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Modulating effects of zinc on the efficacy of tamoxifen in human breast cancer cells Tsukada, Yoko Ann 2003

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Modulating Effects of Zinc on the Efficacy of Tamoxifen in Human Breast Cancer Cells by Yoko Ann Tsukada B.Sc. (Nutritional Sciences), The University of British Columbia, 2000  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Human Nutrition Program) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 2003 © Yoko Ann Tsukada, 2003  in  presenting this  degree at the  thesis in  University of  partial  fulfilment  of  the  requirements  British Columbia, I agree that the  freely available for reference and study. I further  this thesis for scholarly purposes may be granted  department  or  his  or  her  representatives.  an advanced  Library shall make it  agree that permission for extensive  copying of  by  for  It  is  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  ~\^vjo/W/aVi ^ w V ^ n o v N  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT  Growth can be viewed as the net balance between cell proliferation and cell death. Zinc is essential to cell proliferation and functions as a regulator in apoptosis (genedirected cell death).  Thus zinc status can critically influence overall growth.  We  hypothesized that modulating medium zinc concentration will alter the availability of metabolically active intracellular zinc, which in turn affects the overall growth of human cancer cells. The overall objective of my thesis project was to investigate the modulating effects of zinc on the efficacy of tamoxifen in human breast cancer cells. The cells were first cultured in D M E M (Dublecco's Modified Eagle Media; 10% fetal bovine serum (FBS)) until approximately 70% confluence. Then the cells were cultured in a low zinc medium supplemented with 0, 5, 50, or 150 umol/L zinc for 72h. Upon zinc treatment, the cells were treated with a combination of zinc and tamoxifen (0, 1, 5, or 10 umol/L) for 2, 24, or 48 h followed by a 24 h recovery period. At the end of the recovery period, cells were assessed for overall cell growth by counting cell numbers, cell viability by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay, and the profile of live cell and cell death (both necrotic and apoptotic cell death) using flow cytometry.  To explore the possible mechanisms involved, the m R N A levels of  targeted apoptotic regulatory proteins (p53, gadd45, Bax, and Bcl-2) were determined in human breast cancer MDA-MB-231 cells using reverse transcription-polymerase chain reaction. Either zinc supplementation (e.g. 150 umol/L) or tamoxifen alone suppressed overall cell growth, but had little effect on cell survival and cell death.  In contrast, a  combination of zinc and tamoxifen suppressed overall cell growth, reduced cell viability and cell survival, and increased both necrotic and apoptotic cell death.  ii  In addition, a  combination of zinc and tamoxifen also elevated Bax and gadd45 mRNA levels in human breast cancer MDA-MB-231 cells. Since Bax is a well-known pro-apoptotic protein and gadd45 is involved in both negative growth control and the induction of apoptosis, zinc and tamoxifen-induced apoptosis in the above cell line appeared to be Bax- and gadd45dependent. Overall, these observations suggested that zinc increased the efficacy of tamoxifen as indicated by decreased overall cell growth in breast cancer cells.  iii  TABLE OF CONTENTS  Abstract  1 1  Table o f Contents  iv  List o f Tables  v i i  List o f Figures  v  Acknowledgements  m  1 X  CHAPTER 1. Introduction  1  1. Literature Review  2  1.1. Zinc nutrition  2  1.2.  Cellular zinc  3  1.2.1.  4  Cellular zinc homeostasis  1.2.2. Techniques used for determination o f cellular zinc  6  1.3.  Zinc and cell proliferation  7  1.4.  C e l l death  9  1.4.1.  10  1.5.  1.6.  Apoptosis  1.4.2. Necrosis  14  Zinc and cell death  14  1.5.1.  14  Zinc and apoptosis  1.5.2. Zinc and apoptotic regulatory proteins  17  1.5.3.  Zinc and necrosis  19  Tamoxifen and apoptosis  19  iv  1.7. Summary  i : >  2. Hypothesis  2 6  3. Overall Objectives  26  4. Rationale for focusing on MDA-MB-231 cells in Chapter 2  26  5. Bibliography  28  CHAPTER 2. Zinc Supplementation Increases the Efficacy of Tamoxifen in Human Breast Cancer MDA-MB-231 Cells  48  1. Introduction  48  2. Materials and Methods  50  3. Results  55  4. Discussion  58  5. Bibliography  74  CHAPTER 3. General Discussion and Conclusions  79  1. Human breast cancer cells and breast fibrocystic cells acquired ability to grow in low zinc environment  79  2. Effects of zinc and tamoxifen on human breast cancer cells and human fibrocystic cells  81  3. Overall conclusions and future directions  82  4. Bibliography  84  v  APPENDICES  APPENDIX I  Cell culture system  86  APPENDIX II  Effects of zinc and tamoxifen on human breast cancer MDA-MB231 cells  APPENDIX III  91  Effects of zinc and tamoxifen on human breast cancer MCF-7 cells 96  APPENDIX IV  Effects of zinc and tamoxifen on human breast cancer T47D cells 101  APPENDIX V  Effects of zinc and tamoxifen on human breast fibrocystic MCF-10 cells  107  vi  LIST OF TABLES  Table 2-1 Sequences of PCR primers used  vii  LIST OF FIGURES  Figure 2-1. Effects of zinc supplementation and tamoxifen on cellular zinc concentration in human breast cancer MDA-MB-231 cells  65  Figure 2-2. Effects of zinc supplementation and tamoxifen on cell viability in human breast cancer MDA-MB-231 cells  66  Figure 2-3. Effects of zinc supplementation and tamoxifen on cell number in human breast cancer MDA-MB-231 cells  67  Figure 2-4. Effects of zinc supplementation and tamoxifen on live cells, necrotic cells, and apoptotic cells in human breast cancer MDA-MB-231 cells  68  Figure 2-5. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of p53 and p2-microglobulin mRNA levels in human breast cancer MDA-MB-231 cells  69  Figure 2-6. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gadd45 and |32-microglobulin mRNA levels in human breast cancer MDAMB-231 cells  70  Figure 2-7. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of bax and (32-microglobulin mRNA levels in human breast cancer MDA-MB-231 cells  71  Figure 2-8. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of bcl-2 and (32-microglobulin mRNA levels in human breast cancer MDA-MB-231 cells  72  vm  Acknowledgements  I wish to deeply thank Dr. Zhaoming X u for his guidance and encouragement throughout my thesis project. Thank you very much for teaching me both technical and problem solving skills.  I also like to express thanks to Dr. Brian Ellis and Dr. Jim Thompson for their ideas and comments. Special thanks to Dr. Ryna Revy-Milne for reviewing my thesis and offering suggestions. In addition, thank you, Dr. Susan Barr for chairing my defense.  I wish to deeply thank my parents and my sister for their support during the stressful times. I would have not had the determination to finish my thesis project without their love.  I am grateful to Bella Wu for not only her technical assistance, but also her unconditional encouragement. Many thanks to the members of the Xu's laboratory: Sharon Hsi, Madeline Simpson. Thank you Shirley Paski for teaching me the skills necessary for my project.  I wish to thank Sherman Leung for his patience and understanding.  ix  CHAPTER 1: Introduction  Zinc is an essential micronutrient for animals, including humans.  One of the  well-established physiological functions of zinc is its role in growth. Growth can be viewed as the net balance of cell proliferation and cell death. Zinc is important to both. At the cell proliferation side, zinc is critical to gene expression and regulation, and D N A and R N A syntheses (Vallee and Falchuk, 1993).  In vitro, zinc deficiency results in  reduced D N A synthesis and cell cycle arrest (Springgate et al., 1973; Prasad et al., 1996). In vivo, dietary zinc deficiency suppresses growth in animals, including humans (King and Keen, 1994). At the cell death side, zinc is considered as a physiological regulator of apoptosis, a gene-directed cell death.  Zinc modulates apoptosis both in vitro and in vivo.  In  thymocytes, increasing labile intracellular zinc prevents apoptosis while a reduction of the labile intracellular zinc below a threshold concentration induces apoptosis (Zalewski et al., 1993).  When cellular zinc is increased, induction of apoptosis is blocked in  macrophages and T lymphocytes (Waring et al., 1990), U937 cells (Fukumachi et al., 1998) and P B L cells (Marini & Musiani, 1998).  In rats, zinc deficiency induces  apoptosis in embryonic cells (Rogers et a l , 1995).  The mechanisms by which zinc  regulates apoptosis are not clear. However, the expression of several apoptotic regulatory proteins, such as p53 (Reaves et al., 2000; Fanzo et al., 2001), Bax (Iitaka et al., 2001; Smith et al., 2002), Bcl-2 (Fukumachi et al., 1998; Ishido et al., 1999; Untergasser et al., 2000), and caspase 3 (Perry et a l , 1997; Chai et al, 2000; Ho et al., 2000; Chimienti et  1  al., 2001), are affected by cellular zinc concentration, indicating that these apoptotic regulatory proteins can be the potential molecular targets of zinc in regulating apoptosis. Dysregulation of apoptosis has been implicated in the development of many diseases, including cancer. Some anti-cancer drugs induce apoptosis in malignant cells. One such drug is tamoxifen, an anti-estrogen drug widely used in treating breast cancer. Tamoxifen induces apoptosis in numerous human breast cancer cell lines through altering the expression of some apoptotic regulatory proteins. For example, tamoxifen induces apoptosis in MCF-7 cells through downregulating the expression of Bcl-2 (Zhang et al., 1999). In MDA-MB-231 cells, tamoxifen upregulates the expression of c-myc (Kang et al., 1996) and stimulates the activity of caspase 3, 6, 8, and 9 (Mandlekar et al., 2000). Since both zinc and tamoxifen can influence apoptosis and their actions share some common targets, it is possible that zinc could work together with tamoxifen to enhance its efficacy.  1. Literature Review 1.1. Zinc Nutrition Zinc is an essential micronutrient for humans and animals. The Recommended Dietary Allowance (RDA) for adults is 8 mg/day for women and 11 mg/day for men (Institute of Medicine, 2002). Zinc is typically associated with the protein fraction of foods. Foods that are high in protein (e.g. meat and meat products) are generally high in zinc. Fruits and vegetables are usually low in zinc. Zinc is absorbed from the small intestine, primarily from the duodenum and the jejunum. It is released from the enterocytes at a rate partially dependent on luminal zinc  2  concentration (King and Keen, 1994). In the body, zinc is present as a divalent cation (Zn ). There is approximately 1.5 - 2.5 g of zinc in an adult human body with the bone, 2+  liver, and kidney containing most of the zinc (King and Keen, 1994). If enterocytes contain 20 u.g/g, the plasma zinc is approximately 15 umol/L (Oestreicher and Cousins, 1989).  At the cellular level, about 30-40% is in the nucleus, about 50% is in the  cytoplasm and cellular organelles, and the remaining 10-20% is in the cell membrane (Smeyers-Verbeke et al., 1977). In biological systems, zinc has a stable redox status. This stability of zinc makes it favourable to a vast array of functionally significant interactions. Zinc is an important structural or catalytical component in over 300 metalloenzymes (Vallee and Falchuk, 1993). These zinc metalloenzymes are recognized in all classes of enzymes, including oxidoreductase, hydrolase, lyase, isomerase, and transferase. In addition, zinc is also a structural component of a large number of zinc metalloproteins.  Some of these  metalloproteins are transcription factors involved in the regulation of gene expression. Overt zinc deficiency in humans in North America is not common. However, human zinc deficiency most often occurs when zinc intake is inadequate or poorly absorbed, when there are increased losses of zinc from the body, or when the body's requirement for zinc increases (Hambidge, 1989). The symptoms of zinc deficiency is diverse, including growth retardation, dermatitis, alopecia, poor wound healing, skeletal abnormalities, delayed sexual maturation, hypogeusia, and impaired immune function, etc.  1.2. Cellular Zinc  3  Most of the cellular zinc is tightly bound to enzymes and other proteins, and very little is either loosely bound to proteins or in free ionic form (Zn ) (Vallee and Falchuk, 2+  1993). The loosely bound intracellular zinc and zinc ion are often referred to as the labile zinc. Labile zinc is metabolically active (Zalewski et a l , 1993). Zinc concentration in cells is dependent on the cell type. For example, total level of zinc is higher in kidney cells than lymphocytes (Palmiter and Findley, 1995; Cheek et al., 1984). In general, the total cellular zinc concentration is in the nanomolar range while labile zinc concentration is in the picomolar range.  In lymphocytes, total cellular zinc is 150 nmol/10 cells 6  (Cheek et al., 1984) and the estimated labile zinc concentration is 14-31 pmol/10 cells 6  (Zalewski etal., 1993).  1.2.1.  Cellular zinc homeostasis. Intracellular levels of zinc are highly regulated although the mechanisms involved  are not well understood. In eukaryotes, there is a cellular regulatory system involving zinc transporters that play an important role in zinc metabolism (Gaither and Eide, 2001). This system consists of two basic groups of zinc transporters; one is responsible for zinc uptake and another is responsible for zinc efflux. The Zrt-, Irt-like Proteins (ZIP) family transports zinc from the extracellular environment into the cytoplasm. There are several subfamilies that are divided based on sequence similarities: ZIP I family consists of fungal and plant members; ZIP II family consists of mammalian and nematode proteins; gufA family consists of prokaryotes and eukaryotes; and LIV-1 family consists solely of eukaryotes (Gaither and Eide, 2001). Functional information has been investigated for only two ZIP genes in humans: human Zrt-, ih-like Protein 1 (hZIPl) and human Zrt-,  4  /rt-like Protein 2 (hZIP2).  