Long-Term High-Fat Diet Limits the Protective Effect of Spontaneous Physical Activity on Mammary Carcinogenesis.

Breast cancer is influenced by factors such as diet, a sedentary lifestyle, obesity, and postmenopausal status, which are all linked to prolonged hormonal and inflammatory exposure. Physical activity offers protection against breast cancer by modulating hormones, immune responses, and oxidative defenses. This study aimed to assess how a prolonged high-fat diet (HFD) affects the effectiveness of physical activity in preventing and managing mammary tumorigenesis. Ovariectomised C57BL/6 mice were provided with an enriched environment to induce spontaneous physical activity while being fed HFD. After 44 days (short-term, ST HFD) or 88 days (long-term, LT HFD), syngenic EO771 cells were implanted into mammary glands, and tumour growth was monitored until sacrifice. Despite similar physical activity and food intake, the LT HFD group exhibited higher visceral adipose tissue mass and reduced skeletal muscle mass. In the tumour microenvironment, the LT HFD group showed decreased NK cells and TCD8+ cells, with a trend toward increased T regulatory cells, leading to a collapse of the T8/Treg ratio. Additionally, the LT HFD group displayed decreased tumour triglyceride content and altered enzyme activities indicative of oxidative stress. Prolonged exposure to HFD was associated with tumour growth despite elevated physical activity, promoting a tolerogenic tumour microenvironment. Future studies should explore inter-organ exchanges between tumour and tissues.

Vitamin D supplementation associated with physical exercise promotes a tolerogenic immune environment without effect on mammary tumour growth in C57BL/6 mice.

PURPOSE: High plasma vitamin D (VitD) level and regular exercise (Ex) are known to have anti-cancer and immunomodulatory effects. This study aimed to evaluate the impact of VitD supplementation and imposed physical Ex on mammary tumour growth and immune response in ovariectomised mice fed high-fat (HF) diet. METHODS: Ovariectomised 33-week-old mice C57BL/6 (n = 60), housed in enriched environment (EE), were fed HF diet (450 kcal/100 g) supplemented or not with VitD (HF/HF + D: 125/1225 IU/100 g) for 12 weeks and submitted or not to Ex (HF + Ex; HF + D + Ex) on treadmill (45 min/day, 5 days/week). At w8, syngeneic tumour cells EO771 were orthotopically injected into the 4th mammary gland. Spontaneous activity (SPA), maximal speed (MS) and forelimb grip strength (GS) were measured. Tumour immune cells infiltrate was phenotyped by FACS. Data (mean ± SEM) were analysed by two-way ANOVA + Tukey post-test. RESULTS: Ex (p = 0.01) and VitD (p = 0.05) reduced body weight gain. Exercise decreased visceral fat mass [g: 1.5 ± 0.8 (HF); 1.2 ± 0.65 (HF + Ex); 0.9 ± 0.6 (HF + D + Ex); p = 0.03]. SPA (p < 0.0001) and GS (p = 0.01) were higher in HF + D + Ex mice vs others. No effect of Ex or VitD on tumour growth was detected. In tumour, VitD decreased the proportion of NK (p = 0.03), while Ex increased it (p = 0.03). The Th1/Th2 ratio is lowered by VitD (p = 0.05), while Tc/Treg ratio was not affected either by Exercise or VitD. CONCLUSION: In our experimental conditions, VitD supplementation and physical exercise have synergetic effects reducing the weight gain under HF diet and improving the physical capacities of mice. VitD coupled with exercise induces an immunosuppressive response without effect on tumour growth.

EO771, the first luminal B mammary cancer cell line from C57BL/6 mice.

