Molecular aspects of insulin resistance, cell signaling pathways and breast cancer in relation to obesity.

A growing number of clinical studies validate a relation of insulin resistance and breast cancer in obese patients. We hypothesised that high plasma insulin levels cause aberrant insulin signalling in breast epithelial cells which may be responsible for an increase in cell proliferation, indicative of potential carcinogenesis and increased cancer progression. It was of particular interest to determine any differences of high insulin concentrations in activating the phosphoinositide-3 kinase (PI-3 kinase) pathway or the mitogen-activated protein kinase (MAP kinase) pathway, the latter being linked to increased cell proliferation. We used two cell line models to investigate the carcinogenic (MCF-10A, immortalised breast epithelial cells) and cancer progression (MDA-MB-231, ER-negative breast cancer cells) potential of insulin. Insulin treatment (100 nM, 24 h) increased cell proliferation in MCF-10A cells, but had no cell proliferative effect on MDA-MB-231 cells. Additionally expression of PCNA as marker of proliferation was tested. The use of PI-3 kinase and MAP kinase specific inhibitors (Wortmannin and PD98059, respectively) demonstrated both pathways being responsible for the observed increase in cell proliferation (MCF-10A). Simultaneous treatment with both inhibitors eliminated insulin induced cell proliferation entirely. Phosphorylation of ERK1/2 was examined as specific activity measurement of MAP kinase pathway. Insulin induced higher phosphorylation levels in MCF-10A cells than in MDA-MB-231. These preliminary results suggest that insulin may initiate carcinogenesis of breast epithelial cells by increasing cell proliferation rather than increasing cancer progression of existing tumours. These effects may be mediated by insulin activating both the PI-3 kinase and the MAP kinase signalling pathways

Molecular aspects linking insulin resistance to breast cancer by activation of cell signalling pathways.

Recent findings suggest a connection between obesity and breast cancer. Obesity is linked with higher incidences of insulin resistance as part of the metabolic syndrome, resulting in chronically elevated insulin plasma levels. We examined the effect of high insulin concentrations (100 nM) on estrogen-receptor (ER) negative breast cancer cells (MDA-MB-231) and normal breast epithelial cells (MCF-10a). Treatment with high insulin concentrations increased insulin receptor (IR) phosphorylation significantly in both cell lines. Phosphorylation of protein kinase B (Akt), representative of PI3-kinase cell signalling pathway activation was increased by 101% (p=0.0112) in MDA-MB-231 cells and by 81% (p=0.0031) in MCF-10a cells after 10 min insulin treatment. Phosphorylation of extracellular regulated kinase 1/2 (ERK1/2), representative of MAP-kinase cell signalling pathway activation did not change in both cell lines after 10 min of insulin treatment. Cell proliferation did not change in MDA-MB-231 cells and increased by 75% (p=0.0067) in MCF-10a cells after 24 h insulin treatment. Cell proliferation was decreased in MDA-MB-231 cells by 15% (p=0.0083) after 1 h treatment with PD98059, a MAP-kinase inhibitor. In MCF-10a cells cell proliferation was decreased by 51% (

The role of selenium supplementation in cardiovascular disease prevention: an in vitro study to identify the molecular mechanism(s).