Both are involved in zinc transport across the plasma  membrane, but unlike hZIPl, hZIP2 has only been detected in prostate and uterine tissue (Gaither and Eide, 2000). The cation diffusion facilitator (CDF) family promotes zinc efflux by pumping zinc from the cytoplasm out of the cell or transporting zinc into intracellular organelles. There are also several subfamilies that are divided based on sequence similarities: CDF family I is found in prokaryotes; CDF family II and III is found in eukaryotes and prokaryotes (Gaither and Eide, 2001). In mammals, seven genes have been functionally characterized: zinc transporter-1 (ZnT-1), zinc transporter-2 (ZnT-2), zinc transporter-3 (ZnT-3), zinc transporter-4 (ZnT-4), zinc transporter-5 (ZnT-5), zinc transporter-6 (ZnT6), and zinc transporter-7 (ZnT-7). ZnT-1 is found on the basolateral membrane of both the enterocytes (McMahon and Cousins, 1998b) and the renal tubule surface (McMahon and Cousins, 1998a), suggesting a role in transporting zinc out of the enterocytes and urine into the bloodstream. ZnT-2 and ZnT-3 are involved in intracellular zinc sequestration; the former is localized in the membrane of a lysosomal compartment (Palmiter et al., 1996a) and the latter is localized in neurons of the brain and testis (Palmiter et al., 1996b). ZnT-4 is expressed in mammary gland and transports zinc into milk (Huang and Gitschier, 1997). ZnT-5 is ubiquitously expressed in all tested human tissues, especially in the pancreas and is involved in transporting zinc into secretory granules (Kambe et al., 2002).  The functionality of ZnT-6 has not been firmly  established. It may function in transporting cytoplasmic zinc into the Golgi apparatus as well as into the vesicular compartment (Huang et al., 2002). More recently, ZnT-7 has been shown to facilitate zinc transport from the cytoplasm into the Golgi apparatus  5  (Kirschke and Huang, 2003). It is evident that the intracellular zinc concentration is homeostatically regulated involving many zinc transport proteins, which act in tissue-, cell-, and organelle-specific manners. Cellular zinc status influences the influx or efflux of zinc.  For instance,  expression of hZIPl, which belongs to the ZIP family, was repressed when zinc was added to the media in prostate cell lines LNCap and PC-3 (Costello et al., 1999). mRNA levels of ZnT-1, which belongs to the CDF family, were increased when exposed to zinc in neuronal cells (Kohet al., 1996). Not only are the transporters  important in zinc metabolism, but also  metallothionein, a major zinc-binding protein independently regulates labile zinc (Hamer, 1986). Metallothionein is a small protein containing 60-68 amino acids, of which 20 are highly conserved cysteines. atoms.  Metallothionein is capable of binding up to seven zinc  In response to increased flux of zinc, mRNA transcripts coding for  metallothionein increase,  suggesting zinc is a transcriptional regulator  of the  metallothionein genes (Durnam and Palmiter, 1981). The true physiological function of metallothionein still remains controversial, but it seems to be a physiological regulator of intracellular zinc concentration. For instance, when estrogen receptors, which are zinc finger proteins, are zinc-depleted, metallothionein donates zinc; but when thionein lacks zinc, it extracts zinc from estrogen receptors (Cano-Gauci and Sarkar, 1996).  1.2.2. Techniques used for determination of cellular zinc. Several techniques have been used to determine zinc concentration. For total cellular zinc, atomic absorption spectroscopy is a common technique (Vallee and  6  Falchuk, 1993). For intracellular zinc, different techniques have been employed. However, interestingly, different techniques derive somewhat different concentrations, even factoring in variations among different cell types. For example, using radioactive 65  Z n , the labile zinc concentration is estimated at 24 pmol/L in red blood cells (Simons  1991).  In contrast, using fura-2 fluorescent probe, the labile zinc concentration is  estimated at 1 nmol/L in cardiac myocytes (Atar et al., 1995).  Thus, the precise  concentration of the labile zinc has not been established (Beyersmann and Haase, 2001).  1.3. Zinc and Cell Proliferation One of the well known physiological functions of zinc is its role in cell proliferation. Some of the zinc metalloproteins and metalloenzymes are critical to gene expression and regulation, and D N A and R N A syntheses (Vallee and Falchuk, 1993). In some zinc metalloproteins, zinc is an essential structural component of the domain, which is required for binding to single-stranded D N A , known as zinc-finger protein (Vallee and Falchuk, 1993). Some of the zinc-finger proteins are nuclear transcription factors, which work together with other transcription factors to control cell proliferation, differentiation, and death (Urrutia, 1997). For example, replication protein A (RPA) is a zinc-finger protein that is necessary for D N A replication (Iftode et al., 1999). Poly (ADP-ribosyl) polymerase (PARP) is another zinc-finger protein, which is rapidly activated during cellular response to D N A damage and protects the cell from genotoxic damage (de Murcia etal., 1994). Zinc plays an essential role in cell proliferation, including the cascades of intracellular signalling pathways involved in D N A synthesis (Springgate et a l , 1973),  7  transcription (Wu et al., 1992), and ribosomal function (Hard et al., 2000). Being part of the zinc-finger structure of many transcription factors, zinc also plays a role in controlling the activity of genes responding to growth factors (Berg and Shi, 1996). The effects of zinc on cell proliferation are zinc-concentration dependent and tend to be biphasic. Both zinc deficiency and zinc excess may inhibit proliferation. Prasad et al. (1996) showed that zinc deficient T helper cells have a slower cell doubling time and do not undergo mitosis. Also, after 48 h in a low zinc medium, there is suppression in cell proliferation of fibroblast 3T3 cells that is observed through a decline in cell numbers (Paski and X u , 2001). On the other hand, high zinc concentrations also suppress cell proliferation. In prostate carcinoma LNCaP cells, zinc reduces viability at 500 umol/L (Iguchi et al., 1998). Similarly, Liang et al. (1999) observed that, in prostate carcinoma LNCaP and PC-3 cells, zinc at 1 ug/ml (15 umol/L) induced cell cycle arrest at G 2 / M phase and characteristic morphological changes of apoptosis, such as blebbing of the membrane. Yet, the concentration of zinc that affects cell proliferation varies among cell lines. For instance, culturing lymphoid (Molt-3 and Raji) and myeloid (HL-60) cells under zinc deficient conditions 1.4 umol/L results in complete loss of proliferative capacity (Martin et al., 1991). Evidence shows that zinc is involved in the regulation of D N A replication. In fibroblast 3T3 cells, supplementation with zinc promoted D N A synthesis and cell proliferation (Paski and X u , 2001). A combination of zinc and calcium synergistically stimulates D N A synthesis and mitogenic signalling in fibroblast 3T3 cells (Huang et al., 1999). As well, cells need zinc and growth factors to enter the S phase of the cell cycle (Chester and Boyne, 1991).  For example, zinc deficiency (20 umol/L) specifically  8  causes diminished insulin-like growth factor (IGF-1) levels in rats (Roth and Kirchgessner, 1994), indicating zinc could be involved in IGF-1 mediation of the G l to S phase transition (Campisi and Pardee, 1984). When rats are fed zinc deficient diets for 5 d, there is a decrease in thymidine incorporation into D N A in the kidney and spleen (Williams and Chester, 1970). The activity of thymidine kinase increases dramatically during G l and early S phase of the cell cycle and is often used as a marker of cell proliferation. Thymidine kinase is not a zinc metalloenzyme, but its transcription appears to be regulated by zinc availability (Prasad et al., 1996).  Similar effects are seen in  cultured cells. When diethylenetrinitrilopentaacetate (DTPA), an extracellular divalent cation chelator, is used to remove zinc from the media, transcription of the thymidine kinase gene is impaired (Chester et a l , 1990) and thymidine incorporation into D N A is inhibited in fibroblast 3T3 cells (Chester et al., 1989). However, thymidine uptake can be restored with simultaneous treatment of DTP A (600 umol/L) and zinc (400 umol/L), but not other divalent cations (e.g. iron, calcium, or cadmium) in 3T3 cells (McDonald et al., 1998). These observations clearly demonstrate that this DTPA-induced inhibition on thymidine uptake is zinc specific.  1.4. Cell Death There are two types of cell death: apoptosis and necrosis. These two types of death are distinctly different, both morphologically and biochemically. The mechanisms by which these processes are controlled are far from being clear, but increasing evidence shows that they both play an important role in the development and progress of diseases such as cancer.  9  1.4.1. Apoptosis. Apoptosis is a gene-directed cell death. During embryonic and fetal development, and in immune response, apoptosis is more prominent (Cohen et al., 1992; Truong-Tran et al., 2000). Upon the induction of apoptosis, cells undergo a series of characteristic morphological changes, separating the dying cells from the healthy "neighbouring" cells. As the cells loose contact with adjacent cells, shrinkage of the cell occurs due to net outward movement, which is caused by the inhibition of the N a - K - C l " cotransporter +  system (Wilcock and Hickman,  1988).  +  Other morphological changes include  translocation of phosphatidylserine from inner membrane to outer membrane, membrane blebbing, mitochondria leakage, condensation of the cytoplasm and nucleus, and D N A fragmentation. Convolutions of nuclear and plasma membrane occur simultaneously with chromatin condensation. As the blebbing process proceeds, nuclear fragments are found in large, oval cytoplasmic fragments and budding of the cell occurs to form "apoptotic bodies". These apoptotic bodies are then phagocytosed (Kerr et al., 1987; Wyllie, 1980). Apoptotic cell death is a highly regulated process involving a growing number of apoptotic regulatory proteins, including p53, gadd45, and members of the Bcl-2 and caspases families. p53.  p53, a zinc-finger protein, is a tumour suppressor and a modulator in  apoptotic pathways. It is a nuclear DNA-binding phosphoprotein that normally exists as a homotetramer or as complex tetramers. Since it has a rapid turnover rate, p53 is present at low levels in normal conditions. p53 synthesis can be activated by carcinogens, radiation, ultraviolet light, or hypoxia (Kirsch and Kastan, 1998). In response to these cellular stresses, p53 synthesis is upregulated. Subsequently, downstream responses are  10  activated, resulting in either cell cycle arrests allowing D N A repair to occur (Kastan et al., 1991) or apoptosis leading to cell death (Bellamy, 1997). In the case of cell cycle arrest, p53 causes cell growth arrest at certain stages in the cell cycle, thought to be late Gi arrest. As for the possible mechanisms behind p53-induced apoptosis, p53 activates Bax, a pro-apoptotic protein. Bax forms heterodimers with anti-apoptotic Bcl-2 proteins, leading to apoptosis (Miyashita and Reed, 1995). Yet, Bax is not essential for p53induced apoptosis because Bax-deficient mice still undergo p53-induced apoptosis following D N A damage (Knudson et al., 1995). Loss or mutation in p53 is common in human cancers, since it is vulnerable to dysfunction caused by even a single base change in the coding sequence (Bellamy, 1997). P53 mutation is usually a functional mutation rather than a deletion. In mouse mammary tumorigenesis, dysregulated p53 include a dominant negative mutant of p53 (TM4 cells), p53-null phenotype (TM2H cells), and overexpression of p53 (DI cells) (Medina et al., 1998). Regardless o f the mutation, radiation and certain cytostatics can still induce apoptosis via the p53-independent pathway, whereby cell death does not require functional p53 (Clarke et al., 1993). However, the mechanisms of p53-independent apoptosis are still unknown. Growth arrest and DNA damage protein (gadd45). Gadd45 is an apoptotic regulatory protein downstream of p53. There are three isoforms of gadd45: gadd45-oc, (3, and -y. It is expressed following treatment of cells with most DNA-damaging agents and during growth arrest conditions (Fornace et al., 1992). Its induction has been shown to be both p53-dependent and -independent in various cell lines (Papathanasiou et al., 1991). Induction of gadd45 by ionizing radiation is dependent on normal p53 function (Zhan et a l , 1994b), but induction of gadd45 by non-ionizing radiation DNA-damaging  11  agents such as U V radiation does not require p53 (Zhan et al., 1998). In addition, gadd45 is also a cell cycle regulated nuclear protein that reaches maximal levels in Gi phase of the cell cycle (Hall et al., 1995). For example, gadd45-a-null mice are more susceptible to radiation-induced carcinogenesis (Hollander et al., 1999). Similarly, when cells lack gadd45-a genes, there are multiple chromosome abnormalities, showing that the gene contributes to the maintenance of genomic stability (Hollander et al., 1999). On the other hand, overexpression of gadd45 has been reported to suppress cell growth in several different tumour cell lines (Zhan et al., 1994a), but the mechanisms involved are still unclear. Bcl-2 family. Bcl-2 is a family of apoptotic regulatory proteins.  Some of the  members in the family function as pro-apoptotic proteins (e.g. Bax and Bad) while the others function as anti-apoptotic proteins (e.g. Bcl-2 and Bcl-xL) (Pellegrini and Strasser, 1999). Many of these proteins interact with each other through a complex network of homo- and hetero-dimers. The exact nature of the dimerization is unclear, but the ability of the pro-apoptotic members to induce apoptosis is dependent on their ability to bind and antagonize pro-survival members (Kelekar and Thompson, 1998). However, the opposite does not apply (Yin et al., 1994). Expression of Bcl-2 can be regulated by cytokines and the expression of Bax is upregulated when p53 is activated (Miyashita and Reed, 1995). It is thought that the ratio of anti-apoptotic versus pro-apoptotic dimmers (e.g. Bcl-2:Bax ratio) is important in determining resistance of a cell to apoptosis (Konopleva et al., 1999). In the case of cancer, there is an imbalance in the expressions of the above proteins and a disruption in the rate of apoptosis declines. For instance, in lymphocytic leukemia, translocation of the Bcl-2 gene causes its overexpression, decreasing apoptosis  12  compared to cells with a normal Bcl-2 gene (Adams and Cory, 1998). Bax has also been noted to be mutated in gastrointestinal tumours and leukemias (Rampino et a l , 1997). Caspases. Caspases is another family of apoptotic regulatory proteins consisting of at least 14 cysteine proteases (Miura et al., 1997) that have distinct roles in apoptosis. Activation of caspases involves proteolytic processes  between  domains of the  proenzymes, followed by association of large and small subunits to form heterodimers (Thornberry and Lazebnik, 1998). Caspases play a key important role in apoptosis. For example, initiator caspases such as caspase 8 are activated by specific ligand-receptors interaction such as Fas-Ligand (Huppertz et al., 1999). Initiator caspases subsequently activate a second group of caspases known as executioner caspases. Caspase 3 is known as a key "executioner", which inevitably cleaves many key proteins involved in apoptosis. Caspase 6 is downstream to the initiator caspases such as caspase 3 (Huppertz et al., 1999).  