Impact du régime obésogène sur la migration des cellules immunitaires dans un modèle murin de carcinogenèse mammaire

Impact du régime obésogène sur la migration des cellules immunitaires dans un modèle murin de carcinogenèse mammaire. Journée NACRe/Partenariat 201

Impact of leptin on ROS production in human mammary epithelial cells is dependent of neoplasic

Presenting author: Adrien Rossary The Second International Congress of Translational Research in Human Nutrition is organised by the Research Centre in Human Nutrition (CRNH) of Auvergne, of which INRA is a member, in collaboration with NuGO, European Association of universities and research institutes in the field of nutrigenomicsNutritional status and hormonal factors, such as leptin, an adipokine highly regulated inobesity, induce cellular signaling pathways, some of which involving reactive oxygen species (ROS) asintracellular messenger. High levels of ROS contribute to oxidative stress, cellular damages andpathogenesis. That’s why ROS production associated to obesity could be a major risk factor ofmammary carcinogenesis.This study aimed to determine leptin effects on ROS production in 3 human epithelial mammary cellmodels which present different neoplasic status (healthy primary (HP) cells, MCF-7 and MDAMB-231). ROS production is measured by fluorescence in presence of two leptin concentrations (10ng/ml close to physiological values, 100 ng/ml as obesity level) with several probes at 2μM(Dichlorofluorescein (DCF) for total cellular ROS, Diaminofluorescein (DAF) for NO,Dihydrorhodamine (DHR) for mitochondrial ROS and Dihydroethidine (DHE) for cytosolic superoxydeanion (O2°-). Gene expression and catalytic activities of ROS production systems (NADPH oxidases,NO synthases, Dual oxidases) and of anti-oxidant enzymes (glutathione reductase, heme oxygenase,glutathione S-transferase) are performed.Whatever the cell model and the leptin concentration, a slight increase of total cellular ROSproduction is observed. This increase is independent of mitochondrial activity as DHR signalremained stable for HP cells (5.51 ± 0.40 RFU) and decreased for MCF7 and MDAMB-231 cells.Inversely, this ROS increase is dependent of cytosolic O2°- production as shown by DHE signalenhanced for HP cells (0.66 ± 0.01 to 0.81 ± 0.01 RFU), for MCF7 (0.79 ± 0.02 to 0.89 ± 0.03 RFU) andfor MDAMB-231 (0.82 ± 0.01 to 0.89 ± 0.02 RFU). Interestingly, this ROS production is dependent ofthe NADPH oxidase 5 (Nox5) expression and contributes to a different antioxidative response inregard to the neoplasic cell status. Leptin activates, only in HP cells, the antioxidative enzymesexpression and activities such as heme oxygenase or glutathione reductase.These data suggest that leptin could modulate the oxidative status of epithelial mammary cells indifferent ways according to the neoplasic cell status. Therefore leptin induces a similar ROSproduction for the 3 cell models whereas the anti oxidant cell response is not modified in MCF-7 andMA-MB-231 cells. This study highlights lower capacities of neoplasic cells to fight against oxidativestress

