Honors Thesis Proposal by Meredith C. Davis (’00)
For the Academic Year: 1999-2000
Submitted September 27, 1999
Thesis Advisor: Dr. Shyamal K. Majumdar
Raloxifene (marketed as “Evista” by Eli Lilly and Company) belongs to a class of selective estrogen receptor modulators (SERM) and was approved by the FDA in December 1997 for the treatment of osteoporosis in postmenopausal women. Raloxifene has a high affinity for estrogen receptors due to strategically placed phenols (Black et al., 1982) and has been shown to be an effective inhibitor of the growth of breast cancer cells in culture (Jiang et al., 1993). Recent studies from the Multiple Outcomes of Raloxifene Evaluation (MORE) indicate that after a three year treatment period with raloxifene the incidence of breast cancer compared to a placebo group was reduced by 76% (Cummings et al., 1999). The Study of Tamoxifen and Raloxifene (STAR) and are phase III clinical trials currently underway to determine the effectiveness of tamoxifen and raloxifene in preventing the occurrence of breast cancer and also to compare the net effects and risks of both drugs (Jordan and Morrow, 1999).
Tamoxifen was the first antiestrogen approved by the FDA in 1977 for the treatment of advanced breast cancer in postmenopausal women (Lerner and Jordan, 1990). Tamoxifen works by blocking estrogen from binding to estrogen receptors. In estrogen receptor positive cases tamoxifen is effective in preventing breast cancer, whereas in estrogen receptor negative cases, tamoxifen has virtually no effect (Jordan and Jaspan, 1976). Studies on BB-88 murine erythroleukemic and HeLa (human cervical cancer) cell lines have shown that tamoxifen inhibits cell multiplication with a drastic effect at 10, 15 and 20ug/mL concentrations. After 120 hours of treatment at 15 and 20ug/mL there were no viable HeLa cells. 20ug/mL of tamoxifen was shown to be toxic to BB-88 cells, as there were no viable cells after 24 hours (Bonita, et al., 1999).
Tamoxifen and raloxifene are known as antiestrogens because they have the ability to work as an estrogen agonist in bone and in the cardiovascular system while acting as an estrogen antagonist in breast and uterine tissue. Raloxifene acts as an estrogen antagonist in the same manner as tamoxifen by blocking receptors on breast and uterine cells from receiving estrogen (Zou et al., 1999). Unfortunately tamoxifen has been shown to increase the incidence of endometrial cancer 2-3 fold (report in The Lancet,1998), and thus far there is no evidence to suggest that raloxifene increases the risk for uterine cancer (Jordan and Morrow 1999).
Cancer is a life-threatening disease that is characterized by uncontrolled cell growth. Normal cells in culture are programmed to undergo a definite number of divisions, known as the Hayflick limit (50 generations), and once they have reached this limit, they undergo apoptosis (programmed cell death) and die. Cancerous cells are immortal and remain undifferentiated, which allows them to continue to divide, not following the Hayflick limit. Breast cancer can be caused by the presence of two autosomal dominant predisposition genes, BRCA 1 which is located on chromosome 17q21 (Miki et al., 1994) and BRCA2, located on chromosome 13q12-13 (Wooster et al., 1994). These two genes also cause an increased risk in ovarian cancer, which is estimated at 10% by age 60 and lower in BRCA2 carriers (King et al., 1993).
Apoptosis is a highly regulated process by which the cell programs itself to die. This process occurs naturally in the regulation of tissue homeostasis (Verhaegen, 1998) and when it is beneficial to the organism, such as when DNA has been damaged or the cell has been infected by a contaminant. If the mechanism fails and the cell does not receive a signal to undergo apoptosis, the cell will continue to divide indefinitely which can lead to tumor formation. Apoptosis can be characterized by blebbing, a decrease in cell volume, oligosomal fragmentation, chromatin condensation and the formation of apoptotic bodies (Brown, 1999; Kerr et al., 1972; Majumdar and Valdellon, 1999). The apoptotic bodies are then phagocytosed by parenchymal cells or macrophages (Verhaegen, 1998). Apoptosis is distinctly different from necrosis, a type of cell death caused by injury to the cell resulting in rupturing of the plasma membrane and leakage of the cellular components into extracellular space. This then initiates inflammatory responses in nearby cells (Earnshaw, 1995).
In healthy mitochondrial cells, energy from respiration accumulates and is stored as an electrochemical gradient across the mitochondrial membrane. This gradient drives the cell to synthesize more ATP and a disruption in this gradient has been shown to be one of the first intracellular changes in apoptosis (Cossarizza et al., 1995). Once the cell begins apoptosis, a cascade of caspases is activated. Caspases are proteases that are directly involved in the demise of the cell during apoptosis. Each caspsase has a specific role in apoptosis as either effectors or inhibitors. Effectors are caspases that are directly responsible for proteolytic cleavages that lead to cell disassembly and inhibitors are those involved in upstream regulatory events (Thornberry and Lazebnik, 1998). Once a cell has entered the apoptotic pathway and the cascade of caspases has been initiated, linker DNA between nucleosomes in chromatin is digested and fragmented by endonucleases, freeing mononucleosomes and oligonucleosomes (Kerr et al., 1972). Using this theory, a Nucleosome ELISA assay has been developed that uses a biotin-labeled antibody to bind to the histone component of mononucleosomes and oligonucleosomes to quantitate the amount of apoptotic cells in vitro (Oncogene, Cambridge, MA).