Obesity is a worldwide epidemic, with two thirds of the UK population either overweight or obese (body mass index (BMI) 25–29·9 and >30 kg/m2 respectively). Obesity is characterised by systemic oxidative stress (OS), which itself results from chronically high levels of reactive oxidative species (ROS) and reduced antioxidant status. OS is considered to play a key role in cardiovascular disease (CVD) development by initiating atherosclerosis. In particular, increased monocyte ROS generation instigates atherosclerotic plaque formation by increasing the recruitment, binding and transmigration of monocytes across arterial endothelial cells into the arterial wall. An increased intake of dietary antioxidants or up-regulation of endogenous antioxidant enzymes may counteract this OS state and therefore lower CVD risk. Selenium is an essential dietary micronutrient which is incorporated within the catalytic site of endogenous antioxidant Glutathione Peroxidase (GPx) enzymes and protects cells from OS and consequent cell damage. There is, however, a lack of knowledge concerning both the effect of selenium supplementation in an OS state representative of sedentary overweight/obese individuals and the mechanisms involved. The aim of this work was to investigate the effect of selenium supplementation in modulating, in vitro, monocyte cell viability, ROS generation and antioxidant enzymes gene expression (GPx1/GPx4) when under OS. U937 monocyte cells were either supplemented, or not with sodium selenite (Na2SeO3; 100nM or 200nM) and cultured for 48 hours at 37°C. Paraquat (1 mM) and S-Nitroso-N-acetyl-DL-penicillamine (0·7 mM) (SNAP) were added to cells overnight to induce OS. Cell viability was assessed via MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, ROS generation was determined by Flow Cytometry using CM-H2DC-FDA, while GPx1/GPx4 gene expression was quantified by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). PQ/SNAP treatment significantly reduced U937 cell viability and increased ROS generation compared to untreated control U937 cells; confirming the induction of OS. Supplementation with 100nM Na2SeO3, before PQ/SNAP treatment, significantly increased cell viability by 33% (p 0·05). Furthermore, 100nM Na2SeO3 supplementation significantly reduced ROS generation by 32% (p < 0·001) in cells treated with PQ/SNAP. Correspondingly, this increased GPx1 by 146% (p < 0·01) and GPx4 gene expression by 77% (p < 0·05), when compared to un-supplemented PQ/SNAP treated cells. This study indicates that selenium supplementation may be effective in counteracting the detrimental effects of OS by significantly increasing the expression of antioxidant genes; reinforcing endogenous antioxidant protection to quench ROS generation more effectively and improve cell viability. This preliminary data, in monocyte cells, indicates that GPx1 gene expression is affected considerably more by both selenium supplementation and OS when compared with GPx4. This suggests GPx1 may play a more important role in CVD prevention, with regards to selenium supplementation in overweight/obese individuals

Selenoprotein expression in the rat colon during Se deficiency.

Selenium is an essential trace element, which is present in several proteins, called seleneproteins, that have various biological roles

Insulin-induced gene expression changes in breast cancer cells and normal breast epithelial cells.

Obesity increases breast cancer incidence rates in postmenopausal women. Chronic high levels of insulin, present in the majority of obese and insulin resistant patients, may provide the growth promoting stimulus to explain this connection. In this work, the cancer progression and cancer initiating properties of high insulin levels were examined in breast cancer cells (MDA-MB-231) and breast epithelial cells (MCF-10a), respectively. High insulin levels (100 nM) induced differential changes in cell proliferation in the two cell lines used. Human Cancer PathwayFinder DNA Microarrays (SABiosciences) were used to examine gene expression changes after insulin treatment. High insulin levels increased expression of genes involved in cell cycle control (e.g. cyclin D1) and DNA damage repair (e.g. ATM) in MDA-MB 231 cells and in MCF-10a cells (e.g. cyclin E1, CDC25a). Expression of genes responsible for mediating apoptosis and cell senescence (e.g. APAF, BAD, bcl-X) was decreased after insulin treatment in MDA-MB 231 cells but the expression of the same group of genes did not change in MCF-10a cells. High insulin levels increased expression of genes encoding for signal transduction molecules (e.g. AKT1) and transcription factors (e.g. FOS, JUN, MYC), and of genes responsible for invasion and metastasis (e.g. MMP2) in MCF-10a cells whereas gene expression of the same groups of genes did not change or was decreased in MDA-MB 231 cells. These results suggest a role for insulin resistance in breast cancer initiation and progression, aggravating the potential of breast cancer to evade apoptosis, to metastasise and may promote carcinogenesis of healthy epithelial cells

Selenoprotein gene expression in an intestinal cell line during selenium depletion: a macroarray approach indicates effects on SelW and glutathione peroxidise 1.

The micronutrient selenium (Se) is incoprprated into a renage of selenoproteins involved in numerous biochemical processes within the body

Study into the polyphenol content and antioxidant activity of rapeseed pomace extracts.

