Elucidating the role of the gut microbiota in the physiological effects of dietary fiber.

BACKGROUND: Dietary fiber is an integral part of a healthy diet, but questions remain about the mechanisms that underlie effects and the causal contributions of the gut microbiota. Here, we performed a 6-week exploratory trial in adults with excess weight (BMI: 25-35 kg/m2) to compare the effects of a high-dose (females: 25 g/day; males: 35 g/day) supplement of fermentable corn bran arabinoxylan (AX; n = 15) with that of microbiota-non-accessible microcrystalline cellulose (MCC; n = 16). Obesity-related surrogate endpoints and biomarkers of host-microbiome interactions implicated in the pathophysiology of obesity (trimethylamine N-oxide, gut hormones, cytokines, and measures of intestinal barrier integrity) were assessed. We then determined whether clinical outcomes could be predicted by fecal microbiota features or mechanistic biomarkers. RESULTS: AX enhanced satiety after a meal and decreased homeostatic model assessment of insulin resistance (HOMA-IR), while MCC reduced tumor necrosis factor-α and fecal calprotectin. Machine learning models determined that effects on satiety could be predicted by fecal bacterial taxa that utilized AX, as identified by bioorthogonal non-canonical amino acid tagging. Reductions in HOMA-IR and calprotectin were associated with shifts in fecal bile acids, but correlations were negative, suggesting that the benefits of fiber may not be mediated by their effects on bile acid pools. Biomarkers of host-microbiome interactions often linked to bacterial metabolites derived from fiber fermentation (short-chain fatty acids) were not affected by AX supplementation when compared to non-accessible MCC. CONCLUSION: This study demonstrates the efficacy of purified dietary fibers when used as supplements and suggests that satietogenic effects of AX may be linked to bacterial taxa that ferment the fiber or utilize breakdown products. Other effects are likely microbiome independent. The findings provide a basis for fiber-type specific therapeutic applications and their personalization. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract.

Inhibition of Autotaxin with GLPG1690 Increases the Efficacy of Radiotherapy and Chemotherapy in a Mouse Model of Breast Cancer.

Autotaxin catalyzes the formation of lysophosphatidic acid, which stimulates tumor growth and metastasis and decreases the effectiveness of cancer therapies. In breast cancer, autotaxin is secreted mainly by breast adipocytes, especially when stimulated by inflammatory cytokines produced by tumors. In this work, we studied the effects of an ATX inhibitor, GLPG1690, which is in phase III clinical trials for idiopathic pulmonary fibrosis, on responses to radiotherapy and chemotherapy in a syngeneic orthotopic mouse model of breast cancer. Tumors were treated with fractionated external beam irradiation, which was optimized to decrease tumor weight by approximately 80%. Mice were also dosed twice daily with GLPG1690 or vehicle beginning at 1 day before the radiation until 4 days after radiation was completed. GLPG1690 combined with irradiation did not decrease tumor growth further compared with radiation alone. However, GLPG1690 decreased the uptake of 3'-deoxy-3'-[18F]-fluorothymidine by tumors and the percentage of Ki67-positive cells. This was also associated with increased cleaved caspase-3 and decreased Bcl-2 levels in these tumors. GLPG1690 decreased irradiation-induced C-C motif chemokine ligand-11 in tumors and levels of IL9, IL12p40, macrophage colony-stimulating factor, and IFNγ in adipose tissue adjacent to the tumor. In other experiments, mice were treated with doxorubicin every 2 days after the tumors developed. GLPG1690 acted synergistically with doxorubicin to decrease tumor growth and the percentage of Ki67-positive cells. GLPG1690 also increased 4-hydroxynonenal-protein adducts in these tumors. These results indicate that inhibiting ATX provides a promising adjuvant to improve the outcomes of radiotherapy and chemotherapy for breast cancer.

Repeated Fractions of X-Radiation to the Breast Fat Pads of Mice Augment Activation of the Autotaxin-Lysophosphatidate-Inflammatory Cycle.

