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Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y‐box as a cis‐acting element. As a parallel analysis of database records for 149 non–small‐cell lung cancer (NSCLC) cancer patients, we screened for trans‐acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer‐binding protein γ (CEBPγ) or Y‐box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide‐dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y‐box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1. [ABSTRACT FROM AUTHOR]

Sphingolipids constitute a class of bio‐reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non–small‐cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR‐101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1‐positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis. [ABSTRACT FROM AUTHOR]

In cancer cells the small compounds erastin and RSL3 promote a novel type of cell death called ferroptosis, which requires iron-dependent accumulation of lipid reactive oxygen species. Here we assessed the contribution of lipid peroxidation activity of lipoxygenases ( LOX) to ferroptosis in oncogenic Ras-expressing cancer cells. Several 12/15- LOX inhibitors prevented cell death induced by erastin and RSL3. Furthermore, si RNA-mediated silencing of ALOX15 significantly decreased both erastin-induced and RSL3-induced ferroptotic cell death, whereas exogenous overexpression of ALOX15 enhanced the effect of these compounds. Immunofluorescence analyses revealed that the ALOX15 protein consistently localizes to cell membrane during the course of ferroptosis. Importantly, treatments of cells with ALOX15-activating compounds accelerated cell death at low, but not high doses of erastin and RSL3. These observations suggest that tumor ferroptosis is promoted by LOX-catalyzed lipid hydroperoxide generation in cellular membranes. [ABSTRACT FROM AUTHOR]

The effects of silyl and hydrophilic groups on the photodynamic properties of tetraphenylporphyrin (TPP) derivatives have been studied in vitro and in vivo. Silylation led to an improvement in the quantum yield of singlet oxygen sensitization for both sulfo and carboxy derivatives, although the silylation did not affect other photophysical properties. Silylation also improved the cellular uptake efficiency for both sulfo and carboxy derivatives, enhancing the in vitro photodynamic activity of the photosensitizer in U251 human glioma cells. The carboxy derivative (SiTPPC4) was found to show higher cellular uptake efficiency and in vitro photodynamic activity than the corresponding sulfo derivative (SiTPPS4), which indicates that the carboxy group is a more promising hydrophilic group than the sulfo group in the silylated porphyrin. SiTPPC4 was found to show high selective accumulation efficiency in tumors, although almost no tumor selectivity was observed for the nonsilylated porphyrin. The concentration of SiTPPC4 in tumors was 13 times higher than that in muscle 12 h after drug administration. We also studied tumor response after treatment and found that silylation enhanced in vivo photodynamic activity significantly. SiTPPC4 shows higher photodynamic activity than NPe6 with white light irradiation. [ABSTRACT FROM AUTHOR]