Rapeseed pomace (RSP) is a waste product obtained after edible oil production from Brassica napus. This study aimed to determine the polyphenol content, radical scavenging activity, ferric iron reducing antioxidant power and the oxygen-radical absorbance capacity assay, of RSP extracts, with regard to their potential application in the treatment or prevention of neurodegenerative diseases. High performance liquid chromatography/mass spectrometry was applied to determine ubiquitous polyphenolic compounds. Three different extraction methods (Soxhlet-, ultra sonic assisted- and accelerated solvent extractions) were applied on 2 harvest years (2012, 2014). From the extracts obtained, the total phenolic content (Folin–Ciocalteu assay) was found to be between 5·54 (SD 0·28) and 2·48 (SD 0·06) gallic acid equivalents/100 mg dry weight. The ferric iron reducing antioxidant power (FRAP assay) is ranging from 0·834 (SD 0·01) to 0·34 (SD 0·01) Trolox equivalents/100μg. The IC50 values for the radical scavenging (DPPH) assay were found to be between 49·23 (SD 14·00) and 180·30 (SD 16·16) μg/mL. Liquid chromatography-mass spectrometry of the RSP extracts showed substantial presence of several phenolic compounds, the most abundant one being sinapic acid (7496·7 (SD198·9) − 1923·3 (SD18·4) mg/kg). Significant differences (2-way ANOVA, p < 0·0001) were found between the 3 different extraction techniques for all the tested antioxidant properties. The strong antioxidant properties demonstrated by the RSP extracts in this study warrants further investigation for their potential use in the treatment or prevention of oxidative stress related diseases

Açaí (Euterpe oleracea Martius) supplementation in the diet during gestation and lactation attenuates liver steatosis in dams and protects offspring.

Purpose: Maternal high-fat diet affects offspring and can induce metabolic disorders such as non-alcoholic fatty liver disease (NAFLD). New therapeutic strategies are being investigated as way to prevent or attenuate this condition. The objective of this study was to evaluate the effect of açaí supplementation in the maternal high-fat diet on dams and offspring lipid metabolism. Methods: Female Fisher rats were divided in four groups and fed a control diet (C), a high-fat diet (HF), an açaí supplemented diet (CA) and a high-fat diet supplemented with açaí (HFA) 2 weeks before mating, during gestation and lactation. The effects of açaí were evaluated in the male offspring after birth (P1) and weaning (P21). Results: HFA reduced relative liver weight, fat and cholesterol liver content in dams and improved liver steatosis as confirmed by histological analyses. HFA increased serum cholesterol and expression of Srebpf1 and Fasn genes. In offspring, HFA decreased relative liver weight, and serum cholesterol only in P21. An increase in the Sirt1, Srebpf1 and Fasn genes expression was observed in P21. Conclusions: These results suggest that açaí supplementation may attenuate NAFLD in dams and protect offspring from the detrimental effects of lipid excess from a maternal high-fat diet

Association between Polymorphisms in Glutathione Peroxidase and Selenoprotein P Genes, Glutathione Peroxidase Activity, HRT Use and Breast Cancer Risk.

Breast cancer (BC) is one of the most common cancers in women. Evidence suggests that genetic variation in antioxidant enzymes could influence BC risk, but to date the relationship between selenoproteins and BC risk remains unclear. In this report, a study population including 975 Danish cases and 975 controls matched for age and hormone replacement therapy (HRT) use was genotyped for five functional single nucleotide polymorphisms (SNPs) in SEPP1, GPX1, GPX4 and the antioxidant enzyme SOD2 genes. The influence of genetic polymorphisms on breast cancer risk was assessed using conditional logistic regression. Additionally pre-diagnosis erythrocyte GPx (eGPx) activity was measured in a sub-group of the population. A 60% reduction in risk of developing overall BC and ductal BC was observed in women who were homozygous Thr carriers for SEPP1 rs3877899. Additionally, Leu carriers for GPX1 Pro198Leu polymorphism (rs1050450) were at ∼2 fold increased risk of developing a non-ductal BC. Pre-diagnosis eGPx activity was found to depend on genotype for rs713041 (GPX4), rs3877899 (SEPP1), and rs1050450 (GPX1) and on HRT use. Moreover, depending on genotype and HRT use, eGPx activity was significantly lower in women who developed BC later in life compared with controls. Furthermore, GPx1 protein levels increased in human breast adenocarcinoma MCF7 cells exposed to β-estradiol and sodium selenite.In conclusion, our data provide evidence that SNPs in SEPP1 and GPX1 modulate risk of BC and that eGPx activity is modified by SNPs in SEPP1, GPX4 and GPX1 and by estrogens. Our data thus suggest a role of selenoproteins in BC development