Breast cancer patients are usually treated with multiple fractions of radiotherapy (RT) to the whole breast after lumpectomy. We hypothesized that repeated fractions of RT would progressively activate the autotaxin-lysophosphatidate-inflammatory cycle. To test this, a normal breast fat pad and a fat pad containing a mouse 4T1 tumor were irradiated with X-rays using a small-animal "image-guided" RT platform. A single RT dose of 7.5 Gy and three daily doses of 7.5 Gy increased ATX activity and decreased plasma adiponectin concentrations. The concentrations of IL-6 and TNFα in plasma and of VEGF, G-CSF, CCL11 and CXCL10 in the irradiated fat pad were increased, but only after three fractions of RT. In 4T1 breast tumor-bearing mice, three fractions of 7.5 Gy augmented tumor-induced increases in plasma ATX activity and decreased adiponectin levels in the tumor-associated mammary fat pad. There were also increased expressions of multiple inflammatory mediators in the tumor-associated mammary fat pad and in tumors, which was accompanied by increased infiltration of CD45+ leukocytes into tumor-associated adipose tissue. This work provides novel evidence that increased ATX production is an early response to RT and that repeated fractions of RT activate the autotaxin-lysophosphatidate-inflammatory cycle. This wound healing response to RT-induced damage could decrease the efficacy of further fractions of RT.

Mixed Lipid, Fish Oil, and Soybean Oil Parenteral Lipids Impact Cholestasis, Hepatic Phytosterol, and Lipid Composition.

OBJECTIVES: In parenteral nutrition-dependent infants and children, intestinal failure (IF)-associated liver disease (IFALD) remains an important problem. A comparative study was undertaken of parenteral mixed lipid (ML), ω-3 predominant fish oil (FO), and ω-6 predominant soybean oil (SO) emulsions in regards to hepatic phytosterol, neutral lipid, fatty acid (FA) content, and the relationship to cholestasis in piglets. METHODS: Neonatal piglets received parenteral nutrition, varying in lipid dose (5 or 10 g·â€Škg ·â€Šday) and formulation: SO5 (n = 5), SO10 (n = 5), FO5 (n = 5), and ML10 (n = 5). On day 14, liver chemistry, bile flow, histology and neutral lipid staining were assessed. Hepatic triglyceride FA content was determined using thin layer and gas chromatography, and phytosterol content was assessed using gas chromatography-mass spectrometry. RESULTS: SO groups had higher prevalence of biochemical cholestasis (P < 0.04) and lower bile flow (P < 0.0001). Hepatic campesterol, stigmasterol, and ß-sitosterol were highest in SO10 (P < 0.0001). Hepatic FA (P < 0.03) and ω-6/ω-3 FA ratio (P < 0.0001) were higher in the SO groups. Neutral lipid accumulation (P = 0.3) and liver histology (P = 0.16) were not different between groups. Univariate predictors of bile flow were: campesterol (r = -0.77, P = 0.001), ß-sitosterol (r = -0.74, P = 0.002), stigmasterol (r = -0.74, P = 0.002), ω-6 FA (r = -0.72, P = 0.002), and ω-3 FA (r = 0.59, P = 0.02). Only campesterol independently predicted bile flow. CONCLUSIONS: ML and FO lipid emulsions reduce cholestasis in association with lowered hepatic phytosterol and lipid content. Lower hepatic phytosterol and ω-6 FA content, and higher ω-3 FA content are hepatoprotective. Multivariate analysis suggests reduced phytosterol accumulation may best explain the hepatoprotective effect of fish oil-containing lipids.

A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt-/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt-/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt-/- mice compared with those of Pemt+/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt-/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Dexamethasone decreases the autotaxin-lysophosphatidate-inflammatory axis in adipose tissue: implications for the metabolic syndrome and breast cancer.

