In FA transport and the response to a fat diet and specific defect in FA uptake in the proximal intestine of CD362/2 mice is associated with reduced incorporation of FA in TG and a diminished TG secretion. This concept was however AP24534 challenged. Published observations have shown that CD36 genetic deletion does not affect intestinal lipid uptake and the efficient participation of CD36 in LCFA intestinal uptake was questioned. It was suggested that CD36 functions as a FA sensor and stimulates events that control FA metabolism rather than being directly involved in the lipid transit. In any case, our findings show that small inhibitors of the CD36 binding functions can significantly reduce the postprandial BAY 73-4506 hypertriglyceridemia which follows a gastric olive challenge. Again, when compared to a complete deletion of the gene, which favor redundant mechanisms, a partial inhibition of CD36 functions may have different consequences. Our findings demonstrate that a selective down regulation of CD36 in the intestine reduces lipid intake and is beneficial to postprandial hypertriglyceridemia. In conclusion, CD36 is generally recognized as an important lipid and FA receptor which plays a role in the metabolic syndrome and its associated cardiac events. The pleiotropic activity and the various molecular associations of this scavenger in different cells and tissues have however questioned its potential as a safe therapeutic target. Different published observations have indeed suggested that CD36 down regulation might not been beneficial due to redundant mechanisms or potential toxicity. The present study shows that it is possible to identify small molecules that can block the CD36 binding and uptake functions and that such antagonism can reduce atherosclerosis, postprandial hypertriglyceridemia and be beneficial for type II diabetes. Particularly, elevated postprandial hypertriglyceridemia is a metabolic parameter which is now recognized to be strongly associated with cardiovascular events and is independent of traditional cardiovascular risk factors. Thus, CD36 might represent an attractive therapeutic target. Its expression can be induced by a broad spectrum of growth factors, hormones, or stress stimuli, and it is associated with various chronic conditions. Studies in mice have revealed that Mig-6 is required for skin morphogenesis and lung development and that it plays an important role in maintaining joint homeostasis. As a cytoplasmic scaffolding adaptor, MIG-6 has several important protein-protein interaction motifs that may mediate interaction with signaling molecules downstream of receptor tyrosine kinases. One of the most prominent roles of MIG-6 in regulating signal transduction comes from its ability to directly interact with epidermal growth factor receptor and other ErbB family members, inhibiting their phosphorylation and downstream signaling in a negative feedback fashion. MIG-6 can be induced by hepatocyte growth factor and functions as a negative feedback regulator of HGF-MET signaling, indicating that it has broad role as a signal checkpoint for modulating activated RTK pathways in a timely manner. The evidence that MIG-6 is a tumor suppressor gene is compelling. It is located in chromosome 1p36, a locus that frequently has loss of heterozygosity in several human cancers including lung cancer, melanoma, and breast cancer. Indeed, down-regulation or loss of MIG-6 expression has been reported in cancers and is often associated with poor prognosis. MIG-6 down-regulation in non-small cell lung cancer is associated with increased EGFR signaling and poorly differentiated cancer, while loss of its expression in ErbB2-amplified breast carcinoma renders the cancer cells more resistant to Herceptin, the neutralizing antibody against ErbB2.