Immunomodulatory effects of Vitamin D: Focus In the gut

SESSION 1 - EPIDEMIOLOGY - NUTRITIONAL PREVENTION AND CANCER RISKIntroduction:Vitamin D deficiency is observed in more than 80% of the elderly in France and is correlated with the appearance of pathophysiological processes related to aging (cancers and inflammatory diseases). Owing to the expression of its receptor (VDR) in immuno-competent cells (immune cells and barrier cells), vitamin D has the capacity to modulate innate and adaptive immune responses. The richness of intestinal tissue in immuno-competent cells (GALT: Gut Associated Lymphoid Tissue) makes this organ as a preferred target of the anti-inflammatory effects of vitamin D. Several epidemiological studies have yet established a correlation between vitamin D deficiency and the risk of developing a Chronic Inflammatory Bowel Disease and colorectal cancer.Objectives:This study aims to assess immunomodulatory effects of vitamin D in in the ileum of depleted and supplemented young and old rats.Methods:After 2 weeks acclimatization, 6 male Wistar rats aged 2 months and 8 rats aged 18 months were divided into 3 groups: Young control group (n = 6): 1 IU vitD / g diet; Elderly control group (n = 4): 1 IU vitD / g diet;- Elderly supplemented group (n = 4): 10 IU vitD / g diet.The rats were sacrificed after 3 months of diet and ileum was removed. These tissues were disrupted and subjected to a transcriptomic analysis targeted on genes of the metabolism of vitamin D and immunity in order to characterize the immune and inflammatory profile of intestine.Results:Our data demonstrated an overexpression of CYP24A1 with aging. This hydroxylases is known to inactivate vitamin D. Supplementation of vitamin D reduced its expression. Nevertheless, there was not significant difference of VDR expression between young and elderly rats, which remained at the same level after supplementation. mRNA expression levels of cathelicidin, IL6 and NFkB (p65) significantly increased in the elderly group. Supplementation of vitamin D significantly decreased expression of these pro-inflammatory molecules.Conclusion:Aging is accompanied by a majored degradation of active form of vitamin D and an inflammation status at the intestinal level. This inflammation could contribute to a major risk of carcinogenesis. Supplementation of vitamin D is able to limit the pathophysiological effects associated with aging, such as the appearance of low grade inflammatory status. A better understanding of the regulatory mechanisms induced by vitamin D in gut will enable us to acquire new knowledge useful in the prospects of a re-evaluation of the vitamin D requirements of the elderly population or even the systematic administration of a complementatio

Mammary tumor growth limits spontaneous physical activity in high-fat diet fed mice housed in an enriched environment

Aim:Excess weight and several pathological diseases such as cancer alter the total energy expenditureincluding both the resting and the activity energy expenditure. Moreover, the regular physical activity is considered as a protective factor in numerous physio-pathological situations. This study was designed to assess whether physical and social environment enrichment induces differential effects on tumor growth, and to determine their relation to metabolic consequences.Methods: 33-week-old C57BL/6 mice were randomly assigned to a high fat diet (HFD: 4.3 kcal/g, 45% of lipids, n=10/group/2 groups) or a standard diet (SD, 3.4 kcal/g, 10% lipids, n=10/group/2 groups). Among each diet group, mice were housed in an enriched environment (EE) or in standard laboratory environment (SE). After, 4 weeks of diet, the syngeneic EO771 spontaneous mammary adenocarcinoma cell line (5x105 cells) was orthotopically transplanted into the fourth right mammary gland. Tumor was allowed to growth during 1 month. Body weight, body composition (indirect calorimetry) and tumor growth were measured throughout the experiment. Mice spontaneous activity was evaluated by the PhenoMaster/LabMaster system (TSE System, Bad Homburg, Germany) connected to the calorimetric cages. Total spontaneous activity corresponded to the sum of vertical (z) and horizontal positions (xy). The distance covered by the mice were recorded during 1 week. Cages were or not equipped with a nest fixed in height or a running wheel to mimic respectively the EE and SE conditions. Data are presented as mean ± SD, statistical analysis were made using the Mann Whitney test.Results:In SE housing, individual horizontal movements were of 22 ± 8 m/h and vertical moving were 240 ± 60 passing/h In the EE conditions, horizontal movements decreased to 11 ± 5 m/h/mouse whereas vertical passages increased to 556 ± 83 passages/h/mouse (p<0,05). This effect was related to the presence of the nest in height favoring and limiting z and yz movements. Expressed in % of the ES conditions, the total spontaneous activity was enhanced by 40% (p<0.05). HFD comparatively to the SD was associated to a 39% reduction in the spontaneous locomotor activity (p<0.05) and to an increase in fat mass (30 vs 11%, p<0.01). After tumor implantation, HFD induced a higher tumor growth (1114 ± 793 vs 384 ± 339 mm3 at day 16, p<0.05) and a less spontaneous physical activity. In this condition, EE restricted both the tumor growth (663 ± 192 mm3 vs 1114 ± 793, p<0.05) and the loss in physical activity (p<0.05). However, tumor bearing reduced significantly the distance covered during the nocturnal period independently of the diets and the housing conditions. Whatever the diet and the housing condition, the tumor reduced the spontaneous locomotor activity of the diurnal period. So tumor development affects nocturnal/diurnal locomotor cycle.Conclusion: Both diet-induced-overweight and tumor reduce the mice spontaneous locomotor activity. In this context of metabolic perturbations, the enrichment of the housing allow to maintain a locomotor activity which is associated to a health benefit. This mouse model will allow to explore over time the metabolic and molecular mechanisms associated to the physical activity and its impact in the mammary carcinogenesi