Lysophosphatidate (LPA) signaling through 6 receptors is regulated by the balance of LPA production by autotaxin (ATX) vs. LPA degradation by lipid phosphate phosphatases (LPPs). LPA promotes an inflammatory cycle by increasing the synthesis of cyclooxygenase-2 and multiple inflammatory cytokines that stimulate further ATX production. We aimed to determine whether the anti-inflammatory glucocorticoid (GC) dexamethasone (Dex) functions partly by decreasing the ATX-LPA inflammatory cycle in adipose tissue, a major site of ATX secretion. Treatment of human adipose tissue with 10-1000 nM Dex decreased ATX secretion, increased LPP1 expression, and decreased mRNA expressions of IL-6, TNF-α, peroxisome proliferator-activated receptor (PPAR)-γ, and adiponectin. Cotreatment with rosiglitazone (an insulin sensitizer), insulin, or both abolished Dex-induced decreases in ATX and adiponectin secretion, but did not reverse Dex-induced decreases in secretions of 20 inflammatory cytokines and chemokines. Dex-treated mice exhibited lower ATX activity in plasma, brain, and adipose tissue; decreased mRNA levels for LPA and sphingosine 1-phosphate (S1P) receptors in brain; and decreased plasma concentrations of LPA and S1P. Our results establish a novel mechanism for the anti-inflammatory effects of Dex through decreased signaling by the ATX-LPA-inflammatory axis. The GC action in adipose tissue has implications for the pathogenesis of insulin resistance and obesity in metabolic syndrome and breast cancer treatment.-Meng, G., Tang, X., Yang, Z., Zhao, Y., Curtis, J. M., McMullen, T. P. W., Brindley, D. N. Dexamethasone decreases the autotaxin-lysophosphatidate-inflammatory axis in adipose tissue: implications for the metabolic syndrome and breast cancer.

Hepatic Expression of PEMT, but Not Dietary Choline Supplementation, Reverses the Protection against Atherosclerosis in Pemt-/-/Ldlr-/- Mice.

Background: Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine to phosphatidylcholine. Pemt-/-/low density lipoprotein receptor (Ldlr)-/- mice have significantly reduced plasma lipids and are protected against atherosclerosis. Recent studies have shown that choline can be metabolized by the gut flora into trimethylamine-N-oxide (TMAO), which is an emerging risk factor for atherosclerosis. Objective: The objective of this study was to determine whether ectopic hepatic PEMT expression or choline supplementation would promote atherosclerosis in Pemt-/-/Ldlr-/- mice. Methods: Male 8- to 10-wk-old Pemt+/+/Ldlr-/- (SKO) and Pemt-/-/Ldlr-/- (DKO) mice were injected with an adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or human PEMT and fed a Western diet (40% of calories from fat, 0.5% cholesterol) for 8 wk. In a separate experiment, 8- to 10-wk-old SKO and half of the DKO male mice were fed a Western diet with normal (3 g/kg) choline for 12 wk. The remaining DKO mice [choline-supplemented (CS) DKO] were fed a CS Western diet (10 g choline/kg). Plasma lipid concentrations, choline metabolites, and aortic atherosclerosis were measured. Results: Plasma cholesterol, plasma TMAO, and aortic atherosclerosis were reduced by 60%, 40%, and 80%, respectively, in DKO mice compared with SKO mice. AAV-PEMT administration increased plasma cholesterol and TMAO by 30% and 40%, respectively, in DKO mice compared with AAV-GFP-treated DKO mice. Furthermore, AAV-PEMT-injected DKO mice developed atherosclerotic lesions similar to SKO mice. In the second study, there was no difference in atherosclerosis or plasma cholesterol between DKO and CS-DKO mice. However, plasma TMAO concentrations were increased 2.5-fold in CS-DKO mice compared with DKO mice. Conclusions: Reintroducing hepatic PEMT reversed the atheroprotective phenotype of DKO mice. Choline supplementation did not increase atherosclerosis or plasma cholesterol in DKO mice. Our data suggest that plasma TMAO does not induce atherosclerosis when plasma cholesterol is low. Furthermore, this is the first report to our knowledge that suggests that de novo choline synthesis alters TMAO status.

