UNIVERSITI PUTRA MALAYSIA

LOCAL V4-UPM STRAIN NEWCASTLE VIRUS-INDUCED CELL DEATH

OF BRAIN TUMOR CELL LINES

ROHAYA BINTI IBRAHIM

IB 2011 33

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LOCAL V4-UPM STRAIN NEWCASTLE VIRUS-INDUCED CELL DEATH OF BRAIN TUMOR

CELL LINES

By

ROHAYA BINTI IBRAHIM

Thesis Submitted to the School of Graduate Studies,

Universiti Putra Malaysia in Fulfillment of the Requirement for the

Degree of Master of Science

August 2011

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of

the requirement for the degree of Master of Science

LOCAL V4-UPM STRAIN NEWCASTLE VIRUS-INDUCED CELL DEATH OF

BRAIN TUMOR CELL LINES

By

ROHAYA BINTI IBRAHIM

August 2011

Chair: Professor Abdul Manaf Ali, PhD

Faculty: Institute of Bioscience

V4-UPM strain is a local heat resistant variant of the avirulent Australian V4 (QUE)

strain which has higher thermostabilities of infectivity and haemagglutination than the

original. The aims of this study were to analyze the cytolytic effect and mode of cell

death induced by V4-UPM strain of NDV on two human brain tumor cell lines,

glioblastoma multiforme (DBTRG.05MG) and anaplastic astrocytoma cells (U87MG).

In-vitro cytotoxic activity has been performed on both brain tumor cells via standard

MTT assay. At 72 hours post-incubation, the IC50 values obtained were 30 haem

agglutination (HA) unit/ml and 5 HA unit/ml for DBTRG.05MG and U87MG cells,

respectively. In addition, V4-UPM strain inhibited the proliferation of both

DBTRG.05MG and U87MG cells via the same method. Morphological changes of the

treated cells also have been observed sequentially under the inverted light, fluorescence

and electron microscopes. The virus-infected cells showed apoptotic features such as the

cell shrinkage, cell blebbing and formation of apoptotic bodies under the inverted light

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microscope. The differential uptake of acridine orange and propidium iodide dyes

demonstrated the occurrence of apoptosis, mainly by chromatin condensation and DNA

fragmentation that were observed under fluorescence microscope. The next

morphological observation was based on color and nuclear morphology where the nuclei

of treated-cells possessed DNA fragmentation were stained dark brown by 3’,3’-

diaminobenzidine. The distinct ultrastructural changes occurred in the nucleus, cellular

organelles and plasma membrane structures were confirmed to be the apoptotic features

via transmission electron microscopic (TEM) observation. The result of flow cytometric

analyses based on annexin V-FITC that binds phosphatidylserine residue also suggested

the onset of apoptosis for both groups. Meanwhile, the cell cycle was arrested at S phase

followed by apoptosis. Based on these findings, it was confirmed that the V4-UPM

strain induced apoptotic cell death on DBTRG.05MG and U87MG cells. Thus, this

strain has the potential to be developed as an antitumor agent for future treatment

particularly in grade-III brain tumor patients.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Master Sains

STRAIN TEMPATAN V4-UPM DARIPADA VIRUS PENYAKIT NEWCASTLE

MENYEBABKAN KEMATIAN KE ATAS SEL TUMOR OTAK

Oleh

ROHAYA BINTI IBRAHIM

Ogos 2011

Pengerusi: Profesor Abdul Manaf Ali, PhD

Fakulti: Institut Biosains

Strain V4-UPM adalah variasi resistan haba tempatan daripada strain V4 (QUE)

Australia yang tidak berbisa. Ia mempunyai fungsi hemagglutinin and infektiviti yang

lebih tinggi daripada induk asal. Tujuan utama kajian ini adalah untuk melihat kesan

sitolitik dan cara kematian sel yang diakibatkan oleh strain V4-UPM ke atas kedua-dua

sel tumor otak manusia iaitu ‘glioblastoma multiforme’ (DBTRG.05MG) dan ‘anaplastic

astrocytoma’ (U87MG). Aktiviti sitotoksik dijalankan secara in-vitro ke atas kedua-dua

sel tumor otak tersebut, melalui asai MTT piawai. Pada jam ke-72 masa inkubasi, sel

