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UNIVERSITI PUTRA MALAYSIA

EFFECTS OF NEWCASTLE DISEASE VIRUS ON GENE EXPRESSION PROFILING, AND NITRIC OXIDE AND GLUTATHIONE PRODUCTION IN

MCF-7 BREAST CANCER CELL LINE

MOHAMED KALID ALI

FBSB 2011 14

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ii

EFFECTS OF NEWCASTLE DISEASE VIRUS ON GENE EXPRESSION

PROFILING, AND NITRIC OXIDE AND GLUTATHIONE PRODUCTION IN

MCF-7 BREAST CANCER CELL LINE

By

MOHAMED KALID ALI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of

Science

APRIL 2011

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Specially dedicated to,

My beloved Ikram, Abshiro, Khalid, Uncle

Abdulahi and Aunty Fadumo, Brother In-law

Said Hussen Id.

AND

The rest of my Family and Friends

For their invaluable love, understanding,

patience, support and care

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science.

EFFECTS OF NEWCASTLE DISEASE VIRUS ON GENE EXPRESSION

PROFILING, AND NITRIC OXIDE AND GLUTATHIONE PRODUCTION IN

MCF-7 BREAST CANCER CELL LINE

By

MOHAMED KALID ALI

APRIL 2011

Chairman: Profesor Datin Paduka Khatijah Mohd Yusoff, PhD

Faculty: Biotechnology and Biomolecular Sciences

Breast cancer is a major cause of death for many women around the world.

Progress has been made in the survival of breast cancer patients due to

improved understanding of the molecular processes, diagnostic techniques and

knowledge of the treatment using chemotherapy, radiotherapy, and virotherapy.

The field of virotherapy has emerged from the past decade by using oncogenic

viruses to selectively kill cancerous cells without harming normal cells. Newcastle

disease virus (NDV), an avian paramyxovirus, induces apoptosis in a variety of

human malignant cells such as breast cancer cells. In this study, the cytolytic

properties of NDV strain AF2240 on gene expression profiling, measurement of

Nitric oxide (NO) free radical and Glutathione antioxidant (GSH) were

investigated by using RT-PCR, capillary electrophoresis, flow cytometry both

under normoxic and hypoxic condition.

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Multiplex Gene Expression Kit was used whereby the relative expressions of 25

different genes were measured at the mRNA level. The results showed that

treatment of MCF7 cells with NDV caused apoptosis, NO production and GSH

depletion and changed the expression level of most of the genes involved in

tumour progression, cell cycle regulation, cell growth and differentiation,

apoptosis, cancer suppression, and DNA damage, as measured by RT-PCR and

capillary electrophoresis. Since the mode of apoptosis by NDV is NO and GSH

dependent, comparisons of NDV with NO-Donors such as DETA-NONOate and

NO-Scavengers like cPTIO were also studied. Increased production of Nitric

oxide (NO) and depleted levels of Glutathione (GSH) after treatment of NDV at

normoxic condition compared NDV at hypoxic condition were observed.

However, addition of DETA-NONOate to MCF-7 cells has induced NO

production, GSH depletion. Moreover, NO production and apoptosis were

attenuated, and GSH increased after addition of cPTIO either alone or with

combination of either NDV or DETA-NONOate to the MCF-7 cells. In addition to

the above, co-treatment of NDV+DETA-NONOate depleted NO production and

cell death compared to the treatment of NDV alone to cells. From this study, it

was concluded that NDV induces apoptosis, Gene expression, NO production

and GSH depletion in MCF-7 cells which makes it an effective anticancer agent

due to its ability to kill breast cancer cell lines in normoxic and hypoxic conditions.

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Abstrak thesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

KEBERKESANAN NEWCASTLE DISEASE VIRUS DALAM PROFIL EKSPRESI GEN DAN PENGELUARAN NITRIK OKSIDA SEL KANSER PAYU

DARA MCF-7

Oleh

MOHAMED KALID ALI

APRIL 2011

Pengerusi : Profesor Datin Paduka Khatijah Mohd Yusoff, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Kanser payu dara merupakan pembunuh terbesar bagi wanita di seluruh dunia.

Peningkatan kadar kelansungan hidup bagi penghidap kanser payu dara mula

ditunjukkan apabila kefahaman mengenai proses molekul, teknik diagnostik dan

pengetahuan mengenai rawatan seperti kemoterapi, radioterapi dan viroterapi

semakin berkembang. Kaedah viroterapi mula dikenali sejak sedekad yang lepas

apabila virus-virus onkogenik mula digunakan sebagai sasaran untuk

membunuh sel kanser tanpa menggangu sel-sel yang lain. Newcastle Disease

Virus (NDV) yang tergolong dalam kumpulan Avian Paramyxovirus,

menyebabkan kematian sel dalam pelbagai jenis kanser yang merbahaya seperti

kanser payu dara. Dalam kajian ini, kebolehan membunuh sel kanser oleh NDV

AF2240 melalui “gene expression profiling”, penyukatan radikal bebas Nitrik

Oksida (NO), dan “Glutathione” anti oksida telah dikaji dengan menggunakan

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teknik RT-PCR, “ capillary electrophoresis”, “flow cytometry” di bawah kondisi