Activation of caspase 3 can result in activation of pro-caspase-6, but  activation of pro-caspase-6 can in turn result in activation of caspase 3 (Srinivasula et al., 1996) . Together, these 2 caspases form a protease amplification cycle. Activation of caspases results in cleavage of numerous targets and morphological changes, such as the formation of cell surface blebs observed in apoptotic cells (Wolf and Eastman, 1999). Proteins in the Bcl-2 family are targets of caspases. Upon cleavage, these proteins are inactivated and fragmented. 1997) .  The fragments then promote apoptosis (Xue and Horvitz,  Another mechanism by which caspase kills cells is by destroying the cell  structures such as the nuclear lumina (Takahashi et al., 1996). The lumina are rigid structures that are part of the nuclear membrane and allow for chromatin organization.  13  1.4.2. Necrosis. Necrosis is another type of cell death that occurs when cells are exposed to serious physical or chemical damage. The damage leads to rapid incapacitation of major functions and to collapse of internal homeostasis (Kerr et al., 1972). A T P depletion is the critical precursor to the morphological changes.  This type of cell death is a passive  degenerative phenomenon, meaning it does not require energy for its occurrence. Swelling of cells and disruption of membranes are prominent features of necrosis, and the nuclear chromatin undergoes lysis (Hawkins et al., 1972). The swelling is due to the disappearance of ion-pumping activity. The disruption of membranes is amplified by phospholipase and proteases that are activated by cytosolic C a  2+  (Wyllie, 1981). The  nuclear chromatin also condenses like apoptosis but as smaller-sized flocculent densities in an ill-defined, irregular pattern (Wyllie, 1981). In final stages of necrosis, chromatin scatters into many loosely associated particles. Cytoplasmic contents including lysosomal enzymes are released into the extracellular space, which triggers inflammation in necrotic tissue with extensive damage in vivo.  1.5. Zinc and Cell Death 1.5.1. Zinc and apoptosis. Numerous studies have shown modulatory effects of zinc on apoptosis in vitro and in vivo. Incubation of prostate carcinoma cell lines in 15 umol/L zinc, results in G2/M arrest and an increase in apoptosis as indicated by D N A fragmentation (Liang et al., 1999). In premyelocytic leukemia cells cultured in low zinc media (0.5 umol/L) for 30 h, there was a decrease in intracellular zinc and disruption in the mitochondrial  14  transmembrane potential, which is a marker of early phase apoptosis (Duffy et al., 2001). In mouse thymocytes, zinc supplementation (80 - 200 umol/L), induces apoptosis (Telford and Fraker, 1995). However, when media zinc concentration reaches above 200 umol/L, zinc has an inhibitory effect on apoptosis in mouse thymocytes.  Similarly, zinc  deficiency (1.4 umol/L) also induces apoptosis in lymphoid (Molt-3 and Raji) cells while zinc repletion (50 umol/L) inhibits apoptosis (Martin et al., 1991).  In contrast to  lymphoid cells, myeloid (HL-60) cells cultured in zinc-deficient media reduces cell viability, but do not undergo apoptosis (Martin et al., 1991). Depletion of zinc by using chelators also induces apoptosis.  For instance, when neural cells are treated with  ethylenediaminetetraacetic acid (EDTA), apoptosis is detected (Sakabe et al., 1998). In thymocytes, increasing labile intracellular zinc by using zinc ionophore prevents apoptosis while a chelator-induced reduction of the labile intracellular zinc induces apoptosis (Zalewski et al., 1993). Feeding zinc deficient diets to experimental animals also induces apoptosis. In rats, apoptotic embryonal cell death is aroused within several days after female rats are fed on a severe zinc deficient diet (0.5 ug zinc/g diet) (Rogers et al., 1995; JankowskiHennig et al., 2000). Similarly, feeding a zinc deficient diet (1 ug zinc/g diet) induced apoptosis in the thymus, testis, kidney and liver (Nodera et al., 2001). Apoptosis was first induced in the thymus only 1 wk after feeding the zinc deficient diet. In contrast, apoptosis was not induced in the testis, kidney and liver till 3,13 and 34 wk, respectively, after feeding the zinc deficient diet. This latency in the induction of apoptosis suggests that the susceptibility to zinc deficiency-induced apoptosis is tissue-specific in rats. In juvenile chickens, zinc deficiency (10 mg/kg diet) also induced apoptosis in the  15  epiphyseal growth plate (Wang et al., 2002).  Besides zinc deficiency, zinc  supplementation has also been shown to induce apoptosis. For example, zinc deficiency in  rats  increased  esophageal  cell  proliferation  and  the  nitrosomethylbenzylamine (NMBA)-induced esophageal tumours.  incidence  of N -  Zinc replenishment  rapidly induced apoptosis in esophageal cells and thereby reduced the development of NMBA-induced esophageal cancer (Fong et al., 2001).  Clearly, zinc is capable of  modulating apoptosis in animals. This modulating effect of zinc on apoptosis appears to be zinc concentration- and tissue type-dependent. With zinc supplementation, incubated cells become more resistant to the induction of apoptosis.  For example, zinc supplementation at 5 mmol/L blocked  sporidesmin-induced apoptosis in macrophages and T lymphocytes (Waring et al., 1990), and dexamethasone-induced apoptosis in thymocytes (Barbieri et al, 1992). Similarly, zinc supplementation also suppresses hydrogen peroxide-induced apoptosis in peripheral blood lymphocytes (10 umol/L zinc; Marini and Musiani, 1998) and in human premonocytic U937 cells (1 mmol/L zinc; Fukumachi et al., 1998). A supplement of zinc in culture medium also prevented radiation-induced apoptosis in human keratinocytes (50 umol/L zinc; Parat et a l , 1997), human fibroblasts cells 99.4 umol/L; Leccia et al., 1999), and human premyeloctic leukaemic cells (1 mmol/L zinc; McGowan et al., 1994). In some cases, when zinc is supplied simultaneously with compounds such as diethyldithiocarbamate (DTC), which is a thiol-containing compound with radioprotective properties, the zinc concentration needed to inhibit radiation-induced apoptosis is reduced (Mathieu et al., 1996). Furthermore, supplementation of chelator-treated  16  endothelial cells with zinc also reduces linoleic acid- and tumour necrosis factor-induced apoptosis (Meerarani et al., 20000).  1.5.2. Zinc and apoptotic regulatory proteins. The mechanisms by which zinc influences apoptosis are not known. Scattered evidence suggests that some of the regulatory proteins involved in apoptosis are the possible cellular targets of zinc-dependent inhibition of apoptosis.  For instance, in  HepG2 cells, a hepatoblastoma cell line, zinc-deficiency resulted in a depletion of total cellular zinc along with an increased p53 expression (187%) compared with the control (Reaves et al., 2000).  When human bronchial epithelial cells were exposed to zinc  supplementation ( 4 - 1 6 umol/L), p53 mRNA and gadd45 m R N A abundance were elevated, but caspase-3 activity was decreased (Fanzo et al., 2001). At high concentrations such as 100 - 300 umol/L, zinc functions as a potent inhibitor of caspase 3 (Perry et al., 1997). A n increase in intracellular zinc suppressed caspase 3 activity while a decrease in intracellular zinc by N,N,N'N'-tetrakis(2pyridylmethyl)ethylenediamine (TPEN) increased its activity (Chai et al., 2000; Ho et al., 2000; Chimienti et al., 2001). Evidence obtained from in vivo studies also supports the notion that zinc is important to the caspase 3 activity. In embryos, zinc deficiency resulted in an increased caspase 3 activity along with an increased cell death rate (Jankowski-Hennig et al., 2000). However, zinc supplementation-induced attenuation of caspase 3 activity does not always lead to blockage of apoptotic cell death. For example, doxorubin is an important anticancer agent known to induce apoptosis in a wide variety of tumour cells. In adenocarcinoma cells (HeLa), zinc supplementation (100 umol/L)  17  inhibits doxoubin-dependent induction of caspase 3 activity, but does not block doxorubin-induced apoptosis and overall cell death (Lambert et al., 2001). Therefore, inhibiting the activity of caspase 3 does not necessary result in a block of apoptosis. Another potential molecular target of zinc are the members of Bcl-2 family. Zinc supplementation (50 umol/L) stimulated anti-apoptotic Bcl-2 expression in porcine kidney L L C - P K , cells (Ishido et al., 1999) and U937 cells (Fukumachi et al., 1998), resulting in an higher resistance of the cells to apoptosis. In contrast, high levels of zinc (1 mmol/L) led to an increased degradation of Bcl-2 protein, resulting in an increased apoptosis in prostate epithelial cells (Untergasser et al., 2000). Besides, zinc also affects the pro-apoptotic proteins (e.g. Bax and Bad) in the Bcl-2 family. Iitaka et al. (2001) has shown that treating the human thyroid cancer cell line 85053 with zinc (150 umol/L) reduced the levels of pro-apoptotic proteins Bax and Bad. When lymphoma cells were exposed to DNA-damaging agent VP-16, the intracellular zinc concentration was increased, which was paralleled with an increase in p53 protein level and Bax : Bcl-2 ratio (Smith et al., 2002). Cytochrome c is a pro-apoptotic mediator.  Release of cytochrome c from  mitochondria into the cytoplasm can be stimulated by low zinc concentration (10 nmol/L; Jiang et al., 2001). In human peripheral blood T lymphocytes, zinc deficiency induces the translocation of cytochrome c from the mitochondrial intramembranous space into the cytoplasm with subsequent activation of caspase 3 (Kolenko et al., 2001).  Likewise,  deprivation of intracellular zinc by T P E N induces apoptosis in mouse cortical cell cultures (Ahn et al., 1998) and in neuronal cells (Ahn et al., 2000). This zinc deprivationinduced apoptosis is mediated through the release of cytochrome c. Interestingly, zinc  18  supplementation induces apoptosis in HL-60 human promyelocytic leukemia cells (0.01 3 mmol/L; Wolf and Eastman, 1999) and in B P H human malignant prostate cells (15 umol/L; Feng et al., 2002) that is also mediated through the release of cytochrome c. Evidently, the release of cytochrome c is important to zinc regulated apoptosis, but the mechanisms involved are not known. Clearly, there is an increasing body of evidence showing that zinc plays an important role in the induction and regulation of apoptosis in a variety of experimental systems.  However, the mechanisms by which zinc modulates apoptosis are far from  clear. Moreover, the effects of zinc in human breast cancer and the mechanisms involved have not been reported.  1.5.3. Zinc and necrosis. Zinc also plays an important role in necrosis.  In zinc deficient conditions,  myeloid cells die primarily via necrosis - identified by the flocculated state of their chromatin as well as decreased basophilia of their cytoplasm (Martin et al., 1991). Depleting zinc using intracellular zinc chelator TPEN (15 umol/L) induces necrosis in renal carcinoma cell lines that are resistant to apoptosis induced by T P E N (Kolenko et al., 1999).  On the other hand, supplementing zinc at 100-300 umol/L induces both  necrosis and apoptosis in acute lymphoblastic leukemic cell lines, but the induction of necrosis is at greater frequency (Hamatake et al., 2000). Similarly, supplementing zinc at 500 umol/L also induces necrosis in prostate carcinoma cell lines (Iguchi et al., 1998).  1.6. Tamoxifen and apoptosis.  19  It has been shown that a variety of anticancer drugs induce apoptosis in malignant cells in vitro and in vivo.  One such drug is tamoxifen, an estrogen antagonist drug.  Chemically, tamoxifen is a triphenylethylene that has been shown to act as a chemotherapeutic agent for the treatment of breast cancer.  Steady state plasma  concentrations of tamoxifen can be up to 1 umol/L and mean intra-tumour concentrations are higher, about 4 umol/L, when breast cancer patients are treated with daily dosage of 20mg for at least 3 m (MacCallum et al., 2000). Clinical studies have shown that most women with estrogen receptor (ER) -positive breast cancer benefit from tamoxifen therapy. Tamoxifen therapy is usually carried out at a daily dosage ranging from 12 to 20 mg for 3 m to (short term regimen) and 5 y (long term regimen) (Osbourne et al., 1980; Williams et al., 1987; Early Breast Cancer Trialists Collaborative Group, 1998; Cameron et al., 2000). In general, tamoxifen is well tolerated. Hot flushes are the most commonly reported side effects; about 15-20% women receiving tamoxifen develop hot flushes attributable to the drug (Decensi and Costa, 2000) Tamoxifen therapy in breast cancer patients has resulted in an increase in apoptosis in ER-positive tumours (Ellis et al., 1997; Cameron et al., 2000; Farczadi et al., 2002). Tumours taken from elderly women with ER-positive breast cancer exemplified that the expression of Bcl-2 was inversely related to apoptosis (Cameron et al., 2000). After 3 m of tamoxifen treatment (20 mg/d), 54 % of the tumours had a reduction in Bcl2 expression during the therapy.  In these tumours, there was a correlation between  response to therapy and an increase in apoptosis. In these ER-positive tumours in which Bcl-2 expression was not reduced by therapy, there was a correlation between response and a decrease in mitosis. The authors suggested that there are at least two mechanisms  20  for tamoxifen therapy: increased apoptosis through a reduction in Bcl-2 expression or a decrease in proliferation (Cameron et al., 2000).  