Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation.

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects.

Circulating levels of MCP-1 and IL-8 are elevated in human obese subjects and associated with obesity-related parameters.

BACKGROUND: Chemotactic cytokines, referred to as chemokines, play an important role in leukocyte trafficking. The circulating levels of chemokines have been shown to increase in inflammatory processes including obesity-related pathologies (e.g. atherosclerosis and diabetes). However, little is currently known about the relationship between chemokines and human obesity. In the present study, we investigated the circulating levels of selected chemokines (monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), leukotactin-1, interleukin-8 (IL-8)) and the association between the chemokine levels and obesity-related parameters: body mass index (BMI), waist circumference, fasting glucose and insulin levels, lipids profile, and the level of C-reactive protein (CRP). METHODS: A total of 100 subjects, 50 obese (BMI>or=25 kg/m2) and 50 who were not obese (BMI<25 kg/m2) participated in the present study. The levels of chemokines and CRP were measured in a fasting state serum by sandwich enzyme-linked immunosorbent assay. Total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, glucose, and insulin levels were measured by enzymatic analysis and immunoassay. RESULTS: The circulating levels of MCP-1 and IL-8 in the serum were significantly (P<0.05) higher in obese subjects (BMI>30 kg/m2) compared with those of nonobese controls (BMI<25 kg/m2). The levels of CRP were positively correlated with BMI (P<0.001) or waist circumference (P<0.0001). The levels of MCP-1 and IL-8 were positively related to BMI (MCP-1, P<0.02; IL-8, P<0.01) and/or waist circumference (MCP-1, P<0.009; IL-8, P<0.03). The levels of MCP-1 were positively related to the levels of CRP (P<0.007) or interleukin-6 (IL-6) (P<0.0001), and negatively related to the levels of HDL-cholesterol (P<0.01). Homeostasis model assessment (HOMA) score was positively related to the levels of MCP-1 (P<0.02) or IL-8 (P<0.03) in obese subject. DISCUSSION: Our data demonstrated that the circulating levels of MCP-1 and IL-8 are related to obesity-related parameters such as BMI, waist circumference, CRP, IL-6, HOMA and HDL-cholesterol. These findings suggest that the circulating MCP-1 and/or IL-8 may be a potential candidate linking obesity with obesity-related metabolic complications such as atherosclerosis and diabetes.

Contrasting effects of t10,c12- and c9,t11-conjugated linoleic acid isomers on the fatty acid profiles of mouse liver lipids.

The purpose of this study was to examine the effects of two purified isomers of CLA (c9,t11-CLA and t10,c12-CLA) on the weights and FA compositions of hepatic TG, phospholipids, cholesterol esters, and FFA. Eight-week-old female mice (n = 6/group) were fed either a control diet or diets supplemented with 0.5% c9,t11-CLA or t10,c12-CLA isomers for 8 wk. Weights of liver total lipids and those of individual lipid fractions did not differ between the control and the c9,t11-CLA groups. Livers from animals fed the t10, c12-CLA diet contained four times more lipids than those of the control group; this was mainly due to an increase in the TG fractions (fivefold), but cholesterol (threefold), cholesterol esters (threefold), and FFA (twofold) were also significantly increased. Although c9,t11-CLA did not significantly alter the weights of liver lipids when compared with the control group, its intake was associated with significant reductions in the weight percentage (wt% of total FAME) of 18:1n-9 and 18:1n-7 in the TG fraction and with significant increases in the weight percentage of 18:2n-6 in the TG, cholesterol ester, and phospholipid fractions. On the other hand, t10,c12-CLA intake was linked with a significant increase in the weight percentage of 18:1n-9 and a decrease in that of 18:2n-6 in all lipid fractions. These changes may be the result of alterations in the activity of delta9-desaturase (stearoyl CoA desaturase) and the enzymes involved in the metabolism of 18:2n-6. Thus, the two isomers differed not only in their effects on the weights of total liver lipids and lipid fractions but also on the FA profile of the lipid fractions.

Trans-10,cis-12 CLA increases liver and decreases adipose tissue lipids in mice: possible roles of specific lipid metabolism genes.

