UNIVERSITI PUTRA MALAYSIA

MOLECULAR AND CYTOSKELETAL CHANGES IN BREAST CANCER CELL LINES TREATED WITH VELOGENIC NEWCASTLE DISEASE

VIRUS STRAIN AF2240

ZOLKAPLI ESHAK

IB 2006 16

MOLECULAR AND CYTOSKELETAL CHANGES IN BREAST CANCER CELL LINES TREATED WITH VELOGENIC NEWCASTLE DISEASE VIRUS

STRAIN AF2240

By

ZOLKAPLI ESHAK

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirement for the Degree of Master of Science

Mac 2006

I would like to dedicate this thesis to my parents, I thank you for the unconditional love and sacrifices you made for me; to my wife, who has put up

with me during the writing of this thesis; and to my sons and daughter who bring joy and happiness to my heart.

ii

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

fulfilment of the requirement for the degree of Master of Science

MOLECULAR AND CYTOSKELETAL CHANGES IN BREAST CANCER CELL LINES TREATED WITH NEWCASTLE DISEASE VIRUS STRAIN

AF2240

By

ZOLKAPLI BIN ESHAK

March 2006 Chairman : Associate Professor Fauziah Othman, PhD Institute : Bioscience

The study was carried out to investigate the oncolytic effect of Newcastle

disease virus (NDV) strain AF2240 on the MCF-7, MDA-MB-231 breast cancer

cell lines and 3T3 fibroblast. Studies were conducted to investigate the

cytoskeletal protein structure and the molecular changes of the oncogenes. The

AF2240 strain of NDV was propagated in 11 days old embryonated eggs for 72

hours. The virus in the allantoic fluid was harvested, purified and stored at

-80ºC. The haemagglutination (HA) test was conducted on the purified virus to

determine the HA titre of the NDV strain AF2240 which was 16384 HA units.

The inhibition concentration of AF2240 towards several types of breast cancer

cell lines was carried out using microculture tetrazolium (MTT) assay via two

methods; monolayer and co-culture techniques to determine the inhibition

concentration (IC50) value. The IC50 values for MDA-MB-231 breast cancer cell

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lines treated with NDV strain AF2240 were 8 and 2 HA units for the monolayer

and co-culture techniques respectively, whereas the IC50 value for MCF-7 was 2

HA units for both techniques. NDV strain AF2240 has no oncolytic effect

towards 3T3 mouse fibroblast. Further on confocal microscopy was carried out

to observe the localization of the virus in the cells. For detection of the virus,

polyclonal antibody and anti-chicken conjugated with fluorescein isothiocynate

(FITC) were used. The virus particles were detected in the cytoplasm of both

breast cancer cell lines after 24 and 48 hours post treatment. Budding-off of the

virus was detected after 72 hours post treatment. Further study using TdT-

mediated dUTP nick-end labelling (TUNEL) assay was conducted to label and

quantify the percentage of apoptotic cells. By using independent t-test, the

analysis revealed that NDV strain AF2240 works better towards MDA-MB-231

cells compared to MCF-7 (p ≤ 0.05). These methods confirmed that NDV causes

cell death to the breast cancer cells via apoptosis. The finding also suggesting

that NDV react better towards MDA-MB-231 cells compared to MCF-7 cell

(P≤0.05). The immunolabelling of the cytoskeletal proteins, namely,

microfilaments, microtubules and intermediate filaments was conducted by

using FL Phallicidin, monoclonal anti-α-tubulin FITC and monoclonal anti-

vimentin Cy3. The cytoskeletal proteins of the cell lines were disrupted after 72

hours post treatment. However, the number of cells with disrupted cytoskeletal

proteins was much higher in MDA-MB-231 cells compared to MCF-7 cells. The

study of oncogenes was conducted by using reverse transcriptase polymerase

iv

chain reaction (RT-PCR) method. The expressions of c-myc, c-erb-2 and c-fos

oncogenes were detected at pre and post-treatment in the MCF-7 and MDA-MB-

231 breast cancer cell lines. These results prove that cells which had undergone

apoptosis due to NDV strain AF2240 treatment did not suppress the oncogenes.

This study concluded that even though strain AF2240 of NDV have significant

cytotoxic effect towards MCF-7 breast cancer cell lines, the number of apoptotic

cells were higher in MDA-MB-231 cell line and therefore, further study is

needed to understand the underlying mechanism in making the NDV strain of

AF2240 as an anti-cancer agent.

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

PERUBAHAN MOLEKULAR DAN SITOSKELETAL SEL KANSER

PAYUDARA YANG DI RAWAT DENGAN VIRUS PENYAKIT NEWCASTLE STRAIN AF2240

Oleh

ZOLKAPLI ESHAK

Mac 2006

Pengerusi : Profesor Madya Fauziah Othman, PhD Institut : Biosains

Kajian ini dijalankan untuk menyelidik kesan onkolitik virus penyebab

Newcastle disease (NDV) strain AF2240 ke atas sel kanser payudara MCF-7,

MDA-MB-231 dan 3T3 fibroblast. Kajian ini dijalankan untuk menyelidik

perubahan strukur protein sitoskeletal dan perubahan molekular onkogen.

Propagasi NDV telah dilakukan di dalam telur ayam berembrio berusia 11 hari

selama 72 jam. Virus yang terdapat dalam cecair alantoik kemudiannya

dikumpul, ditulenkan dan disimpan pada -80ºC. Ujian penggumpalan (HA)

telah dijalankan ke atas virus yang telah ditulenkan untuk menentukan titer HA

virus NDV strain AF2240 yang mana HA unitnya ialah 16384. Kesan NDV

AF2240 ke atas sel kanser payudara telah dilakukan mengguna kaedah

mikrokultur tetrazolium (MTT) esei melalui dua cara; teknik monolayer dan ko-

kultur untuk menentukan nilai IC50. Nilai IC50 untuk sel payudara MDA-MB-231

yang dirawat dengan NDV strain AF2240 adalah masing-masing 8 dan 2 HA

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unit untuk teknik monolayer dan ko-kultur, manakala nilai IC50 untuk MCF-7

adalah 2 HA unit untuk kedua-dua teknik. NDV strain AF2240 tidak

mempunyai kesan onkolitik terhadap sel fibroblast 3T3. Kajian selanjutnya telah

dilakukan dengan menggunakan mikroskop konfokal untuk melihat taburan

virus tersebut di dalam sel. Untuk mengesan virus tersebut, antibodi poliklonal

dan anti-ayam fluorescein isothiocynate (FITC) telah digunakan. Virus tersebut

telah dikesan di dalam sitoplasma kedua-dua jenis sel payudara selepas dirawat

selama 24 dan 48 jam. Walaubagaimanapun, pelepasan virus dari sel perumah

selepas 72 jam dirawat hanya dapat dikesan di dalam sel kanser payudara.

Kajian selanjutnya menggunakan asai TdT- mediated dUTP nick-end labelling

(TUNEL) telah dijalankan untuk melabel dan mengira peratusan sel apoptotik.

Dengan menggunakan ujian-t berdikari, analisis menunjukkan bahawa NDV

strain AF2240 bertindak balas lebih baik terhadap sel MDA-MB-231

dibandingkan dengan MCF-7 (p ≤ 0.05). Cara ini mengesahkan bahawa NDV

menyebabkan sel payudara mati melalui apoptosis. Kajian ini juga

mencadangkan bahawa NDV bertindak balas lebih baik terhadap sel MDA-MB-

231 berbanding dengan MCF-7 (P≤0.05). Penglabelan imuno ke atas protein

sitoskeletal seperti filamenmikro, tubulmikro dan filamen pertengahan telah

dijalankan dengan menggunakan FL Phallicidin, monoklonal anti-α-tubulin

FITC dan monoklonal anti-vimentin Cy3. Protin sitoskeletal sel rosak selepas 72

jam dirawat. Walau bagaimanapun, jumlah sel yang mengalami kerosakan

protein sitoskeletal lebih tinggi di dalam sel MD-MB-231 berbanding sel MCF-7.

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Kajian mengenai onkogen dilakukan menggunakan kaedah ‘reverse

transcriptase polymerase chain reaction’ (RT-PCR). Ekpresi c-myc, c-erb-2 dan c-

fos onkogen telah dikesan sebelum dan selepas rawatan di dalam sel MCF-7 dan

MDA-MB-231. Keputusan ini menunjukkan bahawa apoptosis yang di sebabkan

NDV strain AF2240 tidak menindas onkogen. Kajian ini menyimpulkan

bahawa, walaupun NDV strain AF2240 mempunyai lebih kesan sitotoksik

terhadap sel MCF-7, sel MDA-MB-231 mempunyai bilangan sel apoptotic yang

lebih banyak dan dengan itu kajian yang lebih mendalam diperlukan untuk

memahami mekanisma yang terselindung dalam menjadikan NDV strain

AF2240 sebagai agen anti-kanser.