Even following a 7 day-tamoxifen  treatment, expression of HER-2, which is a growth factor, and p53 decreased; however, Bcl-2 expression remained unchanged (Farczadi et a l , 2002). Tamoxifen therapy seems to be less effective for ER-negative breast cancer than ER-positive tumours. As termed the estrogen antagonist, in ER-positive tumours, tamoxifen seems to bind and inhibit the estrogen receptors.  In ER-negative tumours, there seems to be a variety of proposed  mechanisms such as the induction of caspase 3 (Mandlekar and Kong, 2001). It appears that, in vitro, tamoxifen has both ER-dependent and ER-independent cytostatic activities. It induces chromatin condensation around the nuclear periphery in both cell types (Reddel et al., 1985; Perry et al., 1995). Tamoxifen induces apoptosis in both ER-positive and ER-negative human breast cancer cells via different mechanisms. In most cases, ER-negative cells are less sensitive to tamoxifen than ER-positive cells (Perry etal., 1995). Many ER-positive breast cancer cell lines have been used as experimental systems for understanding breast cancer. The MCF-7 cell line is one of the most studied ER-positive cell lines. When compared to the untreated control human breast carcinoma cells, 60 nmol/L of tamoxifen was sufficient to significantly enhance the percentage of cells undergoing apoptosis (Candi et al., 1995).  While tamoxifen strongly increases  apoptosis in MCF-7 cells, there is a decrease in net growth rate and final cell pool size, which is the number of cells per cm of flask (Budtz, 1999). Mechanistically, it has been 2  shown that, in MCF-7 cells, tamoxifen (0.1-10 umol/L) down-regulates Bcl-2 expression at both the transcription and translation levels, but does not affect the expression of Bax,  21  B c l - X and p53 (Zhang et a l , 1999). Tamoxifen (10 umol/L) -induced D N A cleavage in L  MCF-7 cells is inhibited by the protein synthesis inhibitor cycloheximide, the R N A synthesis inhibitor actinomycin D, and by 17P-estradiol (Perry et al., 1995). From these observations, it appears that tamoxifen-induced apoptosis in MCF-7 cells requires the synthesis of new proteins and mRNAs. In ER-negative cell lines such as MDA-MB-231 cells, tamoxifen (1 umol/L) transcriptionally upregulated the expression of c-myc, which is a proto-oncogene involved in apoptosis (Kang et al., 1996). Moreover, fibroblasts cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. (Evan et al., 1992). Similarly, tamoxifen strongly stimulated caspase 3 activity, and, to a lesser extent, caspase 6, 8, and 9 activities in MDA-MB-231 cells (Mandlekar et a l , 2000). Tamoxifen (10 umol/L) -induced D N A cleavage in MDA-MB-231 cells is inhibited by protein synthesis inhibitor cycloheximide, partially inhibited by actinomycin D, but not inhibited by 17p-estradiol (Perry et al., 1995). These observations suggest that tamoxifen-induced apoptosis in MDA-MB-231 cells is protein synthesis-dependent  and partially R N A  synthesis-dependent. Tamoxifen is a known E R modulator. It binds to estrogen receptors and induces apoptosis. Usually ER-positive cells are more sensitive to tamoxifen, but resistance to the drug occurs when E R are no longer functional.  Administration of 1 umol/L 4-OH-  tamoxifen, which is an active metabolite of tamoxifen, induced a Bcl-2 up-regulation in MCF-7ras cells, which is a transfected non-functional E R cell line (Adam et al., 1997). When MCF-7 cells are transfected with Ha-ras, although E R are expressed, unlike normal MCF-7 cells, tumours can form in the absence of estrogen (Kasid et al., 1985). Estrogen  22  initiates gene transcription by inducing activity of two activating factors, A F I and AF2; tamoxifen inhibits estrogen activity at AF2 but stimulates it at A F I . Resistance towards tamoxifen's effect on A F 2 may stimulate the cell growth via A F I : encouraging tumour growth. (Tonetti and Jordan, 1995). The resistance to the drug may develop through induction of Bcl-2 (Adam et al., 1997). Some of the anticancer effects of tamoxifen observed in both in vivo and in vitro are independent of ER. A possible mechanism has been proposed in which tamoxifen inhibits ATP-dependent acidification in mammalian cells, suggesting that the drug may act directly on the lipid bilayer by increasing the proton permeability (Chen et al., 1999). Tamoxifen partitions  into acidic vesicles and binds protons,  leading to rapid  neutralization. In addition, there are several isoforms of protein kinase C (PKC) that either cooperate or exert opposing effects on the process of apoptosis. Tamoxifen induces translocation of specific P K C isoforms to the mitochondrial compartment, triggering cytochrome c release that contributes to apoptosis (Horgan et al., 1986; Mandlekar and Kong, 2001). Moreover, depending on the cell line, tamoxifen can induce apoptosis, cell cycle arrest, or reduction in cell proliferation (Mandlekar and Kong, 2001) through induction of c-myc expression, but the mechanisms involved are unknown (Mandlekar and Kong, 2001). As well, tamoxifen induces increased intracellular accumulation of ceramide by virtue of its inhibitory activity on glycosylceramide synthase (GCS) (Maurer et al., 1999). G C S , which catalyzes the synthesis of glucosylceramide, is central in glycosphingolipid metabolism. Glycosphingolipids have obligatory functions in cell proliferation and tumour progression. Ceramide serves as a second messenger for  23  programmed cell death; ceramide may inactivate Bcl-2 by dephosphorylation (Ruvolo et a l , 1999). Although tamoxifen may be successful in inducing apoptosis, subsequent development of cancer and resistance to the drug have been the major concerns in tamoxifen therapy. One of the predictors for subsequent risk of developing breast cancer is p53 expression. B y suppressing p53 expression by retro viral-mediated expression of human papillomavirus type-156 E6 protein (HPV-16 E6) in human mammary epithelial cells, there was a marked increase in the sensitivity towards the drug as 1 umol/L of tamoxifen rapidly induced apoptosis (Dietze et al., 2001; Seewaldt et al., 2001). These cells decreased mitochondrial membrane potential, mitochondrial condensation, and caspase activation followed by morphological changes that are characteristic of apoptosis; processes that may be involved in the early event in the induction of apoptosis by tamoxifen (Dietze et al., 2001). On the contrary, cells with normal p53 expression treated with tamoxifen underwent cycle arrest but not apoptosis.  Unfortunately,  resistance to apoptosis rapidly developed after 10 passages in vitro. Breast cancer cells are relatively resistant to the induction of apoptosis. With tamoxifen and the calmodulin antagonists trifluoperazine, cells undergo rapid and intensive apoptosis (Frankfurt et al., 1995). Inhibition of the Ca -calmodulin signalling 2+  pathway has been shown to be partially responsible for ER-independent cytotoxicity of tamoxifen (Rowlands et a l , 1990). Also, a combination of anticancer drugs, docetaxel and tamoxifen, was more effective in inducing D N A fragmentation and morphological changes than a single drug exposure in ER-negative cell lines (Ferlini et al., 1997). A poor response to anticancer drug therapy has been correlated with the loss of Bax m R N A  24  (Bargou et al., 1995). Bax restoration experiments have shown that Bax sensitizes M C F 7 cells to drug-induced apoptosis (Wagener et al., 1996). Conversely, overexpression of Bcl-2 and B c l - X has been shown to result in suppression of apoptosis in response to L  numerous anticancer drugs (Thompson, 1995). This change in expression may be due to overexpression of growth factors such as HER2 (also known as c-neu), for tamoxifeninduced apoptosis in MCF-7 cells (Kumar et al., 1996).  1.7. Summary In summary, it has been shown that anti-breast cancer drugs, such as tamoxifen, induce apoptosis in breast cancer cells. Tamoxifen-induced apoptosis appears to be both ER-dependent and ER-independent. proteins (e.g. Bcl-2).  Its actions involve several apoptotic regulatory  Zinc is considered as a physiological regulator of apoptosis  (Sunderman 1995), but presently there is no reported study concerning the effects of zinc in breast cancer cells.  Rapid advances in research of apoptosis reveal that some  modulators and effectors in apoptosis are either zinc-finger proteins (e.g. p53) or their gene expression (e.g. Bcl-2) or activity (e.g. caspase 3) is influenced by zinc.  Since  tamoxifen and zinc share some common targets, it is possible that zinc could work together with these tamoxifen to enhance their efficacy.  2. Hypothesis Based on the existing evidence, we hypothesized that modulating medium zinc concentration will alter the availability of metabolically active intracellular zinc, which in turn affects the overall growth of human breast cancer cells.  25  3. Overall Objectives The overall objective of my thesis project was to investigate the modulating effects of zinc on the efficacy of tamoxifen in human breast cancer cells. The specific objectives were: 1. To investigate the effects of zinc on the growth of human breast cancer cells treated with tamoxifen. 2. To explore the possible mechanisms by which zinc modulates the efficacy of tamoxifen in human breast cancer cells.  4. Rationale for focusing on MDA-MB-231 cells in Chapter 2 This thesis project studied the effect of zinc on the efficacy of tamoxifen in human breast cancer cells. Since this was the first study in the area, we employed three human breast cancer cell lines (MCF-7, T47D, and MDA-MB-231).  In addition, a  human breast fibrocystic cell line (MCF-10) was also included for the purpose of comparison between cancer cells and non-cancer cells. These cell lines were chosen because of the differences in the gene profile of zinc-related apoptotic regulatory proteins (Table 1-2). Upon treating the cells with zinc or tamoxifen alone, or a combination of zinc and tamoxifen, the responses measured by overall growth, cell viability, cell survival, and cell death, including both necrosis and apoptosis, to the treatment varied in a cell line-specific manner (Appendix II, Appendix III, Appendix IV, Appendix V). Overall, MDA-MB-231 cells were more responsive to the treatments compared to the other cell lines. Therefore,  26  this cell line was used to explore the possible mechanisms by which combination and tamoxifen reduced the overall cell growth and promoted cell death.  27  5. Bibliography Adam L . , Crepin M . , Israel L . (1997) Tumor growth inhibition, apoptosis, and Bcl-2 down-regulation of MCF-7ras tumors by sodium phenylacetate  and tamoxifen  combination. Cancer Research. 57: 1023-1029. Adams J.M., Cory S. 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Zhang G., Kimijima I., Onda M . , Kanno M . , Sato H., Watanabe T., Tsuchiya A., Abe R., Takenoshita S. (1999) Tamoxifen-induced apoptosis in breast cancer cells relates to down-regulation of bcl-2, but not bax and bcl-X , without alteration of p53 protein L  levels. Clinical Cancer Research. 5: 2971-2977.  47  CHAPTER 2: Zinc Supplementation Increases the Efficacy of Tamoxifen in Human breast cancer MDA-MB-231 cells  1  1. Introduction Zinc is known as a physiological regulator of apoptosis. The effects of zinc on apoptosis are zinc concentration-dependent and biphasic.  In general, zinc at low  concentrations induces apoptosis while at high concentrations, zinc suppresses apoptosis. The concentration at which zinc either induces or suppresses apoptosis is cell type specific. For example, in lymphoid (Molt-3 and Raji) cells, zinc deficiency (0.09 ug/mL or 1.4 umol/L) induces apoptosis while zinc repletion (50 umol/L) inhibits apoptosis (Martin et al., 1991). In contrast, in mouse thymocytes, zinc supplementation at 80 umol/L induces apoptosis, however further increase in zinc supplementation to 200 umol/L suppressed apoptosis (Telford and Fraker, 1995).  In thymocytes, increasing  labile intracellular zinc prevents apoptosis while a reduction of the labile intracellular zinc below a threshold concentration induces apoptosis (Zalewski et al., 1993), demonstrating that the concentration of labile intracellular zinc is critical in modulating apoptosis. Besides its direct involvement in modulating apoptosis, zinc is also capable of modulating apoptosis induced by apoptosis-inducing agents. For example, zinc blocks sporidesmin-induced apoptosis in macrophages and T lymphocytes (Waring et al., 1990). Similarly, zinc also suppresses hydrogen peroxide-induced apoptosis in U937 cells  1  A version of the chapter is in preparation for submission to Breast Cancer Research and  Treatment.  48  (Fukumachi et al., 1998) and P B L cells (Marini & Musiani, 1998), and radiation-induced apoptosis in several other cell lines (Parat et al., 1997; Leccia et al., 1999). In rats, apoptotic embryonal cell death occured within 4 d after the dam was fed a zinc deficient diet (Rogers et al., 1995). Evidently, zinc is capable of modulating apoptosis and this modulating effect of zinc on apoptosis is a function of zinc concentration. Scattered evidence suggests that some of the regulatory proteins involved in apoptosis are possible cellular targets of zinc-dependent modulation of apoptosis. In the HepG2 hepatoblastoma cell line zinc-deficiency depletes total cellular zinc and induces p53 expression compared to that in the control (Reaves et al., 2000). As well, zinc at 50 umol/L stimulates Bcl-2 expression in porcine kidney L L C - P K i cells (Ishido et al., 1999) and U937 cells (Fukumachi et al., 1998). Caspase 3 is another apoptotic regulatory protein affected by zinc. At high concentrations (100-300 umol/L), zinc functions as a potent inhibitor of caspase 3 (Perry et al., 1997).  A n increase in intracellular zinc  suppresses caspase 3 activity while a decrease in intracellular zinc increases its activity (Ho et al., 2000). The mechanisms by which zinc modulates apoptosis are far from clear. Tamoxifen is a non-steroidal anti-estrogen drug used to treat breast cancers. Tamoxifen induces apoptosis in breast cancer cells. For example, tamoxifen (50 umol/L) induces apoptosis in both MCF-7 and MDA-MB-231 cells (Fattman et al., 1998). Similarly, tamoxifen at concentrations up to 25 umol/L induces apoptosis in a dosedependent manner (Mandlekar et al., 2000). Tamoxifen-induced apoptosis appears to involve numerous apoptotic regulator proteins.  