ABSTRACT Previous studies on the metabolic fate of resatorvid (TAK-242) have shown that species differences in the pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate (M-III), a metabolite of TAK-242, between rats and dogs are mainly attributable to the urinary excretion process. In the present study, the renal uptake mechanism of M-III was investigated using kidney slices and Xenopus laevis oocytes expressing rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 ( Slc22a8). The uptake of p-aminohippuric acid (PAH), a substrate for Oats, by kidney slices from rats and dogs increased at 37 °C and M-III inhibited the uptake. The initial uptake clearance of M-III by rat kidney slices was 0.295 and 0.0114 ml/min/g at 37 °C and 4 °C, respectively. The Eadie-Hofstee plot of M-III uptake at 37 °C revealed two-component transport processes with Km values being 6.48 and 724 µmol/l. The uptake was inhibited by probenecid (PBC), PAH and benzylpenicillin (PCG). In contrast, in dog kidney slices, the initial uptake clearance of M-III was 8.70 × 10-3 and 9.00 × 10-3 ml/min/g at 37 °C and 4 °C, respectively, and the uptake was not inhibited by PBC. Furthermore, rOat1- and rOat3-expressing oocytes mediated M-III uptake and the uptake was inhibited by PAH and PCG, respectively. These results suggest that rOat1 and rOat3 are responsible for the renal uptake of M-III in rats. Moreover, it is speculated that Oat(s) is unable to transport M-III in dogs and that the difference in the substrate recognition of Oat(s) contributes to the species difference in the pharmacokinetics of M-III between rats and dogs. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We recently reported that TAK-593, a novel imidazo[1,2- b]pyridazine derivative, is a highly potent and selective inhibitor of the vascular endothelial growth factor ( VEGF) and platelet derived growth factor ( PDGF) receptor tyrosine kinase families. Moreover, TAK-593 exhibits a uniquely long-acting inhibitory profile towards VEGF receptor 2 ( VEGFR2) and PDGF receptor β ( PDGFRβ). In this study, we demonstrated that TAK-593 potently inhibits VEGF- and PDGF-stimulated cellular phosphorylation and proliferation of human umbilical vein endothelial cells and human coronary artery smooth muscle cells. TAK-593 also potently inhibits VEGF-induced tube formation of endothelial cells co-cultured with fibroblasts. Oral administration of TAK-593 exhibited strong anti-tumor effects against various human cancer xenografts along with good tolerability despite a low level of plasma exposure. Even after the blood and tissue concentrations of TAK-593 decreased below the detectable limit, a pharmacodynamic marker (phospho VEGFR2) was almost completely suppressed, indicating that its long duration of enzyme inhibition might contribute to the potent activity of TAK-593. Immunohistochemical staining indicated that TAK-593 showed anti-proliferative and pro-apoptotic effects on tumors along with a decrease of vessel density and inhibition of pericyte recruitment to microvessels in vivo. Furthermore, dynamic contrast-enhanced magnetic resonance imaging revealed that TAK-593 reduced tumor vessel permeability prior to the onset of anti-tumor activity. In conclusion, TAK-593 is an extremely potent VEGFR/ PDGFR kinase inhibitor whose potent anti-angiogenic activity suggests therapeutic potential for the treatment of solid tumors. [ABSTRACT FROM AUTHOR]

Prior to secretion, regulated peptide hormones are selectively sorted to secretory granules ( SGs) at the trans-Golgi network ( TGN) in endocrine cells. Secretogranin III ( SgIII) appears to facilitate SG sorting process by tethering of protein aggregates containing chromogranin A ( CgA) and peptide hormones to the cholesterol-rich SG membrane ( SGM). Here, we evaluated the role of SgIII in SG sorting in AtT-20 cells transfected with small interfering RNA targeting SgIII. In the SgIII-knockdown cells, the intracellular retention of CgA was greatly impaired, and only a trace amount of CgA was localized within the vacuoles formed in the TGN, confirming the significance of SgIII in both the tethering of CgA-containing aggregates and the establishment of the proper SG morphology. Although the intracellular retention of proopiomelanocortin ( POMC) was considerably impaired in SgIII-knockdown cells, residual adrenocorticotropic hormone ( ACTH)/ POMC was still localized to some few remaining SGs together with another granin protein, secretogranin II ( SgII), and was secreted in a regulated manner. Biochemical analyses indicated that SgII bound directly to the SGM in a cholesterol-dependent manner and was able to retain the aggregated form of POMC, revealing a latent redundancy in the SG sorting and retention mechanisms, that ensures the regulated secretion of bioactive peptides. [ABSTRACT FROM AUTHOR]

There is urgent need for biomarkers that provide early detection of pancreatic ductal adenocarcinoma (PDAC) as well as discrimination of autoimmune pancreatitis, as current clinical approaches are not suitably accurate for precise diagnosis. We used mass spectrometry to analyze protein profiles of more than 300 plasma specimens obtained from PDAC, noncancerous pancreatic diseases including autoimmune pancreatitis patients and healthy subjects. We obtained 1063 proteomic signals from 160 plasma samples in the training cohort. A proteomic signature consisting of 7 mass spectrometry signals was used for construction of a proteomic model for detection of PDAC patients. Using the test cohort, we confirmed that this proteomic model had discrimination power equal to that observed with the training cohort. The overall sensitivity and specificity for detection of cancer patients were 82.6% and 90.9%, respectively. Notably, 62.5% of the stage I and II cases were detected by our proteomic model. We also found that 100% of autoimmune pancreatitis patients were correctly assigned as noncancerous individuals. In the present paper, we developed a proteomic model that was shown able to detect early-stage PDAC patients. In addition, our model appeared capable of discriminating patients with autoimmune pancreatitis from those with PDAC. [ABSTRACT FROM AUTHOR]

