Fatty acids modulate the colorectal cancer immune microenvironment via regulating the interaction and transactivation of PPARα/δ and P53.

Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu(6) was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.

Resistance of tumors to cell death signals poses a complex clinical problem. We explored the therapeutic potential and in vivo toxicity of a combination of bortezomib, a proteasome inhibitor, and MD5-1, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor (DR5) agonist monoclonal antibody, in mouse carcinomas. METHODS; Mice bearing Renca-FLAG (renal) or 4T1 (mammary) tumors were treated with bortezomib and/or MD5-1 and examined for lung metastases (Renca-FLAG: n = 93; 4T1: n = 40) or monitored for survival (Renca-FLAG: n = 143). Toxicity was assessed by histopathology and hematology. Viability and apoptotic signaling in Renca-FLAG and 4T1 cells treated with bortezomib alone or in combination with TRAIL were analyzed using 3-[4,5-dimethyiazol-2-yl-5]-[3-carboxymethyloxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium assay and by measuring mitochondrial membrane depolarization and caspase-8 and caspase-3 activation. All statistical tests were two-sided. Bortezomib (20 nM) sensitized Renca-FLAG and 4T1 cells to TRAIL-mediated apoptosis (mean percent decrease in numbers of viable cells, bortezomib + TRAIL vs TRAIL: Renca-FLAG, 95% vs 34%, difference = 61%, 95% confidence interval [CI] = 52% to 69%, P < .001; 4T1, 85% vs 20%, difference = 65%, 95% CI = 62% to 69%, P < .001). Sensitization involved activation of caspase-8 and caspase-3 but not mitochondrial membrane depolarization, suggesting an amplified signaling of the extrinsic cell death pathway. Treatment with bortezomib and MD5-1 reduced lung metastases in mice carrying Renca and 4T1 tumors (mean number of metastases, bortezomib + MD5-1 vs MD5-1: Renca-FLAG, 1 vs 8, difference = 7, 95% CI = 5 to 9, P < .001; 4T1, 1 vs 12, difference = 11, 95% CI = 9 to 12, P < .001) and increased median survival of mice bearing Renca-FLAG tumors (bortezomib + MD5-1 vs bortezomib + control isotype antibody: 22 of 30 [73%] were still alive at day 180 vs median survival of 42 days [95% CI = 41 to 44 days, P < .001]) in the absence of obvious toxicity. Bortezomib combined with DR5 agonist monoclonal antibody may be a useful treatment for metastatic solid tumors.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), a cytokine belonging to the TNF (tumor necrosis factor) family, is currently regarded as a potential anti-cancer agent. Nevertheless, several types of cancer cells display a low sensitivity to TRAIL or are completely resistant to this pro-apoptotic cytokine. TRAIL signalling is dependent on four receptors. Two of them, death receptors 4 and 5 (DR4 and DR5), induce apoptosis, whereas decoy receptors 1 and 2 (DcR1 and DcR2) are unable to evoke cell death upon TRAIL binding. TRAIL resistance may be related to the expression of TRAIL decoy receptors. TRAIL has been proposed as a novel therapeutic agent for the treatment of haematological disorders, including acute myeloid leukaemia (AML). Surprisingly, however, very limited information is available concerning the expression of TRAIL receptors in AML blasts. Here, we have evaluated, using flow cytometry, TRAIL receptor surface expression and sensitivity to TRAIL-dependent apoptosis of AML blasts from 30 patients. We observed frequent expression of TRAIL DcR1 and DcR2, while expression of DR4 and DR5 was less frequent. Nevertheless, the expression of DR4 or DR5 in leukaemic cells was always matched by a similar expression of one of the decoy receptors. Leukaemic blasts were invariably resistant, even to a high concentration (1000 ng/ml) of TRAIL. We suggest that AML blasts are resistant to TRAIL apoptosis in vitro. Therefore, it is unlikely that TRAIL alone might be used in the future as an innovative pharmacological agent for the treatment of AML.

The adipocyte-derived secretory protein adiponectin functions as an insulin-sensitizing agent. In plasma, adiponectin exists as low, medium, and high molecular weight oligomers. Treatment with trans-10, cis-12 conjugated linoleic acid (t-10, c-12 CLA) reduces levels of adiponectin as well as triglyceride (TG) in mice and adipocyte cell culture models. The aim of this study was to determine whether the effects of t-10, c-12 CLA on adiponectin and TG are mediated through modulation of the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). 3T3-L1 cells were treated either during or after differentiation into adipocytes with 100 microM t-10, c-12 CLA with or without 10 microM troglitazone, a PPARgamma agonist, or 1 microM GW9662, a PPARgamma antagonist, and adiponectin and TG levels were analyzed. Treatment with t-10, c-12 CLA reduced TG as well as cellular and secreted adiponectin levels and impaired the assembly of adiponectin oligomers. These changes were accompanied by decreases in PPARgamma mass. Troglitazone was able to reverse the t-10, c-12 CLA-mediated decrease in TG levels and restore the assembly of adiponectin oligomers but was unable to restore adiponectin synthesis. Conversely, treatment with GW9662 decreased TG mass and impaired adiponectin oligomer assembly but did not decrease total adiponectin mass. In a reporter assay, t-10, c-12 CLA appeared to be a partial PPARgamma agonist and prevented the stimulation of reporter activity by troglitazone. Therefore, the t-10, c-12 CLA isomer appears to alter adipocyte adiponectin metabolism through PPARgamma-dependent and PPARgamma-independent mechanisms.