49  In MCF-7 cells, tamoxifen-induced  apoptosis occurrs via downregulation of Bcl-2 expression at both the transcription and translation levels, but the expression of Bax, BC1-XL and p53 is not affected (Zhang et al., 1999). In MDA-MB-231 cells, transcriptional upregulation of the c-myc expression is responsible for tamoxifen induced apoptosis (Kang et al., 1996). Caspase 3 (Mandlekar et al., 2000) and Bax (Fattman et al., 1998) also play a role in tamoxifen-induced apoptosis in breast cancer cells. In addition, increased oxidative stress (Fernili et al., 1999) and intracellular C a  2+  signals (Kim et al., 1999) have been suggested to mediate tamoxifen-  induced apoptosis.  Clearly, tamoxifen-induced apoptosis is under complex regulation  and our understanding in this area is unclear. Since both zinc and tamoxifen are capable of inducing apoptosis, we hypothesized that a combination of zinc and tamoxifen will increase the efficacy of tamoxifen against breast cancer cells. To test this hypothesis, human breast cancer MDA-MB-231 cells were treated with tamoxifen in the presence of various concentrations of zinc supplementation. Results reported herein showed that zinc supplementation, especially at higher concentrations, in combination with tamoxifen, decreased cell viability, reduced cell numbers, and increased both apoptotic and necrotic cell deaths, suggesting that zinc supplementation enhanced the efficacy of tamoxifen against human breast cancer M D A MB-231 cells.  2. Materials and Methods Cell culture systems and treatments Human breast cancer MDA-MB-231 cells (HTB-26; A T C C , Manassas, Virginia) were maintained at 37°C in Dublecco's Modified  50  Eagle Media ( D M E M ;  Life  Technologies, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Grand Island, New York), 584 mg/L L-glutamine, 110 mg/L sodium pyruvate and pyridozine hydrochloride, 1.5 g/L sodium bicarbonate, 50 ug/mL streptomycin, and 50 \xg/mL penicillin in an atmosphere containing 5% C O 2 . To manipulate medium zinc concentration, FBS was treated with Chelex-100 (Biorad, Mississauga, ON) using the procedure described before (Paski and X u , 2001) except using centrifugation instead of a column to separate Chelex-100 from the serum. After treating the FBS with Chelex-100, the serum was centrifuged at 400 x g, 4°C for 15 min to separate Chelex-100 from the serum.  Since Chelex-100 is also capable of  chelating divalent cations besides zinc, a mixture of A l , As, Ca, Cd, Cu, Fe, Mg, Mn, and Mo was added to the Chelex-100-treated FBS to achieve their corresponding pretreatment concentrations. To formulate the low zinc medium, D M E M was supplemented with 10% Chelex-100-treated FBS. Zinc concentration in the low zinc medium was 0.2 umol/L. The low zinc medium was supplemented with zinc at 5 umol/L to mimic the zinc concentration present in the normal medium ( D M E M supplemented with 10% FBS). In addition, the low zinc medium was also supplemented with zinc at 50 or 150 umol/L to formulate high zinc media. Cells (passage 35-40) were cultured in D M E M supplemented with 10% FBS at an initial seeding density of 1 x 10 cells per T25 flask for 72 h. For those studies using 965  well plates, the initial seeding density was 5 x 10 cells per well. The cells were then 3  cultured in the test media for another 72h. At the end of this period, cells were treated with tamoxifen (0, 5, or 10 umol/L; Sigma, St. Louis. Mo., U.S.A.) in the presence of zinc at concentrations indicated above in the culture media. After 2, 24, or 48 h of zinc-  51  tamoxifen treatment, cells were rinsed with phosphate buffered saline (PBS) (pH 7.4) followed by culturing in D M E M supplemented with 10% FBS for 24 h. The cells were then used for biochemical analyses. At the time of harvesting, the cells had not reach total confluency.  Cell viability assay and cell number counting Cell viability was assessed  by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-  diphenyl-tetrazolium bromide (MTT) assay (Molecular Probe, Eugene, OR, U.S.A.) in 96-well plates according to the manufacturer's instructions. Briefly, at the end of the culture period, 100 uL of culture medium was replaced with fresh D M E M supplemented with 10% FBS.  In addition, 10 uL M T T reagent (12 mmol/L) was also added to each  well. After incubation for 4 h at 37°C in 5% CO2, 100 uL of 10% sodium dodecyl sulfate (SDS) was added to each well.  After an overnight incubation at 37°C in 5% CO2,  absorbance was recorded at 570 nm using a microplate reader (Spectramax Plus 384, Molecular Device, Sunnyvale, C A , U.S.A.). The absorbance was then subtracted from the background, which was the absorbance of 10 uL of the M T T solution and 100 uL of 10% SDS in 100 uL of the culture medium. Cell numbers were counted using a Coulter Counter ( Z l , Beckman, Miami, Florida, U.S.A.) to assess cell growth.  Flow cytometery assay of apoptotic and necrotic cells Apoptosis and necrosis were assessed by using annexin-V-FLUOS staining kit (Roche, Indianapolis, IN, U.S.A.) according to the manufacturer's instructions. At the end of the culture period, cells were harvested and washed with PBS (pH 7.4) followed  52  by centrifugation at 200 x g for 5 min. The cell pellets were resuspended in 100 uL of Annexin-V-FLUOS and propidium iodide labelling solution. After adding the HEPES buffer (400 uL), the cells were analyzed by flow cytometry within 30 min. The fluorescence was determined by flow cytometery using F A S C A N (Becton Dickinson, Franklin Lakes, NJ, U.S.A.).  RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) Total RNAs were isolated using the R N A isolation Mini kit (Qiagen, Mississauga, ON). RT-PCR was performed using the ThermoScript RT-PCR System plus Platinum Taq D N A Polymerase (Invitrogen, Burlington, O N , Canada)  according to the  manufacturer's instructions. Total R N A (2 ug) was used for reverse transcription. The resulting c D N A (4 uL, which is equivalent to 0.4 of total R N A ) was subsequently amplified for assessing p53 m R N A level with P C R (GeneAMP P C R system 2400, Perkin Elmer, Norwalk, CT, USA) and for assessing gadd45, Bax, and Bcl-2 mRNA levels with a thermocycler (Mastercycler Gradient, Brinkman Instruments, Westbury, N Y , U.S.A.). PCR conditions used were adopted from the conditions described earlier with modifications (Zhang et al., 1999). Briefly, after a hot start (95°C/2 min), the samples were amplified at 94°C/30 sec; 58°C/30 sec; 72°C/30 sec followed by a final extension at 72°C/10 min for p53 and gadd45, and 95°C/40 sec; 60°C/40 sec; 72°C/120 sec followed by a final extension at 72°C/7 min for bax and bcl-2. For each target gene, preliminary experiments were conducted to determine the relationship between amplification efficiency and the number of cycles. Based on these preliminary experimental results, the number of P C R cycles for p53, gadd45, bax, and bcl-2 was 31, 36, 39, 39,  53  respectively, to ensure the amplification was within the linear phase of the amplification curve and the amplification did not yield non-specific P C R product. The primer pairs used in this study for P C R amplification are shown in Table 2-1. A c D N A blank and a primer blank were included as the negative controls for each P C R amplification. P2microglobulin was co-amplified with the target primer to verify the integrity of the R N A and to serve as the internal control for PCR. Following gel electrophoresis, optical density of the bands were obtained using Scion Image (Release Beta 4.02, Scion Co., Maryland).  The level of target mRNA was normalized to the optical density of the  corresponding p2-microglobulin bands.  Cellular zinc concentration After treating the cells with tamoxifen for 48 h, the harvested cells were rinsed three times with PBS to prevent potential zinc contamination from the residual culture media. The cells were then suspended in PBS (5 mL). A n aliquot of the cells (0.6 mL) was used for counting cell numbers and the remaining cells were acid digested (0.5 mL of concentrated nitric acid) at room temperature.  The digested samples were then  quantitatively transferred to acid-washed volumetric flasks and brought to volume with double-deionized water. The samples were further diluted with 0.1 N nitric acid to an appropriate concentration for the determination of total cellular zinc content using flame atomic absorption spectrophotometer (Perkin Elmer, Model 2380, Norwalk, CT). Total cellular zinc contents were normalized on a per 10 cells basis. 6  Statistical analyses.  54  The differences among the means of the treatment groups were analyzed using one-way A N O V A followed by Tukey's Honest test (The SAS System for Windows Release 6.12; p < 0.05).  3. Results  Zinc supplementation increased cellular zinc concentration In the absence of tamoxifen treatment, supplementation of the low zinc media with zinc concentrations of 150 umol/L increased the total cellular zinc concentration 3.6 fold (Figure 2-1).  Similarly, zinc supplementation (150 umol/L) also increased the total  cellular zinc concentration in the cells treated with 10 umol/L of tamoxifen by 3.8 fold. However, tamoxifen treatment had no effect on the total cellular zinc concentration regardless the medium zinc concentration.  Zinc supplementation decreased cell viability and overall cell growth The effects of zinc supplementation on the cell viability were first assessed within each level of tamoxifen treatment. After 2 h, cell viability was not affected by zinc supplementation alone (Figure 2-2A). When the cells were treated with tamoxifen at 5 umol/L, cell viability was unaffected at a zinc supplementation of 5 umol/L, but was reduced by 24 and 42% when zinc supplementation was increased to 50 and 150 umol/L, respectively. When the cells were treated with tamoxifen at 10 umol/L, higher levels of zinc supplementation (150 umol/L) reduced cell viability by 59%. After 24 h, cell viability was not affected by zinc supplementation alone (Figure 2-2B). When the cells  55  were treated with tamoxifen at 5 umol/L, cell viability was also unaffected at low levels of zinc supplementation (5 and 50 umol/L), but was reduced by 36% when zinc supplementation was increased to 150 umol/L.  When the cells were treated with  tamoxifen (10 umol/L), zinc supplementation at 5, 50, and 150 umol/L resulted in a 32, 47, and 90% reduction in cell viability, respectively.  After 48 h, cell viability was  reduced by about 30% at the high level of zinc supplementation (150 umol/L) in the absence of tamoxifen (Figure 2-2C). When the cells were treated with tamoxifen at 5 umol/L, cell viability was not affected by zinc supplementation. When the cells were treated with tamoxifen at 10 umol/L, zinc supplementation at 50 and 150 umol/L resulted in a 30 and 57% reduction in cell viability, respectively. The effect of zinc supplementation on the cell viability was then assessed within each level of zinc supplementation. After 2 h, treating the cells with tamoxifen at 10 umol/L, but not 5 umol/L, significantly reduced cell viability, regardless of the level of zinc supplementation (Figure 2-2A). After 24 h, treating the cells with tamoxifen at 5 umol/L significantly reduced cell viability at all three levels of zinc supplementation (Figure 2-2B). When tamoxifen level was increased from 5 to 10 umol/L, cell viability was further reduced regardless of the level of zinc supplementation. tamoxifen  After 48 h,  treatment at 10 umol/L, but not 5 umol/L, reduced cell viability in  combination of 50 or 150 umol/L of zinc supplementation (Figure 2-2C). These results revealed that a combination of higher levels of zinc supplementation (50 or 150 umol/L) and tamoxifen treatment (10 umol/L) consistently reduced the cell viability in M D A - M B 231 cells regardless of the treatment duration.  56  The effects of tamoxifen treatment with or without the presence of tamoxifen on cell number were shown in Figure 2-3.  In the absence of tamoxifen, zinc  supplementation at 150 umol/L reduced cell number by 41%.  In the presence of  tamoxifen, zinc supplementation reduced cell number by 46%. Tamoxifen treatment alone reduced cell number by 22%. Interestingly, zinc supplementation alone resulted in a further 24% reduction in cell number compared to tamoxifen treatment alone.  A  combination of zinc supplementation and tamoxifen treatment resulted in a 58% decrease in cell number.  A combination of zinc supplementation and tamoxifen reduced cell survival and promoted cell death To elucidate the possible causes of the reduced cell number observed, the cells were sorted for live cells or dead cells, including both necrotic and apoptotic cells (Figure 2-4). In the cells cultures that received no zinc or tamoxifen treatment, the cell survival rate was about 88%. Both necrosis and apoptosis were constitutively present at a rate of about 5%. In comparison, zinc supplementation or tamoxifen alone had no effect on the cell survival and cell death.  However, a combination of zinc supplementation and  tamoxifen reduced live cells by 17, 16, and 13% compared to the cells treated with no zinc supplementation and tamoxifen, zinc supplementation alone, and tamoxifen alone, respectively (Figure 2-4A).  Furthermore, a combination of zinc supplementation and  tamoxifen increased necrotic cell death by 1.5 fold compared to the cells that received either no treatment, or zinc supplementation alone or tamoxifen alone (Figure 2-4B). Furthermore, a combination of zinc supplementation and tamoxifen also increased  57  apoptotic cell death by 3.3, 2.3 and 1.9 fold compared to the cells that received no treatment, zinc supplementation alone, and tamoxifen alone, respectively (Figure 2-4C).  A combination of zinc supplementation and tamoxifen altered the expression of apoptotic regulatory proteins  Treating the cells with either zinc supplementation or tamoxifen alone had no effect on p53 mRNA level.  However, a combination of zinc supplementation and  tamoxifen reduced p53 mRNA level by 10% compared to that in cells that received no treatment (Figure 2-5). Treating the cells with a combination of zinc supplementation and tamoxifen reduced p53 mRNA level compared to that in the cells treated with zinc supplementation (16%) or tamoxifen (12%) alone. In contrast, gadd45 mRNA level was increased by 8% in response to zinc supplementation, but was unaffected by tamoxifen alone (Figure 2-6).  