The Response of Breast Cancer Cell-Proliferation to Photoperiod Manipulationsthe Role of Zebularine, a Methyl Transferase Inhibitor

• Main Authors: Fares, Eman, פארס, אימאן
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2012
• Subjects: Amino acids; Apoptosis; Biochemistry; Biology; Cell growth; Cellular biology; Chemistry; Circadian rhythm; Disease; DNA methylation; Drug dosages; Endocrinology; Enzymes; Heart; Hormones; Liver; Medicine; Metabolism; Morphology; Mutation; Neurosciences; Physiology; Public health; Signal transduction; Statistical analysis; Toxicity; Tumors; Variance analysis; Womens health
• ISBN: 9798471188686

Background: Breast cancer (BC) is the most common cancer among women worldwide. The risk of BC has been increasing for many decades suggesting a putative association with industrialization and urbanization in the etiology of the disease. Extended exposure to light-at-night (LAN) is one of the most common environmental and occupational features of modern life. Several studies support a positive correlation between night-shifts, exposure to LAN and BC risk .Melatonin (MLT) is a pleiotropic neurohormone produced and secreted from the pineal gland at night under dark conditions and therefore, it might be the mediator of LAN effects on BC. MLT has been shown to reduce the incidence of tumors and to exert direct oncostatic and antiproliferative effects on neoplastic cells, particularly on hormone sensitive cancers.It is known too, that the BC carries with it a significant number of physiological and emotional stressors which operate mechanisms of stress reactions and stimulate the hypothalamus. One of the signs of instability in the endocrine system to a stress reaction is an abnormal circadian rhythm of cortisol.Zebularine a cytidine analogue containing a 2-(1H)-pyrimidinone ring, originally synthesized as a cytidinedeaminase inhibitor. Besides being an effective inhibitor of DNA methylation, zebularine has been shown to cause demethylation and reactivation of a silenced and hypermethylated p16 gene in human bladder tumor cells grown in NUDE mice and it was also demonstrated to be minimally cytotoxic in vitro and in vivo . Since zebularine is a non-specific, genome-wide inducer of demethylation, it is imperative that the precise effects of the chemical be determined for each cancer type.Hypothesis: On the basis of existing knowledge we suggest that “If Zebularine reduces viability of 4T1 BC cells by hypomethylation and acclimation to long night increases MLT, the combination of zebularine and MLT will result in no significant epigenetic change.”Methods: The in vitro model were murine breast cancer cells, line employed was 4T1. The effect of zebularine and cortisol and their combination on cell viability was examined via XTT viability assay. Zebularine and Cortisol toxicity was examined by measuring LDH level using a spectrophotometric method. The in vivo model was C57BL/6 female mice, after acclimation of two weeks to short day (SD) photoperiod, mice were s.c. inoculated with 700000 4T1 cells/mice and then they were divided to two groups: (1) control group: SD mice. (2) Treated group, injected daily with 500 mg/kg of zebularine for four weeks. Every two days body mass was measured. Then, animals were sacrificed and blood samples were taken to test heart, liver, and kidney functions. The following variables were measured: body and tumor mass. Tumors, heart, liver and mammary glands were removed; genomic DNA was extracted for testing global methylation by Methy Flash Methylated DNA Quantification kit.Results: The in vitro results indicate that LDH levels measured in the media of control and all treatments with zebularine (350 µM), cortisol (10-9M, 10-5M), zebularine and cortisol (10-9 M+350 µM, 10-5+350 µM) remained almost unchanged. Treatments of 4T1 cells with 350 µM zebularine for 72h resulted in a significant inhibition of cell viability by 60%, while exposure to physiological or pharmacological concentrations, 10-9 or 10-5 (M) of cortisol, respectively, resulted in a significant increase in cell viability by 11% and by 25% respectively. Exposure of 4T1 cells to the combination of physiological or pharmacological concentrations, 10-5 or 10-9 (M) of cortisol, with Zebularine 350 µM for 72h, resulted in a significant inhibition of cell viability by 48.27% and 57% respectively.The in vivo results indicate that daily treatment of 500 mg/kg zebularine has no effect on liver and kidney function. Zebularine has an effect on heart function it was detected by measuring Creatine phosphokinase (CPK) was six times higher than the control group.The body mass of the treated mice decreased significantly after nine days, in contrast the body mass of the control group increased, and 100% of the mice treated with zebularine developed tumors.The tissues extracted from the treated mice did not exhibit significant changes in their global DNA methylation levels compared to the tissues extracted from the control mice; however, the standard errors were rather large.Conclusions: Our in vitro results indicated that zebularine and cortisol at the effective doses are not toxic. Cortisol increased while zebularine decreased cell viability of 4T1 cells in vitro. However, exposure of 4T1 cells to their combination had no inhibitory effect of zebularine on cell viability and zebularine was still effective in decreasing cell viability in the presence of cortisol.The in vivo studies indicate that the effective dose of zebularine is not toxic, while treatment with high dose of zebularine such as 500 mg/kg may cause muscle injury.