UNIVERSITI PUTRA MALAYSIA

LEE SZE YEN

FBSB 2013 35

EFFECTS OF ZINC SUPPLEMENT ON TUMORIGENIC (MCF-7) AND

NON-TUMORIGENIC (MCF-10A) MAMMARY EPITHELIAL CELLS UNDER DIFFERENT OXYGEN CONDITIONS

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EFFECTS OF ZINC SUPPLEMENT ON TUMORIGENIC (MCF-7) AND NON-TUMORIGENIC (MCF-10A) MAMMARY EPITHELIAL CELLS

UNDER DIFFERENT OXYGEN CONDITIONS

By

LEE SZE YEN

Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of Master of Science

November 2013

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COPYRIGHT

All material contained within the thesis, including without limitation text, logos,

icons, photographs and all other artwork, is copyright material of Universiti Putra

Malaysia unless otherwise stated. Use may be made of any material contained

within the thesis for non-commercial purposes from the copyright holder.

Commercial use of material may only be made with the express, prior, written

permission of Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

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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

EFFECTS OF ZINC SUPPLEMENT ON TUMORIGENIC (MCF-7) AND

NON-TUMORIGENIC (MCF-10A) MAMMARY EPITHELIAL CELLS

UNDER DIFFERENT OXYGEN CONDITIONS

By

LEE SZE YEN

November 2013

Chairman : Norazizah Shafee, PhD

Faculty : Biotechnology and Biomolecular Sciences

Zinc is an important trace element which is obtained through our daily food intake.

Its imbalance causes a range of human diseases from simple headaches to cancer.

Insufficient zinc is addressed with the use of zinc supplements mostly in the form of

tablets. The importance of zinc level in the normal homeostasis of the human body

highlights the need for further understanding of zinc involvement in cellular

regulation. Changes in human physiology also affect the outcome of zinc functions.

These changes in women are more common due to conditions such as pregnancy,

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lactation and menopausal changes. Earlier studies reported that malignant breast

tissues were commonly found to correlate with high cellular zinc concentration.

Further studies on the outcome of high cellular zinc concentration on the cellular

functionhowever were never reported until now. To address this issue, this study was

conducted to investigate the effects of zinc supplement on tumorigenic MCF-7 and

non-tumorigenic MCF-10A mammary epithelial cells. It was hypothesized that zinc

supplement will cause the cell cycle control mechanisms of breast epithelia to be

deregulated. Results obtained showed that the cells responded differently to zinc

treatment beginning at 100 µM zinc, but not at lower concentrations. MCF10-A was

found to be arrested at the G2/M cell phase at higher level compared to the MCF-7

cells. Conditions of normoxia and hypoxia did not drastically affect the way that the

MCF-7 and MCF-10A responded to zinc treatment. The G2/M arrest was found to be

associated with the increase in Cyclin A in both cell lines. In MCF-10A, p21CIP1/WAF1

protein was increased but not in MCF-7. This perhaps contributed to the higher G2/M

population in zinc-treated MCF-10A. Interestingly, a hypoxia-inducible factor alpha

(HIF-1α), a monomer of the HIF-1 transcription factor also became accumulated in

the presence of zinc, in both MCF-7 and MCF-10A cells. The HIF-1 complex was

found to be active via detection of the CAIX expression, its specific transcriptional

target. Overall, data obtained from this study contribute to further understanding of

zinc in the regulation of breast epithelial cells. The information can be used to assist

future research in the correlation of zinc and the development of breast cancer as well

as other types of cancers in human.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

KESAN ZINK TAMBAHAN TERHADAP SEL EPITELIUM MAMARI YANG

BERBARAH (MCF-7) DAN TIDAK BERBARAH (MCF-10A) BAWAH

KANDUNGAN OKSIGEN YANG BERBEZA

Oleh

LEE SZE YEN

November 2013

Pengerusi : Norazizah Shafee, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Zink merupakan unsur surih penting yang dapat diperolehi melalui pemakanan harian.

