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