“hypoxic” dan “normoxic”. “Multiplex Gene Expression Kit” telah digunakan dan

sementara itu, ekspresi 21 gen yang berhubung-kait boleh diukur pada peringkat

mRNA. Keputusan kajian ini menunjukkan bahawa sel MCF-7 yang dirawat

dengan menggunakan NDV telah menyebabkan kematian sel secara

berprogram, tiada pengurangan atau pengeluaran GSH dan perubahan ekspresi

sebahagian besar gen yang terlibat dalam pembesaran tumor, kawalan kitaran

hidup sel, pertumbuhan dan diferensiasi sel, kematian sel secara berprogram,

pembantutan pertumbuhan kanser dan kerosakan DNA seperti yang diukur

melalui RT-PCR and “capillary electrophoresis”. Memandangkan cara NDV

membunuh sel bergantung kepada NO and GSH, perbandingan NDV dengan

NO-Donors seperti DETA-NONOate dan NO-Scavengers seperti cPTIO turut

dijalankan. Peningkatan pengeluaran Nitrik Oksida (NO) and penurunan kadar

Glutathione (GSH) selepas dirawat dengan NDV di bawah kondisi normoxic

berbanding hypoxic turut diamati. Walau bagaimanapun, penambahan. DETA-

NONOate terhadap MCF-7 telah menyebabkan pengeluaran NO dan

pengurangan GSH. Tambahan lagi, pengeluaran NO dan kematian sel secara

berprogram berkurangan dan GSH meningkat apabila ditambah cPTIO sama

ada secara sendiri atau digabungkan dengan NDV atau DETA-NONOate

terhadap MCF-7. Malah, gabungan rawatan NDV+DETA-NONOate

mengurangkan NO dan kematian sel berbanding dengan rawatan NDV

sendirian.

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Daripada kajian ini dapatlah dirumuskan bahawa NDV menyebabkan kematian

sel secara berprogram, ekspresi gen, pengeluaran NO dan pengurangan GSH di

dalam sel MCF-7 yang mana membuatkan ia sebagai rawatan anti kanser yang

efektif kerana mampu membunuh sel kanser payu dara di bawah kondisi

normoxic dan hypoxic.

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Acknowledgements

First and foremost, Alhamdulilah, all praise is for Allah who made this thesis

possible. It would have also not been possible without the guidance and the help

of several individuals who have one way or another contributed and extended

their valuable assistance in the preparation and completion of this study.

My utmost gratitude to Professor Datin Paduka Dr. Khatijah Mohamed Yusoff,

Deputy Secretary General (Science), Ministry of Science, Technology and

Innovation Malaysia. I would never forget the first day when I stepped into her

office when she was the Deputy Vice-chancellor at UPM. Moments after I came

out at her office I felt that I had met someone who will be of great help to me.

Thank you for your encouragement, funding, support and guidance that had

made my Masters possible.

Besides my Advisor, I would like to thank the rest of my supervisory committee,

Dr. Firoozeh Jahanshiri, Professor. Abdul Rahman Omar, and Dr. Norazizah

Shafee, for their patience, motivation, enthusiasm, discussion and immense

knowledge. Their guidance helped me throughout the time of research and

writing of this thesis.

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I thank all my friends at Virology Lab 143, Puan Rodiah for her immediate

attention for processing quotations and orders to the companies, Min and Ain,

Aini, Hidayah, Wei Choong, Halimi, Eddie, Wannie, Hafifi, Kahfai, Rajik and

Nicole.

Last but not the least; I would like to thank my Family and my other friends for

their unflagging love and support throughout my life. I am indebted to my father

(Khaalid Ali Xundhur) for his care and love, he worked industriously to support

the family and spare no effort to provide the best possible environment for me to

grow up. I cannot ask for more from my mother Abshiro, and my grandmother

Hawa. I remember their constant support when I encountered difficulties. Mother

(Hooyo) I love you. My brothers Ahmed, Abdulahi, Abdul Rahman and sisters

Faiza, Ikram, Deeqa, Halima and Hawa, for your love. Friends like Said Hussein

Id, Mawlid bashir, Said Toldo and Dr Abdiwali.

Special thanks to my beloved Ikram Bashaash, it is difficult to find words to

express my gratitude and giving me part of your mind to think it more. Your

motivation has made what I am today.

The generous support from the Islamic Development Bank is greatly appreciated.

In retrospect, I am amazed by the confidence from the trustees. I am sure my

application was not the most outstanding one, but they still granted me the

scholarship. Mr.Lkadkadi for his immediate replies and patience.