Ketidakseimbangan zink mengakibatkan pelbagai penyakit dari simptom ringan

seperti sakit kepala ke penyakit barah yang kronik. Kekurangan zat zink diatasi

dengan pengambilan zink tambahan selalunya dalam bentuk pil. Kepentingan zink

dalam pengawalaturan homeostasis tubuh manusia menekankan keperluan

pemahaman lanjut terhadap penglibatan zink dalam pengawalaturan sel. Perubahan

fisiologi manusia juga menjejaskan fungsi zink. Perubahan ini adalah lebih ketara di

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kalangan wanita disebabkan oleh faktor-faktor seperti kehamilan, laktasi dan

perubahan putus haid. Kajian sebelum ini melaporkan bahawa tisu payu dara

malignan biasanya dihubungkaitkan dengan kepekatan selular zink yang tinggi.

Walaubagaimanapun, tiada kajian lanjut mengenai kesan kepekatan selular zink yang

tinggi terhadap fungsi selular pernah diterbitkan. Oleh itu, kajian ini dijalankan untuk

mengkaji kesan zink tambahan pada sel mamari epitelium yang berbarah (MCF-7)

dan tidak berbarah (MCF-10A). Zink tambahan dihipotesiskan akan menyebabkan

penyahkawalan dalam mekanisme kitaran sel. Keputusan yang diperolehi

menunjukkan bahawa sel-sel bertindakbalas secara berbeza terhadap rawatan zink

dengan kepekatan zink pada 100 µM dan ke atas, tetapi tidak pada kepekatan yang

lebih rendah. Kitaran sel MCF-10A didapati ditahankan pada fasa G2/M dengan

magnitud yang lebih tinggi berbanding MCF-7. Keadaan normosia dan hipoksia tidak

menjejaskan tindakbalas MCF-7 dan MCF-10A terhadap rawatan zink secara drastik.

Penahanan kitaran sel pada fasa G2/M didapati berhubungkait dengan peningkatan

dalam Cyclin A dalam kedua-dua sel yang digunakan dalam kajian ini. Peningkatan

protein p21CIP1/WAF1

dalam MCF-10A tidak diperhatikan dalam MCF-7. Peningkatan

ini mungkin menyumbang kepada populasi G2/M yang lebih tinggi dalam MCF-10A

yang menerima rawatan zink. Menariknya, peningkatan subunit factor induksi

hipoksia 1α (HIF-1α) juga diperhatikan dalam MCF-7 dan MCF-10A dengan

kehadiran zink. Kompleks HIF-1 tersebut dibuktikan aktif dari segimen jalankan

fungsi transkripsi melalui ekspresi CAIX yang merupakan sasaran khusus transkripsi

HIF-1α. Secara keseluruhan, data kajian ini menyumbang kepada pemahaman lanjut

tentang peranan zink dalam pengawalaturan sel epithelium mamari. Maklumat kajian

ini juga dapat membantu penyelidikan masa depan yang berkaitan hubungan zink

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tambahan dengan pembentukkan kanser manusia secara am dan kanser payu dara

secara khusus.

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ACKNOWLEDGEMENTS

First of all, I would like to express my deepest gratitude to my project supervisor

Assoc. Prof. Dr. Norazizah Shafee for her continuous support and advice throughout

my Master study. Her patience and guidance throughout the thesis writing process are

also highly appreciated. I could not imagine the difficulties that I would face without

her assistance.

Besides my project supervisor, I would like to thank all my co-supervisors: Prof. Dr.

Eric J. Stanbridge and Datin Paduka Prof. Dr. Khatijah Mohd Yusoff for their

encouragement, critical suggestions and comments which improved the research and

this thesis.

I am grateful to have kind and helpful labmates for the discussion and assistance in

the past three years. Thank you all for sharing the hard time, stress, and also fun

which cheered me up when I was demotivated by undesired research outcomes.

Last but not least, I would like to thank my family and friends for their understanding

and support throughout my life generally and along my Master study specifically.

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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:

Norazizah Shafee, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Khatijah Mohd Yusoff, PhD

Professor Datin Paduka

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

Eric J. Stanbridge, PhD

Distinguished Research Professor

School of Medicine

University of California, Irvine, USA

(Member)

________________________

BUJANG 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 institutions.

