Adrien Rossary

Effects of Enriched Environment on COX-2, Leptin and Eicosanoids in a Mouse Model of Breast Cancer

Cyclooxygenase-2 (COX-2) and adipokines have been implicated in breast cancer. This study investigated a possible link between COX-2 and adipokines in the development of mammary tumors. A model of environmental enrichment (EE), known to reduce tumor growth was used for a syngeneic murine model of mammary carcinoma. 3-week-old, female C57BL/6 mice were housed in standard environment (SE) or EE cages for 9 weeks and transplanted orthotopically with syngeneic EO771 adenocarcinoma cells into the right inguinal mammary fat pad. EE housing influenced mammary gland development with a decrease in COX-2 expressing cells and enhanced side-branching and advanced development of alveolar structures of the mammary gland. Tumor volume and weight were decreased in EE housed mice and were associated with a reduction in COX-2 and Ki67 levels, and an increase in caspase-3 levels. In tumors of SE mice, high COX-2 expression correlated with enhanced leptin detection. Non-tumor-bearing EE mice showed a significant increase in adiponectin levels but no change in those of leptin, F2-isoprostanes, PGF2α, IL-6, TNF-α, PAI-1, and MCP-1 levels. Both tumor-bearing groups (SE and EE housing) had increased resistin, IL-6, TNF-α, PAI-1 and MCP-1 levels irrespective of the different housing environment demonstrating higher inflammatory response due to the presence of the tumor. This study demonstrates that EE housing influenced normal mammary gland development and inhibited mammary tumor growth resulting in a marked decrease in intratumoral COX-2 activity and an increase in the plasma ratio of adiponectin/leptin levels.

Leptin, adipocytes and breast cancer: Focus on inflammation and anti-tumor immunity

More than one million new cases of breast cancer are diagnosed worldwide each year and more than 400,000 deaths are caused by the disease. The origin of this pathology is multifactorial and involved genetic, hormonal, environmental and nutritional factors including obesity in postmenopausal women. The role played by the adipose tissue and their secretions, ie adipokines, is beginning to be recognized. Plasma adipokine levels, which are modulated during obesity, could have “remote” effects on mammary carcinogenesis. Breast cancer cells are surrounded and locally influenced by an adipocyte microenvironment, which is probably more extensive in obese people. Hence, leptin appears to be strongly involved in mammary carcinogenesis and may contribute to the local pro-inflammatory mechanisms, especially in obese patients, who have increased metastatic potential and greater risk of mortality. This review presents the multifaceted role of leptin in breast cancer development and the different molecular pathways involved such as inflammation, oxidative stress and antitumor immunity.

Leptin modulates dose-dependently the metabolic and cytolytic activities of NK-92 cells.

Leptin, a hormone‐cytokine produced primarily in the adipose tissue, has pleiotropic effects on many biological systems and in several cell types, including immune cells. Hyperleptinemia is associated with immune dysfunction and carcinogenesis. Natural killer (NK) cells are critical mediators of anti‐tumor immunity, and leptin receptor deficiency in mice leads to impaired NK function. It was thus decided to explore the in vitro effects of leptin on human NK cell function. NK‐92 cells were cultured during 48 h with different leptin concentrations [absence, 10 (physiological), 100 (obesity), or 200 ng/ml (pharmacology)]. Their metabolic activity was assessed using the resazurin test. NK‐92 cell cytotoxicity and intracellular IFN‐γ production were analyzed by flow cytometry. NK‐92 cell mRNA and protein expression levels of cytotoxic effectors were determined by RT‐qPCR and Western blot. In our conditions, leptin exerted a dose‐dependent stimulatory effect on NK‐92 cell metabolic activity. In addition, high leptin concentrations enhanced NK‐92 cell cytotoxicity against K562‐EGFP and MDA‐MB‐231‐EGFP target cells and inversely reduced cytotoxicity against the MCF‐7‐EGFP target. At 100 ng/ml, leptin up‐regulated both NK cell granzyme B and TRAIL protein expressions and concomitantly down‐regulated perforin expression without affecting Fas‐L expression. In response to PMA/ionomycin stimulation, the proportion of IFN‐γ expressing NK‐92 cells increased with 100 and 200 ng/ml of leptin. In conclusion, leptin concentration, at obesity level, variably increased NK‐92 cell metabolic activity and modulated NK cell cytotoxicity according to the target cells. The underlying mechanisms are partly due to an up‐regulation of TRAIL and IFN‐γ expression and a down‐regulation of perforin. J. Cell. Physiol. 228: 1202–1209, 2013.