In vitro L-arginine deprivation impairs natural killer cell functions

National audienc

Effects of 6 weeks of betaine or C-phycocyanin supplementation associated or not with wheel running on redox statusEffet de six semaines de supplémentation en bétaïne ou C-phycocyanine associée ou non à une activité physique sur le statut redox

Purpose : The effect of 6 weeks of betaine (3.88 ± 0.49 g by kg of body weight per day) or C-phycocyanin (0.34 ± 0.0 g by kg of body weight per day) supplementation alone or in association with voluntary wheel running was tested on redox status.Methods : Thirty Sprague-Dawley rats were randomly assigned to 6 groups: control, wheel activity, betaine with and without wheel activity, C-phycocyanin with and without wheel activity. At the end of the treatment, gastrocnemius, plasma and serum were collected on sacrificed animals. The levels of antioxidant activities (superoxide dismutase, catalase, glutathione peroxidase, total glutathione) and the myokine irisin were evaluated. Furthermore, the oxidative stress was quantified through the thiobarbituric acid reaction, and inflammation through Cyclooxygenase-2.Results : Median running distance ranged from 4 to 6 km.day−1 for the entire duration of the study, whatever the group. Results showed no effect of wheel running on antioxidant markers and oxidative stress, but an increased inflammation. Elevated activity of antioxidant enzymes was observed in betaine- or C-phycocyanin–treated rats both in plasma and gastrocnemius; however the Thiobarbituric acid reactive substance levels remained stable. For the wheel-running-coupled-to-diet group, the reduction of serum lipid peroxidation was stronger than the reduction observed in the diet-alone group. In any of the 6 groups, there were no correlations between irisin concentrations and lipid peroxidation or antioxidant parameters.Conclusion : The diet supplementations alone appear to have stronger effects on redox balance than the exercise training alone. It could be interesting to evaluate whether betaine or C-phycocyanin could modulate the oxidative stress and inflammation, which occurs during pathologies such as cancer.Objectifs : L’effet de six semaines d’une supplémentation en bétaïne (3.88 ± 0.49 g par kg de poids par jour) ou en C-phycocyanine (0.34 ± 0.0 g by par kg de poids par jour) seule ou associée à la pratique d’un exercice volontaire (roue d’activité) a été testé sur le statut redox.Méthodes : Trente rats Sprague-Dawley ont été assignés en 6 groupes : contrôle, roue d’activité, betaïne avec ou sans roue d’activité, C-phycocyanine avec ou sans roue d’activité. À la fin du traitement, le gastrocnémien, le plasma et le sérum ont été collectés sur les rats euthanasiés. L’activité des marqueurs antioxydants (superoxyde dismutase, catalase, glutathion peroxydase, glutathion total) et de la myokine nommée irisine ont été évalués ainsi que le niveau des TBARS (reflet de la peroxydation lipidique). La cyclooxygénase-2 a été utilisée comme marqueur de l’inflammation.Résultats : La distance moyenne parcourue était entre 4 et 6 km.jour−1 durant toute l’étude, quel que soit le groupe. Les résultats n’ont pas montré d’effet de l’activité physique sur les marqueurs antioxydants et du stress oxydatif. On constate par contre une augmentation de l’inflammation musculaire. Une élévation des concentrations des enzymes antioxydantes a été observée chez les rats traités à la bétaïne ou à la C-phycocyanine dans le plasma et dans le gastrocnémien, sans modification des TBARS. Pour les groupes où l’activité physique était associée à une supplémentation nutritionnelle, la diminution de la peroxydation lipidique était plus importante que la diminution observée dans les groupes uniquement supplémentés. Quel que soit le groupe, aucune corrélation entre les concentrations en irisine et la peroxydation lipidique ou les paramètres antioxydants n’a été observée.Conclusion : Les supplémentations nutritionnelles seules semblent avoir de plus importants effets sur la balance redox que l’exercice seul. Il serait intéressant de voir si la bétaïne ou la C-phycocyanine pourraient moduler l’activité oxydative et l’inflammation dans des conditions pathologiques telles que le cancer