____________________

LEE SZE YEN

Date: 29 November 2013

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TABLE OF CONTENT

Page

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vii

APPROVAL viii

DECLARATION x

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF ABBREVIATIONS xvii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4

2.1 The importance of zinc 4

2.1.1 Zinc and cancer 4

2.1.1.1 Zinc influences the level of oxidative 4

stress in cells

2.1.1.2 Zinc affects cellular signal transduction 6

pathways

2.1.1.3 Involvement of zinc in cancer development 7

2.2 Cell cycle 9

2.2.1 Cyclin 11

2.2.2 Cyclin-dependent kinase (CDK) 13

2.2.3 Cyclin-dependent kinase inhibitor (CKI) 13

2.2.4 Cell cycle events 15

2.2.5 Cell cycle restriction points and checkpoints 18

2.2.6 Cell cycle and cancer 20

2.2.6.1 Cyclin 20

2.2.6.2 CDK 21

2.2.6.3 CKI 21

2.3 Oxygen and cancer 22

2.3.1 Hypoxia inducible factor (HIF) 22

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2.3.2 Biochemical and biological properties of HIF 23

2.3.3 HIF and cancer 24

3 MATERIALS AND METHODS 28

3.1 Source of cell lines 28

3.2 Maintenance of cell culture 28

3.2.1 Reconstitution of cells from liquid nitrogen 28

3.2.2 Culturing of cells 29

3.2.3 Confirmation of cells' mycoplasma status 29

3.2.4 Subculturing and propagation of cells 30

3.2.5 Cryopreservation of cells 31

3.2.6 Cell counting and seeding 31

3.2.7 Treatment of cells with zinc 32

3.3 Cell viability assay by flow cytometry 32

3.4 Protein analysis 33

3.4.1 Protein harvesting in the form of cell lysate 33

3.4.2 Protein quantitation 34

3.4.3 Sodium dodecyl sulfate polyacrylamide gel 36

electrophoresis

3.4.4 Western blotting and immunodetection 37

3.5 Cell cycle analysis 39

3.6 Confocal immunofluorescence microscopy 40

3.7 Ribonucleic acid (RNA) analysis 41

3.7.1 RNA extraction and purification 41

3.7.2 Confirmation of RNA concentration and purity 42

3.7.3 Reverse transcriptase polymerase chain reaction 43

(RT-PCR)

3.7.4 Agarose gel electrophoresis 47

3.8 Statistical analyses 47

4 RESULTS AND DISCUSSION 48

4.1 Confirmation of mycoplasma status 48

4.2 Optimisation of zinc concentration 50

4.2.1 Cellular morphological changes following zinc 51

treatment

4.2.2 Cell viability assay using flow cytometry 54

4.3 Cell cycle analysis using flow cytometry 60

4.3.1 Zinc interferes with cell cycle regulation 60

4.3.2 Hypoxia released zinc induced cell cycle arrest 66

in MCF-7 but enhanced it in MCF-10A

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4.4 Evaluation of cell cycle regulatory protein level in zinc- 67

treated cells

4.4.1 Effects of zinc on cyclin 67

4.4.2 Effects of zinc on CKI, p21CIP1/WAF1

71

4.5 Effects of hypoxia on cell cycle regulation on zinc- 73

treated cells

4.5.1 Zinc treatment still caused CyclinA accumulation 74

in hypoxic MCF-7 and MCF-10A cells

4.5.2 p21CIP1/WAF1's fold-difference remained increased 77

in zinc-treated hypoxic MCF-10A but no increase

in hypoxic MCF-10A

4.6 Effects of zinc treatment on HIF-1α and CAIX 79

4.6.1 Zinc treatment caused HIF-1α accumulation 79

leading to increased expression of CAIX

4.6.2 Zinc caused HIF-1α accumulation at its post- 84

translational level

5 CONCLUSION AND RECOMMENDATIONS 87

FOR FUTURE RESEARCH

REFERENCES 90

APPENDICES 101

BIODATA OF STUDENT 103

PUBLICATIONS 104