HDL protects against doxorubicin-induced cardiotoxicity in a scavenger receptor class B type 1-, PI3K-, and Akt-dependent manner.

Doxorubicin is a widely used chemotherapeutic with deleterious cardiotoxic side effects. HDL has been shown to protect cardiomyocytes in vitro against doxorubicin-induced apoptosis. Scavenger receptor class B type 1 (SR-B1), a high-affinity HDL receptor, mediates cytoprotective signaling by HDL through Akt. Here, we assessed whether increased HDL levels protect against doxorubicin-induced cardiotoxicity in vivo and in cardiomyocytes in culture and explored the intracellular signaling mechanisms involved, particularly the role of SR-B1. Transgenic mice with increased HDL levels through overexpression of human apolipoprotein A1 (apoA1Tg/Tg) and wild-type mice (apoA1+/+) with normal HDL levels were treated repeatedly with doxorubicin. After treatment, apoA1+/+ mice displayed cardiac dysfunction, as evidenced by reduced left ventricular end-systolic pressure and +dP/d t, and histological analysis revealed cardiomyocyte atrophy and increased cardiomyocyte apoptosis after doxorubicin treatment. In contrast, apoA1Tg/Tg mice were protected against doxorubicin-induced cardiac dysfunction and cardiomyocyte atrophy and apoptosis. When SR-B1 was knocked out, however, overexpression of apoA1 did not protect against doxorubicin-induced cardiotoxicity. Using primary neonatal mouse cardiomyocytes and human immortalized ventricular cardiomyocytes in combination with genetic knockout, inhibitors, or siRNA-mediated knockdown, we demonstrated that SR-B1 is required for HDL-mediated protection of cardiomyocytes against doxorubicin-induced apoptosis in vitro via a pathway involving phosphatidylinositol 3-kinase and Akt1/2. Our findings provide proof of concept that raising apoA1 to supraphysiological levels can dramatically protect against doxorubicin-induced cardiotoxicity via a pathway that is mediated by SR-B1 and involves Akt1/2 activation in cardiomyocytes. NEW & NOTEWORTHY We have identified an important role for the scavenger receptor class B type 1 in facilitating high-density lipoprotein-mediated protection of cardiomyocytes against stress-induced apoptosis and shown that increasing plasma high-density lipoprotein protects against the deleterious side effects of the chemotherapeutic and cardiotoxic drug doxorubicin.

High-Speed Counter-Current Chromatography (HSCCC) Purification of Antifungal Hydroxy Unsaturated Fatty Acids from Plant-Seed Oil and Lactobacillus Cultures.

Hydroxy unsaturated fatty acids (HUFA) can function as antifungal agents. To investigate the antifungal spectrum, that is, the scope of the in vitro fungal-inhibition activities of HUFA and their potential applications, three HUFA were produced by microbial transformation or extracted from plant-seed oils; these compounds included coriolic acid (13-hydroxy-9,11-octadecadienoic acid) from Coriaria seed oil, 10-hydroxy-12-octadecenoic acid from cultures of Lactobacillus hammesii, and 13-hydroxy-9-octadecenoic acid from cultures of Lactobacillus plantarum TMW1.460Δlah. HUFA were purified by high-speed counter-current chromatography (HSCCC), characterized by LC-MS and MS/MS, and their antifungal activities were evaluated with 15 indicator fungal strains. The HUFA had different antifungal spectra when compared with unsaturated fatty acids with comparable structures but without hydroxy groups. The inhibitory effects of HUFA specifically targeted filamentous fungi, including Aspergillus niger and Penicillium roqueforti, whereas yeasts, including Candida spp. and Saccharomyces spp., were resistant to HUFA. The findings here support the development of food applications for antifungal HUFA.