The hallmark of the dengue hemorrhagic fever/dengue shock syndrome is hematologic abnormality. The pathogenesis of dengue hemorrhagic fever/dengue shock syndrome remains unknown. Our work showed that the dengue virus serotype-2 induced apoptosis in human umbilical vein endothelial cells. Fas (CD95), Tumor Necrosis Factor receptors, and Tumor Necrosis Factor-related apoptosis-inducing ligand receptors are the most common death receptors, which can induce apoptosis. Compared with the untreated human umbilical vein endothelial cells, Fas expression was increased both in the mRNA level and on the surface of infected human umbilical vein endothelial cells. FasL was expressed at similar levels on human umbilical vein endothelial cells over a course of dengue virus serotype-2 infection, but the expression in mRNA level was increased in infected human umbilical vein endothelial cells. It is possible that there is soluble FasL secreted from human umbilical vein endothelial cells in the supernatant. Tumor Necrosis Factor-related apoptosis-inducing ligand receptor 1 and Tumor Necrosis Factor receptors 1-2 were constantly very low, whereas Tumor Necrosis Factor-related apoptosis-inducing ligand receptors 2-4 decreased after dengue virus serotype-2 infection. This result suggested that dengue virus serotype-2 may inhibit Tumor Necrosis Factor-related apoptosis-inducing ligand receptors-induced apoptosis. The apoptotic rates in human umbilical vein endothelial cells were decreased upon the addition of caspase family inhibitors. In addition, activated caspase 8 and caspase 3 were also observed by Western blot following dengue virus serotype-2 infection. Thus, it is shown that the Fas/FasL pathway may participate in dengue virus-induced apoptosis of vascular endothelial cells in vitro.

Apoptosis is a cardinal feature of liver injury. Death receptors are major mediators of the apoptotic pathway in the liver and have been implicated in the pathogenesis of diverse human diseases. More importantly, several studies have demonstrated a link between apoptosis and hepatic fibrosis; the latter is the most ominous consequence of chronic liver injury. In this article, we focus on the four death receptors: Fas, tumor necrosis factor receptor 1, tumor necrosis factor-related apoptosis-inducing ligand receptor 1, and tumor necrosis factor-related apoptosis-inducing ligand receptor 2. Although the death receptors have similar structures, they also possess distinct characteristics in their signaling pathways. Fas is abundantly expressed by all cells in the liver and plays a central role in variety of liver diseases. Tumor necrosis factor receptor 1 can induce both proapoptotic and prosurvival pathways. Tumor necrosis factor-related apoptosis-inducing ligand receptors likely trigger selective cell death in malignant and viral infected cells. Understanding the mechanism of liver injury caused by death receptors will enable therapeutic strategies to ameliorate human liver diseases.

HIV/hepatitis C virus (HCV) coinfection causes accelerated liver disease compared to HCV monoinfection, and only 30-60% of HIV/HCV-coinfected individuals respond to HCV therapy with pegylated interferon and ribavirin. There are currently no biomarkers that predict treatment response in these coinfected patients. We investigated whether there is an association between HCV treatment response and SNPs of apoptosis-related genes during HIV/HCV coinfection. Genomic DNA from 53 HIV/HCV-coinfected individuals was analyzed for 82 SNPs of 10 apoptosis-related genes. We found that the presence of the rs4242392 SNP in tumor necrosis factor receptor superfamily, member 10a (TNFRSF10A), which encodes for tumor necrosis factor-related apoptosis-inducing ligand receptor 1, predicts poor outcome to HCV therapy, in HIV/HCV-co-infected patients [odds ratio 5.91 (95% confidence interval 1.63-21.38, P = 0.007)]. The rs4242392 SNP of the tumor necrosis factor-related apoptosis-inducing ligand receptor 1 gene predicted poor interferon-based HCV treatment response in HIV/HCV-coinfected patients.