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ACKNOWLEDGEMENTS

I would like to thank to all people who supported me and were involved in one

way or another in the preparation of this thesis. I gratefully appreciate the

guidance of my supervisor, Assoc. Prof. Dr. Fauziah Othman who inspired and

provided me the purpose of my study. I am grateful to the support and

mentoring of my co-supervisors, Prof. Dr. Aini Ideris, for her depth insightful

knowledge of the virus and to Assoc. Prof. Dr. Asmah Rahmat, for her guidance

in tissue culture and molecular works. Many special thanks to Assoc. Prof. Dr.

Rozita Rosli from the Faculty of Medicine and Health Sciences and Assoc. Prof.

Dr. Abdul Rahman Omar from Faculty of Veterinary Medicine, who gave me

the chance to work on this topic in their lab.

I wish to thank Dr. Abdah Md Akim who mentored me on TUNEL assay and

molecular work. I also want to thank Dessy Arisanty for her knowledge in

analytical analysis, Tengku Shahrul for his invaluable lesson in computer

software and Hadiyatul Hanim who is always there when help is needed. I

would like to acknowledge the support of my lab mates and friends: Hernani,

Mahani, Norhayati, Sally, and Phang who helped me with the editing of the

current text. I also would like to thank the staff of Microscopy Imaging and

Nanoscience Unit for helping me out with the microscopy imaging and their

excellence services. With the biggest contribution to this thesis, I would like to

thank Ministry of Science and Technology (MOSTE) and Majlis Kanser Nasional

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(MAKNA) for sponsoring the research. Without their financial support, this

research would be impossible.

Last but not the least; I want to thank my wife, Rohhannie, and my sons and

daughter, Irfan Zunnurrain, Irfan Zikry and Aleeya for their understanding,

support, and unconditional love. Their love and support has been and will

continue to be my inspiration, and I am so blessed to have such a caring and

supporting family.

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I certify that an Examination Committee has met on 20th Mac 2006 to conduct the final examination of Zolkapli Eshak on his Master of Science thesis entitled “Molecular and Cytoskeletal Changes in Breast Cancer Cell Lines Treated With Newcastle Disease Virus Strain AF2240” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Nasaruddin Abdul Aziz, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Mohd Hair Bejo, PhD Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) Azian Abd. Latiff, PhD Lecturer Faculty of Medicine Universiti Kebangsaan Malaysia (External Examiner)

__________________________________

HASANAH MOHD GHAZALI, PhD Professor/Deputy Dean

School of Graduate Studies Universiti Putra Malaysia

Date:

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This thesis 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 are as follows:

Fauziah Othman, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Aini Ideris, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Asmah Rahmat, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member)

______________________ AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

___________________________ ZOLKAPLI ESHAK

Date:

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TABLE OF CONTENTS Page

DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xix LIST OF FIGURES xxiii LIST OF ABBREVIATIONS xxvii

CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW

2.1 Breast Cancer 5 2.2 Risk Factors 6 2.3 Oncogenes 8 2.3.1 Her2/Neu/C-erbB-2 9 2.3.2 c-myc 10 2.33 c-fos 12 2.4 Types of Breast Cancer 12 2.4.1 Carcinoma in Situ 13 2.4.2 Infiltrating Ductal Carcinoma 14 2.4.3 Infiltrating Lobular Carcinoma 14 2.4.4 Medullary Carcinoma 15 2.4.5 Colloid Carcinoma 15 2.4.6 Tubular Carcinoma 15 2.4.7 Inflammatory Breast Cancer 16 2.5 Commercialized Breast Cancer Cell Line 16 2.5.1 Staging of Breast Cancer 17 2.6 Breast Cancer Treatment 20 2.6.1 Breast-Conserving Surgery 21 2.6.2 Mastectomy 21 2.6.3 Lymph Node Surgery 22 2.6.4 Radiation Therapy 22 2.6.5 Chemotherapy 23 2.6.6 Hormonal Therapy 23 2.7 Apoptosis 24