BACKGROUND: Despite decades of therapeutic trials, effective diagnosis, many drugs available and numerous studies on breast cancer, it remains the deadliest cancer in women. In order to choose the most appropriate treatment and to understand the prognosis of the patients, breast cancer is divided into different subtypes using a molecular classification. Just as there remains a need to discover new effective therapies, models to test them are also required. METHODS: The EO771 (also named E0771 or EO 771) murine mammary cancer cell line was originally isolated from a spontaneous tumour in C57BL/6 mouse. Although frequently used, this cell line remains poorly characterized. Therefore, the EO771 phenotype was investigated. The phenotype was compared to that of MCF-7 cells, known to be of luminal A subtype and to express estrogen receptors, as well as MDA-MB-231 cells, which are triple negative. Their sensitivity to hormonal treatment was evaluated by viability tests. RESULTS: The EO771 were estrogen receptor α negative, estrogen receptor ß positive, progesterone receptor positive and ErbB2 positive. This phenotype was associated with a sensitivity to anti-estrogen treatments such as tamoxifen, 4-hydroxy-tamoxifen, endoxifen and fulvestrant. CONCLUSIONS: On account of the numerous results published with the EO771 cell line, it is important to know its classification, to facilitate comparisons with corresponding types of tumours in patients. Transcriptomic and protein analysis of the EO771 cell line classified it within the luminal B subtype. Luminal B cancers correspond to one of the subtypes most frequently encountered in patients and associated with a poor prognosis.

Activation of antioxidant defences of human mammary epithelial cells under leptin depend on neoplastic state.

BACKGROUND: Obesity is associated with oxidative stress, a major factor in carcinogenesis, and with high leptin concentration. The aim of this study was to determine the effects of leptin on the antioxidant response in three human mammary epithelial cells each presenting a different neoplastic status: healthy human mammary epithelial cells (HMEC), oestrogen-receptor positive MCF-7 cells and triple-negative MDA-MB-231 cells. METHODS: This in vitro kinetic study characterized the cell antioxidant response after 1, 6 and 24 h in the presence of leptin (10 or 100 ng/ml).The antioxidant response was defined in terms of cell glutathione content, gene expression and catalytic activity of antioxidant enzymes (i.e. glutathione peroxidase 1 (Gpx1), glutathione reductase (GR), glutathione S transferase (GST), heme-oxygenase 1 (HO-1) and cyclooxygenase-2 (COX-2)). Oxidative stress occurrence was assessed by lipid hydro peroxide (HPLIP) and isoprostane concentrations in culture media at 24 h. RESULTS: At both concentrations used, leptin induced ROS production in all cell models, contributing to various antioxidant responses linked to neoplastic cell status. HMEC developed a highly inducible antioxidant response based on antioxidant enzyme activation and an increase in cell GSH content at 10 ng/ml of leptin. However, at 100 ng/ml of leptin, activation of antioxidant response was lower. Conversely, in tumour cells, MCF-7 and MDA-MB-231, leptin did not induce an efficient antioxidant response, at either concentration, resulting in an increase of lipid peroxidation products. CONCLUSIONS: Leptin can modulate the oxidative status of mammary epithelial cells differently according to their neoplastic state. These novel results shed light on oxidative status changes in mammary cells in the presence of leptin.

Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in Murine Primary Splenocytes.

Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation, and survival. Leptin receptor is integrated inside cholesterol-rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl-ß-cyclodextrin (ß-MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/ml). Unlike ß-MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. J. Cell. Physiol. 232: 101-109, 2017. © 2016 Wiley Periodicals, Inc.

Does aging affect the immune status? A comparative analysis in 300 healthy volunteers from France, Austria and Spain.

BACKGROUND: As the European population is getting older, there is growing need in scientific data on how to achieve healthy and successful aging. A decline in immune function with age is unanimously supported by many epidemiological and clinical observations, with a decrease in T-cell mediated function encompassing a large part of this alteration. In the EU-funded VITAGE project, the effects of aging on biomarkers of immune status are being studied in three European countries. According to strict inclusion/exclusion criteria, a cohort of 300 healthy male non-smoking 20-75 years old volunteers were enrolled in France (n = 99), Spain (n = 100) and Austria (n = 101). In each country, the volunteers were classified as a function of age (one age group per decade). Biomarkers of immune status were determined including delayed-type hypersensitivity tests, measurement of lymphocyte surface markers, and serum determinations of interleukin-2, complement fractions and immunoglobulins. RESULTS: There were moderate differences in the biomarkers of immune status of the VITAGE study volunteers among the three European centres. The percentage of Natural Killer (NK) cells was 156% and 142% higher in Spain as compared to France and Austria, respectively (p < 0.0001), and this increase was observed at any age group above 30 years. Comparison between age-groups showed that in Spain, but not in France or Austria, older individuals had significantly a lower B lymphocyte distribution and conversely, a higher NK cell distribution. Moreover, the CD4/CD8 ratio was positively correlated with age in Austrian subjects (p < 0.0001). CONCLUSION: Our results provide evidence of an increased NK cell distribution in the elderly, especially in the Spanish population. NK cell status may predict morbidity and mortality in the elderly, emphasizing the importance of innate as well as adaptive immunity in ensuring healthy longevity and cancer resistance, possibly in link with the Mediterranean diet.