DBTRG.05MG dan U87MG masing-masing memberikan nilai sebanyak 30 haem

agglutination (HA) unit/ml dan 5 HA unit/ml. Melalui kaedah yang sama, proliferasi sel

untuk kedua-dua sel DBTRG.05MG dan U87MG juga telah dikenalpasti. Seterusnya,

perubahan morfologi sel yang dijangkiti diperhatikan menerusi rangkaian mikroskop

cahaya inverted, fluoresen dan elektron. Menerusi mikroskop cahaya inverted, sel yang

dijangkiti virus menunjukkan ciri-ciri apoptotik seperti pengecutan sel, ‘blebbing’ dan

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ACKNOWLEDGEMENTS

In the name of Allah, the Most Gracious, the Most Merciful.

I would like to take this opportunity to thank all the special peoples who have

contributed and helped me through the completion of my Master’s thesis successfully.

Without them, this project would not have been possible. First and foremost, I would

like to express my deepest and heartiest gratitude to my supportive supervisor, Prof. Dr.

Abdul Manaf Ali, for his sumptuous guidance, limitless patience, suggestion, advice and

encouragement throughout the course of this study. Special thanks also go to my cosupervisor,

Prof. Dr. Aini Ideris and Prof. Dr. Abdul Rahman Omar, the other projectcommittee member. I have been

fortunate to receive sponsorship from Majlis Kanser

Nasional (MAKNA) for my master’s program and would like to extend my sincere

thanks to MAKNA, particularly to Yg. Berbahagia Dato’ Farid Ariffin, President of

MAKNA and Yg. Berbahagia Dato’ P. Venugopal, the Chief Executive Officer of MAKNA.

My utmost appreciation is conferred to my beloved family, especially my mother who

supported me during writing process. I am grateful to have my lovely buddies especially

Dr. Chan Kok Meng, Dr. Asmah Hamid, Dr. Yeap Swee Keong, Puan Nori,

Jeevanathan, Atiqah, Idah, Ifah, Shahzuwan, Ain and Sabrina for being my helping

hand, and for the valuable contributions and estimable advices. Their friendship and

supports throughout everything are highly appreciated. I also want to thank my seniors

who work in the same field in veterinary medicine and biochemical department for their

assistance and cooperation regarding this project.

Special thanks to Dr. Noorjahan Banu Mohd Alitheen from Molecular Biology

Department, Faculty of Biotechnology and Biomolecule Sciences, UPM for her

technical assistance regarding the flow cytometry method. This appreciation also goes to

all staff of Electron Microscopy Unit, Faculty of Medical, University Malaya especially

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Pn. Zu, Phang, Vijaya and Elsi for guiding me with the transmission electron

microscope method and for their friendships that made working in the lab enjoyable.

Wassalam…

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I certify that a Thesis Examination Committee has met on 1 August 2011 to conduct the

final examination of Rohaya Ibrahim on her Master of Science thesis entitled “Cytolytic

effect of local V4-UPM strain of Newcastle disease virus of brain tumor cell lines” in

accordance with Universities and University Colleges Act 1971 and the Constitution of

the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee

recommends that the student be awarded the Master of Science.

Members of the Thesis Examination Committee are as follows:

Rasedee @ Mat bin Abdullah, PhD

Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Chairman)

Zeenathul Nazariah binti Allaudin, PhD

Associate Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Internal Examiner)

Noorjahan Banu binti Mohammed Alitheen, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Internal Examiner)

Nor Fadilah Rajab, PhD

Associate Professor

Faculty of Allied Health Sciences

Universiti Kebangsaan Malaysia

(External Examiner)

________________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 2 March 2012

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Abdul Manaf Ali, PhD

Professor

Deputy Vice Chancellor

(Research and Innovation)

Universiti Sultan Zainal Abidin

(Chairman)

Aini Ideris, PhD

Professor

Deputy Vice Chancellor

(Academic and International Affairs)

Universiti Putra Malaysia

(Member)

Abdul Rahman Omar, PhD

Professor

Institute of Bioscience

Universiti Putra Malaysia

(Member)

__________________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations, which

have been duly acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other

institution.

