PI3K pathway activation on gene expression regulated by triiodothyronine and estradiol in breast cancer cell lines

Abstract

It is known that estrogen (E2) and hormonal status of the patient are important for the proliferation and treatment of breast cancer (CaM). About thyroid hormone (T3), although epidemiological studies are still contradictory in relation to its influence on breast cancer, laboratory studies demonstrate its ability to increase the proliferation of cells with CaM E2 receptor (ER) positive, inducing expression genes normally stimulated by E2 (PR, TGFA). Although T3 exert many actions by the classical genomic regulation of gene transcription, a number of T3 effects occurs rapidly and are not affected by inhibitors of protein synthesis. Non-genomic actions of thyroid hormones are described in the plasma membrane, cytoplasm and cell organelles. In vitro, independent of protein synthesis, thyroxine (T4) induces inositol triphosphate (IP3) and calcium signaling by enhancing the effects of interferon-g via PKC and PKA. Recently our group has demonstrated that genes Amphiregulin, FBLN1, CLDN6, were stimulated by TGFA T3 and E2 in MCF7 cells, while HIF1A gene expression was augmented by T3 through non-genomic way merge. Therefore, we hypothesized that thyroid hormone alters the expression of genes AREG, FBLN1, CLDN6, TGFA e HIF1A without translocation to the nucleus, or via the activation of phosphatidylinositol 3-kinase (PI3K). Therefore, we intend to elucidate the mechanism of action of thyroid hormones in gene activation of these targets in cell lines of breast adenocarcinoma. Methods: Cell lines Breast Cancer MCF-7 and MDA are plated at intervals of 10 minutes, 30 minutes, 1 hour and 4 hours with E2 (10-7M), T3 (10-8M) E2 (10 - 7M) + ICI (1¼M), T3 (10-8M) + ICI (1¼M), ICI (1¼M). All treatments are performed in triplicate employees. In a second group, MCF-7 and MDA-MB-231 will pass through the same treatments in the presence of Actinomycin D (5.0 pg / ml) inhibitor of mRNA synthesis, to verify the expression of AREG, FBLN1, CLDN6, TGFA and HIF1A are dependent gene expression prior. In the third group these cells are subjected to treatment with cicloexamida (50mM) inhibitor of protein expression, to determine whether the expression of these genes is dependent on another protein expressed previously. The treatments in which change occurs in gene expression, compared to control, will be repeated association with LY294002 (50¼M), which blocks the PI3K pathway and shows whether the action is dependent on this pathway. It will be performed for total RNA extraction to obtain cDNA - Reverse transcription (RT) of RNA and real time PCR for expression of genes in every treatment. (AU)