Treating the cells with a combination of zinc supplementation and  tamoxifen increased gadd45 mRNA level compared to that in the cells that received no treatment (20%) or tamoxifen alone (18%). Similarly, Bax mRNA levels were also not affected by zinc supplementation or tamoxifen alone (Figure 2-7).  However, a  combination of zinc supplementation and tamoxifen increased Bax mRNA level compared to that in the cells received no treatment (56%), or zinc supplementation (28%) or tamoxifen (43%) alone. In contrast, Bcl-2 mRNA level was not affected by zinc supplementation or tamoxifen alone, or a combination of zinc supplementation and tamoxifen (Figure 2-8).  4. Discussion  58  Zinc increased the efficacy of tamoxifen In this study, supplementing the low zinc medium with zinc at 150 umol/L lowered the number and viability of MDA-MB-231 cells while cell survival and cell death, including both necrotic and apoptotic cell deaths were not affected.  Similarly,  treating the cells with tamoxifen at 10 umol/L also lowered cell number, but had no effect on cell viability, cell survival, and cell death. In contrast, a combination of zinc and tamoxifen resulted in a lower cell number, cell viability, and cell survival while both necrotic and apoptotic cell deaths were increased, demonstrating that treating the cells with a combination of zinc and tamoxifen was far more effective than either zinc or tamoxifen alone. Evidently, zinc increased the efficacy of tamoxifen in treating breast cancer cells.  Zinc and tamoxifen additively suppressed overall cell growth via promoting apoptosis Treating the cells with a combination of zinc and tamoxifen resulted in a reduced overall cell number and in an increased cell death.  Moreover, cell viability and cell  survival were also reduced. These observations collectively suggest that reduced overall cell growth can be attributed to an increased cell death. Furthermore, flow cytometery analyses revealed that a larger portion of the cells underwent apoptosis (16%) than underwent necrosis (9%), suggesting that apoptosis was the major cause of cell death and that a combination of zinc and tamoxifen suppressed overall growth of MDA-MB-231 cells through promoting apoptosis.  59  Although it has not been reported before, it is not surprising that zinc and tamoxifen additively induce apoptosis, because there is sufficient evidence showing that zinc (Telford and Fraker 1995; Manev et al., 1997; Garvy et al., 1998) or tamoxifen (Candi et al., 1995; Ferlini et al., 1997; 1999; Mandlekar et al., 2000) alone induces apoptosis in a variety of cells.  For example, Manev et al. (1997) observed a dose-  dependent increase of apoptosis in rat cerebellar granule cells treated with 100 - 500 umol/L of zinc.  Similarly, Garvy et al. (1998) found that zinc (100 - 200 umol/L)  induced apoptosis in the mouse B220 IgM B-lineage cells in bone marrow and I g M B +  +  +  cells in the spleen. In human breast cancer MCF-7 cells, treatment with tamoxifen (1 umol/L) for 12 d induced apoptosis (Chen et al., 1996). Fattman et al. (1998) observed that tamoxifen (50 umol/L; 16 h) increased apoptosis by about 24- and 9-folds in MCF-7 and MDA-MB-231 cells, respectively. It is important to point out that the flow cytometery assay used in this study tends to underestimate the incidence of apoptosis. This is because the flow cytometery assay used two dyes: Annexin-V-FLUOS and propidium iodide (PI).  Annexin-V-FLUOS  stains cells by binding to phosphatidylserine (PS), which is normally present in the inner face of the cell membrane and is translocated to the outer face of the cell membrane during the early phase of apoptosis.  When cells are stained by Annexin-V-FLUOS  alone, they are counted as apoptotic cells. During necrosis, in contrast to apoptosis, cell membranes are ruptured (Allen et al., 1997). This rupture of the cell membrane provides Annexin-V-FLUOS access to the PS located in the inner face of the cell membrane and allows the D N A to be stained by dyes such as PI. Thus, when cells are stained with both Annexin-V-FLUOS and PI, they are counted as necrotic cells. During the late stages of  60  apoptosis, cell membranes are ruptured.  This cell membrane rupture provides an  opportunity for both Annexin-V-FLUOS and PI staining. As a result, apoptosis tends to be underestimated while necrosis is likely overestimated. Therefore, the actual number of cells that died via apoptosis might be higher than was indicated by the data. To explore the possible mechanisms involved in the zinc and tamoxifen induced apoptosis, the expression profile of several zinc-related apoptotic regulatory proteins was determined. In response to the treatment by a combination of zinc and tamoxifen, p53 mRNA level was decreased while Bax mRNA level was increased. The mRNA level of Bcl-2 was unaffected. These observations are in line with an earlier report by Fattman and co workers (1998) who observed that tamoxifen treatment (50 umol/L) increased the level of Bax protein in MDA-MB-231 cells, but not breast cancer MCF-7 cells. However, they reported that p53 and Bcl-2 protein levels were not affected by tamoxifen treatment in either cell lines. MDA-MB-231 cells expresses a mutant p53 (codon 280: Arg —» Lys; Elledge et al., 1995). It is unlikely that the decreased p53 mRNA level observed in the present study in zinc and tamoxifen treated cells has functional significance. Since Bax is a well established pro-apototic protein and Bax mRNA level was elevated by about 50%, it is possible that the observed increase in apoptosis in the MDA-MB-231 cells treated with a combination of zinc and tamoxifen is Bax-dependent, but further studies are needed to confirm this speculation.  Potential key role of gadd45 Gadd45 is a member of the growth arrest D N A damage gene family. Gadd45 has been shown to play pivotal roles in growth control (Fornace et al., 1992; Liebermann and  61  Horrman 1998; Zhang et al., 1999), D N A repair (Vairapandi et al., 1996; Smith et al., 1994; 2000), and apoptosis (Harkin et al., 1999; Zhang et al., 2001).  In this study,  treating cells with a combination of zinc and tamoxifen resulted in a reduced overall cell growth and an increased apoptosis while gadd45 mRNA level was elevated. Because gadd45 has been implicated in both growth control and induction of apoptosis, an elevated gadd45 m R N A level suggests that gadd45 may be central in mediating the effects of zinc and tamoxifen on suppressing overall growth and in inducing apoptosis in MDA-MB-231 cells. Moreover, since gadd45 is regulated by both p53-dependent and independent mechanisms in mammalian cells (Smith and Fornace, 1996) and M D A - M B 231 cells express a mutant p53, elevated gadd45 mRNA level is likely regulated via a p53-independent mechanism, but the exact mechanism remains to be elucidated.  Effective zinc acquiring ability ofMDA-MB-231 cells Zinc concentrations in regular culture media, e.g. D M E M supplemented with 10% of FBS, is about 5 umol/L. In the low zinc medium, zinc concentration was only about 4% of the concentration present in regular culture media. However, the cell viability was unaffected, suggesting that this low level of zinc concentration was well tolerated by MDA-MB-231 cells. More importantly, total cellular zinc concentration was also the same in cells cultured in the low zinc medium and cells cultured in the medium supplemented with zinc at 5 umol/L.  Lee et al. (2003) have shown that zinc is  accumulated in N-methyl-N-nitrosourea-induced rat mammary tumours.  This zinc  accumulation results from an altered zinc homeostasis, which was characterized by increased expression of metallothionein, a major cellular zinc-binding protein, coupled  62  with decreased expression of ZnT-1, a zinc exporter. Thus, it is possible that M D A - M B 231 cells are capable of efficiently acquiring zinc from their surroundings to support their growth. Further studies are warranted to elucidate the mechanisms involved.  In summary, supplementation of zinc at high concentration (e.g. 150 umol/L) suppressed overall cell growth and reduced cell viability, but had no effect on cell survival and cell death. Similarly, tamoxifen alone also reduced overall cell growth, but had no effect on cell viability, cell survival and cell death. In contrast, a combination of zinc and tamoxifen suppressed overall cell growth, reduced cell viability and cell survival, and increased both necrotic and apoptotic cell death. Collectively, these results showed that zinc increased the efficacy of tamoxifen in suppressing overall cell growth through promoting cell death. In addition, treating cells with a combination of zinc and tamoxifen also elevated Bax and gadd45 mRNA levels. Since Bax is a well-known proapoptotic protein and gadd45 is involved in both negative growth control and the induction of apoptosis, zinc and tamoxifen-induced apoptosis may to be Bax- and gadd45-dependent.  63  J3 Ol  co  „  <7 ro  a6 o< ou << uu au  «s 3  a cu  a I  V  CU  s I  PH CU  o H o o H o o b  u H  o H  u o o° <;  a <n o < ua a HU H o H 6 ° a H  o  < <  i-i CD  H  a  I  •a  uo CD  pi  8.8  PH  CD  H  oo <C  CU  o  CU  CO  1  00  c cu  00  CU  , 1999 CU  (-H  co ro  6 o H U U  u  5  H  <  < V< H  a u < HH u OU  y <  au ou a a H  O H H U U H  R5  ou  a H ou  H  IT)  U rj U < O U  uo  o  cu  H H  H  H  u H U o o H H o u o o U  yB 9 o < u H  H H  O  i ^ wo w> 1  CU  00  00  00 00  U  cu  CU  cu  <  H  a  01  c oo CU  cu  oo  00  a  cu H  <  S  oo • —  cu  oo  3  x> cu  DC  «  H  u U o  ^  a00  -4—»  ro co i  00  o CT CU  -*-> cu  CD  oo C!  u  CU  Audi  OH O  al, 2001  CD  OH O  VJaUl  CU  Gaul schi  a  schi  ON ON  ON ON  al., 2001  a) u  IH  o I-I  uo  o  CN  ro uo  DH  I  O  PQ  ffl  CN CQ.  5 <  Figure 2-1. Effects of zinc supplementation and tamoxifen on total cellular zinc concentrations in human breast cancer MDA-MB231 cells. Cells were cultured in media containing 0 (open bar), 5 (dotted bar), or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO) or 10 umol/L tamoxifen for 48 h. Mean ± S E M (n=6). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asteriks are statistically different (p<0.05).  65  Tamoxifen (umol/L) Figure 2-2. Effects of zinc supplementation and tamoxifen on viability of human breast cancer MDA-MB231 cells . Cells were cultured in media containing 0 (open bar), 5 (dotted bar), 50 (shaded bar), or 150 (filled bar) umol/L zinc for 72 h followed by treatement with 0 (DMSO), 5, or 10 umol/L tamoxifen for 2 (A), 24 (B), or 48 (C) h. Mean ± S E M (n=5). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different (p<0.05).  66  • 0 umol/L Zn • 150 umol/L Zn  a  Tamoxifen (umol/L) Figure 2-3. Effects of zinc supplementation and tamoxifen on number of human breast cancer MDA-MB231 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO) or 10 yi m< tamoxifen for 48 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different (p<0.05).  67  Tamoxifen (umol/L) Figure 2-4. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer MDA-MB-231 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO) or 10 umol/L tamoxifen for 48 h. Mean ± SEM. (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different (p<0.05).  68  1  2  3  4  5  6  7  8  Zinc Tamoxifen  +  9  10  11  12  13  14  +  +  -  +  15  B  10 Tamoxifen (u,mol/L)  Figure 2-5. RT-PCR analysis of p53 mRNA level in human breast cancer MDA-MB231 cells. Cells were cultured in media containing 0 or 150 umol/L zinc for 72 h followed by treatment with 0 (DMSO) or 10 umol/L tamoxifen for 48 h. Total RNAs were isolated from the cells and reverse transcribed. P C R products were subjected to agarose (2%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing the representative p53 mRNA levels. Lane 1: D N A ladder; lane 2: negative control (no primer); lane 3: negative control (no cDNA); lane 4-6: 0 umol/L zinc and 0 (DMSO) umol/L tamoxifen; lane 7-9: 0 umol/L zinc and 10 umol/L tamoxifen; lane 10-12:150 umol/L zinc and 0 (DMSO) umol/L tamoxifen; lane 13-15: 150 umol/L zinc and 10 umol/L tamoxifen. (B) Relative p53 mRNA level. 0 (open bar) or 150 (filled bar) umol/L zinc. Values represent mean ± S E M (n=4). Zinc -: 0 umol/L; zinc +: 150 umol/L; tamoxifen -: 0 (DMSO) umol/L; tamoxifen +: 10 umol/L. (32 mg: p2-microglobulin. Means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different (p<0.05). Means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different (p<0.05).  69  300 b p  Gadd45  200 b p  P2 mg  12  3  4  5  6  7  8  9  10 11 12 13 14 15  +  Zinc Tamoxifen  +  +  13  0  10 Tamoxifen (umol/L)  Figure 2-6. R T - P C R analysis o f gadd45 m R N A level i n human breast cancer M D A - M B 2 3 1 cells. Cells were cultured in media containing 0 or 150 u m o l / L zinc for 72 h followed by treatment with 0 ( D M S O ) or 10 umol/L tamoxifen for 48 h. Total R N A s were isolated from the cells and reverse transcribed. P C R products were subjected to agarose (2%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing the representative gadd45 m R N A levels. Lane 1: D N A ladder; lane 2: negative control (no primer); lane 3: negative control (no c D N A ) ; lane 4-6: 0 umol/L zinc and 0 ( D M S O ) u m o l / L tamoxifen; lane 7-9: 0 umol/L zinc and 10 umol/L tamoxifen; lane 10-12:150 u m o l / L zinc and 0 ( D M S O ) umol/L tamoxifen; lane 13-15: 150 umol/L zinc and 10 u m o l / L tamoxifen. (B) Relative gadd45 m R N A level. 0 (open bar) or 150 (filled bar) u m o l / L zinc. Values represent mean ± S E M (n=4). Zinc -: 0 umol/L; zinc +: 150 umol/L; tamoxifen -: 0 ( D M S O ) umol/L; tamoxifen +: 10 umol/L. P2 mg: p2-microglobulin. Means among different concentrations o f zinc within the same concentration o f tamoxifen with different lower-case letters are statistically different (p<0.05). Means among different concentrations o f tamoxifen within the same concentration o f zinc with different asterisks are statistically different (p<0.05).  70  1  2  3  4  5  6  7  8  9  10  11  12 13  +  Zinc Tamoxifen  15  + +  +  B  14  • 0 umol/L Zn  • 150 umol/L Zn b  X  10  0 Tamoxifen (u,mol/L)  Figure 2-7. RT-PCR analysis of Bax mRNA level in human breast cancer MDA-MB231 cells. Cells were cultured in media containing 0 or 150 umol/L zinc for 72 h followed by treatment with 0 (DMSO) or 10 umol/L tamoxifen for 48 h. Total RNAs were isolated from the cells and reverse transcribed. P C R products were subjected to agarose (2%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing the representative bax mRNA levels. Lane 1: D N A ladder; lane 2: negative control (no primer); lane 3: negative control (no cDNA); lane 4-6: 0 umol/L zinc and 0 (DMSO) umol/L tamoxifen; lane 7-9: 0 umol/L zinc and 10 umol/L tamoxifen; lane 10-12:150 umol/L zinc and 0 (DMSO) umol/L tamoxifen; lane 13-15: 150 umol/L zinc and 10 umol/L tamoxifen. (B) Relative bax mRNA level. 