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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
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