Based upon my literature review, very little is known about the effects of raloxifene in vitro, whereas research into the cytotoxic effects of tamoxifen has been performed and is better established (Bonita, et al., 1999; Brown, 1999; Valdellon, 1999). Because raloxifene holds the possibility of reducing the risk of breast cancer without stimulating endometrial cancer unlike tamoxifen (Jordan and Morrow, 1999), further research in this area should be conducted. My preliminary findings show that raloxifene has a toxic effect on BB-88 cell multiplication. With increasing concentrations of raloxifene (5, 10 and 20ug/mL), the number and percentage of viable cells decreased. BB-88 cells treated with 20ug/mL declined in number after only 24 hours and continued to decrease over 120 hours. This investigation will study the effects of raloxifene on BB-88, HeLa, and WI-38 cell multiplication. Research will also be conducted to investigate the mechanism of cell death upon treatment with raloxifene.
Murine erythroleukemic (BB-88), human cervical cancer (HeLa) and human diploid lung (WI-38) cell lines were purchased from the American Type Culture Collection (Rockville, MD). BB-88 cells are virally induced murine erythroleukemic cells (MEL) that grow in suspension with a generation time of 10-12 hours. (Ruan and Lilly, 1992). HeLa cells are human cervical cancer cells that adhere to surfaces and grow by forming a multilayer. HeLa cells have a generation time of 11-12 hours. HeLa and MEL are well-studied cell lines that are commonly used in cancer research. WI-38 is a human diploid cell line derived from normal embryonic lung tissue and has a finite lifetime of approximately 50 population doublings with a generation time of 17 hours. These cells do not form tumors and are free from viruses. WI-38 cells adhere to surfaces forming a multilayer.
Raloxifene (Evista) was obtained from Eli Lilly & Co. (Indianapolis, IN) through Lafayette College Infirmary. The drug, in the form of a 60mg tablet, was dissolved and diluted with sterile water and filtered to obtain a stock solution of 400ug/mL and stored at -20oC.
All cell lines are maintained in Dulbecco¹s Modified Eagles Medium supplemented with 10% fetal calf serum (DME-10). The cells will be grown in 25cm2 tissue culture flasks in a 37oC humidified incubator with 7.5% CO2 in air.
Cell multiplication studies will be performed on BB-88, HeLa and WI-38 to determine the dose curve of raloxifene for each cell line. Exponentially growing cells will be treated with 0, 1, 5, 10 or 20ug/mL of raloxifene, counted, and viability will be determined every 24 hours for 144 hours using the trypan blue exclusion method (Freshney, 1994). Statistical analyses such as Student¹s T-test, ANOVA and regression and correlation will be performed.
Two different assays will be used to understand the mechanism of cell death in raloxifene treated and untreated cells: the Trevigen Mitochondrial Membrane Potential Disruption Assay (Gaithersburg, MD) and a Nucleosome ELISA (Oncogene, Cambridge, MD).
The Trevigen Mitochondrial Membrane Potential Disruption Assay (Gaithersburg, MD) will detect via fluorescence a change in membrane potential of the mitochondria that indicates occurrence of apoptosis. This procedure will be performed according to the provided protocol. Cells will be treated with 0, 1, 5, 10, 15 or 20ug/mL raloxifene and incubated at 37oC in 7.5%CO2. The media will be removed and cells will be treated with DePsipher solution, which is a lipophilic cation that acts as a mitochondrial activity marker, and incubated at 37oC in 7.5% CO2 in air for 15-20 minutes. After incubation, cells will be washed with Reaction Buffer and observed under the fluorescence microscope. In healthy cells the mitochondria will appear red at 585/590nm due to the aggregation of the DePsipher solution within the mitochondria. In dying or nonviable cells, the dye remains in its monomeric form in the cytoplasm and appears green at 510/527nm.
The Nucleosome ELISA (Oncogene, Cambridge, MA) will be used to detect DNA damage via biotin-labeled antibodies that bind to histones, quantitating free nucleosomes in apoptotic cells. The procedure will be followed according to the protocol provided with the kit. Cells will be counted and treated with various concentrations of raloxifene and incubated at 37oC in 7.5%CO2. Using the components of the kit, the cells will be lysed followed by centrifugation. The supernatant will be removed and saved for analysis. All samples and standards will be diluted to a final concentration of 0.2mM, transferred into the appropriate wells of a 96-well plate and incubated. A Detector Antibody is then added to each well to bind to the mononucleosomes and oligonucleosomes and incubated, after which each well is washed and a Conjugate Solution is added to bind to the Detector Antibodies and incubated. Each well is washed and Substrate Solution is then added to elicit a color change proportional to the amount of detected mononucleosomes and oligonucleosomes. After a brief incubation period, Stop Solution is added to stop the reaction and absorbance is measured in each well using a spectrophotometer at 450/595nm.