2.7.1 Morphological and Chemical Changes During Apoptosis

25

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2.7.2 Apoptosis and Necrosis 27 2.8 Cytoskeletal Proteins 30 2.8.1 Microfilaments 31 2.8.2 Microtubules 32 2.8.3 Intermediate Filaments 33 2.8.4 Cytoskeleton and Apoptosis 34 2.9 Newcastle Disease Virus 35 2.9.1 Characterization of NDV 37

2.9.2 Mechanism of NDV Inducing Apoptosis

40

2.9.3 Newcastle Disease Virus Strain AF2240 42

3 INHIBITION CONCENTRATION OF NEWCASTLE DISEASE VIRUS TOWARDS BREAST CANCER CELL LINES

43

3.1 Introduction 43 3.2 Materials and Methods 45 3.2.1 Propagation of the virus 45 3.2.2 Embryonated chicken egss 45 3.2.3 Preparation of seed virus dilution 46 3.2.4 Inoculation of the virus 47 3.2.5 Harvesting 48

3.2.6 Clarification and Purification of the virus

48

3.2.7 Preparation of Chicken Red blood cell (RBC) for virus titration

49

3.2.8 Haemagglutination Test (HA) 50

3.2.9 Maintaining Cell Culture 50

3.2.10 Cell Sub Culturing 51

3.2.11 Mono-layer Method 52 3.2.12 Co-culture Method 53 3.2.13 Microculture Tetrazolium (MTT) Assay 54

3.3 Results 55 3.4 Discussion and Conclusion 58

4 IMMUNOLABELING OF THE CYTOSKELETAL PROTEIN, NEWCASTLE

60

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DISEASE VIRUS AND THE APOPTOTIC CELLS

4.1 Introduction 60 4.2 Materials and Methods 63 4.2.1 Maintaining Cells on Cover slips 63

4.2.2 Immunolabelling of The Virus and the Cytoskeletal Proteins

63

4.2.3 Detection of apoptotic and viable cells using TdT-mediated dUTP nick-end labeling (TUNEL) assay

65

4.2.4 Quantification 66 4.3 Results 67

4.3.1 Confocal Microscopy of Untreated MCF-7 and MDA-MB-231 cells stained by TUNEL technique

67

4.3.2 Confocal Microscopy of Treated MCF-7 and MDA-MB-231 Cells Stained by TUNEL Technique

67

4.3.3 Quantification of the Apoptotic Cells 68 4.3.4 Confocal Microscopy of Untreated MCF-7

and MDA-MB-231 Cells Stained With Antibody Against F- actin

70

4.3.5 Confocal Microscopy of Treated MCF-7 and MDA-MB-231 Cells Stained With Antibody Against F- actin

71

4.3.6 Confocal Microscopy of Untreated MCF-7 and MDA-MB-231 Cells Stained With Antibody Against α- tubulin Conjugated

With FITC

71

4.3.7 Confocal Microscopy of Treated MCF-7 and MDA-MB-231 Cells Stained With Antibody Against α-

tubulin Conjugated With FITC

71

4.3.8 Confocal microscopy of untreated MCF-7 and MDA-MB-231 Cells Stained With Antibody Against

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Vimentin Conjugated With Cy3 4.3.9 Confocal microscopy of treated MCF-7 and MDA-MB-231 Cells Stained With Antibody Against

Vimentin Conjugated With Cy3

72

4.3.10 Confocal Microscopy of Untreated MCF-7

and MDA-MB-231 Cells Labeled With Polyclonal Antibody Against NDV and Anti-chicken FITC

73

4.3.11 Confocal microscopy of treated MCF-7 and MDA-MB-231 cells

labeled with polyclonal antibody against NDV and anti-chicken FITC

73

4.3.12 Quantification of Disrupted Cytoskeletal Proteins

73

4.3.13 Statistical Analysis 75 4.4 Discussion and Conclusion 92

5 REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR) STUDY OF THE ONCOGENES EXPRESSION

99

5.1 Introduction 99 5.2 Materials and Methods 100 5.2.1 Treatment of The Cells 100 5.2.2 Extraction of mRNA 100 5.2.3 Preparation of the Mastermix 102 5.2.4 Extraction of c-myc oncogene 102

