Essam E. Enan, Ph.D.
University of California, Davis

Initial Award Abstract

Recent epidemiological evidence suggests that there is a relationship between the incidence of breast cancer and the presence of organochlorine insecticides in the body. There are many study results indicating that environmental factors have strong influence on the occurrence of breast cancer and that estrogenic actions of various chemicals are one of the most strongly suspected contributing factors. The overall objective of this project is to understand the cellular mechanisms by which organochlorine pesticides act to cause cancer-like transformation of human breast cancer cells under laboratory (in vitro) conditions.

The role of environmental organochlorines in breast cancer is the subject of continuing debate and research. Epidemiological studies have reported numerous environmental chemical contaminants to be present in normal and cancerous breast tissue, including organochlorine insecticides and polychlorinated biphenyls. The proposed study will address the interaction between estrogen and insecticides in human breast cancer cells and normal cells. Specifically, we will study the role of growth factors and the process of transmitting the growth cues from outside of the cell to the cellular proteins (signal transduction) in the insecticide-induced foci formation (a biomarker for cancer promotion).

Our preliminary study identified two components involved in the signal transduction pathway in human breast cancer cells that are activated by organochlorine insecticide treatment. These two components are c-Neu kinase and c-src kinase. We believe that the interaction between these two enzymes is a key point at which organochlorine insecticides promote breast cancer. Our study will address whether the blocking of c-Neu and c-src kinases signaling using specific inhibitors for these two enzymes will prevent the induction of foci formation by organochlorine insecticides in cells. In addition, we will examine the interaction of DDT and Lindane with the estrogen response pathway in breast cancer cells. Furthermore, it is the goal of this proposal to identify and establish a biomarker for early detection of breast cancer promotion. We will compare the level of pesticide residues found in breast tissues of cancer patients with the levels that are required to affect breast cancer cells in the laboratory.

Why are these basic mechanistic studies on cell transformation necessary? The major benefits of these studies are to provide the logical basis for understanding the action of pesticides on breast tissue and to identify other environmental chemicals that may increase the risk of breast cancer in exposed women.

Final Report (1998)

Breast cancer incidences has been steadily rising during the past two or three decades, a trend characterized by increasing rates among estrogen-responsive tumors, by continuing increases among older women, and by growing numbers in both developed and developing countries. The incidence of breast cancer in the United States has increased a modest 8% among women under 50 years of age, while it has risen 32.1% among women in the age group of 50 years or older. This upward shift is consistent with the historical pattern of accumulation of organochlorine residues in the environment. The overall objective of this proposal was to understand cellular mechanisms by which organochlorine pesticides act to cause transformation in human breast cancer cells (MCF-7) under laboratory conditions. Our data showed that among several test organochlorine insecticides, lindane-like chemicals and DDT-like chemicals increased MCF-7 cell proliferation as judged by cell count and ³H-thymidine incorporation (a measure of DNA replication). This effect was dose-dependent and it is likely to involve both an estrogen receptor-dependent pathway and an estrogen receptor-independent pathway as judged by the effect of DDT-like chemicals that bind to the estrogen receptor and lindane-like chemicals that do not bind to the estrogen receptor on MCF-10A (a breast cell linethat does not have estrogen receptors) vs. MCF-7 (a cell line that a lot of these receptors). Our data also showed that beta-HCH, a relative of lindane, significantly decreased PKA activity and significantly increased the oncogene protein, cSrc kinase activity in MCF-10A. An opposite effect for beta-HCH on these two signaling proteins was found in MCF-7 cells. More importantly, this toxicant decreased the amount of the tumor suppressor protein p53 and increased the DNA binding activity of AP-1. The functional status of the wild-type p53 activity and AP-1 subunits is still under investigation in our laboratory.

In our effort to block the proliferative effect of this type of toxicant on MCF-7 cells, geldanamycin, the c-Src kinase inhibitor was applied at different concentrations with and without beta-HCH. At high concentrations (10 ng geldanamycin/ml), the effect of beta-HCH on ³H-thymidine incorporation was decreased by 35%. Lower concentrations of geldanamycin did not affect beta-HCH action. The disadvantages of using 10 ng geldanamycin/ml are: 1) it is toxic to the cells after 5-7 days of treatment; 2) it is not highly specific to the c-Src kinase; and 3) it has a toxic effect on steroid hormone receptors, particularly those receptors that form a heterodimer with the heat shock protein 90 (hsp90).

Therefore, we used another signaling protein modulator, forskolin, that induces the intracellular production of cAMP. The data showed that forskolin at physiological concentration (5 m M) antagonized the proliferative effects of beta-HCH without any effect on cell viability as was found with geldanamycin. From these data it is obvious that signaling protein components play a key role in the proliferative and estrogenicity of several environmental toxicants. Therefore, an understanding of the role of signal transduction pathways in the environmental toxicants may provide important clues for developing chemical therapies to retard their toxic action.

http://www.ucop.edu/srphome/bcrp/progressreport/abstracts/biology/3pb0095.html