25-Hydroxyvitamin D potentializes extracellular cathelicidin release from human PBMC stimulated ex vivo with either bacterial (LPS) or viral (P: IC) mimetics.

Human cathelicidin refers to the cationic antimicrobial peptide hCAP18/LL-37. LL-37 is formed by cleavage of the propeptide hCAP18 coded by the CAMP gene. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)D), has been shown to induce the CAMP gene expression through promoter activation. We previously failed to demonstrate in a clinical trial that supplementation of 25-hydroxyvitamin D (25(OH)D) improves LL-37 serum levels. The aim of this work was to evaluate the impact of 25(OH)D supplementation on intracellular expression of CAMP and secretion of LL-37 in an ex vivo model using the peripheral blood mononuclear cells (PBMC). PBMC collected from healthy donors and incubated with different concentrations of 25(OH)D (0 ng/ml: control (D0); 25 ng/ml: deficient (D25); 75 ng/ml: physiological (D75); 125 ng/ml: supraphysiological (D125)) were stimulated or not with lipopolysaccharide (LPS, 100 ng/ml) or synthetic double-stranded RNA Poly (I: C) (PIC, 10 µg/ml). The intracellular expressions of the CAMP gene and the hCAP18 peptide were measured respectively after 24-h and 48-h incubation periods. The concentration of LL-37 was determined in the culture medium after 48-h incubation. 25(OH)D significantly induced CAMP gene expression at 24 h with a maximum effect at a dose of D125 in either unstimulated (tenfold expression) or stimulated (LPS: 100-fold expression; PIC: 15-fold expression) conditions. Intracellular hCAP18 peptide was overexpressed at 48 h under unstimulated (1.5-fold, D125) and stimulated conditions, LPS (twofold, D125) and PIC (2.5-fold, D125). The secretion of LL-37 in the culture medium was significantly induced by 25(OH)D only in both stimulated (LPS and PIC) conditions in a dose-dependent manner. Our results demonstrate that 25(OH)D incubation increases intracellular expression of CAMP and hCAP18, but extracellular secretion of LL-37 antimicrobial peptide is increased by 25(OH)D only when PBMC from healthy donors were stimulated with bacterial or viral immune mimetic.

Impact of Vitamin D Supplementation on Influenza Vaccine Response and Immune Functions in Deficient Elderly Persons: A Randomized Placebo-Controlled Trial.

Background: Immunosenescence contributes to reduced vaccine response in elderly persons, and is worsened by deficiencies in nutrients such as Vitamin (Vit-D). The immune system is a well-known target of Vit-D, which can both potentiate the innate immune response and inhibit the adaptive system, and so modulate vaccination response. Objective: This randomized placebo-controlled double-blind trial investigated whether Vit-D supplementation in deficient elderly persons could improve influenza seroprotection and immune response. Design: Deficient volunteers (Vit-D serum <30 ng/mL) were assigned (V1) to receive either 100,000 IU/15 days of cholecalciferol (D, n = 19), or a placebo (P, n = 19), over a 3 month period. Influenza vaccination was performed at the end of this period (V2), and the vaccine response was evaluated 28 days later (V3). At each visit, serum cathelicidin, immune response to vaccination, plasma cytokines, lymphocyte phenotyping, and phagocyte ROS production were assessed. Results: Levels of serum 25-(OH)D increased after supplementation (D group, V1 vs. V2: 20.7 ± 5.7 vs. 44.3 ± 8.6 ng/mL, p < 0.001). No difference was observed for serum cathelicidin levels, antibody titers, and ROS production in D vs. P groups at V3. Lower plasma levels of TNFα (p = 0.040) and IL-6 (p = 0.046), and higher ones for TFGß (p = 0.0028) were observed at V3. The Th1/Th2 ratio was lower in the D group at V2 (D: 0.12 ± 0.05 vs. P: 0.18 ± 0.05, p = 0.039). Conclusions: Vit-D supplementation promotes a higher TGFß plasma level in response to influenza vaccination without improving antibody production. This supplementation seems to direct the lymphocyte polarization toward a tolerogenic immune response. A deeper characterization of metabolic and molecular pathways of these observations will aid in the understanding of Vit-D's effects on cell-mediated immunity in aging. This clinical trial was registered at clinicaltrials.gov as NCT01893385.