While MIG-6 down-regulation is reported in a high percentage of papillary thyroid cancers, high MIG-6 expression correlates with longer survival and is associated with favorable surgical outcomes for those patients. Decreased MIG-6 expression has also been reported in skin cancer, endometrial cancer, and PR-171 hepatocellular carcinomas. Moreover, even though such events are rare, three mutations in the MIG-6 gene have been identified in human lung cancer and one in neuroblastoma. Further evidence supporting MIG-6 as a tumor suppressor gene arose from mouse studies; Mig-6deficient mice are prone to develop epithelial hyperplasia or tumors in organs including the lung, skin, uterus, gallbladder, and bile duct. Epigenetic alteration, one of the most well-known mechanisms leading to inactivation of a tumor suppressor gene, can result from DNA methylation or histone deacetylation in the gene’s promoter. Given that down-regulation of MIG-6 is frequently observed in many human cancers, we asked whether MIG-6 expression was affected by DNA methylation and histone deacetylation. Here, we show that the MIG-6 PI-103 promoter itself is neither hypermethylated nor affected by histone deacetylation. However, its expression is induced by the DNA methyltransferase inhibitor 5-aza-29-deoxycytidine in melanoma cell lines and by the histone deacetylase inhibitor trichostatin A in lung cancer lines. By dissecting its promoter regulatory region using a luciferase reporter assay, we identified a minimal TSA-response element in exon 1 of MIG-6 that is essential for its induction by TSA in lung cancer cells. Compared with the wild-type P reporter, mutation in the m4 and m5 elements resulted in a significant decrease of reporter activity in response to TSA, while mutation in other elements had lesser effect. This result agrees with the deletion analyses, as the m4 and m5 elements are within the distal 20nucleotide segment that, when deleted, resulted in a steep drop-off in TSA response. We generated another mutant reporter m11 in which half of the sequences in both m4 and m5 were mutated. We found that the m11 mutant had a much greater reduction in TSA response, indicating that those sequences are essential for the binding of a yet to be identified transcription factor which regulates MIG-6 gene expression induced by TSA in the lung cancer. MIG-6, a tumor suppressor gene, has been found downregulated in many human cancers. To determine if downregulation of MIG-6 expression was affected by epigenetic modification in its promoter, we treated lung cancer and melanoma cell lines with inhibitors of methylation and histone deacetylation and then determined how those inhibitors influenced MIG-6 expression. Intriguingly, we found that DNMT inhibitor 5aza-dC specifically induced MIG-6 expression in melanoma cells but not in lung cancer cells, while the HDAC inhibitor TSA induced the reverse pattern. Despite both inductions being regulated at transcriptional level, we were surprised to find that the MIG-6 promoter was neither hypermethylated nor directly affected by histone deacetylation, indicating that an indirect mechanism might be responsible for differential induction. In fact, 5-aza-dC has also been reported to induce the expression of several other genes whose promoters are not directly affected by methylation in leukemia cells, suggesting that 5-aza-dC might have a broader influence on regulating gene expression via a methylation-independent manner. Many DNMT inhibitors and HDAC inhibitors are currently in clinical trials for their anti-cancer properties.

While both wt and shFKBP5 xenograft mice were able to benefit from combination therapy with gemcitabine and the Akt inhibitor, triciribine, shFKBP5 mice showed a greater effect after combination treatment. We recently reported that FKBP5 is a scaffolding protein that can enhance PHLPP-Akt interaction. The functional consequence of this interaction results in negative regulation of Akt activity. Down regulation of FKBP5 results in decreased PHLPP-Akt interaction and increased Akt phosphorylation at the Ser473 site, suggesting that FKBP5 may function as a tumor suppressor, an important fact contributing to chemoresistance. Based on our previous findings with FKBP5 and its role in chemoresistance, we tested this hypothesis in vivo using a xenograft mice model. We found that tumors in shFKBP5 mice were more resistant to gemcitabine treatment and also exhibited a faster tumor growth rate. This phenomenon appeared to involve the regulation of Akt activation, as determined by phosphorylated Akt and downstream signaling molecules. Since Akt is activated when FKBP5 is Selumetinib knocked down,we hypothesized that the addition of inhibitors targeting this pathway might reverse the drug resistance phenotype. The PI3K-Akt pathway has multiple drugable