__

________________________

ROHAYA BT IBRAHIM

Date: 1 August 2011

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TABLE OF CONTENTS

DEDICATION ii

ABSTRACT iii

ABSTRAK v

ACKNOWLEDGEMENTS vii

APPROVAL viii

DECLARATION x

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF ABBREVIATIONS xx

CHAPTER

INTRODUCTION

1.1 General Introduction 1

1.2 Problem Statement and Hypothesis 3

1.3 Objectives 4

LITERATURE REVIEW

2.1 BRAIN TUMORS 5

2.1.1 Epidemiology of Brain Tumors 6

2.1.2 Brain Tumor in Malaysia 7

2.1.3 Brain Tumor Grading System 8

2.1.4 Brain Tumor Types

2.1.5 Central Nervous System Tumors 8

2.1.5.1 Pathology of Central NervousSystemTumors 9

2.1.5.2 Tumors of Neuroepithelial Origin 10

2.1.6 High-Grade Astrocytomas 11

2.1.7 Treatment of Brain Tumors

2.2 NEWCASTLE DISEASE VIRUS (NDV)

2.2.1 Newcastle Disease 14

2.2.2 Morphology 14

2.2.3 Replication 16

2.2.4 Typing of Strains 19

2.3 ONCOLYTIC VIRUSES

2.3.1 What is Oncolytic Viruses?

2.3.2 Mechanisms of Oncolytic Viruses

2.4 HUMAN/CLINICAL STUDIES

2.4.1 Virus-Cancer Approaches

2.4.2 Clinical Studies on High-Grade Gliomas

2.5 MALAYSIAN ISOLATES OF NDV

2.5.1 Other Malaysian Oncolytic NDV Strains

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2.5.2 V4-UPM Strain Of NDV

2.6 CELL DEATH

2.6.1 Apoptosis

2.6.2 Necrosis

MATERIALS AND METHODS

3.1 VIRUS WORK

3.1.1 Virus Isolates

3.1.2 Virus Propagation

3.1.3 Virus Inoculation

3.1.4 Virus Harvesting

3.1.5 Virus Clarification

3.1.6 Virus Purification

3.1.7 Virus Quantification

3.2 CELL CULTURE WORK

3.2.1 Tumor and Non-Tumoral Brain Cells

3.2.2 Test Compounds

3.2.3 Cell Reviving and Maintenance

3.2.4 Cryo-Preservation and Trypsinization

3.3 CYTOTOXICITY ASSAY

3.3.1 Cytotoxicity Methods

3.3.2 Microculture Tetrazolium Assay

3.3.3 Proliferation Assay

3.4 MORPHOLOGICAL ANALYSES

3.4.1 Acridine Orange/Propidium Iodide (AO/PI)

Staining

3.4.2 Terminal Deoxynucleotidyl Transferasemediated

dUTP Nick End-Labeling

(TUNEL) Assay

3.4.3 Transmission Electron Microscopy (TEM)

Technique

3.5 FLOW CYTOMETRIC ANALYSES

3.5.1 Cell Cycle Analyses

3.5.2 Annexin V-FITC/Propidium Iodise Staining

RESULTS

4.1 VIRUS TITRATION

4.2 CYTOTOXIC ASSAY

4.2.1 V4-UPM Strain versus Tumor Cells

4.2.2 V4-UPM versus Non-Tumoral Neuronal-Like

Cells, HCN-2

4.2.3 Compounds versus Tumor Cells

4.3 PROLIFERATION PROFILES

4.3.1 V4-UPM Strain versus Tumor Cells

4.3.2 Goniothalamin versus Tumor Cells

4.4 MORPHOLOGICAL STUDY

4.4.1 Light-Inverted Cellular Profiles

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4.4.2 Fluorescent Cellular Profiles

4.4.3 Cell Scoring

4.4.4 In-situ Apoptotic Cell Labelling by the

TUNEL Method

4.4.5 Transmission Electron Microscopy (TEM)

Analysis

4.5 FLOW CYTOMETRY ANALYSES

4.5.1 Cell Cycle Analyses

4.5.2 Detection of Apoptosis Events

DISCUSSION

CONCLUSION

REFERENCES 117

APPENDICES 131

BIODATA OF THE AUTHOR 149