5.2.5 Extraction of c-erb-2 and c-fos oncogenes

103

5.2.6 Reverse Transcriptase polymerase Chain Reaction (RT- PCR)

104

5.3 Results 105 5.3.1 MDA-MB-231 Breast Cancer Cell Line

105

5.3.2 MCF-7 Breast Cancer Cell Line 106 5.4 Discussion and Conclusion 113

6 GENERAL DISCUSSION AND CONCLUSION 115

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REFERENCES 123 APPENDICES 152 BIODATA OF THE AUTHOR 175

LIST OF TABLES

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Table Page 1 TNM category for breast cancer staging.

20

2 Morphological differences between apoptosis and necrosis.

29

3 NDV isolate examined phylogenetically as country of origin and year of isolation with pathotype designation.

39

4 Percentage of apoptotic cells of the MCF-7 and MDA-MB-231 cells treated with AF2240 strain of NDV.

68

5 The statistical analysis of mean number of apoptotic cells in the effect of AF2240 strains of NDV on the MCF-7 and MDA-MB-231 breast cell lines.

69

6 The statistical analysis of mean number of viable cells of the untreated MCF-7 and MDA-MB-231 breast cell lines

70

7 Percentage of disrupted F-actin cytoskeletal protein of the MCF-7 and MDA-MB-231 cells treated with AF2240 strain of NDV.

73

8 Percentage of disrupted α-tubulin cytoskeletal protein of the MCF-7 and MDA-MB-231 cells treated with AF2240 strain of NDV.

74

9 Percentage of disrupted vimentin cytoskeletal protein of the MCF-7 and MDA-MB-231 cells treated with AF2240 strain of NDV.

74

10 The statistical analysis of mean number of disrupted F-actin cytoskeletal protein in the effect of AF2240 strain of NDV on the MCF-7 and MDA-MB-231 breast cell lines.

75

11

The statistical analysis of mean number of

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disrupted F-actin cytoskeletal protein of untreated cells of the MCF-7 and MDA-MB-231 breast cell lines

12 The statistical analysis of mean number of disrupted α-tubulin cytoskeletal protein in the effect of AF2240 strain of NDV on the MCF-7 and MDA-MB-231 breast cell lines.

78

13 The statistical analysis of mean number of disrupted α-tubulin cytoskeletal protein of the untreated MCF-7 and MDA-MB-231 breast cell lines

79

14 The statistical analysis of mean number of disrupted vimentin cytoskeletal protein in the effect of AF2240 strain of NDV on the MCF-7 and MDA-MB-231 breast cell lines.

80

15 The statistical analysis of mean number of

disrupted vimentin cytoskeletal of the untreated MCF-7 and MDA-MB-231 breast cell lines.

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16 PCR condition for c-myc and c-fos oncogenes.

103

17 Independent t-test to compare the number of apoptotic cells after 24 hours post treatment between different types of breast cells.

155

18 Independent t-test to compare the number of viable cells of untreated cells after 24 hours between different types of breast cells.

156

19 Independent t-test to compare the number of apoptotic cells after 72 hours post treatment between different types of breast cells

157

20 Independent t-test to compare the number of viable cells of untreated cells after 72 hours between different types of breast cells.

158

21 Independent t-test to compare the number of disrupted F-actin of apoptotic cells after 24 hours post treatment between different types of breast

159

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cells

22 Independent t-test to to compare the number of disrupted F-actin of apoptotic of untreated cells after 24 hours between different types of breast cells.

160

23 Independent t-test to to compare the number of disrupted F-actin of apoptotic cells after 72 hours post treatment between different types of breast cells.

161

24 Independent t-test to to compare the number of disrupted F-actin of apoptotic cells of untreated cell after 72 hours between different types of breast cells.

162

25 Independent t-test to compare the number of disrupted α-tubulin of apoptotic cells 24 hours post treatment between different types of breast cells.

163

26 Independent t-test to compare the number of disrupted α-tubulin of apoptotic cells after 24 hours of untreated cell between different types of breast cells.

164

27 Independent t-test to compare the number of disrupted α-tubulin of apoptotic cells 72 hours treatment between different types of breast cells.

165

28 Independent t-test to compare the number of disrupted α-tubulin of apoptotic cells after 72 hours of untreated cell between different types of breast cells.