Feeding a Mixture of Choline Forms to Lactating Dams Improves the Development of the Immune System in Sprague-Dawley Rat Offspring.

Dietary choline is essential during lactation, but few studies have examined the implications of feeding a mixture of choline forms on immune function. This study investigates the impact of feeding lactating dams different mixtures of choline forms, similar to those in human diets, on the development and later immune function of suckled offspring. Sprague-Dawley lactating dams (n = 6/diet) were randomized to consume one of three diets, containing 1 g/kg choline: Control (100% free choline (FC)), Mixed Choline (MC: 50% phosphatidylcholine (PC), 25% FC, 25% glycerophosphocholine (GPC)), or High GPC (HGPC: 75% GPC, 12.5% PC, 12.5% FC). At weaning, female pups (n = 2/dam) were fed the Control diet until 10 weeks. At 3 weeks, MC and HGPC pups were heavier and their splenocytes had a higher proportion of helper T cells expressing CD25 and CD28 and produced less interferon gamma (IFN-γ) and tumor-necrosis factor-α (TNF-α) after Concanavalin A stimulation vs. Control pups (p < 0.05). At 10 weeks, MC and HGPC offspring had a lower proportion of macrophages and dendritic cells and produced less interleukin (IL)-1ß but more IL-10 after lipopolysaccharide stimulation vs. Control pups (p < 0.05). In summary, feeding mixed choline diets during lactation improved T cell phenotype/function at the end of suckling and programmed a less inflammatory response later in life.

Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation.

BACKGROUND: We previously discovered that tetracyclines increase the expression of lipid phosphate phosphatases at the surface of cells. These enzymes degrade circulating lysophosphatidate and therefore doxycycline increases the turnover of plasma lysophosphatidate and decreases its concentration. Extracellular lysophosphatidate signals through six G protein-coupled receptors and it is a potent promoter of tumor growth, metastasis and chemo-resistance. These effects depend partly on the stimulation of inflammation that lysophosphatidate produces. METHODS: In this work, we used a syngeneic orthotopic mouse model of breast cancer to determine the impact of doxycycline on circulating lysophosphatidate concentrations and tumor growth. Cytokine/chemokine concentrations in tumor tissue and plasma were measured by multiplexing laser bead technology. Leukocyte infiltration in tumors was analyzed by immunohistochemistry. The expression of IL-6 in breast cancer cell lines was determined by RT-PCR. Cell growth was measured in Matrigel™ 3D culture. The effects of doxycycline on NF-κB-dependent signaling were analyzed by Western blotting. RESULTS: Doxycycline decreased plasma lysophosphatidate concentrations, delayed tumor growth and decreased the concentrations of several cytokines/chemokines (IL-1ß, IL-6, IL-9, CCL2, CCL11, CXCL1, CXCL2, CXCL9, G-CSF, LIF, VEGF) in the tumor. These results were compatible with the effects of doxycycline in decreasing the numbers of F4/80+ macrophages and CD31+ blood vessel endothelial cells in the tumor. Doxycycline also decreased the lysophosphatidate-induced growth of breast cancer cells in three-dimensional culture. Lysophosphatidate-induced Ki-67 expression was inhibited by doxycycline. NF-κB activity in HEK293 cells transiently expressing a NF-κB-luciferase reporter vectors was also inhibited by doxycycline. Treatment of breast cancer cells with doxycycline also decreased the translocation of NF-κB to the nucleus and the mRNA levels for IL-6 in the presence or absence of lysophosphatidate. CONCLUSION: These results contribute a new dimension for understanding the anti-inflammatory effects of tetracyclines, which make them potential candidates for adjuvant therapy of cancers and other inflammatory diseases.

An LC/MS/MS method for the simultaneous determination of individual sphingolipid species in B cells.