0 (open bar) or 150 (filled bar) umol/L zinc. Values represent mean ± S E M (n=4). Zinc -: 0 umol/L; zinc +: 150 umol/L; tamoxifen -: 0 (DMSO) umol/L; tamoxifen +: 10 umol/L. P2 mg: p2-microglobulin. Means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different (p<0.05). Means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different (p<0.05).  71  1  2  3  4  5  6  Zinc Tamoxifen  7  8  9  10  11  12 13  +  • 0 umol/L Zn  0  15  +  +  B  14  +  • 150 umol/L Zn  10 Tamoxifen (umol/L)  Figure 2-8. RT-PCR analysis of bcl-2 mRNA level in human breast cancer MDA-MB231 cells. Cells were cultured in media containing 0 or 150 umol/L zinc for 72 h followed by treatment with 0 (DMSO) or 10 umol/L tamoxifen for 48 h. Total RNAs were isolated from the cells and reverse transcribed. P C R products were subjected to agarose (2%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing the representative bcl-2 mRNA levels. Lane 1: D N A ladder; lane 2: negative control (no primer); lane 3: negative control (no cDNA); lane 4-6: 0 umol/L zinc and 0 (DMSO) umol/L tamoxifen; lane 7-9: 0 umol/L zinc and 10 umol/L tamoxifen; lane 10-12:150 umol/L zinc and 0 (DMSO) umol/L tamoxifen; lane 13-15: 150 umol/L zinc and 10 umol/L tamoxifen. (B) Relative bcl-2 mRNA level. 0 (open bar) or 150 (filled bar) umol/L zinc. Values represent mean ± S E M (n=4). Zinc -: 0 umol/L; zinc +: 150 umol/L; tamoxifen -: 0 (DMSO) umol/L; tamoxifen +: 10 umol/L. P2 mg: p2-microglobulin. Means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different (p<0.05). Means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different (p<0.05).  72  5. Bibliography Allen R.T., Hunter W J . 3rd, Agrawal D.K. (1997) Morphological and biochemical characterization and analysis of apoptosis. Journal of Pharmacological and Toxicological Methods. 37: 215-228. Candi E., Melino G., De Laurenzi V., Piacentini M . , Guerrieri P., Spinedi A., Kinght R.A. (1995) Tamoxifen and somatostatin affects tumours by inducing apoptosis. Cancer Letters. 96: 141-145. Chen H , Tritton T.R., Kenny N . , Absher M . , Chiu J.F. (1996) Tamoxifen induces TGFbeta 1 activity and apoptosis of human MCF-7 breast cancer cells in vitro. Journal of Cellular Biochemistry.61: 9-17. Elledge R . M . , Lock-Lim S., Allred D.C., Hilsenbeck S.G., Cordner L. (1995) p53 mutation and tamoxifen resistance in breast cancer. Clinical Cancer Research. 1: 12031208. Fattman C.L., A n B., Sussman L., Dou Q.P. (1998) p53-independent dephosphorylation and cleavage of retinoblastoma protein during tamoxifen-induced apoptosis in human breast carcinoma cells. Cancer Letters 130: 103-113 Ferlini C , Scambia G., Distefano M . , Filippini P., Isola G., Riva A., Bomardelli E., Fattorossi A., Panici A . B . , Mancuso S. (1997) Synergistic antiproliferative activity of tamoxifen and docetaxel on three oestrogen receptor-negative cancer cell lines is mediated by the induction of apoptosis. British Journal of Cancer. 75: 884-891. Ferlini C, Scambia G , Marone M , Distefano M , Gaggini C, Ferrandina G , Fattorossi A , Isola G, Benedetti Panici P, Mancuso S. (1999) Tamoxifen induces oxidative stress and  73  apoptosis in oestrogen receptor-negative human cancer cell lines. British Journal of Cancer. 79: 257-263 Fornace A.J., Jackman Jr. J., Hollander M.C., Hoffman-Liebermann B., Lierbmann D.A. (1992) Genotoxic-stress-response genes and growth-arrest genes, gadd, M y D , and other genes induced by treatments eliciting growth arrest. Annals of New York Academy of Sciences. 663: 139-153. Fukumachi Y . , Karasaki Y . , Sugiura T., Itoh H., Abe T., Yamamura K., Higashi K . (1998) Zinc suppresses apoptosis of U937 cells induced by hydrogen peroxide through an increase of the Bcl-2/Bax ratio. Biochemical and Biophysical Research Communications. 246: 364-369. Garvy B.A., Telford W.G., King L.E., Fraker P J . (1993) Glucocorticoids and irradiationinduced apoptosis in normal murine bone marrow B-lineage lymphocytes as determined by flow cytometry. Immunology. 79: 270-277. Harkin D.P., Bean J.M., Miklos D., Song Y . H . , Truong V . B . , Englert C , Christians F . C , Ellisen L.W., Maheswaran S, Oliner J.D., Haber D.A. (1999) Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of B R C A 1 . Cell. 97: 575-586. Ho L . H . , Ratnaike R.N., Zalewski P.D. (2000) Involvement of intracellular labile zinc in suppression of DEVD-caspase activity in human neuroblastoma cells. Biochemical and Biophysical Research Communications. 268: 148-154. Ishido M . , Suzuki T., Adachi T., Kunimoto M . (1999) Zinc stimulates D N A synthesis during its antiapoptotic action independently with increments of an antiapoptotic  74  protein Bcl-2, in porcine kidney L L C - P K j cells. Journal of Pharmacology and Experimental Therapeutics. 290: 923-928. Kang Y., Cortina R., Perry R.R. (1996) Role of c-myc in tamoxifen induced apoptosis in estrogen-independent breast cancer cells. Journal of National Cancer Institute. 88: 279284. K i m J.A., Kang Y.S., Jung M.W., Lee S.H., Lee Y.S. (1999) Involvement of C a  2 +  influx  in the mechanism of tamoxifen-induced apoptosis in HepG2 human hepatoblastoma cells. Cancer Letters. 147: 115-123. Leccia M . , Richard M . , Favier A., Beani J. (1999) Zinc protects against ultraviolet A l induced D N A damage and apoptosis in cultured human fibroblasts. Biological Trace Element Research. 69: 177-190. Lee R., Woo W., Wu B., Kummer A., Duminy H., X u Z. (2003) Zinc accumulation in N methyl-N-nitrosourea-induced rat mammary tumors is accompanied by an altered expression of ZnT-1 and metallothionein. Experimental Biology and Medicine. (Accepted) Liebermann D A , Hoffman B. (1998) M y D genes in negative growth control. Oncogene. 17: 3319-3329. Mandlekar S., Y u R., Tan T., Kong A.T. (2000) Activation of caspase-3 and c-Jun N H 2  terminal kinase-1 signaling pathways in tamoxifen-induced apoptosis of human breast cacner cells. Cancer Research. 60: 5995-6000. Manev H., Kharlamov E., U z T., Mason R.P., Cagnoli C M . (1997) Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells. Experimental Neurology. 146: 171-178  75  Marini M . , Musiani D . (1998) Micromolar zinc affects endonucleolytic activity in hydrogen peroxide-mediated apoptosis. Experimental Cell Research. 239: 393-398. Martin S.J., Mazdai G., Strain J.J., Cotter T.G., Hannigan B . M . (1991) Programmed cell death (apoptosis) in lymphoid and myeloid cell lines during zinc deficiency. Clinical and Experimental Immunology. 83: 338-343. Parat M . , Richard M . , Pollet S., Hadjur C , Favier A., Beani J. (1997) Zinc and D N A fragmentation in keratinocyte apoptosis: its inhibitory effect in U V B irradiated cells. Journal of Photochemistry and Photobiology B: Biology. 37: 101-106. Paski S . C , X u Z. (2001) Labile intracellular zinc is associated with 3T3 cell growth. Journal of Nutritional Biochemistry. 12: 655-661. Perry D.K., Smyth M.J., Stennicke H.R., Salvesen G.S., Duries P., Poirier G.G., Hannun Y . A . (1997) Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. Journal of Biological Chemistry. 272: 18530-18533. Reaves S.K., Fanzo J . C , Arima K., Wu J.Y., Wang Y.R., Lei K . Y . (2000) Expression of the p53 tumor suppressor gene is up-regulated by depletion of intracellular zinc in HepG2 cells. Journal of Nutrition. 130: 1688-1694. Rogers J.M., Taubeneck M.W., Daston G.P., Sulik K . K . , Zucker R . M . , Elstein K., Jankowski M . A . , Keen C L . (1995) Zinc deficiency causes apoptosis but not cell cycle alerations in organogenesis-stage rat embryos: effect of varying duration of deficiency. Teratology. 52: 149-159. Smith M . L . , Ford J.M., Hollander M . C , Bortnick R.A., Amundson S.A., Seo Y.R., Deng C , Hanawalt P . C , Fornace A.J. Jr. (2000) p53-Mediated D N A Repair Responses to  76  U V Radiation: Studies of Mouse Cells Lacking p53, p21, and/or gadd45. Genes Molecular and Cellular Biology. 20: 3705-3714 Smith, M.L., Fornace, A.J. (1996) Mammalian D N A damage-inducible genes associated with growth arrest and apoptosis. Mutation Research/Reviews in Genetic Toxicology. 340: 109-124. Smith M.L., Chen I.T., Zhan Q., Bae I., Chen C.Y., Gilmer T.M., Kastan M.B., O'Connor P.M., Fornace A.J. Jr. (1994) Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 266: 1376-1380. Telford W.G., Fraker P.J. (1995) Preferential induction of apoptosis in mouse C D 4 C D 8 a p T C R C D 3 s thymocytes by zinc. Journal of Cellular Physiology. 164: +  +  l0  10  259-270. Vairapandi M . , Balliet A . G . , Fornace A.J. Jr, Hoffman B., Liebermann D.A. (1996) The differentiation primary response gene M y D l 18, related to GADD45, encodes for a nuclear protein which interacts with P C N A and p 2 1 W A F l / C I P l . Oncogene. 12: 25792594. Waring P., Egan M . , Braithwaite A., Mullbacher A., Sjaarda A. (1990) Apoptosis induced in macrophages and T blasts by the mycotoxin sporidesmin and protection by Zn2+ salts. International Journal of Immunopharmacology. 12: 445-457. Zalewski P.D., Forbes I.J., Betts W.H. (1993) Correlation of apoptosis with change in intracellular labile Zn(II) using Zinquin [(2-methyl-8-p-toluenesulphonamido-6quinolyloxy)acetic acid], a new specific fluorescent probe for Zn(II). Biochemical Journal. 296: 403-408.  77  Zhang G., Kimijima I., Onda M . , Kanno M . , Sato H., Watanabe T., Tsuchiya A., Abe R., Takenoshita S. (1999) Tamoxifen-induced apoptosis in breast cancer cells relates to down-regulation of bcl-2, but not bax and bcl-X , without alteration of p53 protein L  levels. Clinical Cancer Research. 5: 2971-2977. Zhang W., Hoffman B., Liebermann D.A. (2001) Ectopic expression of MyD118/Gadd45/CR6 (Gadd45beta/alpha/gamma) sensitizes neoplastic cells to genotoxic stress-induced apoptosis. International Journal of Oncology. 18: 749-57. Zhang W., Bae I., Krishnaraju K., Azam N . , Fan W., Smith K., Hoffman B., Liebermann D.A. (1999) CR6: A third member in the M y D l 18 and Gadd45 gene family which functions in negative growth control. Oncogene. 18: 4899-4907.  78  CHAPTER 3: General Discussion and Conclusions  1. Human breast cancer cells and breastfibrocysticcells acquired ability to grow in low zinc environment. Unlike in humans and animals, the amount of zinc required for supporting optimum growth in cultured cells, including the three human breast cancer cell lines and the human breast fibrocystic cell lines used in this project, are not established. Most cell culture systems routinely include 10% FBS for supporting optimum growth. FBS, like other serum, contains zinc. Since most of the cell culture media such as D M E M used in this project does not contain zinc, FBS is the main source of zinc in these culture systems. The chemical composition of FBS, including zinc concentration, is often not well defined and varies from batch to batch. The zinc concentration in the low zinc medium used in my thesis project was 0.2 umol/L.  Because there is no established zinc requirement for supporting optimum  growth in these cell lines, there is no base to determine whether a particular zinc concentration is adequate to support optimum growth. However, a zinc concentration of 0.2umol/L is extremely low compared to the plasma zinc concentrations of 10 to 15 umol/L in humans (King and Keen, 1994) and to the zinc concentration of 4-5 umol/L in regular cell culture media (Table 1-2). In our lab, we have previously shown that when 3T3 fibroblast cells are cultured in a similar low zinc medium, both D N A synthesis and cell proliferation are significantly lower than in cells cultured in media supplemented with 5 umol/L of zinc (Paski and Xu, 2001). Interestingly, cell viability in all the human breast cancer and fibrocystic cell types cultured in the low zinc medium was the same as  79  in the cells cultured in the media supplemented with 5 umol/L of zinc (Figure III-1, IV-1, V - l ) , showing that human breast cancer cells and fibrocystic cells can survive in a low zinc environment well.  The reasons as why and how these cell lines can tolerate  seemingly extremely low zinc environment is not known. It is noteworthy that although the zinc concentration in the low zinc medium was 4% of that in the medium supplemented with 5 umol/L of zinc, the total cellular zinc concentration in these cells was the same. Moreover, zinc is accumulated in chemically induced mammary tumours through reduced expression of the zinc exporter ZnT-1 and increased expression of metallothionein, a major intracellular zinc-storage protein (Lee et al., 2003).  In addition, zinc homeostasis is achieved on multiple levels including  intracellular sequestration in zinc storing vesicles, known as zincosomes (Beyersmann and Haase, 2001). The zinc is transported into the vesicles by ZnT-2, a zinc transportor. ZnT-2 is localized on the vesicles and allows the zinc-sensitive hamster kidney B H K cells to accumulate zinc (Palmiter et al., 1996); their existence in MDA-MB-231 cells is unknown. This intracellular storage of zinc is responsible for storage of zinc during extremely low levels of zinc in the culture media (Nasir et al., 1999). The previously stored zinc may be released from the vesicles to self sustain its cellular growth when it is needed. When the supply of zinc is low, cells may employ a mechanism by which the uptake and accumulation of zinc increases. Human malignant prostate LNCaP and PC-3 cells possess the ability to accumulate high levels of zinc (Costello et al., 1999). The most plausible explanation is that a plasma membrane zinc transporter permits the rapid uptake of zinc from the extracellular environment.  