Date of Award

12-2009

Degree Name

Master of Science

Department

Pharmacology

First Advisor

Tischkau, Shelley

Abstract

The physiological and behavioral functions of the body are coordinated into daily patterns that are synchronized with the earth's light/dark cycles. This patterning of function is referred to as circadian rhythms. A central pacemaker located in suprachiasmatic nucleus of the brain serves to coordinate the body's rhythms with the light/dark cycle. Disturbances in normal circadian rhythm have been shown to increase the risk of certain types of cancer, including breast cancer. This effect is so significant that the World Health Organization has recently classified shift work as a probable carcinogen. One effect of alteration in the light/dark cycle such as that experienced by shift workers is a change in the production of the pineal hormone melatonin (N-acetyl-5-methoxytryptamine). Secretion of melatonin is regulated by the suprachiasmatic nucleus. Melatonin has a wide variety of functions including physiological regulation of sleep, modulation of the immune system and antioxidant action. Recent studies have determined that melatonin has oncostatic actions in a variety of cancers, including breast, prostate and endometrial cancer. Thus, this thesis explores the effects of melatonin on clock gene expression and growth of a mouse mammary tumor cell line, EMT6. I hypothesized that growth inhibitory actions of melatonin involve alteration in clock gene expression, induction of apoptosis and cell cycle arrest. Thus, this thesis investigates the modulatory effects of melatonin on clock genes, cell cycle parameters and apoptosis. Western blot analysis and immunocytochemistry confirmed expression of melatonin receptors in the EMT6 cell line. I investigated the circadian rhythm of EMT6 cells by measuring the clock gene expression pattern over a 24 hour period. I found a significant rhythm in Per1 and Per2 transcripts. Expression of estrogen and progesterone receptors was measured as they are putative clock controlled genes involved in the development of breast cancer. The results showed significant rhythm in Per1 (p=0.05), Per2 (p=0.03) and estrogen receptors ERalpha (p=0.001) and ERbeta (p=0.028). Peak expression for Period genes and ERalpha is found at 16 hrs and 20 hrs after serum shock, respectively. Peak expression for ERbeta is found at 24 hours after serum shock. Other steroid hormone receptors such as progesterone receptors PRB and PRA+B were not rhythmic. Treatment with melatonin in a concentration range from 10µM to 1nM inhibited growth in the cells. The antiproliferative effect of melatonin was dose and time dependent. At the end of 48 hours, melatonin at a concentration of 10-7 M induced apoptosis in EMT6 cells as indicated by caspase-3 immunocytochemistry. Furthermore, this same treatment caused an upregulation of the clock gene and putative tumor suppressor gene, Per2. These studies provide evidence that melatonin alters growth of EMT6 cells by inducing caspase-3 and apoptosis, which may be regulated through induction of Per2. Thus, disturbance in rhythmic secretion of melatonin may promote tumor progression in breast cancer.

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