Although consumption of CLA mixtures has been associated with several health effects, less is known about the actions of specific CLA isomers. There is evidence that the t10,c12-CLA isomer is associated with alterations in body and organ weights in animals fed CLA, but the mechanisms leading to these changes are unclear. The purpose of this study was to determine the effects of two commonly occurring isomers of CLA on body composition and the transcription of genes associated with lipid metabolism. Eight-week-old female mice (n = 11 or 12/group) were fed either a control diet or diets supplemented with 0.5% c9,t11-CLA or t10,c12-CLA isomers or 0.2% of the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist fenofibrate for 8 wk. Body and retroperitoneal adipose tissue weights were significantly lower (6-10 and 50%, respectively), and liver weights were significantly greater (100%) in the t10,c12-CLA and the fenofibrate groups compared with those in the control group; body and tissue weights in the c9,t11-CLA group did not differ from those in the control group. Livers from animals in the t10,c12-CLA group contained five times more lipids than in the control group, whereas the lipid content of the fenofibrate group did not differ from that in the control group. Although fenofibrate increased the mRNA for PPARalpha, t10,c12-CLA decreased it. These results suggest that PPARalpha did not mediate the effects of t10,c12-CLA on body composition. The CLA isomers and fenofibrate altered mRNA levels for several proteins involved in lipid metabolism, but the most striking difference was the reduction of mRNA for leptin and adiponectin in the t10,c12-CLA group. These initial results suggest that changes associated with energy homeostasis and insulin action may mediate the effects of t10,c12-CLA on lipid metabolism.

Modulation of body composition and immune cell functions by conjugated linoleic acid in humans and animal models: benefits vs. risks.

We have reviewed the published literature regarding the effects of CLA on body composition and immune cell functions in humans and in animal models. Results from studies in mice, hamsters, rats, and pigs generally support the notion that CLA reduced depot fat in the normal or lean strains. However, in obese rats, it increased body fat or decreased it less than in the corresponding lean controls. These studies also indicate that t10,c12-CLA was the isomer that reduced adipose fat; however, it also increased the fat content of several other tissues and increased circulating insulin and the saturated FA content of adipose tissue and muscle. Four of the eight published human studies found small but significant reductions in body fat with CLA supplementation; however, the reductions were smaller than the prediction errors for the methods used. The other four human studies found no change in body fat with CLA supplementation. These studies also report that CLA supplementation increased the risk factors for diabetes and cardiovascular disease including increased blood glucose, insulin, insulin resistance, VLDL, C-reactive protein, lipid peroxidation, and decreased HDL. Most studies regarding the effects of CLA on immune cell functions have been conducted with a mixture of isomers, and the results have been variable. One study conducted in mice with the purified c9,t11-CLA and t10,c12-CLA isomers indicated that the two isomers have similar effects on immune cell functions. Some of the reasons for the discrepancies between the effects of CLA in published reports are discussed. Although significant benefit to humans from CLA supplementation is questionable, it may create several health risks in both humans and animals. On the basis of the published data, CLA supplementation of adult human diets to improve body composition or enhance immune functions cannot be recommended at this time.

Similar effects of c9,t11-CLA and t10,c12-CLA on immune cell functions in mice.

Published results regarding the effects of CLA on immune cell functions have ranged from stimulation to inhibition. In those studies, a mixture of CLA isomers were used, and food intake was not controlled. We have examined whether the discrepancies in the results of earlier studies may be due to the lack of controlled feeding and whether the two isomers of CLA may differ in their effects on immune cell functions. Three groups of C57BL/6 female mice were fed either a control, c9,t11-CLA-, or t10,c12-CLA (0.5 wt%)-supplemented diet, 5 g/d, for 56 d. At the end of the study, the number of immune cells in spleens, bone marrows, or in circulation; proliferation of splenocytes in response to T and B cell mitogens; and prostaglandin secretion in vitro did not differ among the three groups. Both CLA isomers significantly increased in vitro tumor necrosis factor alpha and interleukin (IL)-6 secretion and decreased IL-4 secretion by splenocytes compared to those in the control group. Thus, the two CLA isomers had similar effects on all response variables tested. The discrepancies among the results from previous studies did not seem to be caused by the differences in the isomer composition of CLA used.

Prostaglandin E(2) modulation of vascular endothelial growth factor production in murine macrophages.

We have previously shown that dietary (n-3) fatty acids decrease mammary tumor vascularization and PGE(2) production. One possible mechanism may be the modulation of vascular endothelial growth factor (VEGF) production by PGE(2). Macrophages are major producers of VEGF, and thus we assessed the role of PGE(2) in vitro and in vivo on their VEGF production. When added to macrophages, pharmacological (10(-7) M) but not physiological (10(-9) to 10(-11) M) concentrations of PGE(2) increased VEGF mRNA and protein levels. That increased expression was relatively rapid and sustained up to 8 hrs, but declined by 24 hrs. Similarly, dibutryl cAMP increased production of VEGF protein which was completely inhibited by H89. Addition of cAMP-elevating agents further potentiated the production of VEGF by PGE(2). Next, (n-3) and (n-6) fatty acids were added to macrophages in vitro or provided in the diet. Macrophages of mice fed safflower oil (n-6) had 2- to 4-fold greater copy number of VEGF transcripts after lipopolysaccarhide (LPS) stimulation compared to fish oil (n-3). A decreasing trend was seen in LPS-induced VEGF secretion from macrophages in vitro after docosahexaenoic acid or eicosapentaenoic acid incubation compared to arachidonic acid. While pharmacological concentrations of PGE(2) modulate VEGF expression, physiological alterations did not alter VEGF protein production by macrophages.