Intérêt de l’activité physique spontanée en situation d’obésité sur la cancérogenèse mammaire : approche expérimentale chez la souris C57/bl6

Cette année les assises sont en partenariat avec le Pôle de compétitivité Lyonbiopôle et le cluster Nutravita, des symposiums thématiques portant sur les interactions entre la recherche académique, l’innovation et l’industrie seront proposésObjet: L’obésité est reconnue comme étant un facteur de risque du cancer du sein après la ménopause. Il est établi que les sécrétions adipokiniques modulent la capacité de prolifération des cellules épithéliales mammaires en culture. Par ailleurs, l’activité physique est un élément régulateur des sécrétions adipokiniques. Le but de cette étude est de caractériser in vivo l’impact de l’activité physique, décrite comme un élément protecteur, sur la sécrétion de leptine et le métabolisme énergétique de l’animal, lors de la croissance tumorale en situation d’obésité. Méthode: Des souris femelles C57/bl6 âgées (28 semaines) ovariectomisées et placées ou non en environnement enrichi, pour favoriser l’activité physique et les interactions sociales (n = 10), sont nourries pendant 12 semaines avec un régime hyper-lipidique (HL : 4,3 kcal/g, lipides 45% des AET). Après 8 semaines, les cellules tumorales mammaires syngèniques (lignée EO 771) sont implantées dans la quatrième paire de glande mammaire par la technique «fat pad». La prise alimentaire, la prise de poids, l’activité physique, la composition corporelle des animaux et la croissance tumorale sont mesurées tout au long de l’expérimentation. Enfin, un bilan métabolique et hormonal est réalisé sur le plasma au bout de 12 semaines après le sacrifice des animaux. Résultats : La prise énergétique journalière est de 12,8 ± 0,4 calories par jour et s’accompagne d’une prise de masse grasse significative (p<0,05) au bout de 8 semaines (10,2 ± 3,7 vs 2,7 ± 0,3 g), qui est limitée par l’activité physique (8,4 ± 2,9 g). Après implantation des cellules tumorales, l’environnement enrichi permet de limiter la perte d’activité physique des animaux qui est significativement plus élevée que dans l’environnement standard (p<0,05). Par ailleurs, la croissance tumorale est limitée en environnement enrichi conduisant à un volume tumoral de 663 ± 192 mm3 vs 1222 ± 482 mm3 (p<0,05) à 18 jours. Au sacrifice, les animaux hébergés en environnement enrichi montrent une moindre augmentation de la leptinémie, une normalisation de la glycémie, ainsi qu’une diminution de l’insulino-résistance objectivée par les concentrations d’insuline et de résistine (p<0,05). Enfin, il est noté une diminution de la concentration circulante en interleukine 6 (p<0,05). Conclusion: Un régime hyper-lipidique associé à l’ovariectomie favorise l’augmentation de masse grasse et la croissance tumorale. Dans ce contexte, l’augmentation d’activité physique due à l’environnement enrichi limite à la fois la prise de masse grasse et la croissance tumorale. Ces modifications s’accompagnent d’une diminution de l’inflammation à bas bruit associée à l’obésité. Ces données confirment le rôle bénéfique de l’activité physiqu

Impact de la carcinogenèse mammaire et du surpoids sur l’activité physique spontanée et le rythme circadien chez la souris C57BL/6 placée ou non en environnement enrichi

Impact de la carcinogenèse mammaire et du surpoids sur l’activité physique spontanée et le rythme circadien chez la souris C57BL/6 placée ou non en environnement enrichi. 43. Colloque AFSTAL 201

Impact of physical activity on tumour immunity in C57BL/6 mouse syngeneic model of mammary cancer