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I certify that a Thesis Examination Committee has met on 4 April 2011 to conduct the final examination of Mohamed Kalid Ali on his thesis entitled “Effects of Newcastle disease virus on Gene Expression Profiling, and Nitric Oxide and Glutathione Production in MCF-7 Breast Cancer Cell Line” 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 degree.

Members of the Examination Committee were as follows: Muhajir Hamid, PhD

Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Noorjahan Banu Binti Mohammed Alitheen, PhD

Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Janna Ong binti Abdullahi, PhD

Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Subha Bhasu, PhD

Senior Lecturer Faculty of Science Universiti Malaya (External Examiner) NORITAH OMAR, PhD

Associate Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 23 August 2011

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows. KhatijahYusoff, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Abdul Rahman Omar, PhD Professor Institute of Biosciences University Putra Malaysia (Member) Norazizah Shafee, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Fatemah Jahanshiri, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

___________________

HASANAH MOHAMED GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 4 April 2011

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DECLARATION

I declare that the thesis is my original work except the 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 UPM or at any other Institution.

_________________ Mohamed Kalid Ali

Date: 4 April 2011

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TABLE OF CONTENTS

Page

DEDICATION ii.

ABSTRACT iii.

ABSTRAKT vii.

ACKNOWLEDGEMENTS viii.

APPROVAL x.

DECLARATION xii.

LIST OF TABLES xvi.

LIST OF FIGURES xvii.

LIST OF ABBREVIATIONS xviii.

CHAPTER 1 INTRODUCTION 1

2 LITRATURE REVIEW

2.1 Cancer 4

2.2 Breast Cancer 4

2.3 Cancer Genes 5

2.4 Cancer Therapy 7

2.4.1 Cancer Gene Therapy 7

2.4.2 Cancer Chemotherapy 8

2.4.3 Cancer Virotherapy 9

2.5 Virotherapy Vs Chemotherapy 10

2.6 Mechanism of Oncolytic Viruses to Destruct Tumour Cells. 11

2.7 Replication of Oncolytic Virus in Tumour Cells 12

2.8 Newcastle Disease Virus. 14

2.9 NDV Induces Apoptosis in Cancer 16

2.10 Nitric Oxide 17

2.10.1 Therapeutic Application of NO-donors on Cancer Cells 17

2.10.2 Nitric Oxide in Hypoxia 18

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2.10.3 NO as p53 activator 19 2.10.4 NO as a Chemosensitizer and as Radiosensitizer 20

2.10.5 Effect of NO in Newcastle Disease Virus 20

2.10.6 Glutathione Production in Cancer (GSH) 21

3 GENE EXPRESSION PROFILING IN NDV INFECTED (MCF-7) BREAST CANCER CELL LINE

22

3.1 Introduction 22

3.2 Materials and Methods 23

3.2.1 Sources of Viruses and Cell 23 3.2.2 Sources of Chemicals and Biochemical 23

3.2.3 Newcastle Disease Virus Cultivation 23

3.2.4 Titration of the Virus 25

3.2.5 Breast Cancer Cell Lines (MCF-7) 26

3.2.6 Cell Viability 27

3.2.7 Infection of NDV in MCF-7 Cell Culture 27 3.2.8 RNA extraction and DNase Treatment 28

3.2.9 Reverse Transcriptase. 28

3.2.10 Polymerase Chain Reaction 29

3.2.11 RT-minus Control and No-template 29

3.2.12 Pre-dilution Step 29

3.2.13 Flow Cytometry 30

3.2.14 GenomeLab GeXP Genetic analysis 30

3.2.15 Normalization Against Genes of Interest 31

3.3 Results and Discussion 32

3.3.1 ESR1 36

3.3.2 KR18 36

3.3.3 OXCT1 37

3.3.4 MYBL2 and TGF β-1 38

3.3.5 PRC1 39

3.3.6 CDC42BPA 39

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3.3.7 KNTC2 40

3.3.8 RAB6B 41

3.3.9 BBC3 or PUMA 41

3.3.10 AP2β1 42

3.3.11 WISP1 43

3.3.12 HSC4A2 43

3.3.13 RFC4 44

3.3.14 EGL nine1 44

3.4 Conclusion 45

4 INDUCTION OF NITRIC OXIDE AS A MECHANISM OF ACTION OF NEWCASTLE DISEASE VIRUS IN BREAST CANCER

47

4.1 Introduction 47

4.2 Materials and Methods 49

4.2.1 Sources of Viruses and Biochemicals 49

4.2.2 NDV Propagation, MCF-7 Maintainance 49

4.2.3 Determination of Nitric Oxide Production 50

4.2.4 Determination of Reduced Glutathione (GSH)

4.3 Results and Discussion 55

4.3.1 Effect of NO production, NO-DONOR, NO-Scavenger Treated cells

55

4.3.2 Determination of Anti-Oxidant Glutathione 72

4.4 Conclusion

77

5 GENERAL CONCLUSION AND FUTURE RECOMMENDATION 79 REFERENCES

81

APPENDICES 97 BIODATA OF THE STUDENT 105 PUBLICATION 106