Aim: Nestin, an intermediate filament originally identified as a marker of neural progenitor cells, is transiently expressed in endothelial cells and tubuloepithelial cells during kidney development. However, in adult kidneys, podocytes are the only cells that express nestin. In this study, we examined tubulointerstitial nestin expression in human glomerulonephritis. Methods: Renal biopsy specimens obtained from 41 adult patients with immunoglobulin (Ig)A nephropathy were studied. Nestin expression was determined by immunohistochemical staining and estimated by digital image analysis. To identify the phenotype of nestin-positive cells, a double immunofluorescent study was performed for nestin and CD34 (a marker for endothelial cells) or α-smooth muscle actin (α-SMA, a marker for myofibroblasts). Results: In normal kidney, nestin expression was restricted to the podocytes and was not detected in tubular cells and tubulointerstitial cells. In contrast, increased nestin expression was observed at tubulointerstitial areas of IgA nephropathy. The degree of tubulointerstitial nestin expression was positively correlated with tubulointerstitial fibrosis ( r = 0.546, P < 0.001). The double immunofluorescent study showed that most nestin-positive cells in the interstitium were co-stained with CD34 or α-SMA, suggesting that peritubular endothelial cells and tubulointerstitial myofibroblasts express nestin during the progression of tubulointerstitial injury. In addition, strong nestin expression was associated with deterioration of renal function. Conclusion: Nestin expression is associated with tubulointerstitial injury and predicts renal prognosis in IgA nephropathy. Nestin could be a new marker for peritubular endothelial cell injury and tubulointerstitial fibrosis. [ABSTRACT FROM AUTHOR]

Phogrin is an integral glycoprotein primarily expressed in neuroendocrine cells. The predominant localization of phogrin is on dense-core secretory granules, and the lumenal domain has been shown to be involved in its efficient sorting to the regulated secretory pathway. Here, we present data showing that a leucine-based sorting signal [EExxxIL] within the cytoplasmic tail contributes its steady-state localization to secretory granules. Deletion mutants in the tail region failed to represent granular distribution in pancreatic β-cell line, MIN6, and anterior pituitary cell line, AtT-20. A sorting signal mutant with two glutamic acids substituted into alanines (EE/AA) is primarily accumulated in the Golgi area instead of secretory granules, and another mutant (IL/AA) is trapped at the plasma membrane due to a defect in endocytosis. We further demonstrate that the leucine-based sorting signal of phogrin specifically interacts with both adaptor protein (AP)-1 and AP-2 clathrin adaptor complexes in vitro. These observations, along with previous studies, suggest that distinct domains of phogrin mediate proper localization of this transmembrane protein on secretory granules. [ABSTRACT FROM AUTHOR]

Rab GTPases regulate the membrane transport pathways by recruiting their specific effector proteins. Melanophilin, a putative Rab effector, has recently been identified as a gene that is mutated in leaden mice, in which peripheral localization of melanosomes is impaired in melanocytes. Genetic studies suggest that three coat-color mutation genes, dilute (MyoVad), ashen (Rab27aash), and leaden (Mlphln), act in the same or overlapping pathways. Here we have cloned and characterized a human melanophilin homolog, which belongs to the rabphilin3/granuphilin-like Rab effector family. Cosedimentation assays using recombinant proteins reveal that melanophilin directly binds to Rab27a and myosin Va through its N-terminal and its first C-terminal coiled-coil region, respectively. Moreover, we show that Rab27a, melanophilin, and myosin Va form a ternary complex in the human melanocyte cell line HMV-II. These findings suggest that melanophilin has a role in bridging Rab27a on melanosomes and myosin Va on actin filaments during melanosome transport. We also propose that the Rab-binding region conserved in a novel rabphilin3/granuphilin-like Rab effector family constitutes an α-helix-based coiled-coil structure. [Copyright &y& Elsevier]