166

29 Independent t-test to compare the number of disrupted vimentin of apoptotic cells 24 hours treatment between different types of breast cells.

167

30 Independent t-test to compare the number of disrupted vimentin of apoptotic cells after 24 hours of untreated cell between different types of

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breast cells

31 Independent t-test to compare the number of disrupted vimentin of apoptotic cells 72 hours post treatment between different types of breast cells.

169

32 Independent t-test to compare the number of disrupted vimentin of apoptotic cells after 72 hours of untreated cell between different types of breast cells.

170

LIST OF FIGURES

Figure Page

xxii

1 Work flow for NDV propagation and purification

45

2 MTT assay work flow

52

3 Percentage of cytotoxicity of MCF-7 cells inoculated with AF 2240 strain of NDV in mono-layer and co-culture methods at 72 hours post-inoculation.

56

4 Percentage of cytotoxicity of MDA-MB-231 cells inoculated with AF2240 strain of NDV in mono-layer and co-culture methods 72 hours post-inoculation

56

5 Percentage of cytotoxicity of 3T3 cells inoculated with AF 2240 strain of NDV in mono-layer and co-culture methods at 72 hours post-inoculation.

57

6 Confocal micrographs of MCF-7 cells treated with AF2240 strains of NDV and stained by TUNEL technique: (a) control (b-d) treated for 24, 48 and 72 hours. Cells were double stained with fluorescein-12-dUTP and propidium iodide. Viable (V) cells show orange to red nuclei, whereas apoptotic (a) cells show yellow to green nuclei. Note the fragmented nucleus (B-D). Mag. (A-D): 40x, inserted picture (B-D): 60x.

82

7 Confocal micrographs of MDA-MB-231 cells treated with AF2240 strains of NDV and stained by TUNEL technique: (A) control (B-D) treated for 24, 48 and 72 hours. Cells were double stained with fluorescein-12-dUTP and propidium iodide. Viable (V) cells show orange to red nuclei, whereas apoptotic (a) cells show yellow to green nuclei. Note the fragmented nucleus (inserted) B-D. Mag. (A-D): 40x, inserted picture (B-D): 60x.

83

8 Confocal micrograph of untreated (A) and treated (B-D) of F-actin of MCF-7 breast cancer cell lines for 24, 48 and 72 hours stained with biodipy

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phallicidin. F-actin is still intact in untreated MCF-7 span throughout the plasma membrane. Even after 24 post-treatment (B), F-actin is still intact and disruption of F-actin (arrow) is only noted after 48 and 72 hours post treatment causing the cells to loose its integrity (C and D). Mag. (A-B): 60X, (C,D): 120X.

9 Confocal micrograph of α-tubulin of MCF-7 breast

cancer cell lines stained with monoclonal anti-α-tubulin conjugated with FITC for untreated (A) and treated for 24, 48 and 72 hours (B, C and D respectively). Disruption of α-tubulin (white arrows) is noted after 48 and 72 hours post treatment (C-D). Mag. (A-B) 60X, (C-D) 120X.

85

10 Confocal micrograph of vimentin of MCF-7 breast cancer cell lines stained with monoclonal anti-vimentin conjugated with cy3 for untreated (A) and treated for 24, 48 and 72 hours (B, C and D respectively). Formations of ‘ring’ shape (yellow arrows) of vimentin around the perinuclear zone were noted as soon as 24 hours post treatment. Mag. (A-C) 60X, (D) 120X.

86

11 Confocal micrograph of F-actin for untreated (A) and treated MDA-MB-231 cell line for 24, 48 and 72 hours (B, C and D respectively). The F-actin is still intact even after 48 hours treatment (C). The F-actin forms a spherical shape 72 hours post treatment. Mag. (A-C) 60X, (D) 120X.

87

12 Confocal micrograph of α-tubulin of MDA-MB-231 for control (A) and treated for 24, 48 and 72 hours (B, C and D respectively). Even after 48 hours post-treatment, the α-tubulin is still intact (C) and deteriorated after 72 hours treatment (arrow). Mag. 60X (A, B, D), 100X (C).

88

13 Confocal micrograph of vimentin for control (A) and treated MDA-MB-231 cell line for 24, 48 and 72 hours (B, C and D respectively). The F-actin is

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