Comprehensive profiling of sphingolipids is of great importance for clinical and pharmaceutical studies. An LC/MS/MS method was established for the simultaneous separation and quantification of individual sphingolipid species including ceramides, dihydroceramides, glucosylceramides, sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate. All target individual sphingolipid species were separated and quantified in a single chromatographic run of <20min. Method validation results indicated that calibration curves were linear in the range of 2.5-10,000nM for ceramides and glucosylceramides, 10-10,000nM for dihydroceramides, 5-10,000nM for sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate, respectively. The limits of detection ranged from 0.5nM to 5nM. Accuracies of 92.5-113% with precisions of 0.3-8.0% RSD were obtained for all of the standards over a wide range of concentrations. The application of this method was demonstrated using B cells collected from Chronic Lymphocytic Leukemia patients (n=5) and healthy donors (n=4). 17 sphingolipid species were successfully characterized and quantified in the lipid extract. This is a rapid method that could be readily adapted to lipidomic investigations of sphingolipids in other bio-fluids and tissues.

The Form of Choline in the Maternal Diet Affects Immune Development in Suckled Rat Offspring.

BACKGROUND: Lipid-soluble phosphatidylcholine (PC) and aqueous free choline are absorbed and metabolized differently, but the metabolic effects of feeding these 2 forms of choline have not been thoroughly investigated. OBJECTIVE: We sought to compare the effects of PC and free choline in the maternal diet on the development of the offspring's immune system. METHODS: During lactation, Sprague-Dawley dams (n= 10) were randomly assigned to 1 of 2 diet groups containing the same concentration of total choline (1 g/kg diet) as free choline (choline bitartrate) or PC (egg lecithin). The splenocytes of pups aged 21 d were isolated and stimulated ex vivo with concanavalin A (ConA) or lipopolysaccharide (LPS), and the choline concentrations of stomach content, plasma, and the spleen were measured. RESULTS: Pups from PC-fed dams had a lower proportion of cells involved in antigen presentation but produced 54% more interleukin (IL)-2, 163% more IL-6, and 107% more IFN-γ after ConA stimulation and 110% more IL-6 and 43% more tumor necrosis factor (TNF)-α after LPS stimulation (allP< 0.05). The PC concentrations were significantly higher in the plasma and spleen of pups from PC-fed dams (P< 0.05). Increasing the supply of PC in the form of lysophosphatidylcholine to splenocytes in vitro increased the rate of proliferation and IL-2 production and the surface expression of CD25, CD28, CD71, and CD152 on CD8+ T cells, suggesting 1 possible mechanism. CONCLUSIONS: The results of this study demonstrate that providing choline to rats in the form of PC (compared to free choline), possibly by increasing the supply of PC to the suckling pups, promotes maturation and improves function of the offspring's immune system.

Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 µg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs.

Oxidative stress contributes to the tamoxifen-induced killing of breast cancer cells: implications for tamoxifen therapy and resistance.

Tamoxifen is the accepted therapy for patients with estrogen receptor-α (ERα)-positive breast cancer. However, clinical resistance to tamoxifen, as demonstrated by recurrence or progression on therapy, is frequent and precedes death from metastases. To improve breast cancer treatment it is vital to understand the mechanisms that result in tamoxifen resistance. This study shows that concentrations of tamoxifen and its metabolites, which accumulate in tumors of patients, killed both ERα-positive and ERα-negative breast cancer cells. This depended on oxidative damage and anti-oxidants rescued the cancer cells from tamoxifen-induced apoptosis. Breast cancer cells responded to tamoxifen-induced oxidation by increasing Nrf2 expression and subsequent activation of the anti-oxidant response element (ARE). This increased the transcription of anti-oxidant genes and multidrug resistance transporters. As a result, breast cancer cells are able to destroy or export toxic oxidation products leading to increased survival from tamoxifen-induced oxidative damage. These responses in cancer cells also occur in breast tumors of tamoxifen-treated mice. Additionally, high levels of expression of Nrf2, ABCC1, ABCC3 plus NAD(P)H dehydrogenase quinone-1 in breast tumors of patients at the time of diagnosis were prognostic of poor survival after tamoxifen therapy. Therefore, overcoming tamoxifen-induced activation of the ARE could increase the efficacy of tamoxifen in treating breast cancer.