Altered functions of natural killer cells in response to L-Arginine availability

L-Arginine (L-Arg) availability is crucial in the regulation of immune response. Indeed, L-Arg deficiency induces T-cell dysfunction and could modulate the properties of natural killer (NK) cells involved in the early host defense against infections and tumors. We explored the impact of L-Arg depletion on NK cell functions using two models - an NK-92 cell line and isolated human blood NK cells. Below 5mg/L of L-Arg, NK-92 cell proliferation was decreased and a total L-Arg depletion reduced NK-92 cell viability. NK cell cytotoxicity was significantly inhibited in presence of low L-Arg concentration (2.5 mg/L). L-Arg depletion reduced the expression of NK-92 activating receptors, NKp46 and NKp30, the expression of NK ζ chain and the NK-92 intracellular production of IFN-γ. Whatever the L-Arg concentrations tested, no significant variation in the gene expression of transporters and enzymes involved in L-Arg metabolism was found. Thus, L-Arg availability modulates the phenotypic and functional properties of NK cells.

Dietary fat without body weight gain increases in vivo MCF‐7 human breast cancer cell growth and decreases natural killer cell cytotoxicity

High‐calorie (HC) diet contributes to the increased incidence of obesity, which is a risk factor for breast cancer in postmenopausal women, and in particular for estrogen receptor (ER) positive tumors. This study investigated whether an HC diet increases human ER‐positive breast cancer progression and modulates natural killer (NK) cell functions. Four‐week‐old female BALB/c athymic nude mice were fed a HC diet (5320 kcal/kg) or standard calorie diet (SC, 2820 kcal/kg) for 6 mo. After 5 mo, the mice were randomly implanted with MCF‐7 breast cancer cells (SCT and HCT) or received an isovolumic injection (SC and HC) in both inguinal fat pads. Tumor growth was greater in the HCT group than in the SC group without change in body weight. The HC diet decreased the tumor expression of genes involved in the citrate cycle and in adiponectin and lipid metabolism but increased that of genes controlling glycolysis and angiogenesis. The tumor expression level of Ki67 was increased while that of the cleaved caspase 3 and the ER‐β and progesterone receptors was reduced. Tumor development in response to the HC diet was associated with smaller numbers and lower cytotoxicity of splenic NK cells. These results indicate that an HC diet without body weight gain increases ER‐positive breast cancer cell proliferation and reduces tumor apoptosis. The underlying mechanisms might involve a downexpression of tumor hormonal receptor and reduced NK cell functions, and might also result in the regulation of genes involved in several cellular functions.

Does Consumption of Two Portions of Salmon Per Week Enhance the Antioxidant Defense System in Pregnant Women

Salmon is a rich source of marine n-3 fatty acids, which may increase oxidative stress and, in turn, could affect the antioxidant defense system in blood plasma and erythrocytes of pregnant women. The Salmon in Pregnancy Study provided two meals of salmon per week to pregnant women from week 20 of gestation; the control group maintained their habitual diet low in oily fish. Higher selenium and retinol plasma concentrations were observed after dietary salmon supplementation. Besides, a concomitant increase in selenium and glutathione concentration as well as glutathione peroxidase and reductase activities were detected as pregnancy progressed. However, tocopherols, retinol, β-carotene, and coenzyme Q(10) decreased in late pregnancy. Collectively, our findings lead to the hypothesis that increased farmed salmon intake may increase antioxidant defenses during pregnancy. Clinical trials identifier NCT00801502.

Increased consumption of salmon during pregnancy partly prevents the decline of some plasma essential amino acid concentrations in pregnant women

BACKGROUND & AIMS Oily fish is a good source of n-3 long-chain polyunsaturated fatty acids. Since these fatty acids may change efficiency of amino acid (AA) absorption, we determined whether increased salmon consumption influences plasma AA concentrations in pregnant women and their newborns. METHODS Pregnant women were randomly allocated to remain on their habitual diet (n = 61; control group) or to consume two 150 g farmed salmon portions per week from 20 weeks pregnancy until birth (n = 62; salmon group). Plasma AA concentrations were determined in women at w20, w34 and w38 of pregnancy and in umbilical cord at delivery. RESULTS Concentrations of arginine, valine, leucine and lysine were affected by both time of pregnancy and salmon intake (p < 0.05), with a smaller gestation-associated decrease in the salmon group. Total essential AA concentrations were similar in both groups at w20, but at w38 were higher in salmon group (p < 0.05). Cord plasma AA concentrations, higher than in maternal plasma (p < 0.01), were similar in the two groups (p > 0.05). CONCLUSIONS Two portions/wk of oily fish increased plasma essential AA concentrations during pregnancy and could contribute to a maternal health benefit. Two portions/wk of salmon did not affect plasma AA concentrations in the newborn. CLINICAL TRIALS IDENTIFIER NCT00801502.