targets, so we tested a series of inhibitors targeting PI3K, Akt and mTOR. In addition to the role of FKBP5 in chemoresistance, based on our xenograft models it could also function as a tumor suppressor through negative regulation of the Akt pathway. As shown in Figures 3 and 5A, activity of the Akt pathway is significantly higher in FKBP5 knockdown SU86 xenografts than that in wild type SU86 xenografts and these observations correlated with higher tumor growth rates in shFKBP5 mice. Therefore, probably because of the higher basal levels of Akt activity, shFKBP5 xenografts responded better to combination treatment, which was seen as enhanced inhibition of tumor growth. This phenomenon was also reflected by decreased Akt 473 phosphorylation levels after gemcitabine and TCN treatment. The shFKBP5 xenografts showed a more dramatic decrease in Akt 473 phosphorylation levels wt xenografts. Our in vivo results further confirmed findings observed using the cell lines. Those studies demonstrated that lack of expression of FKBP5 led to increased Akt phosphorylation at the regulatory S473 amino acid residue as well as for downstream genes in the Akt pathway such as phosphorylated FOXO1 and GSK3b. Therefore, FKBP5 could be a tumor suppressor in pancreatic cancer and it could also be a biomarker for response to chemotherapy, especially gemcitabine therapy, a first line treatment for pancreatic cancer. Our findings that a specific Akt inhibitor can reverse resistance to gemcitabine in FKBP5 knockdown cells and xenografts indicate that FKBP5 levels might be used to stratify patients into different treatment arms, such as gemcitabine or gemcitabine plus an Akt inhibitor. Future clinical studies will be needed to test this hypothesis. In addition, the mechanisms underlying differences between the effects of PI3K inhibition, mTOR inhibition and Akt inhibition in combination with gemcitabine need to be explored further. PI3K activation causes phosphatidylinositol-3,4,5-triphosphate dependent membrane localization of Akt and PDK1, in which the latter can phosphorylate Akt 308. Therefore, the inhibition of PI3K might have less effect on 473 phosphorylation. Rapamycin can potentially LY294002 in vivo activate Akt 473 phosphorylation in an mTOR-2 dependent manner due to relief of feedback inhibition of IGF-1R signaling. That may explain why treatment with rapamycin plus gemcitabine failed to show a significant reduction of Akt 473 phosphorylation.

Unity from the Sybyl package was used for pharmacophore filtering. Pharmacophoric points were defined protein based code 2v8p) with default settings to include the desired directionalities for hydrogen bonding. The initial pharmacophore search was performed with flexible ligand molecules, allowing rotation and conformational changes to match the required features. At least two of the possible four features had to be fulfilled to pass this filter. For filtering the docking poses, the docked ligands were kept rigid and no translations and rotations were allowed. A database containing all compounds passing the pharmacophore filter step in a format suitable for docking and considering multiple protonation states and tautomers was prepared as described previously. The AaIspE crystal structure was the receptor for docking. Four different setups were prepared taking into account the possible tautomers of His25, and the presence or absence of the co-factor. Polar hydrogen atoms were added to the receptor and their positions minimised using the MAB force field as implemented in MOLOC. Partial charges for the co-factor were calculated using AMSOL. Spheres as matching points for docking were placed around the cytidine heterocycle of the bound substrate. The sphere set defining the buried region of the binding site was generated around the whole substrate and cofactor. Grids to store information about excluded volumes, electrostatic and van der Waals potential, and solvent occlusion were calculated as described earlier. Each docking pose which did not place any atoms in areas occupied by the receptor was scored for electrostatic and van der Waals complementarity and Lapatinib penalised according to its estimated partial desolvation energy. The docking setup was validated by successful predictions of the binding modes of CDP, CDP-ME, and cytosine. For each compound in the screening database, only the best-scoring representation was stored in the final docking hit list. Cytidine analogues such as gemcitabine are widely used to treat a variety of cancers. Gemcitabine remains standard therapy for pancreatic cancer in the adjuvant and palliative settings. However, the gemcitabine response rate is very low in pancreatic cancer, with only an 18% 1 year survival rate. This poor survival rate is primarily because of the lack of early detection and frequent