Dietary supplementation with conjugated linoleic acid increased its concentration in human peripheral blood mononuclear cells, but did not alter their function.

The purpose of this study was to examine if conjugated linoleic acid (CLA) supplementation of diets would alter fatty acid (FA) composition and function of peripheral blood mononuclear cells (PBMC). Seventeen women, 20-41 yr, participated in a 93-d study conducted at the Metabolic Research Unit. The same diet (19, 30, and 51% energy from protein, fat, and carbohydrate, respectively) was fed to all subjects throughout the study. Seven subjects (control group) supplemented their diet with six daily capsules (1 g each) of placebo oil (sunflower) for 93 d. For the other 10 subjects (CLA group), the supplement was changed to an equivalent amount of Tonalin capsules for the last 63 d of the study. Tonalin provided 3.9 g/d of a mixture of CLA isomers (trans-10,cis-12, 22.6%; cis-11,trans-13, 23.6%; cis-9,trans-11, 17.6%; trans-8,cis-10, 16.6%; other isomers 19.6%), and 2.1 g/d of other FA. PBMC isolated on study days 30 and 90 were used to assess intracellular cytokines by flow cytometry, secreted cytokines, and eicosanoid by enzyme-linked immonosorbent assay, and FA composition by gas-liquid chromatography. After supplementation, total CLA concentration increased from 0.012 to 0.97% (P < 0.0001) in PBMC lipids, but it did not significantly alter the concentration of other FA. CLA supplementation did not alter the in vitro secretion of prostaglandin E2, leukotriene B4, interleukin-1beta (IL-1beta), or tumor necrosis factor alpha (TNFalpha) by PBMC simulated with lipopolysaccharide, and the secretion of IL-2 by PBMC stimulated with phytohemagglutinin. Nor did it alter the percentage T cells producing IL-2, interferon gamma, and percentage of monocytes producing TNFalpha. The intracellular concentration of these cytokines was also not altered. None of the variables tested changed in the control group. Our results show that CLA supplementation increased its concentration in PBMC lipids, but did not alter their functions.

Georgamide, a new cyclic depsipeptide with an alkynoic acid residue from an Australian cyanobacterium.

A cyclic depsipeptide, georgamide (1), was isolated from an Australian cyanobacterium and characterized by spectroscopic means. The constituent units were five amino acid residues, one each of L-Thr, L-Pro, L-Val, N-Me-L-Val, and N-Me-L-Phe, and two hydroxy carboxylic acids, 2(S)-hydroxy-3(R)-methylpentanoic acid and the unusual 2,2-dimethyl-3-hydroxy-7-octynoic acid. The stereochemistry was determined by hydrolysis of the peptide followed by derivatization and HPLC comparison with standard samples.

Ma'iliohydrin, a cytotoxic chamigrene dibromohydrin from a Philippine Laurencia species.

A new cytotoxic tribrominated chamigrene with a dibromohydrin functionality was isolated from an undetermined species of red alga, Laurencia sp. Its structure was determined by spectroscopic methods.

Polychlorinated acetamides from the cyanobacterium Microcoleus lyngbyaceus.

Several new compounds were isolated from the organic extract of the cyanobacterium Microcoleus lyngbyaceus, and their structures were determined by spectroscopic means. Polychlorinated acetamidoalkynes and alkanes were the major metabolites. 6-Acetamido-1,1,1-trichloroundecane, a positional isomer of the naturally occurring 5-acetamido-1,1,1-trichloroundecane, was synthesized in six steps from delta-decanolactone.

Differential mRNA expression of prostaglandin receptor subtypes in macrophage activation.