Phospho-proteomic approach to identify new targets of leucine deprivation in muscle cells.

The aim of this study was to optimize a protocol that allows identifying changes at the phosphorylation level of specific proteins in response to cell stimulation by leucine starvation. To make possible the identification of differentially phosphorylated proteins by the combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we prepared fraction enriched in phosphoproteins. For that purpose, we adapted the immobilized metal affinity chromatography (IMAC) technique to make it compatible with 2D-PAGE. On the whole, this procedure allowed identifying regulated targets of leucine deprivation: molecular chaperones glucose-regulated protein 58 kDa (GRP58) and BiP (GRP78), RNA helicase DEAD box polypeptide 3, and eukaryotic translation initiation factor 4B (eIF4B).

Food Chemicals Disrupt Human Gut Microbiota Activity And Impact Intestinal Homeostasis As Revealed By In Vitro Systems.

Growing evidence indicates that the human gut microbiota interacts with xenobiotics, including persistent organic pollutants and foodborne chemicals. The toxicological relevance of the gut microbiota-pollutant interplay is of great concern since chemicals may disrupt gut microbiota functions, with a potential impairment of host homeostasis. Herein we report within batch fermentation systems the impact of food contaminants (polycyclic aromatic hydrocarbons, polychlorobiphenyls, brominated flame retardants, dioxins, pesticides and heterocyclic amines) on the human gut microbiota by metatranscriptome and volatolome i.e. "volatile organic compounds" analyses. Inflammatory host cell response caused by microbial metabolites following the pollutants-gut microbiota interaction, was evaluated on intestinal epithelial TC7 cells. Changes in the volatolome pattern analyzed via solid-phase microextraction coupled to gas chromatography-mass spectrometry mainly resulted in an imbalance in sulfur, phenolic and ester compounds. An increase in microbial gene expression related to lipid metabolism processes as well as the plasma membrane, periplasmic space, protein kinase activity and receptor activity was observed following dioxin, brominated flame retardant and heterocyclic amine exposure. Conversely, all food contaminants tested induced a decreased in microbial transcript levels related to ribosome, translation and nucleic acid binding. Finally, we demonstrated that gut microbiota metabolites resulting from pollutant disturbances may promote the establishment of a pro-inflammatory state in the gut, as stated with the release of cytokine IL-8 by intestinal epithelial cells.

Serum from rats fed red or yellow tomatoes induces Connexin43 expression independently from lycopene in a prostate cancer cell line.

Epidemiologic studies suggested a protective effect of tomatoes against prostate cancer brought by lycopene, a carotenoid conferring the red colour of tomatoes. However, intervention studies on patients have shown that the preventive effect of tomato was more potent than that of lycopene. The aim of this study was to compare the effects of red tomato, yellow tomato (devoid of lycopene) and lycopene on Connexin43 (Cx43) expression, a protein regulating cell growth, on a prostate cancer cell line expressing the androgen receptor. Cells were incubated with serum from rats fed a control diet (CS) or control diet supplemented with red tomato (RTS), yellow tomato (YTS) or lycopene beadlets (LBS). After exposure of the cells to RTS or YTS for 48h, the expression of Cx43 was significantly increased compared to cells exposed to CS. Whereas LBS effect was not significantly different. The cells incubated with RTS and LBS had similar levels of lycopene, while those incubated with YTS contained no lycopene. These data first show that serum nutritionally enriched with red and yellow tomatoes could up-regulate Cx43 turn-over in PC3AR cells independently from lycopene level. Within the physiological approach used in the present study, it can be concluded that compounds other than lycopene contribute to the preventive effect of tomatoes.

Differential effects of lycopene consumed in tomato paste and lycopene in the form of a purified extract on target genes of cancer prostatic cells.