One of such zinc transporter, hZIPl, is  expressed in LNCaP and PC-3 cells; the zinc-accumulating trait is believed to be specific  80  for prostate cells (Costello et al., 1999). Although involvement of the hZIPl transporter in breast cancer cells is uncertain, other zinc transporters may be involved in the zinc uptake. Since both human breast cancer cells and fibrocystic cells are not normal cells, it is possible that these cells have altered zinc homeostasis, which permits efficient scavenging of zinc from its surrounding environment to support their growth needs of zinc.  2. Effects of zinc and tamoxifen on human breast cancer cells and human fibrocystic cells In this study, three breast cancer cell lines and one human breast fibrocystic cell line were used. The responses of these cell lines to zinc supplementation and tamoxifen varies. For example, after 48 h, MDA-MB-231 cells responded to zinc supplementation and tamoxifen with a decrease in cell viability and an increase in apoptosis compared to either zinc supplementation or tamoxifen alone. Similarly, T47D cells also responded with decreased cell viability (Figure IV-1) and increased apoptosis (Figure IV-4) compared to the controls. In contrast, both MCF-7 (Figure III-l and III-4) and MCF-10 cells (Figure V - l and V-4) responded with reduced cell viability and no change in both necrotic and apoptotic cell death compared to the controls. Besides differences in the general trend, the magnitude of their responses to zinc supplementation and tamoxifen also varies from cell line to cell line.  For example,  treating MCF-7 cells with 10 umol/L of tamoxifen for 48 h reduced cell viability to about 33% of that in the cells that received no tamoxifen treatment. In T47D cells, tamoxifen reduced cell viability to about 69% of that in the cells that received no tamoxifen  81  treatment. In MCF-10 cells, tamoxifen reduced cell viability to about 27% of that in the cells that received no tamoxifen treatment. In contrast, the same treatment regime had no effect on cell viability in MDA-MB-231 cells (Figure 2-1). These differences in their response to zinc supplementation and tamoxifen treatment may reflect differences in their gene expression profile (Table 1-1).  For  example, both MDA-MB-231 and T47D cells express mutant p53 while both MCF-7 and MCF-10 cells expressing wide-type p53 (Table 1-1). MDA-MB-231 and MCF-7 cells express both Bcl-2 and Bax while expression of these two apoptotic regulatory proteins in T47D and MCF-10 cells is still controversial. In addition, tamoxifen is a known estrogen modulator. In most cases, ER-negative breast cancer cells are less sensitive to tamoxifen than are ER-positive cells (Perry et al., 1995). Since both MCF-7 and T47D cells are ER-positive cells, they would presumably be more responsive to tamoxifen treatment than ER-negative cells such as MDA-MB-231 cells. However, my study is made more complicated by incorporating zinc supplementation, especially the effects of zinc on breast cancer cell growth have not been reported before. Based on the results reported herein, it appears that ER-negative cells are more sensitive to the treatment of a combination of zinc supplement and tamoxifen, suggesting that the efficacy of tamoxifen in suppressing breast cancer cells growth was more greatly enhanced in ER-negative cells than that in ER-positive cells.  3. Overall conclusions and future directions Zinc supplementation in the cell culture media directly influenced total cellular zinc concentrations. Zinc supplementation or tamoxifen alone reduced cell viability and  82  promoted cell death in a cell line-specific fashion. In contrast, a combination of zinc supplementation and tamoxifen reduced cell viability and promoted cell death, especially apoptosis, to a greater extend. These observations suggested an additive effect of zinc and tamoxifen supplementation in suppressing overall growth of human breast cancer cells and fibrocystic cells. Exploratory studies on apoptotic regulatory proteins illustrated that increased apoptosis by zinc and tamoxifen treatment apparently involved several regulatory proteins in the apoptosis pathway. Based on the results that a combination of zinc and tamoxifen improved the efficacy of tamoxifen, as indicated by a reduced cell viability and increased apoptosis, it is necessary to further investigate the molecular mechanisms by which this combination influences apoptosis.  The effects of zinc and tamoxifen on the gene expression of  apoptotic regulatory proteins in the remaining three cell lines should be investigated. In addition, it is also important to establish that the activity of the proteins encoded by these genes are also affected by the zinc and tamoxifen treatment, since changes at the transcriptional level are not necessarily reflected in functional differences.  Another  limitation of this project is that, as all in vitro experiments, it is very difficult to apply the findings to in vivo situations.  In animals, zinc metabolism and apoptosis are highly  regulated involving many systems interacting with each other.  On the other hand,  culturing transformed cells represents highly isolated environment. obtained from my thesis project should be further tested in vivo.  83  Thus, findings  4.  Bibliography  Beyersmann D., Haase H . (2001) Functions of zinc in signalling, proliferation and differentation of mammalian cells. BioMetals. 14: 331-341. Costello L . C , Liu Y . , Zou J., Franklin R.B. (1999) Evidence for a zinc uptake transporter in human prostate cancer cells which is regulated by prolactin and testosterone. Journal of Biological Chemistry. 274: 17499-17504. King JC, Keen CL. Zinc. In: Shils M E , Olson JA, Shike M . eds. Modern nutrition in health and disease, 8 edition. Philadelphia: Lea and Febiger, 1994: 214-230. th  Lee R., Woo W., Wu B., Kummer A., Duminy H., X u Z. (2003) Zinc accumulation in N methyl-N-nitrosourea-induced rat mammary tumors is accompanied by an altered expression of ZnT-1 and metallothionein. Experimental Biology and Medicine. (Accepted) Nasir, M . S., Fathrni, C. J., Suhy, D. A., Kolodsick, K . J., Singer, C. P., and O'Halloran, T. V . (1999) The chemical cell biology of zinc: structure and intracellular fluorescence of a zinc-quinolinesulfonamide complex. Journal of Biological Inorganic Chemistry, 4: 775-783. Palmiter R.D., Cole T.B., Findley S.D. (1996) ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. European Molecular Biology Organization Journal. 15: 1784-1791. Paski S . C , X u Z. (2001) Labile intracellular zinc is associated with 3T3 cell growth. Journal of Nutritional Biochemistry. 12: 655-661.  84  Perry R.R., Kang Y . , Greaves B. (1995) Effects of tamoxifen on growth and apoptosis of estrogen-dependent and -independent human breast cancer cells. Annals of Surgical Oncology. 2: 238-245.  85  APPENDIX I Cell Culture System  86  Table 1-1. Gene profile of apoptotic regulatory proteins in human breast cancer cells (MCF-7, MDA-MB-231, and T47D) and human breast fibrocystic cells (MCF-10).  Cell Line  p53  Bcl-2  MCF-7 MDA-MB231 T47D MCF-10  +  + + -/+ -/+  mutant mutant +  87  Bax/Bak + + -/+ -/+  Table 1-2. Total zinc concentration of the culture media used for culturing human breast cancer cells (MCF-7, MDA-MB-231, and T47D) and human breast fibrocystic cells (MCF-10). Cell Line MCF-7 MDA-MB231 T47D MCF-10  Culture Media D M E M + 10%FBS D M E M + 10%FBS RPMI1640+ 10%FBS DMEM/F12 + 5%HS  88  [Zn] (umol/L) 4.9 4.8 4.0 2.6  Table 1-3. Mineral composition of fetal bovine serum (FBS) and Chelex-100-treatedfetal bovine serum (CFBS). Elements Al As Ca Cd Co Cr Cu Fe Mg Mn Mo Ni P Se Zn  FBS (ug/ml) 0.027 0.034 14.368 0.001 0.000 0.000 0.021 0.287 3.240 0.004 0.004 0.000 12.911 0.000 0.305  89  C F B S (ug/ml) 0.012 0.003 0.267 0.000 0.000 0.000 0.000 0.164 0.054 0.000 0.002 0.002 7.285 0.000 0.010  Table 1-4. Mineral composition of horse serum (HS) and Chelex-100-treated  Elements Al As Ca Cd Co Cr Cu Fe Mg Mn Mo Ni P Se Zn  HS (ue/ml)  C H S (ue/ml)  0.043 0.077 11.602 0.002 0.001 0.001 0.116 0.213 1.823 0.000 0.009 0.001 10.304 0.000 0.030  0.000 0.024 0.167 0.000 0.000 0.001 0.044 0.115 0.000 0.000 0.001 0.000 5.403 0.000 0.008  90  APPENDIX II Effects of Zinc and Tamoxifen on Human Breast Cancer MDA-MB-231 Cells  91  • 0 umol/L Zn 100  • 150 umol/L Zn  n  95 H 0>  s»  90  10  H  n  B a  ^  a  b  n J ri ri  U o u  10 n "a*  ^  5  a. o a 0  1 5 Tamoxifen (umol/L)  10  Figure II-l. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer MDA-MB-231 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 2 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05).  92  • 0 umol/L Zn • 150 umol/L Zn  20  B -I  15-1 CU  U u  10  •—  © U  u  cu  z  5A  *  *  a  a  5  rl  r l  »  rl  10 Tamoxifen (umol/L) Figure II-2. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer MDA-MB-231 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 24 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with asterisks are statistically different. (p<0.05)  93  • 0 umol/L Zn • 150 umol/L Zn 100 90 g  8  60  l m m rk  30  cu  o  **  **  °-«  CU  £  **  n  B b  a  20  *  *  U  a  b  a  *  **  1 5 Tamoxifen (umol/L)  10  *  **  **  b  o CU  z  30 i cu  u  20 ^  io  H  o  0  Figure II-3. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer MDA-MB-231 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 48 h. Mean ± SEM (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  94  Table II-4. The number of cycles tested to determine the optimum conditions for reverse transcriptase polymerase reaction (RT-PCR).  Cell Line  MDA-MB-231  Number of Cycles Tested 28, 32, 34 p53 36, 37, 39 gadd45 36, 39,40 bcl-2 36, 39,40 bax 36,38, 39 b-microglobulin Target  95  R  2  0.997 0.84 0.975 0.99 0.952  Number of Cycles Used 31 36 39 39  -  APPENDIX III Effects of Zinc and Tamoxifen on Human Breast Cancer MCF-7 Cells  96  0  1  5  10  Tamoxifen (pmol/L) Figure III-l. Effects of zinc supplementation and tamoxifen on cell viability in human breast cancer MCF-7 cells . Cells were cultured in media containing with 0 (open bar), 5 (dotted bar), 50 (shaded bar), or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 2 (A), 24 (B), or 48 (C) h. Mean±SEM (n=5). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are differently different (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are differently different. (p<0.05)  97  I • 0 umol/L Zn  M150 umol/L Zn  30.i £  b  b  i°'rl ri rl rl 30  -i  o 15 a o a,  <  b  b  b  *  **  * **  5  **  0 0  1 5 Tamoxifen (umol/L)  10  Figure HI-2. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer MCF-7 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 2 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  98  I • 0 umol/L Zn 100  S 80  • 150 umol/L Zn 1  n  H  B  15 n  15 n  1 5 Tamoxifen (p.mol/L)  10  Figure III-3. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer M C F - 7 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 24 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  99  0  1 5 Tamoxifen (umol/L)  10  Figure III-4. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer M C F - 7 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 48 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are not statistically different. (p<0.05)  100  APPENDIX IV Effects of Zinc and Tamoxifen on Human Breast Cancer T47D Cells  101  • 0 umol/L Zn • 50 umol/L Zn  • 5 umol/L Zn • 150 umol/L Zn  c  Tamoxifen (umol/L) Figure IV-1. Effects of zinc supplementation and tamoxifen on cell viability in human breast cancer T47D cells . Cells were cultured in media containing with 0 (open bar), 5 (dotted bar), 50 (shaded bar), or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 2 (A), 24 (B), or 48 (C) h. Mean±SEM (n=5). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are differently different (p<0.05).The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are differently different. (p<0.05)  102  1 DO umol/L Zn  • 150 umol/L | b  3  90  H  15 n  15 n g  ft o ,<!  1  0  5H 0  1 5 Tamoxifen (umol/L)  10  Figure IV-2. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer T47D cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 2 h. Mean ± SEM (n=3).The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05).The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  103  ^  20  n  1 5 Tamoxifen (umol/L) Figure IV-3. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer T47D cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 24 h. Mean ± SEM (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  104  • 0 umol/L Zn  0  1 5 Tamoxifen (umol/L)  • 150 umol/L Zn  10  Figure IV-4. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast cancer T47D cells." Cellswere cultured" in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 48 h. Mean ± SEM (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  105  Figure IV-5. Effects of zinc supplementation and tamoxifen on cell number in human breast cancer T47D cells. Cells were cultured in media containing 0 (open bar)or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO) or 10 umol/L tamoxifen for 48 h. Mean ± SEM (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different (p<0.05).  106  APPENDIX V Effects of Zinc and Tamoxifen on Human Breast Fibrocystic M C F - 1 0 Cells  107  0  1 5 Tamoxifen (u,moI/L)  10  Figure V - l . Effects of zinc supplementation and tamoxifen on cell viability in human breast firocystic MCF-10 cells . Cells were cultured in media containing with 0 (open bar), 5 (dotted bar), 50 (shaded bar), or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L tamoxifen for 2 (A), 24 (B), or 48 (C) h. Mean±SEM (n=5). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are differently different (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are differently different. (p<0.05)  108  • 0 umol/L Zn • 150 umol/L Zn  B 30-1 *  I 2020 • u  iu  u cu  z  a  •Tk ri rl mM  io  ^30i  0  1 5 Tamoxifen (umol/L)  Figure V-2. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast fibrocystic MCF-10 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L of tamoxifen for 2 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05) The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  109  0  1 5 Tamoxifen (pmol/L)  10  Figure V-3. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast fibrocystic MCF-10 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L of tamoxifen for 24 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  110  • 0 umol/L Zn • 150 umol/L Zn  100 i  8 n  B  1-ni  0  n n rl 1 5 Tamoxifen (umol/L)  Figure V-4. Effects of zinc supplementation and tamoxifen on live cells (A), necrotic cells (B), and apoptotic cells (C) in human breast fibrocystic MCF-10 cells. Cells were cultured in media containing 0 (open bar) or 150 (filled bar) umol/L zinc for 72 h followed by treatment with 0 (DMSO), 1, 5, or 10 umol/L of tamoxifen for 48 h. Mean ± S E M (n=3). The means among different concentrations of zinc within the same concentration of tamoxifen with different lower-case letters are statistically different. (p<0.05). The means among different concentrations of tamoxifen within the same concentration of zinc with different asterisks are statistically different. (p<0.05)  111  

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