Glucagon-Like Peptide-2 Alters Bile Acid Metabolism in Parenteral Nutrition--Associated Liver Disease.

BACKGROUND: We aim to study the mechanisms underlying our previous finding that exogenous glucagon-like peptide-2 (GLP-2) treatment in a preclinical model of neonatal parenteral nutrition-associated liver disease (PNALD) improves cholestasis. METHODS: Neonatal piglets received 17 days of parenteral nutrition (PN) therapy and either saline control (PN/Saline n = 8) or GLP-2 treatment at 11 nmol/kg/d (PN/GLP-2, n = 7). At terminal laparotomy, bile and liver samples were collected. The relative gene expression of enzymes involved in bile acid synthesis, regulation, and transport was measured in liver by reverse-transcriptase quantitative polymerase chain reaction. Bile acid composition in bile was determined using tandem mass spectrometry. Data were analyzed using 1-way analysis of variance (ANOVA) or Kruskal-Wallis ANOVA. RESULTS: GLP-2 increased the expression of bile acid export genes: multidrug resistance-associated proteins 2 (MRP2) (P = .002) and 3 (MRP3) (P = .037) over saline control. GLP-2 increased expression of Farnesoid X receptor (FXR) (P < .001) and CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1) (P = .03). GLP-2 treatment was associated with decreased concentrations of taurohyocholic acid and conjugates of toxic lithocholic acid (P < .01). GLP-2 treatment increased the liver bile acid content. CONCLUSIONS: GLP-2 treatment was associated with alterations in the hepatic expression of genes involved in bile acid metabolism. The transcriptomic results indicate the mechanisms at the transcriptional level acting to regulate bile acid synthesis and increase bile acid export. Differences in bile acid profiles further support a beneficial role for GLP-2 therapy in PNALD.

Autotaxin is an inflammatory mediator and therapeutic target in thyroid cancer.

Autotaxin is a secreted enzyme that converts extracellular lysophosphatidylcholine to lysophosphatidate (LPA). In cancers, LPA increases tumour growth, metastasis and chemoresistance by activating six G-protein coupled receptors. We examined >200 human thyroid biopsies. Autotaxin expression in metastatic deposits and primary carcinomas was four- to tenfold higher than in benign neoplasms or normal thyroid tissue. Autotaxin immunohistochemical staining was also increased in benign neoplasms with leukocytic infiltrations. Malignant tumours were distinguished from benign tumours by high tumour autotaxin, LPA levels and inflammatory mediators including IL1ß, IL6, IL8, GMCSF, TNFα, CCL2, CXCL10 and platelet-derived growth factor (PDGF)-AA. We determined the mechanistic explanation for these results and revealed a vicious regulatory cycle in which LPA increased the secretion of 16 inflammatory modulators in papillary thyroid cancer cultures. Conversely, treating cancer cells with ten inflammatory cytokines and chemokines or PDGF-AA and PDGF-BB increased autotaxin secretion. We confirmed that this autotaxin/inflammatory cycle occurs in two SCID mouse models of papillary thyroid cancer by blocking LPA signalling using the autotaxin inhibitor ONO-8430506. This decreased the levels of 16 inflammatory mediators in the tumours and was accompanied by a 50-60% decrease in tumour volume. This resulted from a decreased mitotic index for the cancer cells and decreased levels of vascular endothelial growth factor and angiogenesis in the tumours. Our results demonstrate that the autotaxin/inflammatory cycle is a focal point for driving malignant thyroid tumour progression and possibly treatment resistance. Inhibiting autotaxin activity provides an effective and novel strategy for decreasing the inflammatory phenotype in thyroid carcinomas, which should complement other treatment modalities.