BACKGROUND: Prospective studies indicate that tomato consumers are protected against prostate cancer. Lycopene has been hypothesized to be responsible for tomato health benefits. OBJECTIVE: Our aim was to differentiate the effects of tomato matrix from those of lycopene by using lycopene-rich red tomatoes, lycopene-free yellow tomatoes, and purified lycopene. DESIGN: Thirty healthy men (aged 50-70 y old) were randomly assigned to 2 groups after a 2-wk washout period. In a crossover design, each group consumed yellow and red tomato paste (200 g/d, which provided 0 and 16 mg lycopene, respectively) as part of their regular diet for 1 wk separated by 2 wk of washout. Then, in a parallel design, the first group underwent supplementation with purified lycopene (16 mg/d) for 1 wk, whereas the second group received a placebo. Sera collected before and after the interventions were incubated with lymph node cancer prostate cells to measure the expression of 45 target genes. RESULTS: Circulating lycopene concentration increased only after consumption of red tomato paste and purified lycopene. Lipid profile, antioxidant status, prostate-specific antigen, and insulin-like growth factor I were not modified by consumption of tomato pastes and lycopene. We observed significant up-regulation of IGFBP-3 and Bax:Bcl-2 ratio and down-regulation of cyclin-D1, p53, and Nrf-2 after cell incubation with sera from men who consumed red tomato paste when compared with sera collected after the first washout period, with intermediate values for yellow tomato paste consumption. Cell incubation with sera from men who consumed purified lycopene led to significant up-regulation of IGFBP-3, c-fos, and uPAR compared with sera collected after placebo consumption. CONCLUSION: Dietary lycopene can affect gene expression whether or not it is included in its food matrix. This trial was registered by the French Health Ministry at http://www.sante-sports.gouv.fr as 2006-A00396-45.

Amino-acid limitation induces the GCN2 signaling pathway in myoblasts but not in myotubes.

There is a growing body of evidence that suggests that amino acids play an important role in controlling gene expression, but the cell specificity of the amino-acid-mediated regulation of gene expression in mammals remains unknown. Using a model of muscle cells (C2C12) at two stages of differentiation, i.e. myoblasts and myotubes, we employed transcriptional profiling to show that amino-acid deficiency does not regulate the same set of gene in differentiated and non-differentiated cells. Furthermore, in myotubes, the GCN2 pathway is not activated by amino-acid starvation due to an amino-acid supply from intracellular proteolysis associated with a low GCN2 expression.

Regulation of protein synthesis by leucine starvation involves distinct mechanisms in mouse C2C12 myoblasts and myotubes.

Leucine modulates protein translation in higher eukaryotes by affecting phosphorylation and the function of proteins that regulate the initiation and/or elongation steps. These include the initiation factor 4E binding protein 1 (4E-BP1), initiation factor 4E (eIF4E), initiation factor 2 (eIF2alpha), ribosomal S6 kinases (S6K1/2), and elongation factor 2 (eEF2). The alteration of protein translation by leucine starvation was studied during myogenic differentiation using the mouse C2C12 cell line as well as the role of rapamycin-sensitive mTOR (mammalian target of rapamycin) in the signaling of leucine in myotubes. A time course study showed that 1 h of leucine starvation decreased protein synthesis and S6K1 phosphorylation in myoblasts, whereas 3-5 h of starvation were necessary to induce such an alteration in myotubes. Although S6K1 phosphorylation was reduced in leucine-deprived myotubes, S6K2 and S6 phosphorylation were not affected. In contrast, rapamycin decreased the phosphorylation of S6K2 and S6 in myotubes. It is therefore likely that under the conditions present, the rapamycin-sensitive mTOR was not affected by leucine starvation. S6K1 dephosphorylation may thus be mTOR independent, and the functional mTOR/S6K2 pathway may maintain S6 phosphorylation. An increased phosphorylation of eEF2 in myoblasts and myotubes indicated that global protein synthesis was reduced via a decrease in translation elongation. An increased association between 4E-BP1 and eIF4E, and increased phosphorylation of eIF2alpha also contributed to decreasing protein synthesis in leucine-starved myoblasts. In contrast, in leucine-starved myotubes, there were no change in the 4E-BP1-eIF4E association or eIF2alpha phosphorylation, suggesting that these factors were not rate limiting for decreasing protein synthesis in leucine-deprived myotubes.