RESUMO
Phytoestrogens are a group of plant-derived compounds with an estrogen-like activity. In mammalians, phytoestrogens bind to the estrogen receptor (ER) and participate in the regulation of cell growth and gene transcription. There are several reports of the cytotoxic effects of phytoestrogens in different cancer cell lines. The aim of this study was to measure the phytoestrogen activity against breast cancer cells with different levels of ER expression and to elucidate the molecular pathways regulated by the leader compound. Methods used in the study include immunoblotting, transfection with a luciferase reporter vector, and a MTT test. We demonstrated the absence of a significant difference between ER+ and ER- breast cancer cell lines in their response to cytotoxic stimuli: treatment with high doses of phytoestrogens (apigenin, genistein, quercetin, naringenin) had the same efficiency in ER-positive and ER-negative cells. Incubation of breast cancer cells with apigenin revealed the highest cytotoxicity of this compound; on the contrary, naringenin treatment resulted in a low cytotoxic activity. It was shown that high doses of apigenin (50 µM) do not display estrogen-like activity and can suppress ER activation by 17ß-estradiol. Cultivation of HER2-positive breast cancer SKBR3 cells in the presence of apigenin resulted in a decrease in HER2/neu expression, accompanied by cleavage of an apoptosis substrate PARP. Therefore, the cytotoxic effects of phytoestrogens are not associated with the steroid receptors of breast cancer cells. Apigenin was found to be the most effective phytoestrogen that strongly inhibits the growth of breast cancer cells, including HER2-positive ones.
RESUMO
The main goal of this work was to study the intracellular signaling pathways responsible for the development of hormone resistance and maintaining the autonomous growth of breast cancer cells. In particular, the role of PAK1 (p21-activated kinase 1), the key mitogenic signaling protein, in the development of cell resistance to estrogens was analyzed. In vitro studies were performed on cultured breast cancer cell lines: estrogen-dependent estrogen receptor (ER)-positive MCF-7 cells and estrogen-resistant ER-negative HBL-100 cells. We found that the resistant HBL-100 cells were characterized by a higher level of PAK1 and demonstrated PAK1 involvement in the maintaining of estrogen-independent cell growth. We have also shown PAK1 ability to up-regulate Snail1, one of the epithelial-mesenchymal transition proteins, and obtained experimental evidence for Snail1 importance in the regulation of cell proliferation. In general, the results obtained in this study demonstrate involvement of PAK1 and Snail1 in the formation of estrogen-independent phenotype of breast cancer cells showing the potential role of both proteins as markers of hormone resistance of breast tumors.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/genética , Quinases Ativadas por p21/genética , Antineoplásicos Hormonais/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Estradiol/farmacologia , Feminino , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail , Tamoxifeno/farmacologia , Fatores de Transcrição/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
The loss of hormonal dependency in breast tumor cells is often accompanied by epithelial-mesenchymal transition (EMT) features and an increase in cell metastasizing and invasiveness. Here we studied the role of transcription factors Snail1--the central mediator of EMT, in the progression of hormonal resistance of breast cancer cells. The experiments were performed on the estrogen receptor(ER)-positive estrogen-dependent MCF-7 breast cancer cells, ER-positive estrogen-resistant MCF-7/LS subline generated through long-term cultivation of the parental cells in steroid-free medium, and ER-negative estrogen-resistant HBL-100 breast cancer cells. We found that decrease in the estrogen dependency of breast cancer cells is accompanied by an increase in Snail1 expression and activity, and demonstrated the Snail1 involvement in the negative regulation of ER. NF-kappaB was found to serve as a positive regulator of Snail1 in breast cancer cells, and simultaneous inhibition of NF-kappaB and Snail1 by RNA interference resulted in marked increase of cell response to antiestrogen tamoxifen. In general, the results obtained demonstrate that direct inhibition of NF-kappaB and Snail1 partially restores the estrogen receptor machinery, and show that Snail1 and NF-kappaB may serve as an important targets in the treatment of breast cancer.
