Molecular mechanisms of oestrogen action on growth of human breast cancer cells in culture

Growth responses to oestrogen can be reproducibly obtained using a selection of oestrogen-receptor-containing human breast cancer cell lines, and molecular mechanisms have been shown to include modulation to growth factor/receptor/signalling pathways, cell-cycle proteins, apoptosis, differentiation, adhesion, motility and migration. Considerable progress has been made in understanding the molecular basis of oestrogen action on gene expression through the ligand-activated transcription factors human oestrogen receptor α (ERα) and ERβ and the resulting effects on global gene expression patterns, but the full profile of coordination of the alterations, which brings about changes in cell growth through genomic and non-genomic mechanisms remain to be fully elucidated. Oestrogen regulation of cell growth involves a complex cross-talk between oestrogen receptor and growth factor signalling pathways such that inhibition of one pathway may lead to stimulation of another, which may explain the remarkable ability of human breast cancer cells to escape from any mode of imposed growth inhibition be it oestrogen deprivation or administration of antioestrogen. Although studies on cell growth have focused to date on the effects of physiological oestrogens, many hundreds of environmental chemicals with oestrogenic properties have now been measured in the human breast. Whether or not the weight of evidence eventually establishes any causal link of complex mixtures of environmental oestrogenic chemicals with breast cancer, the presence of so many oestrogenic chemicals in the breast must influence resulting oestrogenic responses, and the impact of this additional oestrogenic burden needs to be taken into account in future studies on growth regulation of human breast cancer cells.

Collagen or collagen-related peptide cause (Ca2+)i elevation and increased tyrosine phosphorylation in human megakaryocytes.

Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in megakaryocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.

The effect of proteolysis on the induction of cell death by monomeric alpha-lactalbumin.

α-Lactalbumin (α-la) is a major whey protein found in milk. Previous data suggested that α-la has antiproliferative effects in human adenocarcinoma cell lines such as Caco-2 and HT-29. However, the cell death inducing α-la was not a naturally occurring monomer but either a multimeric variant or an α-la:oleic acid complex (HAMLET/BAMLET). Proteolysis showed that both human and bovine α-la are susceptible to digestion. ELISA assays assessing cell death with the native undigested α-la fractions showed that undigested protein fractions did have a significant cell death effect on CaCo-2 cells. Bovine α-la was also more effective than human α-la. A reduction in activity corresponded with lower concentrations of the protein and partial digestion and fragmentation of the protein using trypsin and pepsin. This suggests that the tertiary structure is vital for the apoptotic effect.

Exposure to parabens at the concentration of maximal proliferative response increases migratory and invasive activity of human breast cancer cells in vitro

Alkyl esters of p–hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen-responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. Since proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen-responsive human breast cancer cell lines (MCF-7, T-47-D, ZR-75-1). Cells were maintained short-term (1 week) or long-term (20±2 weeks) in phenol-red-free medium containing 5% charcoal-stripped serum with no addition, 10-8M 17-oestradiol, 1-5x10-4M methylparaben, 10-5M n-propylparaben or 10-5M n-butylparaben. Long-term exposure (20±2 weeks) of MCF-7 cells to methylparaben, n-propylparaben or n-butylparaben increased migration as measured using a scratch assay, time-lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E-cadherin and -catenin in the long-term paraben-exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long-term paraben-exposed T-47-D and ZR-75-1 cells using a scratch assay and time-lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells.