Identification Of Genes Involved In The Action Of Tamoxifen And Estrogen In The Human Breast Cancer Cells Using Microarray And Real-time PCR Analysis

Supervisor: Prof. Wang Youde Estrogen plays an important role in the growth and progression of human breast cancer, but the mechanism by which this hormone elicits its regulatory function still remains largely unknown. While one possible mechanism may include the formation of reactive oxygen species (ROS) and / or a receptor-mediated pathway, other reports suggest that estradiol (E2), the most active member of the estrogen family of female sex hormone, could act as an antioxidant that could enhance anti-apoptotic activity. Tamoxifen (TAM) is well known as an adjuvant treatment in estrogen-dependent breast cancer. The main mechanism of action of TAM is believed to be as the estrogen-receptor (ER) agonist, which would inhibit estrogen's function. And there exist other mechanisms of TAM as anti-tumor drug, such as modulation of signaling proteins, oxidative stress, etc. Our experiments are mainly focused on the characterization and quantification of the genes related to ER function, and others related to the antioxidant and anti-apoptotic functions. We have used human ER-positive (ZR-75-1) and ER-negative (MDA-MB-231) breast cancer cell lines as in vitro models to study alterations in the expressions of the following classes of genes in response to TAM and E2 treatments: (1) protooncogenes, (2) Tumor suppress genes, (3) Estrogen related protein genes, and (4) Antioxidant enzyme genes. We have quantified gene expressions using real-time PCR. In this manner, we have attempted to elucidate the antioxidant and antiapoptotic roles of E2 and TAM. The findings that we present here serve to enlarge our vision in understanding the TAM and estradiol networks and their interactions. The reasons that prompted us to undertake the research reported here were as follows: (a) estrogen is important in the progression and development of breast cancer; (b) TAM constitutes an important arsenal in adjuvant endocrine therapy in the treatment of breast cancer in estrogen receptor positive postmenopausal women; and (c) although there is considerable information on the etiological and therapeutic roles of estrogen and TAM, not much detailed information is available on the genes that mediate carcinogenesis, apoptosis and ROS nutralisation pathways and their mutual interactions. In this context, we believed it was very important to quantify the expressions of the related genes in breast cancer cells, regulated by tamoxifen and estrogen, using a reliable methodology such as the real-time PCR. Quantification of gene expressions by real-time PCR has evolved during the last few years as a valuable tool in molecular diagnostics and has proved to be extremely well suited for this purpose. Real-time PCR allows continuous monitoring of product formation. As the protocol is developed today, the technique is quite well established for a number of applications including scanning for gene mutations and quantification of gene expressions.Our observations lead to the following major conclusions: (1) Both estradiol and tamoxifen were more toxic to the ER-positive (ZR-75-1) breast cancer cells and reduced their viability to a higher degree as compared to the ER-negative (MDA-MB-231) cells. This suggests that the effects of these agents on the viability of ER-negative cells may be mediated by a combination of different toxic mechanisms, while their effects on ER-positive cells may be mediated through more specific pathways, such as targeted apoptosis or initiation of abnormal gene interactions. (2) There were marked differences, spanning a range of several hundred fold either positively or negatively, in the basal levels of expression of different genes between the ER-positive and ER-negative cell lines. An examination of members of gene classes involved indicated that this may be reflective of fundamental genetic mechanisms related to the estradiol receptor function. For example, a marked underexpression of SOD2 and GPx1 in ER-positive ZR-75-1 cells was compensated for by an overexpression of SOD1 and CAT which mediate the same sequence of reactions leading to conversion of the superoxide radical, first to hydrogen peroxide and then to water. (3) Treatment with estradiol and tamoxifen, either individually or in combination, produced much greater effects on the ER-positive cells than on the ER-negative cells. The protooncogene c-erbB2 and the mitochondrial antioxidant enzyme gene, SOD2, were the most affected, resulting in striking upregulations ranging over several tens of folds.