´s action after LBH589 treatment in autophagy competent and

deficient cells is given in Fig. 32. LBH589 treatment simultaneously induces DAPK-

dependent autophagy but DAPK-independent apoptosis in human colon tumor cells.

Inhibition of autophagy sensitized the tumor cells to treatment-induced apoptosis and DAPK

may be responsible to initiate apoptosis if the capacity for autophagy is insufficient. This

indicates that under autophagy inhibition DAPK could act to switch between autophagy and

apoptosis.

Fig. 32. Schematic model illustrating that DAPK commits cells to apoptosis under autophagy-deficient conditions.

5.7 Conclusion and outlook

In conclusion, our data clearly demonstrated that histone deacetylase inhibitor LBH589

simultaneously induces apoptosis and autophagy in human colon tumor cells in a DAPK-

independent and -dependent manner, respectively. One of the major findings of our study is

that LBH589 up-regulates and activates the tumor suppressor DAPK by dephosphorylating

the inactive pser308 form. In our study, we have shown that the DAPK mRNA expression can

be reinforced by inhibiting the histone deacetylation. Our data suggest that manipulation of

autophagy could improve the efficacy of anticancer therapies and should be taken into

account in tumor cells that overexpress DAPK, e.g. in non-methylated tumors.

In near future, our further studies will focus on combination drug treatment approaches such

as DNMT inhibitors or irradiation or traditional chemotherapeutic agents (5-FU, irinotecan,

oxaliplatin, cetuximab, leucovorin or bevacizumab) in the combination with HDAC inhibitors

in order to attain better therapeutic efficacy especially in the resistant cell line HT-29.

Combination therapies with these agents/drugs might be very interesting as they might yield

Discussion

74

reversal of methylated genes and/or reinforcement of gene expression of tumor suppressor

genes such as DAPK. In addition, the combination of drugs might augment survival rate and

also decrease the toxicity/side effects. The current study is verifying the high potential of

LBH589 for the treatment of colorectal cancer. Finally, we provide a new insight into

LBH589-induced cell death pathways in human colon tumor cells.

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7. Curriculum Vitae First Name: Muktheshwar Surname: Gandesiri 7.1 List of Original Research Publications 1. Chakilam S, Gandesiri M, Rau TT, Agaimy A, Mahadevan V, Ivanovska J, Wirtz R, Schulze-Luehrmann J, Benderska N, Wittkopf N, Chellappan A, Ruemmele P, Vieth M,

Rave-Fränk M, Christiansen H, Hartmann A, Neufert C, Atreya R, Becker C, Steinberg P, Schneider-Stock R. Death associated protein kinase (DAPK) controls STAT3 activity in intestinal epithelial cells (Accepted; American Journal of Pathology). 2. Gandesiri M, Chakilam S, Ivanovska J, Benderska N, Ocker M, Di Fazio P, Feoktistova M, Gali-Muhtasib H, Rave-Fränk M, Prante O, Christiansen H, Leverkus M, Hartmann A, Schneider-Stock R. DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions. Apoptosis. 2012 Dec; 17(12):1300-15. 3. Kunisch E, Chakilam S, Gandesiri M, Kinne RW. IL-33 regulates TNF-α dependent effects in synovial fibroblasts. Int J Mol Med. 2012 Apr; 29(4):530-40. 4. Benderska N, Chakilam S, Hugle M, Ivanovska J, Gandesiri M, Schulze-Lührmann J, Bajbouj K, Croner R, Schneider-Stock R. Apoptosis Signalling Activated by TNF in the Lower Gastrointestinal Tract - Review. Curr Pharm Biotechnol. 2011 May 24. 5. El-Najjar N, Chatila M, Moukadem H, Vuorela H, Ocker M, Gandesiri M, Schneider-Stock R, Gali-Muhtasib H. Reactive oxygen species mediate thymoquinone-induced apoptosis and activate ERK and JNK signaling. Apoptosis. 2010 Feb;15(2):183-95.

6. Kunisch E* , Gandesiri M* , Fuhrmann R, Roth A, Winter R, Kinne RW. Predominant activation of MAP kinases and pro-destructive/pro-inflammatory features by TNF alpha in early-passage synovial fibroblasts via TNF receptor-1: failure of p38 inhibition to suppress matrix metalloproteinase-1 in rheumatoid arthritis. Ann Rheum Dis. 2007 Aug; 66(8):1043-