metastasis of primary tumors into lymph nodes and surrounding organs, such as the liver and stomach. As a step toward individualized gemcitabine therapy in order to achieve better outcomes, we previously performed a genome wide association study using 197 individual lymphoblastoid cell lines and identified a protein, FKBP5, that showed a significant effect on gemcitabine response in tumor cells by negatively regulating Akt phosphorylation at serine 473. Phosphorylation of Akt activates the Akt pathway, which plays a critical role in tumorigenesis and chemoresistance. Therefore, low FKBP5 expression renders tumor cells resistant to many chemotherapeutic agents, including gemcitabine. In addition, FKBP5 expression is low or lost in many pancreatic cancer cell lines and pancreatic cancer patient samples, correlating with increased Akt Ser473 phosphorylation. These results suggested that FKBP5 might be a tumor suppressor and that levels of FKBP5 might determine patients’ response to TWS119 chemotherapy. If that is correct, patients with low levels of FKBP5 and Akt hyperactivation might benefit from the addition of inhibitors targeting the Akt pathway. In the current study, we tested that hypothesis by using an FKBP5 knockdown pancreatic cancer xenograft mouse model and the results of these experiments may form a foundation for future clinical translational studies.

The plasma glycome which revealed very high variability in the composition of plasma glycome in the population and identified individuals having significantly aberrant glyco-phenotypes, some of which could be BAY-60-7550 associated with specific diseases. To test potential therapeutic usefulness of epigenetic inhibitors TSA, sodium butyrate and zebularine in inducing a reversal of undesired glyco-phenotypes, we developed an HPLCbased method for the determination of glycan structures from cells embedded in polyacrylamide gels. In addition, we specifically investigated the preservation of altered glycan profiles over a prolonged period of time in a drug-free environment. Our results emphasize the importance of epigenetic control in the regulation of N-glycosylation, but also suggest the stability of complex biosynthetic pathways responsible for the establishment of glycan profiles in human cells in culture. Indeed, it has been shown that the overexpression of glycoproteins with oligomannose N-glycans at the cell surface induces proliferative and adhesive properties of cancer cells and potentially facilitates loss of contact inhibition, a hallmark feature of majority of cancer cells. Since glycans can also be obtained following disruption of cellular integrity, we decided to compare the methodological precision of N-glycan analysis from embedded cells to the total Nglycome obtained by the analysis of HeLa cell lysates. The observed glycan profiles were somewhat different, with particular glycan groups being selectively more abundant in the lysates or the embedded cell fraction. This is not surprising since Golgi and ER, which get disrupted during cell lysis, contain high amounts of both complete and partly synthesized glycans and these can alter the proportion of a particular glycan group. Each of the two glycan release methods was repeated six times to estimate their reproducibility. For nearly all glycan peaks, we observed higher coefficients of variation when glycome was analyzed from cell lysates. Especially high experimental variation was observed in highly branched glycan structures, which are important for the regulation of membrane half-life of many receptors. This increased variation of complex structures from experiment to experiment probably reflects their small contribution to the total glycome. Since glycans function as regulators of the activity of membrane proteins, many of them are also attached to proteins on the luminal side of various cytoplasmic vesicles, including the Golgi apparatus where posttranslational glycan processing occurs. Homogenization of cells results in mixing of these two compartments, containing physiologically separate fractions of glycans, and consequently leads to masking of relatively subtle, but functionally important, differences in the cell membrane Nglycome. Therefore, we believe that the analysis of Nglycome from embedded cells results in improved analytical precision due to elimination of the homogenization step from the procedure. Based on these observations we decided to further exploit the method of glycan analysis from embedded cells. We validated this hypothesis by mannosidase treatment, which resulted in Tubulin Acetylation Inducer almost complete disappearance of the corresponding chromatographic peaks, thus confirming the contribution of free oligomannose glycans to the pool of glycans released by PNGase F from glycoproteins associated with the cell membrane.