Gene and protein expressions induced by 17beta-estradiol and parathion in cultured breast epithelial cells

Abstract

Cancer of the breast is the most common form of malignant disease occurring among women of the western world and environmental substances seem to be involved in the etiology of this disease. Many studies have found an association between human cancer and exposure to agricultural pesticides and among them parathion, the organophosphorous pesticide used in agriculture to control mosquito plagues. The association between breast cancer and prolonged exposure to estrogens suggests that this hormone also may have a role in such process. However, the causative factors for breast carcinogenesis remain an enigma. The objective of this study was to determine the effects of 17beta-estradiol (E2) and parathion on cell transformation of human breast epithelial cells in vitro. The results of this study showed that parathion alone and in combination with E2 induced malignant transformation of an immortalized human breast epithelial cell line, MCF-10F, and the malignant feature was confirmed by anchorage independency and invasive capabilities. Parathion alone efficiently elevated the expression of EGFR, c-Kit, Trio, Rac 3, Rho-A, and mutant p53 proteins. Analysis of gene expression using commercially available human cell cycle array revealed transcriptional alterations in 22 out of a total of 96 genes. Among them, nine genes involved in the regulation of cell cycle were altered. These included cyclins (A1, A2, C, G1, G2, and H), cyclin-dependent kinases (CDKs), and minichromosome maintenance deficient (MCM). Results suggest that parathion has the potency to cause malignant transformation of breast epithelial cells through modulation of expression of cell cycle regulated genes.

Figure 1

(A) The parental MCF-10F cell line was treated with 17β-estradiol (E2) 10⁻⁸ M, parathion (100 ng/mL) and combination of E2 plus parathion during 20 passages. AIA: Anchorage Independence Assay. IA: Invasion Assay. Positive signs (+): referred to the lack of anchorage independent growth and invasiveness. Negative signs (−): referred to the lack of both characteristics. (B) MCF-10F cell line did not form colonies in agar. (C) Colony formed by parathion-treated cells. (D) Colony formed by combination of E2- and parathion-treated cells. Phase contrast microscopy (4X). (E) Invasive characteristics of control and treated-MCF-10F cell line were scored 20 h after plating onto matrigel basement membranes using modified Boyden’s chambers constructed with multi-well cell culture plates and cell culture inserts. Values represent the invasive characteristics of MCF-10F, E2, parathion, and a combination of E2- and parathion-treated cells obtained by scoring the number of cells that crossed the filters.

Figure 2

Bars represent the average and standard error of (A) EGFR, (B) c-Kit, (C) Trio and (D) Rac 3 of MCF-10F, E2, parathion and a combination of both. Representative immunofluorescence- stained images of (A) EGFR, (B) c-Kit, (C) Trio and (D) Rac 3 protein expression of MCF-10F, E2, parathion and a combination of both. Protein expression was determined by immunofluorescent staining and quantified using confocal microscopy and a computer program, which gives the area and the intensity of the staining as described in the text. The primary antibodies used were: rabbit polyclonal for EGFR, c-Kit and Trio and goat polyclonal for Rac 3 antibody (Biotechnology Inc., Santa Cruz, CA, USA).

Figure 3

Bars represent the average and standard error of (A) Rho-A and (B) mutant p53 protein expression of MCF-10F, E2, parathion and combination of both. Representative immunofluorescence stained images of analyzing (A) Rho-A and (B) mutant p53 and protein expression of MCF-10F, E2, parathion and combination of both. The primary antibodies used were mouse monoclonal antibody (Biotechnology Inc.).