Assessing the regulation of macrophage receptors for prostaglandin (PGE2) is essential to understanding the control which that potent lipid mediator has in modulating macrophage activities. The purpose of this study was to assess the differential mRNA expression of PGE2 receptor subtypes (EP) during macrophage exposure to activating and transducing agents. RAW 264.7 macrophages constitutively expressed mRNA for EP2,EP3 and EP4 receptor subtypes. Messenger RNA for EP4 was expressed at a much higher level when compared to EP2 in unstimulated macrophages as assessed by kinetic quantitative RT-PCR. When macrophages were stimulated with LPS, EP2 m RNA levels were 12-fold higher when compared to unstimulated macrophages, while EP4 m RNA remained unchanged. Conversely, mRNA levels of both EP2 and EP4 receptors were lower after macrophages were treated with IFN-gamma. Messenger RNA levels of both receptors were lower in macrophages after treatment with PGE2 or dibutyryl (db) cAMP Addition of the PKA inhibitor H89 reversed the effects of PGE2 and dbcAMP to varying degrees. Proteosome and p38 MAP kinase inhibitors blocked the LPS-stimulated increase in EP2 mRNA levels. Those inhibitors had no effect on EP4 mRNA.Thus, activating agents such as LPS and IFN-gamma may differentially regulate mRNAfor PGE2 receptor types in macrophages but the ligand and its associated signal transducing factors probably have similar regulatory effects.

Regulation of cellular differentiation and apoptosis by fatty acids and their metabolites.

We have reviewed the literature regarding the effects of fatty acids and their metabolites on cellular differentiation and apoptosis. Results obtained in different studies have been variable, but some generalizations can be made. Differentiation was increased by incubation of cells with arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), or leukotriene D4 (LTD4). Effects of these agents on differentiation could be magnified with the simultaneous addition of other differentiation-inducing agents like dimethylsulfoxide or retinoic acid. AA and gamma-linolenic acid increased apoptosis while the effects of n-3 fatty acids (EPA and DHA) and of eicosanoids varied from stimulation to inhibition. These inconsistencies are attributed to the differences in methods used to evaluate differentiation and apoptosis, concentrations of fatty acids and serum, exposure time and the cell models used. Studies using the physiological concentrations of the fatty acids and standardized experimental conditions need to be conducted to establish effects of fatty acids and their metabolites on these cellular processes.

Dietary conjugated linoleic acid did not alter immune status in young healthy women.

The purpose of this study was to examine whether conjugated linoleic acid (CLA) supplementation in human diets would enhance indices of immune status as reported by others for animal models. Seventeen women, 20-41 yr, participated in a 93-d study conducted in two cohorts of 9 and 8 women at the Metabolic Research Unit of Western Human Nutrition Research Center. Seven subjects were fed the basal diet (19, 30, and 51% energy from protein, fat, and carbohydrate, respectively) throughout the study. The remaining 10 subjects were fed the basal diet for the first 30 d, followed by 3.9 g CLA (Tonalin)/d for the next 63 d. CLA made up 65% of the fatty acids in the Tonalin capsules, with the following isomeric composition: t10, c12, 22.6%; c11, t13, 23.6%; c9, t11, 17.6%; t8, c10, 16.6%; and other isomers 19.6%. Most indices of immune response were tested at weekly intervals, three times at the end of each period (stabilization/intervention); delayed-type hypersensitivity (DTH) to a panel of six recall antigens was tested on study day 30 and 90; all subjects were immunized on study day 65 with an influenza vaccine, and antibody titers were examined in the sera collected on day 65 and 92. None of the indices of immune status tested (number of circulating white blood cells, granulocytes, monocytes, lymphocytes, and their subsets, lymphocytes proliferation in response to phytohemagglutinin, and influenza vaccine, serum influenza antibody titers, and DTH response) were altered during the study in either dietary group. Thus, in contrast to the reports with animal models, CLA feeding to young healthy women did not alter any of the indices of immune status tested. These data suggest that short-term CLA supplementation in healthy volunteers is safe, but it does not have any added benefit to their immune status.

Conjugated linoleic acid supplementation in humans: effects on circulating leptin concentrations and appetite.

Conjugated linoleic acid (CLA) has been demonstrated to reduce body fat in animals. However, the mechanism by which this reduction occurs is unknown. Leptin may mediate the effect of CLA to decrease body fat. We assessed the effects of 64 d of CLA supplementation (3 g/d) on circulating leptin, insulin, glucose, and lactate concentrations in healthy women. Appetite was assessed as a physiological correlate of changes in circulating leptin levels. Analysis of plasma leptin concentrations adjusted for adiposity by using fat mass as a covariate showed that CLA supplementation significantly decreased circulating leptin concentrations in the absence of any changes of fat mass. Mean leptin levels decreased over the first 7 wk and then returned to baseline levels over the last 2 wk of the study in the CLA-treated group. Appetite parameters measured at around the time when the greatest decreases in leptin levels were observed showed no significant differences between supplementation and baseline determinations in the CLA-supplemented group or between the CLA and placebo-supplemented groups. There was a nonsignificant trend for mean insulin levels to increase toward the end of the supplementation period in CLA-treated subjects. CLA did not affect plasma glucose and lactate over the treatment period. Thus, 64 d of CLA supplementation in women produced a transient decrease in leptin levels but did not alter appetite. CLA did not affect these parameters in a manner that promoted decreases of adiposity.