The total cost of this project will be approximately $600. This amount will cover the cost of the Trevigen Mitochondrial Membrane Potential Disruption Kit (Gaithersburg, MD), a Nucleosome ELISA Kit (Oncogene, Cambridge, MA) and other associated laboratory supplies.
Black L.J., C.D. Jones, J.H. Clark, et al., (1982). LY156758: a unique antiestrogen displaying high affinity for estrogen receptors, negligible estrogenic activity, and nearly total estrogen antagonism in vivo. Breast Cancer Res Treat.2:279.
Bonita, Diane, N. Eraydin, M. Nyako, et al., (1999). Response of non-breast cancer cells to tamoxifen in presence and absence of beta-estradiol. The NucleusVol. 42 (in press).
Brown, Kimberly (1999). An Investigation of the Mechanism of HeLa Cell Death Induced by Tamoxifen. Honors Thesis: Lafayette College Biology Department. Easton, PA.
Cossarizza A., C. Franceschi, D. Monti, et al., (1995). Protective effect of N- acetylcysteine in tumor necrosis factor-alpha-induced apoptosis in U937 cells: the role of mitochondria. Exp. Cell Res.220:232-40.
Cummings, S.R., S. Eckert, K.A. Kruege, et al. (1999). The Effect of Raloxifene on Risk of Breast Cancer in Postmenopausal Women: Results From the MORE Randomized Trial. J. Amer. Med. Assn.281:2189-2197.
Early Breast Cancer Trialists¹ Collaborative Group (1998). Tamoxifen for early breast cancer: an overview of the randomized trials. The Lancet351:1451-1467.
Earnshaw, W.C. (1995). Nuclear Changes in Apoptosis. Current Opinion in Cell Biology7:337-343.
Freshney, R. Ian (1994). Culture of Animal Cells: A manual of Basic Technique. New York: Wiley-Liss.
Jiang S.Y., C.J. Parker, and V.C. Jordan (1993). A model to describe how a point mutation of the estrogen receptor alters the structure function relationship of antiestrogens. Breast Cancer Res Treat.26:139-147.
Jordan V.C. and T. Jaspan (1976). Tamoxifen as an antitumour agent: oestrogen binding as a predictive test for tumour response. J. Endocrinology68:453-460.
Jordan V.C. and M. Morrow (1999). Tamoxifen, raloxifene, and the prevention of breast cancer. Endocrine Reviews20:253-278.
Kerr, J.F.R, A.H. Wylie, and A.R. Currie (1972). Apoptosis: a Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics.Br. J. Cancer 26:239-257.
King M.C., S. Rowell, and S.M. Love (1994). Inherited breast and ovarian cancer. What are the risks? What are the choices? JAMA269:175-180.
Lerner L.J. and V.C. Jordan (1990). Development of antiestrogens and their use in breast cancer. B.F. Cain Memorial Lecture. Cancer Res.50:4177-4189.
Majumdar, S.K. and J. Valdellon (1999). Surface morphological changes of HeLa cells exposed to tamoxifen and taxol. Microsc. Microanal.5: 1270-1271.
Miki Y, J. Swen, D. Shattuck-Eidens, et al., (1994). A strong candidate for the breast and ovarian cancer susceptibility gene, BRCA1. Science266:66-71.
Ruan, Ke-San and Frank Lilly (1992). Approach to a Retrovirus Vaccine: Immunization of Mica against Friend Virus Disease with a Replication-Defective Friend Murine Leukemia Virus. Proc. Natl. Acad. Sci., USA89:12202-12206.
Thornberry, Nancy A and Yuri Lazebnik (1998). Caspases: the enemy within. Science281:1312-1316.
Valdellon, Jennifer (1999). Microscopal Studies of the Effects of Tamoxifen on Cancer Cell Lines. Honors Thesis: Lafayette College Biology Department. Easton, PA.
Verhaegen S. (Jan 1998). Microscopal study of cell death via apoptosis. Amer. Microsc. Analysis19-21.
Wooster R., S.L. Neuhausen, J. Mangion, et al., (1994). Localization of a breast cancer Susceptibility gene, BRCA2, to chromosome 13q12-13. Science265:2088-2090.
Zou A., K.B. Marschke, K.E. Arnold, et al. (1999). Estrogen Receptor beta activates the Human retinoic acid receptor alpha-1 promoter in response to tamoxifen and other estrogen receptor antagonists, but not in response to estrogen. Mol. Endocinol.3:418-430.