Feed is the main factor impacting the composition and quality of milk of dairy animals. Therefore, the present review explores the effects of feed and nutrition on milk fat content and levels of healthy fatty acids (FA) in milk consumed by humans. Milk and dairy products are two main sources of healthy and unhealthy FA in human nutrition. The concentrations of FA in milk depend mainly on diets; therefore, milk FA concentrations and ratios can be greatly altered by some feeding strategies. Dietary supplementation of the diets of dairy livestock with vegetable seeds or oils, microalgae and phytogenic feed additives, and feeding of some grasses can enhance the contents of healthy FA, including n-3 FA, α-linolenic acid, conjugated linoleic acid (CLA) and, generally, unsaturated FA in milk and dairy products. Enrichment of milk with healthy FA may make milk a source of anticarcinogens (CLA and polyphenols) for human health. This review, therefore, focusses on the current research findings on enrichment of milk with healthy FA and summarizes some effective supplementation strategies to alter milk FA profile.

Conjugated linoleic acid (CLA), a mixture of isomers of linoleic acid, has previously been shown to be able to decrease porcine subcutaneous (SC) adipose tissue levels while increasing the count of intramuscular (IM) adipose tissue in vivo. However, the underlying mechanisms through which it acts are poorly understood. The objective of this study was to investigate the different effects of CLA on adipogenesis in cultured SC adipose tissue and IM stromal vascular cells obtained from neonatal pigs. As shown here, trans-10, cis-12 CLA decreased the expression of adipocyte-specific genes as well as adipose precursor cell numbers and the accumulation of lipid in cultured SC adipose tissue stromal vascular cells. However, the cis-9, trans-11 CLA did not alter adipogenesis in SC cultures. On the other hand, both CLA isomers increased the expression of adipocyte-specific genes in IM cultures, together with the increasing accumulation of lipid and Oil Red O-stained cells. Collectively, these data show that CLA decreases SC adipose tissue but increases IM adipose tissue by different regulation of adipocyte-specific gene expression. These results suggest that adipogenesis in IM adipocytes differs from that in SC adipocytes.

Conjugated linoleic acid (CLA) causes insulin resistance and hepatic steatosis in conjunction with depletion of adipokines in some rodent models. Our objective was to determine whether the maintenance of adipokines, mainly leptin and adiponectin, by either removing CLA from diets or using an adiponectin enhancer, rosiglitazone (ROSI), could attenuate CLA-induced insulin resistance. Male C57BL/6 mice were consecutively fed two experimental diets containing 1.5% CLA mixed isomer for 4 weeks followed by a diet without CLA for 4 weeks. CLA significantly depleted adiponectin but not leptin and was accompanied by hepatic steatosis and insulin resistance. These effects were attenuated after switching mice to the diet without CLA along with restoration of adiponectin. To further elucidate the role of adiponectin in CLA-mediated insulin resistance, ROSI was used in a subsequent study in male ob/ob mice fed either control (CON) or CLA diet. ROSI maintained significantly higher adiponectin levels in CON- and CLA-fed mice and prevented the depletion of epididymal adipose tissue and the development of insulin resistance. In conclusion, we show that insulin resistance induced by CLA may be related more to adiponectin depletion than to leptin and that maintaining adiponectin levels alone either by removing CLA or using ROSI can attenuate these effects.

A meningioma is the most common primary intracranial tumor in adults. Here, we investigated the therapeutic potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in 37 meningiomas. Freshly isolated primary meningioma cells were treated with TRAIL with or without different sensitizing protocols, and apoptotic cell death was then quantified. Mechanisms of TRAIL sensitization were determined by a combination of Western blotting, flow cytometry, receptor complex immunoprecipitation, and siRNA-mediated knockdown experiments. Tumor necrosis factor-related apoptosis-inducing ligand receptor expression was analyzed using immunohistochemistry and quantified by an automated software-based algorithm. Primary tumor cells from 11 (29.7%) tumor samples were sensitive to TRAIL-induced apoptosis, 12 (32.4%) were intermediate TRAIL resistant, and 14 (37.8%) were completely TRAIL resistant. We tested synergistic apoptosis-inducing cotreatment strategies and determined that only the proteasome inhibitor bortezomib potently enhanced expression of the TRAIL receptors TRAIL-R1 and/or TRAIL-R2, the formation of the TRAIL death-inducing signaling complex, and activation of caspases; this treatment resulted in sensitization of all TRAIL-resistant meningioma samples to TRAIL-induced apoptosis. Bortezomib pretreatment induced NOXA expression and downregulated c-FLIP, neither of which caused the TRAIL-sensitizing effect. Native TRAIL receptor expression could not predict primary TRAIL sensitivity. This first report on TRAIL sensitivity of primary meningioma cells demonstrates that TRAIL/bortezomib cotreatment may represent a novel therapeutic option for meningiomas.

The peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated transcription factors within the broad nuclear receptor superfamily. The PPAR family includes three members encoded by distinct genes: α, β/δ, and γ (see reviews1,2). PPARα was originally identified as the intracellular receptor for a class of nongenotoxic rodent hepatocarcinogens, which includes the hypolipidemic drug clofibrate, a potent inducer of hepatic peroxisomal proliferation and hypolipidemic agent. The three PPARs are now distinguished by tissue- and developmental-specific patterns of expression and by the distinct, albeit overlapping, nature of ligands capable of activating each receptor. PPARα, which is abundant in tissues with high rates of mitochondrial fatty acid oxidation, such as heart, liver, and kidney, regulates a wide variety of target genes involved in cellular lipid catabolism. In contrast, PPARγ, an adipose-enriched nuclear receptor, directs the expression of genes involved in adipocyte differentiation and fat storage. The function of the ubiquitously expressed PPARβ/δ, is not well understood although some evidence suggests that it exerts actions on the epidermis and activates antiinflammatory programs. Ligand activation of PPARs leads to obligate heterodimerization with the 9- cis retinoic acid-activated receptor, RXR, promoting binding of the complex to cognate DNA response elements within PPAR target gene promoter regions (Figure). A variety of natural and synthetic compounds including fatty acids, eicosanoids, and arachidonic acid derivatives can serve as activators of the PPARs, some in a receptor-specific manner. However, the true endogenous ligands have not been identified. PPAR transcriptional regulatory complex. PPARs bind to sequence-specific target elements (PPREs) as a liganded heterodimer with the retinoid X receptor (RXR). Major functions based on known target genes and examples of relevant tissues (in parentheses) for each member of the PPAR family are shown. The question mark indicates that target genes for PPARβ/δ are largely unidentified. PPARα has been shown to …

Recent studies suggest that a circulating protein called TRAIL (TNF-related apoptosis inducing ligand) may have an important role in the treatment of type 2 diabetes. It has been shown that TRAIL deficiency worsens diabetes and that TRAIL delivery, when it is given before disease onset, slows down its development. The present study aimed at evaluating whether TRAIL had the potential not only to prevent, but also to treat type 2 diabetes. Thirty male C57BL/6J mice were randomized to a standard or a high-fat diet (HFD). After 4 weeks of HFD, mice were further randomized to receive either placebo or TRAIL, which was delivered weekly for 8 weeks. Body weight, food intake, fasting glucose, and insulin were measured at baseline and every 4 weeks. Tolerance tests were performed before drug randomization and at the end of the study. Tissues were collected for further analyses. Parallel in vitro studies were conducted on HepG2 cells and mouse primary hepatocytes. TRAIL significantly reduced body weight, adipocyte hypertrophy, free fatty acid levels, and inflammation. Moreover, it significantly improved impaired glucose tolerance, and ameliorated non-alcoholic fatty liver disease (NAFLD). TRAIL treatment reduced liver fat content by 47% in vivo as well as by 45% in HepG2 cells and by 39% in primary hepatocytes. This was associated with a significant increase in liver peroxisome proliferator-activated receptor (PPAR) γ (PPARγ) co-activator-1 α (PGC-1α) expression both in vivo and in vitro, pointing to a direct protective effect of TRAIL on the liver. The present study confirms the ability of TRAIL to significantly attenuate diet-induced metabolic abnormalities, and it shows for the first time that TRAIL is effective also when administered after disease onset. In addition, our data shed light on TRAIL therapeutic potential not only against impaired glucose tolerance, but also against NAFLD.

Obesity affects the cardiovascular system at many different levels, including the heart muscle itself. Clinical and experimental studies have shown an accumulation of triglycerides and other lipid species in cardiomyocytes. Analogous to hepatic steatosis, investigators have introduced the term “cardiac steatosis”. The present review addresses the complex relationships between cardiac fuel homeostasis, insulin resistance, and proposed mechanisms of damage to cardiomyocytes in different models of obesity, insulin resistance, and lipotoxicity. Specifically, the review weighs the evidence whether there is a heart muscle disorder in human obesity. It discusses how adipokines can modulate cardiac metabolism, and it focuses on the metabolic remodeling accompanying increased fatty acid supply in the heart of rodent models of lipotoxicity, with special attention to the role played by mitochondrial uncoupling and futile cycling. We stress the notion that, in spite of the many proposed mechanisms, cardiac lipotoxicity is still a hypothesis rather than an established pathophysiologic principle. Although the concept of a “lipotoxic cardiomyopathy” seems attractive, we propose instead a series of steps on a path from adaptation to maladaptation of the heart in obesity. A case in point is insulin resistance of the heart which may be both adaptive (protecting the heart from excess fuel) or maladaptive (associated with reactive oxygen species formation and activation of signaling pathways of programmed cell death). The present literature reflects an extraordinary complexity of the heart’s metabolic, functional and structural changes in obesity.

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.