A metabolic shift favoring sphingosine 1-phosphate at the expense of ceramide controls glioblastoma angiogenesis.

Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite sphingosine 1-phosphate (S1P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S1P over ceramide, which increases with increasing cancer grade. S1P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C18 ceramide, was on average 5-fold lower. Increased S1P content in the tumors was significantly correlated with increased sphingosine kinase 1 (SPHK1) and decreased sphingosine phosphate phosphatase 2 (SGPP2) expression. Inhibition of S1P production by cultured glioblastoma cells, using a highly potent and selective SPHK1 inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S1P balance is an important feature of human cancers and support the development of SPHK1 inhibitors as antiangiogenic agents for cancer therapy.

The discovery of novel isoflavone pan peroxisome proliferator-activated receptor agonists.

Twenty three dual PPARα and γ molecules of natural product origin, previously reported by our group, were further investigated for pan PPAR transactivation against PPARδ. The in vitro cell toxicity profile, as well as, in silico study of the most active molecules within this new class of pan PPAR agonists are also described. 3',5' Dimethoxy-7 hydroxyisoflavone 6, Ψ-baptigenin 7, 4' fluoro-7 hydroxyisoflavone 8, and 3' methoxy-7 hydroxyisoflavone 9 were identified as the most potent molecules studied within the set compared to the commercially available pan PPAR agonist, bezafibrate 1. These novel active molecules may thus be useful as future leads in PPAR-related disorders, including type II diabetes mellitus and metabolic syndrome.

7-Hydroxy-benzopyran-4-one derivatives: a novel pharmacophore of peroxisome proliferator-activated receptor alpha and -gamma (PPARalpha and gamma) dual agonists.

Design, synthesis, and in vitro bioevaluation of a new class of potential dual PPARalpha and gamma agonists discovered through a structure-driven design paradigm are described. The 7-hydroxy-benzopyran-4-one moiety (includes flavones, flavanones, and isoflavones) is the key pharmacophore of these novel molecules, exhibiting similarity to the core structure of both fibrates and thiazolidinediones. New lead PPAR ligands were identified from "natraceuticals" and synthetic analogues. In total, 77 molecules, including chalcones, flavones, flavanones, isoflavones, and pyrazole derivatives, were screened and structure-activity relationship studies of the dual agonists undertaken. Compounds 68, 70, 72, and 76 were identified as novel and potent dual PPARalpha and gamma agonists. These novel molecules may have the potential to be the future leads in PPAR-related disorders, including type II diabetes mellitus and metabolic syndrome.

A fluorescence-based assay for indoleamine 2,3-dioxygenase.

A rapid and sensitive fluorescence-based bioassay for determination of indoleamine 2,3-dioxygenase (IDO) activity has been developed. This assay relies on the quantification of the amount of kynurenine produced in the assay medium by fluorescence and complements the standard absorbance and high-performance liquid chromatography (HPLC) assay methods. The fluorescence method has limits of detection similar to those of the standard assay methods. Measured activities of IDO, including in the presence of tryptophan-based inhibitors, were in statistical agreement with the absorbance and HPLC assay methods. The fluorescence-based assay was also suitable for assessment of IDO inhibition by compounds that are incompatible with the absorbance method.

Optimised expression and purification of recombinant human indoleamine 2,3-dioxygenase.

The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its role in the pathogenesis of many diseases. Here, we report attempts to improve soluble expression and purification of hexahistidyl-tagged recombinant human IDO from Escherichia coli (EC538, pREP4, and pQE9-IDO). Significant formation of inclusion bodies was noted at the growth temperature of 37 degrees C, with reduced formation at 30 degrees C. The addition of the natural biosynthetic precursor of protoporphrin IX, delta-aminolevulinic acid (ALA), coupled with optimisation of IPTG induction levels during expression at 30 degrees C and purification by nickel-agarose and size exclusion chromatography, resulted in protein with 1 mol of heme/mol of protein and a specific activity of 160 micromol of kynurenine/h/mg of protein (both identical to native human IDO). The protein was homogeneous in terms of electrophoretic analysis. Yields of soluble protein (3-5 mg/L of bacterial culture) and heme content are greater than previously reported.