SESSION 4 - BREAST CANCER: FROM EXPERIMENTAL APPROACH TO CLINICALBackground: Obesity, an increasingly prevalent health problem, is a major risk factor for recurrence and morbidity of breast cancer in postmenopausal women. Moreover, obesity increases therapy resistance and tumour escaping. To limit the obesity adverse effects, one of the health recommendations is regular physical practice. To clarify the physical activity effects on obesity and breast cancer development, it is important to consider the physical activity impact not only on metabolism but also on tumour immunity balance between pro and anti-tumour immune response.Objectives: The purpose of this study was to examine the influence of spontaneous physical activity, promoted by an enriched environment (EE), on systemic biological markers and on tumour immune response in C57BL/6 mice (33 weeks old) ovariectomized and fed with a high fat (HFD). Systemic and tissue biological assays evaluated energetic substrates (i.e. glucose, cholesterol, triglycerides) and signalling molecules (i.e. chemokines, cytokines, adipokines). Flow cytometric analysis permitted to phenotype immunity balance in terms of pro tumour cells (myeloid derivate suppressive cells (MDSC), regulator T lymphocytes (Treg)) and anti-tumour cells (Natural Killer (NK), cytotoxic T lymphocytes (Tc)). Immune trafficking between lymphatic tissue and tumour completed this approach.Results: Ovariectomized old mice feeding with HFD presented higher body weight and adiposity (fat mass: +5 ± 0.5g, p<0.05) associated with higher plasma levels for leptin, estradiol and glycaemia. This fat phenotype was associated with a decrease in spontaneous physical activity (-22 ± 5%, p<0.01). At the opposite, housing in the enriched environment reduced adiposity, leptin, estradiol and glycaemic levels and increased spontaneous physical activity of the mice (p<0.05). After tumour implantation, a significant decrease of spontaneous physical activity was observed in all groups, but less important in the enriched environment(-56 ± 10% vs -39 ± 10%, p<0.05). In addition, tumour growth was enhanced by HFD and linked to a worse survey. Housing in the enriched environment reduced tumour growth and increased the survey (p<0.05). HFD-tumour immune phenotyping presented an imbalance between pro- and anti-tumour cells in favour of pro-tumour cells. A significant increase of MDSC and Treg appeared (24.7 and 21.5% of the immune infiltrate) in contrast to a decline of Tc content (5.3%). Immune trafficking from spleen to inguinal lymph nodes reflected these tumour immunity modifications EE changed tumour immune phenotype, resulting in an equilibrate immune response with a higher Tc infiltration (19.9%) and a lesser MDSC and Treg content (11.1 and 1.4%).Conclusion: These data confirmed that HFD imbalanced the anti-tumour immunity response and enhanced fat mass, leptinemia and tumour growth. Interestingly, they highlighted the beneficial effect of spontaneous physical activity to limit these carcinogenesis disorde

Leptin induces ROS via NOX5 in healthy and neoplastic mammary epithelial cells

NADPH oxidase (NOX) complexes (a family of seven isoforms) drive cellular ROS production in patho-logical processes such as cancer. NOX-driven ROS production is involved in cell mechanisms from signalling to oxidative stress. Leptin, an adipokine overexpressed in obese patients, has been investigated in studies on breast carcinogenesis, but its effects on oxidative stress remain largely unexplored, especially in breast cancer. The study used three human mammary epithelial cell models presenting different neoplastic status (healthy primary HMECs, neoplastic MCF-7 cells and neoplastic MDA-MB-231 cells) to determine the effects of leptin on short-term ROS production and to characterize the enzymes involved. All three cell models significantly expressed NADPH oxidase isoform 5 (NOX5) in our culture conditions. All models showed induced ROS production regardless of leptin concentration (10 ng/ml mimicking good health, 100 ng/ml mimicking obesity). Cell treatment with either siRNA against NOX5, NOX inhibitor DPI or a calcium channel blocker (verapamil) confirmed the putative involvement of the NOX5 isoenzyme in ROS production. Moreover, cell treatments suppressed ROS production under leptin at both concentrations. Neoplastic cells appeared unable to downregulate NOX5 mRNA expression under leptin. Leptin emerged as a potential activator of ROS production in human epithelial mammary cells, where the ROS production was apparently linked to NOX5 activation. This novel finding could shed light on the potential role of obesity-associated hyperleptinemia in mammary cells via the activation of NOX enzymes