Regulation of autotaxin expression and secretion by lysophosphatidate and sphingosine 1-phosphate.

Autotaxin (ATX) is a secreted enzyme, which produces extracellular lysophosphatidate (LPA) from lysophosphatidylcholine (LPC). LPA activates six G protein-coupled receptors and this is essential for vasculogenesis during embryonic development. ATX is also involved in wound healing and inflammation, and in tumor growth, metastasis, and chemo-resistance. It is, therefore, important to understand how ATX is regulated. It was proposed that ATX activity is inhibited by its product LPA, or a related lipid called sphingosine 1-phosphate (S1P). We now show that this apparent inhibition is ineffective at the high concentrations of LPC that occur in vivo. Instead, feedback regulation by LPA and S1P is mediated by inhibition of ATX expression resulting from phosphatidylinositol-3-kinase activation. Inhibiting ATX activity in mice with ONO-8430506 severely decreased plasma LPA concentrations and increased ATX mRNA in adipose tissue, which is a major site of ATX production. Consequently, the amount of inhibitor-bound ATX protein in the plasma increased. We, therefore, demonstrate the concept that accumulation of LPA in the circulation decreases ATX production. However, this feedback regulation can be overcome by the inflammatory cytokines, TNF-α or interleukin 1ß. This enables high LPA and ATX levels to coexist in inflammatory conditions. The results are discussed in terms of ATX regulation in wound healing and cancer.

Choline is required in the diet of lactating dams to maintain maternal immune function.

Choline demands during lactation are high; however, detailed knowledge is lacking regarding the optimal dietary intake during this critical period. The present study was designed to determine the effects of varying intakes of choline on maternal immune function during lactation. Primiparous Sprague-Dawley rats (n 42) were randomised 24-48 h before birth and fed the following diets for 21 d: choline-devoid (0 g choline/kg diet; D, n 10); 1·0 g choline/kg diet (C1, n 11); 2·5 g choline/kg diet (C2·5, n 10); 6·2 g choline/kg diet (C6, n 11). Splenocytes were isolated and stimulated ex vivo with concanavalin A, lipopolysaccharide (LPS) or CD3/CD28. D and C6 dams had lower final body weight, spleen weight and average pup weight than C1 dams (P< 0·05). There was a linear relationship between free choline concentration in pup stomach contents with maternal dietary choline content (P< 0·001, r² 0·415). Compared with C1 and C2·5, D spleens had a lower proportion of mature T cells and activated suppressor cells, and this resulted in reduced cytokine production after stimulation (P< 0·05). Feeding 6·2 g choline/kg diet resulted in a higher cytokine production after stimulation with CD3/CD28 (P< 0·05). Except for a higher IL-6 production after LPS stimulation with cells from the C2·5 dams (P< 0·05), there were no differences between the C1 and C2·5 dams. For the first time, we show that feeding lactating mothers a diet free of choline has substantial effects on their immune function and on offspring growth. Additionally, excess dietary choline had adverse effects on maternal and offspring body weight but only minimal effects on maternal immune function.

Stability of dicyclohexylamine and fumagillin in honey.

Fumagillin is extensively used to control nosema disease in apiculture. In the commercial formulation, fumagillin is present as a salt in an equimolar quantity with dicyclohexylamine (DCH). In this study DCH was observed to be significantly more resistant to degradation in honey than fumagillin using LC-MS/MS analysis. Observed half-lives for DCH ranged from a minimum of 368 days when kept at 34 °C in darkness, to a maximum of 852 days when stored at 21 °C in darkness. A maximum half-life of 246 days was observed for fumagillin in samples kept in darkness at a temperature of 21 °C. The observed half-life of fumagillin was estimated to be 3 days when exposed to light at 21 °C, and complete decomposition was observed after 30 days under the same conditions. The stability of DCH, combined with its genotoxicity and tumorigenic properties make it an important potential contaminant in honey destined for human consumption.