51.* both authors contributed equally to this work

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7.2 Poster /Oral Presentations: (Selected only) Oral presentation - S.Chakilam, A.Agaimy, R.Atreya, M. Gandesiri, J. Ivanovska, M. Rave-Fränk, H.Christiansen, T.T.Rau, R. Schneider-Stock; “TNF induces STAT3-mediated inflammation in normal colon epithelial cells”. 96th Jahrestagung der Deutschen Gesellschaft für Pathologie, 31 May-03 June 2012, Berlin, Germany. Oral presentation - Benderska N, Gandesiri M, Ivanovska J, Ziesche E, Chakilam S, Schulze-Lührmann J, Fischer T, Agaimy A, Schneider-Stock R; “DAPK-dependent HSF1 phpsphorylation triggers TNF-induced apoptosis in colon cancer cells”. 95th Jahrestagung der Deutschen Gesellschaft für Pathologie, 16-19 June 2011, Leipzig, Germany. Poster presentation - Jelena Ivanovska, Alexandra Tregubova, Mahadevan Vijayalakshmi, Saritha Chakilam, Muktheshwar Gandesiri, Natalya Benderska, Benjamin Ettle, Arndt Hartmann, Stephan Soeder, Elisabeth Ziesche, Thomas Fischer, Lena Lautscham, Ben Fabry, Gabriela Segerer, Antje Gohla, and regine schneider-stock; “LIMK/cofilin/DAPK complex triggers TNF-induced apoptosis in colorectal tumor cells” 16th International joint meeting of signal transduction, 5-7 November 2012, Weimar, Germany. Poster presentation - Saritha Chakilam, Muktheshwar Gandesiri, Jelena Ivanovska, M. Rave-Fränk, H.Christiansen, Regine Schneider-Stock; “TNF induces STAT3-mediated inflammation in normal colon epithelial cells”. The 4th Annual Meeting on Cancer and Control of Genomic Integrity, 30 Sept-2 Oct 2011, Zandvoort, The Netherlands. Poster presentation - Muktheshwar Gandesiri, Saritha Chakilam, Jelena Ivanovska, Matthias Ocker, Pietro Di Fazio, Martin Leverkus, Feoktistova Masha, Margret Rave-Fränk, Hans Christiansen, Arndt Hartmann, Regine Schneider-Stock; “DAPK-mediated panobinostat-induced autophagy and apoptosis in human colon cancer cells”. The 4th Annual Meeting on Cancer and Control of Genomic Integrity, 30 Sept-2 Oct 2011, Zandvoort, The Netherlands. Poster presentation - Muktheshwar Gandesiri, Saritha Chakilam, Irina Tolstov, Matthias Ocker, H. Christiansen, M. Rave-Fränk, Martin Leverkus, Sonja Rauchschwalbe, Jelena Ivanovska, Arndt Hartmann, Regine Schneider-Stock; “DAPK-dependent apoptosis induction after treatment of colorectal tumor cells with the histone deacetylase inhibitor panobinostat (LBH589)”. AACR, 17-21 April, 2010, Washington DC, USA. Poster presentation - Muktheshwar Gandesiri, Saritha Chakilam, M.Ocker, Jelena Ivanovska, M. Rave-Fraenk, H. Christiansen, Roland Hartig, Markus Schirmer, Regine Schneider-Stock; DAPK-dependent apoptosis induction after treatment with the histone deacetylase inhibitor LBH589; The 2nd Annual Meeting on Cancer and Control of Genomic Integrity, 21-23 August 2009, Stockholm, Sweden. Oral presentation - Saritha Chakilam, M. Rave-Fraenk, Muktheshwar Gandesiri, Jelena Ivanovska, Roland Hartig, Markus Schirmer, H.Christiansen, Regine Schneider-Stock; Resistance to TNF-α induced apoptosis in immortalised human colon epithelial cells; The 2nd Annual Meeting on Cancer and Control of Genomic Integrity, 21-23 August 2009, Stockholm, Sweden. Oral presentation - Jelena Ivanovska, Maria Feoktistova, Kathrin Haase, Muktheshwar Gandesiri, Saritha Chakilam, Martin Leverkus, Regine Schneider-Stock; The use of an inducible lentiviral overexpression vector for Death associated protein kinase (DAPK): a

90

versatile tool for the study of DAPK-dependent signalling pathways; Symposium “Tumor Immunology meets Oncology”, 15-16 May 2009, Halle-Wittenberg, Germany. Poster presentation - Gandesiri M, Chakilam S, Kinne R.W, Kunisch E; Cross-talk between JNK and p38/ERK MAPKs in synovial fibroblasts, 36. Kongress der Deutschen Gesellschaft für Rheumatologie, 24-27 September 2008, Berlin, Germany. Poster presentation - Chakilam S, Gandesiri M, Ukena B, Kinne R.W, Kunisch E; Low concentrations of TNF-α induce a pro-destructive phenotype in synovial fibroblasts; 35th Kongress der Deutschen Gesellschaft für Rheumatologie, 19-22 September 2007, Hamburg, Germany. Poster presentation - Heger J, Schiegnitz E, Muktheshwar G, Asif AR, Piper HM, Euler G; Matrix metalloproteinsases act as repressor of hypertrophy in ventricular cardiomyocytes of rat, Physiologen Tagung, Hannover 2007. Poster presentation - Kunisch E, Gandesiri M, Fuhrmann R, Roth A, Kinne R.W; Alterations of TNF-α/TNF-Receptor 1 induced MAP kinase signal transduction may contribute to the destructive phenotype of RA-SFB; 26th European Workshop for Rheumatology Research, 23-26 Februrary 2006, Hereklion, Crete, Greece. Poster presentation - Kunisch E, Gandesiri M, Lux S, Jansen A, Kinne R.W; Striktly time-dependent regulation of the mRNA expression of pro-inflammatory/pro-destructive genes in SFB by TNF-α; 34. Kongress der Deutschen Gesellschaft für Rheumatologie, 18-21 October 2006, Wiesbaden, Germany. Poster presentation - Heger J, Gandesiri M, Piper HM, Euler G; Characterization of DNA binding protein which acts as a natural repressor of cardiac hypertrophy, American Heart Association, Dallas, USA 2005. Poster presentation - Gandesiri M , Fuhrmann R, Roth A, Winter R, Kinne R.W, Kunisch E; Alterations of TNF-α/TNF-R1-i nduced Jun kinase phosphorylation in RA-SFB may contribute to the pathogenesis of RA; 33. Kongress der Deutschen Gesellschaft für Rheumatologie, 14-17 September 2005, Dreseden, Germany. Oral presentation - Kunisch E, Gandesiri M, Fuhrmann R, Roth A, Winter R, Kinne R.W; RA synovial fibroblasts are partially resistant to p38 MAP kinase inhibition of TNF-alpha/TNF-receptor-1 induced pro-destructive functions; 33. Kongress der Deutschen Gesellschaft für Rheumatologie, 14-17 September 2005, Dreseden, Germany.

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