Micronutrients and innate immunity.

Micronutrients such as zinc, selenium, iron, copper, beta-carotene, vitamins A, C, and E, and folic acid can influence several components of innate immunity. Select micronutrients play an important role in alteration of oxidant-mediated tissue injury, and phagocytic cells produce reactive oxidants as part of the defense against infectious agents. Thus, adequate micronutrients are required to prevent damage of cells participating in innate immunity. Deficiencies in zinc and vitamins A and D may reduce natural killer cell function, whereas supplemental zinc or vitamin C may enhance their activity. The specific effects of micronutrients on neutrophil functions are not clear. Select micronutrients may play a role in innate immunity associated with some disease processes. Future studies should focus on issues such as age-related micronutrient status and innate immunity, alterations of micronutrients in disease states and their effect on innate immunity, and the mechanisms by which micronutrients alter innate immunity.

Probiotic immunomodulation in health and disease.

Probiotics, microorganisms that have a favorable influence on physiologic and pathological processes of the host by their effect on the intestinal flora, may play a role in improving human health. One of the putative effects is the modulation of immune function. Thus, the mucosal immune system and methods to assess its function are reviewed briefly. Probiotic modulation of humoral, cellular and nonspecific immunity is reviewed, with emphasis placed on immune response in disease models. There are very few reports of human intervention studies with probiotics. However, some of the possible future directions for research with respect to probiotics, immunity, and human health are discussed. Although the application of probiotics has demonstrated trends with respect to altered aspects of immune response, the underlying mechanisms by which that occurs are unclear.

Reduction of murine mammary tumor metastasis by conjugated linoleic acid.

Recent studies have shown that conjugated linoleic acid (CLA) can inhibit the initiation and thus, incidence of mammary tumors in rodents. The concentration of CLA required for these effects was as low as 0.1% of the diet, with no increased effects above 1%. To date, there is little evidence that CLA has any effect on growth or metastasis of mammary tumors. In this report, we demonstrate that CLA, at the concentrations used in previous studies, had a significant effect on the latency, metastasis, and pulmonary tumor burden of transplantable murine mammary tumors grown in mice fed 20% fat diets. The latency of tumors from mice fed CLA was significantly increased when compared with the 0% CLA control diet. The volume of pulmonary tumor burden, as a result of spontaneous metastasis, decreased proportionately with increasing concentrations of dietary CLA. With 0.5 and 1% CLA, pulmonary tumor burden was significantly decreased compared to mice treated with the eicosanoid inhibitor, indomethacin and fed diets containing no CLA. Tumors of mice fed as little as 0.1% CLA and as much as 1% had significantly decreased numbers of pulmonary nodules when compared with diets containing no CLA. The decrease in the number of pulmonary nodules by CLA was nearly as effective as indomethacin, a known suppressor of tumor growth and metastasis in this malignant model. These data suggest that effects of CLA on mammary tumorigenesis may go beyond the reported alterations in tumor incidence and effect later stages, especially metastasis.

Modulation of murine mammary tumor vasculature by dietary n-3 fatty acids in fish oil.

We have previously shown that mice fed a high (n-3) fatty acid-containing diet with 20% (w/w) total fat had significantly slower mammary tumor growth, decreased numbers of metastatic pulmonary nodules, and decreased total metastatic load. In this study we sought to determine whether tumor vascularization was altered in mice fed diets varying in concentrations of (n-3) and (n-6) fatty acids. Several direct or indirect parameters of vascularization were tested. With 20% dietary fat, fish oil (FO) or a mixture of FO and safflower oil (FS) significantly reduced blood vascular area, mast cell number and macrophage infiltration in solid mammary tumors compared to tumors grown in mice fed safflower oil (SO). A decreasing trend was seen in the percent area of vessels positive for CD31 and vascular endothelial growth factor (VEGF) in the 20% FO and 20% FS compared to the 20% SO dietary groups. VEGF concentrations were twice as high in smaller tumors (100 mm3) from all dietary groups as compared to larger tumors (500 mm3). A two-fold increase in VEGF levels was found in the 20% SO dietary group compared to the 20% FO group in 100-mm3 but not larger tumors. We conclude that at 20% total fat, the n-3 fatty acids found in fish oil may inhibit primary mammary tumor growth through modulation of select determinants of vascularization.