UNIVERSITI PUTRA MALAYSIA

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1 UNIVERSITI PUTRA MALAYSIA EFFECTS OF ZINC SUPPLEMENT ON TUMORIGENIC (MCF-7) AND NON-TUMORIGENIC (MCF-10A) MAMMARY EPITHELIAL CELLS UNDER DIFFERENT OXYGEN CONDITIONS LEE SZE YEN FBSB

2 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

3 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

4 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, ii

5 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 G 2 /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 G 2 /M arrest was found to be associated with the increase in Cyclin A in both cell lines. In MCF-10A, p21 CIP1/WAF1 protein was increased but not in MCF-7. This perhaps contributed to the higher G 2 /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. iii

6 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 iv

7 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 G 2 /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 G 2 /M didapati berhubungkait dengan peningkatan dalam Cyclin A dalam kedua-dua sel yang digunakan dalam kajian ini. Peningkatan protein p21 CIP1/WAF1 dalam MCF-10A tidak diperhatikan dalam MCF-7. Peningkatan ini mungkin menyumbang kepada populasi G 2 /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 v

8 tambahan dengan pembentukkan kanser manusia secara am dan kanser payu dara secara khusus. vi

9 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. vii

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11 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: ix

12 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 x

13 TABLE OF CONTENT ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS CHAPTER Page ii iv vii viii x xiv xv xvii 1 INTRODUCTION 1 2 LITERATURE REVIEW The importance of zinc Zinc and cancer Zinc influences the level of oxidative 4 stress in cells Zinc affects cellular signal transduction 6 pathways Involvement of zinc in cancer development Cell cycle Cyclin Cyclin-dependent kinase (CDK) Cyclin-dependent kinase inhibitor (CKI) Cell cycle events Cell cycle restriction points and checkpoints Cell cycle and cancer Cyclin CDK CKI Oxygen and cancer Hypoxia inducible factor (HIF) 22 xi

14 2.3.2 Biochemical and biological properties of HIF HIF and cancer 24 3 MATERIALS AND METHODS Source of cell lines Maintenance of cell culture Reconstitution of cells from liquid nitrogen Culturing of cells Confirmation of cells' mycoplasma status Subculturing and propagation of cells Cryopreservation of cells Cell counting and seeding Treatment of cells with zinc Cell viability assay by flow cytometry Protein analysis Protein harvesting in the form of cell lysate Protein quantitation Sodium dodecyl sulfate polyacrylamide gel 36 electrophoresis Western blotting and immunodetection Cell cycle analysis Confocal immunofluorescence microscopy Ribonucleic acid (RNA) analysis RNA extraction and purification Confirmation of RNA concentration and purity Reverse transcriptase polymerase chain reaction 43 (RT-PCR) Agarose gel electrophoresis Statistical analyses 47 4 RESULTS AND DISCUSSION Confirmation of mycoplasma status Optimisation of zinc concentration Cellular morphological changes following zinc 51 treatment Cell viability assay using flow cytometry Cell cycle analysis using flow cytometry Zinc interferes with cell cycle regulation Hypoxia released zinc induced cell cycle arrest 66 in MCF-7 but enhanced it in MCF-10A xii

15 4.4 Evaluation of cell cycle regulatory protein level in zinc- 67 treated cells Effects of zinc on cyclin Effects of zinc on CKI, p21 CIP1/WAF Effects of hypoxia on cell cycle regulation on zinc- 73 treated cells Zinc treatment still caused CyclinA accumulation 74 in hypoxic MCF-7 and MCF-10A cells p21 CIP1/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 Zinc treatment caused HIF-1α accumulation 79 leading to increased expression of CAIX 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 xiii