Inflammatory F2-isoprostane, prostaglandin F2α, pentraxin 3 levels and breast cancer risk: The Swedish Mammography Cohort

INTRODUCTION Breast cancer is a common cancer among women. Identifying cellular participation of F2-isoprostane, prostaglandin F2α (PGF2α) and pentraxin 3 (PTX3) in cancer we evaluated whether their prediagnostic systemic levels that originate from different inflammatory pathways were associated with breast cancer risk. METHODS Seventy-eight breast cancer cases diagnosed after blood collection and 797 controls from the Swedish Mammography Cohort were analysed for urinary F2-isoprostane, PGF2α and plasma PTX3 levels. RESULTS None of the biomarkers investigated were significantly associated with breast cancer risk. However, there was the suggestion of an inverse association with PTX3 with multivariable adjusted ORs (95% CI) of 0.56 (95% CI=0.29-1.06) and 0.67 (95% CI=0.35-1.28) for the second and third tertiles, respectively (ptrend=0.20). No associations were observed between F2-isoprostane (OR=0.87; 95% CI=0.48-1.57; ptrend=0.67) and PGF2α metabolite (OR=1.03; 95% CI=0.56-1.88; ptrend=0.91) comparing the top to bottom tertiles. CONCLUSIONS The systemic levels of F2-isoprostane, PGF2α and PTX3 witnessed in women who later developed breast cancer may not provide prognostic information regarding tumor development in spite of their known involvement in situ cellular context. These observations may indicate that other mechanisms exist in controlling cellular formation of F2-isoprostane, PGF2α and PTX3 and their systemic availability in breast cancer patients.

Adipocyte/breast cancer cell crosstalk in obesity interferes with the anti-proliferative efficacy of tamoxifen

Background Obesity is a well-known risk factor of breast cancer in post-menopausal women that also correlates with a diminished therapeutic response. The influence of adipocytes and their secretome, i.e. adipokines, on the efficacy of hormone therapy has yet to be elucidated. Methods We investigated, ex vivo, whether mature adipocytes, differentiated from adipose stem cells of normal-weight (MA20) or obese (MA30) women, and their secretions, were able to counteract the effects of tamoxifen (Tx) which is known to decrease neoplastic cell proliferation. Results In a tridimensional model and in a model of co-culture, the anti-proliferative effect of Tx on MCF-7 cancer cells was counteracted by MA30. These two models highlighted two different specific gene expression profiles for genes encoding cytokines or involved in angiogenesis based on the adipocyte microenvironment and the treatment. Thus it notably showed altered expression of genes such as TNFα that correlated with IL-6. In addition, leptin, IL-6 and TNFα, at concentrations reflecting plasma concentrations in obese patients, decreased the anti-proliferative efficacy of 4-hydroxytamoxifen (a major active metabolite of Tx). Conclusions These findings bring insights on adipocytes and mammary cancer cell interactions in Tx therapy, particularly in overweight/obese people. Indeed, patient’ adipokine status would give valuable information for developing individual strategies and avoid resistance to treatment.

NMR metabolomic signatures reveal predictive plasma metabolites associated with long-term risk of developing breast cancer

Background Combination of metabolomics and epidemiological approaches opens new perspectives for ground-breaking discoveries. The aim of the present study was to investigate for the first time whether plasma untargeted metabolomic profiles, established from a simple blood draw from healthy women, could contribute to predict the risk of developing breast cancer within the following decade and to better understand the aetiology of this complex disease. Methods A prospective nested case-control study was set up in the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) cohort, including 206 breast cancer cases diagnosed during a 13-year follow-up and 396 matched controls. Untargeted nuclear magnetic resonance (NMR) metabolomic profiles were established from baseline plasma samples. Multivariable conditional logistic regression models were computed for each individual NMR variable and for combinations of variables derived by principal component analysis. Results Several metabolomic variables from 1D NMR spectroscopy were associated with breast cancer risk. Women characterized by higher fasting plasma levels of valine, lysine, arginine, glutamine, creatine, creatinine and glucose, and lower plasma levels of lipoproteins, lipids, glycoproteins, acetone, glycerol-derived compounds and unsaturated lipids had a higher risk of developing breast cancer. P-values ranged from 0.00007 [odds ratio (OR)T3vsT1=0.37 (0.23-0.61) for glycerol-derived compounds] to 0.04 [ORT3vsT1=1.61 (1.02-2.55) for glutamine]. Conclusion This study highlighted associations between baseline NMR plasma metabolomic signatures and long-term breast cancer risk. These results provide interesting insights to better understand complex mechanisms involved in breast carcinogenesis and evoke plasma metabolic disorders favourable for carcinogenesis initiation. This study may contribute to develop screening strategies for the identification of at-risk women for breast cancer well before symptoms appear.